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PMC9624034 | Peng Song,Ying Li,Feng Wang,Lingxiao Pu,Linsen Bao,Hengfei Gao,Chuandong Zhu,Meng Wang,Liang Tao | Genome-wide screening for differentially methylated long noncoding RNAs identifies LIFR-AS1 as an epigenetically regulated lncRNA that inhibits the progression of colorectal cancer | 31-10-2022 | LIFR-AS1,Methylation markers,Prognosis,Colorectal cancer | Background Aberrant DNA methylation is an epigenetic marker that has been linked to the pathogenesis of colorectal cancer (CRC). Long noncoding RNAs (lncRNAs) have been increasingly identified to be associated with tumorigenic processes of CRC. Identifying epigenetically dysregulated lncRNAs and characterizing their effects during carcinogenesis are focuses of cancer research. Methods Differentially methylated loci and expressed lncRNAs were identified by integrating DNA methylome and transcriptome analyses using The Cancer Genome Atlas database. Bisulfite sequencing PCR (BSP) was performed to analyze LIFR-AS1 promoter methylation status. The functional roles of LIFR-AS1 in CRC were determined by in vitro and in vivo experiments. Results We identified a novel hypermethylated lncRNA, LIFR-AS1, that was downregulated and associated with tumorigenesis, metastasis, and poor prognosis in CRC. High methylation burden of LIFR-AS1 indicated a poor survival of CRC patients. Promoter hypermethylation of LIFR-AS1 in tumor tissues was confirmed by BSP. Functional assays revealed that LIFR-AS1 could competitively bind to hsa-miR-29b-3p, and repressed colon cancer cell proliferation, colony formation and invasion. LIFR-AS1 also inhibited tumor growth in a mouse xenograft model of CRC. Conclusions Our results showed that the identified DNA methylation-dysregulated lncRNAs may be potential biomarkers and highlighted a role for LIFR-AS1 as a tumor suppressor in CRC. Supplementary Information The online version contains supplementary material available at 10.1186/s13148-022-01361-0. | Genome-wide screening for differentially methylated long noncoding RNAs identifies LIFR-AS1 as an epigenetically regulated lncRNA that inhibits the progression of colorectal cancer
Aberrant DNA methylation is an epigenetic marker that has been linked to the pathogenesis of colorectal cancer (CRC). Long noncoding RNAs (lncRNAs) have been increasingly identified to be associated with tumorigenic processes of CRC. Identifying epigenetically dysregulated lncRNAs and characterizing their effects during carcinogenesis are focuses of cancer research.
Differentially methylated loci and expressed lncRNAs were identified by integrating DNA methylome and transcriptome analyses using The Cancer Genome Atlas database. Bisulfite sequencing PCR (BSP) was performed to analyze LIFR-AS1 promoter methylation status. The functional roles of LIFR-AS1 in CRC were determined by in vitro and in vivo experiments.
We identified a novel hypermethylated lncRNA, LIFR-AS1, that was downregulated and associated with tumorigenesis, metastasis, and poor prognosis in CRC. High methylation burden of LIFR-AS1 indicated a poor survival of CRC patients. Promoter hypermethylation of LIFR-AS1 in tumor tissues was confirmed by BSP. Functional assays revealed that LIFR-AS1 could competitively bind to hsa-miR-29b-3p, and repressed colon cancer cell proliferation, colony formation and invasion. LIFR-AS1 also inhibited tumor growth in a mouse xenograft model of CRC.
Our results showed that the identified DNA methylation-dysregulated lncRNAs may be potential biomarkers and highlighted a role for LIFR-AS1 as a tumor suppressor in CRC.
The online version contains supplementary material available at 10.1186/s13148-022-01361-0.
Colorectal cancer (CRC) is the third most common malignancy and the second highest cancer-related cause of death in the world [1]. CRC is caused by the accumulation of multiple genetic and epigenetic alterations in the genome [2, 3]. Over the past decade, research has focused on better understanding of cancer epigenetics, particularly regarding aberrant DNA methylation, microRNA and long noncoding RNA (lncRNA) deregulation, to identify prognostic and predictive factors for cancer [3]. DNA methylation is a reversible and regulatory modification, and changes in DNA methylation can occur in the early stage of cancer development. DNA methylation has also been shown to be a candidate biomarker in cancer [4]. For example, SEPT9 gene methylation has been implicated as a biomarker for predicting CRC [5]. Methylated SEPT9 gene in serum is closely related to the advanced stage of CRC [6]. Increasing studies have revealed cancer-linked aberrant methylation of protein-coding gene promoters. However, the genome-wide identification of differential DNA methylation and expression of lncRNAs with functional importance in CRC is still lacking. LncRNAs are crucial regulators at the transcriptional and post-transcriptional levels and are involved in diverse biological functions [7]. Aberrant lncRNA expression in cancers can be caused by alteration of epigenetic patterns, such as changes in DNA methylation. For instance, Lin et al. reported that lncRNA DLX6-AS1 hypermethylation was present in colorectal neoplasms at all stages and increased during colorectal carcinogenesis [8]. Hypermethylation of DLX6-AS1 was also detected in cell-free DNA samples from CRC patients. Mamivand et al. identified an epigenetically deregulated lncRNA OBI1-AS1 with decreased expression in glioblastoma multiforme. Hi-C and ChIP-Seq analysis showed that methylation of the CTCF binding site blocked the expression of OBI1-AS1 by influencing chromatin interactions [9]. LncRNAs also function as a scaffold for the recruitment of chromatin modifiers to target promoters. In CRC cells, linc00337 recruited DNA methyltransferase 1 (DNMT1) to the promoter region of CNN1 and restricted its transcription, promoting tumor growth and angiogenesis [10]. DNA methylation aberrations in cancer and the crosstalk with lncRNAs are research hotspots. The availability of high-throughput sequencing technology has facilitated the exploration of epigenetic changes across the genome. Therefore, here we used a reannotation strategy to construct the DNA methylation profile of lncRNAs in CRC. In this study, we screened and identified methylation-driven differentially expressed lncRNAs in CRC, with the aim of improving diagnosis and personalized treatment of CRC patients.
The DNA methylation array data (Illumina Infinium Human Methylation 450 BeadChip) and level 3 RNA-sequencing data (HTSeq-Counts and HTSeq-FPKM-UQ) along with clinicopathological information were downloaded from the UCSC Xena browser (https://xenabrowser.net/). Level 3 miRNA-seq data were obtained through TCGA Genomic Data Commons portal (GDC). We also downloaded RNA sequencing data of CRC from the Gene Expression Omnibus (GEO, www.ncbi.nlm.nih.gov/geo) database (GSE156451). An in-house dataset including 92 CRC tissues and 43 normal tissues from patients who underwent surgery at Nanjing Drum Tower Hospital (The Affiliated Hospital of Nanjing University Medical School, Nanjing, China) was also used for analysis. All patients were pathologically diagnosed with colon adenocarcinoma following the American Joint Committee on Cancer’s criteria. None of the patients received preoperative chemotherapy or radiotherapy. The adjacent normal tissues were collected > 5 cm from the tumor margins. All patients provided written informed consent, and this study was approved by the Research Ethics Committee of Nanjing Drum Tower Hospital.
To identify differentially expressed lncRNAs, RNA-seq read count tables mapped on the hg38 human genome with GENCODE v22 as gene annotation were imported into three statistically-based expression analysis tools (edgeR [11], limma [12], and DESeq2 [13]). Differentially methylated CpG sites between CRC samples and adjacent tissues were identified using the “minfi” package. Conjoint analysis of the methylome and transcriptome of lncRNAs was performed as previously described [14]. Briefly, the genomic coordinates of each CpG site and individual lncRNA were extracted from hm450.hg38.manifest and GENCODE v22 files, respectively [14]. We combined both information using the above genomic location, taking the differentially methylated loci within promoter regions (DNA sequences between −2500 and 1000 bp relative to the putative transcription start site) into account.
Cellular genomic DNA was isolated from fresh frozen tissues using the Genomic DNA extraction kit (TIANamp Genomic DNA Kit, DP304). The purified DNA was bisulfite-treated using the EpiTect Fast DNA Bisulfite Kit (Qiagen) following the manufacturer’s protocol. The bisulfite-modified promoter regions were amplified by BSP primers (forward primer: GGAGGAAAAATTTTATTTTATTAAGA, reverse primer: ACCRAACCCAAACAAATCCTC). Amplified sequences were cloned into the pMD18-T vector and sequenced. The sequencing results were compared to the original sequence using the QUMA website (http://quma.cdb.riken.jp/). To determine the methylation rate, 10 clones were required for each sample.
Total RNA was extracted from tissues or cells using TRIzol reagent (Invitrogen, USA), and reverse transcription was conducted with the Primescript RT Reagent Kit (TaKaRa, Japan) following company protocols. RT-qPCR was performed on the 7900 Real-Time PCR System (Applied Biosystems, USA) using the SYBR Premix Ex Taq Kit (TaKaRa, China). The primers used for amplification were as follows: F: 5′- AAGTTTCAGGCTCCTGACAGC -3′ and R: 5′- TTCGCCCACGTTCTTCTCGC -3′ for LIFR-AS1, F: 5′- TGGAACGACAGGGGTTCAGT -3′ and R: 5′- GAGTTGTGTTGTGGGTCACTAA -3′ for LIFR, and F: 5′- AGAAGGCTGGGGCTCATTTG -3′ and R: 5′- AGGGGCCATCCACAGTCTTC -3′ for GAPDH.
The human colon cancer cell lines LOVO, HCT116, SW480, SW620, DLD-1, HT-29 and T84 were purchased from the Chinese Academy of Sciences, China. Cells were cultured in RPMI 1640 medium (Gibco, USA), and supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin. For overexpression of LIFR-AS1, the sequence of LIFR-AS1 was synthesized and subcloned into the pcDNA3.1 plasmid. HT-29 and T84 cells were transfected with the LIFR-AS1 expression plasmid (oe-LIFR-AS1) or the empty vector (NC) as control using Lipofectamine 3000 (Invitrogen, USA). Overexpression was evaluated by RT-qPCR.
Amplification of cDNA fragments of LIFR-AS1 wild type (WT) and mutant (MUT) containing binding sites of hsa-miR-29b-3p were cloned into a psiCHECK-2 vector (Promega, USA). The LIFR-AS1_WT vector or LIFR-AS1_MUT vector and hsa-miR-29b-3p mimics were co-transfected into HT-29 and T84 cells by Lipofectamine 3000 (Invitrogen, USA). After 24 h of culture, the luciferase intensity was assessed by Promega Dual-Luciferase Reporter Assay System.
For colony formation assays, approximately 1 × 103 cells in DMEM medium supplemented with 10% FBS were cultured at 37 °C and 5% CO2 for two weeks. Cell colonies were photographed and counted. For CCK-8 assays, transfected cells were plated into 96-well plates (approximately 1 × 104/well) and incubated with CCK-8 solution (Dojindo, Japan) for 1 h at 37 °C. The optical density value at 450 nm was measured. For invasion assays, cells at a density of 1 × 105 cells/ml were seeded in the upper chambers of 24-well transwell systems (Corning, USA) coated with a polycarbonate membrane, and 600 ml DMEM medium containing 15% FBS was added into the lower chamber. Cells were cultured for 24 h. After removing non-invading cells, the remaining cells were fixed with 4% paraformaldehyde and stained with 0.1% crystal violet (Beyotime, China) at room temperature for 30 min. The invaded cells were counted under five randomly selected views using a phase-contrast microscope (Nikon, Tokyo, Japan).
Transfected HT-29 cells (1 × 107/ml) were injected into 6-week-old female BALB/C nude mice. Tumor growth (determined by measuring length and width) was monitored and recorded. Tumor volume was calculated by the formula (length × width2 × 1/2). The mice were euthanized after 4 weeks, and the tumors were extracted and analyzed. This study was approved by the Animal Ethics Committee of Experimental Animal Center of Nanjing Drum Tower Hospital.
The R package ClusterProfiler was used to perform Gene Set Enrichment Analysis (GSEA) and plot the results [15]. The input data was ranking metric using the value of log2 (Fold Change) calculated by “DESeq2” package. The reference gene sets were retrieved from the C2 collection in the Molecular Signatures Database. Single-cell RNA-sequencing (scRNA-seq) data for CRC (GSE144735) were obtained from the GEO database [16]. Seurat package was used for downstream analysis, and the t-SNE algorithm was used for nonlinear dimension reduction of the scRNA-seq data [17].
The Student’s t test or Mann–Whitney test (for continuous variables) and Pearson’s χ2 test (for categorical variables) were used to examine differences between two groups. Kaplan–Meier method and log-rank test were employed to evaluate gene expression or methylation level on the survival of patients. Multivariate Cox regression analysis was used to estimate adjusted hazard ratios and 95% confidence intervals for LIFR-AS1 expression. All statistical analysis was conducted on R programming language v4.1.2, and two-sided and P < 0.05 was defined as statistically significant.
We first performed differential expression analysis for lncRNAs using RNA-seq data of 471 primary colon tumors and 41 normal tissues from The Cancer Genome Atlas (TCGA). Using three algorithms (edgeR, limma and DESeq2), a total of 1137 lncRNAs (825 upregulated lncRNAs and 312 downregulated lncRNAs) were identified using the criteria of false discovery rate (FDR) < 0.05 and absolute fold change (FC) ≥ 2 (Fig. 1A, B). Long intergenic noncoding RNAs (lincRNAs) accounted for most (45.03%) of all differentially expressed lncRNAs, followed by antisense transcripts (36.59%) (Additional file 1: Fig. S1). To explore the DNA methylation pattern of lncRNAs in CRC, we then compared the differentially methylated CpG sites in 309 tumor and 38 normal tissues. A total of 16,266 CpG sites with FDR < 0.05 were obtained in the promoters of lncRNAs (Fig. 1C). We further identified 432 differentially methylated regions from the following parameters: resamples = 100, cut-off = 0.2, and probe number ≥ 2. These two omics data (methylome and transcriptome) were combined for further analysis. By associating the 16,266 CpG sites to 1137 lncRNAs, 276 pairs of methylation-driven lncRNAs were identified (Additional file 2: Table S1). The top five hypermethylated lncRNAs (BVES-AS1, ZNF582-AS1, FGF14-AS2, NOVA1-AS1 and LIFR-AS1) and top five hypomethylated lncRNAs (AC017002.2, LINC00152, RP1-140K8.5, LINC00460 and RP11-474D1.4) in CRC are shown in Fig. 1D.
The highest levels of methylation were observed in LIFR-AS1. Therefore, we performed further analysis to explore its biological function. Ten CpG sites in the LIFR-AS1 promoter (cg05923785, cg08392199, cg12587766, cg20699036, cg18174928, cg03723506, cg12602374, cg11841722, cg18848688 and cg01369082) were significantly correlated with LIFR-AS1 expression via MEXPRESS (correlation coefficients from − 0.159 to − 0.246, Fig. 2A, Additional file 3: Fig. S2). Colon tumors exhibited significantly higher levels of hypermethylation compared with normal tissues (Fig. 2B). Among the above 10 CpG sites, cg12587766 showed prominent methylation with a mean delta beta value of 0.559. Kaplan–Meier curves showed that four CpG sites (cg12587766, cg18174928, cg12602374, and cg18848688) were associated with the overall 5-year relative survival rate of CRC patients, which indicated that high methylation burden group had significantly poor survival (Fig. 2C). LIFR-AS1 is located on chromosome 5 (38,556,786–38,671,216) and encodes two different transcripts: NR_103554.1 is 3386 bp in length with nine exons and NR_103553.1 is 3803 bp in length and contains three exons. RNA-seq is widely used for transcript quantification of gene isoforms. We used RNA expression data available from GSE156451 to identify the products transcribed from this locus in CRC [18]. Consistent with our previous results, the expression of LIFR-AS1 was downregulated in CRC (Fig. 3A). As shown in Fig. 3B, most aligned reads were mapped within the exons in NR_103554.1. We thus chose this sequence for further analysis. We ranked 18,524 genes from CRC samples in the TCGA dataset by their relative LIFR-AS1 expression in the top 10th percentile vs. the bottom 10th percentile for GSEA. The “MIKKELSEN_IPS_ICP_WITH_H3K4ME3_AND_H327ME3”, “NABA_CORE_MATRISOME” and “SABATES_COLORECTAL_ADENOMA_DN” sets were enriched in the LIFR-AS1 high expression group, which implied that this lncRNA might be involved in colorectal carcinogenesis (Fig. 3C). Using the threshold selection of absolute correlation coefficient > 0.1 and P value < 0.05, we found that 7681 genes were positively correlated and 3387 genes were negatively correlated with the expression of LIFR-AS1 (Fig. 3D). As scRNA-seq technology could not detect the expression of LIFR-AS1, we then investigated the expression of LIFR-AS1-related genes (LIFR, EDIL3, EPHA3, MRPS5, LETM1 and SNRPF) in 1678 epithelial cells from CRC (Fig. 3E). Interestingly, despite a low level of expression overall, EDIL3 was highly expressed in normal epithelial cells compared with CRC cells. In addition, elevated levels of LETM1 and SNRPF were detected in malignant epithelial cells (Fig. 3F). The LIFR-AS1 sequence overlaps with the LIFR gene (Fig. 4A). LIFR was downregulated in CRC tumors compared with paired normal tissues in TCGA dataset (Fig. 4B). Both TCGA and GSE156451 databases analyses showed that the expression level of LIFR-AS1 in CRC tissues was positively correlated with LIFR expression (Fig. 4C). The 10 CpG sites were also negatively correlated with the expression of LIFR (correlation coefficients from − 0.020 to − 0.385, Fig. 4D). However, overexpression of lncRNA LIFR-AS1 did not affect the mRNA level of LIFR in colon cancer cells (Fig. 4E). Analysis of scRNA-seq data revealed that LIFR was markedly increased in stromal cells and its expression was apparent in epithelial cells (Fig. 4F). LIFR-AS1 also harbors a microRNA, miR-3650, that is transcribed in an antisense orientation. We speculated whether miR-3650 suppressed LIFR; whereas miR-3650 was not detected in CRC (Fig. 4G). Based on the differential analysis by the Wilcoxon test, we identified 517 miRNAs (202 upregulated miRNAs and 315 downregulated miRNAs) as significantly differentially expressed in CRC tissue compared with the normal samples in TCGA database (Additional file 4: Fig. S3). Then the downstream upregulated miRNAs targeted by LIFR-AS1 in CRC were determined using StarBase [19]. Correlation analysis showed that the expression levels of six target miRNAs (hsa-miR-30b-5p, hsa-miR-30e-5p, hsa-miR-4677-3p, hsa-miR-374b-5p, hsa-miR-29b-3p and hsa-miR-144-3p) were negatively correlated with LIFR-AS1 (Fig. 4H, Additional file 5: Fig. S4). We selected the most significantly associated miRNA (hsa-miR-29b-3p) into the further study. As expected, hsa-miR-29b-3p mimic prominently decreased luciferase activity in LIFR-AS1_WT group in comparison with LIFR-AS1_MUT group in both HT-29 and T84 cell lines, which indicated that hsa-miR-29b-3p is a target of lncRNA LIFR-AS1 (Fig. 4I).
The CpG island and 33 CpG sites were also predicted by MethPrimer (Fig. 5A). Therefore, the BSP approach was applied to determine the methylation status of the LIFR-AS1 promoter region in 18 colorectal tumor tissues and adjacent normal tissues (Fig. 5B, C). We observed markedly elevated average DNA methylation levels with a mean of 36.1% in CRC specimens compared with 5.5% in paired adjacent normal tissues. Consistent with our bioinformatic analysis, the LIFR-AS1 promoter region was significantly hypermethylated (P < 0.001, Fig. 5D). To determine the diagnostic potential of LIFR-AS1 promoter methylation status, ROC curve analysis was performed. The optimal cut-off of LIFR-AS1 promoter methylation (12.3%) was defined by maximizing the Youden index (sensitivity + specificity − 1). This cut-off discriminated between CRC and normal tissues with a sensitivity of 77.8%, a specificity of 88.9%, and an area under the curve (AUC) value of 0.872 (Fig. 5E). Moreover, we found a potential correlation between DNA methylation in the promoter region of LIFR-AS1 and LIFR with their mRNA expression, respectively (Fig. 5F).
We measured LIFR-AS1 expression in our cohort of 43 tumor and paired adjacent normal tissues using RT-qPCR. The 2 − ΔΔCT method was used to calculate FC for LIFR-AS1 compared with the internal control GAPDH mRNA. LIFR-AS1 was downregulated in CRC compared with normal tissues (FC = − 1.50, P = 0.008, Fig. 6A). We also found that LIFR-AS1 expression positively correlated with TNM stage and lymph node metastasis in 92 CRC patients (Fig. 6B). Moreover, forest plot from multivariate regression analysis demonstrated that high LIFR-AS1 related to increased overall survival (P < 0.05, Fig. 6C). Kaplan–Meier analysis revealed poor survival for patients with low expression of LIFR-AS1 compared with patients with high expression of LIFR-AS1 (P = 0.010, Fig. 6D). Thus, these findings suggest that LIFR-AS1 might be an independent predictor of CRC aggressiveness.
We next investigated the potential roles of LIFR-AS1 in CRC in vitro and in vivo. RT-qPCR demonstrated that the expression of LIFR-AS1 in colon cancer cells (HT-29 and T84) was appreciably lower than that in other cells (LOVO, HCT116, SW480, SW620 and DLD-1) (Fig. 7A). We thus chose HT-29 and T84 cells for further analysis and overexpressed LIFR-AS1 using oe-LIFR-AS1 (Fig. 7B). Both HT-29 and T84 cells overexpressing LIFR-AS1 displayed lower cell viabilities than the respective control cells (Fig. 7C). Stable overexpression of LIFR-AS1 induced a significant decrease in colony formation and cell invasion (Fig. 7D, E). To confirm the above observed phenotype, xenograft mouse models were established. The results showed that overexpression in LIFR-AS1 cells decreased the average volume and weight of tumors (Fig. 7F–H). Together, these findings indicate that LIFR-AS1 functions as a tumor suppressor in CRC.
Recent multi-omics analysis has demonstrated that cancer involves a complex regulatory network that harbors both genetic and epigenetic abnormalities, contributing to escape from chemotherapy and host immune surveillance [20, 21]. Advances in high-throughput sequencing technologies have led to the identification of individual molecular heterogeneity. Epigenetics including DNA methylation and histone modifications are a new research focus in cancer [22]. Methylation features were found to be closely linked to CRC patient prognosis. For example, methylation levels in the intragenic regions of oncogenes (PDX1, EN2, and MSX1) levels could predict CRC patient prognosis [23]. In this study, we performed an integrated methylome and transcriptome analysis to identify potential lncRNAs regulated by aberrant DNA methylation in CRC. By precisely mapping altered DNA methylation to the promoter regions of lncRNAs, 276 epigenetically deregulated lncRNAs in CRC were identified. To confirm the accuracy of the approach, BSP assay was performed, and the results showed high methylation status of LIFR-AS1 promoter region. Four CpG sites (cg12587766, cg18174928, cg12602374 and cg18848688) and the expression of LIFR-AS1 were related to the prognosis of CRC. Overexpression of LIFR-AS1 was shown to remarkably suppress colon cancer cell proliferation, growth and invasion in vitro and in vivo. LIFR-AS1 is located on chromosome 5p13.1 and transcribed in an antisense manner from the LIFR gene. Several human solid tumors have been shown to exhibit aberrant expression of LIFR-AS1 [24–26]. Liu et al. found that LIFR-AS1 and miR-29a negatively regulated each other through direct binding in PDT-treated HCT116 cells [24]. LIFR-AS1 knockdown reduced the effect of PDT on proliferation and apoptosis of CRC cells, implying that LIFR-AS1 may act as a tumor suppressor via interacting with miR-29a. Pan et al. observed that the expression levels of LIFR-AS1 were significantly increased in gastric tumor tissues and cells compared with normal adjacent tissue samples and GSE1 cells [26]. LIFR-AS1 modulates COL1A2 to promote gastric cancer cell proliferation and migration by miR-29a-3p. Chen et al. reported that METTL3-mediated m6A hyper-methylation induced the upregulation of LIFR-AS1 in pancreatic cancer by enhancing METTL3 mRNA stability, resulting in increased expression of VEGFA by directly interacting with miR-150-5p [25]. LIFR-AS1 has been shown to act as a sponge for miR-942-5p in lung cancer [27], for miR-29a in CRC [24] and osteosarcoma [28], for miR-31-5p in thyroid carcinoma [29], for miR-4262 in glioma [30], for miRNA-150-5p in pancreatic cancer [25], for miR-197-3p in breast cancer [31], and for miR-29a-3p [26] and miR-4698 [32] in gastric cancer. These results prompted us to investigate the ceRNA function of LIFR-AS in CRC. Interestingly, we found that LIFR-AS1 could interact with hsa-miR-29b-3p through luciferase reporter gene in colon cancer cells. Considering the heterogeneity of LIFR-AS1 in cancers, we performed scRNA-seq on CRC and non-tumor cells of the single-cell expression to characterize the functional role of LFIR-AS1 indirectly. Intriguing, SNRPF, which is negatively linked to LIFR-AS1, was highly expressed in CRC cells. A previous study showed that SNRPN was highly expressed in CRC tissues and high SNRPN expression indicated a poor prognosis [33]. Additionally, the GSEA-mined “Genes downregulated in colorectal adenoma compared to normal mucosa samples” gene set was related to LIFR-AS1. Colorectal adenomas are often precursor lesions of CRC. These phenomena suggested LIFR-AS1 is an important tumor-suppressive lncRNA during carcinogenesis. A strong association was observed between LIFR-AS1 and LIFR in CRC, whereas LIFR expression was not detected in epithelial cells. Rockman et al. demonstrated that colonic epithelial cells express LIF protein but not LIFR. Conversely, pericryptal fibroblasts express LIFR but not LIF protein [34]. This is consistent with the results of scRNA-analysis in detecting LIFR expression in stromal cells. Meanwhile, overexpression of LIFR-AS1 did not affect the expression of LIFR in colon cancer cells. Differences in tissue distribution led to little biological correlation between LIFR-AS1 and LIFR. As a result, the displayed relationship between LIFR-AS1 and LIFR might due to the methylation status in the promoter region. Methylation of cytosines in the human genome is a critical epigenetic modification that functions in transcriptional silencing [35]. DNA methylation regulates tissue-specific gene expression [36]. However, the genome-wide identification of abnormal DNA methylation in a specific lncRNA region with functional importance is lacking. In this study, we found hypermethylation of a CpG island located in the promoter region of the tumor suppressor gene LIFR-AS1 that enhanced cancer progression. The methylation level of LIFR-AS1 had high sensitivity and specificity for the diagnosis of CRC. Recent studies indicate that aberrant DNA methylation is an early and frequent event in carcinogenesis [37]. We found that early CRC tumors had high levels of LIFR-AS1 methylation, and four CpG sites were associated with prognosis. Detection of the methylation level of LIFR-AS1 might be a promising biomarker for CRC screening. Combining bioinformatic analysis and experimental verification, our research highlighted the role of LIFR-AS1 in the progression of CRC. However, our study has several limitations. First, conjoint analysis identified a series of methylation-driven lncRNAs, and we only chose LIFR-AS1 for validation. Second, LIFR-AS1 appeared to play a significant role in determining the developing of colon cancer both in vitro and in vivo, and recent studies reported that LIFR-AS1 could interact with other molecules in multiple cancers. Thus, LIFR-AS1 may be involved in other cancers in addition to CRC. Third, whether the methylation status of LIFR-AS1 functions as an independent diagnostic and prognostic marker for CRC remains to be investigated and requires clinical multicenter studies with larger samples to confirm our findings. Fourth, the FC in LIFR-AS1 overexpression experiments was much higher than that in RNA-seq or our validation cohort and might not reflect changes in the human body. In conclusion, by using integrative analysis and molecular experiments, we revealed that the promoter region of LIFR-AS1 was hypermethylated in CRC and was negatively associated with the expression of LIFR-AS1. Furthermore, LIFR-AS1 was correlated with poor outcome of CRC patients and repressed tumor cell growth and metastasis. Overall, our results indicate that aberrant DNA methylation mediates downregulation of LIFR-AS1 to promote the progression of colon cancer.
Additional file 1: Figure S1. Pie chart shows the number of differentially expressed lncRNAs in each category.Additional file 2: Table S1. The characteristics of methylation-driven lncRNAs.Additional file 3: Figure S2. Correlation (P values derive from Spearman’s correlation) between DNA methylation and the expression of LIFR-AS1 in CRC samples.Additional file 4: Figure S3. Volcano plot showing the log2 fold change of 517 significantly differentially expressed miRNAs in CRC patients from the TCGA database.Additional file 5: Figure S4. Prediction of 6 miRNAs (miR-29b-3p, miR-4677-3p, miR-144-3p, miR-30b-5p, miR-30e-5p and miR-374b-5p) targeting LIFR-AS1 in CRC. | true | true | true |
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PMC9624087 | Virginie Ravigné,Nathalie Becker,François Massol,Erwan Guichoux,Christophe Boury,Frédéric Mahé,Benoit Facon | Fruit fly phylogeny imprints bacterial gut microbiota | 03-05-2022 | community ecology,long‐read sequencing,metabarcoding | A bstract One promising avenue for reconciling the goals of crop production and ecosystem preservation consists in the manipulation of beneficial biotic interactions, such as between insects and microbes. Insect gut microbiota can affect host fitness by contributing to development, host immunity, nutrition, or behavior. However, the determinants of gut microbiota composition and structure, including host phylogeny and host ecology, remain poorly known. Here, we used a well‐studied community of eight sympatric fruit fly species to test the contributions of fly phylogeny, fly specialization, and fly sampling environment on the composition and structure of bacterial gut microbiota. Comprising both specialists and generalists, these species belong to five genera from to two tribes of the Tephritidae family. For each fly species, one field and one laboratory samples were studied. Bacterial inventories to the genus level were produced using 16S metabarcoding with the Oxford Nanopore Technology. Sample bacterial compositions were analyzed with recent network‐based clustering techniques. Whereas gut microbiota were dominated by the Enterobacteriaceae family in all samples, microbial profiles varied across samples, mainly in relation to fly identity and sampling environment. Alpha diversity varied across samples and was higher in the Dacinae tribe than in the Ceratitinae tribe. Network analyses allowed grouping samples according to their microbial profiles. The resulting groups were very congruent with fly phylogeny, with a significant modulation of sampling environment, and with a very low impact of fly specialization. Such a strong imprint of host phylogeny in sympatric fly species, some of which share much of their host plants, suggests important control of fruit flies on their gut microbiota through vertical transmission and/or intense filtering of environmental bacteria. | Fruit fly phylogeny imprints bacterial gut microbiota
One promising avenue for reconciling the goals of crop production and ecosystem preservation consists in the manipulation of beneficial biotic interactions, such as between insects and microbes. Insect gut microbiota can affect host fitness by contributing to development, host immunity, nutrition, or behavior. However, the determinants of gut microbiota composition and structure, including host phylogeny and host ecology, remain poorly known. Here, we used a well‐studied community of eight sympatric fruit fly species to test the contributions of fly phylogeny, fly specialization, and fly sampling environment on the composition and structure of bacterial gut microbiota. Comprising both specialists and generalists, these species belong to five genera from to two tribes of the Tephritidae family. For each fly species, one field and one laboratory samples were studied. Bacterial inventories to the genus level were produced using 16S metabarcoding with the Oxford Nanopore Technology. Sample bacterial compositions were analyzed with recent network‐based clustering techniques. Whereas gut microbiota were dominated by the Enterobacteriaceae family in all samples, microbial profiles varied across samples, mainly in relation to fly identity and sampling environment. Alpha diversity varied across samples and was higher in the Dacinae tribe than in the Ceratitinae tribe. Network analyses allowed grouping samples according to their microbial profiles. The resulting groups were very congruent with fly phylogeny, with a significant modulation of sampling environment, and with a very low impact of fly specialization. Such a strong imprint of host phylogeny in sympatric fly species, some of which share much of their host plants, suggests important control of fruit flies on their gut microbiota through vertical transmission and/or intense filtering of environmental bacteria.
Agro‐ecosystems comprise a significant proportion of land use and harbor a non‐negligible fraction of biodiversity (Pimentel et al., 1992; Tilman et al., 2011). More than many others, these ecosystems suffer from intense structural anthropogenic alterations. Conflicting imperatives to intensify production while simultaneously reducing environmental impacts increasingly drive short‐term and fine‐scale ecological and evolutionary processes (Thrall et al., 2011), demanding greater capacity to predict and manage their consequences (Gilligan, 2008). One promising avenue for reconciling the goals of crop production and ecosystem preservation consists in manipulating quantitatively and/or qualitatively beneficial biotic interactions (Gaba et al., 2014; Massol & Petit, 2013). Over the last decade, this strategy has taken a new turn by considering risks and opportunities associated with plant and insect microbiota. In particular, microbes associated with phytophagous insect are thought to offer great potential for improved management of economically important pests (Crotti et al., 2012). For instance, gut bacteria can be used to reverse radiation‐induced fitness decrease in sterile males used in the sterile insect technique, to produce new bacterial odoriferous attractants for insect traps, or to stimulate insect behaviors such as feeding or oviposition (Noman et al., 2020; Raza et al., 2020). Yet, identification of the associated microbial species, and of their respective role in plant–insect interactions and dynamics, is still far from complete. There is now good agreement on the idea that microbes may play an important role in host adaptation (Macke et al., 2017). In particular, one of the major arenas for host–microbe interactions is the insect gut, which is typically colonized by a large number of diverse microbes, among which bacterial associations predominate (Engel & Moran, 2013). Empirical evidence accumulates, showing that insect gut microbiota can affect host fitness by contributing to development, host immunity, nutrition, or behavior (Kolodny et al., 2020). Gut microbes have even been suspected to be the hidden key player of plant exploitation by their insect pests, as, for example, for the olive fly Bactrocera oleae (Ben‐Yosef et al., 2014) and the coffee berry borer Hypothenemus hampei (Ceja‐Navarro et al., 2015). Gut microbiota are complex, heterogeneous, and variable communities of microbes. First, they assemble within each host generation through different transmission routes. Specifically, gut microbes are mainly acquired via horizontal transfer from the surrounding environment (Broderick & Lemaitre, 2012). However, a number of mechanisms exist for inoculating progeny with microbial symbionts, increasing rates of vertical transmission, and enabling long‐term associations (Engel & Moran, 2013). For example, in some flies eggshells are contaminated with parental bacteria (Capuzzo et al., 2005; Raza et al., 2020). Even when acquired horizontally at each generation, gut communities are not random assemblages of bacteria from the food or local environment, due to host filtering and promoting specific bacteria (Engel & Moran, 2013). Second, insect species vary immensely in their dependence on gut microbes: Some almost lack them entirely, while others have developed obligate dependence (Moran et al., 2019). Third, host–microbiota interactions extend along the parasite–mutualist continuum and the exact position may change according to the cost–benefit balance resulting from interactions between bacteria composing the microbiota (Mushegian & Ebert, 2016). Fourth, gut microbiota are often considered as having a multilayered structure (Shapira, 2016). One layer would be the so‐called core microbiota, which would tend to be under host genetic and immune control, reliably transmitted across generations, and sharing evolutionary interests with the host (Macke et al., 2017). Some of these microbes may be beneficial to the host and contribute to essential functions or provide long‐term adaptation to stable features of the host niche (Nougué et al., 2015). A second layer would be composed of a flexible, environment‐modulated pool of microbes, varying within the course of individual life and exhibiting high interindividual variation. Because of possibly divergent evolutionary interests, microbes from this second layer could either be beneficial or detrimental to the host (Macke et al., 2017), potentially depending on the rest of the gut microbiota members (Mushegian & Ebert, 2016). In relation to this important variability of insect–microbe associations, understanding the role of gut microbiota in plant–insect interactions may benefit from deciphering the determinants of gut microbiota composition and structure. Gut microbiota are affected by many factors, including host phylogeny and host ecology (Spor et al., 2011). First, the environment in which insects develop and live strongly determines the set of bacteria, with which they will have an opportunity to associate. In phytophagous insects, the environments encountered are not random. They depend on insect ecology, a major feature of which is host range, that is, the host plant species an insect uses. For instance, one could expect that insect species specialized on different host plants encounter different initial microbe pools and that generalist insect species encounter a more diverse set of microbes than specialist species (Deb et al., 2019). Second, host phylogeny could potentially structure insect gut microbiota through different mechanisms ranging from active filters (constrained by host development, immune function morphology, and physiology), to the sharing of similar microbe pools (through social interactions or similarity in diet; Brooks et al., 2016). While host phylogeny, host specialization, and sampling environment factors are all considered as potential determinants of gut microbial communities, their relative importance is still a matter of debate, not only because it probably varies across taxa but also because of the associated technical challenge. Studies generally compare gut microbiota among related host species with contrasting ecologies in natural environments (Ivens et al., 2018), and through broad phylogenetic sampling of animals with both divergent and convergent feeding ecologies (Nishida & Ochman, 2018). However, in addition to their differences in phylogenetic history and level of specialization, surveyed host species may differ in their geographic ranges, thus experiencing different microbial species pools in their local environment. Controlled or laboratory environments, used for studies of closely related host taxa (Erlandson et al., 2018; Kohl et al., 2018), may partially reduce this bias. However, sampled microbial pools are unlikely to be representative of those encountered in the wild. This limitation can be overcome by analyzing microbiota in sympatric species of known ecology and phylogenetic history (Martinson et al., 2017). Reunion, a small island in South‐West Indian Ocean, harbors a community of eight sympatric fruit flies, considered as the main actors in the local guild of fruit‐eating phytophagous arthropods (Quilici & Jeuffrault, 2001), which could constitute a convenient system to tackle this question. These species belong to five genera from two tribes of the Tephritidae family (Moquet et al., 2021): Three species are Ceratitinae (Ceratitis capitata, Ceratitis quilicii, and Neoceratitis cyanescens), and five species are Dacinae (closely related species Bactrocera dorsalis, Bactrocera zonata, and Zeugodacus cucurbitae on the one hand, and Dacus ciliatus and Dacus demmerezi, on the other). They differ in their level of specialization: Four are generalist species (the Ceratitis and Bactrocera species, commonly found on more than 30 plant species of several distant plant families), three are specialists of Cucurbitaceae (the Dacus and Zeugodacus species), and one is a specialist of Solanaceae (N. cyanescens). Most importantly, both tribes comprise specialist and generalist species. Gut microbiota of Tephritidae have received great attention among those of phytophagous insects due to their promises for innovative pest management strategies (Deutscher et al., 2018; Noman et al., 2020), and because Tephritidae, which have a worldwide distribution, include some of the most economically damaging fruit and vegetable crop pests (Qin et al., 2015). The functional role of some particular bacterial taxa has been investigated within Tephritidae revealing links with nutritional provisioning (Behar et al., 2005), resistance to pathogenic bacteria (Behar, Yuval, et al., 2008), social interactions (Hadapad et al., 2016), pesticide resistance (Cheng et al., 2017), and foraging behavior (MacCollom et al., 2009). More recently, metabarcoding studies using next‐generation sequencing have helped describe the diversity and structure of the gut bacterial communities associated with wild Tephritid flies (Noman et al., 2020). These studies have uncovered a substantial diversity of gut bacteria with a strong predominance of the Proteobacteria phylum, including many genera of the Enterobacteriaceae family. Some conclusions, such as the lower diversity of microbial communities harbored in laboratory‐reared insects compared with field‐collected ones (Liu et al., 2016; Ras et al., 2017), and a core microbiota found only at the family level (De Cock et al., 2020; Deutscher et al., 2019), were shared by most studies. However, these studies also came to contrasted conclusions about the relative importance of host plants (Behar, Jurkevitch, et al., 2008; Majunder et al., 2019; Malacrinò et al., 2018; Ventura et al., 2018) or fruit fly species (De Cock et al., 2020; Morrow et al., 2015) in determining the composition and variation of gut bacterial communities in natural populations. Here, we aimed at using the fruit fly community of Reunion Island to test the contributions of fly phylogeny, fly specialization, and fly sampling environment on the composition and structure of their bacterial gut microbiota. To do so, for each species, whose precise host range and phylogenetic history are known, we studied bacterial gut communities in samples from two contrasted environments (field vs. laboratory). Assessing the amount of network variation driven by different environmental and biological factors is still an experimental and statistical challenge (Joffard et al., 2019). Here, bacterial inventories were conducted using 16S metabarcoding with the Oxford Nanopore Technology, reported to confer a greater taxonomic resolution than Illumina at the genus level (Matsuo et al., 2021; Nygaard et al., 2020), and hence a key feature to dig into the diversity of Enterobacteriaceae. Moreover, meaningful network analyses relied on the framework recently proposed by Massol et al. (2021), based on two methods: (i) group decomposition followed by canonical correspondence analysis (CCA) and (ii) singular value decomposition (SVD) followed by redundancy analysis (RDA).
Details on each sample are provided in Table S1, Appendix S2. Field samples were collected in several localities between April and June 2018. When possible, flies were caught with pheromone traps in places where several host plants coexist. For species with no efficient trap, flies were collected from sets of infested fruits from a given locality (details in Table S1, Appendix S2). As pheromone traps only attract males, only male individuals were included in the study. Differences in gut composition between the sexes have been found nonsignificant in a preliminary study on C. capitata and B. dorsalis (not shown), and in previous studies on B. dorsalis (Andongma et al., 2015; Liu et al., 2018) and on another Tephritid species (Bactrocera carambolae, Yong et al., 2017). Laboratory flies were collected using mouth aspirator in populations maintained in the laboratory of Plant Populations and Bio‐agressors in Tropical Ecosystems Joint Research Unit (Saint‐Pierre, Reunion Island). All flies were stored for at least 48 h in fresh 90% ethanol at −30°C in a 10× liquid/fly volume ratio to optimize washing and dilution of any external bacteria. One hour prior to dissection, flies were rinsed at ambient temperature by successive buffers providing three more washes (75% ethanol, 50% ethanol, and 25% ethanol, 5 min each), while ensuring a progressive rehydration of the abdominal tissues for dissection. Dissection of the abdominal gut portion was performed on a sterilized glass slide with a pair of sterile tweezers under a stereomicroscope. The abdominal gut portion includes the midgut and the ileum of the hindgut, excluding anterior thoracic crop, foregut, and posterior rectum. For each sample, guts from around 30 males were dissected under sterile conditions and pooled. DNA extraction from dissected guts was performed using the DNeasy Blood & Tissue Kit (Qiagen) following the manufacturer's instructions, adding 0.5% N‐lauroyl sarkosyl (Merck KGaA) for 30’, 65°C at the end of the lysis step. DNA was subsequently checked for quantity and quality with a NanoDrop 2000 (Thermo Fisher Scientific Inc.).
For each sample, ~10 ng of DNA was amplified using specific primers that target the whole 16S rRNA gene (27F 5′‐AGAGTTTGGATCMTGGCTCAG‐3′; 1492R 5′‐GGTTACCTTGTTACGACTT‐3′), as well as subsequent specific barcodes using a 16S Barcoding Kit (SQK‐RAB204; Oxford Nanopore Technologies). After bead purification for removal of excess primers, amplification products were attached to rapid sequencing adapters before being loaded on a MinION flow cell for real‐time sequencing. Samples were analyzed in three separate experiments (RUN1, RUN2C, and RUN3 in barcodes cited in Table S1), each containing a mock community sample (more details in Appendix S1).
Basecalling, demultiplexing, and chimera removal were performed using Guppy v4.0.11 (https://community.nanoporetech.com). Reads were trimmed (only nucleotides between positions 60 and 1460 bp of the 16S rRNA gene were kept) and filtered (only sequences longer than 900 bp and above quality score Q10 were kept) using Nanofilt (De Coster et al., 2018), leading to a total of 268,960 sequences (ranging from 4693 to 36,902 across the 16 samples). Taxonomy was assigned by confronting reads to the Silva 138 database (Quast et al., 2013; Yilmaz et al., 2014) using VSEARCH 2020.8.0 (Rognes et al., 2016) embedded in QIIME 2 2020.8 (Bolyen et al., 2019), with a percentage of identity of 90%. A phyloseq object was produced and imported in R (McMurdie & Holmes, 2013; R Core Team, 2020). Examining mock samples revealed correct identification of mock taxa at all taxonomic levels, with relative abundances both very constant across runs and very close to the expectation (Figure S1 in Appendix S1). Among all reads, the percentage of successful assignment (proportion of total reads assigned to a taxon identified in the reference database) was 78.1% at phylum, class, order, and family levels. It dropped to 74.9% at the genus level and 34.0% at the species level. For further analyses, features were merged at the genus level, constituting an incidence table of 105 genera in 16 fruit fly samples. As the maximal relative abundance of a false‐positive taxon was 0.001 in mock community samples, the incidence table was filtered of taxa below this threshold relative abundance. This led to a final incidence table of 46 genera (list provided in Table S2) in 16 fruit fly samples (Table S3), used for all following statistical analyses.
Community diversity was described as “effective numbers” (Hill, 1973; Jost, 2006, 2007) of bacterial genera within and among sample groups. The total (gamma) diversity of each group was multiplicatively partitioned into two components: (i) alpha diversity, the within‐group component; and (ii) beta diversity, the among‐group component, that is, the effective number of completely distinct communities present (Jost, 2006). Diversity decomposition was performed using inext (Hsieh et al., 2016) and the multipart() function of package vegan in R (Oksanen et al., 2020). To approximate uncertainty around diversity estimates, hierarchical bootstrapping was used. Further exploration of the variability of gut microbiota was conducted by nonmetric multidimensional scaling (NMDS) applied to the Bray–Curtis dissimilarity scores (Bray & Curtis, 1957).
To determine to what extent gut community structure is driven by fruit fly phylogeny, specialization, or sampling environment, we applied two network analysis methods exposed in Massol et al. (2021). To account for fly phylogeny, samples were divided into four groups based on fly genus: Neoceratitis, Ceratitis, Dacus, and the group formed by Bactrocera and Zeugodacus. The two latter genera are considered very close, to the point that until recently Z. cucurbitae was called Bactrocera cucurbitae (Virgilio et al., 2015; Zhang et al., 2010). Specialization groups are based on known host ranges in Reunion as inferred from long‐term observational data (Moquet et al., 2021) and divide samples into three groups: generalists (Bactrocera and Ceratitis species), specialists of Cucurbitaceae (Z. cucurbitae and Dacus species), and specialist of Solanaceae (N. cyanescens). Sampling environment opposes laboratory versus field samples. The first method is based on inferring groups within the observed network. It compares this grouping of nodes (here samples) with groups based on factors at stake (here fly phylogeny, fly specialization, and fly sampling environment). The second method assesses the link between multivariate explanatory variables and network structure using redundancy analyses after SVD of the incidence matrix. In both methods, the significance of effects can be gauged through randomization. Read counts can be poor proxies of abundances due to distortions inherited from the PCR process itself, and to representation biases of bacteria in reference databases (Brooks et al., 2016; Pollock et al., 2018). Therefore, it is generally considered safer to use presence–absence data. Here mock samples suggested both repeatable and moderate biases in abundance estimates from read counts. Thus we systematically conducted all community analyses on two versions of the sample × bacterial taxa incidence matrix: the weighted matrix, containing raw read counts; and presence–absence matrices, obtained by applying a threshold after rarefaction of the weighted matrix. While presence–absence matrices are generally considered to enable coping with uncertainty on relative abundance inference, they give rare taxa more weight into the analysis, as compared to weighted matrices. Importantly, because rarefaction is a random process, all analyses were applied on a distribution of presence–absence matrices, a safety step rarely done in microbiome studies.
Presence–absence matrices can be obtained from read count data by setting a read count threshold below which a taxon is considered absent. Such threshold will only be meaningful if samples are first rarefied to a common total read count. However, rarefaction is a random process generating different matrices each time it is applied (examples are provided in Figure S2, Appendix S2). To account for this variability, we conducted community analyses on 1000 binary matrices. Each binary matrix was obtained by rarefying the read count matrix to 3000 reads (the smallest read count was 3250 for a C. capitata sample) and applying a threshold of three reads. This threshold value was determined by rarefying the mock samples to 3000 reads as well, and observing that false positives were never above three reads. For each observed binary matrix, a search of groups was conducted by maximizing network modularity with the leading eigenvector algorithm (Newman, 2006) using the R package igraph (Csardi & Nepusz, 2006). The membership of each fruit fly sample to inferred groups was summed up into a binary adjacency matrix (16 samples × 16 samples) with zero if samples belonged to two different groups and one if they belonged to the same group. The probability that two samples belong to the same group was then obtained as the proportion of the 1000 binary matrices leading to group these samples together.
Subsequent analyses required producing null distributions of network statistics. Following Massol et al. (2021), we produced a null model, called the configuration model, using the “curveball” algorithm (Strona et al., 2014), with functions “simulate” and “nullmodel” of R package vegan. In theory, each observed binary matrix is associated with a specific null distribution, which can only be approached by simulating multiple networks. For the sake of computation time, in the following, each “curveball”‐based test was performed using 1000 simulated networks for each of 100 observed binary matrices.
We assessed the effect of sampling environment, fly phylogeny, and fly specialization on gut bacterial community structure. We first proceeded one factor at a time and tested the congruence of sample classifications obtained through community‐search algorithms with those associated with external categorical variables, using the Normalized Mutual Information Index (NMI) (Astegiano et al., 2017) available through the function “compare” in the R package igraph (Csardi & Nepusz, 2006). The NMI takes values between zero, indicating no congruence, and one, corresponding to perfect congruence. One NMI value can be obtained for each rarefied matrix, and its associated significance can be inferred from 1000 corresponding matrices simulated under the null model as explained above. Here, mean NMI values were obtained on 1000 rarefied matrices, and the mean associated p‐value was obtained by comparing 100 rarefied matrices with 1000 corresponding simulations each. To extend the same logic to multiple factors, we used CCA (ter Braak, 1986) using the function “cca” in the R package vegan. CCA allowed decomposing the variation of the community‐based classification of samples relatively to fly phylogeny, fly specialization and sampling environment. CCA can classically test the significance of a given “fraction” (e.g., chi‐square explained by factors X or Y once the effect of Z has been removed) by comparing the obtained F‐statistic with those yielded by randomizations of data rows (Peres‐Neto et al., 2006). Using the null model matrices, we could further test whether an effect that is deemed significant based on classical row permutations is purely due to heterogeneity in node degrees between communities (i.e., not significantly different from edge‐permuted expectation; richness effect) or not (affinity effect). Again, the whole CCA was conducted on 100 rarefied matrices, using 1000 corresponding simulations each.
As a complementary approach, we also modeled the effects of fly phylogeny, fly specialization, and sampling environment on network structure using SVD coupled with RDA as explained in Massol et al. (2021). Any given n × p bipartite network can be approximated as two matrices (L and R) with a low number of columns and as many rows as nodes (n in L, p in R). Matrices L and R can be analyzed through a RDA to gauge how much variation among rows is explained by external variables. The number of vectors to keep after SVD was fixed after examining the congruence between communities inferred from SVD‐approximated networks with those inferred from the original network. SVD‐approximated networks were obtained by multiplying matrices L and R and setting a threshold for interaction prediction. Congruence between communities was obtained using the NMI between module partitions on a number of rarefied matrices.
A similar approach was applied to the weighted (read counts) incidence matrix, with the following differences. First, with weighted matrices, it is recommended to proceed through latent block models (LBMs) rather than modularity maximization to look for groups of nodes in networks (Leger et al., 2015). We therefore inferred groups using LBM with the R package sbm (Chiquet et al., 2021). We used a Gaussian distribution to model log‐transformed read counts. The best grouping was selected based on ICL criterion (Integrated Complete‐data Likelihood, a penalized likelihood criterion suited for clustering; Biernacki et al., 2000). Second, as no rarefaction step was used, analyses were conducted only once. Third, the null model comprised 10,000 matrices produced by Gaussian sampling on the outer product of margins of the log‐transformed weighted incidence matrix.
The full bacterial composition of samples is provided in Figure 1 and Table S3 (Appendix S2). Rarefaction curves for each sample are provided in Figure S3 (Appendix S2). The 46 bacterial genera identified in the global dataset belonged to three phyla: Proteobacteria (97.7%), including two classes, eight orders, 15 families, and 36 genera; Firmicutes (2.0%), all of class Bacilli and order Lactobacillales, with eight genera in six families; and Bacteroidota (0.3%), only represented by two genera (Table S2). The genera above 1% in total abundance belonged to two classes (Bacilli and Gamma‐Proteobacteria), with an overrepresentation of the latter (97.1%). Among the 11 detected orders, four were above 1% in total abundance (Enterobacterales, Lactobacillales, Orbales, and Pseudomonadales), with an overrepresentation of Enterobacterales in all samples (52.5%–99.9%). Only five families (Enterobacteriaceae, Enterococcaceae, Morganellaceae, Orbaceae, and Pseudomonadaceae) were above 1% in total abundance, with an overrepresentation of Enterobacteriaceae (69.1%) in all fly species but N. cyanescens, dominated by the phylogenetically close Morganellaceae. Only nine genera were above the 1% threshold (Enterobacter, Klebsiella, Citrobacter, Providencia, Morganella, Raoultella, Gilliamella, Pseudomonas, and Enterococcus; Table S3). Some bacterial taxa have preferential associations with fly phylogenetic groups. The Bacteroidota phylum and the Alpha‐Proteobacteria class tended to associate with samples of Dacus. The Firmicutes phylum associated with Bactrocera and Zeugodacus samples. Some bacterial taxa had variable prevalence across sampling environments as well. Examples of genera with variable prevalence between laboratory and field samples include Enterobacter, Morganella, and Citrobacter. Finally, for some bacteria, the prevalence seemed determined by both fly phylogeny and sampling environment, such as the Orbales class, mainly found in field samples of Bactrocera and Zeugodacus.
The total (gamma) diversity of the 16 samples was 8.40 (95% CI 5.72–10.18) genus equivalents. The alpha diversity of samples ranged from 1.41 (95% CI 1.41–1.45, for field C. capitata) to 6.86 (95% CI 6.84–7.11, laboratory D. ciliatus) genus equivalents, with a mean of 4.01 (SE 0.41) (Figure 2). Average alpha diversity of laboratory (3.87, SE 0.54) and field samples (4.14, SE 0.65) was close. For all Dacinae samples but D. ciliatus, the field sample was more diverse than the laboratory sample, whereas in Ceratitinae, the laboratory sample was more diverse than the field sample (Figure 2). Among laboratory samples, there was no clear link between alpha diversity and the number of generations spent by populations in the laboratory prior to sampling (Appendix S2, Figure S4). Alpha diversity did not seem to particularly correlate with specialization (Figure 2): Diversity was not greater in generalists (3.45, SE 0.63) than in specialists of Cucurbitaceae and Solanaceae (5.06, SE 0.48 and 3.08, SE 0.31, respectively). In contrast, sample diversity tended to differ between phylogenetic groups (Figure 2). Dacinae samples had an average of 4.83 (SE 0.47) genus equivalents, while Ceratitinae samples had only 2.64 (SE 0.27). Pairwise beta diversity between samples ranged between 1.03 (between laboratory B. dorsalis and field C. capitata) and 1.91 (between laboratory N. cyanescens and D. ciliatus). Differentiation among bacterial communities was not particularly structured by sampling environment, as beta diversity between laboratory and field samples was 1.12. In contrast, beta diversity, even though estimated on the whole dataset (i.e., with both laboratory and field samples), tended to be higher between specialization groups (1.70) and between host phylogenetic groups (1.83). Nonmetric multidimensional scaling attained a stress value of 0.1932. It tended to group samples by phylogenetic group, rather than by sampling environment or fly specialization (Figure 3), a result also observed in NMDS ordination of presence–absence matrices (Figure S5 in Appendix S2). Dacus samples seemed to distinguish from other samples by higher relative abundance of Bacteroidota (genera Elizabethkingia and Sphingobacterium), lower relative abundance of Firmicutes (eight genera, all of class Bacilli, order Lactobacillales), and higher relative abundance of several genera from two orders of Alpha‐Proteobacteria (Rhizobiales and Burkholderiales). Field Bactrocera and Zeugodacus samples tended to preferentially associate with the Lactobacillales Streptococcus, Lactobacillus, and Vagococcus, and among Gamma‐Proteobacteria, with three Orbales genera (Frischella, Gilliamella, and Orbus) and some Enterobacterales genera. Field Ceratitinae mainly differed from others by their association with Enterobacterales genera such as Kosakonia and Pantoea and with the Burkholderiales genus Herbaspirillum.
Applying the leading eigenvector community‐search algorithm to the 16 samples over 1000 observed presence–absence matrices led to identify 4.203 groups of nodes in the network on average (SE = 0.057, Figure 4a and Figure S6 in Appendix S2), with a relatively high and significant modularity score (Q = 0.301, SE = 0.0005, 95% PI = 0.262–0.328, left panel of Figure S7, Appendix S2). Over a random subset of 100 observed binary matrices, the p‐value of the observed modularity had a mean of 0.005 (SE = 0.002, 95% PI = 0.000–0.0461, right panel of Figure S7, Appendix S2), suggesting that observed matrices were more structured than expected under the null model. All binary matrices separated at least two relatively stable groups (Figure 4a). The first group tended to split into two subgroups: (i) all Dacus samples, whatever their environment, most frequently grouped together (72%–95% of observed binary matrices), and (ii) field samples of Ceratininae species (genera Ceratitis and Neoceratitis). Field Ceratitis species were associated with 80% of observed binary matrices. Neoceratitis was less frequently associated with them (68% of observed binary matrices). Samples of both subgroups (Dacus and field Ceratitinae) were associated with 22.4%–63.6% of observed binary matrices. The second group was also composed of two main subgroups, with more variable composition: (i) Zeugodacus samples and field Bactrocera samples (percentages varying from 51% to 84%), and (ii) all remaining samples, that is, laboratory Bactrocera and Ceratitinae samples. The same community‐search algorithm also revealed an average of 3.45 (SE = 0.02) groups among field samples only and 3.81 (SE = 0.03) groups among laboratory samples, with congruent compositions with the 16‐sample grouping (Figure 4b,c). On the whole weighted incidence matrix, LBM identified three groups of samples (Table S4, Figure S8 in Appendix S2, and Figure 5): one with field samples of Zeugodacus and Bactrocera species, one with all Dacus samples, and the remaining samples (all Ceratitinae samples and laboratory samples of Zeugodacus and Bactrocera). On field samples only, two groups of samples were found: one with Dacinae species (Bactrocera, Dacus, and Zeugodacus) and one with all Ceratitinae species (Ceratitis, and Neoceratitis) (Figure S9, Appendix S2). On laboratory samples, no group was identified (Figure S9, Appendix S2).
Distributions of the congruence indices (NMI) are provided in Table 1. The communities found in the whole network were most congruent with genus‐level fly phylogeny (mean p‐value < 0.05). Other classifications of samples, that is, based on higher‐level fly phylogeny (Ceratitinae vs. Dacinae), sampling environment, or fly specialization, were not statistically more congruent with gut microbiota‐based clustering than expected by chance (see Figure 6 for an illustration).
The results of CCA applied to the communities found on both presence–absence and read count data confirmed the results found by congruence comparisons. Fly phylogeny significantly explained gut bacterial communities, irrespective of the removal of the effects of environment, host specialization, or both (Table 2A,B). On presence–absence data, none of the models omitting phylogeny or removing the effect of phylogeny was significant (Table 2A). Significant models were all doubly significant (with permutation tests on rows and edges), indicating both node richness and affinity differences between groups. On read count data, the models associated with the lowest p‐values included both phylogeny and sampling environment (Table 2B). Most significant effects were not significant under edge permutations, indicating an effect mainly driven by differences in gut microbiota richness between groups of nodes.
As a first step, the number of vectors required to faithfully approximate incidence matrices was determined by estimating the congruence between groups obtained from the approximated matrices with groups obtained on the full matrix. On presence–absence data, congruence tended to increase with the number of vectors retained, but the first local maximum occurred between two and four vectors retained depending on the rarefied matrix. On read counts, a single local maximum was observed at four vectors. Adjusted R 2 values of individual fractions are given for these various options (Figure 7) and are all very congruent. Residual error (i.e., variance not explained by fly phylogeny, fly specialization of sampling environment) increased steadily with the number of vectors, but remained high (>36%). For any given number of vectors, fly phylogeny had the highest adjusted R 2, followed by the interaction between fly phylogeny and fly specialization. Sampling environment explained a marginal part of variance on read counts only.
The gut bacterial microbiota of eight Tephritidae species were described using Oxford Nanopore MinION full‐length 16S metabarcoding. At taxonomic levels ranging from phylum to family, the abundance of bacterial taxa was found congruent with former descriptions obtained with Illumina MiSeq data from other Tephritidae species (for a review, see Noman et al., 2020 and Raza et al., 2020), and from some of these species in other geographic area (De Cock et al., 2020; Hadapad et al., 2016; Malacrinò et al., 2018). Enterobacteriaceae, identified as the most prevalent family in nearly all samples, are reportedly transferred vertically in some species (Aharon et al., 2013; Lauzon et al., 2009; Majunder et al., 2019) and thus are considered important for Tephritid development and physiology. At genus level, existing published studies exhibit substantial variability in descriptions of abundant bacteria. Here, thanks to the higher resolution of long‐read metabarcoding, 46 genera were found, the most abundant of which have also been described in other Tephritidae studies, including Enterobacter, Klebsiella, Citrobacter, Providencia, Morganella, and Raoultella (for a review, see Noman et al., 2020 and Raza et al., 2020). In contrast, some genera mentioned as frequent in other Tephritid studies were only found at low abundances here, as for example, Acetobacter, Escherichia, Pectobacterium, and Serratia. Whether these discrepancies are due to methodological issues or natural variability cannot be fully deciphered here. In the present study, only one pooled sample by fly species and fly sampling environment was studied, hampering considerations on natural intraspecific variability in gut microbiome composition. For some abundant taxa, results of functional studies monitoring fruit fly fitness are worth mentioning. For instance, Enterobacter and Klebsiella enhance larval nutrition (Noman et al., 2020 and references herein). An addition of Klebsiella in controlled conditions increases pathogen resistance of C. capitata (Ben‐Ami et al., 2010). In the same way, Cheng et al. (2017) have described the resistance of Citrobacter to resist trichlorfon insecticide in B. dorsalis. Finally, Enterobacter, Raoultella, Klebsiella, Citrobacter, and Providencia may also play a role in sexual and host plant attractiveness (Raza et al., 2020 and references herein). In contrast, Providencia and Morganella have been described as potential pathogens of fruit flies (M'Saad Guerfali et al., 2018; Salas et al., 2017), thus able to decrease fruit fly fitness. The recent accumulation of sequence data from microbial communities has made some authors plead for an extension of community analyses beyond the exploration of alpha‐ and beta‐diversity patterns in order to detect robust associations between microorganisms and hosts (Barberán et al., 2012; Burns et al., 2016). Here, classic diversity analyses were supplemented with network‐based clustering techniques (Massol et al., 2021) using either the leading eigenvector of presence–absence matrices (Csardi & Nepusz, 2006) or LBMs for the read count matrix (Chiquet et al., 2021). Such techniques may help cluster bacterial taxa according to their pattern of association with host flies and gut samples based on their microbial community profiles. Clustering methods may thus provide a natural way of revisiting the notion of core microbiome. Here, the use of various clustering analyses (on all, only laboratory or only field samples, and on read count vs. presence–absence data) supported at least three congruent groups of samples: all Dacus samples, field Bactrocera and Zeugodacus samples, and other samples (Ceratitinae and laboratory Bactrocera and Zeugodacus). Within this latter group, presence–absence matrices suggested possible subgrouping of Ceratitinae vs. the Dacinae Bactrocera and Zeugodacus. Clustering of bacteria highlighted a group of bacterial genera accounting for more than half of the bacterial prevalence in all samples: the Enterobacteriaceae Citrobacter, Enterobacter, and Klebsiella, and the Morganellaceae Providencia. This group, also supported by numerous studies of Tephritidae microbiota (Behar, Jurkevitch, et al., 2008; Hadapad et al., 2016; Liu et al., 2016; Morrow et al., 2015; Ventura et al., 2018; for a review, see Noman et al., 2020 and Raza et al., 2020), could be considered as a core microbiota at the scale of the Tephritidae family. A second group of bacterial genera, common in field Bactrocera and Zeugodacus samples, was rare in Dacus samples and of variable abundance in other samples. This group included Enterobacterales (Kluyveria, Morganella, Serratia), two Orbales (Gilliamella and Orbus), and all the Lactobacillales (represented by Enterococcus, Lactococcus, and Vagococcus). Associations between Lactococcus and B. zonata have already been described (De Cock et al., 2020). The third group of bacteria was preferentially associated with Dacus samples: the Alpha‐Proteobacteria genera Rhizobium and Ochrobactrum, the Bacteroidia genera Elizabethkingia and Sphingobacterium, and among Gamma‐Proteobacteria, genera belonging to diverse orders (the Burkholderiales Comamonas and Delftia, the Pseudomonadales Acinetobacter and Pseudomonas, the Xanthomonadales Stenotrophomonas, and the Enterobacterales Raoultella). Other bacteria fell in a fourth cluster, with no obvious association profile, likely due to their low abundances. These nonrandom associations of bacterial taxa with fly samples were further confirmed by NMDS. Interestingly, some preferential associations occurred at higher taxonomic scales. For instance, Bacteroidota and Alpha‐Proteobacteria were mainly associated with Dacus samples. In contrast, Firmicutes were completely absent from Dacus samples, as well as from field Ceratitis samples. Many preferential associations involved different families of Gamma‐Proteobacteria and different genera within the Enterobacteriaceae family, raising the need for a finer taxonomic resolution within this key bacterial family. Because of the genuine sympatry of the eight species, the highlighted clusters could not be considered as determined by geographic differentiation in microbial pools, and therefore offer candidate taxa for subsequent functional analyses. The different methods used to evaluate the relative importance of fly phylogeny, fly specialization, and fly sampling environment converged to the conclusion that fly phylogeny was the main factor explaining microbial profile. In contrast, host ecology (i.e., fly specialization and sampling environment) did not imprint significantly gut microbial communities. For instance, samples of the species Z. cucurbitae, a specialist of Cucurbitaceae host plants, systematically grouped with the Bactrocera samples, which correspond to generalist species, and not with Dacus samples, which share the same host range but are more distant phylogenetically. Samples of both Ceratitis generalist species tended to group with the other Ceratitinae species, N. cyanescens, a specialist of Solanaceae, rather than with Bactrocera samples, which share this generalist niche. The methodological robustness of our results was achieved by the observation of both read count (which tend to give more weight to very abundant species) and of presence–absence (which are more affected by rare taxa) data. Our results, suggesting that microbial profiles are affected by host phylogeny rather than host ecology, are thus unsupportive of the hypothesis formulated by Zhao et al. (2016), according to which the Tephritidae gut community membership would be controlled by host genetics, while bacterial abundance would be driven by nongenetic factors. Phylogenetic determinism of gut microbial communities has been observed in a diversity of taxonomic groups, including nematodes, numerous insect clades, fish, mammals, and hominids (Moran et al., 2019). Such a pattern may indicate a shared, faithful history between hosts and their microbes (Brooks et al., 2016). This process, sometimes referred to as “phylosymbiosis,” has been observed in Nasonia wasps, and is prone to co‐adaptations between hosts and microbes (Brucker & Bordenstein, 2012). Alternatively, this same pattern may very well be driven by physiological, morphological, ecological, or behavioral similarities in closely related hosts that lead to similar environmental filtering of microbial pools (Moran & Sloan, 2015). In Tephritidae species of Reunion Island, host ecology likely determines social interactions. Bactrocera and Ceratitis species on the one hand, and Dacus and Zeugodacus species on the other hand, are often found developing in the same fruits (Facon et al., 2021). We do not find such clustering when analyzing their gut microbiota, which suggests that such social interactions unlikely contribute to the structure of gut microbiota in the studied species. The present results do not conform to the hypothesis that generalist species should have more diverse gut microbial communities (Deb et al., 2019), as confirmed in scavengers and omnivores (Shukla et al., 2016; Yadav et al., 2015; Yun et al., 2014). The generalist Ceratitis species had lowest gut microbial diversity (around two genus equivalents). Their relative specialist of Solanaceae, N. cyanescens, had slightly higher microbial diversity, noticeably due to a relatively high abundance of Morganellaceae. The specialists of Cucurbitaceae (Dacus and Zeugodacus species) had the highest microbial diversity (around five genus equivalents), whereas their relative generalists of genera Bactrocera had less diverse gut content (around four genus equivalents). The observation that fruit fly specialization does not significantly imprint gut microbial communities is rather a surprise (but see De Cock et al., 2020, for a first mention). Plants present numerous nutritional and defensive challenges to phytophagous insects. A growing body of research emphasizes the potential contribution of symbiotic microbes to phytophagous diets (Feldhaar, 2011; Felton & Tumlinson, 2008; Oliver et al., 2010). Nevertheless, the accumulated evidence is mixed and requires further sampling and functional analyses of the fruit fly gut microbiota. Gut microbiota respond more to host phylogeny rather than to host ecology in aphids (McLean et al., 2019) and in lycaenid butterflies (Whitaker et al., 2016). The reverse has been observed in both fruit‐feeding and mycophagous drosophilid species (Adair et al., 2020), ants (Anderson et al., 2012), and beetles (Blankenchip et al., 2018). In interaction with phylogeny, the environment of sampling (here field vs. laboratory) had a detectable moderate effect on gut communities, in terms of both diversity and composition. In most Dacinae species (all but D. ciliatus, the most recent laboratory population), laboratory populations had less diverse gut microbiota as compared to natural populations. This observation has been made repeatedly in Tephritidae, such as B. tryoni (Morrow et al., 2015), B. oleae (Ras et al., 2017), and a range of arthropod species (Belda et al., 2011; Ng et al., 2018; Pérez‐Cobas et al., 2015; Staubach et al., 2013; Xiang et al., 2006). In clear contrast with these observations, we found that Ceratitinae laboratory populations were more diverse than field ones. Besides, except for Dacus samples, laboratory and field populations tended to differ strongly in terms of composition. In the present study, laboratory populations almost missed the class of Orbales (as already observed by Martinson et al., 2017), several Enterobacterales and Lactobacillales. Laboratory samples were also less dominated by the genus Klebsiella. Three genera were only present in the laboratory (Aeromonas, Fructobacillus, and Pluralibacter), and some genera very rare in nature had important relative abundance in the laboratory, such as the Yersiniaceae Serratia and the Morganellaceae Morganella. Some differences between laboratory and field populations contrasted with former observations, for example, describing a dominance of Providencia or Acinetobacter in laboratory populations (Ben‐Yosef et al., 2015; Kounatidis et al., 2009). The laboratory populations are occasionally supplemented with field individuals so that this differentiation cannot be explained by pure drift. This suggests a genuine recomposition of gut microbiota in laboratory populations in response to local conditions (missing nutriments, antifungal treatment…). Interestingly, while laboratory populations did share very similar conditions, the constraint of phylogeny on microbial communities was still much apparent. Such a result has implications for fruit fly management strategies based on sterile insect techniques, as well as for ecological and evolutionary studies using laboratory populations. Many studies have mentioned a loss of competitiveness of laboratory flies vs. field individuals. It is possible that part of this lesser fitness is due to gut microbiota modifications, that it could be attenuated by working of microbiota restoration, and that the intensity of this effect is species‐specific. Overall, gut microbiota were strongly imprinted by fly phylogeny, but could be subject to important restructuring in the face of new environmental conditions. As a consequence, the observed lack of correlation between gut microbiota and both fly specialization and fly sampling environment is a surprise and needs to be addressed. It is possible that most gut microbes have functions other than fruit digestion (Ben‐Ami et al., 2010; Cheng et al., 2017; Hadapad et al., 2016) or that there is functional redundancy; that is, microbial functions can be ensured by different taxa (Moya & Ferrer, 2016). Importantly only adults were studied here. In fruit flies, adults do not eat much, and only larvae feed on fruit. Yet, adult gut bacteria are the ones with a chance to be vertically transmitted. It could then be advantageous for flies that adults keep and transmit bacteria beneficial to larvae, including bacteria associated with plant use. Alternatively, it is possible that some useful gut bacteria are transitorily acquired by larvae in the fruit they grow in, before being eliminated at metamorphosis. Such ability to select and breed useful bacteria in the environment would confer an adaptive plastic response to host plants. These bacteria, which would likely differ across sampling environments, would not be detectable in studies focused on adults as here. And studying the contribution of gut microbiota to fly host range would require studying larvae as well. In Tephritidae, comparisons between larval and adult gut content are too rare and divergent for any conclusion to be drawn as to whether or not larvae acquire essential bacteria in the fruit, which would be released upon metamorphosis. Evidence from comparisons between larvae and fruits does not point toward this hypothesis. In B. tryoni, larval gut microbiota were more diverse than those of fruits and not influenced by fruit (Majunder et al., 2019). But in other flies, such as drosophilid flies, host ecology seems to have detectable impact on larval gut microbiota (Chandler et al., 2011). Another possible factor affecting gut microbiota composition and transmission might be the effect of larval diet on adult immunity (Fellous & Lazzaro, 2010). Adult immunity is likely the final gate filtering microbial taxa inherited by their progeny, and thus factors affecting immunity, including diet and other environmental conditions, could explain phylosymbiosis (or the lack thereof). Besides, the interactions between the host and a given microbe could be highly dependent on the other microbes constituting the microbiota. In such cases, a high rate of vertical transmission for a given microbe could greatly influence the rest of the microbiota. Dissecting the contribution of niche‐based processes in the assembly of the gut microbiota is therefore still an important challenge for future research using both field samples and gnotobiotic animals in controlled conditions.
The authors declare that there is no conflict of interest.
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PMC9626330 | 36193770 | Jing Wang,Shuanghui Li,Xiujuan Zhang,Ning Zhu,Ruzetuoheti Yiminniyaze,Liang Dong,Chengwei Li,Wumaier Gulinuer,Jingwen Xia,Jing Li,Daibing Zhou,Xinning Liu,Youzhi Zhang,Yuanyuan Zhang,Shengqing Li | Protein tyrosine phosphatase PTPL1 suppresses lung cancer through Src/ ERK / YAP1 signaling | 04-10-2022 | non‐small cell lung cancer,proliferation,PTPL1 | Abstract Background To reveal the function of protein tyrosine phosphatase‐L1 (PTPL1) in lung adenocarcinoma. Methods Lung cancer cell lines were transfected with short hairpin RNA against PTPL1 (shPTPL1 group) or negative control (shmock group). Quantitative real‐time polymerase chain reaction (qRT‐PCR) and western blotting were used to verify the transfection efficacy. Cell proliferation was analyzed by ethynyldeoxyuridine (EdU), Cell counting kit 8 (CCK8), and colony formation assay after PTPL1 or PTPL1 and yes‐associated protein (YAP1) knockdown. The effect of PTPL1 on tumor growth was examined in a xenograft lung cancer model. Results PTPL1 was downregulated in various types of lung cancer cell lines. The EdU, CCK8, colony formation assays and investigation using a xenograft lung cancer model indicated that PTPL1 knockdown increased the proliferation of lung cancer cells. Mechanistically, PTPL1 knockdown induced the activation of the Proto‐oncogene tyrosine‐protein kinase SRC (Src)/Extracellular regulated MAP kinase (ERK) pathway and thereby promoted yes‐associated protein (YAP1) nuclear translocation and activation. Conclusions In our study, PTPL1 played a crucial suppressive role in the pathogenesis of lung cancer potentially through counteracting the Src/ERK/YAP1 pathway. | Protein tyrosine phosphatase PTPL1 suppresses lung cancer through Src/ ERK / YAP1 signaling
To reveal the function of protein tyrosine phosphatase‐L1 (PTPL1) in lung adenocarcinoma.
Lung cancer cell lines were transfected with short hairpin RNA against PTPL1 (shPTPL1 group) or negative control (shmock group). Quantitative real‐time polymerase chain reaction (qRT‐PCR) and western blotting were used to verify the transfection efficacy. Cell proliferation was analyzed by ethynyldeoxyuridine (EdU), Cell counting kit 8 (CCK8), and colony formation assay after PTPL1 or PTPL1 and yes‐associated protein (YAP1) knockdown. The effect of PTPL1 on tumor growth was examined in a xenograft lung cancer model.
PTPL1 was downregulated in various types of lung cancer cell lines. The EdU, CCK8, colony formation assays and investigation using a xenograft lung cancer model indicated that PTPL1 knockdown increased the proliferation of lung cancer cells. Mechanistically, PTPL1 knockdown induced the activation of the Proto‐oncogene tyrosine‐protein kinase SRC (Src)/Extracellular regulated MAP kinase (ERK) pathway and thereby promoted yes‐associated protein (YAP1) nuclear translocation and activation.
In our study, PTPL1 played a crucial suppressive role in the pathogenesis of lung cancer potentially through counteracting the Src/ERK/YAP1 pathway.
Despite the advances in medication and therapeutics, lung cancer remains the leading cause of cancer death worldwide. Non‐small cell lung cancer (NSCLC) is the most common subtype of lung cancer, accounting for 85% of all cases. The overall 5‐year survival rate is less than 20%, and less significant improvement has been made over the past decades. Recently, although recent targeted therapies and immunotherapy have improved the situation, the long‐term survival of advanced patients is still poor. Since lung cancer is a molecularly heterogeneous disease and most patients are already in the advanced stage at the time of diagnosis, early diagnosis could remarkably reduce the mortality of patients. An in‐depth understanding of lung cancer pathogenesis may potentially lead to new effective treatments. It is well known that protein tyrosine phosphorylation plays an important role in intracellular signaling and cell behaviors including proliferation, apoptosis, invasion, and drug resistance. , , It has been determined that the deregulation of the aforementioned signal pathways is related to the development and progression of cancer. In contrast to kinases that catalyze phosphorylation of the signal transducers, the roles of protein tyrosine phosphatases (PTPs) have not been clearly depicted in the past decades. So far, only a few known PTPs have been established as oncogenes or tumor suppressors. , Human genome sequencing identified 107 PTP genes, 81 of which encode active protein phosphatases. PTP represents an enzyme superfamily subdivided into classical PTPs, dual‐specificity PTPs, and low molecular weight PTPs 13, 14. The catalytic domains of the PTP superfamily are highly conserved. In accordance with their critical involvement in orchestrating canonical intracellular signal pathways, PTP mutations have been frequently documented in various malignancies. The classical PTPs are further grouped into receptor and nonreceptor PTPs. Of the receptor PTPs, protein tyrosine phosphatase receptor type T (PTPRT)‐regulated signal transducer and activation of transcription 3 (STAT3) signaling and paxillin phosphorylation seem to be critical for head and neck or colorectal tumorigenesis. Protein tyrosine phosphatase receptor type D (PTPRD) suppresses tumors of various tissue origins by inducing apoptosis of cells. Protein tyrosine phosphatase receptor type K (PTPRK) can counteract HER2‐induced proliferation of breast cancer cells by inhibiting human epidermal growth factor receptor 2 (HER2) phosphorylation. Protein tyrosine phosphatase receptor type M (PTPRM) is mainly involved in regulating the proliferation and migration of breast and brain cancer cells, while protein tyrosine phosphatase receptor type J (PTPRJ) may be a candidate colon tumor suppressor gene. Protein tyrosine phosphatase receptor type B (PTPRB) regulates endothelial cell polarity and vascular permeability through the dephosphorylation of substrates such as receptor‐2 during angiogenesis. The mitogen‐activated protein kinases (MAPK) pathway has been documented as a regulatory target of PTPs. In particular, PTPN11 inactivates MAPK signaling through dephosphorylating Src or Sprouty, the inhibitor of reticular activating system (RAS). Protein‐tyrosine phosphatase‐L1 (PTPL1, also known as tyrosine‐protein phosphatase nonreceptor type [PTPN13], fas‐associated phosphatase 1 [FAP‐1], protein‐tyrosine phosphatase‐Basophil [PTP‐BAS], protein tyrosine phosphatase 1E [PTP1E], and protein tyrosine phosphatase LE [PTPLE]) is a nonreceptor PTP with 2485 amino acid residues and has the largest molecular weight of the known PTPs. PTPL1 is composed of several important domains including the kinase noncatalytic C‐leaf domain (KIND), the 4.1/ezrin/radixin/moesin (FERM) domain that usually exists in the peripheral membrane protein family, and the carboxy‐terminal catalytic domain. Five PSD‐95/CD large/ZO‐1 (PDZ) domains located between the FERM domain and the carboxy‐terminal catalytic domain. As a protein/protein interaction domain, the PDZ domain catalyzes the dephosphorylation of different protein tyrosine sites and participates in the occurrence and development of various diseases. Previous studies have found that PTPL1 has mutations or low expression in a variety of tumors, including lung cancer, colon cancer, breast cancer, liver cancer, head and neck tumors. , , , , Interestingly, it acts as a tumor suppressor gene in most tumor types. PTPL1 directly dephosphorylates Src on the active protease 419 (Y419) to regulate the invasiveness of breast cancer cells, suggesting its function of suppressing breast cancer. On the contrary, PTPL1 can protect pancreatic cancer cells from CD95‐mediated apoptosis. In addition, PTPL1 can also induce chemoresistance in head and neck cancer. Wang et al. found that PTPL1 inhibited the progression of ovarian cancer by dephosphorylating and stabilizing phospho‐inhibitor of kappa Balpha (IκBα) and thus impairing nuclear translocation of Nuclear factor kappaB NF‐κB. The activation of STAT3 inhibits the expression of PTPN13 in squamous cell lung cancer by recruiting histone deacetylase 5 (HDAC5). MicroRNA‐30e‐5p negatively regulates PTPL1 to promote the growth of lung adenocarcinoma cells, which eventually affects the survival and recurrence rate of patients. The present study aimed to investigate the impact of PTPL1 on the proliferation of lung cancer cells. To explore the signaling responsible of its role in carcinogenesis, the activation of MAP kinse‐ERK kinase (MEK)/ERK pathway and the expression of yes‐associated protein (YAP1), a key oncogenic transcription factor, were examined in PTPL1 knockdown cells. Both in vivo and in vitro investigations have proved that the knockdown of PTPL1 increases the proliferation of lung cancer cells and induces the activation of the Src/ERK/YAP1 signaling pathway.
Human NSCLC cell lines A549, NCI‐H1975(H1975), BEAS‐2B and human embryonic kidney cell HEK293T were purchased from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China). Cells were cultured in growth medium (GM) consisting of Dulbecco's Modified Eagle Medium (DMEM, Gibco) and Roswell Park Memorial (RPMI‐ 1640 medium, Gibco) supplemented with 10% fetal bovine serum (FBS, Gibco), 100 μg/ml streptomycin and 100 U/ml penicillin under a humidified atmosphere of 5% CO2 at 37°C.
Total RNA extraction was performed using a RNase kit (Yeasen), according to the manufacturer's protocol. RNA purity and concentration were assessed with the Nano Drop 1000 Spectrophotometer (Thermo Fisher Scientific). For each RT‐PCR reaction, first‐strand cDNA was generated from 1 μg of total RNA with the RT master mix (Takara). The SYBR Green Master mix (Roche) was used to perform real‐time PCR following instructions stipulated by the manufacture. All data were normalized to the expression levels of housekeeping genes glyceraldehyde‐3‐phosphate dehydrogenase (GAPDH) according to the (2−ΔΔCt ) method.
Total protein extraction mainly involved placing the treated cells in radioimmunoprecipitation assay (RIPA) lysis buffer (Solarbio) supplemented with ProtLytic protease and phosphatase inhibitor cocktail (NCM Biotech) for 30 min at 4°C. The cytoplasmic protein was extracted using a cytoplasmic protein extraction kit (Beyotime Biotechnology). Protein concentration was assessed using a bicinchoninic acid (BCA) protein assay kit (Thermo Fisher Scientific). Lysates were diluted in 5 × laemmli loading buffer, loaded onto 10% sodium dodecyl sulfate‐polyacrylamide gel electrophoresis (SDS‐PAGE) gels, proteins were transferred to polyvinylidene fluoride membranes. The blots were placed in 5% BSA blocking solution for 1 h and incubated with primary antibodies at 4°C overnight. The primary antibodies used for western blotting included PTPL1 (Proteintech, 25 944‐1‐AP), Src (Abcam, ab109381), anti‐Src (phosphoY419) (Abcam, ab185617), Phospho‐p44/42 MAPK (Erk1/2) (CST, #4370), p44/42 MAPK (Erk1/2) (CST, #4695), MEK (CST, #4694), Phospho‐MEK1/2 (Ser217/221) (CST, #9154), YAP (CST, #14074), Phospho‐YAP (Ser127) (CST, #13008). Tris‐buffered saline plus tween 20 (TBST) was used to wash the membranes three times, and horseradish peroxidase (HRP)‐conjugated secondary antibody was incubated for 1.5 h at room temperature. TBST was used to wash the excess secondary antibody, and the western blot was visualized by electrogenerated chemiluminescent (ECL) solution, and finally the chemiluminescence imaging system was used for imaging (Tanon). Band densities were quantified by ImageJ software (Wayne Rasband, National Institutes of Health).
Cells were seeded into 96‐well dish at an initial density of 2 × 103 –3 × 103 cells/well. At 0, 24, 48, or 72 h post‐transfection, the culture medium was changed to serum‐free medium containing the same doses of CCK8 (10 μl/well) for an additional 2 h for 37°C. The absorbance at the wavelength of 450 nm using Thermo Multiskan GO (Thermo Scientific) was then recorded to assess cell proliferation.
Cells were collected in the logarithmic growth phase, and a suspension prepared with a concentration of 2000 cells/6‐well plate. The culture dish was placed in an incubator at 37°C and 5% carbon dioxide. After culturing in complete medium for 7 days, the cells were fixed with 4% paraformaldehyde for 30 min, and then stained with 0.1% crystal violet solution (Sigma‐Aldrich). After washing the excess dye with phosphate buffered saline (PBS), a microscope was used to detect the total number of colonies with a minimum of 80 cells, and the data recorded and analyzed.
Ethynyldeoxyuridine (EdU) detection kit (Beyotime Biotechnology) was used to assess cell proliferation according to the manufacturer's instructions. Cells were cultured at 1 × 105 cells/well in a 6‐well dish. EdU‐labeled medium was added to the 6‐well dish, and then incubated for 2 h at 37°C and 5% carbon dioxide. Cells were fixed with 4% paraformaldehyde for 30 min at room temperature, followed by permeabilization in 0.5% Triton X‐100 at room temperature. Then, 500 μl of anti‐EdU working solution was added to each well and incubated for 30 min under light‐shading conditions. After washing three times with PBS, the nuclei were counterstained with Hoechst 333 42 for 10 min at room temperature. Finally, EdU‐labeled cells were observed by a fluorescence microscope (Bio‐Rad). The EdU incorporation rate was calculated as the ratio of EdU positive cells (red cells) to the total number of Hoechst 333 42 positive cells (blue cells).
To silence PTPL1 and YAP1, cells were transduced with short hairpin (shRNA) lentivirus targeting the human PTPN13 gene (Gene ID: 5783) and YAP1 (Gene ID: 10413) with lentivirus recombinant interfering plasmid (pGCSIL)‐green fluorescent protein (GFP) for transduction rate evaluation. Lentivirus lacking the shRNA insert was used as a control. Cells were seeded into a 6‐well dish at a density of 1 × 105 cells/well and transduced with shRNA‐PTPL1 (6 × 108 TU/ml) shRNA‐YAP1 (6 × 108 TU/ml) or shRNA‐mock lentivirus (6 × 108 TU/ml). After transfection for 48 h, the cells were imaged under a fluorescence microscope and further selection by puromycin. Seven days post‐infection, PTPL1 and YAP1 silencing was verified through qRT‐PCR analysis and western blotting.
BALB/c mice were purchased from the Shanghai Slack Laboratory Animal Center. Fifteen 8‐week‐old nude mice were randomly assigned to three groups, and five mice in each group were independently reared in a 12‐h light/dark system to provide sufficient water and clean food. For tumorigenicity assays, 1 × 106 shmock, shPTPL1, or shPTPL1 + shYAP1 cells were resuspended in 100 μl of PBS. Underarm injections of 8 weeks old male or female nude mice as described previously. The bodyweight and tumor size were measured every 3 days, and the tumor size was measured manually using calipers. All animals were used in compliance with the guidelines approved by the Institutional Animal Care and Use Committee. On the 30th day, the mice were anesthetized to obtain tumor tissue samples and stored in liquid nitrogen. Tumor volume calculation formula: Tumor volume = 0.5 × length × width2 mm.
Statistical analysis was performed using GraphPad Prism8.0 (GraphPad Software, Inc.). Each experiment was repeated three times and all values are expressed as mean ± SD. Image J software was used for Western blot pattern gray‐scale scanning. A t‐test was used to compare the data between the two groups, and the analysis of variance was used to compare the data between multiple groups. p < 0.05 was considered statistically significant.
To examine the function of PTPL1 in lung cancer, we assessed its expression in a normal human bronchial epithelial cell line, Human Normal Lung Epithelial cells(BEAS‐2B), and NLCSC cell lines, H1299, H1650, PC‐9, H1975 and A549. We found that PTPL1 knockdown caused significantly improved Src activation and concurrently PTPL1 levels were significantly lower in NSCLC cells than in BEAS‐2B cells (Figure 1), indicating a potential role in NSCLC pathogenesis. We next infected cells with recombinant lentiviruses expressing PTPL1‐targeted short hairpin RNAs (sh‐PTPL1), and validated efficient PTPL1 knockdown via qRT‐PCR and Western blot (Figure 2a,b). Consequently, cell proliferation was enhanced by knockdown of PTPL1 in these cells, as revealed by CCK8 assay (Figure 3a). We also used clone formation and Ethynyldeoxyuridine (EdU) assays to investigate the role of PTPL1 in the proliferation of lung cancer cells. Compared with the control group, PTPL1 knockdown significantly increased the proliferation of cells (Figure 3b,c). Collectively, these results indicate that PTPL1 downregulation contributes to the over‐proliferation of NSCLC cells.
PTPL1 was reported to repress cell proliferation through dephosphorylating Src, a non‐receptor tyrosine kinase known to activate canonical signal pathways including MEK/ERK, and YAP1 is a key oncogenic transcription factor regulated by various signaling such as MEK/ERK pathway. We thus investigated the effects exerted by PTPL1 on the phosphorylation status of Src, MEK, ERK and YAP1 in NSCLC cells. We observed that PTPL1 knockdown A549 cells exhibited consistently increased levels of phosphorylated Src and ERK when cells were exposed to serum or EGF (Figure 4a). In parallel, with the increased phosphorylation of Src and MEK, nuclear YAP1 levels were significantly upregulated after knockdown of PTPL1 in A549 cells (Figure 4b). Thus, PTPL1 knockdown promotes YAP1 nuclear translocation and activation possibly via MEK/ERK pathway in NSCLC cells.
To examine the potential role of YAP1 in the pathogenesis of NSCLC and figure out whether there is an interaction relationship between YAP1 and PTPL1. Next, we infected cells with recombinant lentivirus expressing YAP1 targeting short hairpin RNA (sh‐YAP1) and verified the efficient knockout of YAP1 by qRT PCR and Western blot (Figure 5). Therefore, as shown by CCK8 assay, knockdown of YAP1 in these cells reversed cell proliferation in the short hairpin RNA against PTPL1 (shPTPL1 group) (Figure 6a). We also used clonogenic and EdU analysis to investigate the role of YAP1 in lung cancer cell proliferation. Compared with shmock, PTPL1 knockdown significantly increased cell proliferation, while the excessive proliferation of cells was reversed after YAP1 knockdown (Figure 6b,c). Taken together, these results indicate that YAP1 downregulation reverses the hyperproliferation of shPTPL1 cells.
We next evaluated whether PTPL1 and downstream signaling played a role in in vivo lung cancer development. Nude mice were subcutaneously injected with control A549 cells or those subjected to PTPL1 or concurrent YAP1 knockdown. Within 30 days, all mice developed visible subaxillary solid tumors and exhibited comparable bodyweights (Figure 7a,b) While PTPL1 knockdown significantly promoted the growth of xenograft tumors, further silencing of YAP1 counteracted the effect of PTPL1 knockdown on tumor development (Figure 7b,c). These data suggest that PTPL1 represses in vivo development of lung cancer via downregulation of YAP1.
In this study, we demonstrated that the expression of the PTP, PTPL1, was reduced in lung cancer cells compared with normal lung epithelial cells. Moreover, we evaluated the impact of PTPL1 in lung cancer cell proliferative using in vitro and animal models and dissected the molecular mechanism responsible for this effect. We found that PTPL1 impaired the proliferation of lung cancer cells, and silencing of PTPL1 led to increased tumor growth in athymic mice. These results suggest that PTPL1 functions as a potent suppressor in the occurrence of NSCLC. This is also consistent with previous reports that PTPs plays an important role in inhibiting or controlling growth as a tumor suppressor. Although it is largely uncharacterized how PTPL1 fine‐tunes intracellular signaling that controls mitosis, this has been addressed by several previous studies. PTPL1 was found to regulate the cell cycle and upregulate genes related to invasion in prostate cancer cells. PTPL1 acts as a negative regulator of the insulin‐like growth factor (IGF)‐1R/IRS‐1/Akt pathway in breast cancer cells and inhibits fat formation. The loss of PTPL1 in prostate cancer cells may contribute to apoptosis resistance and tumor progression. Depletion of PTPL1 in mice caused increased incidence of breast cancer and accelerated tumor growth in vivo. These findings unraveled a multifaceted role of PTPL1 in suppressing tumorigenesis, highlighting the importance of identifying novel players in PTPL1‐regulated network in the context of vital malignancies like lung cancer. The Hippo pathway is one of the most frequently altered signaling pathways in human cancer. Targeting the core YAP1/PDZ‐binding motif‐TEA domain family member 1(TAZ‐TEAD) complex in this pathway has the potential to remarkably suppress tumorigenesis. TEAD activity is necessary for maintaining cell proliferation and renewal, and the blockade of YAP1/TAZ binding to TEAD leads to the activation of the Kruppel‐like factor 4 (KLF4) transcription network. Furthermore, KLF4 knockout mice showed an increased incidence of cancer. YAP1 has been established to function as a critical oncoprotein in NSCLC. Here, the phenotypic results of the double knockdown cell lines showed that YAP1 promoted the proliferation of NSCLC cells. In vitro experiments showed that YAP1 knockdown reduced the tumorigenic ability of nude mice. Previous studies have also shown that YAP1 was related to the proliferation of various cell types. The N6‐methyladenosine demethylase AlkB homolog5 (ALKBH5) inhibited tumor growth by reducing YTH domain‐containing‐proteins (YTHDFs)‐mediated YAP1 expression and inhibiting miR‐107/large tumor suppressor kinase 2 (LATS2)‐mediated YAP1 activity in NSCLC. The long non‐coding RNA, MALAT1, can upregulate YAP1 through sponging miR‐194‐3p, and thereby induce drug resistance of NSCLC. YAP/TAZ was also found to play an important role in regulating alveolar regeneration and alleviation of lung inflammation. In line with these observations, we found that YAP1 could be upregulated via MEK/ERK signaling in response to PTPL1 deficiency. Although the detailed mechanisms remain elusive, ERK has been documented to serve as a crucial regulator of YAP1. , Since YAP1 has also been reported to activate the MEK/ERK pathway, it is likely that they form a feedback regulatory loop to reinforce oncogenic signaling. Finally, our findings do not rule out the probability that other mediators also play indispensable roles in the downstream of PTPL1/ERK signaling in NSCLC. Nonetheless, we currently describe a new mechanism by which PTPL1 inhibits pulmonary tumorigenesis, and thus provide candidate targets for clinical therapy and novel biomarkers for prognostic assessment of lung cancer.
JW and SHL performed the experiments, XJZ and RY analyzed data and help conduct partial experiment. NZ, LD and CWL provided helpful discussion and reviewed the manuscript. WG, JWX, JL, DBL and XNL provided guidance on experimental technology and gave suggestions. YZZ and YYZ supervising the study. Shengqing Li designed this study and supervised the manuscript. All authors read and approved the final manuscript.
The authors have no conflicts of interest to declare. | true | true | true |
PMC9626554 | Marie A. C. Depuydt,Femke D. Vlaswinkel,Esmeralda Hemme,Lucie Delfos,Mireia N. A. Bernabé Kleijn,Peter J. van Santbrink,Amanda C. Foks,Bram Slütter,Johan Kuiper,Ilze Bot | Blockade of the BLT1-LTB4 axis does not affect mast cell migration towards advanced atherosclerotic lesions in LDLr−/− mice | 01-11-2022 | Mast cells,Atherosclerosis,Inflammation | Mast cells have been associated with the progression and destabilization of advanced atherosclerotic plaques. Reducing intraplaque mast cell accumulation upon atherosclerosis progression could be a potent therapeutic strategy to limit plaque destabilization. Leukotriene B4 (LTB4) has been reported to induce mast cell chemotaxis in vitro. Here, we examined whether antagonism of the LTB4-receptor BLT1 could inhibit mast cell accumulation in advanced atherosclerosis. Expression of genes involved in LTB4 biosynthesis was determined by single-cell RNA sequencing of human atherosclerotic plaques. Subsequently, Western-type diet fed LDLr−/− mice with pre-existing atherosclerosis were treated with the BLT1-antagonist CP105,696 or vehicle control three times per week by oral gavage. In the spleen, a significant reduction in CD11b+ myeloid cells was observed, including Ly6Clo and Ly6Chi monocytes as well as dendritic cells. However, atherosclerotic plaque size, collagen and macrophage content in the aortic root remained unaltered upon treatment. Finally, BLT1 antagonism did not affect mast cell numbers in the aortic root. Here, we show that human intraplaque leukocytes may be a source of locally produced LTB4. However, BLT1-antagonism during atherosclerosis progression does not affect either local mast cell accumulation or plaque size, suggesting that other mechanisms participate in mast cell accumulation during atherosclerosis progression. | Blockade of the BLT1-LTB4 axis does not affect mast cell migration towards advanced atherosclerotic lesions in LDLr−/− mice
Mast cells have been associated with the progression and destabilization of advanced atherosclerotic plaques. Reducing intraplaque mast cell accumulation upon atherosclerosis progression could be a potent therapeutic strategy to limit plaque destabilization. Leukotriene B4 (LTB4) has been reported to induce mast cell chemotaxis in vitro. Here, we examined whether antagonism of the LTB4-receptor BLT1 could inhibit mast cell accumulation in advanced atherosclerosis. Expression of genes involved in LTB4 biosynthesis was determined by single-cell RNA sequencing of human atherosclerotic plaques. Subsequently, Western-type diet fed LDLr−/− mice with pre-existing atherosclerosis were treated with the BLT1-antagonist CP105,696 or vehicle control three times per week by oral gavage. In the spleen, a significant reduction in CD11b+ myeloid cells was observed, including Ly6Clo and Ly6Chi monocytes as well as dendritic cells. However, atherosclerotic plaque size, collagen and macrophage content in the aortic root remained unaltered upon treatment. Finally, BLT1 antagonism did not affect mast cell numbers in the aortic root. Here, we show that human intraplaque leukocytes may be a source of locally produced LTB4. However, BLT1-antagonism during atherosclerosis progression does not affect either local mast cell accumulation or plaque size, suggesting that other mechanisms participate in mast cell accumulation during atherosclerosis progression.
The mast cell, a cell type of our innate immune system that acts in the first line of defence against pathogens, has been shown to promote the development and progression of atherosclerosis. Upon activation, mast cells secrete the proteases chymase and tryptase and pro-inflammatory cytokines such as IFN-γ, which have been shown to promote atherogenesis and to lead to destabilization of the plaque. Systemic activation of mast cells in dinitrophenyl hapten (DNP)-challenged apolipoprotein E (apoE)−/− mice for example resulted in an increased plaque size and incidence of intraplaque haemorrhage, whereas mast cell deficiency was shown to reduce atherosclerotic plaque development. In humans, mast cells have been shown to accumulate in advanced and ruptured coronary plaques. More recently, the association of mast cells with disease progression in cardiovascular disease patients was established, as significantly increased serum tryptase levels were observed in patients with acute coronary syndromes and intraplaque mast cell numbers were seen to increase upon atherosclerotic plaque destabilization. In addition, in that study an independent association of intraplaque mast cell numbers in carotid plaques with the incidence of clinical cardiovascular events was revealed. In patients with systemic mastocytosis, a disease characterized by the accumulation of mast cells in different organs, the prevalence of cardiovascular events was increased, despite a reduction in circulating low density lipoprotein levels. Together, the contribution of mast cells and their activation to atherosclerosis has been well established as has also been reviewed, however it remains elusive what factors contribute to mast cell migration towards these plaques. Apart from various chemokines and cytokines that have been implicated in mast cell migration, lipid mediators have been described to provoke a chemotactic response in mast cells. Leukotriene B4 (LTB4) is a pro-inflammatory lipid mediator well known for its chemotactic effect on myeloid and lymphoid cells. Intracellular biosynthesis of LTB4 occurs in a two-step enzymatic reaction in which arachidonic acid is metabolized by 5-lipoxygenase (5-LOX), 5-lipoxygenase activating protein (FLAP) and LTA4 hydrolase (LTA4H). Monocytes, macrophages and mast cells are able to release LTB4 in response to stimulation with factors such as Complement component 5a (C5a), Interleukin-1 (IL-1), Leukemia Inhibitory Factor (LIF) and Tumor Necrosis Factor α (TNFα). Synthesis and subsequent release of LTB4 by these leukocytes will elicit a directed migration of vascular smooth muscle cells, neutrophils, eosinophils, basophils, monocytes, macrophages, dendritic cells and T cells but also of mast cell progenitors through binding with its receptor BLT1. Previous in vitro studies showed an autocrine manner of mast cell migration towards LTB4, which mainly recruits mast cell progenitors from the bone marrow as BLT1 expression is significantly reduced upon mast cell maturation. LTB4 and its associated mediators have been suggested to participate in atherogenesis. Heterozygous deficiency of 5-LOX for example resulted in a 95%-decrease in lesion size in Low Density Lipoprotein receptor (LDLr)−/− mice. Moreover, BLT1 deficiency in apoE−/− mice resulted in decreased plaque development. Aiello et al. showed that antagonism of BLT1 through CP105,696 caused a decrease in the infiltration of monocytes into the lesions as well as reduced monocyte activation. In these studies however, the authors primarily assessed initial lesion development and the number of mast cells in these lesions was not assessed. As mast cell numbers particularly accumulate in advanced atheromatous human plaques, it may be of therapeutic interest to identify whether the LTB4-BLT1 axis is involved in mast cell recruitment to advanced atherosclerosis. In our study, we thus first aimed to determine whether cells in the advanced plaque are able to produce LTB4 and next investigated whether LTB4 participates in the chemotaxis of mast cells towards pre-established atherosclerotic plaques and would thereby contribute to further progression of the disease. Here, we administered the BLT1-antagonist CP105,696 to LDLr−/− mice with pre-existing atherosclerosis and analysed its effect on plaque progression. In this study, treatment with CP105,696 resulted in reduced splenic myeloid cell content, but did not affect plaque morphology and mast cell accumulation in advanced atherosclerosis.
Blockade of mast cell recruitment to atherosclerotic lesions may be a promising intervention target to reduce plaque destabilization. Mast cells have previously been described to be involved in their own recruitment, in which LTB4 can act as an autocrine chemoattractant in multiple diseases. Moreover, transcriptome analysis of ex vivo skin mast cells using deep-CAGE sequencing revealed expression of ALOX5 (5-LOX), ALOX5AP (FLAP) and LTA4H (LTA4H), the rate-limiting enzymes needed for biosynthesis of LTB4. Yet, it remains unknown whether plaque cells, among which mast cells, can produce LTB4 in atherosclerotic lesions. In a previous study, we performed single-cell RNA sequencing on human atherosclerotic plaques obtained from carotid endarterectomy surgery from 18 patients. Fourteen different cell clusters were found, of which one distinctly represented mast cells (cluster 13, Fig. 1A). Within this data set, we analysed the expression of the aforementioned genes involved in LTB4 biosynthesis. Indeed, we observed expression of ALOX5, ALOX5AP and LTA4H in several myeloid cell populations, including mast cells, from human atherosclerotic plaques. Whereas ALOX5AP and LTA4H was ubiquitously expressed by all leukocytes, mast cells showed the highest expression of ALOX5 (Fig. 1B). To assess whether these genes are also expressed in murine atherosclerotic plaques, we examined a publicly available murine single-cell RNA sequencing data set by Cochain et al.. In this study, single-cell RNA sequencing was performed on CD45+ cells isolated from aortas from LDLr−/− mice fed a chow diet, 11 weeks high fat diet and 20 weeks high fat diet, representing respectively the healthy aorta, early atherosclerotic and advanced atherosclerotic aortas. Integrating data from all three mouse models revealed 17 different clusters (Figure S1A–B). Alox5, Alox5ap and Lta4h were mainly found in the Cd14+ Cd68+ Itgam+ myeloid cell clusters (Figure S1C). Therefore, we isolated all myeloid cells (clusters 0, 2, 3, 4, 6, 8, 13, 15 and 16) and examined whether expression of these three genes differed per plaque stage. Alox5 was mainly expressed in myeloid cells of healthy aorta’s compared to atherosclerotic aorta’s (Figure S1D). Expression of Alox5ap and Lta4h showed no differences between plaque stages. Finally, we confirmed the expression of Alox5, Alox5ap and Lta4h in murine bone-marrow derived mast cells (BMMCs) (Fig. 1C). Together, these data imply that LTB4 can be produced locally in the plaque and that intraplaque mast cells may be its main source in the human plaque. We next aimed to determine whether blockade of the LTB4 receptor affects mast cell migration towards the advanced atherosclerotic lesion.
Next, we studied the effects of BLT1-antagonist CP105,696 treatment in our LDLr−/− mouse model with pre-existing atherosclerosis (Fig. S2). CP105,696 treatment did not affect total body weight throughout the experiment (t = 9 weeks, Control: 24.0 ± 0.7 g vs. CP105,696: 23.9 ± 0.5 g; Fig. 2A). Serum total cholesterol levels were decreased in both groups during treatment (w5 vs. w9, Control: 1809.7 ± 98.6 mg/dL vs. 1354.8 ± 44.1 mg/dL, p = 0.0002 and CP105,696: 1866.2 ± 92.2 mg/dL vs. 1351.9 ± 42.7 mg/dL, p = 0.00002), but no differences were found between control and CP105,696 (Fig. 2B). The decline in cholesterol level can be ascribed to the Tween 80 in the solvent as polysorbates were previously reported to induce cholesterol lowering. No differences in serum triglyceride levels were detected (Fig. 2C). The BLT1-LTB4 axis has previously been described to affect migration of multiple myeloid cells, including monocytes and dendritic cells. To confirm that CP105,696 inhibited the BLT1 receptor in vivo, we used flow cytometry to measure myeloid subsets in blood and spleen. In the circulation, no differences were found in the total percentage of myeloid cells (CD11b+; Fig. 3A). The percentage of the different monocyte subtypes (CD11b+Ly6Chi, CD11b+Ly6Cmid and CD11b+Ly6Clo) and of CD11b+CD11c+ cells, which predominantly characterize dendritic cells, also remained unaltered upon treatment (Fig. 3B–E). In the spleen we observed a clear effect of treatment with the BLT1-antagonist. The percentage of total myeloid cells was significantly reduced upon treatment with CP105,696 (Control: 11.3 ± 0.48% vs CP105,696: 9.4 ± 0.56%; p = 0.016; Fig. 3F). Both the percentage of inflammatory monocytes (Ly6Chi; Control: 0.96 ± 0.067% vs. CP105,696: 0.69 ± 0.069%; p = 0.011; Fig. 3G,K) and that of the patrolling monocytes (Ly6Clo; Control: 0.80 ± 0.032 vs. CP105,696: 0.71 ± 0.36; p = 0.07; Fig. 3I,K) showed respectively a significant decrease and a trend towards a decrease after BLT1 inhibition, whereas Ly6Cint monocyte levels did not differ between groups (Fig. 3H,K). Furthermore, we observed a reduced accumulation of CD11b+CD11c+ dendritic cells in the spleen of treated mice (Control: 4.8 ± 0.16 vs. CP105,696: 4.2 ± 0.17; p = 0.016; Fig. 3J).
Next, we assessed whether BLT1-antagonism affected the size and morphology of advanced lesions. CP105,696 treatment did not affect aortic root plaque area (Control: 3.7 ± 0.3*105 μm2 vs. CP105,696: 3.9 ± 0.2*105 μm2; p = 0.60) and the percentage of Oil Red O staining in the plaque (Control: 34.5 ± 3.3% vs. CP105,696: 32.3 ± 5.1%; p = 0.18; Fig. 4A). The degree of stenosis (Control: 36 ± 2% vs. CP105,696: 39 ± 1%; p = 0.233) as well as the total lesion area and plaque volume (Control: 1212 ± 86*105 µm3 vs. CP105,696: 1264 ± 64*105 µm3; p = 0.633) were unaltered by BLT1 antagonism (Figure S3A–C). We also examined collagen content and necrotic core size by Sirius Red staining. Both parameters did not change upon treatment with CP105,696 (Fig. 4B). Furthermore, no differences in macrophage content were observed between the groups (Fig. 4C). In addition, flow cytometry analysis of the atherosclerotic aortic arch did not reveal any differences in the percentage of live CD45+ leukocytes of the single cell population and the percentage of total lymphocytes in the CD45+ cell population (data not shown) between the groups. Also, the percentage of CD11b+ myeloid cells and that of CD11b+CD11c+ dendritic cells in the CD45+ cell population (Figure S4A–C) were not affected by CP105,696 treatment. Thus, BLT1-antagonism did not affect plaque morphology or myeloid cell content in a model of pre-existing atherosclerosis.
Subsequently, we aimed to investigate whether LTB4 is a chemoattractant for the recruitment of mast cells towards the atherosclerotic plaque. We first assessed the percentage of mast cell progenitors (MCps) in blood and thereby examined whether BLT1-antagonism affected their migration. No differences were observed in CD34+Lin-CD127-CD16/32+CD117+FCεRI+ MCps in blood between groups (Control: 0.010 ± 0.002% vs. CP105,696: 0.012 ± 0.002%; p = 0.34; Fig. 5A). We also determined mast cell numbers in the aortic root of treated and non-treated mice. In line with the data on mast cell progenitors, no differences were found in total mast cell numbers (Control: 16.0 ± 1.5 mast cells/mm2 vs. CP105,696: 16.5 ± 1.3 mast cells/mm2; p = 0.80; Fig. 5B,D) and in the percentage of activated mast cells (Control: 46 ± 2% vs. CP105,696: 51 ± 2%; p = 0.12; Fig. 5C) in the aortic root.
Mast cells actively contribute to progression and destabilization of advanced atherosclerotic lesions. Prevention of mast cell recruitment to the atherosclerotic lesion could thus be a promising therapeutic strategy to limit plaque destabilization. In this study, we aimed to investigate whether inhibition of the BLT1-LTB4 axis via BLT1-antagonism limits mast cell accumulation in advanced atherosclerosis and via this way prevent plaque instability. Although we observed expression of genes involved in LTB4 biosynthesis in the human atherosclerotic plaque, including prominent expression in mast cells, inhibition of BLT1 in vivo did not affect plaque morphology and the number of mast cells in the advanced atherosclerotic plaque. The LTB4 biosynthesis pathway has previously been examined in atherosclerosis. ALOX5 (5-LOX), ALOX5AP (FLAP) and LTA4H have been found to be expressed in human atherosclerotic plaques, and were generally seen to colocalize with macrophages. As we show here, these genes are also detected in the murine single-cell RNA sequencing data, while protein expression of ALOX5 and LTA4H has been established in murine plaques and adventitial macrophages as well. These proteins have also been targeted in experimental atherosclerosis studies. Similar to CP105,696, promising effects on atherosclerosis development have been shown with FLAP inhibitors like MK-886 and BAYx1005 Studies examining 5-LOX in atherosclerosis have been less evident. 5-LOX deficiency alone was not sufficient to limit atherosclerosis development, only in combination with 12–15-LO deficiency an effect was observed. Furthermore, in humans, 5-LOX inhibition using VIA-2291 gave rather conflicting results. Gaztanaga et al. reported that treatment with VIA-2291 was not associated with significant reductions in vascular inflammation in patients after an acute coronary syndrome, while Matsumoto et al. showed that VIA-2291 resulted in slower plaque progression as measured by CT-angiography. It must be noted that all experimental studies in mice focussed on the effects of treatment during lesion initiation. As our single-cell RNA sequencing data of advanced human plaques revealed that mast cells have high expression of the LTB4 biosynthesis-related genes, we aimed to examine the effect of LTB4 inhibition in advanced atherosclerosis. In our study, we observed a significant reduction in splenic myeloid cells, of which the most pronounced effects were found on both the inflammatory and patrolling monocyte subsets as well as on dendritic cells. This may be a result of direct inhibition of BLT1 on these cell subsets, but may also be indirectly related as LTB4 has previously been shown to upregulate Monocyte Chemoattractant Protein (MCP-) 1. Huang et al. showed that in human monocytes, LTB4 interacts with BLT1 to upregulate mRNA expression and active synthesis of MCP-1 by monocytes to induce a feed-forward amplification loop for their chemotaxis. Furthermore, it was shown that LTB4 induced increased avidity and/or affinity of β1-integrin and β2-integrin to their endothelial ligands, further stimulating firm arrest of monocytes under physiologic flow. Moreover, in both studies pharmacological inhibition of the LTB4-BLT1 axis abrogated these effects. In line with these reported findings, CP105,696 reduced splenic myeloid cell and dendritic cell levels, of which the most pronounced effects were found on inflammatory and patrolling monocytes, which both have been found to migrate towards MCP-1. In atherosclerosis, BLT1 has already been a target in multiple studies. In line with the chemotactic effects on monocytes, Heller et al. showed that BLT1 deficiency resulted in a significant reduction in lesion size as well as macrophage content in apoE deficient mice. Furthermore, in both apoE and LDLr deficient mice, a 35-day treatment with BLT1-antagonist CP105,696 led to a significant reduction in plaque size and CD11b+ cells in the circulation. In these studies however, intervention in the LTB1-BLT4 axis occurred immediately upon lesion initiation. As mast cells are known to accumulate in later stages of disease, we aimed to assess the effect of BLT1 blockade on pre-existing plaques. However, we did not observe any differences in plaque morphology as neither plaque and necrotic core size, nor plaque collagen and macrophage content were affected by BLT1 antagonism. Furthermore, flow cytometry analysis of the atherosclerotic aortic arch did not reveal any effects on total leukocyte and myeloid cell content upon CP105,696 treatment. Apparently, blockade of BLT1 is not sufficient to affect plaque size and composition at this stage of the disease. Indeed, Aiello et al. described that more distinct effects of CP105,696 were observed in apoE−/− mice of 15 weeks old, with smaller and thus less complex lesions as compared to 24 weeks old mice with more advanced atherosclerosis. In addition, in a BLT1−/−apoE−/− mouse model on a western type diet, differences in lesion size were only detected after 4 weeks of diet, whereas after 8 weeks and 19 weeks of diet, lesion size was similar in both BLT1−/−apoE−/− and apoE−/− mice. This may be explained by the fact that monocyte influx into the plaque is a less dominant mechanism in advanced atherosclerosis as compared to early atherogenesis. In addition, macrophage egress has been shown to decrease with atherosclerosis progression. Combined, this results in differences in myeloid cell dynamics between early and advanced atherosclerosis and effects observed upon BLT1-antagonism may thus be disease stage specific. Alternatively, in later stages of disease, other factors in the plaque microenvironment may contribute to the recruitment of different cell types to the plaque, which means that the decrease in myeloid content in the spleen due to BLT1-antagonism may not directly translate into effects in the plaque. LTB4 has previously been described to induce directed migration of mast cells and their progenitors. In vitro, LTB4 eluted from the supernatant of activated BMMCs elicited chemotaxis of immature mast cells. In mice, increased recruitment of CMFDA-labelled mast cell progenitors was detected upon injection of LTB4 into the dorsal skin. LTB4 was shown to be a potent chemoattractant for immature c-kit+ human umbilical cord blood-derived mast cells (CBMCs), whereas mature c-kit+ mast cells remained unresponsive. Nevertheless, in our study we did not observe any differences in the percentage of circulating mast cell progenitors after 4 weeks of treatment with CP105,696. Moreover, the total number of mast cells and the percentage of activated mast cells in the aortic root also remained unaffected, suggesting that LTB4 does not act as a chemoattractant for mast cells in advanced atherosclerosis via the LTB4 receptor BLT1. Assessment of mast cell accumulation in other sites of advanced atherosclerosis may be warranted to confirm our findings. In vitro cultured murine and human mast cells have however also shown expression of the LTB4 low affinity receptor BLT2. Lundeen et al. showed that inhibition with a selective BLT2 antagonist dose-dependently reduced migration of mast cells towards LTB4 in vitro, suggesting that this interaction of BLT2 and LTB4 could be involved in mast cell chemotaxis. As CP105,696 is a selective BLT1-antagonist, we cannot exclude BLT2-induced mast cell recruitment towards the plaque in our experiment. Future studies may aim to investigate whether BLT2 is involved in mast cell chemotaxis to the advanced plaque independent of BLT1. Although we did not see any differences in mast cell recruitment towards the atherosclerotic plaque in CP105,696 treated mice compared to our controls, single-cell RNA sequencing of human carotid atherosclerotic lesions suggests that mast cells may contribute to the local LTB4 concentrations in the plaque as intraplaque mast cells highly expressed genes involved in LTB4 biosynthesis. Interestingly, ALOX5, encoding for 5-LOX, was most prominently expressed in intraplaque mast cells as compared to other plaque cell types. In line, Spanbroek et al. showed that 5-LOX colocalized with tryptase+ mast cells in carotid arteries and that the number of 5-LOX expressing cells increased in later stages of disease. Furthermore, 5-LOX expression was mainly found in the shoulder regions of atherosclerotic lesions, where mast cells have also been shown to reside and accumulate. Together, this suggest that although mast cells may not induce an autocrine loop for their recruitment towards murine atherosclerotic lesions, that they may induce migration of other leukocytes towards the lesion via LTB4. We cannot exclude species-induced differences here, however based on current literature, we do not expect any differences as both mouse and human mast cells in culture were seen to migrate towards LTB4. To conclude, here we show that BLT1-antagonism does not affect plaque size and morphology during advanced stages of atherosclerosis, which suggests that LTB4 is not involved in the progression of advanced atherosclerotic lesions. Moreover, we show that LTB4 does not seem to be involved in mast cell migration towards atherosclerotic plaques, but that mast cells are able contribute to local LTB4 production in the lesion. To identify novel therapeutic intervention strategies, further research should be aimed at the elucidation of mechanisms that induce directed migration of mast cells towards the advanced atherosclerotic plaque.
Human carotid artery plaques were collected from 18 patients (14 male, 4 female) that underwent carotid endarterectomy surgery as part of AtheroExpress, an ongoing biobank study at the University Medical Centre Utrecht (Study approval number TME/C-01.18, protocol number 03/114). Single cells were obtained and processed for single-cell RNA sequencing as previously described. All studies were performed in accordance with the Declaration of Helsinki. Informed consent was obtained from all subjects involved in the study. Murine single-cell RNA sequencing data sets were obtained from Cochain et al. and processed as previously described. Briefly, data sets were processed using the SCTransform normalization method, integrated using rpca reduction and subsequently clustered, all according to the Seurat “scRNA-seq integration” vignette. All data analyses were executed in R-4.0.2.
All animal experiments were performed in compliance with the guidelines of the Dutch government and the Directive 2010/63/EU of the European Parliament. The experiment was approved by the Ethics Committee for Animal Experiments and the Animal Welfare Body of Leiden University (Project 106002017887, Study number 887, 1–103). Female 7–10 week old LDLr−/− mice (C57BL/6 background) (n = 15/group) that were bred in-house were provided with food and water ad libitum. From the start of the experiment, the mice were fed a cholesterol-rich western-type diet (0.25% cholesterol, 15% cocoa butter, Special Diet Services, Essex, UK), which continued for 9 weeks in total. At week 5, the mice were randomized in groups based on age, weight and serum cholesterol levels. Previous work from our group showed that mast cell accumulation in the aortic root starts at approximately 6 weeks after the start of western-type diet feeding. Therefore, from week 5 onwards, mice received either 20 mg/kg of BLT1-antagonist CP105,696 (Sigma-Aldrich) or vehicle control (0.6% Tween 80, 0.25% methylcellulose in phosphate-buffered saline (PBS)) three times per week via oral gavage for 4 weeks (n = 15 per group). A detailed schedule of the experimental setup is provided in Figure S2. Blood was drawn by tail vein bleeding at week 5 and week 7. At week 9, the mice were sacrificed upon subcutaneous administration of anaesthetics (ketamine (40 mg/mL), atropine (0.1 mg/mL) and xylazine (8 mg/mL)). Blood was collected via orbital bleeding, after which the mice were perfused with PBS through the left cardiac ventricle. Next, organs were collected for analysis.
Serum was collected through centrifugation at 8000 rpm for 10 min at 4 °C and stored at − 80 °C until further use. Total cholesterol levels were determined through an enzymatic colorimetric assay (Roche/Hitachi, Mannheim, Germany). Triglyceride levels in serum were measured by an enzymatic colorimetric assay (Roche Diagnostics). For both assays, Precipath standardized serum (Roche Diagnostics) was used as an internal standard.
Blood samples were lysed with ACK lysis buffer (0.15 M NH4Cl, 1 mM KHCO3, 0.1 mM Na2EDTA, pH 7.3) to obtain a single white blood cell suspension. Spleens were passed through a 70 μm cell strainer (Greiner, Bio-one, Kremsmunster, Austria) and splenocytes were subsequently lysed with ACK lysis buffer. Aortic arches were cut into small pieces and enzymatically digested in a digestion mix containing collagenase I (450 U/mL), collagenase XI (250 U/mL), DNAse (120 U/mL), and hyaluronidase (120 U/mL; all Sigma–Aldrich) for 30 min at 37 °C while shaking. After incubation, all samples were passed through a 70 µm cell strainer (Geiner, Bio-one, Kremsmunster, Austria). Single cell suspensions were then used for flow cytometry analysis.
Single cell suspensions from blood and spleen were extracellularly stained with a mixture of selected fluorescent labelled antibodies for 30 min at 4 °C. The antibodies used for flow cytometry are listed in Table S1. All measurements were performed on a Cytoflex S (Beckman and Coulter, USA) and analysed with FlowJo v10.7 (Treestar, San Carlos, CA, USA).
After euthanasia, the hearts were dissected, embedded and frozen in Tissue-Tek OCT compound (Sakura). 10 μm cryosections of the aortic root were prepared for histological analysis. Mean plaque size and the percentage plaque area of total vessel area (vessel occlusion) were assessed by Oil-Red-O (ORO) staining. From the first appearance of the three aortic valves, 5 consecutive slides with 80 µm distance between the sections were analysed for total lesion size within the three valves, after which the average lesion size was calculated. Average lesion size was also calculated in relation to distance from the start of the three-valve area. Subsequently, plaque volume was calculated as area under the curve. Collagen content of the plaque was measured using a Sirius Red staining after which fluorescent staining of three section per mouse was analysed and averaged. Similarly, the average necrotic core size was measured using the Sirius Red staining by measuring the acellular debris-rich areas of the plaque of three sections per mouse. Macrophage content was determined by using MOMA-2 antibody (1:1000; rat IgG2b; Bio-Rad). Naphthol AS-D chloroacetate staining (Sigma-Aldrich) was performed to manually quantify resting and activated mast cells in the plaques. Mast cells were identified and counted in the perivascular tissue of the aortic root at the site of atherosclerosis. A mast cell was considered resting when all granula were maintained inside the cell, while mast cells were assessed as activated when granula were deposited in the tissue surrounding the mast cell. Sections were digitalised using a Panoramic 250 Flash III slide scanner (3DHISTECH, Hungary). Analysis was performed using ImageJ software.
Bone marrow-derived mast cells (BMMCs) from 7 to 10 weeks LDLr−/− mice were cultured in RPMI 1640 containing 25 mM HEPES (Lonza) and supplemented with 10% fetal calf serum, 1% L-glutamine (Lonza), 100 U/mL mix of penicillin/streptomycin (PAA), 1% sodium pyruvate (Sigma-Aldrich), 1% non-essential amino acids (MEM NEAA; Gibco) and 5 ng/mL IL-3 (Immunotools). Cells were incubated at 37 °C and 5% CO2 and were kept at a density of 0.25*106 cells per mL by weekly subculturing. BMMCs were cultured for 4 weeks in total to obtain mature mast cells.
RNA isolation from 1*106 mast cells was performed using the guanine isothiocyanate method. Using RevertAid M-MuLV reverse transcriptase cDNA was isolated according to the manufacturer’s instructions. Quantitative gene expression analysis was performed with the SYBR Green Master Mix technology on a QuantStudio 6 Flex (Applied Biosystems by Life Technologies). A list of qPCR primers can be find in Table S2.
The data are presented as mean ± SEM and analysed in GraphPad Prism 9. Shapiro-Wilkson normality test was used to test data for normal distribution. Outliers were identified by a Grubbs’ test. Data was analysed using an unpaired two-tailed Student t-test or Mann–Whitney test. p < 0.05 was considered to be significant.
All experiments have been performed in accordance with the ARRIVE guidelines. Human samples The study was conducted according to the guidelines of the Declaration of Helsinki, and approved by the Medical Ethical Committee of the University Medical Centre Utrecht (UMCU). All samples were included in the Athero-Express Study (www.atheroexpress.nl), an ongoing biobank study at the UMCU. Informed consent was obtained from all subjects involved in the study. Animal studies: All animal experiments were performed in compliance with the guidelines of the Dutch government and the Directive 2010/63/EU of the European Parliament. The experiment was approved by the Ethics Committee for Animal Experiments and the Animal Welfare Body of Leiden University (project 106,002,017,887, study number 887,1–103).
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PMC9626557 | Ashley A. Rowe,Xin Chen,Emily R. Nettesheim,Yacine Issioui,Thomas Dong,Yuhui Hu,Souad Messahel,Saima N. Kayani,Steven J. Gray,Katherine J. Wert | Long-term progression of retinal degeneration in a preclinical model of CLN7 Batten disease as a baseline for testing clinical therapeutics | 29-10-2022 | Neuronal ceroid lipofuscinoses,Mfsd8,Bipolar cell degeneration,Photoreceptor synapse,Electroretinography,Optical coherence tomography | Summary Background Batten disease is characterized by cognitive and motor impairment, retinal degeneration, and seizures leading to premature death. Recent studies have shown efficacy for a gene therapy approach for CLN7 Batten disease. This gene therapy approach is promising to treat cognitive and motor impairment, but is not likely to delay vision loss. Additionally, the natural progression of retinal degeneration in CLN7 Batten disease patients is not well-known. Methods We performed visual examinations on five patients with CLN7 Batten disease and found that patients were far progressed in degeneration within their first five years of life. To better understand the disease progression, we characterized the retina of a preclinical mouse model of CLN7 Batten disease, through the age at which mice present with paralysis and premature death. Findings We found that this preclinical model shows signs of photoreceptor to bipolar synaptic defects early, and displays rod-cone dystrophy with late loss of bipolar cells. This vision loss could be followed not only via histology, but using clinical live imaging similar to that used in human patients. Interpretation Natural history studies of rare paediatric neurodegenerative conditions are complicated by the rapid degeneration and limited availability of patients. Characterization of degeneration in the preclinical model allows for future experiments to better understand the mechanisms underlying the retinal disease progression in order to find therapeutics to treat patients, as well as to evaluate these therapeutic options for future human clinical trials. Funding Van Sickle Family Foundation Inc. , 10.13039/100000002 NIH P30EY030413, Morton Fichtenbaum Charitable Trust and 5T32GM131945-03. | Long-term progression of retinal degeneration in a preclinical model of CLN7 Batten disease as a baseline for testing clinical therapeutics
Batten disease is characterized by cognitive and motor impairment, retinal degeneration, and seizures leading to premature death. Recent studies have shown efficacy for a gene therapy approach for CLN7 Batten disease. This gene therapy approach is promising to treat cognitive and motor impairment, but is not likely to delay vision loss. Additionally, the natural progression of retinal degeneration in CLN7 Batten disease patients is not well-known.
We performed visual examinations on five patients with CLN7 Batten disease and found that patients were far progressed in degeneration within their first five years of life. To better understand the disease progression, we characterized the retina of a preclinical mouse model of CLN7 Batten disease, through the age at which mice present with paralysis and premature death.
We found that this preclinical model shows signs of photoreceptor to bipolar synaptic defects early, and displays rod-cone dystrophy with late loss of bipolar cells. This vision loss could be followed not only via histology, but using clinical live imaging similar to that used in human patients.
Natural history studies of rare paediatric neurodegenerative conditions are complicated by the rapid degeneration and limited availability of patients. Characterization of degeneration in the preclinical model allows for future experiments to better understand the mechanisms underlying the retinal disease progression in order to find therapeutics to treat patients, as well as to evaluate these therapeutic options for future human clinical trials.
Van Sickle Family Foundation Inc., 10.13039/100000002NIHP30EY030413, Morton Fichtenbaum Charitable Trust and 5T32GM131945-03.
Research in contextEvidence before the studyBatten disease is characterized by death of the neuronal cells, such as those of the brain and retina. Patients show signs of disease as infants or young children, and these include movement impairment, neurological deficits, blindness and seizures, leading to premature death. Research studies have discovered the genes involved in Batten disease, although the precise mechanisms of action underlying the disease are not yet known. A recent study has found a gene therapy treatment that has moved forward to a clinical trial to treat the brain and spinal cord degeneration, but is not likely to treat the vision loss. The natural progression of vision loss needs to be understood in order to find and test therapeutic approaches for treatment in these patients.Added value of this studyIn this study, we performed visual examinations on five patients with CLN7 Batten disease and found that these patients are far progressed in vision loss in early childhood. To better define the disease progression, we examined the eyes of a preclinical mouse model of CLN7 Batten disease using both histological analysis as well as clinical imaging that is also performed in human patients. We found that the preclinical model showed early signs of synaptic defects, rod-cone photoreceptor degeneration, and late bipolar cell death, resulting in blindness.Implications of all available evidenceThe knowledge of the retinal cells affected during CLN7 Batten disease progression allows for therapeutics to be tested that target these specific cell types. Furthermore, our study has provided a baseline for CLN7 Batten disease vision loss that can be used when testing therapeutics in this preclinical model, to lead to future human clinical trials for treating vision loss associated with CLN7 Batten disease.
Death of the post-mitotic neuronal cells of the body have deleterious effects on health and viability, and are particularly harmful early in post-natal development. Neuronal ceroid lipofuscinoses (NCLs) are the most prevalent form of neurodegenerative disorders in paediatric patients, and most fall under diseases known collectively as Batten disease., These are autosomal recessive disorders characterized by their age-of-onset to infantile, late-infantile, or juvenile Batten disease.3, 4, 5 Patients present with cognitive and motor impairment, retinal dystrophy, and epileptic seizures that result in premature death.,, In recent decades, studies have determined thirteen causal genes involved in Batten disease, however there remains a critical need to understand the definitive functions of these genes to provide better therapeutic outcomes for patients. Currently, there are no available treatments to alleviate clinical symptoms for the majority of Batten disease., In the case of CLN2 Batten disease, research studies have shown that a recombinant human tripeptidyl peptidase-1 (TPP1) can be delivered into the cerebrospinal fluid every two weeks.10, 11, 12, 13 While positive results have been seen with this form of enzyme replacement therapy, it has a high incremental cost-utility ratio and the frequent delivery causes distress for paediatric patients. As Batten disease is monogenic and caused by recessive inheritance, gene therapy vectors are a promising treatment approach. Recently, a clinical trial has emerged to test the delivery of gene therapy for the treatment of CLN7 Batten disease (NCT04737460). The CLN7 form of Batten disease is caused by more than thirty-five mutations in the major facilitator superfamily domain containing 8 (MFSD8) gene, leading to protein reduction, or an inactive form of the CLN7 protein., Clinical signs of disease present during the late-infantile stage, with most patients dying in late childhood to teenaged years. In addition, some compound heterozygous patients have been identified with late-onset disease characterized by retinal dystrophies, including non-syndromic macular dystrophies with central cone involvement. Although this new gene therapy approach shows promise, it is not expected to pass the blood-retina barrier to efficiently prevent retinal degeneration in these CLN7 patients. Studies are needed that examine the efficiency and efficacy of this gene therapy vector, or other potential therapeutic approaches, on retinal degeneration for CLN7 Batten disease. In order to examine the therapeutic efficiency and efficacy, there need to be phenotypic outcomes that can be monitored with and without treatment. Thus, an understanding of the natural progression of retinal degeneration in these patients is necessary. In this study, we performed visual examinations and clinical imaging on five patients with CLN7 Batten disease. We found that patients were far progressed in retinal degeneration within their first five years of life. Therefore, we utilized a preclinical model of CLN7 Batten disease and characterized the long-term progression of retinal degeneration. Along with the rod degeneration detectable early in disease that replicated the histological findings previously published, we found a possible defect in photoreceptor to bipolar cell synaptic connectivity, early loss of both rod and cone function, and a loss of the inner nuclear layer (INL) at late stages of disease. The ability to follow the degeneration using clinical imaging in this preclinical model provides testing outcomes that can be used to monitor the potential efficacy of therapeutic approaches to treat retinal degeneration caused by CLN7 Batten disease.
Patients were enrolled in one of two Institutional Review Board (IRB)-approved clinical studies at the Children's Research Hospital and the University of Texas Southwestern Medical Center (UTSW; see Ethics). Patient I is a female that underwent clinical evaluation at four years and two months of age. She is compound heterozygous for an early stop mutation (c.1444C>T) and frameshift mutation (c.1206del) in MFSD8. Patient II is a male that underwent clinical evaluation at four years and eight months of age. He carries a homozygous missense mutation (c.1373C>A) in MFSD8. Patient III is a female that underwent clinical evaluation at five years and two weeks of age. She carries a homozygous early stop mutation (c.103C>T) in MFSD8. Patient IV is a female that underwent clinical evaluation at five years and eleven months of age. She carries a homozygous splice donor mutation (c.198+2T>C) in MFSD8. Patient V is a male that underwent clinical evaluation at six years and eleven months of age. He is compound heterozygous for a frameshift mutation (c.1036delG) and a splice acceptor mutation (c.440-2A>T) in MFSD8. See Table 1 for more details. The normal control patient was eighteen years of age at clinical evaluation and presented with intracranial hypertension but normal retinal function and no known mutations in MFSD8.
Visual acuity exams were attempted with variations of method based on the communication ability of the patients. Electroretinography (ERG) was conducted in accordance with international standards set by the International Society for Clinical Electrophysiology of Vision (ISCEV) and recorded on a Diagnosys Espion Electrophysiology System (Diagnosys LLC, Littleton, MA, USA). Recordings for scotopic ERG with a flash intensity of 3.0 Hz were followed by photopic ERG with a flash intensity of 3.0 Hz. Optical coherence tomography (OCT) was attempted using the Heidelberg HRA/OCT Spectralis (Heidelberg Engineering, Heidelberg, Germany).
Mfsd8 knockout (KO) mice (RRID: MGI:6388452) were obtained, carrying a deletion of exon 2 as previously described., Heterozygous Mfsd8 mice were bred to obtain litters of wild-type, heterozygous, and homozygous Mfsd8 KO mice, herein referred to as wild-type, Cln7 heterozygous, or Cln7 KO, respectively. C57BL/6J mice (Jackson Laboratory, Bar Harbor ME; RRID: MSR_JAX:000664) were used as additional controls. Euthanasia was performed by cervical dislocation or CO2 asphyxiation followed by secondary cervical dislocation. All mice were maintained in approved animal facilities at the UTSW and were kept on a normal light–dark cycle (12/12 h). Food and water were available ad libitum throughout the experiment.
Mice were sacrificed, and the eyes enucleated and processed as previously described., Quantification of retinal nuclei in either the outer nuclear layer (ONL) or inner nuclear layer (INL) was conducted on several sections that contained the optic nerve, as follows: the distance between the optic nerve and ciliary body was divided into three, approximately equal, regions on each side of the retina. The number of nuclei in four columns were counted within each region. These counts were used to determine the average thickness of the ONL and INL for each individual animal and within each region of the retina, spanning from the ciliary body to the optic nerve head (ONH) and back out to the ciliary body. Each section contained upper and lower retina as well as the posterior pole. N = five eyes per group and five sections per eye were used for analysis. Statistics were performed with a multiple comparisons test with Holm-Šidák's correction.
Mouse eyes were prepared and sectioned for IHC as previously described. Primary antibodies were GFAP (1:500, catalog Mab360, MilliporeSigma, RRID: AB_11212597), PSD95 (1:100, catalog ab238135, Abcam, RRID: AB_2895158), Red/Green Opsin (1:250, catalog AB5405, MilliporeSigma, RRID: AB_177456), PKCα (1:250, catalog sc8393, Santa Cruz Biotech, RRID: AB_628142), Blue Opsin (1:250, catalog AB5407, MilliporeSigma, RRID: AB_177457), Glutamine Synthetase (1:250, catalog MA5-27749, Invitrogen, RRID: AB_2735204), RBPMS (1:250, catalog ab152101, Abcam, RRID: AB_2923082), IBA1 (1:500, catalog ab178846, Abcam, RRID: AB_2636859) and VGLUT1 (1:250, catalog 48-2400, Thermo Fisher Scientific, RRID: AB_2533843). Secondary antibodies were Goat anti-Mouse Alexa Fluor 488 (1:1000, catalog A11001, Thermo Fisher Scientific, RRID:AB_2534069) and Goat anti-Rabbit Alexa Fluor 594 (1:1000, catalog A11012, Thermo Fisher Scientific, RRID:AB_2534070). Slides were imaged on a Leica SP8 laser scanning confocal microscope using a 25× water immersion objective lens or a 63× oil immersion objective lens. N = three mice and three or more sections per eye were used for analysis.
Retina tissue was collected as previously described., Samples represent both the left and right retinas from a mouse combined. Samples were lysed in RIPA buffer (Thermo Fisher Scientific, catalog 89900) with protease and phosphatase inhibitor added (Thermo Fisher Scientific, catalog 87786). During lysis, samples were sonicated in 5 s pulses a total of three times. Analysis was performed by running samples on either a 4–12% Bis-Tris gradient gel (Thermo Fisher Scientific, NW04125BOX) or a 3–8% Tris Acetate Gel (Thermo Fisher Scientific, EA03755BOX) and then transferred to a nitrocellulose membrane using the iBlot2 dry transfer system (Thermo Fisher Scientific, IB21001). Transfer was done in a three-step sequence: 20 V for 1 min, 23 V for 4 min, and 25 V for 2 min. Following transfer, membranes were blocked for 1 h at room temperature in 5% milk in TBST. Primary antibodies were diluted in 5% BSA in TBST and placed on the membrane and left shaking in a 4 °C fridge overnight. Primary antibodies were: PSD95 (1:2000, catalog ab238135, Abcam, RRID: AB_2895158), Red/Green Opsin (1:1000, catalog AB5405, MilliporeSigma, RRID: AB_177456), PKCα (1:1000, catalog sc8393, Santa Cruz Biotech, RRID: AB_628142), Blue Opsin (1:1000, catalog AB5407, Millipore Sigma, RRID: AB_177457), RBPMS (1:250, catalog ab152101, Abcam, RRID: AB_2923082), IBA1 (1:500, catalog ab178846, Abcam, RRID: AB_2636859), VGLUT1 (1:1000, catalog 48-2400, Thermo Fisher Scientific, RRID: AB_2533843), PROX1 (1:2000, catalog AB5475, Millipore Sigma, RRID: AB_177485), and GAPDH (1:2000, catalog GTX82560, GeneTex, RRID: AB_11174663). The next day, the membranes were washed three times in TBST followed by a 45 min incubation in secondary antibodies (LICOR, catalog 926-32211, RRID: AB_621843; catalog 926-68070, RRID: AB_10956588) diluted in 5% milk-TBS. Membranes were then washed three times in TBST, followed by one wash in TBS. Membranes were kept protected from light during the antibody staining process once secondary antibody had been added. Imaging and quantification were performed using the LI-COR Odyssey CLx. N = three mice and at least two technical replicates per mouse per antibody were used for analysis. Statistics were performed with one-way ANOVA with Tukey's multiple comparisons test.
Infrared (IR) and autofluorescence (AF) imaging were obtained with the Spectralis OCT scanning laser confocal ophthalmoscope (OCT-SLO Spectralis; Heidelberg Engineering, Franklin, MA, USA) as previously described.21, 22, 23, 24, 25, 26 Images were taken with a 55-degree wide focus lens (Heidelberg Engineering). The optic nerve was positioned in the centre of the image and 100 image sweeps were averaged to obtain the composite. For OCT, the IR image was used to select a plane through the centre of the eye, transecting the ONH. OCT images were taken by averaging one hundred image sweeps. N ≥ five mice per group.
For each experimental group and time point, five mouse eyes from separate mice were analysed for changes in OCT retinal thickness. The measurements were taken at six regions, three on each side of the optic nerve (Fig. S1). The first location was determined as being the closest to the optic nerve. A second location was located at the outer edge of the image and the third measurement was taken at the halfway point between the first two locations. Thickness of the neural retina was measured from the retinal nerve fiber layer (RNFL) to the retinal pigmented epithelium (RPE). Measurements were taken in micrometers using the Heidelberg Eye Explorer Software (Franklin, MA, USA). Statistics were performed with two-way ANOVA with Tukey's multiple comparisons test.
Scotopic ERG recordings were collected as previously described., Briefly, mice were dark-adapted for at least 12 h, manipulations were conducted under dim red-light illumination, and recordings were made using either the Celeris ERG system by Diagnosys LLC (Lowell, MA, USA) or the Phoenix MICRON Ganzfeld ERG System (Phoenix Technology Group, Pleasanton, CA, USA). On the Ganzfeld ERG, retinal responses were recorded at four different green light intensity settings: −1.7, −1.1, 1.9, and 2.5 log cd.s/m2. On the Celeris ERG, retinal responses were recorded at three different white light intensity settings: 0.01, 0.1 and 1.0 cd.s/m2. A minimum of seven measurements were recorded and averaged for each light setting. Photopic ERG was recorded using the Celeris ERG by first exposing the mouse eyes to 10 min of white light, followed by flashes at two white light settings: 3.0 and 10.0 Hz, each one averaging at least fifteen sweeps. Flicker response was measured at 10 Hz and 30 Hz, each averaging fifty sweeps. N ≥ eight eyes per group. Statistics were performed using two-way ANOVA with Šidák's multiple comparisons test, one-way ANOVA with Tukey's multiple comparisons test, or unpaired student's t-test (see Results and Figure Legends for specific details).
Mice were dark adapted for 12 h before eyes were dilated and animals were anesthetized via an IP injection of anaesthesia [1 mL ketamine 100 mg/mL (Ketaset III, Fort Dodge, IA, USA) and 0.1 mL xylazine 20 mg/mL (Akorn Inc., Lakeforest, IL, USA) in 8.9 mL PBS]. PERG and VEP recordings were taken simultaneously with a PERG stimulator on one eye, a second eye used as a corneal ground reference and additional electrodes in the forehead, cheek, and tail. Six hundred sweeps were averaged. N = five mice per group. Statistics were performed using the unpaired student's t-test.
Data are reported as mean ± SEM unless otherwise noted. GraphPad Prism Software (version 9.0) was used to generate graphs and perform statistical analysis. We calculated the sample sizes based on an effect size of 1 with standard deviation (SD) of 0.5. This calculation was based on our previously published work with mouse models of retinal degeneration using similar experimental methods. For example, for live imaging, this was based on an SD of 50 μV when looking for an effect of 100 μV, or during late degeneration an SD of 10 μV when looking for an effect of 20 μV. This indicated a sample size of approximately four mice to achieve 80% power when significance is set to P < 0.05 to compare two groups with two-tailed t-test. For the precise calculation, we used the formula: sample size = 2SD2 (1.96 + 0.842)2/d2. We did increase our mouse number used for the majority of our experimental methods, to ensure that we had an appropriate number of mice of both sexes and littermates from more than one litter included in the data results. Data comparing two groups were analysed via two-tailed t-test. Data comparing more than two groups were analysed using one-way ANOVA followed by Tukey's post-hoc multiple comparison's test. Data comparing two groups over multiple time points were analysed using multiple two-tailed t-tests with the Holm-Šidák's method to correct for multiple comparisons. Data with two independent variables were analysed using two-way ANOVA followed by Tukey's post hoc multiple comparison's test. Normal distribution and homogeneity of variance was determined by graphical analysis. See individual methods sections, results and figure legends for specific testing methods. No mice were excluded from the analysis. A P value of less than 0.05 was considered significant and measurements were done blinded to experimental groups (i.e. genotypes).
The study protocol was approved by the IRB for Human Subjects Research at UTSW (STU#2020-0640 and #2018-0226), was HIPAA compliant, and adhered to the tenets of the Declaration of Helsinki. Informed consent was obtained prior to any study procedures starting. Parents consented for their children to participate, and no assent was obtained as children were below the age of 10 years. Informed consent was written consent. All animal experiments were performed in accordance with the ARVO Statement for the Use of Animals in Ophthalmic and Visual Research and were approved by the Animal Care and Use Committee at UTSW (APN#2019-102840).
P30 EY030413 for the Department of Ophthalmology at UTSW was used to support the running of equipment under the Microscopy and Digital Imaging Module. Partial support for the project was provided by a kind gift to SJG from the Morton Fichtenbaum Charitable Trust. No other funding source had any roles in this project. No funding source had any role in study design, data collection, data analyses, data interpretation, or the writing of the manuscript.
Five patients carrying homozygous or compound heterozygous mutations in MFSD8 that were pathogenic for CLN7 Batten disease underwent visual examination and clinical imaging (Table 1). During testing of visual acuity, three out of five patients were uncooperative and no results were able to be obtained by the clinician. Patient III completed fix and follow examination. Patient I completed an LEA symbol examination which showed a reduction in visual acuity (Table 2). Scotopic and photopic electroretinography (ERG) was performed to test for retinal function. Difficulty in patient cooperation led to artifacts and noise upon ERG analysis, with Patient III unable to continue photopic testing due to elevated distress (Fig. 1). However, all ERG results displayed a loss in visual function for each patient (Fig. 1). Interestingly, Patient I - the youngest in the study - displayed an electronegative scotopic ERG with detectable, but reduced, a-wave OU (141.3 μV OD and 197.9 μV OS) at a 3.0 Hz flash intensity that was also detected at 10 Hz. Optical coherence tomography (OCT) was attempted in all five patients. Due to the inability for patients to cooperate, no reliable OCT images were obtained for either eye (Table 2). Thus, reliance on clinical imaging to characterize retinal degeneration in patients with CLN7 Batten disease is not ideal. However, determining the phenotypic progression of retinal neurodegeneration in CLN7 Batten disease is necessary for understanding the natural history of disease to be able to define the potential efficacy of clinical therapeutics.
To address this critical need, we utilized an available preclinical model for CLN7 Batten disease: the Mfsd8 knockout (KO) mouse model. The Cln7 KO mouse has previously been shown to have rod degeneration through four months of age. Additionally, this preclinical model was used to provide efficacy of intrathecal gene therapy injections, leading to the onset of a human clinical trial for CLN7 Batten disease patients (NCT04737460). However, this preclinical mouse model did not display detectable loss of the cone photoreceptors or bipolar cells in the four month period of analysis. It is possible that the loss of additional retinal cells occurs after four months of age in the Cln7 KO mice. Therefore, we characterized the progression of retinal degeneration via histological analysis through six months of age, the time in which the mice present with seizures, neurological degeneration, and premature death. As expected for the recessive inheritance, heterozygous Cln7 mice displayed similar retinal morphology compared to wild-type controls at six months of age (Fig. 2a). Quantification of the outer nuclear layer (ONL; Fig. 2b) and inner nuclear layer (INL; Fig. 2c) showed no significant differences between wild-type (black) and heterozygous Cln7 mice (blue) [Multiple comparisons test with Holm-Šidák's correction]. In contrast, the Cln7 KO mice displayed a loss of the ONL beginning at one month of age, with rapid degeneration between one and two months of age, and continuing to decline through six months of age (Fig. 2d). Quantification of ONL thickness showed a significant reduction (approximately 50–65% depending on the region of the retina examined based on distance from the optic nerve head) in the Cln7 KO mice (orange) compared to heterozygous controls (blue) at three months of age (Fig. 2e) [P < 0.0001; multiple comparisons test with Holm-Šidák's correction]. No significant differences were noted in the INL thickness between the Cln7 KO mice and heterozygous controls at three months of age (Fig. 2f), similar to previously published research [Multiple comparisons test with Holm-Šidák's correction]. However, at six months of age, Cln7 KO mice not only had lost approximately 82–89% of ONL thickness of heterozygous controls (Fig. 2g), but also had a significant - approximately 37% - loss of INL thickness (Fig. 2h) [∗∗∗P = 0.00018 and P = 0.00017; ∗∗∗∗P < 0.0001; multiple comparisons test with Holm-Šidák's correction]. This is the first time that the loss of the INL, similar to that seen in human CLN7 Batten disease patients, has been shown in this mouse model.
To investigate these changes further, we examined the Cln7 KO retinas at early- (two months) and late- (five months) stages of disease compared to wild-type controls (Fig. 3). As expected, the Cln7 KO mice showed elevated glial activation by immunostaining for glutamine synthetase (GS) and glial fibrillary acidic protein (GFAP) at both two and five months of age, indicative of retinal stress during both the early- and late-stages of disease (Fig. 3; Fig. S2). Microglial activation was also elevated at both the early- and late-stages of disease in the Cln7 KO mice compared to wild-type controls, and ionized calcium-binding adaptor molecule 1 (IBA1) protein was found to be significantly elevated in the Cln7 KO mice compared to controls upon Western blot analysis (Fig. 3, Fig. 4; Fig. S2) [∗∗P = 0.0032 for comparison to two month wild-type and 0.0028 for comparison to five month wild-type; ∗∗∗∗P < 0.0001; one-way ANOVA with Tukey's multiple comparisons test]. No change was noted for the retinal ganglion cells, either by immunostaining or Western blot analysis for RNA binding protein with multiple splicing (RBPMS; Fig. 3, Fig. 4; Fig. S3) [One-way ANOVA with Tukey's multiple comparisons test]. The bipolar cells, stained with protein kinase c alpha (PKCα), appeared similar between wild-type and early-stage Cln7 KO mice upon immunostaining analysis (Fig. 3; Fig. S3). There was a slight significant elevation in PKCα between five month wild-type and two month Cln7 KO retinas with Western blot analysis (Fig. 4) [∗P = 0.013; one-way ANOVA with Tukey's multiple comparisons test]. However, there was a thinning of the bipolar cell layer present by late-stage of disease in the Cln7 KO mice (Fig. 3; Fig. S3), as would be expected with the reduction in INL thickness seen at six months of age (Fig. 2h). Western blot analysis of PKCα showed a significant decline between the early- and late-stage Cln7 KO mice, although it did not show significance against the wild-type controls (Fig. 4) [∗∗P = 0.0038; one-way ANOVA with Tukey's multiple comparisons test]. This trend was also detected, without significance, by examining prospero homeobox 1 (PROX1) protein, which represents the amacrine cells as well as some bipolar and horizontal cells of the INL (Fig. 4) [One-way ANOVA with Tukey's multiple comparisons test]. Since Patient I indicated that there may be a defect in photoreceptor to bipolar synaptic signalling prior to the loss of phototransduction (Fig. 1), we examined the wild-type and Cln7 KO retinas for synaptic markers (Fig. 3). Interestingly, post-synaptic density protein-95 (PSD95) in the outer plexiform layer (OPL) appeared similar between early-stage Cln7 KO mice and wild-type controls, even though ONL thickness is severely reduced by this time (Fig. 3; Fig. S4). However, Western blot analysis did show a significant loss of PSD95 in the early-stage Cln7 KO mice compared to wild-type controls (Fig. 4) [P < 0.0001; one-way ANOVA with Tukey's multiple comparisons test]. At late-stage, a reduction in PSD95 was detectable via immunostaining in the Cln7 KO mice, likely reflecting the loss of the photoreceptors. This was also significant upon protein analysis (Fig. 4) [P < 0.0001; one-way ANOVA with Tukey's multiple comparisons test]. Vesicular glutamate transporter 1 (VGLUT1) is known to be required by the photoreceptors to signal to the second- and third-order neurons of the retina, such as the bipolar cells.27, 28, 29 We found that there is a slight reduction in VGLUT1 at the OPL, but not the inner plexiform layer (IPL), early in the Cln7 KO mice compared to controls. However, Western blot analysis of the early-stage Cln7 retinas showed a slight elevation of VGLUT1 compared to wild-type controls (Fig. 4) [P = 0.028; one-way ANOVA with Tukey's multiple comparisons test]. VGLUT1 was still present in both the OPL and IPL at late-stage of disease with no significant changes (Fig. 3, Fig. 4; Fig. S4) [One-way ANOVA with Tukey's multiple comparisons test].
In order to establish a natural history of retinal degeneration in the Cln7 KO mice that would provide a baseline to test clinical therapeutics, we performed clinical imaging over six months in the wild-type, Cln7 heterozygous, and Cln7 KO mice. Infrared (IR) and autofluorescence (AF) imaging was performed at both three and five months of age. Compared to controls, IR imaging in the Cln7 KO mice displayed vasculature attenuation and hyperfluorescent regions at three months of age that persisted through five months of age (Fig. 5a). AF imaging displayed hyperfluorescent puncta in the Cln7 KO mice, but not in either control, at three months of age (Fig. 5b). Some wild-type and heterozygous Cln7 control mice did display some hyperfluorescence at five months of age, likely due to natural aging.
To investigate the retinal degeneration in each cellular layer, we performed OCT imaging at both three and five months of age in the wild-type, Cln7 heterozygous, and Cln7 KO mice. Reduced thickness of the retina was detected in the Cln7 KO mice compared to both controls, with a noticeable loss of the ONL (Fig. 5c). Quantification of the retinal thickness displayed an approximate 20–25% thinning at three months of age (Fig. 5d) [∗P = 0.041; ∗∗P = 0.0013 and 0.0031; ∗∗∗∗P < 0.0001; two-way ANOVA with Tukey's multiple comparisons test] which progressed to an approximate 33% thinning by five months of age (Fig. 5e) [∗P = 0.014; ∗∗P = 0.0022; ∗∗∗∗P < 0.0001; two-way ANOVA with Tukey's multiple comparisons test].
Since we observed a loss of retinal thickness in the Cln7 KO mice on OCT analysis, as well as detected signs of photoreceptor degeneration on IR and AF imaging, we then investigated the function of the retinal cells. Scotopic electroretinography (ERG) was performed at post-natal day (P)21 and P28, to examine retinal function early in disease progression. Dim, white light (0.01 cd.s/m2 flash intensity), ERG traces displayed a loss of the b-wave amplitudes in the Cln7 KO mice compared to controls at both P21 and P28, indicating reduced function of the rod photoreceptor cells (Fig. 6a, b). Quantification of dim-light, scotopic ERG amplitudes confirmed the results seen in the representative traces, where the Cln7 KO mice displayed a significant loss of the b-wave at P21 (143 μV compared to 275 μV for the control) and P28 (113 μV compared to 374 μV for the control; Fig. 6c) [∗∗P = 0.0027; ∗∗∗∗P < 0.0001; two-way ANOVA with Šidák's multiple comparisons test]. At bright light (1.0 cd.s/m2 flash intensity), ERG traces displayed a loss of both the a- and b-wave amplitudes in the Cln7 KO mice compared to controls at both P21 and P28, indicating reduced function of the neural retina (Fig. 6d, e). Quantification of bright-light, scotopic ERG amplitudes confirmed the results seen in the representative traces, where the Cln7 KO mice displayed a significant loss of the a-wave at P21 (−127 μV compared to −251μV for the control) and P28 (−71 μV compared to −268μV for the control; Fig. 6f) [P < 0.0001; two-way ANOVA with Šidák's multiple comparisons test]. The Cln7 KO mice displayed a significant loss of the b-wave at P21 (270 μV compared to 518 μV for the control) and P28 (223 μV compared to 561 μV for the control; Fig. 6g) [P < 0.0001; two-way ANOVA with Šidák's multiple comparisons test]. To follow the natural progression of retinal function loss, scotopic ERG was performed at two, four, and six months of age in the wild-type, Cln7 heterozygous, and Cln7 KO mice. Scotopic ERG traces displayed a loss of both the a- and b-wave amplitudes in the Cln7 KO mice compared to controls at two months of age (Fig. 7a). Both the a- and b-wave amplitudes continued to degenerate in the Cln7 KO mice at four (Fig. 7b) and six months of age (Fig. 7c), with Cln7 heterozygous mice remaining similar to wild-type throughout the six month analysis. Quantification of scotopic ERG amplitudes confirmed the results seen in the representative traces, where the Cln7 KO mice displayed a significant loss of the a-wave (−45 μV compared to approximately −150μV for both control groups) at two months of age (Fig. 7d) [P < 0.0001; one-way ANOVA with Tukey's multiple comparisons test]. This decline continued to be significant at both four (−22 μV; Fig. 7e) and six (−16.13 μV; Fig. 7f) months of age [P < 0.0001; one-way ANOVA with Tukey's multiple comparisons test]. Additionally, the a-wave amplitude significantly declined within the Cln7 KO mouse cohort over the six months, providing insight into the natural progression of retinal degeneration in CLN7 Batten disease (Fig. 7m) [∗∗P = 0.0042; ∗∗∗P = 0.0003; one-way ANOVA with Tukey's multiple comparisons test]. Investigation of the b-wave displayed similar results to that noted for the a-wave (Fig. 7g–i). Cln7 KO mice showed a significant reduction in b-wave amplitudes at all times analysed (approximately 166.54 μV, 127.73 μV, and 58.11 μV compared to approximately 305.38 μV for both controls) [∗∗P = 0.0012 compared to wild-type and P = 0.0033 compared to heterozygous mice; ∗∗∗∗P < 0.0001; one-way ANOVA with Tukey's multiple comparisons test]. The Cln7 KO preclinical model also displayed a statistically significant decline in b-wave amplitudes over six months (Fig. 7n) [∗P = 0.014; ∗∗∗∗P < 0.0001; one-way ANOVA with Tukey's multiple comparisons test]. Since we found a reduction in the INL by six months of age in the Cln7 KO mice on histological analysis, and an indication of this loss at five months of age by protein analysis, we tested whether or not the INL signalling was compromised over time. We found that the oscillatory potentials (OPs) were similar between the Cln7 KO mice and control groups at two months of age [∗P = 0.0495 and 0.034 for P5; two-way ANOVA with Tukey's multiple comparisons test], but that the OPs significantly declined by four months of age (Fig. 7j and k) [∗P = 0.033 for P3 and P = 0.011 for P5; ∗∗P = 0.0098; ∗∗∗P = 0.0002; two-way ANOVA with Tukey's multiple comparisons test] and continued to significantly decline through six months (Fig. 7l) [∗P = 0.044; ∗∗∗∗P < 0.0001; two-way ANOVA with Tukey's multiple comparisons test]. As the Cln7 KO mice displayed indications of photoreceptor degeneration on histology and clinical imaging, we examined the function of the individual photoreceptors. Dim, green-light, scotopic ERGs were used to examine rod function at two, four and six months of age. At two months of age, rod function at this light setting was similar between the Cln7 KO mice and their controls (Fig. 8a, d) [P = 0.022 between wild-type and Cln7 heterozygous mice; one-way ANOVA with Tukey's multiple comparisons test]. However, a significant reduction in the rod ERG response was detected by four months of age (86.83 μV compared to approximately 149.8 μV for both controls; Fig. 8b, e) [∗P = 0.029; ∗∗P = 0.002; one-way ANOVA with Tukey's multiple comparisons test] and at six months of age (41.63 μV compared to approximately 206.65 μV for both controls; Fig. 8c, f) [P < 0.0001; one-way ANOVA with Tukey's multiple comparisons test]. Rod function significantly declined over the six month analysis within the Cln7 KO cohort (Fig. 8g) [∗∗P = 0.0084; ∗∗∗∗P < 0.0001; one-way ANOVA with Tukey's multiple comparisons test]. Previous reports did not note cone degeneration in the Cln7 KO mice, but analysis was halted by four months of age. Since we found that rod function declined over time, and photoreceptor degeneration was shown to progress through our six month analysis, we hypothesized that cone degeneration may be detectable after four months of age. To test this hypothesis, we looked at cone function using flash and flicker photopic ERG at early-stage disease (P21 and P28) as well as late-stage disease (five months of age) in wild-type and Cln7 KO mice. Surprisingly, we found that flash photopic ERG responses were significantly decreased in the early-stage Cln7 KO mice examined with 10.0 Hz light intensity (69 μV for the Cln7 KO mice compared to 116 μV for controls at P21, and 58 μV for the Cln7 KO mice compared to 114 μV for controls at P28; Fig. 9a, c) [P = 0.0006 at P21 and P = 0.0002 at P28; two-way ANOVA with Tukey's multiple comparisons test]. Flicker photopic ERG responses showed a similar trend, with a significant reduction in the Cln7 KO mice compared to wild-type controls at a 30.0 Hz light intensity beginning at P28 (9 μV for the Cln7 KO mice compared to 13 μV for controls at P21, and 5 μV for the Cln7 KO mice compared to 21 μV for controls at P28; Fig. 9b, e) [P = 0.0051; two-way ANOVA with Tukey's multiple comparisons test]. At late-stage of disease, we found that flash photopic ERG responses were significantly decreased in the Cln7 KO mice (25.37 μV for the Cln7 KO mice compared to 55.82 μV for controls at 3.0 Hz light intensity, and 34.75 μV for the Cln7 KO mice compared to 92.64 μV for controls at 10.0 Hz light intensity; Fig. 9a, d) [P < 0.0001; unpaired student's t-test]. Flicker photopic ERG responses showed a similar trend, with a significant reduction in the Cln7 KO mice compared to wild-type controls (4.96 μV for the Cln7 KO mice compared to 33.95 μV for controls at 10.0 Hz light intensity, and 3.53 μV for the Cln7 KO mice compared to 6.33 μV for controls at 30.0 Hz light intensity; Fig. 9b, f) [∗P = 0.028; ∗∗∗P = 0.0004; unpaired student's t-test]. As we detected a loss of cone function before one month of age but it persisted through at least five months of age, we examined the retinas of Cln7 KO mice for cones at both two and five months of age. Blue opsin staining was present in the Cln7 KO retinas, but displayed a collapsed morphology compared to wild-type controls (Figs. 9g and S5). Upon analysis of red/green opsin, the Cln7 KO mice also showed the collapsed morphology (Fig. 9g; Fig. S5). Protein analysis in the Cln7 KO retinas showed a significant elevation of both blue opsin [P = 0.0004; one-way ANOVA with Tukey's multiple comparisons test] and red/green opsin [P = 0.039 for comparison to two month wild-type, 0.038 for five month wild-type compared to two month KO, and 0.049 for comparison of five month wild-type and five month KO; one-way ANOVA with Tukey's multiple comparisons test] compared to wild-type controls, possibly reflecting a larger portion of cone protein when rods are lost in the Cln7 KO retinas (Fig. 4).
Since CLN7 Batten disease leads to neuronal degeneration in the brain as well as retina, we hypothesized that downstream visual processing will be affected in the Cln7 KO preclinical model. To test this hypothesis, we performed pattern ERG (PERG) on the Cln7 KO mice and wild-type controls at five months of age. The PERG waveform was normal but significantly reduced in the Cln7 KO mice, indicating transmission defects in ganglion cells (Fig. S6a, c) [P = 0.023; unpaired student's t-test]. We also tested pattern visually evoked potentials (VEPs) and found that these were reduced but not significantly altered in the Cln7 KO mice (Fig. S6b, d) [Unpaired student's t-test].
A loss of Mfsd8 causes lysosomal dysfunction, and it is likely to impact the degradation and metabolism of various compounds., In our study, we found that wild-type, Cln7 heterozygous, and Cln7 KO mice had similar body weights (in grams), respective of their sex and age (Fig. 10a, b) [two-way ANOVA with Tukey's multiple comparisons test]. However, we discovered that the Cln7 KO mice required significantly greater doses of ketamine anaesthetic in order to undergo clinical imaging, and that this requirement increased over time to levels that were lethal in wild-type and Cln7 heterozygous mice (Fig. 10c) [∗∗P = 0.0049 compared to wild-type and P = 0.0086 compared to heterozygous mice; ∗∗∗∗P < 0.0001; two-way ANOVA with Tukey's multiple comparisons test]. At two months of age, the Cln7 KO mice required approximately 116% of the amount of ketamine per body weight required for the control mice. By six months of age, this level had increased to be 144% of the age-matched controls. Although these mice required heavy dosage of IP anaesthetic to undergo clinical imaging, no adverse complications were detected and all mice recovered normally.
Batten disease is a fast-progressing, paediatric condition characterized by impaired vision, seizures, loss of motor control and premature death. The ability to use gene therapy advances to treat patients is promising. However, there remains a need to understand the natural progression of retinal degeneration in these patients in order to define clinical therapeutic outcomes and efficacy to treat vision loss. For instance, the ability to gather natural history information on vision loss was critical for setting the baselines for therapeutic efficacy in the gene therapy clinical trials for Leber Congenital Amaurosis (LCA) caused by mutations in RPE65, which led to the first Food and Drug Administration-approved in vivo gene therapy. Studies investigating human patients and mammalian models for this early-onset childhood retinal degenerative disease found that ERGs were reduced or non-detectable at early ages, indicating that therapeutic intervention is necessary at an early stage of disease.33, 34, 35, 36 In this study, we performed visual examinations on five patients with CLN7 Batten disease. Although this is a cohort with a small sample size, data obtained showed that these patients were far progressed in retinal degeneration within their first five years of life. This is expected to represent the general population of CLN7 patients, as the disease presents within the late-infantile age with an early retinal degenerative phenotype. We did find that the youngest patient displayed an electronegative ERG with reduced, but present, a-waves OU. This indicates the possibility of an early photoreceptor to bipolar synaptic defect. However, there was no ERG data collected at this earlier age for the other four patients, and it may be an outcome specific to this single case, and not CLN7 Batten disease patients as a whole. Other mammalian models for the study of NCLs have provided insight into the mechanisms underlying retinal degeneration.37, 38, 39, 40, 41 For instance, the retinal degeneration noted in the Cln3Δex7/8 mouse model for CLN3 Batten disease displays a loss of the bipolar cells, and this can be rescued using gene therapy approaches targeting the bipolar cells. Surprisingly, in the Cln6nclf mouse model of CLN6 Batten disease, the main retinal cell loss is in the photoreceptors, similar to that shown in the CLN7 mouse model. However, gene therapy vectors targeting the photoreceptor cells were unable to delay retinal degeneration in the Cln6nclf mouse, but vectors that targeted the bipolar cells delayed photoreceptor degeneration. This indicates a possible critical role for the bipolar cells in preserving retinal function in Batten disease. To further investigate the progression of retinal degeneration during CLN7 Batten disease, we utilized the Cln7 KO mouse model., This model has its limitations as the neurological deficits arise much later than seen in human patients. However, retinal degeneration was noted in this preclinical model before one month of age, similar to human patients. No clinical imaging had been performed and no analysis beyond four months of age was carried out for the Cln7 KO mice. In our study, we followed this preclinical model of CLN7 Batten disease through six months of age, the time in which the mice present with seizures, paralysis and premature death. We not only found a rapid rod degeneration as previously shown, but a significant loss of cone photoreceptor function before one month of age, although the cells persisted over time. This ability to monitor both rod and cone survival and function over time provides a baseline for testing the efficacy of potential clinical therapeutics on the photoreceptor cells and visual preservation. Furthermore, human CLN7 Batten disease patients show a loss of the bipolar cells of the inner nuclear layer (INL), which has not been previously detected in the Cln7 KO mouse. In our study, we detected a significant loss of the INL on histological analysis at six months of age, and found that beginning at four months of age there was a significant and progressive decline in the oscillatory potentials. These indicate signalling loss in the inhibitory amacrine and bipolar cells. Immunostaining analysis showed a possible reduction in VGLUT1 in the outer plexiform layer (OPL) at two months of age, suggestive of a defect in photoreceptor to bipolar synaptic signalling. Protein analysis of VGLUT1 did not show a reduction in the Cln7 KO mice at five months of age, but this may be due to the fact that it detected both VGLUT1 in the IPL and OPL, and the change visible on immunostaining in the OPL was not drastic enough to detect with Western blot of whole retinas. There was a similar trend for the bipolar cells, where a reduction was visible by immunostaining at five months of age in the Cln7 KO mice, but this was significant on Western blot analysis only between early- and late-stage Cln7 KO mice, and not when compared to wild-type controls. We did find variability in the rate of degeneration within Cln7 KO mice over time, with some progressing to a significant loss of the ONL and INL by five months of age, while others required six months or longer to show a significant reduction in the INL. In our study, we examined single dependent variables (i.e. scotopic visual response, outer nuclear layer thickness, etc.). There is a potential for confounding variables to play a role on the variables examined in this study. For instance, the cage location within the animal facility where the mice are housed as well as the nesting and social arrangement of the mice within a single cage can impact the light exposure to a given mouse, possibly affecting the rate of retinal degeneration. Additionally, the precise time of day for experimental methods, such as ERG, can play a role in the electrical response obtained. To reduce the likelihood of these confounding factors, we housed our mice in the same row and rack of the same animal facility room, as well as performed ERGs on controls and experimental littermates on the same day and same window of time (morning or afternoon). All of this data opens up the possibility that early in CLN7 Batten disease, defects arise in the synaptic connections to the bipolar cells. These would likely occur prior to the onset of rod and cone functional loss, followed by rod degeneration and secondary death of the cones with a late-onset degeneration of the INL, likely the bipolar cells. As mentioned, recent gene therapy approaches in a preclinical model of CLN6 Batten disease - also caused by a loss of the rod photoreceptors - determined that targeting the bipolar cells was necessary for therapeutic efficacy. Future studies are needed to test gene therapy vectors targeted specifically to either photoreceptor or bipolar cells using this Cln7 KO mouse model. One surprising outcome of our study was the sensitivity to intraperitoneally (IP) injected anaesthetics. Cerebral and retinal impaired lysosomal function has been shown in Cln7 KO mice,,,44, 45, 46 which can impact the ability to metabolize drug compounds. We find that care should be taken in treatment of human CLN7 Batten disease patients when prescribing drugs or considering anaesthetic options and routes of delivery. Additionally, lysosomal function is known to be important in controlling autophagy pathways in the retina and has implications in the progression of age-related macular degeneration (AMD). Some compound heterozygous CLN7 Batten disease patients present with non-syndromic retinal dystrophies, with the most common presentation that of macular dystrophy with central cone involvement. We found that our Cln7 KO mouse model has an early loss of cone function, supporting its involvement in macular dystrophy. CLN7 has also recently been found to function as an endolysosomal chloride channel, potentially playing a role in chloride permeability and regulation of lysosomal function in the cell. Future experiments can explore the potential link between CLN7 lysosomal dysfunction and macular degeneration. Overall, we provide a natural history of retinal degeneration in the preclinical model for CLN7 Batten disease, and define clinical indicators for testing therapeutic outcomes. We found that this preclinical model mimics the human patient retinal disease progression, with a rod-cone dystrophy followed by a late loss of the bipolar cells. Thus, this preclinical model can be followed with clinical imaging to test the efficiency and efficacy of therapeutics to treat retinal degeneration in CLN7 Batten disease.
XC, SJG and KJW conceived and designed the study. AAR, XC, ERN, TD, YH, SM, and SNK acquired the data. AAR, XC, and YI provided statistical analyses. AAR and KJW analysed and interpreted the data. AAR and KJW drafted the manuscript. SJG and KJW obtained funding. SJG and KJW supervised the study. AAR and KJW verified the underlying data. All authors had full access to the data in the study and have read and approved the final version of the manuscript.
All data associated with this study are present in the paper or in the Supplementary Materials.
Ms. Rowe has nothing to disclose. Dr. Chen has nothing to disclose. Ms. Nettesheim has nothing to disclose. Mr. Issioui has nothing to disclose. Dr. Dong has nothing to disclose. Ms. Hu has nothing to disclose. Dr. Messahel has nothing to disclose. Dr. Kayani has nothing to disclose. Dr. Gray has nothing to disclose. Dr. Wert has nothing to disclose. | true | true | true |
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PMC9626891 | Irina Gilyazova,Elizaveta Ivanova,Mikhail Sinelnikov,Valentin Pavlov,Elza Khusnutdinova,Ilgiz Gareev,Aferin Beilerli,Ludmila Mikhaleva,Yanchao Liang | The potential of miR-153 as aggressive prostate cancer biomarker | 13-10-2022 | Gene expression,Aggressive prostate cancer,miR-153,Target gene mutations,Oncogenesis,Biomarker | Introduction Prostate cancer (PC) is one of the most frequently diagnosed cancers in males. MiR-153, as a member of the microRNA (miRNA) family, plays an important role in PC. This study aims to explore the expression and possible molecular mechanisms of the miR-153 action. Methods Formalin-fixed paraffin-embedded (FFPE) tissues were collected from prostatectomy specimens of 29 metastatic and 32 initial stage PC patients. Expression levels of miR-153 were measured using real-time reverse transcription polymerase chain reaction (qRT-PCR). 2−ΔΔCT method was used for quantitative gene expression assessment. The candidate target genes for miR-153 were predicted by TargetScan. Mutations in target genes of miR-153 were identified using exome sequencing. Protein-protein interaction (PPI) networks, Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses were performed to investigate the potential molecular mechanisms of miR-153 in PC. Results MiR-153 was significantly up-regulated in PC tissues compared to non-cancerous tissues. The analysis of correlation between the expression level of miR-153 and clinicopathological factors revealed a statistically significant correlation with the stage of the tumor process according to tumor, node, metastasis (TNM) staging system (p = 0.0256). ROC curve analysis was used to evaluate the predictive ability of miR-153 for metastasis development and it revealed miR-153 as a potential prognostic marker (AUC = 0.85; 95%CI 0.75–0.95; sensitivity = 0.72, specificity = 0.86)). According to logistic regression model the high expression of miR-153 increased the risk of metastasis development (odds ratios = 3.14, 95% CI 1.62–8.49; p-value = 0.006). Whole exome sequencing revealed nonsynonymous somatic mutations in collagen type IV alpha 1 (COL4A1), collagen type IV alpha 3 (COL4A3), forkhead box protein O1 (FOXO1), 2-hydroxyacyl-CoA lyase 1 (HACL1), hypoxia-inducible factor 1-alpha (HIF-1A), and nidogen 2 (NID2) genes. Moreover, KEGG analysis revealed that the extracellular matrix–receptor (ECM-receptor) interaction pathway is mainly involved in PC. Conclusion MiR-153 is up-regulated in PC tissues and may play an important role in aggressive PC by targeting potential target genes. | The potential of miR-153 as aggressive prostate cancer biomarker
Prostate cancer (PC) is one of the most frequently diagnosed cancers in males. MiR-153, as a member of the microRNA (miRNA) family, plays an important role in PC. This study aims to explore the expression and possible molecular mechanisms of the miR-153 action.
Formalin-fixed paraffin-embedded (FFPE) tissues were collected from prostatectomy specimens of 29 metastatic and 32 initial stage PC patients. Expression levels of miR-153 were measured using real-time reverse transcription polymerase chain reaction (qRT-PCR). 2−ΔΔCT method was used for quantitative gene expression assessment. The candidate target genes for miR-153 were predicted by TargetScan. Mutations in target genes of miR-153 were identified using exome sequencing. Protein-protein interaction (PPI) networks, Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses were performed to investigate the potential molecular mechanisms of miR-153 in PC.
MiR-153 was significantly up-regulated in PC tissues compared to non-cancerous tissues. The analysis of correlation between the expression level of miR-153 and clinicopathological factors revealed a statistically significant correlation with the stage of the tumor process according to tumor, node, metastasis (TNM) staging system (p = 0.0256). ROC curve analysis was used to evaluate the predictive ability of miR-153 for metastasis development and it revealed miR-153 as a potential prognostic marker (AUC = 0.85; 95%CI 0.75–0.95; sensitivity = 0.72, specificity = 0.86)). According to logistic regression model the high expression of miR-153 increased the risk of metastasis development (odds ratios = 3.14, 95% CI 1.62–8.49; p-value = 0.006). Whole exome sequencing revealed nonsynonymous somatic mutations in collagen type IV alpha 1 (COL4A1), collagen type IV alpha 3 (COL4A3), forkhead box protein O1 (FOXO1), 2-hydroxyacyl-CoA lyase 1 (HACL1), hypoxia-inducible factor 1-alpha (HIF-1A), and nidogen 2 (NID2) genes. Moreover, KEGG analysis revealed that the extracellular matrix–receptor (ECM-receptor) interaction pathway is mainly involved in PC.
MiR-153 is up-regulated in PC tissues and may play an important role in aggressive PC by targeting potential target genes.
Prostate cancer (PC) is a commonly diagnosed condition, with an estimated 1414.3 thousand new cases of PC and 375.3 thousand deaths from the disease in 2020 [1]. In one third of PC patients the tumor progresses (perineural and stromal invasion) after initial regression in response to androgen deprivation therapy [2]. Despite current advances in surgical, chemotherapeutic and radiological methods of treatment, the five-year survival rate in castration-resistant patients is approximately 31.0% [3]. Most PC-related deaths occur due to the inability of existing treatments to prevent tumor spread [4]. Rectal examination and prostate specific antigen (PSA) levels are used to diagnose PC. However, the medical and scientific communities have questioned routine PSA testing. PSA has high sensitivity but very low specificity for PC. It can be elevated in the presence of benign prostate disease, infection, inflammation or benign hyperplasia [5]. Routine PSA testing leads to a high percentage of false-positive results. Moreover, PSA levels correlate poorly with the stage of the disease, leading to misdiagnoses and overtreatment of indolent forms of PC [6]. Considering all the above, there is a need for new molecular genetic markers capable of both detecting the disease at the earliest stage and predicting its course. MicroRNAs (miRNAs) are considered to be one of the most promising markers for various diseases including PC. The discovery of miRNAs provided a conceptual breakthrough in cancer research. MiRNAs are non-coding RNAs (ncRNAs) (19–22 nucleotides) which are involved in post-transcriptional regulation of gene expression. MiRNAs are increasingly associated with the initiation, development and progression of malignancies. Aberrant miRNAs expression has been identified in a variety of malignant tumors, with recent evidence suggesting that miRNAs function as tumor suppressor genes and/or oncogenes [7]. Recent studies have various miRNAs expression profiles in PC tissues [[8], [9], [10], [11]]. MiR-153 was found to be downregulated in various cancers, such as breast cancer (BC), gastric cancer (GC) and oral cancer (OC) [15]. However, there are few investigations devoted to miR-153 and its role PC [[12], [13], [14], [15]]. In this study, we evaluate the expression levels of miR-153 in PC tissue specimens and adjacent normal tissue specimens, the association of miR-153 with clinical characteristics and mutations in target genes of miR-153 and identified the pathways involved in PC progression. Our study was aimed to evaluate and understand the expression level of miR-153 in malignant tumor and normal prostate tissue in patients with metastatic PC and initial stages of the disease and possible molecular mechanism of the miR-153.
In this study, 61 PC patients (average age 60, range 41–80 years) who underwent surgical treatment between 2008 and 2020 in Bashkir State Medical University hospital were included. Ethical approval for this study was obtained from Institute of Biochemistry and Genetics Bioethics Committee. All samples investigated in this study were obtained with written informed consents of the participants. We collected formalin-fixed paraffin-embedded (FFPE) tissues from prostatectomy material including 29 metastatic and 32 localized (stages I, II) PC patients. The samples were classified using the tumor, node, metastasis (TNM) staging system from clinical stages I-IV.
After tissue section were obtained, Hematoxylin and Eosin (H&E) staining was performed and the slides were examined by two independent experienced pathologists. DNA and RNA were isolated from tumor regions and normal prostate tissue from each patient. The healthy region of the prostate without tumor cells in the selected FFPE block, was taken as a source of normal RNA and DNA. Total RNA and DNA extraction was performed using Quick-DNA/RNA™ FFPE Kits (Zymo Research) following the manufacturer's protocol. The process consisted of simple tissue deparaffinization in deparaffinization solution, proteinase K treatment followed by RNA and DNA isolation in spin columns.
The PCR amplification for the quantification of the miR-153 and U6 RNA was performed using a TaqMan MicroRNA Reverse Transcription Kit (Applied Biosystems; Life Technologies Corp) and TaqMan Human MicroRNA Assay Kit (Applied Biosystems; Life Technologies Corp). Expression levels were measured using CFX96™ PCR detection system (BioRad). All reactions were performed three times for each sample. The 2−ΔΔCT method was used for quantitative gene expression assessment. The 2−ΔΔCT method is based on the assumption that the cycle threshold difference (ΔCt) between target gene and reference gene is proportional to relative target gene expression. The relative expression of miR-153 was shown as fold difference relative to U6 RNA.
Patients with metastatic adenocarcinoma were chosen for exome sequencing to identify mutations in target genes of miR-153. Target genes were selected using TargetScan. DNA fragmentation, library preparation, and exome capture were conducted according to the manufacturer's recommendations. Selection of specific DNA fragments was conducted using the SureSelect system followed by concurrent sequencing of the obtained libraries using Illumina HiSeq 2000. All the reads were aligned with the reference genome using Burrows-Wheeler Alignment (BWA) software. We used the human genome sequence (Genome Reference Consortium Human Build 37 (GRCh37-hg19)) as a reference. Identification of the variants was conducted using the Genome Analysis Tool Kit (GATK). The identified variants were annotated by ANNOVAR software using the scripts table_annovar.pl and annotate_variation.pl, which allows to compare single nucleotide substitutions with the number of specialized databases and to annotate prognostic functional significance of the revealed alterations using six in silico software programs (SIFT, PolyPhen-2, LRT, Mutation Assessor, MutationTaster, phyloP, and GERP++) from dbNSFP v.3.0а. Additionally, we used CLINVAR and CADD (Combined Annotation Dependent Depletion) softwares. Exome sequencing procedure and bioinformatics were performed as previously described by Gilyazova et al. [16].
The network of mutated genes was generated using STRING-DB 9.1 [17]. Minimum required interaction scores were set to “high confidence” (0.700). The Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis was performed using online KEGG tools (http://www.kegg.jp/).
The obtained data was analyzed using R-studio program by calculating average values, standard deviation, and arithmetic mean error. The data are presented as means ± standard deviation. To assess the significance of differences, the Mann–Whitney U test was used. The predictive ability of miR‐153 was determined by the receiver operating characteristic (ROC) curves. A logistic regression prediction model was applied to calculate prediction scores for individual samples. Changes were considered reliably significant at p ≤ 0.05.
The expression level of miR-153 was analyzed in 61 samples of tumor tissue (Fig. 1) and 61 adjacent normal prostate tissue (Fig. 2) using qRT-PCR. MiR-153 expression showed to be increased in tumor tissue (mean ± SEM:1.36 ± 0.23) over normal prostatic tissue (mean ± SEM:0.66 ± 0.09) with p-value = 0.03 (Fig. 3).
Patients were divided into subgroups with high (n = 31) expression and low (n = 30) expression of miR-153. The median miR-153 expression value defined the threshold value. The analysis of correlation between miR-153 expression and clinicopathological factors (Table 1) revealed a significant correlation with the stage of the tumor process according to TNM classification (p = 0.001). Expression levels of miR-153 in different TNM stages of PC is presented (Fig. 4). It was shown that miR-153 expression significantly higher (mean ± SEM: 2.29 ± 0.41) in metastatic PC tumors compared to non-metastatic tumors (mean ± SEM: 0.53 ± 0.12) with p < 0.0001.
In addition, the receiver-operating characteristic (ROC) curve was drawn to further calculate the area under the curve (AUC) and authenticate the diagnostic ability of miR-153. Results revealed that miR-153 expression presented low diagnostic ability for tumor and normal prostate tissue discrimination. As shown at ROC curve of the miR-153 presented in Fig. 5, the AUC was 0.61 (95% CI 0.52–0.71; sensitivity = 0.66, specificity = 0.53). Furthermore, logistic regression analysis revealed that the miR-153 was independent predictor for PC (odds ratios = 1.57, 95% CI 1.13–2.36; p = 0.014). Further the ROC curve analysis was used to evaluate the predictive ability of miR-153 for metastasis development. As shown at Fig. 6 the AUC was 0.85 (95%CI 0.75–0.95; sensitivity = 0.72, specificity = 0.86) that let suggest miR-153 as potential prognostic marker. According to logistic regression model the high expression of miR-153 increased the risk of metastasis development (odds ratios = 3.14, 95% CI 1.62–8.49; p-value = 0.006).
In order to identify mutations in miR-153 target genes we performed exome sequencing in all patients with metastatic PC. We identified nonsynonymous somatic mutations in genes collagen type IV alpha 1 (COL4A1), collagen type IV alpha 3 (COL4A3), forkhead box protein O1 (FOXO1), 2-hydroxyacyl-CoA lyase 1 (HACL1), hypoxia-inducible factor 1-alpha (HIF-1A), and nidogen 2 (NID2). No pathogenic mutations were revealed in the coding regions of the phosphatase and tensin homolog deleted on chromosome 10 (PTEN) gene. None of the pathogenic mutations in miR-153 target genes was seen in all patients. This may be due to the fact that shared somatic mutations were not the cause of metastatic PC. The most deleterious germline and somatic mutations are summarized in Table 2.
From 23 proteins, consists of 75 nodes, 23 edges (PPI enrichment value 0.0568, and average local clustering coefficient of 0.253) a PPI network was constructed. Gene Ontology (GO) analysis showed that overlapping miR-153 target genes were mainly enriched in collagen type IV trimer, basement membrane, extracellular matrix and cytoplasm. Regarding molecular function classification, miR-153 target genes were enriched in these functions: transforming growth factor beta binding, ATPase-coupled intramembrane lipid transporter activity, nucleoside monophosphate kinase activity, collagen binding and ATPase activity, coupled to movement of substances. The extracellular matrix–receptor (ECM-receptor) pathway was the most commonly seen enrichment pathway in KEGG (FDR = 0.03) (Supplementary Table S1). STRING analysis of protein-protein interaction network of metastatic PC is presented in Fig. 7.
Patients who have undergone a radical prostatectomy remain at risk of biochemical recurrence after surgery, even though they have increased survival rates [18]. PC prognostic biomarkers are important to facilitate optimization of existing treatment strategies. Recently, it has been shown that miRNAs are intimately related to the development of several different types of cancers and may be useful to determine therapeutic targets for effective treatment strategies in a variety of cancers [19,20]. Xie et al. evidenced that miR-520a inhibits non-small cell lung cancer (NSCLC) progression through suppression of ribonucleoside-diphosphate reductase subunit M2 (RRM2) and Wnt signaling pathways [21]. Dong et al. show that miR-369 expression is reduced in hepatocellular carcinoma (HCC) tissues [22]. MiR-516a-3p expression is suppressed in PC tissue, and loss of miR-516a-3p expression promotes PC progression through ATP binding cassette subfamily C member 5 (ABCC5) targeting [23]. In 2020 Wang et al. presented evidence supporting the fact that miR-1231 expression is decreased in PC tissues and cell lines and that reduced expression of this miRNA had significant association with presence LNM, TNM stage, and clinical stage [24]. This research suggests that miRNAs play an important role in the process of cancer progression. Previous studies have shown that miR-153 is aberrantly expressed in several common cancers. Zhang et al. performed miRNAs profiling and showed that miR-153 is overexpressed in tissues of advanced colorectal cancer (CC). The miRNAs upregulation was further noted in primary CC compared, unlike normal colorectal epithelium [25]. Further studies showed miR-153 plays a role in promotion of colorectal malignancy progression through invasion and chemotherapy resistance enhancement. Hua et al. showed miR-153 to be activated in HCC cells with correlation between increased expression and poor outcome [26]. In our study we compare expression levels of miR-153 in tumor and normal prostate tissues in patients with metastatic PC and initial stages of the disease and identify specific pathogenic mutations in miR-153 gene targets in PC patients. We showed that miR-153 expression rates are significantly increased in PC tissue, when compared to normal prostate tissue. Moreover, high expression of miR-153 was found significantly associated with TNM stage. Our results suggest that it may act as an oncogene in PC and may be involved in the development of PC. MiR-153 overexpression is able to stimulate the transcriptional activity of β-catenin, which leads to cycle progression of cells, activation of proliferation and HCC cell colony formation. However, decreased expression of miR-153 is observed in some other cancers. Interestingly, Zhao et al. show that miR-153 expression is suppressed glioma cells compared to normal glial cells [27]. The authors showed that miR-153 suppresses cell invasion by regulating the expression of the snail family transcriptional repressor 1 (SNAI1), which is the target of miR-153. Guo et al. present findings showing that miR-153 is significantly overexpressed in patients with nasopharyngeal carcinoma (NPC), more so this miRNA affects NPC progression through the transforming growth factor-beta 2 (TGF-β2)/Smad2 signaling pathway [28]. Wang et al. showed that miR-153 was overexpressed in BC tissue samples and MDA-MB-231 cells [29]. Our study revealed that miR-153 is in fact overexpressed in PC tissues. Our results are consistent with Wu et al., who identified miR-153 to be overexpressed in PC and showed that miR-153 plays a crucial role in increased proliferation of human PC cells and via a process of miRNA-mediated suppression of PTEN expression in PC cells [8]. So far, there is very limited data on clinical significance of miR-153 and its role in PC. Bi et al. found that high miR-153 expression in PC tissues closely correlated with burdened clinical manifestations [11]. They presented data suggesting that PC patients with high levels of miR-153 expression had a lower five-year survival rate, when compared with patients with low miR-153 expression levels. Importantly, the authors use multivariate Cox regression analysis to show that miR-153 rates of expression are independent factors in predicting 5-year overall outcome in patients with PC. Thus, based on data of the present study and the results of previous publications, we can assume that miR-153 may serve as an available biomarker for PC prognosis. In our study we built upon existing reports of miR-153 significant by identifying specific somatic mutations in miR-153 target genes (Table 2). One of the mutated genes in metastatic PC patient is COL4A1 gene. COL4A1 is involved in epithelial-mesenchymal transformation (EMT). In previous studies it was found that depending on age of PC patients and Gleason score altered expression of COL4A1 together with 7 other genes may be an EMT marker among PC patients [30]. Mapelli et al. generated a 10-gene predictive classifier which showed that COL4A1, a low-luminal marker, supports the association of attenuated luminal phenotype with metastatic disease [31]. We also found mutations in the FOXO1 gene in metastatic PC patients. The forkhead box O (FOXO) has a common conserved DNA-binding “fork-box” domain and in mammals consists of four members: FOXO1, forkhead box class O 3a (FOXO3a), forkhead box class O 4 (FOXO4), and forkhead box class O 6 (FOXO6). All FOXO factors are involved in a wide range of biological processes, including cell cycle arrest, apoptosis, DNA repair, glucose metabolism, resistance to oxidative stress and longevity [32]. The biological activity of FOXO factors mainly depends on posttranslational modification of phosphorylation, acetylation or ubiquitination, thereby determining their intracellular transport [33]. Dong et al. demonstrated that FOXO1A inhibits androgen receptor (AR)-mediated gene regulation and cell proliferation in PC [34]. Another found mutation in PC cells, minichromosome maintenance complex component 4 (MCM4), belongs to the minichromosomal maintenance (MCM) protein complex which consists of six highly conserved proteins (MCM2-7) collectively interacting to promote DNA replication and DNA unwinding through replicative helicase activity disinhibition [35]. Cancers arising in different anatomic sites are also associated with minichromosome maintenance complex component 2 (MCM2), MCM4, and minichromosome maintenance complex component 6 (MCM6) overexpression, but there is not much information about the role of MCM4 in PC [[35], [36], [37]]. It is known that PSA may mediate MCM4 to promote the initiation and progression of PC and confirmed that PSA knockdown induce the upregulation of MCM4 [38]. Another important tumor microenvironment component is the hypoxia-inducible factor (HIF) pathway. HIF1A was also mutated in metastatic PC patient. There are some articles devoted to analysis of mutations in HIF1A and their role in PC, although ultimately its role in PC remains unknown [39,40]. KEGG analysis showed that a key signaling pathway in metastatic PC is the extracellular matrix (ECM)-receptor interaction signaling. The ECM is a non-cellular component of the stroma of tumor. ECM represent a complex network of macromolecules which undergo extensive reconstruction during tumor progression. Such remodeling of the extracellular matrix during cancer progression causes changes in its density and composition [41]. Damage to the ECM structure leads to reactive growth of tumor cells due to switching of intracellular signaling processes and cell cycle changes [42]. Proliferation increases, normal tissue architectonics is lost, local migration of tumor cells and invasion into surrounding stromal tissue occurs. One of the main factors determining the degree of tumor malignancy is the process of EMT characterized as loss of epithelial phenotype by epithelial cells and acquisition of mesenchymal phenotype associated with the ability to migrate through basal membrane. ECM is accompanied by loss of cell adhesion molecule E-cadherin, cytokeratins, increased N-cadherin, fibronectin, and vimentin [43]. ECM proteins provide biochemical signals to induce EMT. In turn, EMT becomes an inducer of metastasis, triggering various transcription factors. Thus, transformation plays an important role in tumor progression and metastasis, involving various transcription factors (TFS) and signaling processes [43,44].
Our results show that high miR-153 expression is associated with TNM stage increase in PC patients. Several potential PC related genes and pathways were identified in the study, which will improve our understanding of the molecular mechanisms which support prostate cancer progression and development. A key signaling pathway, the ECM-receptor interaction signal pathway, was identified as possibly involved in the development of PC. Further investigations are needed to perform in-depth analysis on gene ontology, browser tracks, and expression levels of miR-153 targets in metastatic PC patients.
Ethical approval for this study was obtained from Institute of Biochemistry and Genetics Bioethics Committee. The study was carried out in accordance to Helsinki Declaration and local guidelines.
This work was supported by the Megagrant from the 10.13039/501100017638Government of Russian Federation № 075-15-2021-595, the 10.13039/501100003443Ministry of Education, and Science of the Russian Federation № 122041400169-2. In the study, DNA samples from the “Collection of Biological Materials of Human Beings” of the IBG UFRC RAS were used, supported by the Program of Bioresource Collections of the 10.13039/501100013176FASO of Russia [Agreement No.007–030164/2]. This work was supported by the Bashkir State Medical University Strategic Academic Leadership Program (PRIORITY-2030).
All samples investigated in this study were obtained with written informed consents of the participants.
All supporting data and materials are available from the corresponding author upon reasonable request.
Irina Gilyazova: conceptualization, writing–original draft, and project administration. Elizaveta Ivanova: writing–review and editing, investigation. Mikhail Sinelnikov: formal analysis, writing–review and editing, methodology. Ilgiz Gareev: resources and data curation. Aferin Beilerli, Ludmila Mikhaleva, and Yanchao Liang: validation and data curation. Valentin Pavlov and Elza Khusnutdinova: validation and visualization. Valentin Pavlov and Elza Khusnutdinova: supervision and funding acquisition. All authors have read and agreed to the published version of the manuscript.
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. | true | true | true |
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PMC9627156 | Yaqiang Sun,Jiawei Luo,Peien Feng,Fan Yang,Yunxiao Liu,Jiakai Liang,Hanyu Wang,Yangjun Zou,Fengwang Ma,Tao Zhao | MbHY5-MbYSL7 mediates chlorophyll synthesis and iron transport under iron deficiency in Malus baccata | 19-10-2022 | Malus baccata,iron deficiency,chlorophyll synthesis,Fe transporter,regulatory network,MbHY5 | Iron (Fe) plays an important role in cellular respiration and catalytic reactions of metalloproteins in plants and animals. Plants maintain iron homeostasis through absorption, translocation, storage, and compartmentalization of iron via a cooperative regulative network. Here, we showed different physiological characteristics in the leaves and roots of Malus baccata under Fe sufficiency and Fe deficiency conditions and propose that MbHY5 (elongated hypocotyl 5), an important transcription factor for its function in photomorphogenesis, participated in Fe deficiency response in both the leaves and roots of M. baccata. The gene co-expression network showed that MbHY5 was involved in the regulation of chlorophyll synthesis and Fe transport pathway under Fe-limiting conditions. Specifically, we found that Fe deficiency induced the expression of MbYSL7 in root, which was positively regulated by MbHY5. Overexpressing or silencing MbYSL7 influenced the expression of MbHY5 in M. baccata. | MbHY5-MbYSL7 mediates chlorophyll synthesis and iron transport under iron deficiency in Malus baccata
Iron (Fe) plays an important role in cellular respiration and catalytic reactions of metalloproteins in plants and animals. Plants maintain iron homeostasis through absorption, translocation, storage, and compartmentalization of iron via a cooperative regulative network. Here, we showed different physiological characteristics in the leaves and roots of Malus baccata under Fe sufficiency and Fe deficiency conditions and propose that MbHY5 (elongated hypocotyl 5), an important transcription factor for its function in photomorphogenesis, participated in Fe deficiency response in both the leaves and roots of M. baccata. The gene co-expression network showed that MbHY5 was involved in the regulation of chlorophyll synthesis and Fe transport pathway under Fe-limiting conditions. Specifically, we found that Fe deficiency induced the expression of MbYSL7 in root, which was positively regulated by MbHY5. Overexpressing or silencing MbYSL7 influenced the expression of MbHY5 in M. baccata.
Although iron content is very abundant in the earth, its main existing form is ferric iron (Fe3+), which is insoluble and difficult for plants to uptake (Jeong and Guerinot, 2009). Iron (Fe) is one of the most essential micronutrients in plants and plays an important role in whole-life processes, including chlorophyll synthesis, electron transfer, and respiration (Kobayashi and Nishizawa, 2012). Also, iron can affect physiological processes such as nitrogen metabolism, carbohydrate, and organic acid metabolism in plants (Curie and Briat, 2003; Hell and Stephan, 2003; Kobayashi and Nishizawa, 2012). Fe deficiency can cause a series of problems in fruit production (Tagliavini et al., 1995; Alvarez-Fernandez et al., 2003; Hao et al., 2022). Therefore, revealing the sophisticated mechanism of Fe2+ uptake, transport, and homeostasis in fruit plants is important for fruit yield and quality. Fe deficiency affects a variety of physiological and biochemical reactions in the leaves and roots of fruit plants. One of the most prominent symptoms in plant is interveinal chlorosis, or veins yellowing, which leads to a reduced photosynthetic performance of fruit trees (Curie and Briat, 2003; Hao et al., 2022). About 80% of the total iron was stored in chloroplasts; although iron is not a component of chlorophyll, it is an indispensable catalyst for chlorophyll synthesis (Yang et al., 2022). Previous studies have shown that the number of thylakoid membranes decreased in the lamellar structure of the chloroplast under iron deficiency (TerBush et al., 2013). Roots under iron deficiency can form root tip swellings or increase lateral roots and/or root hairs (Morrissey and Guerinot, 2009). Iron content in plants mainly depends on the uptake and transport of exogenous iron by roots. In plants, there are two distinct strategies for root iron uptaking (Ivanov et al., 2012). Plant species belonging to the dicot and non-graminaceous monocot lineages use Strategy I, which consists of three steps: first, proton efflux from plant cells was mediated by the P-type ATPase to decrease the pH of the rhizosphere soil, which leads to soil acidification and an increase of iron solubility. Meanwhile, Fe(III) is also chelated and mobilized by coumarin-family phenolics exported by an ABC transporter PDR9 from the cortex to the rhizosphere (Tsai and Schmidt, 2017). Next, Fe(III) is reduced to Fe(II) by ferric reduction oxidase 2 (FRO2) localized on the plasma membrane. Third, the divalent iron Fe(II) was taken up into epidermal cells by metal transporter IRT1 (Eide et al., 1996; Santi and Schmidt, 2009). Subsequently, nicotianamine synthase (NAS), yellow stripe-like (YSL), and other transporters helped Fe(II) transport to vacuoles, chloroplasts, and other organs and organelles for further utilization (Walker and Connolly, 2008). Strategy II plants (grasses) synthesize and secrete phytosiderophores (PS) which form chelates with Fe(III) in roots, and this complex was then transported into cells by YSL transporters (Curie et al., 2009). In either way, YSLs play key roles in iron transportation and acquisition. Multiple copies of YSL genes were found in the genomes of angiosperm and gymnosperm species (Chowdhury et al., 2022). AtYSL1, AtYSL3, AtYSL4, and AtYSL6 have been demonstrated to be involved in the transportation of Fe and Zn from leaves to seeds through the phloem (Murata et al., 2006; Ishimaru et al., 2010; Kumar et al., 2019). The expression of AtYSL2 was downregulated in response to iron deficiency (Zang et al., 2020). In addition, YSLs have been proposed as transporters of iron from xylem to phloem and then to young tissues (Le Jean et al., 2005; Morrissey and Guerinot, 2009). YSL2 and YSL7 have been found to be associated with the movement of Fe/Zn-NA complexes to maintain Fe homeostasis in Arabidopsis (Khan et al., 2018). HY5 (elongated hypocotyl 5) is a member of the basic leucine zipper (bZIP) transcription factors, which is known for its key roles in light reception and transmission (Gangappa and Botto, 2016; Li et al., 2020). Moreover, HY5 has been shown to be a positive regulator in nitrate absorption, phosphate response, and copper signaling pathways (Zhang et al., 2014; Huang et al., 2015; Chen et al., 2016; Gao et al., 2021). Arabidopsis HY5 mutants contain less chlorophyll content (Oyama et al., 1997; Holm et al., 2002; Xiao et al., 2022). A recent study has shown that HY5 can bind the promoter of the FER gene in roots, which is required for the induction of iron mobilization genes, thus providing us a new perspective in understanding the regulatory mechanism of iron uptake in plants (Guo et al., 2021). However, few studies have reported the correlation of HY5 and chlorophyll synthesis genes under Fe-deficient conditions. Moreover, no report has yet been published on the regulative role of HY5 to YSL iron transporters in response to iron stress in Malus. Malus baccata has been widely used as a cold-resistant apple rootstock, especially in Northeast China. However, M. baccata is sensitive to iron deficiency. In this study, we compared the physiological characteristics and the transcriptive features of M. baccata under Fe-sufficient/deficiency conditions in the leaves and the roots and explored the regulative role of MbHY5 to chlorophyll metabolic genes and iron transporters (MbYSL). Our results provide insight into the molecular mechanism of iron deficiency response in M. baccata.
M. baccata in vitro shoots were cultured on MS medium (0.5 mg/l 6-BA and 0.5 mg/l IBA) for 30 days (Hao et al., 2022). Next, seedlings (with a height ~5 cm) were transported to the rooting medium (0.5 mg/l IBA) and cultured for 30 days. Rooted seedlings were transplanted into an improved-Hoagland nutrient solution and cultured for 3 weeks. Seedlings were cultivated at 25 ± 2°C day/21 ± 1°C night with a 16-h day/8-h night photoperiod.
Seedling leaves grown on Fe-sufficient (+Fe, 40 μM) and Fe-deficient (-Fe, 0 μM) for 0, 24, 72, and 144 h were sampled, respectively. Leaves were cut into pieces after cleaning and removal of the veins. Next, 0.2 g tissues was mixed with quartz sand, calcium carbonate, and 95% ethanol. The absorbance of the filtrate was measured using a spectrophotometer (Shimadzu, Kyoto, Japan) at 663 and 645 nm. The rhizosphere pH was measured using a pH meter.
FCR activity was determined by the Ferrozine assay. The roots were first cultivated under +Fe and -Fe conditions for 0, 72, and 144 h and were then submerged into a chromogenic medium (0.5 mM ferrozine, 0.5 mM FeNa-EDTA, 0.5 mM CaSO4, and 0.7% (w/v) agar (Schmidt et al., 2000)) and incubated in the dark for 1 h. All measurements were performed at room temperature with a Shimadzu spectrophotometer (Kyoto, Japan).
Fresh root, stem, and leaf tissues were collected and placed in a small box (2 cm*2 cm*2 cm), which contains an appropriate amount of OCT, with tissues submerged by an embedding agent. Next, the bottom of the box was exposed to liquid nitrogen for quick freezing. Finally, the embedded blocks were placed on a freezing microtome for slicing, with continuous slicing of 10~20 μm. Perls staining was conducted using a Prussian Blue Iron Stain Kit (Solarbio, 60533ES20). Micro-tissues were transferred into Perls solution and stained for 0.5~1 h, then they were washed with deionized water and incubated in the methanol solution (Sun et al., 2020). Imaging was performed with a volume microscope (BA210, Motic) (Jia et al., 2018).
The roots and leaves of the M. baccata seedlings treated under +Fe and -Fe conditions (see above) at different times were sampled 1 g for each sample. The samples were first dried at 105°C for 30 min then were placed at 80°C for 72 h till the samples were completely dry. Inductively coupled plasma–optical emission spectrometry was used to determine the active iron contents (Zheng et al., 2021).
Total RNA was extracted from the roots of M. baccata seedlings and was purified using the RNAprep Pure Plant Kit (TIANGEN, Beijing, China) according to the manufacturer’s instructions. cDNA was prepared from total RNA using the HiScript II 1st Strand cDNA Synthesis Kit (+gDNA wiper) (Vazyme, Nanjing, Chain). The LightCycler® 480 II system (Roche) was used for the qPCR assay, and the primers are listed in Supplementary Table 5 . The relative expression of each gene was calculated based on the 2-△△Ct method. A total of 30 groups of RNA-seq data from a project (PRJNA598053) was used to analyze the expression pattern of chlorophyll synthesis and iron transporter genes under Fe sufficiency and Fe deficiency conditions (0, 24, and 72 h) (Sun et al., 2020) (https://www.ncbi.nlm.nih.gov/bioproject/PRJNA598053/ ). Data for the project were downloaded from the NCBI database, including roots and leaves. The expression abundance of the leaves and roots genes was calculated using the FPKM value, and the relative expression level is shown as log2 (fold change) values.
The full length of the MbHY5-coding sequence was inserted into the PRI101 (AN) vector. The promoters (upstream ~2 kb) of MbYSL7 or MbYSL2 were cloned respectively into the pCAMBIA1391 vector with the GUS reporter (Li et al., 2021b). Histochemical GUS staining of Nicotiana benthamiana leaves was conducted as previously described (Liu et al., 2002; Sun et al., 2020). The samples were incubated for 24 h at 37°C. Chlorophyll was removed by washing the samples with 70% (v/v) ethanol for 2 days. Imaging was performed with a volume microscope (MZ10F, Leica).
The full length of the MbYSL7-coding sequence was amplified without the stop codon using the specific primer pairs ( Supplementary Table 5 ) and was inserted into the PRI101 (AN) vector with the 35S promoter. In order to repress the expression of MbYSL7, the pTRV-MbYSL7 vector was constructed as previously described (Sun et al., 2020; Hao et al., 2022). The MbYSL7-overexpression and VIGS vectors were transformed into Agrobacterium tumefaciens cells (GV3101). Infected apple seedlings were placed in a dark place for 2 days and then were transferred to normal light conditions for 1 day. Seedlings grown on Fe-sufficient and Fe-deficient conditions for 0, 24, 72, and 144 h were sampled and then stored at -80°C for RNA extraction.
The full-length MbHY5 CDS sequence was inserted into pB42AD (AD vector), while the MbYSL7 or MbYSL2 protein-binding sites (CACGTG) were inserted into pLacZi (BD vector). The fusion vectors were transformed into the yeast EYG48 strain (Li et al., 2021b; Wu et al., 2021).
Homologous YSL gene sequences of M. domestica, M. baccata, and Arabidopsis thaliana were aligned using ClustalX version 2.0 (Jeanmougin et al., 1998). The phylogenetic tree was constructed in MEGA (version 11) (Tamura et al., 2021) with the Neighbor-Joining method (bootstrap replicates = 100).
In order to identify key genes involved in Fe deficiency in M. baccata, chlorophyll synthesis-related genes and iron homeostasis-related genes were selected, and their expression patterns under Fe deficiency were investigated based on the transcriptome data. Subsequently, their co-expressed genes were predicted using the AppleMDO database (network analysis) (http://bioinformatics.cau.edu.cn/AppleMDO/) (Da et al., 2019). Finally, these genes (503 genes in the leave samples and 693 genes in root samples) were selected to construct the co-expression network using Cytoscape 3.8.0 (Shannon et al., 2003; Zhao et al., 2017).
Statistical analyses were executed using GraphPad Prism. The correlation of MbHY5 and chlorophyll synthesis- and roots iron homeostasis-related genes was calculated using the Pearson correlation (Lv et al., 2021). All statistical analyses were performed by one-way ANOVA test, with p ≤ 0.05 considered as significantly different among different samples. Diagrams illustrating the mechanism of chlorophyll synthesis and Fe acquisition were created using BioRender (https://biorender.com/) (Therby-Vale et al., 2022).
The chlorophyll content of M. baccata leaves showed a continual decrease from 0 to 144 h ( Figure 1A ) under -Fe treatments. After 144 h, the rhizosphere pH of -Fe treatment was lower than that of +Fe treatment, but with no statistically significant differences ( Figure 1B ). The results indicated that iron deficiency caused lower chlorophyll content in the leaves and a decrease in rhizosphere pH. Meanwhile, as for the content of active Fe in the leaves, it decreased from 104 to 42 mg/kg·DW after 144-h Fe deficiency stress. Similarly, its content in the roots also decreased from 923 to 284 mg/kg·DW ( Figures 1C, D ). We further measured the FCR activity of the roots to better understand the iron acquisition processes. Fe-deficient roots showed higher FCR activity in contrast with Fe-sufficient roots at different treatment times ( Figure 1E ). Moreover, Perls staining results showed that tissues (leave, stem, and root) from Fe-sufficient conditions showed stronger Fe3+ staining than Fe-deficient ones ( Figure 1F ). Interestingly, it also showed that Fe deficiency induces a sharp decrease of Fe3+ in xylem and phloem ( Figure 1F ). In conclusion, these results revealed that iron deficiency induced morphological and biochemical changes in M. baccata, including decreases in chlorophyll content, rhizosphere pH, and active iron content in the leaves and roots.
We hypothesized that the well-known light-responsive gene HY5 or PIF genes may have participated in the regulation of the chlorophyll synthesis process ( Figure 2A ). Indeed, we detected a series of chlorophyll metabolic genes from RNA-seq analysis under Fe deficiency, including Glu-tRNA reductase (HEMA), Glu 1-semialdehyde (GSA), uroporphyrinogen III synthase (UROS), chlorophyll synthase (CHLG), GUN, Chla oxygenase (CAO), protochlorophyllide oxidoreductase (PPO), and divinyl reductase (DVR). The results showed that HEMA1-1, HEMA1-2, CHLG1-1, CHLI, PPO5, CAO1-2, and CRD1 were highly expressed in all treatment times ( Figure 2B ). In contrast, the gene expressions of DVR, CHLG1-2, CLH1-1, UROS, and CLH were significantly lower in leaves ( Figure 2B ). Specially, the expression levels of PPO3, PPO9, CHLM1-1, CLH1, PPO8, GUN, GSA1-2, CHLM1-2, HEMA1-3, CAO1-2, CHLG1-1, and HEMA1-1 were significantly changed under Fe deficiency. We constructed a gene co-expression network to investigate the correlation of HY5 or PIF genes and the chlorophyll biosynthesis-related genes. The results showed that HY5, PIF1, PIF3, HMEA, GSA, and GUN form a complicated co-expression network in regulating chlorophyll biosynthesis ( Figure 2C ; Supplementary Table 1 ). Moreover, the expression levels of HY5 were positively correlated with those of most chlorophyll biosynthesis-related genes, such as HMEA, GSA1-2, CAO, CHLI, PPO, and GUN. The Pearson correlation coefficients between HY5 and these genes ranged from 0.54 to 0.78. In contrast, UROS, CHLG1-2, DVR, and CLH1-1 were only slightly correlated or did not correlate with HY5 ( Figure 2D ).
Under iron deficiency conditions, Malus baccata, similar to other dicots, use Strategy I to acquire Fe in roots. We summarized the key genes reported in transferring and regulating Fe2+ transportation from the rhizosphere into root cells, including AHA2, FRO2, PDR9, IRT1, bHLH100/101, OPT3, and FIT (Ito and Gray, 2006; Satbhai et al., 2017; Khan et al., 2018; Lv et al., 2021; Pei et al., 2022) ( Figure 3A ). Under Fe deficiency, most of these genes were highly induced in roots, especially for PDR1, HY5, YSL7, FDR2, and FER genes ( Figure 3B ). In order to analyze the regulatory network of iron homeostasis genes in roots, a total of 693 iron homeostasis-related genes in roots were selected to construct the co-expression network, and the results showed that MbHY5–bHLH04–FIT–FRO2 constructed the biggest module, indicating that MbHY5 plays an essential role in regulation iron homeostasis in roots ( Figure 3C ; Supplementary Table 2 ). Pearson correlation analysis further showed that iron homeostasis-related genes differentially expressed in root under Fe deficiency were significantly positively related with MbHY5, including OPT3, PDR1, bHLH104, YSL, and AHA10 ( Figure 3D ). The correlation coefficients ranged from 0.45 to 0.78 ( Figure 3D ).
We found that the expressions of YSL2 and YSL7 were highly related to HY5 (r = 0.7693 and 0.7119, respectively, Pearson correlation) ( Figure 4A ). The phylogenetic tree showed that each of the apple YSL genes clustered with its closely related homologous genes in Arabidopsis ( Figure 4B ). Previous studies have shown that HY5 can bind to the promoters of SlFER and AtBTS and induce the expression of a series of iron-uptaken genes under iron-deficient conditions (Guo et al., 2021; Mankotia et al., 2022). A G-box (CACGTG) element was found in each of the promoters of MbYSL2 and MbYSL7, which allows HY5 binding ( Figure 4C ). Y1H analysis showed that MbHY5 can directly bind to the promoter of MbYSL7, but not that of MbYSL2 ( Figure 4D ). Transient transformation of tobacco leaves with proMbYSL7:GUS showed lower GUS activity than co-transformation with 35S:MbHY5 ( Figures 4E, F ). Similarly, co-transformation of 35S:MbHY5 and proMbYSL2:GUS showed slightly higher GUS activity than the transformation of proMbYSL2:GUS only ( Figures 4E, F ). In conclusion, these data suggested that MbHY5 functions as a positive and direct regulator of MbYSL7.
To further investigate whether MbYSL7 was involved in regulating Fe deficiency responses in apple, we made transient transformed lines of apple seedlings with overexpression vector and VIGS vector, respectively. As we can see, compared with the control line, the expression levels of MbYSL7 were highly induced in the transient transformed apple seedling lines of 35S:MbYSL7-1, -2, and -3 ( Figure 5A ). Under –Fe treatment, the expressions of MbYSL7 and MbHY5 were highly increased in MbYSL7 overexpression lines, compared with the control lines ( Figure 5B ). The expression of MbYSL7 was greatly reduced in pTRV : MbYSL7-1 ( Figure 5C ). Specifically, the expression of MbYSL7 slightly increased at the 144-h -Fe treatment, compared with that of the 72-h treatment. In comparison, the expression level of MbHY5 was lowest at the initial -Fe treatment but greatly induced from 24 h onward ( Figure 5D ). Similar to MbHY5, we found that MbYSL7 was positively related with chlorophyll synthesis-related genes as well, including PPO5, GSA1-2, and HEMA ( Supplementary Table 3 ). In addition, we observed that MbYSL7 positively correlated with most of Fe homeostasis genes in root either, such as AHA10, bHLH104, and PDR2; the correlation coefficients ranged from 0.40 to 0.92 ( Supplementary Table 4 ).
In plants, iron deficiency leads to chlorosis caused by a reduced chlorophyll biosynthesis (Li et al., 2021a). Chlorophyll content decreased dramatically in chlorosis leaves under Fe deficiency ( Figure 1 ), which is in agreement with the findings in citrus and grapes (Chen et al., 2004; Jin et al., 2017). Iron deficiency increased ferric chelate reduction (FCR) activity and decreased the rhizosphere pH of the apple roots ( Figure 1 ). Also, we observed a reduction of active Fe content in the leaves and roots under iron deficiency. Perls staining is a reliable chemical method to stain the iron trivalent in tissues; ferric iron reacts with potassium ferrocyanide and generates blue insoluble compounds (Lv et al., 2021; Hao et al., 2022). Under Fe deficiency, a lower ferric iron content was observed compared to that of the Fe-sufficient treatment ( Figure 1 ). HY5 has been found to be involved in the metabolism of nitrogen (N), phosphorus (P), copper (Cu), sulfur (S), etc. (Zhang et al., 2014; Gangappa and Botto, 2016; Yang et al., 2020; Gao et al., 2021). In Arabidopsis, HY5 regulates the expression of key nitrogen signaling genes including NIA1, NIR1, NRT1.1, NRT2.1, and AMT1;2 (Jonassen et al., 2008; Jonassen et al., 2009; Yanagisawa, 2014; Chen et al., 2016; Xiao et al., 2022). In apple, NIA2 and NRT1.1 were positively regulated by HY5 in promoting nitrate assimilation (An et al., 2017). Nevertheless, few studies have reported its function in Fe uptake and homeostasis. In Arabidopsis, HY5 regulates BTS in response to Fe deficiency. Similar results were also found in tomato, in which the HY5-FER pathway could be involved in Fe metabolism (Guo et al., 2021; Mankotia et al., 2022). In the present study, we firstly found that MbHY5 was significantly changed in M. baccata under Fe deficiency. HY5 plays essential roles in photosynthetic pigment synthesis in light responses (Liu et al., 2017; Liu et al., 2020). It regulates the expression of chlorophyll-related genes in leaves, including HEMA1, GUN4, CAO, PORC, and CHLH (Toledo-Ortiz et al., 2014; Job and Datta, 2021). In addition, HY5 can regulate the genes involved in maintaining iron homeostasis, such as FRO2, FIT, IRTI, and PYE in roots (Mankotia et al., 2022). Further analysis found that MbHY5 participated in the regulation of chlorophyll synthesis in the leaves and iron acquisition in the roots under iron deficiency ( Figures 2 and 3 ). Our results enriched the regulatory mechanism of HY5 in plants in response to Fe deficiency. YSL genes have been found to participate in plant metal uptake, such as Cu and Fe (Ishimaru et al., 2010; Zheng et al., 2012; Dai et al., 2018). In Arabidopsis, AtYSL1-3 and AtYSL6-8 were responsive under Fe deficiency conditions; among them, some were characterized as long-distance signaling media or Fe(II)-NA transporters (Waters et al., 2006; Castro-Rodriguez et al., 2021). Previously, MtYSL7, AtYSL7, and GmYSL7 were identified and characterized as peptide transporters without further functional annotation (Castro-Rodriguez et al., 2021; Gavrin et al., 2021). Our results suggested that MbYSL7 plays an important role under Fe deficiency. Interestingly as evidenced by our Y1H and the transient co-transformation assays, MbYSL7 was positively regulated by MbHY5. Overall, we propose that MbHY5-YSL7 was involved in regulating the genes involved in chlorophyll synthesis and iron transportation, in both the leaves and the roots, to alleviate iron deficiency-caused chlorosis and to promote Fe transportation ( Figure 6 ).
Contrasting differences of chlorophyll content and the concentration of active iron were observed under +Fe and -Fe conditions in M. baccata. We propose that MbHY5 functions as a vital transcription factor in regulating chlorophyll synthesis and Fe transportation. Lastly, MbHY5 directly regulates the expression of MbYSL7 in roots under Fe deficiency.
The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found in the article/ Supplementary Material .
YS designed the experiment, analyzed the data, and drafted the manuscript. YS, JWL, and PF prepared the materials and performed the bioinformatics analysis. YL, JKL, and FY helped with the qRT-PCR analysis. YZ, FM, and TZ edited the manuscript. All authors contributed to the article and approved the submitted version.
This work was financially supported by the National Natural Science Foundation of China (32102311 and 32102338), the China Postdoctoral Science Foundation (2021M690129), the Chinese Universities Scientific Fund (2452020265 and 2452021133), and the Xinjiang Production and Construction Corps Key Laboratory of Protection and Utilization of Biological Resources in Tarim Basin (BRZD2105).
The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher. | true | true | true |
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PMC9627563 | Guiyuan Gu,Yuhong Wang,Yucheng Shen,Yan Ma,Hongli Yang,Shan Huang,Lin Hu | A pan‑cancer analysis of RCC2 and its interaction with HMGA2 protein in an in vitro model of colorectal cancer cells | 20-10-2022 | regulator of chromosome condensation 2,high mobility group A2,receiver operating characteristic,biomarkers,cancer prognosis | Regulator of chromosome condensation 2 (RCC2) is highly involved in the development of tumor malignancies. The underlying mechanisms remain to be elucidated. The present study aimed to explore the role of RCC2 in the development of tumor malignancies and explore the underlying mechanisms in colorectal cancer (CRC). RCC2 expression and survival analysis were performed in human pan-cancer. The results of searching its mRNA expression in The Cancer Genome Atlas (TCGA) database showed that RCC2 was highly expressed in different types of cancer. High RCC2 expression levels were significantly correlated with poor survival outcomes by the Kaplan-Meier analysis in the TCGA database. Immunohistochemistry revealed that RCC2 was higher expressed in 36 CRC tissues than in adjacent normal tissues. Co-immunoprecipitation revealed that RCC2 bound to high mobility group A2 (HMGA2). Ectopic expression of RCC2 promoted cell proliferation, migration and invasion, whereas knockdown of HMGA2 exerted the opposite effects. Collectively, the data provided a novel biomarker of RCC2 in various types of cancer. High RCC2 expression levels were correlated with poor prognosis in different types of cancer. In addition, RCC2 may combine with HMGA2 to promote CRC malignancy. | A pan‑cancer analysis of RCC2 and its interaction with HMGA2 protein in an in vitro model of colorectal cancer cells
Regulator of chromosome condensation 2 (RCC2) is highly involved in the development of tumor malignancies. The underlying mechanisms remain to be elucidated. The present study aimed to explore the role of RCC2 in the development of tumor malignancies and explore the underlying mechanisms in colorectal cancer (CRC). RCC2 expression and survival analysis were performed in human pan-cancer. The results of searching its mRNA expression in The Cancer Genome Atlas (TCGA) database showed that RCC2 was highly expressed in different types of cancer. High RCC2 expression levels were significantly correlated with poor survival outcomes by the Kaplan-Meier analysis in the TCGA database. Immunohistochemistry revealed that RCC2 was higher expressed in 36 CRC tissues than in adjacent normal tissues. Co-immunoprecipitation revealed that RCC2 bound to high mobility group A2 (HMGA2). Ectopic expression of RCC2 promoted cell proliferation, migration and invasion, whereas knockdown of HMGA2 exerted the opposite effects. Collectively, the data provided a novel biomarker of RCC2 in various types of cancer. High RCC2 expression levels were correlated with poor prognosis in different types of cancer. In addition, RCC2 may combine with HMGA2 to promote CRC malignancy.
Regulator of chromosome condensation 2 (RCC2), also known as telophase disc-60 (TD60), was initially identified in the anaphase spindle midzone (1). RCC2 is an essential protein in the chromosomal passenger complex (2). It was defined according to the movements from centromeres during early mitosis to the spindle midzone (3,4). The RCC2 protein, encoded by the RCC2 gene, is a guanine exchange factor that activates Ras-related protein RalA, a small GTPase. The RCC2 and RalA proteins are both essential in kinetochore-microtubule functions in early mitotic stages (5). Studies have documented that RCC2 facilitates tumorigenesis and enhances metastasis in different types of tumor. Matsuo et al (6) report that miR-29c downregulates RCC2 and inhibits the proliferation of gastric carcinoma. Micro (miR)-1247 targets RCC2 and suppresses the proliferation of pancreatic cancer (7). miR-331-3p suppresses ovarian cancer metastasis and proliferation by targeting RCC2(8). RCC2 promotes breast cancer proliferation by regulating the Wnt signaling pathways (9). In addition, RCC2 is also been implicated in melanoma recurrence and overall survival outcomes (10). Studies have also revealed that RCC2 promotes the progression of CRC malignancies. Bruun et al (11) report high RCC2 expressions in patients with microsatellite instability (MSI). Impaired RCC2 levels affect clinical endpoints of CRC. High-risk patients with CRC and MSI were identified with cost-effective routine RCC2 assays. Song et al (5) reveal that p53 binds to a palindromic RCC2 motif to act as a transcriptional regulator. However, RCC2 mechanisms in CRC remain to be elucidated. High mobility group A2 (HMGA2) is a small architectural transcription factor and contains three AT-hook DNA-binding motifs (12,13). Higher expression of HMGA2 leads to oncogenesis with increased cell proliferation and metastatic potential (14). Overexpression of HMGA2 promotes malignant progression in various types of tumors especially in CRC (15). The authors previously reported that HMGA2 promotes intestinal tumorigenesis by accelerating the degradation of p53(16). The present study aimed at determining the oncogenic role of RCC2 in various types of cancer by analyzing its expression levels in cancerous and normal tissues. The clinical overall and recurrence-free survival of RCC2 in various types of cancer were also determined. These cancers were stomach adenocarcinoma, CRC, liver cancer, prostate cancer, bladder urothelial carcinoma, renal clear cell carcinoma, head and neck squamous cell carcinoma, lung adenocarcinoma, endometrial cancer, sarcoma, mesothelioma, brain lower grade glioma, pancreatic adenocarcinoma, adrenocortical cancer and renal papillary cell carcinoma. The RCC2 expression levels in CRC and adjacent normal tissues were also determined. Finally, the relationships between RCC2 and HMGA2 were evaluated to ascertain the molecular mechanisms of RCC2 mediated CRC progression.
Human CRC cell lines, including DLD1, HCT116, HCT8, HT29, LOVO, RKO, SW620 and SW480 cell lines, were maintained in Soochow University (Suzhou, China). The HT29 cell line was authenticated by STR identification. The cell lines were maintained at 37˚C in RPMI-1640 supplemented with 10% fetal bovine serum, with the exception of HCT116, which was maintained in DMEM. 1% penicillin and streptomycin antibiotic and an atmosphere of 5% CO2 was used for all cell lines.
RCC2 gene expression levels in various types of cancer and correspondence clinical information were obtained from The Cancer Genome Atlas (TCGA) database. These included stomach adenocarcinoma, colorectal cancer, liver cancer, prostate cancer, bladder urothelial carcinoma, head and neck, squamous cell carcinoma, renal clear cell carcinoma, lung adenocarcinoma, endometrial cancer, sarcoma, mesothelioma, brain lower grade glioma, pancreatic adenocarcinoma, adrenocortical cancer and renal papillary cell carcinoma. The datasets were classified into two cohorts: The expression dataset in cancer tissues and in the adjacent normal tissues. A combination of receiver operating characteristic (ROC) curve analysis, specificity and sensitivity were used to choose a cutoff point. The RCC2 expression levels in patients with cancer were used to generate two groups: High and low expression groups. Based on the overall survival and recurrence-free survival times of patients derived from the clinical datasets, the differences between high and low RCC2 expression groups were analyzed and compared. The 10-year overall survival and recurrence-free survival rates was determined by the Kaplan-Meier analysis (17). The TIMER2 database (http://timer.cistrome.org/) was used to analyze the relationship between RCC2 expression and CD8+ T cells in digestive system tumors (18). Pearson's correlation analysis was performed to reveal the correlations between RCC2 and HMGA2 in digestive system tumors.
CRC cell pellets were lysed by the RIPA lysis buffer and a protease inhibitor cocktail (both Beyotime Institute of Biotechnology) was added. The protein concentration of CRC cell lysates was determined with a BCA kit (Pierce; Thermo Fisher Scientific, Inc.). For western blotting 30 µg of cell lysate protein were analyzed by 6-18% SDS PAGE, transferred onto 0.45-µm PVDF membranes (MillporeSigma). The PVDF membranes were blocked with 5% skimmed mild for 15 min at room temperature. Then the proteins were probed with primary antibodies against RCC2 (1:1,000; cat. no. 5104; CST Biological Reagents Co., Ltd.) and GAPDH (1:10,000; Clone 686613; R&D Systems Inc.) for 12 h at 4˚C. The western blots were incubated with DyLight 680 or DyLight 800 conjugated secondary antibodies (Cell Signaling Technology, Inc.) for 1 h at room temperature and visualized by the Odyssey Imaging System (LI-COR Biosciences). Western blot images were normalized by Image Studio 3.1 software (LI-COR Biosciences).
The RCC2 expression levels in 36 cases of patients with CRC were assessed by immunohistochemistry of paraffin sections, which were from the First Affiliated Hospital of Soochow University (Suzhou, China). Primary RCC2 antibody was obtained from Abcam (cat. no. ab70788; rabbit RCC2 polyclonal antibodies, anti-RCC2; 1:200). This was performed as previously described (16). Briefly, immunohistochemistry was conducted on 2-µm sections using the BenchMark ULTRA automated stainer (Ventana Medical Systems, Inc.) in accordance with the manufacturer's instructions. RCC2 scoring was performed according to the proportion of positive cancer cells (1, 0-25%; 2, 25-50%; 3, 50-75% and 4, >75%) and the staining intensity of cancer cells (negative, 0; light yellow, 1; dark yellow, 2 and brown, 3). Slides were analyzed under bright field microscopy. The formula for obtaining the IHC staining score was: IHC staining score=percentage of positive cancer cells x staining intensity of the cancer cells. Scoring was carried out by two pathologists, independently. Approval for the present study was obtained from the Institutional Ethics Committee of Soochow University (authorization number ECSU-2019000212).
Full-length RCC2 coding sequences (CDSs) were subcloned into pcDNA3.1-FLAG plasmid while HMGA2 was subcloned into pcDNA3.1-Myc plasmid as previously described (16). Vectors with ligated sequences were confirmed by matched DNA sequencing. Lipofectamine® 2000 (Invitrogen; Thermo Fisher Scientific, Inc.) was used to transfect empty vector and pcDNA3.1-FLAG-RCC2 with or without pcDNA3.1-Myc-HMGA2 into 293T cells when the cell density was 70% according to the manufacturer's instructions. The transfection mixtures were pre-incubated for 15 min at room temperature before transfection. A total of 2 µg (1 µg each) of plasmid DNA was used in transfection of each well of a 6-well culture plate and the duration of transfection was 6 h. At 36 h post-transfection, the expression efficiency of RCC2 and HMGA2 was confirmed by western blot analysis.
293T cells were divided into three subgroups. The first subgroup was transfected with pCDNA3.1 empty vector and pcDNA3.1-FLAG-RCC2, the second subgroup was transfected with pCDNA3.1 empty vector and pcDNA3.1-Myc-HMGA2, while the third subgroup was transfected with pcDNA3.1-FLAG-RCC2 and pcDNA3.1-Myc-HMGA2. A total of 4 µg (2 µg each) of plasmid DNA was transfected onto a 6-cm plate when the cell density was 50%. After 48 h, the cell pellets were lysed using lysis buffer [20 mM Tris-HCl (pH 7.4), 150 mM NaCl, 0.5% NP-40, 10% glycerol, 1 mM DTT and complete protease inhibitor cocktail] for 30 min on ice and centrifuged at 20,000 x g for 15 min. Supernatants (300 µg) were immunoprecipitated using M2-FLAG-magnetic beads (cat. no. M8823; MilliporeSigma) according to the manufacturer's instructions. Cell lysates were then analyzed by subjecting them to SDS-PAGE and immunoblotting with indicated antibodies.
In 96-well plates, HCT116 cells were plated at 2,000 per well and cultured for 0, 24, 48, 72 and 96 h. Then, 10 µl of the CCK-8 reagent (cat. no. C0039; Beyotime Institute of Biotechnology) was added into each well and the cells were incubated for another 2 h. The OD values at 450 nm were measured by microplate reader (Synergy 4 Hybrid Multi-Detection Reader; BioTek Instruments, Inc.). For migration and invasion assay, HCT116 cells (5x104/well) resuspended in the serum-free RPMI-1640 were plated onto the upper chamber of a Transwell (cat. no. 3422, Corning, Inc.), with the upper chamber surface precoated with Matrigel for 1 h at 37˚C (cat. no. 356234; BD Biosciences) for invasion assay. The bottom chamber contained RPMI-1640 with 30% FBS. After culturing for 24-48 h, cells transferred through the membrane were fixed with methanol for 30 min at room temperature and stained with 0.1% crystal violet for 20 min at room temperature (cat. no. C0121; Beyotime Institute of Biotechnology) and the cells remaining in the upper chamber were wiped off. The images of three randomly selected fields of view were captured under a microscope at x20 magnification (Eclipse Ti-S; Nikon Corporation). To analyze the fraction of apoptotic cells, the HCT116 cells were detected by Annexin V-APC/7-AAD apoptosis detection kit (Nanjing KeyGen Biotech Co., Ltd.) according to the manufacturer's instructions. All these experiments were performed three times independently.
An analysis of the data was performed using the Statistical Package for Social Sciences (SPSS version 20.0; IBM Corp.). The experimental data are shown as the mean ± standard deviation of triplicate independent sets of experiments. GraphPad Prism 7.0 (GraphPad Software Inc.) was used for graphs. For comparisons between two groups, data were analyzed using an unpaired Student's t-test and comparisons among multiple groups were performed using one-way analysis of variance followed by Tukey's multiple comparisons test. P<0.05 was considered to indicate a statistically significant difference.
RCC2 was found to be significantly high expressed (P<0.001) in digestive system tumors such as stomach adenocarcinoma (Fig. 1A), CRC (Fig. 1B), and liver cancer (Fig. 1C) when compared with the adjacent normal tissues. RCC2 was also found to be a biomarker for digestive system cancers. This was due to its expression being upregulated in cancer tissues and associated with poor overall survival outcomes. In 360 stomach adenocarcinoma patients, the high RCC2 expression group exhibited poor clinical overall survival outcomes (Fig. 1D, P=0.049). In 349 CRC (Fig. 1E) and 332 patients with liver cancer (Fig. 1F), a high RCC2 expression indicated poor recurrence-free survival/overall survival (P=0.022 and P<0.001, for each respective cancer). Furthermore, RCC2 expression was positively correlated with CD8+ T cells in colon adenocarcinoma (Fig. S1A), stomach adenocarcinoma (Fig. S1B) and liver hepatocellular carcinoma (Fig. S1C). RCC2 was positively correlation with cytotoxic T-lymphocyte associated protein 4 (CTLA4) in a number of tumors including colon adenocarcinoma (Fig. S1D), liver hepatocellular carcinoma (Fig. S1E) and stomach adenocarcinoma (Fig. S1F). RCC2 was also positively correlated with CD274 (PDL1) in liver hepatocellular carcinoma (Fig. S1E). Therefore, RCC2 may be a good biomarker for immune checkpoint inhibitor treatment of these tumors. Finally, the RCC2 expression levels was evaluated in different tumor stages including colon adenocarcinoma (Fig. S2A), liver hepatocellular carcinoma (Fig. S2B), and stomach adenocarcinoma (Fig. S2C). There was no difference between RCC2 expression level and tumor progression in the low RCC2 expression group. In the high RCC2 expression group, only in liver hepatocellular carcinoma did RCC2 expression level increase with tumor progression.
The TCGA database exhibited similar results in the urogenital male reproductive system cancers such as prostate cancer (Fig. 2A), bladder urothelial carcinoma (Fig. 2B) and renal clear cell carcinoma (Fig. 2C). There were significantly high RCC2 expression levels (P<0.001) in these cancer tissues compared with matched normal tissues. In 466 patients with prostate cancer (Fig. 2D), 279 patients with bladder urothelial carcinoma (Fig. 2E) and 525 renal clear cell carcinoma patients (Fig. 2F), high RCC2 expression levels (P=0.001, 0.05 and <0.001, respectively) were correlated with poor prognosis for 10 year survival/recurrence-free survival. In prostate cancer, high RCC2 expression levels (P=0.002) were also associated with poor overall survival and recurrence-free survival (Fig. S3A).
The RCC2 mRNA expression levels were found to be significantly high (P<0.001) in head and neck squamous cell carcinoma (Fig. 3A) as well as in lung adenocarcinoma patients (Fig 3B). Furthermore, the head and neck squamous cell carcinoma (Fig. 3C) and lung adenocarcinoma (Fig. 3D) with elevated RCC2 levels (P=0.011 and 0.001, respectively) exhibited worse clinical outcomes in overall survival when compared with low expression levels. In addition, high RCC2 expression levels (P=0.012) were correlated with worse recurrence-free survival outcomes in 350 lung adenocarcinoma patients (Fig. S3B).
RCC2 expression levels were found to be high in endometrial (Fig. 4A) and sarcoma cancer (Fig. 4B) tissues compared with correspondence normal tissues (P<0.001 and <0.05, respectively). High expression levels of RCC2 were associated with worse recurrence-free survival in endometrial cancer (Fig. 4C) and worse overall survival in sarcoma (Fig. 4D; P=0.066 and 0.006, respectively). A high expression was also correlated with poor recurrence-free survival in sarcoma (Fig. S3C). In cholangiocarcinoma (Fig. S3D), breast cancer (Fig. S3E) and esophageal cancer (Fig. S3F), RCC2 was found to be highly expressed in cancer tissues (P<0.001) compared with the adjacent normal tissues. These findings show that RCC2 also serves as a biomarker and can predict clinical overall survival/recurrence-free survival time in endometrial cancer and sarcoma.
In 73 patients with mesothelioma (Fig. 5A), 462 patients with brain lower grade glioma (Fig. 5B) and 163 patients with pancreatic adenocarcinoma (Fig. 5C), high RCC2 expression levels (P=0.022, 0.007 and <0.001 respectively) were correlated with poor overall survival. In the mesothelioma (Fig. 5D), brain lower grade glioma (Fig. 5E) and pancreatic adenocarcinoma (Fig. 5F) cancers, the recurrence-free survival analysis revealed that high RCC2 expression levels were associated with worse 10-year recurrence-free survival for each of the above cancers (P=0.068, 0.002, and 0.006 respectively). In 79 patients with adrenocortical cancer, elevated RCC2 expression was correlated with poor overall survival, (P<0.001; Fig. 5G). In 250 patients with renal papillary cell carcinoma, high RCC2 expression levels were correlated with poor overall survival (P=0.03; Fig. 5H).
RCC2 is a novel cancer biomarker and can be used as a predictor for poor clinical prognosis. To uncover the role of RCC2 in CRC, immunohistochemical staining in 36 paired CRC tissues and adjacent normal tissues were performed. It revealed that RCC2 was highly expressed in CRC tissues (Fig. 6A). The IHC staining score showed that RCC2 expression was significantly high in CRC compared with the adjacent normal tissue (P<0.001; Fig. 6B). It has been documented that HMGA2 promotes CRC malignancy by regulating the translation of fibronectin 1 (FN1) and IL11(19). To promote CRC tumorigenesis, HMGA2 also enhances the degradation of P53(16). The present study revealed that RCC2 and HMGA2 were highly expressed in HCT116, HCT8 and SW620 cell lines (Fig. 6C). In addition, RCC2 was shown to interact with HMGA2 in 293 cells that had been transiently transfected with pcDNA3.1-vector, pcDNA3.1-FLAG-RCC2, pcDNA3.1-Myc-HMGA2 expression plasmids, followed by dual Co-IP assays with an anti-FLAG antibody. The RCC2 and HMGA2 proteins Co-IP reciprocally in these cells (Fig. 6D). The endogenous Co-IP assay also confirmed the interaction between RCC2 and HMGA2 (Fig. 6E). In addition, RCC2 was positively related to HMGA2 in liver hepatocellular carcinoma (Fig. S4B) and stomach adenocarcinoma (Fig. S4C). However, there was no correlation between RCC2 and HMGA2 in colon adenocarcinoma (Fig. S4A). To test whether RCC2 expression in CRC cells affected their proliferation or tumorigenicity through HMGA2, RCC2-WT was ectopically expressed in HCT116 cells using lentiviral constructs and knockdown of HMGA2. In vitro growth kinetics assay revealed that overexpression of RCC2 significantly promoted cell proliferation of HCT116 and knockdown of HMGA2 reduced the proliferation rate of HCT116 (Fig. 6F). Colony formation assay also showed a similar result (Fig. 6G). Migration and invasion experiments were conducted to determine whether RCC2 and HMGA2 contribute to the migratory and invasive characteristics of CRC cells. The results revealed that overexpression of RCC2 significantly increased the migration and invasion of HCT116 cells and this effect was eliminated with downregulation of HMGA2 (Fig. 6H). Overexpression of RCC2 blocked the spontaneous apoptosis of HCT116 cells and this effect was mediated by HMGA2 (Fig. 6I). To test whether RCC2 could regulate the activity of HMGA2, which contributes to colorectal carcinogenesis, the HMGA2 downstream target genes FN1 (Fig. 6J) and IL11 (Fig. 6K) were detected. The results showed that RCC2 could promote the transcriptional activation of HMGA2. Overall, these results suggested that RCC2 promotes CRC malignancy by associating with HMGA2.
RCC2 was first identified as a nuclear protein located at the chromosomal centromeres essential for cell division (1). The role of RCC2 in the establishment and progression of tumors has been studied extensively in recent years. RCC2, as an oncogene, is involved in cancer tumorigenesis and metastasis. RCC2 overexpression promotes cancer malignant progression (20). In breast cancer, RCC2 promotes malignant progression by regulating the Wnt signaling pathways and epithelial-mesenchymal transition (EMT) (9). In lung cancer, RCC2 mediates the effect of long non-coding RNA LCPAT1 on migration, invasion, cell autophagy and EMT (21,22). Conversely, the downregulation of RCC2 mRNA expression leads to opposite effects; miRNAs such as miR-29c, miR-1247 and miR-331-3p inhibit cancer malignancy by targeting RCC2 (6-8). The sarcomas are a group of tumors with a wide variety of localization and the survival rate depends on the affected organ. For example, in head and neck sarcomas the survival rate is influenced by the surgical removal (23). The present study evaluated the role of RCC2 in different types of tumor including stomach adenocarcinoma, CRC, liver cancer, prostate cancer, bladder urothelial carcinoma, renal clear cell carcinoma, head and neck squamous cell carcinoma, lung adenocarcinoma, endometrial cancer, sarcoma, mesothelioma, brain lower grade glioma, pancreatic adenocarcinoma, adrenocortical cancer and renal papillary cell carcinoma. The results showed that RCC2 was upregulated in these types of cancer. Moreover, patients with highly expressed RCC2 exhibited a short overall and recurrence survival rate. To further understand the mechanistic role of RCC2 in CRC. Immunohistochemical staining revealed that RCC2 was highly expressed in patients with CRC. Whole-genome sequencing reveals that RCC2 is one of the commonly mutated genes in CRC (24). RCC2 acts as an oncogene in microsatellite instable tumors, and low level of RCC2 protein expression is associated with poor prognosis of microsatellite stable tumors. One reason is that RCC2 inhibits cancer cell metastasis by regulating integrin α5β1-fibronectin signaling pathway (11,25). Our knowledge of the different roles of RCC2 serves in various phases of tumor progression and metastasis is limited. The present study found that RCC2 and HMGA2 were highly expressed in HCT116, HCT8 and SW620 CRC cell lines. Co-IP assays demonstrated that RCC2 interacted with HMGA2. RCC2 promotes tumor metastasis by interacting with Rac1 and Arf6 (26,27). The present study identified a new RCC2 binding protein that provided new insights into RCC2-mediated tumor progression. It was also found that RCC2 expression is positively related to HMGA2 in liver hepatocellular carcinoma and stomach adenocarcinoma. Although HMGA2, as an architectural transcription factor, has no intrinsic transcriptional activity, it can induce gene transcription by changing chromatin architecture (28,29). It was hypothesized that the interaction between RCC2 and HMGA2 promotes architectural changes in promoter regions of some genes. Ectopic expression of RCC2 promoted cell proliferation, migration and invasion in vitro, whereas knockdown of HMGA2 exerted the opposite effects. In addition, RCC2 promoted the transcriptional activation of HMGA2. Further studies characterizing RCC2 coordination with upstream and downstream functional pathways and functional target proteins are required. Taken together, the results of the present study suggested that RCC2 could be a novel biomarker in human cancer and provide new insights into the mechanisms of RCC2 in CRC progression. | true | true | true |
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PMC9627658 | 36338034 | Tingjun Liu,Yuhan Li,Shengjie Chen,Lulu Wang,Xiaolan Liu,Qingru Yang,Yan Wang,Xiaorong Qiao,Jing Tong,Xintao Deng,Shihe Shao,Hua Wang,Hongxing Shen | CircDDX17 enhances coxsackievirus B3 replication through regulating miR-1248/NOTCH receptor 2 axis | 13-10-2022 | coxsackievirus B3,circular RNAs,miRNAs,NOTCH2,METTL3 | Coxsackievirus B3 (CVB3) was one of the most common pathogens to cause viral myocarditis. Circular RNAs as novel non-coding RNAs with a closed loop molecular structure have been confirmed to be involved in virus infectious diseases, but the function in CVB3 infection was not systematically studied. In this study, we identified that hsa_circ_0063331 (circDDX17) was drastically decreased after CVB3 infection by circRNA microarray. In vivo and in vitro, when cells or mice were infected with CVB3, the expression of circDDX17 was significantly reduced, as demonstrated by quantitative real-time PCR assays. Additionally, circDDX17 enhanced CVB3 replication by downregulating the expression of miR-1248 in HeLa and HL-1 cells, and miR-1248 regulated CVB3 replication through interacting with the gene coding for NOTCH Receptor 2 (NOTCH2), and NOTCH2 could upregulate methyltransferase-like protein 3 (METTL3). Taken together, this study suggested that circDDX17 promoted CVB3 replication and regulated NOTCH2 by targeting miR-1248 as a miRNAs sponge. | CircDDX17 enhances coxsackievirus B3 replication through regulating miR-1248/NOTCH receptor 2 axis
Coxsackievirus B3 (CVB3) was one of the most common pathogens to cause viral myocarditis. Circular RNAs as novel non-coding RNAs with a closed loop molecular structure have been confirmed to be involved in virus infectious diseases, but the function in CVB3 infection was not systematically studied. In this study, we identified that hsa_circ_0063331 (circDDX17) was drastically decreased after CVB3 infection by circRNA microarray. In vivo and in vitro, when cells or mice were infected with CVB3, the expression of circDDX17 was significantly reduced, as demonstrated by quantitative real-time PCR assays. Additionally, circDDX17 enhanced CVB3 replication by downregulating the expression of miR-1248 in HeLa and HL-1 cells, and miR-1248 regulated CVB3 replication through interacting with the gene coding for NOTCH Receptor 2 (NOTCH2), and NOTCH2 could upregulate methyltransferase-like protein 3 (METTL3). Taken together, this study suggested that circDDX17 promoted CVB3 replication and regulated NOTCH2 by targeting miR-1248 as a miRNAs sponge.
Coxsackievirus B3 (CVB3) is an RNA virus belonging to the Enterovirus genus of Picornaviridae. CVB3 has been identified as the most common pathogen for viral myocarditis (VM). CVB3 infections are spread worldwide, and the clinical manifestations are mainly asymptomatic or mild infections, and cold-like symptoms, but newborns and children are more likely to have severe diseases, such as pancreatitis, myocarditis, encephalitis, and type 1 diabetes (Pauschinger et al., 2004; Kühl et al., 2005). Many factors can affect the infection of the virus, like host protein and non-coding RNAs (ncRNAs). ncRNAs include microRNAs (miRNAs), long non-coding RNA (lncRNA), and circular RNA (circRNA; Salmena et al., 2011). Circular RNAs (circRNAs) are a novel class of ncRNAs, originating from pre-mRNAs. CircRNAs are circularized by connecting a 5′ splice site with the 3′ splice site of an upstream exon or intron by a back-splicing reaction (Wang et al., 2016). Most circRNAs are composed of exons and are located in the cytoplasm; they play a significant role in regulating the translation and modification of proteins (Danan et al., 2012). In some research, it has been shown that circRNAs act as sponges for miRNAs; by forming the competing endogenous RNAs loops, circRNAs could direct binding with specific miRNAs to regulate post-transcriptional gene expression events (Memczak et al., 2013). CircBACH1 regulated hepatitis B virus by miR-200a-3p/MAP 3K2 axis (Du et al., 2022). CircSIAE inhibited CVB3 by targeting miR-331-3p (Yang et al., 2021). CircEAF2 reduced Epstein–Barr virus by miR-BART19-3p/APC/β-catenin axis (Zhao et al., 2021). These researches showed that circRNAs play an important role in infectious diseases. The hsa_circ_0063331 (circDDX17) was formed by reverse splicing the linear transcript of exons 2–5 of the DEAD-Box Helicase 17 (DDX17) gene with a length of 451 nucleotides. DDX17 was a member of the DEAD-box helicase family proteins involved in cellular RNA folding, splicing, and translation (Linder and Jankowsky, 2011). Moreover, DDX17 was involved in some virus replication, like by binding to specific stem-loop structures of viral RNA to antivirus (Moy et al., 2014). In another study, it could downregulate the expression of Epstein–Barr virus genes by YTH domain-containing proteins recruiting (Xia et al., 2021). Major studies about circDDX17 were focused on cancer, like circDDX17 as a tumor suppressor in colorectal cancer, breast cancer, and colorectal cancer (Li et al., 2018; Lin et al., 2020; Peng and Wen, 2020; Ren et al., 2020), but its function in the virus was still unclear. N6-methyladenosine (m6A) is intimately associated with three categories of molecular compositions: “writers,” “readers,” and “easers” (Zaccara et al., 2019). Writers are m6A methyltransferases like the methyltransferase-like protein 3 (METTL3) and methyltransferase-like protein 14 (METTL14). Some research showed that METTL3 could regulate virus replication, like METTL3 inhibits Enterovirus 71 by autophagy regulation (Xiao et al., 2021), decreases syndrome coronavirus clade 2 viral load and viral gene expression in host cells (Li et al., 2021), and promotes Epstein–Barr virus infection of nasopharyngeal epithelial cells (Dai et al., 2021). NOTCH1 to 4 are transmembrane receptors that determine cell fate. The NOTCH Receptor 2 (NOTCH2) has been reported to exert distinct functions in regulating tissue homeostasis and cell fate determination (Baron, 2017; Afaloniati et al., 2020). In infectious diseases, NOTCH2 is possibly involved in regulating the Epstein–Barr virus latent/lytic status (Giunco et al., 2015), and 4.3% of hepatitis C virus-positive cells diffuse large B-cell lymphoma have NOTCH2 mutations (Arcaini et al., 2015). Previous studies have shown that NOTCH2 has some relationship with METTL3, like the Notch signaling pathway as an important downstream target of METTL3 in muscle stem cells (Liang et al., 2021), but no data showed that NOTCH2 has a direct relation with METTL3. In this study, we found silence NOTCH2 could downregulate METTL3, and overexpression NOTCH2 could upregulate METTL3. In co-immunoprecipitation analysis, METTL3 was present in the immunoprecipitated complex, and METTL3 was partially co-localized with NOTCH2 in HeLa cells. Here, we study the effect of CVB3 infection on expression levels of circRNAs and investigate potential downstream mechanisms of their involvement in viral processes in vivo and in vitro. We examined the prevalence, regulation, and functional roles of circDDX17 in CVB3 infection. CVB3 infection could decrease the expression level of circDDX17 in cells and mics. CircDDX17 up-regulated CVB3 replication in HeLa and HL-1 cells. Furthermore, CircDDX17 regulated NOTCH2 by target miR-1,248, miR-1,248 could down-regulate NOTCH2 expression and inhibit CVB3 replication.
HeLa and HEK-293T cells were a gift from Dr. Huaiqi Jing (Chinese Center for Disease Control and Prevention). HL-1 cells were stored at the School of Medicine, Jiangsu University. Cells were cultured with Dulbecco’s modified Eagle’s medium (DMEM, Gibco, United States), supplemented with 8% fetal bovine serum (FBS, Gibco, United States), 100 U/ml penicillin, and 100 μg/ml streptomycin in 5% CO2 at 37°C. CVB3 (Nancy; Corsten et al., 2015) was a gift from Professor Ruizhen Chen (Department of Cardiology, Zhongshan Hospital, Shanghai, China). GFP-CVB3, expressing the green fluorescence protein (GFP; Lei et al., 2013; Shuo et al., 2014).
Coxsackievirus B3 (105 PFU/mouse) was injected intraperitoneally into 3-week-old BALB/c male mice. CVB3 was diluted in 100 μl PBS for injection, and an equal volume of PBS was injected into the blank control mice (3 mice per group). This study was conducted according to the recommendations in the Guide to the Care and Use of Experimental Animals-Chinese Council on Animal Care. All protocols were approved by the Animal Care Committee of University Jiangsu (protocol number: UJS-IACUC-AP-20190307087).
PcicR-3.0-circDDX17 (pcircDDX17) for overexpression circDDX17, PcicR-3.0 (pcicR) for its negative control. PcDNA-3.0-NOTCH2 (pNOTCH2) for overexpression NOTCH2, used pcDNA-3.0 (pcDNA) for its negative control. miR-885 mimics (miR-885), miR-1248 mimics (miR-1248), and miR-1279 mimics (miR-1279), negative control (miR-NC); miR-1,248-inhibitor (miR-1,248-in), negative control (NC-in); siRNA-NOTCH2 (si-NOTCH2), negative control (si-NOTCH2-NC) were all synthesized by GenePharma Co., Ltd. (Suzhou, China), the sequences were listed in Table 1. Plasmid and oligonucleotide were transfected using Lipofectamine 3000™ (Invitrogen, United States).
The total RNA was isolated using Trizol reagent (Invitrogen, United States). PrimeScript RT Reagent Kit (Takara, Japan) was used for reverse transcription RNA, and quantitative real-time PCR (RT-qPCR) was performed using TB Green Premix Ex TaqII (Takara, Japan). The RT-qPCR was conducted to examine the expression levels of circDDX17, mRNA levels for GAPDH, NOTCH2, and VP1, and miRNA levels for miR-885, miR-1248, miR-1279, and U6. The divergent primer was synthesized by Sangon (China), and the sequences are listed in Table 2. For RNase treatment, 2 mg of total RNA was incubated with or without 3 U/mg RNase R for 30 min at 37°C.
According to the manufacturer’s instructions, nuclear plasma was extracted with a cytoplasmic nucleus extraction kit (Thermo Fisher, United States); the steps were followed as previously described (Yang et al., 2021).
Cy5-labeled circDDX17 probes (Jima Biotech, China) were detected in HeLa cells using a Fluorescent in Situ Hybridization Kit (Jima Biotech, China) following the manufacturer’s guidelines. Cell nuclei were counterstained with DAPI (Jima Biotech, China). The glass slides were analyzed and images were captured under a fluorescence microscope.
Cells cultured in collagen-coated chamber slides (NEST Biotechnology Co., Ltd., China) were washed and fixed with either 4% paraformaldehyde or with ice-cold methanol. Cells were permeabilized with 0.1% Triton X-100 in PBS, slides were stained with all primary antibodies (anti-METTL3, 1:200, anti-NOTCH2, 1:100), washed three times with PBS, and stained with conjugated Alexa Fluor secondary antibodies Alexa Fluor 488/594 (1200, Genetex, United States), cell nuclei were counterstained with DAPI (Jima Biotech, China). The glass slides were analyzed and images were captured under a fluorescence microscope.
The total proteins of the cells were extracted using the RIPA lysis buffer (Sigma, United States). Samples were subjected to 12% SDS-PAGE and transferred to polyvinylidene fluoride (PVDF) membranes (Millipore, United States). The primary antibodies against NOTCH2 (1:1,000, Sangon, China), VP1 (1:1,000, Genetex, United States), METTL3 (1:2,000, CST, United States), methyltransferase-like protein 14 (METTL14; 1:2,000, CST, United States), and GAPDH (1:20000, Genetex, United States). Membranes were blocked, incubated with secondary antibody (1:20000, Jackson, United States), and detected by electrochemiluminescence (ECL, Millipore, United States).
For coimmunoprecipitation (co-IP) assays, 500 μg HeLa cell lysate protein was reacted with primary antibodies (10 μl) overnight at 4°C and incubated with protein A/G beads at the next day for 4 h at 4°C. Then immunoprecipitated proteins were eluted from the beads for Western blotted with indicated antibodies.
HEK-293 T cells (1 × 105/well) were plated in 24-well plates. Cells were co-transfected with miR-1248 and psiCHECK-2 luciferase reporter to generate psiCHECK-2-circDDX17-wild-type (circDDX17-wt) or psiCHECK-2-circDDX17-mutant (circDDX17-mu) constructs after 48 h. Luciferase activity was determined following a dual-luciferase reporter assay detection kit (Promega, Madison, WI, United States).
Sample supernatants were collected at CVB3 7 h post-infection, serially diluted, and added onto HeLa cells in 24-well plates (1.0 × 105 cells/well). After incubation for 1 h, cells were rewashed with PBS three times, overlaid with 0.75% soft agar medium, and incubated for 3 days. Cells were fixed with glacial acetic acid for 30 min and stained with 1% crystal violet. All the assays were conducted at least in triplicate.
Statistical analysis was performed using Graph Pad Prism 7 (GraphPad Software Inc., San Diego, CA, United States). The group difference was evaluated by Student’s t-test, error bars represent mean ± SD and the difference was statistically significant when *p < 0.05, **p < 0.01, or ***p < 0.001.
The Circbank database showed that circDDX17 is formed by reverse splicing of the linear transcript of exons 2–5 of the DDX17 gene with a length of 451 nucleotides. To further characterize circDDX17, Sanger sequencing was performed to confirm head-to-tail splicing (Figures 1A,B). FISH analysis (Figure 1C) and nuclear separation assay (Figures 1D,E) was conducted to determine the subcellular localization of circDDX17 in HeLa cell lines. To investigate the roles of circDDX17 in CVB3 infection, HeLa and HL-1 cells were infected with CVB3, and circDDX17 levels were examined. In HeLa cells, at the early stages of infection (0–2 h), no significant change in circDDX17 expression was detected between CVB3-infected and mock control cells. At 4–7 h post-infection, the circDDX17 expression was significantly decreased compared to MOCK cells (Figure 1F). The expression level of circDDX17 in HL-1 cells infected with 200 MOI CVB3 was lower than 100 MOI CVB3 at 24 h post-infection (Figure 1G). In VM mice, the circDDX17 expression level was lower than in MOCK mice (Figure 1H).
HeLa cells (Figures 2A,B) and HL-1 cells (Figure 2C) overexpressed circDDX17 were infected with CVB3 to analyze the expression of VP1. The results showed that circDDX17 increased the expression of VP1 after CVB3 infection. HEK-293 T cells co-transfected with pcicR-3.0-circDDX17 (pcircDDX17) and GFP-CVB3, cells overexpressed circDDX17 GFP-positive cell number were observed more than cells co-transfected with pcicR-3.0 (pcicR) and GFP-CVB3 (Figure 2D). To gain further insight into the function of cricDDX17 on CVB3 replication, a viral plaque assay was adopted. The results showed that cricDDX17 overexpression increased viral release compared to the control (Figure 2E). In contrast, circDDX17 silencing decreased the expression of VP1 at CVB3 after CVB3 infection (Figures 2F–H). HEK-293T cells co-transfected with siRNA-circDDX17 (si-circDDX17) and GFP-CVB3 showed lower GFP-positive cells than the cells transfected siRNA-circDDX17-NC (si-circDDX17-NC) and GFP-CVB3 (Figure 2I), and circDDX17 silencing could reduce viral release (Figure 2J).
CircRNAs have been shown to act as a miRNA sponge to regulate gene expression (Guo et al., 2020); therefore, the potential miRNAs associated with circDDX17 were investigated. First, through bioinformatics analysis, three miRNAs (miR-885, miR-1248, and miR-1279) were identified from the overlap of three databases (circBank, miRanda, and CircInteractome) as possible targets for circDDX17. RT-qPCR of HeLa cells transfected with pcircDDX17 or si-circDDX17 showed that circDDX17 downregulates the miRNAs expression (Figures 3A–C). To analyze the role of CVB3 on miRNA expression, total RNA from CVB3 infected cells was collected for RT-qPCR. miR-885, miR-1248, and miR-1279 expression increase with virus infection (Figures 3D–F). To investigate the role of miRNAs in CVB3 infection, HeLa cells were transfected with miRNAs mimics, and the results showed that miR-885, miR-1248, and miR-1279 could inhibit the replication of CVB3, among which miR-1248 had the most obvious effect (Figure 3G), so we chose miR-1248 as the object of study. The dual-luciferase reporter assay confirmed the direct interaction between circDDX17and miR-1248. The circDDX17-wild-type (circDDX17-wt) and circDDX17-mutant (circDDX17-mu) full-length sequences without miR-1248 binding sites were cloned into the luciferase vector. Subsequently, luciferase reporter assays confirmed that miR-1248 mimics markedly reduced the luciferase activity of circDDX17-wt but not that of circDDX17-mu compared to the miR-NC group (Figure 3H). To investigate the signal pathways contributing to the miR-1,248 effect on CVB3 replication, we sought to identify its target genes. Bioinformatic analyses using TargetScan, miRDB, and miRWalk programs showed that NOTCH2 is one of the predicted targets. HeLa cells were transfected with miR-1248 mimics (miR-1248) or miR-1248-inhibitor (miR-1248-in), and the results showed that miR-1248 has a negative regulatory role on NOTCH2 expression (Figure 3I). In previous research, NOTCH2 has a relationship between METTL3 and DNA-methylation (Terragni et al., 2014), we predicted METTL3 as NOTCH2 interaction protein by String, therefore, HeLa cells were transfected with specific si-NOTCH2 or pNOTCH2 to silence or overexpressed NOTCH2. For further verification, we performed coimmunoprecipitation experiments to study the relationship between NOTCH2 and METTL3 in HeLa cells (Figure 3J), the result showed that the METTL3 was present in the immunoprecipitated complex. As shown in Figure 3K, METTL3 and NOTCH2 were distributed in the nucleus although a small fraction of these proteins were also found in the cytoplasm, and METTL3 was partially co-localized with NOTCH2. The Western blot results show that NOTCH2 regulates METTL3 expression, METTL3 increases with the increasing expression level of NOTCH2 and decreases with NOTCH2 decreasing expression level (Figures 3L,M).
To elucidate the mechanism of CVB3 upregulation of host circDDX17, the expressions of DNA-methylation-associated proteins METTL3, and METTL14 were examined. With CVB3 infection, NOTCH2 declined gradually, and METTL3 and METTL14 were elevated (Figures 4A–D). To understand the roles of NOTCH2 in circDDX17-mediated DNA-methylation, we examined the downstream effector gene expression after CVB3 infection in HeLa cells overexpressing or silencing circDDX17. Western blot analysis showed that overexpression of circDDX17 upregulated the expression level of NOTCH2, METTL3, and METTL14 (Figures 4E,F). Conversely, circDDX17 silencing decreases NOTCH2, METTL3, and METTL14 expression levels (Figures 4G,H).
To study the effect of miR-1,248 in NOTCH2, METTL3, and METTL14 expression, HeLa cells were transfected with miRNA mimics or miRNA inhibitors, Western blot showed that miR-1248 played a negative role on NOTCH2 expression, and so did METTL3 and METTL14 expression levels (Figure 5A). Then cells infected with CVB3, miR-1248 decreased VP1 expression while inhibiting miR-1248 increased VP1 expression, and miR-1248 down-regulate the NOTCH2, METTL3, and METTL14 expression (Figure 5B). HeLa cells overexpressing miR-1248 reduced CVB3 replication and cells silencing miR-1248 increased CVB3 replication (Figures 5C,D). At the same time, miR-1248 decreased CVB3 released as detected by viral plaque assay (Figure 5E). To further confirm that miR-1248 downregulation by circDDX17 benefits CVB3 replication, we overexpressed miR-1248 in the presence of circDDX17 by co-transfection, and cells without CVB3 (Figure 5F) or infected CVB3 (Figure 5G). Western blot showed that compared with miR-NC + PcicR, overexpression of miR-1248 inhibited VP1, NOTCH2, METTL3, and METTL14 expression.
To understand the roles of NOTCH2 in circDDX17 and miR-1248-mediated methylation-related pathways, we first confirmed NOTCH2 function on methylation-related proteins. Western blot data showed that independent of CVB3 infection, METTL3, and METTL14 expression was reduced by si-NOTCH2 transfection and induced in pNOTCH2 transfection (Figure 6A). In HeLa cells, VP1 expression was decreased by si-NOTCH2 and increased by pNOTCH2 expression (Figure 6B). Furthermore, in HL-1 cells, overexpression of NOTCH2 increased VP1 expression compared to the negative control (Figure 6C). NOTCH2 could significantly increase VP1 expression level, NOTCH2 deficiency repressed VP1 expression (Figures 6D,E), and overexpression NOTCH2 increased viral release (Figure 6F). To further confirm miR-1248 regulated CVB3 replication by targeting NOTCH2, HeLa cells overexpression miR-1248 and NOTCH2 by co-transfection (Figures 6G,H). Without CVB3 infection, cells transfection miR-NC + pNOTCH2 the METTL3 and METTL14 expression levels were higher than cells transfection miR-1248 + pNOTCH2 (Figure 6G). Seven hours post CVB3 infection, miR-NC + pNOTCH2 increased the production of VP1, METTL3, and METTL14 compared with miR-NC + pcDNA. Co-transfection of pNOTCH2 and miR-1248 reduced the production of VP1, METTL3, and METTL14 compared to miR-NC + pNOTCH2 (Figure 6H).
Coxsackievirus B3 is the commonest pathogen for acute and chronic myocarditis (Pauschinger et al., 2004; Esfandiarei and McManus, 2008). After CVB3 entry into the cardiomyocytes, the virus replicates and induces cell damage, triggering the host immune responses. If the virus cannot be eliminated, myocarditis can become chronic, triggering extensive myocardial fibrosis and the development of dilated cardiomyopathy (Kawai, 1999; Garmaroudi et al., 2015). In our previous study, miR-324-3p inhibits CVB3 replication by targeting the tripartite motif 27 (Liu et al., 2021), but there are fewer studies on circRNA regulation of CVB3 replication. CircRNA can play a role as a miRNA sponge to influence miRNA expression and regulate gene function. This study identified that circDDX17 was a novel regulator of CVB3 replication. MiR-1248, a target miRNA of circDDX17, played a negative role in replicating CVB3 in host cells. Moreover, NOTCH2 was the miR-1248 target gene. NOTCH2 has been involved in cardiac fibrosis, regulating heart development and multiple antiviral immune responses. In addition, NOTCH2 mutations resulted in multiple cardiac diseases and vascular anomalies (Pinkert et al., 2019). Interestingly, NOTCH2 was distributed in m6A modification proteins. m6A was a conserved internal modification found in almost all eukaryotic nuclear RNAs (Jia et al., 2013) and the viral RNA. m6A was dynamic methylation involved in RNA metabolism, splicing, and decay (Roundtree et al., 2017; Zhao et al., 2017). METTL3 could modulate the NOTCH signaling pathway (Wang et al., 2020). In our study, there was a positive correlation between NOTCH2 and METTL3. By the analysis of IP, METTL3 was present in the immunoprecipitated complex, and METTL3 was partially co-localized with NOTCH2, those results showed that METTL3 and NOTCH2 have interaction in cells. METTL3 could negatively regulate type I interferon response by dictating the fast turnover of interferon mRNAs for antivirus (Winkler et al., 2019), and METTL3 boosted Enterovirus 71 replication (Hao et al., 2019), which might explain how NOTCH2 regulates viral replication. In another way, m6A modification was dynamically and reversibly regulated by the “writers” complex (METTL3 and METTL14; Liu et al., 2014). Our study analyzed METTL3 and METTL14 as targets indicating m6A modification changes and function in cells overexpressing or silencing circDDX17 infected with CVB3. The results showed that CVB3 infection could increase the METTL3 and METTL14 expression. METTL14 played an important role in the transcription of IFNs and inflammatory cytokines, and regulates antivirus innate immunology response (Xu et al., 2021). However, in this research, we have not studied the effect of METTL14 on the replication of CVB3. In conclusion, this study reported that circDDX17 promotes CVB3 replication by regulating miR-1248 and NOTCH2/METTL3. These findings enriched our understanding of the functional roles of circRNA in viral replication and provided novel insights into the development of therapeutic strategies.
The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found in the article/Supplementary material.
The animal study was reviewed and approved by Animal Care Committee of University Jiangsu (protocol number: UJS-IACUC-AP-20190307087).
HS and HW conceived and designed the experiments. TL, YL, XL, QY, YW, and XQ performed the experiments. HS, HW, and SS analyzed the data. TL, HS, HW, JT, XD, and SC contributed reagents, materials, and analysis tools. TL, HS, and HW wrote the paper. All authors contributed to the article and approved the submitted version.
The research was supported by National Natural Science Foundation of China, grant no. 81971945 (https://isisn.nsfc.gov.cn/egrantweb/).
The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
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PMC9627666 | 36121123 | Joshua J Peter,Helge M Magnussen,Paul A DaRosa,David Millrine,Stephen P Matthews,Frederic Lamoliatte,Ramasubramanian Sundaramoorthy,Ron R Kopito,Yogesh Kulathu | A non‐canonical scaffold‐type E3 ligase complex mediates protein UFMylation | 19-09-2022 | E3 ligase,enzyme substrate,post‐translational modification,ribosome,ubiquitin‐like modifier,Post-translational Modifications & Proteolysis | Abstract Protein UFMylation, i.e., post‐translational modification with ubiquitin‐fold modifier 1 (UFM1), is essential for cellular and endoplasmic reticulum homeostasis. Despite its biological importance, we have a poor understanding of how UFM1 is conjugated onto substrates. Here, we use a rebuilding approach to define the minimal requirements of protein UFMylation. We find that the reported cognate E3 ligase UFL1 is inactive on its own and instead requires the adaptor protein UFBP1 to form an active E3 ligase complex. Structure predictions suggest the UFL1/UFBP1 complex to be made up of winged helix (WH) domain repeats. We show that UFL1/UFBP1 utilizes a scaffold‐type E3 ligase mechanism that activates the UFM1‐conjugating E2 enzyme, UFC1, for aminolysis. Further, we characterize a second adaptor protein CDK5RAP3 that binds to and forms an integral part of the ligase complex. Unexpectedly, we find that CDK5RAP3 inhibits UFL1/UFBP1 ligase activity in vitro. Results from reconstituting ribosome UFMylation suggest that CDK5RAP3 functions as a substrate adaptor that directs UFMylation to the ribosomal protein RPL26. In summary, our reconstitution approach reveals the biochemical basis of UFMylation and regulatory principles of this atypical E3 ligase complex. | A non‐canonical scaffold‐type E3 ligase complex mediates protein UFMylation
Protein UFMylation, i.e., post‐translational modification with ubiquitin‐fold modifier 1 (UFM1), is essential for cellular and endoplasmic reticulum homeostasis. Despite its biological importance, we have a poor understanding of how UFM1 is conjugated onto substrates. Here, we use a rebuilding approach to define the minimal requirements of protein UFMylation. We find that the reported cognate E3 ligase UFL1 is inactive on its own and instead requires the adaptor protein UFBP1 to form an active E3 ligase complex. Structure predictions suggest the UFL1/UFBP1 complex to be made up of winged helix (WH) domain repeats. We show that UFL1/UFBP1 utilizes a scaffold‐type E3 ligase mechanism that activates the UFM1‐conjugating E2 enzyme, UFC1, for aminolysis. Further, we characterize a second adaptor protein CDK5RAP3 that binds to and forms an integral part of the ligase complex. Unexpectedly, we find that CDK5RAP3 inhibits UFL1/UFBP1 ligase activity in vitro. Results from reconstituting ribosome UFMylation suggest that CDK5RAP3 functions as a substrate adaptor that directs UFMylation to the ribosomal protein RPL26. In summary, our reconstitution approach reveals the biochemical basis of UFMylation and regulatory principles of this atypical E3 ligase complex.
UFM1 is a ubiquitin‐like (Ubl) modifier that contains the characteristic β‐grasp fold found in all UBLs and is highly conserved in eukaryotes (Sasakawa et al, 2006; Van Der Veen & Ploegh, 2012). Like other UBLs, UFM1 is covalently attached to lysine residues on substrates via an enzymatic cascade involving an E1 activating enzyme, UFM1‐activating enzyme 5 (UBA5), E2 conjugating enzyme, UFM1‐conjugating enzyme 1 (UFC1), and an E3 ligase, UFM1 E3 ligase 1 (UFL1; Komatsu et al, 2004; Cappadocia & Lima, 2018). Recent studies have identified that UFMylation of the ribosomal protein RPL26 plays a critical role in endoplasmic reticulum (ER) homeostasis (Walczak et al, 2019; Liang et al, 2020; Wang et al, 2020). Besides ER‐associated roles, UFMylation has also been implicated in other cellular processes including protein translation, DNA damage response, nuclear receptor‐mediated transcription and development (Yoo et al, 2014; Lee et al, 2019; Qin et al, 2019; Wang et al, 2019; preprint: Gak et al, 2020; Liu et al, 2020). Further, loss of or mutations of components of the UFMylation machinery has been linked to many diseases such as cancer, type‐2 diabetes, neurological disorders, and cerebellar ataxia, where the failure of ER homeostasis and protein quality control could be one of the major contributing factors (Yoo et al, 2014; Duan et al, 2016; Liu et al, 2017; Nahorski et al, 2018; Xie et al, 2019). Indeed, UFMylation is an essential post‐translational modification for animal development as loss of UFM1 or any of the UFMylation enzymes results in the failure of erythropoiesis and embryonic lethality (Lemaire et al, 2011; Tatsumi et al, 2011; Cai et al, 2015, 2016). UFM1 is synthesized in a precursor form consisting of 85 amino acids that are then post‐translationally processed by UFM1‐specific proteases (UFSPs) to remove the last two residues at the C‐terminus generating mature UFM1 that contains an exposed C‐terminal glycine, Gly83 (Sung et al, 2007). Mature UFM1 is activated by the E1, UBA5 via the formation of a high energy thioester bond between Cys250 of UBA5 and Gly83 of UFM1 (Komatsu et al, 2004; Oweis et al, 2016). Activated UFM1 is then transferred from UBA5 onto the catalytic Cys116 of UFC1 (Komatsu et al, 2004; Banerjee et al, 2020), which has a canonical UBC fold and an N‐terminal helical extension whose function is not known (Mizushima et al, 2007; Liu et al, 2009). UFC1 lacks many of the features conserved in many E2s such as the catalytic HPN motif suggesting unique modes of action and regulation. Further, some ubiquitin (Ub) E2 enzymes associate with Ub via a backside interaction, which mediates E2 dimerization, helping stabilize E2s in their active‐closed state and enhancing their catalytic efficiency (Brzovic et al, 2006; Buetow et al, 2015; Stewart et al, 2016; Patel et al, 2019). For UFC1, it is unknown whether similar mechanisms exist to regulate its activity as detailed biochemical characterization is lacking. Transfer of UFM1 from UFC1 to substrates is thought to be mediated by UFL1, the only E3 ligase identified in the UFMylation pathway to date (Tatsumi et al, 2010). Deletion of UFL1 results in loss of UFMylation and mice lacking Ufl1 exhibit a failure in hematopoiesis and embryonic lethality suggesting that it could be the main or sole E3 ligase (Cai et al, 2019). Although the role of UFL1 in UFMylation is well established, the mechanism of how it functions as an E3 ligase is not understood (Banerjee et al, 2020). In general, E3 ligases are classified either as scaffold‐type ligases that bring together a UBL‐charged E2 with a substrate for direct transfer of the UBL from the E2 to the substrate, or as Cys‐dependent enzymes where the UBL is first transferred from the E2 to the catalytic Cys of the E3 for subsequent transfer to the substrate (Deol et al, 2019). Examples of scaffold‐type E3s are RING family ligases and RANBP2 whereas HECT, RBR, and RCR enzymes typify the latter type (Deshaies & Joazeiro, 2009; Pichler et al, 2017; Lorenz, 2018; Pao et al, 2018; Walden & Rittinger, 2018). Intriguingly, UFL1 does not possess any conserved sequence or domain features found in known E3 ligases. Hence it is not clear whether UFL1 is a Cys‐dependent E3 enzyme or uses a scaffolding mechanism. UFL1 is predominantly found anchored at the ER through its interaction with an adaptor protein, DDRGK1/UFBP1 (UFM1 binding protein‐1), which has been suggested to be one of its substrates (Tatsumi et al, 2010; Wu et al, 2010). UFBP1 localizes to the ER membrane via an N‐terminal transmembrane segment (Walczak et al, 2019). Intriguingly, loss of UFBP1 affects the stability and expression levels of UFL1 (Tatsumi et al, 2010; Wu et al, 2010; Cai et al, 2015). CDK5RAP3 is another poorly characterized protein commonly associated with UFL1. Several reports have led to speculations that UFL1, UFBP1, and CDK5RAP3 together form an E3 ligase complex (Wu et al, 2010; Banerjee et al, 2020; Witting & Mulder, 2021). Only a handful of substrates of UFL1 have been identified to date and most of them are ER‐localized (Gerakis et al, 2019; Witting & Mulder, 2021). In addition to monoUFMylation, polyUFM1 chains can be assembled, which are mainly linked via K69 (Yoo et al, 2014). In the Ub system, the type of polyUb linkage formed is generally dictated by the last enzyme in the cascade that forms a thioester linkage with Ub before transfer to the substrate. Scaffold‐type E3s such as RING E3 ligases bind to charged E2 (E2~Ub) to mediate the transfer of Ub onto the substrate, and hence, the E2 enzymes are thought to determine the linkage type assembled. By contrast, catalytic Cys containing E3 ligases form an E3~Ub thioester intermediate and dictate the linkage type formed (Deshaies & Joazeiro, 2009; Deol et al, 2019). Given the lack of understanding of the molecular mechanism underpinning the UFM1 conjugation machinery and the mechanisms governing its regulation, here we adopt a rebuilding approach with purified components to reconstitute UFMylation in vitro. Using this approach, we reveal that UFL1 on its own is an unstable protein that cannot support UFMylation. We demonstrate that UFL1 together with UFBP1 forms a functional heterodimeric E3 ligase complex and classify it as a scaffold‐type E3 ligase. Further, we identify CDK5RAP3 to bind to the E3 ligase complex and inhibit UFMylation by preventing the discharge of UFM1 from UFC1. Importantly, by reconstituting UFMylation of ribosomes in vitro, we find that CDK5RAP3 has a regulatory function to restrict UFMylation to bona fide substrates. Our mechanistic insights describe an atypical E3 ligase complex and provide a foundation for investigating its catalytic mechanism, regulation, and substrate specificity.
The interaction of UFC1 with UBA5 and how UFM1 is transferred from UBA5 to the catalytic Cys are well understood (Oweis et al, 2016; Padala et al, 2017; Soudah et al, 2019; Banerjee et al, 2020; Kumar et al, 2021). We therefore focused on subsequent events in the UFMylation cascade. To analyze how the E2 UFC1 works together with the E3 ligase UFL1 to transfer UFM1, we aimed to reconstitute UFMylation reactions using purified components. We expressed 6xHis‐UFL1 in Escherichia coli and purified it by affinity chromatography as the first step. Subsequent size exclusion chromatography (SEC) analysis indicated UFL1 to form soluble aggregates as it was found to elute in the void fraction (Fig EV1A). Our attempts to prevent aggregation using alternative buffers and additives or by incorporating different solubility‐enhancing tags were unsuccessful. As expressing subunits of protein complexes individually may result in aggregation (Warner, 1977; Kihm et al, 2002; Yanagitani et al, 2017), we reasoned that UFL1 might require additional binding partners for its stability and activity. To identify binding partners of UFL1, we performed yeast two‐hybrid (Y2H) screening using a cDNA library derived from the human placenta as prey, which revealed UFBP1 and CDK5RAP3 as interactors (Fig EV1B). We therefore wondered if co‐expressing UFBP1 with UFL1 might confer solubility and stability to UFL1. To achieve this, we used a co‐expression system to express full‐length UFL1 together with UFBP1 lacking its transmembrane sequence and purified the ligase complex with three purification steps (Fig EV1C). Indeed, UFL1 co‐expressed with UFBP1 (UFL1/UFBP1) is well behaved and no longer formed soluble aggregates in SEC (Fig 1A). Mass photometry (Sonn‐Segev et al, 2020) and SEC‐MALS analyses (Figs 1B and EV1D) revealed a stable heterodimeric UFL1/UFBP1 complex. Importantly, the mass photometry measurements are performed at low nanomolar concentrations highlighting the stable nature of the complex. Having obtained soluble UFL1/UFBP1 complexes, we next tested the catalytic activity of UFL1 expressed alone and in complex with UFBP1 using in vitro UFMylation assays containing UBA5, UFC1, and UFM1 and ATP. Whereas UFL1 on its own did not show any appreciable activity, the UFL1/UFBP1 complex is an active ligase as evidenced by UFL1 autoUFMylation and the formation of multiple UFMylated products that correspond to free UFM1 chains and UFMylated UFBP1 and UFC1 (Fig 1C). To check whether UFL1/UFBP1 was also capable of UFMylating substrates in vitro, we used TRIP4/ASC1, a protein reported to be UFMylated in cells (Yoo et al, 2014). Indeed, UFL1/UFBP1 can UFMylate TRIP4 in vitro (Fig 1C, Bottom panel). Intriguingly, the exogenous addition of purified UFBP1 to UFL1 is not sufficient to restore its E3 ligase activity (Fig EV1E and F). The ribosomal subunit RPL26 is one of the best described UFMylation substrates to date with proteomic analyses identifying RPL26 to be the main UFMylated protein in cells (Walczak et al, 2019; Wang et al, 2020). Hence, we set up a cell‐free reconstitution of ribosome UFMylation. Strikingly, the addition of UFL1/UFBP1 to purified 60S ribosomes leads to robust modification of RPL26 (Fig 1D), and in line with previous observations, both mono‐ and di‐UFMylated RPL26 are observed. Taken together, these results show that UFL1 and UFBP1 form an obligate heterodimer required for E3 ligase activity. Since polyUFMylated products are formed in UFMylation reactions, we next analyzed the reaction products by mass spectrometry to determine the linkage types assembled. Interestingly, LC–MS analyses revealed that the polyUFM1 chains formed are predominantly linked via K69 with some K3 and K7 linkages also observed (Figs 1E and EV1G). To validate these observations, we generated a series of K‐R mutants where every Lys in UFM1 was individually mutated to Arg. In line with the mass spectrometry analyses, only UFM1 K69R resulted in near complete loss of polyUFMylated species and unanchored diUFM1, closely resembling reactions with UFM1 K0 in which all Lys were mutated to Arg (Fig 1F, lanes 7 and 8). With UFM1 K69R, mono but not polyUFMylation of UFL1 was observed confirming that UFL1 autoUFMylation involves the formation of K69‐linked polyUFM1 chains (Fig 1F). We next generated Konly UFM1 mutants where all but one Lys is mutated to Arg. All the Konly mutants along with UFM1 K0 mutant abrogate polyUFMylation formation with the sole exception of K69‐only UFM1 (Fig 1G). In summary, our reconstitution experiments reveal that UFL1 together with UFBP1 forms an active E3 ligase complex that can efficiently UFMylate substrates and assemble polyUFM1 chains that are K69‐linked.
Intrinsic reactivity of E2s assayed using free amino acids can give insights into the ability of E2s to transfer UBLs onto substrates and can provide clues about the type of E3 ligase they work with and the nature of the substrate (Stewart et al, 2016; Pao et al, 2018). For instance, E2s such as UBE2D3, which function together with RING/scaffold‐like E3 ligases are capable of aminolysis and are therefore reactive towards the free amino acid Lys (Figs 2A and EV2A; Wenzel et al, 2011). On the other hand, UBE2L3, which functions only with Cys‐dependent E3 ligases is incapable of aminolysis and lacks reactivity to free Lys on its own (Fig EV2A; Wenzel et al, 2011). We therefore analyzed the intrinsic reactivity and stability of the E2~UBL, i.e., UFC1~UFM1 (~ denotes the thioester bond between UFM1G83 and UFC1C116) and compared this to the well‐characterized ubiquitin E2s, UBE2D3, and UBE2L3. When the discharge of UFM1 from UFC1~UFM1 onto free amino acids was analyzed, we observed discharge onto Cys while no discharge was observed in the presence of Lys, Arg, Ser, and Thr (Fig 2B). Under the same experimental conditions, UBE2D3 discharged Ub in the presence of both Cys and Lys (Fig 2B, lanes 2 and 3). Further, using increasing concentrations of free Lys or with prolonged incubation in a time course, we find that UFC1 has negligible Lys reactivity, which is similar to UBE2L3 (Fig EV2B–E). Since UFC1 is reactive only to cysteines, we systematically assessed potential catalytic Cys residues to determine whether UFL1 is a Cys‐dependent enzyme. The N‐terminal region of UFL1 contains four Cys residues, and sequence analysis shows that all four vary in their degree of conservation with C32 being the most conserved (Fig 2C). Based on analysis of predicted folding propensity (Fig EV2F) and secondary structure prediction, we made a C‐terminal truncation of UFL1, UFL11‐410, which when expressed on its own formed soluble aggregates like full‐length UFL1 and required co‐expression of UFBP1 to obtain a heterodimeric complex (Fig EV2G). Importantly, in vitro UFMylation assays showed that UFL11‐410/UFBP1 complex is an active E3 ligase (Fig EV2H). To identify the catalytic Cys in UFL1, we mutated each Cys individually to Ala in the UFL11‐410/UFBP1 complex. To our surprise, in vitro UFMylation assays showed that none of the single mutants affected the autoUFMylation activity of UFL1 or UFMylation of substrates, implying that UFL1 does not contain a catalytic Cys (Fig 2D). Further, we do not observe any transthiolation products of UFL1/UFBP1 demonstrating that UFM1 is not transferred to a Cys residue in the E3 ligase (Fig EV2I). Since UFBP1 does not have any Cys residues, these results suggest that the UFL1/UFBP1 ligase complex may instead employ a scaffolding mechanism to transfer UFM1 onto substrates. The lack of a transthiolation activity raises the possibility that the UFL1/UFBP1 ligase complex could induce aminolysis of UFC1~UFM1. Hence, we compared the discharge of UFM1 from UFC1~UFM1 onto Lys in the absence and presence of the ligase complex. Whereas UFC1 does not discharge UFM1 onto Lys on its own, it was able to do so in the presence of the UFL1/UFBP1 E3 complex (Fig 2E). Based on these observations, we suggest that UFL1/UFBP1 functions as a scaffold‐type E3 ligase that binds to charged UFC1 to promote aminolysis. While inefficient, UFC1 on its own can assemble free UFM1 chains, an activity that is significantly enhanced in the presence of the UFL1/UFBP1 ligase complex (Fig EV2J and K). Interestingly, UFC1 assembles mainly K69 linkages (Fig EV2L) and this linkage specificity is maintained in the presence of UFL1/UFBP1 (Fig 1E). Thus, linkage specificity is determined by the E2 and is not altered by the E3 ligase complex, a feature commonly observed in ubiquitin RING E3 ligases (Deng et al, 2000; Deshaies & Joazeiro, 2009; Branigan et al, 2015). These results further strengthen our conclusion that UFL1/UFBP1 is a scaffold‐type ligase complex.
As UFL1/UFBP1 does not have any obvious sequence or domain features found in any of the known E3 ligases, we attempted to define the minimal catalytic region. As our efforts to experimentally determine the structure of this complex were not successful, we used AlphaFold to predict the structure (Fig 3A; Jumper et al, 2021; Mirdita et al, 2022). Structure prediction of the complex had good predicted aligned error (PAE) scores (Fig EV3A) and shows UFL1 and UFBP1 to form a heterodimer, which are composed of several winged helix (WH) domain repeats (Figs 3A and EV3B and C). These WH domains could be classified as PCI‐like WH domains, named so because they commonly found in components of the proteasome lid, the COP9 signalosome, and the eukaryotic translation initiator, eIF3 (Scheel & Hofmann, 2005; Stewart, 2012). Overall, UFL1 has an N‐terminal helix (a.a 1–25) followed by a partial WH (pWH) domain and five WH domains that extend into a stack of α‐helices at its C‐terminus which we refer to as CTR (C‐terminal region). Likewise, UFBP1 has an N‐terminal transmembrane segment, a long helical region which we refer to as NTR (N‐terminal region) followed by a WH (WH1′) and a partial WH (pWH′) domain. Surprisingly, the partial pWH at the N‐terminus of UFL1 complements the partial pWH′ at the C‐terminus of UFBP1 to form a composite WH (pWH‐pWH′) domain (Fig 3B). The predicted formation of this composite WH domain provides a potential explanation to why UFBP1 is required for the stability of UFL1. The predicted seven WH repeats of the UFL1/UFBP1 complex are arranged such that α‐helix 1 from each WH domain creates a helical backbone. All predicted WH domains of UFL1 and UFBP1 have identical folds except for a β‐strand, which is missing in the composite WH domain, WH1 domain, and WH2 domain (Fig EV3D). The predicted helical nature of UFBP1 and the helical backbone of UFL1 may explain the unusual migration of UFL1/UFBP1 by SEC. To test the predicted structure of the UFL1/UFBP1 complex, we mutated a key residue at the pWH‐pWH′ interface (UFL1 L45R) to disrupt complex formation (Fig 3C). As expected, co‐immunoprecipitation experiments in HEK293 cells expressing tagged versions of UFL1 and UFBP1 revealed that the UFL1 L45R mutant is unable to form a complex with UFBP1 (Fig 3D). Further support to this model is provided by our Y2H screen, which identifies the region spanning a.a 268–298, i.e., the C‐terminal portion of UFBP1 to interact with UFL1. Hence, we conclude that the formation of the composite WH (pWH‐pWH′) domain is essential for complex formation and protein stability. Guided by the insights from the AlphaFold predictions, we made additional truncations to map the minimal catalytic domain of the ligase complex (Fig 3E). The truncated complexes expressed well and were purified to homogeneity (Fig EV3G). We first analyzed the ability of the different truncated complexes to promote the discharge of UFM1 from UFC1~UFM1 onto Lys. All the truncations tested could discharge UFM1 and the smallest region (denoted as Complex I in Fig 3E) that is able to promote discharge contains the predicted composite WH domain and one WH domain each from UFL1 and UFBP1 (Figs 3F and EV4A and B). Many E3 ligases bind to E2~UBL conjugates with higher affinity compared with E2 on their own (Metzger et al, 2014). To investigate the ability of the different UFL1/UFBP1 complexes to bind to the E2 UFC1, we used analytical SEC. Different UFL1/UFBP1 complexes as shown in Fig 3E were incubated with a mixture containing UFC1, UFM1, and a nonreactive UFC1‐O‐UFM1 conjugate where UFM1 is linked to C116S via an oxyester bond (Fig EV4C and D). UFC1‐O‐UFM1 is more stable compared to thioester‐linked UFC1~UFM1 and therefore used for these analyses. Post preparation, we incubated UFC1‐O‐UFM1 with the deUFMylase UFSP2 or 0.2 N NaOH and observed complete collapse only in alkaline conditions confirming that the purified UFC1‐O‐UFM1 is indeed linked via an oxyester linkage (Fig EV4E). Analysis of complex formation by SEC revealed that UFL1/UFBP1 has higher affinity for the UFC1‐O‐UFM1 conjugate compared to UFC1 or UFM1 on its own, and the complex is of high enough affinity to elute as a stable complex (Fig EV3E). Interestingly, even the smallest of the UFL1/UFBP1 variants was able to bind to UFC1‐O‐UFM1 (Fig EV3F). Taken together, the N‐terminal segment of UFL1 (pWH‐WH1) and the C‐terminal region of UFBP1 (WH1′‐pWH′), which we refer to as UFL1/UFBP1min (denoted as I in Fig 3E) is sufficient for both binding to charged UFC1 and activating UFC1~UFM1 for aminolysis may represent the minimal ligase domain. Next, we assayed ligase activity by monitoring diUFM1 formation. Like full‐length UFL1, the different C‐terminal UFL1 truncations co‐expressed with UFBP1 did not exhibit any loss of activity (Fig 3G). Thus, the N‐terminal region of UFL1 (pWH‐WH1) is sufficient for its ligase activity. Similarly, truncation of the N‐terminus region (NTR) of UFBP1 did not affect diUFM1 formation. Lastly, we checked the impact of truncations of different regions of the UFL1/UFBP1 complex on their ability to modify 60S ribosomes (Fig 3H). Deletion of the CTR region on UFL1 did not affect UFMylation of 60S ribosome whereas deletion of WH3, WH4, and part of WH2 completely abolished ribosome UFMylation thus underlining the importance of these regions in ribosome UFMylation. Intriguingly, deletion of the NTR of UFBP1 predominantly led to di‐UFMylation of ribosomes suggesting that this region may play a role in substrate recognition and UFMylation. We then expanded this analysis to other reported substrates such as Histone H4 and MRE11 (Fig EV4F and G). While regions important for MRE11 UFMylation were very similar to that of the 60S ribosome, H4 UFMylation was impaired only by simultaneous deletion of NTR of UFBP1 and all WH domains except pWH and WH1. Also, as observed in the case of 60S ribosome, deletion of the UFBP1 NTR significantly impaired Histone H4 UFMylation (Fig EV4H). This suggests that substrate recognition and UFMylation may follow different modes that are dependent on the substrate. In summary, using systematic truncations based on AlphaFold predictions, we here make the surprising discovery that three tandem WH domains (Fig EV4I) of UFL1/UFBP1 are sufficient for E3 ligase activity, but additional regions are required for substrate modification.
We next analyzed the second hit identified in our Y2H screen, CDK5RAP3, an evolutionarily conserved 53 kDa protein that lacks any known functional domains or motifs. To determine whether CDK5RAP3 can interact with UFL1 in the context of the UFL1/UFBP1 complex, we incubated recombinant full‐length CDK5RAP3 with UFL1/UFBP1 and analyzed complex formation by analytical SEC. Analysis of UV chromatograms and corresponding fractions by SDS PAGE, confirmed that CDK5RAP3 interacts with UFL1/UFBP1 in vitro and forms a stable complex (Fig 4A). We further analyzed the complex by mass photometry, which confirmed the presence of a stable ternary complex with an experimental molecular mass of 192 kDa (Fig EV5A). Since CDK5RAP3 forms an integral complex with UFL1/UFBP1 and CDK5RAP3 has recently been suggested to function as a substrate adaptor (Yang et al, 2019; Stephani et al, 2020), we wondered if it could influence E3 ligase activity or substrate UFMylation. We therefore monitored the E3 ligase activity of UFL1/UFBP1 in the presence of increasing concentrations of CDK5RAP3. Surprisingly, incubation of CDK5RAP3 with UFL1/UFBP1 impaired E3 ligase activity and UFMylation in a concentration‐dependent manner (Fig 4B). Moreover, preformed UFL1/UFBP1/CDK5RAP3 complexes purified by SEC also showed no ligase activity (Fig EV5B). These observations imply that CDK5RAP3 binds to and inhibits the ligase complex. One possibility is that CDK5RAP3 inhibits UFMylation by blocking complex formation between UFL1/UFBP1 and UFC1‐O‐UFM1. However, in pull‐downs and analytical SEC, CDK5RAP3 forms a complex together with UFL1/UFBP1 and UFC1‐O‐UFM1 (Fig EV5C and D). Since CDK5RAP3 does not affect UFC1~UFM1 binding, we next tested if CDK5RAP3 binding influences the discharge of UFM1 from UFC1~UFM1 by monitoring the transfer of UFM1 onto free Lys. While UFM1 is readily discharged onto Lys in the presence of UFL1/UFBP1, this is completely blocked in the presence of CDK5RAP3 (Figs 4C and D, and EV5E). These observations suggest that binding of CDK5RAP3 to the UFL1/UFBP1/UFC1~UFM1 complex prevents activation of UFC1~UFM1 resulting in inhibition of UFMylation. While these in vitro experiments clearly demonstrate that CDK5RAP3 inhibits E3 ligase activity, it raises the question of the role of CDK5RAP3 in substrate UFMylation. Hence, we used the cell‐free reconstitution of ribosome UFMylation described in Fig 1D and monitored UFMylation of RPL26. In the absence of CDK5RAP3, mono‐ and di‐UFMylated RPL26 species are formed, but with increasing concentrations of CDK5RAP3, the di‐UFMylation of RPL26 is completely abolished (Fig 4E). Importantly, even at the highest concentration of CDK5RAP3, monoUFMylation of RPL26 is not affected. By contrast, the addition of increasing concentrations of CDK5RAP3 decreased UFMylation of other substrates such as H4, MRE11, and TRIP4 (Figs 4F and EV5F). Hence, we propose CDK5RAP3 to be a specificity determinant, keeping the activity of the ligase complex inhibited in the absence of substrate and directing ligase activity towards the ribosomal subunit RPL26.
Canonical E2 enzymes contain a conserved HPN motif upstream of the catalytic cysteine (Wu et al, 2003; Cook & Shaw, 2012). The Asn in this motif is indispensable for the transfer of the Ub/UBL from the E2~Ub/UBL onto substrate Lys, while the His forms a hydrogen bond with the Asn to stabilize the architecture of the HPN motif (Cook & Shaw, 2012). Intriguingly, UFC1 lacks the oxyanion hole stabilizing Asn as part of the highly conserved HPN motif found in E2 enzymes, which is instead replaced by a TAK motif at this position (Fig 5A, Appendix Fig S1A). However, the importance of the alternative TAK motif is underscored by the identification of T106I mutations in patients with severe early‐onset encephalopathy (Nahorski et al, 2018). Having established a UFMylation assay with UFL1/UFBP1, we wanted to ascertain whether TAK motif residues are required for UFMylation. While mutation of T106A and A107G did not affect UFMylation, the K108A mutant completely abolished UFMylation (Fig 5B). In contrast to the T106A mutant, which showed no impact on activity, the disease‐causing T106I mutant showed a dramatic impairment of UFMylation (Fig 5C). Indeed, both the UFMylation deficient mutants T106I and K108A were defective in being activated by the E3 ligase complex for aminolysis (Fig 5D, Appendix Fig S1C), which in turn manifested in reduced RPL26 UFMylation (Fig 5E). However, Cys reactivity of UFC1 is not significantly impacted by mutations to T106 and K108 (Fig 5E, Appendix Fig S1D). In addition to the HPN motif, canonical E2s have a conserved negatively charged residue that activates the attacking substrate lysine (Dou et al, 2012; Plechanovová et al, 2012). In UFC1, the equivalent residue is D119, mutation of which to Ala did not show significant defects in UFMylation (Fig 5B–E, Appendix Fig S1B). Together, these results reveal that UFC1 potentially utilizes an alternative mechanism for the transfer of UFM1 to the substrate.
In addition to its core UBC fold, UFC1 has at its N‐terminus a conserved α‐helix (α0) whose function is unknown (Fig 6A and B). Motivated by the fact that previous studies have shown that the N‐ and C‐ terminal extensions on E2s regulate E2 activity (Stewart et al, 2016), we sought to determine whether α0 has a role in regulating UFMylation. We first compared the activity of UFC1WT and UFC1 lacking α0 (UFC1ΔN) in UFMylation assays containing UFC1 with or without UFL1/UFBP1. Surprisingly, UFC1ΔN showed stronger overall UFMylation compared with UFC1WT in the presence of UFL1/UFBP1 (Fig 6C). This suggests an inhibitory role for the N‐terminal helical extension of UFC1. Next, we compared the discharge of UFM1 from UFC1~UFM1 and UFC1ΔN~UFM1 onto free Lys. In the absence of E3 ligase, UFC1ΔN rapidly discharges UFM1 onto Lys (Fig 6D and E) suggesting an increase in its intrinsic lysine reactivity. In the presence of UFL1/UFBP1, the discharge of UFM1 onto Lys is enhanced when α0 of UFC1 is deleted (Fig 6D and F). Taken together with the increase in UFMylation in the presence of UFL1/UFBP1, these results further strengthen the hypothesis of an inhibitory role for the N‐terminal helix of UFC1. Since we identified the N‐terminal α0 of UFC1 to restrain UFMylation, we analyzed if inhibition by CDK5RAP3 was mediated via this helix. Indeed, UFMylation assays together with discharge assays comparing the activity of UFC1WT and UFC1ΔN reveal that the inhibition of E3 ligase activity by CDK5RAP3 requires the N‐terminal helix of UFC1 (Fig 6D and G, Appendix Fig S2). As the inhibition mediated by CDK5RAP3 is relieved in the presence of 60S ribosomes, we examined the role of UFC1 α0 on UFMylation of RPL26 (Fig 6H). While ~50% of RPL26 is UFMylated at 6 min in reactions containing UFC1WT, near complete RPL26 UFMylation is observed at 3 min with UFC1ΔN (Fig 6H, lane 3 vs. 5). Based on these results we propose that CDK5RAP3 may interact with both UFL1/UFBP1 and α0 of UFC1 to clamp the complex in an inhibited state. Together these analyses reveal a previously unappreciated regulatory role for the N‐terminal helix of UFC1 in modulating UFMylation.
In this study, we establish a robust in vitro reconstitution system using purified components of the UFM1 enzymatic pathway to reveal the minimal requirements for UFMylation and mechanistic insights into the ligase machinery. Previous reports have provided conflicting views on the roles of UFBP1 and CDK5RAP3, with some suggesting that they are mainly substrates of UFMylation (Tatsumi et al, 2010; preprint: Gak et al, 2020). Further, it has been suggested that UFBP1 must be UFMylated first at K267 before it can associate with UFL1 to subsequently support UFL1 ligase activity (Yoo et al, 2014). Since both UFL1 and UFBP1 complexes are purified from bacteria, our work unequivocally demonstrates that UFL1 and UFBP1 can associate in the absence of any PTMs and the formation of this complex is essential for it to function as an E3 ligase. Scaffold‐type E3 ligases such as RING E3 ligases bind to both E2 and substrate to bring them together for substrate transfer. Since UFL1 can bind to both UFC1 and its supposed substrate UFBP1 at the same time, it was suggested that UFL1 may be a scaffold‐type E3 ligase (Komatsu et al, 2004). However, direct evidence of whether UFL1 is a scaffold/adaptor‐type E3 ligase or a Cys‐based HECT‐like enzyme was lacking. More recently, a UFMylation assay that relied on UFL1 present in mammalian cell extracts to which in vitro generated biotinylated E2~UFM1 thioesters were added, found that UFMylation occurred even when cell extracts were treated with cysteine‐alkylating reagents, further suggesting that UFL1 could be a scaffold‐type E3 ligase (preprint: Gak et al, 2020). Our mutational analysis showing that the UFL1/UFBP1 complex lacks a single catalytic Cys together with the ability of the E3 ligase to promote UFC1~UFM1 aminolysis firmly establishes that UFL1/UFBP1 is a scaffold‐type E3 ligase. Further reinforcing this conclusion is the observation that K69‐linkage specificity is imparted by the E2 in ligase‐free di‐UFM1 formation, and this K69‐linkage specificity is unaltered by the E3 ligase. Indeed, this is a feature observed in RING E3 ligases where linkage specificity is determined by the E2 enzyme (Deshaies & Joazeiro, 2009; Deol et al, 2019). Since UFL1/UFBP1 promotes aminolysis, we speculate that UFL1/UFBP1 binding induces a closed UFC1~UFM1 conformation akin to RING and atypical SUMO E3 ligases (Pichler et al, 2004; Reverter & Lima, 2005; Plechanovová et al, 2012; Pruneda et al, 2012; Cappadocia et al, 2015). UFC1 differs from prototypical E2s since it lacks the catalytic HPN motif, has an exposed catalytic Cys with a lower pKa, lacks the C‐terminal α‐helix observed in canonical E2s, and has an additional α‐helix at its N‐terminus (α0) (Mizushima et al, 2007; Gundogdu & Walden, 2019; Kumar et al, 2021). Further, it is unclear whether the canonical “backside” interaction can occur in UFC1 (Brzovic et al, 2006; Middleton et al, 2017). These unique features of UFC1 and the unconventional features of the ligase complex make it likely that the UFM1 machinery employs a unique mechanism to transfer UFM1 from UFC1 onto the substrate. Interestingly, the UBE2E enzymes also have an intrinsically disordered N‐terminal extension that has an inhibitory role in restricting Ub transfer thereby limiting polyUb chain formation (Schumacher et al, 2013). Future work will reveal whether E3 binding induces a closed UFM1~UFM1 conformation and whether α0 impedes this process. Nevertheless, our data seem to suggest that α0 has a regulatory role in potentially preventing UFM1 discharge in the absence of the E3 ligase. The recent advances in structure prediction (Jumper et al, 2021) enabled us to identify the regions of UFL1/UFBP1 essential for ligase activity as comprising a tandem repeat of three WH domains—one full WH domain each from UFL1 and UFBP1 and the composite WH domain formed at the point of interaction between the two proteins. As this region can bind to UFC1~UFM1 and activate the E2 for UFM1 discharge, we define this to be the minimal catalytic domain. Further structural studies will be required to reveal why three WH domains are required to form a functional ligase complex and the identification of linchpin residues in UFL1/UFBP1 that play similar roles to RING E3 ligases to activate the E2 for aminolysis (Plechanovová et al, 2012; Pruneda et al, 2012). Intriguingly, the anaphase‐promoting complex (APC) subunit APC2 contains a WHB domain that binds the “backside” of UCBH10/UBE2C, located at a face opposite from the catalytic site and is an allosteric site in several E2s (Brzovic et al, 2003; Brown et al, 2015). This backside binding of the WHB domain is specific to UBE2C and is important for the transfer of Ub onto specific substrates (Brown et al, 2015). Our observations also raise the question of the function of the other WH/PCI domains in UFL1/UFBP1 to ligase function. One possibility is that the WH domains may mediate substrate recognition, enable binding to UFM1, or serve either to either regulate recruitment or processivity of chain formation. Given that the WH domain of Cockayne syndrome group B (CSB) can bind to ubiquitin, it is tempting to speculate that one of the predicted WH domains of UFL1/UFBP1 may bind to UFM1 (Takahashi et al, 2019). WH domains commonly recognize nucleic acids and are typically found in transcription factors or as protein interaction domains (Harami et al, 2013). To our knowledge, this is the first demonstration of catalytic activity mediated by WH domains (Aravind et al, 2005), leading us to propose moonlighting functions for other WH domain‐containing proteins. It also raises the possibility that other WH domains could have E3 ligase activity for UFMylation or other UBLs. Since UFL1 has been proposed to have nuclear functions in telomere maintenance and DNA damage response (Lee et al, 2019; Qin et al, 2019, 2020), another possibility is that the WH domains may mediate DNA binding. A further possibility is the use of the WH domains of UFL1/UFBP1 for recognizing rRNA or translating mRNA during ribosome UFMylation. While our mutational analyses validate the predicted structure of the ligase complex, it is possible that the ligase adopts a completely different conformation when bound to the E2, CDK5RAP3, and substrates. The flexible linkers at the NTR and CTR of UFBP1 and UFL1, respectively (Fig 3A) may enable the complex to adopt alternative conformations. Our in vitro studies show that the formation of UFM1 chains and autoUFMylation is inhibited by CDK5RAP3 (Fig 2C). Indeed, previous studies have observed an altered pattern of UFMylation in the absence of CDK5RAP3 leading to the suggestion that it may be a substrate adaptor (Yang et al, 2019). Interestingly, CDK5RAP3 was suggested to function as a sensor for ER stress to induce autophagic degradation of aberrant proteins formed as a result of ribosomal stalling (Stephani et al, 2020). Several multidomain and multiprotein E3 ligases such as PARKIN and Cullin Ring Ligases (CRLs) are inhibited and their activation is a carefully orchestrated multistep process (Walden & Rittinger, 2018; Baek et al, 2021). Based on our observations, we propose that ligase complexes containing UFL1, UFBP1, and CDK5RAP3 represent an autoinhibited state. The surprising finding that ribosome UFMylation is not abolished in the presence of CDK5RAP3 but rather restricted to monoUFMylation leads us to suggest that CDK5RAP3 regulates ligase activity in the following manner: (i) in the absence of substrate, binding of CDK5RAP3 inhibits E3 ligase activity; (ii) this autoinhibition is relieved when substrates such as the 60S ribosome are encountered. This release from inhibition may involve conformational changes induced upon recognition of structural features on the substrate by UFL1, UFBP1, or CDK5RAP3 to mediate substrate UFMylation. Further, CDK5RAP3 prevents UFM1 from being attached to another UFM1 molecule thereby restricting UFM1 chain formation on substrates. Such multi‐layered regulation possibly prevents spurious UFMylation ensuring ribosome UFMylation only in the right context. While previous studies have suggested roles for UFMylation to facilitate UFL1‐UFBP1 interaction, and UFMylation and phosphorylation to enhance UFMylation (Tatsumi et al, 2010; Lemaire et al, 2011; Yoo et al, 2014; Qin et al, 2019), our minimal reconstitution clearly demonstrates that the ligase complex assembles in the absence of any PTM. Although we cannot rule out a role for phosphorylation in enhancing ligase activity, the rapid UFMylation of ribosomes by the reconstituted ligase suggests that this is unlikely. Our rebuilding approach provides insights into the assembly, minimal requirements, and mechanism of the UFL1/UFBP1/CDK5RAP3 ligase and reveals principles of protein UFMylation. The in vitro reconstitution system we describe here using purified components lays the foundation for future biochemical and structural studies to understand the molecular mechanism of this unusual E3 ligase complex and substrate UFMylation. Further, the cell‐free UFMylation system can be applied to understand the logic of ribosome UFMylation and its relationship to ribosome quality control pathways. Ultimately, extending these studies into a cellular setting will be needed to understand how the ligase complex is activated to attach UFM1 onto ribosomes at the ER and how UFMylation is regulated in cells.
The details of cDNA constructs used in this study are given in Appendix Table S1. Cloning of most of the constructs was performed by MRC‐PPU Reagents and Services (University of Dundee). Briefly, mutagenesis was carried out using Q5 site‐directed mutagenesis kit (NEB) with KOD polymerase (Novagen) according to the manufacturer's protocol. Following mutagenesis, cDNA constructs were amplified using E. coli DH5α and purified using QIAprep spin mini‐prep kit (Qiagen). All cDNA constructs were verified by DNA sequencing and services, the University of Dundee using DYEnamic ET terminator chemistry (Amersham Biosciences) on Applied Biosystems automated DNA sequencers.
Recombinant His6‐3C‐UBA5, His6‐3C‐UFM1WT, His6‐3C‐UFM1Konly, His6‐3C‐UFM1KtoR, His6‐3C‐Cys‐UFM1WT, His6‐3C‐UFC1WT, His6‐3C‐UFC1ΔN, and His6‐3C‐MRE11 were expressed in E. coli BL21 (DE3) and purified using Ni2+‐NTA affinity chromatography as the first step. Briefly, E. coli BL21 cultures expressing His6‐tagged proteins were grown in 2xTY medium at 37°C until OD600 reached 0.6–0.8. Final concentration of 0.3 mM IPTG was added and the cultures were incubated at 18°C for 16 h. Cells were harvested, resuspended, and lysed in lysis buffer containing 25 mM Tris–pH 8, 300 mM NaCl, 10% Glycerol, 2 mM DTT, 1 mM Benzamidine, 1 mM AEBSF, 1× protease inhibitor cocktail (Roche) by ultrasonication. Lysed cells were then clarified by centrifugation at 30,000 g for 30 min at 4°C. The clarified lysate was then incubated with pre‐equilibrated Ni2+‐NTA Agarose beads (Amintra, Abcam) for 2 h in binding buffer containing 25 mM Tris–pH 8, 300 mM NaCl, 10% Glycerol, and 10 mM Imidazole. The beads were then washed extensively using wash buffer containing 25 mM Tris–pH 8, 300 mM NaCl, 10% Glycerol, 2 mM DTT, and 20 mM Imidazole. Finally, the bound protein was eluted using elution buffer containing 25 mM Tris–pH 8, 300 mM NaCl, 10% Glycerol, 2 mM DTT, and 300 mM Imidazole. Wherever necessary, His6‐tags were cleaved off by incubating tagged proteins with PreScission protease at 4°C overnight. A final size exclusion chromatography step was performed using HiLoad™ 16/60 Superdex™ 75 pg and HiLoad™ 16/60 Superdex™ 200 pg columns (GE Healthcare Life Sciences) with buffer containing 25 mM Tris–pH 8.0, 150 mM NaCl, 10% Glycerol, and 2 mM DTT. The purified proteins were then concentrated using Amicon™ Ultra 15 concentrators (MERCK Millipore) and stored in −80°C.
GST‐TEV‐TRIP4 was expressed in E. coli BL21 (DE3) strain as described above. Cells were harvested and lysed in lysis buffer containing 25 mM Tris–pH 7.5, 300 mM NaCl, 10% Glycerol, and 2 mM DTT using ultrasonication. Pre‐equilibrated Glutathione 4B‐sepharose beads (Amintra, abcam) were incubated with clarified lysate for 2 h. The beads were then washed with high salt buffer containing 25 mM Tris–pH 7.5, 500 mM NaCl, 10% glycerol and 2 mM DTT. Further, the beads were washed with low salt buffer containing 25 mM Tris–pH 8, 150 mM NaCl, 10% glycerol, and 2 mM DTT. The protein was then cleaved off the tag by incubation with Precision protease at 4°C overnight. A second ion exchange chromatography step was carried out using Resource Q (6 ml) (GE Healthcare Life Sciences) column with low salt buffer (25 mM Tris–pH 7.5, 150 mM NaCl, 10% glycerol, 2 mM DTT) and high salt buffer (25 mM Tris–pH 7.5, 500 mM NaCl, 10% glycerol, and 2 mM DTT). A final size exclusion chromatography step was performed using HiLoad™ 16/60 Superdex™ 75 pg (GE Healthcare Life Sciences) and the protein was buffer exchanged in buffer 25 mM Tris–pH 7.5, 150 mM NaCl, 10% glycerol, and 2 mM DTT. The purified proteins were then concentrated and stored in −80°C.
His6‐TEV‐UFL1 and StrepII‐3C‐UFBP1(29‐end) were cloned in a pET Duet1 construct and expressed in E. coli BL21 codon plus RIPL (Agilent) cells. Bacterial cultures were grown in 2xTY medium at 37°C until OD600 reached 0.6. Final concentration of 0.3 mM IPTG was added to induce the expression and the cultures were incubated at 18°C for 16 h. Cells were harvested and resuspended in buffer containing 25 mM Tris–pH 8.0, 300 mM NaCl, 2 mM DTT, 1 mM Benzamidine, 1 mM AEBSF, and protease inhibitor cocktails (Roche). Cells were lysed by high pressure homogenization using an Emulsiflex C3 homogenizer (Avestin). The lysate was then clarified by Ultracentrifugation at 30,000 g for 30 min at 4°C. Affinity purification was carried out in two steps. In the first step, the clarified lysate was passed through HisTrap™ FF (GE Healthcare Life Sciences) column. After binding, the column was washed with 10 column volumes (CVs) of binding buffer containing 25 mM Tris–pH 8.0, 300 mM NaCl, 20 mM Imidazole, and 2 mM DTT. After washing, bound proteins were eluted using buffer containing 25 mM Tris–pH 8.0, 300 mM NaCl, 300 mM Imidazole, and 2 mM DTT by applying a concentration gradient of Imidazole. The eluted proteins were then passed through StrepTrap™ (GE Healthcare Life Sciences) column pre‐equilibrated with binding buffer containing 25 mM Tris–pH 8.0, 300 mM NaCl and 2 mM DTT. After 2 CVs of washing with binding buffer, the proteins were eluted using 25 mM Tris–pH 8.0, 300 mM NaCl, 2 mM DTT, and 2.5 mM Desthiobiotin. Finally, purified proteins were passed through HiLoad™ 16/60 Superdex™ 200 pg (GE Healthcare Life Sciences) and the fractions corresponding to the complex were collected. The purified protein was then stored in −80°C in buffer containing 25 mM Tris–pH 8.0, 300 mM NaCl, 5% Glycerol, and 2 mM DTT until further use.
Cys‐UFM11‐83 which contains a Cys residue upstream of M1 was purified as described above and exchanged into a fresh buffer containing 50 mM HEPES pH 7.5, 150 mM NaCl and 0.5 mM TCEP using CentriPure P10 columns (EMP Biotech). Protein was then mixed with IRDye® 800CW Maleimide (LI‐COR®) at a 5:1 molar ratio and incubated for 2 h at RT. Unreacted dye was removed by a three‐step buffer exchange process using CentriPure P10 columns (EMP Biotech) (once) and PD‐10 Sephadex G‐25 column (twice) in a sequential manner according to the manufacturer's instruction. Finally, to remove residual unreacted dye material, the labeled protein was dialyzed into buffer containing 25 mM Tris–pH 8, 150 mM NaCl and 2 mM DTT using a dialysis membrane (Thermo Scientific) (MWCO 3000) overnight at 4°C and stored at −80°C until further use.
To analyze the intrinsic reactivity of UFC1, a single turnover discharge assay was employed. Firstly, UFC1 was charged with UFM1WT or labeled UFM1 by incubating 0.5 μM UBA5, 10 μM UFC1 and 20 μM UFM1 in a buffer containing 50 mM HEPES pH 7.5, 50 mM NaCl, 0.5 mM DTT and10 mM MgCl2. The charging reaction was initiated by the addition of 10 mM ATP and incubation of the reaction mix at 37°C for 20–30 min. To stop further charging of UFM1 onto UFC1, the reaction was quenched by the addition of 50 mM EDTA and subsequent incubation at RT for 10 min. To initiate discharge, the quenched mixture was incubated with 50 mM lysine (pH 8.0) or other amino acids namely Serine, Threonine, Arginine, and Cysteine at 37°C. The reaction was stopped at each of the time points by the addition of SDS Loading dye without any reducing agent to the reaction mix and run on a 4–12% SDS–PAGE under non‐reducing conditions followed by Coomassie staining or visualization using LI‐COR® Odyssey. In reactions involving the UBE2D3 and UBE2L3, 0.5 μM UBE1 was incubated with 10 μM UBE2D3/UBE2L3 and 20 μM Ub in reaction buffer containing 50 mM HEPES pH 7.5, 50 mM NaCl, 10 mM ATP and 10 mM MgCl2 at 37°C for 20 min. The reaction was quenched, and discharge was analyzed as described above. In discharge assays involving UFL1/UFBP1 and CDK5RAP3, the quenched reaction mix was added to a cocktail containing 1 μM UFL1/UFBP1 or 1 μM or 2 μM of CDK5RAP3. Discharge was initiated by the addition of 50 mM lysine (pH 8.0) and incubated at 37°C for indicated time duration. The reaction was stopped and analyzed as described above.
To check for UFMylation in vitro, 0.25 μM UBA5, 5 μM UFC1, 1 μM UFL1, and 20–30 μM UFM1 were incubated in reaction buffer containing 50 mM HEPES 7.5, 10 mM MgCl2, and 5 mM ATP for 1 h at 37°C. The reaction was stopped by the addition of SDS loading buffer (1× final) containing a reducing agent. The reaction products were then separated on a 4–12% SDS–PAGE gel under reducing conditions and analyzed by immunoblotting using indicated antibodies. In UFMylation assays involving substrates, around 1–2 μM of recombinant substrates were incubated with 0.25 μM UBA5, 5 μM UFC1, 1 μM UFL1, and 30 μM of UFM1 in buffer containing 50 mM HEPES 7.5, 10 mM MgCl2, and 5 mM ATP for 1 h at 37°C. Following incubation, the reaction was stopped and analyzed using SDS–PAGE or Immunoblotting using indicated antibodies.
60S ribosomes were a generous gift from the Puglisi lab, and were also purified in‐house as described previously (Johnson et al, 2019; Lapointe et al, 2021).For reconstituting 60S Ribosome UFMylation, approximately 50 nM purified 60S ribosomes were mixed with, 0.5 μM UBA5, 1 μM UFC1, 0.5 μM UFM1, and 100 nM UFL1/UFBP1 in a reaction buffer containing 25 mM HEPES pH 7.5, 100 mM NaCl, 10 mM MgCl2 and 5 mM ATP and incubated at 30°C for 10 min or indicated time duration. The reaction was stopped by the addition of SDS loading buffer (1× final) and run on a 4–12% SDS–PAGE gel under reducing conditions followed by immunoblotting using indicated antibodies. Ribosome UFMylation assay as described in Fig 4E was performed at 30°C with 1 μM UFC1, 0.5 μM UBA5, 0.5 μM UFM1, 0 nM or 75 nM UFL1/UFBP1, 50 nM purified 60S ribosomes, and increasing concentrations of CDK5RAP3 (0, 38, 75, 150 or 375 nM) in a reaction buffer of 25 mM HEPES pH 7.5, 100 mM NaCl, 10 mM MgCl2 and 5 mM ATP. The reaction was quenched by the addition of SDS‐Load buffer (1× final) with reducing agent. Western blots show 10 min reaction time; immunoblots for RPL26 (Abcam, 59567) and UFL1 (Bethyl, A303‐455M) were run on the same gel, which was cut and probed for these proteins separately. Immunoblots for CDK5RAP3 (Bethyl, A300‐871A) and UFM1 (Abcam, Ab109307) were run on separate gels.
UFC1‐O‐UFM1 was prepared by incubating 40 μM UFC1C116S with 40 μM UFM1, 1 μM UBA5 in buffer containing 50 mM Tris–pH 8.8, 10 mM MgCl2, and 5 mM ATP overnight at 25°C. The reaction was incubated briefly with 20 mM DTT to remove any non‐specific di‐sulfide linked adducts formed and passed through HiLoad™ 16/60 Superdex™ 75 pg with buffer containing 25 mM Tris–pH 8.0, 150 mM NaCl, 5% Glycerol and 2 mM DTT. The fractions corresponding to UFC1‐O‐UFM1 were collected and analyzed on 4–12% SDS–PAGE gel. Finally, the fractions containing UFC1‐O‐UFM1 were pooled, concentrated, and stored in −80°C until further use.
To analyze the interaction of the E3 ligase with the E2, pulldown assays were performed. Approximately 10 μM of untagged UFC1 or UFC1‐O‐UFM1 was incubated with 5 μM of full‐length UFL1/UFBP1 in binding buffer containing 25 mM Tris–pH 8.0, 300 mM NaCl, 2 mM DTT for 1 h at 4°C. Following incubation, 30 μl of pre‐equilibrated Streptavidin Sepharose beads (50% slurry) (IBA Life Sciences) was added and further incubated for about 1 h at 4°C. Following binding, centrifuge tubes containing the reaction mix were spun down at 500 g for 3 min and the supernatant was discarded to remove unbound proteins. To remove weakly bound proteins, the beads were washed thrice with 1 ml of ice‐cold binding buffer. Finally, the bound proteins were eluted by the addition of binding buffer containing 2.5 mM Desthiobiotin (pH 8.0) and incubation at 4°C for 30 min. The eluates were then analyzed on 4–12% SDS–PAGE under reducing conditions followed by Coomassie staining.
HEK293T Ufl1 KO cells were transfected with UFBP1 C‐terminally tagged with streptavidin binding peptide (SBP) together with UFL1WT‐3xFLAG or UFL1L45R‐3xFLAG expressing plasmids using Lipofectamine LTX (Thermo Life Sciences) (related to Fig 3C). 0.5% NP‐40 lysates were subjected to pulldown using M2 anti‐FLAG or streptavidin affinity gels (Sigma‐Aldrich), separated by SDS–PAGE, and immunoblotted with UFBP1 & UFL1 antibodies.
For analyzing the interaction between UFL1/UFBP1 and CDK5RAP3, around 30 μg of UFL1/UFBP1 and 15 μg of CDK5RAP3 were mixed and incubated for 30 min on ice. Then, around 50 μl of the sample was loaded on Superdex™ 200 Increase 3.2/300 column (GE Healthcare Lifesciences) pre‐equilibrated with buffer containing 25 mM Tris–pH 8.0, 300 mM NaCl and 2 mM DTT. The fractions corresponding to each peak were collected and further analyzed on a 4–12% SDS–PAGE under reducing conditions followed by Coomassie staining. To check whether UFL1/UFBP1 could be reconstituted in trans, around 15 μg of purified His6‐UFL1 and Untagged UFBP1(29‐end) were mixed and incubated on ice for 2 h. After incubation, the sample was loaded on Superdex™ 200 Increase 3.2/300 column (GE Healthcare Lifesciences) column pre‐equilibrated with buffer containing 25 mM Tris–pH 8.0, 300 mM NaCl, 2 mM DTT, and analyzed as described above.
The structure of UFL1/UFBP1 was predicted using the ColabFold Google Colab notebook “AlphaFold_advanced” (Jumper et al, 2021) (https://github.com/sokrypton/ColabFold). The predicted model with the highest IDDT score is shown in the main figure and the PAE scores for different models ranking 1–5 are shown in Fig EV3A.
DALI server was used to perform structural similarity analysis to identify and compare structurally similar proteins (Holm, 2020). The WH1 domain of UFL1 (a.a 52–115) was used as the query model and searched against PDB25 database. The top 10 models in the order of the Z‐score is shown in Fig EV3B.
For the generation of a composite Foldindex profile, a previously reported method was adapted (Prilusky et al, 2005; Ahel et al, 2020). First, individual Foldindex profiles of UFL1 from different organisms were generated locally using a custom Foldindex program provided by Dr. Juraj Ahel. Then, a composite Foldindex profile was prepared by blending the images in Adobe illustrator with 30% opacity. For the generation of multiple sequence alignments, the protein sequences of target proteins and their homologs were manually downloaded from UNIPROT database in fasta format. Multiple sequence alignment was performed using Jalview version 2.11.1.7 program (MAFFT algorithm module using L‐INS‐I) (Waterhouse et al, 2009; Katoh & Toh, 2010).
Protein samples were prepared in a solution containing 25 mM Tris–pH 8.0, 300 mM NaCl, and 2 mM DTT and stored on ice before loading onto the Refeyn OneMP instrument (Refeyn). Typically, a set of protein standards (NativeMark™ Unstained Protein Standard, Invitrogen) are used for calibration and to generate a standard curve. Following calibration, approximately 10 μl of diluted protein sample in the concentration range of 5–25 nM was introduced into the flow chamber and movies of 60s duration were recorded. Data acquisition was performed using Acquire MP (Refeyn Ltd, v1.1.3) and analyzed using Discover MP software. Data shown here are the representation of at least three independent acquisitions (n ≥ 3).
Size exclusion chromatography and multiangle light scattering (SEC‐MALS) experiments were performed on a Dionex Ultimate 3000 HPLC system with an inline Wyatt miniDAWN TREOS MALS detector and Optilab T‐rEX refractive index detector. In addition, the elution profile of the protein was monitored with UV 280 attached to the Dionex system. For size exclusion chromatography, Superdex™ 200 Increase 10/300 GL column (GE Healthcare LifeSciences) was used. Fifty microliter of the purified UFL1/UFBP1 stored in buffer containing 25 mM Tris–pH 8.0, 300 mM NaCl and 2 mM DTT was loaded into the SEC column with Dionex autoloader at a concentration of 4 mg/ml and a flow rate of 0.3 ml/min was maintained throughout the experiment. Molar masses spanning elution peaks were calculated using ASTRA v6.0.0.108 (Wyatt).
First, an in vitro UFMylation reaction was performed to generate UFMylated products including free UFM1 chains. Then, the reaction products were run on a 4–12% SDS–PAGE gel to separate the products based on electrophoretic mobility. Next, the bands corresponding to di‐UFM1 chains were excised and in‐gel digestion was performed according to the previously described protocol (Shevchenko et al, 2007). Digested peptides were analyzed by LC–MS/MS on an Exploris 480 coupled to an Ultimate 3000 nanoLC system (Thermo Fisher Scientific) for Fig 2D and on an Exploris 240 (Thermo Fisher Scientific) coupled to an Evosep One (Evosep) for Fig 4D. For the analysis performed on the Ultimate 3000, samples were loaded on a 100 μm × 2 cm trap column (Thermo Fisher Scientific #164564‐CMD) and analyzed on a 75 μm × 50 cm analytical column (Thermo Fisher Scientific #ES903) using a gradient from 3 to 35% Buffer B (80% LC–MS grade acetonitrile, 0.08% formic acid in water) over 53 min. The columns were then washed with 95% Buffer B for 2 min prior to equilibration in 97% Buffer A (0.1% formic acid in LC–MS grade water). For the analysis performed on the Evosep One, samples were loaded onto the Evotips as per manufacturer recommendations and analyzed using the 30 SPD Method. Peptides were then analyzed in on either the Exploris 240 or 480 using data dependant with an MS1 resolution of 60,000, AGC target of 300%, and maximum injection time of 25 or 28 ms. Peptides were then fragmented using TOP 2 s method, MS2 resolution of 15,000, NCE of 30 or 32%, AGC of 100%, and maximum injection time of 100 ms. Peptide identification was performed in Mascot using a restricted and frequently updated database containing ~2,000 protein sequences of interest (MRC db). Carbamidomethylation (C) was set at fixed modification and Oxidation (M), Deoxidation (M) and the addition of the dipeptide Glycine‐Valine (K) were set as variable modifications. Peptides were searched using an MS1 tolerance of 10 ppm and MS2 tolerance of 0.06 Da, and a maximum of two missed cleavages were allowed. Only hits identified with an FDR (false discovery rate) <1% were selected and further analyzed in Scaffold viewer V5. For semi‐quantitative analysis of VG modification on individual sites, total ion chromatogram (TIC) values were obtained from the LC–MS and represented graphically.
Joshua J Peter: Conceptualization; formal analysis; validation; investigation; methodology; writing – original draft; writing – review and editing. Helge M Magnussen: Investigation; methodology; writing – review and editing. Paul A DaRosa: Investigation; methodology; writing – review and editing. David Millrine: Investigation; methodology. Stephen P Matthews: Investigation; methodology; writing – review and editing. Frederic Lamoliatte: Formal analysis; investigation; writing – review and editing. Ramasubramanian Sundaramoorthy: Investigation. Ron R Kopito: Funding acquisition; investigation; methodology; writing – review and editing. Yogesh Kulathu: Conceptualization; supervision; funding acquisition; investigation; methodology; writing – original draft; project administration; writing – review and editing.
The authors declare that they have no conflict of interest.
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PMC9628031 | Aya M. Hussein,Nadia M. El-Beih,Menha Swellam,Enas A. El-Hussieny | Pomegranate juice and punicalagin-mediated chemoprevention of hepatocellular carcinogenesis via regulating miR-21 and NF-κB-p65 in a rat model | 02-11-2022 | Apoptosis,HCC,Hepatic antioxidants,miR-21,NF-κB-p65,TNF-α,Pomegranate juice,Punicalagin | Background Hepatocellular carcinoma (HCC) is the most common neoplasm among primary liver malignancies, accounting for 70%–85% of total liver cancer cases worldwide. It is also the second-leading cause of cancer-related death worldwide. Recent research has investigated naturally occurring products high in polyphenolic compounds in the regression and prevention of HCC. This study investigated the chemoprevention effects of pomegranate juice (PJ) and punicalagin (PCG) against diethylnitrosamine (DENA)-induced hepatocarcinogenesis in male albino rats. Methods Animals were randomized into six groups and treated for 11 weeks as follows: group 1 was a negative control group, group 2 was treated orally with 10 mL PJ per kilogram body weight (kg bw), group 3 was treated orally with 18.5 mg PCG/kg bw, and groups 4–6 were injected with an intraperitoneal dose of DENA (50 mg/kg bw) weekly beginning in the third week. Group 4 was a HCC control (DENA-treated group), group 5 was HCC + PJ, and group 6 was HCC + PCG. Results PJ antagonized DENA-induced elevations of ALAT, TNF-α, NF-κB-p65, GST, MDA, and NO and restored total protein, IL-10, SOD, and CAT levels. Moreover, PJ resulted in downregulation of miR-21, Bcl-2, and Bcl-XL and an upregulation of caspase-3 and Bax mRNA expressions. These chemoprevention effects of PJ also alleviated the hepatic preneoplastic lesions induced by DENA. Although PCG treatment induced some modulation in DENA-treated rats, it did not show potent chemoprevention activity and induced some side effects. Conclusion Both of PJ and PCG downregulated miR-21 expression and triggered apoptosis. However, PJ was more effective than pure PCG in alleviating the hepatic antioxidant defense state and the inflammatory status. So, PJ was superior in prevention of DENA-induced hepatocellular carcinogenesis in rats than pure PCG. Graphical Abstract | Pomegranate juice and punicalagin-mediated chemoprevention of hepatocellular carcinogenesis via regulating miR-21 and NF-κB-p65 in a rat model
Hepatocellular carcinoma (HCC) is the most common neoplasm among primary liver malignancies, accounting for 70%–85% of total liver cancer cases worldwide. It is also the second-leading cause of cancer-related death worldwide. Recent research has investigated naturally occurring products high in polyphenolic compounds in the regression and prevention of HCC. This study investigated the chemoprevention effects of pomegranate juice (PJ) and punicalagin (PCG) against diethylnitrosamine (DENA)-induced hepatocarcinogenesis in male albino rats.
Animals were randomized into six groups and treated for 11 weeks as follows: group 1 was a negative control group, group 2 was treated orally with 10 mL PJ per kilogram body weight (kg bw), group 3 was treated orally with 18.5 mg PCG/kg bw, and groups 4–6 were injected with an intraperitoneal dose of DENA (50 mg/kg bw) weekly beginning in the third week. Group 4 was a HCC control (DENA-treated group), group 5 was HCC + PJ, and group 6 was HCC + PCG.
PJ antagonized DENA-induced elevations of ALAT, TNF-α, NF-κB-p65, GST, MDA, and NO and restored total protein, IL-10, SOD, and CAT levels. Moreover, PJ resulted in downregulation of miR-21, Bcl-2, and Bcl-XL and an upregulation of caspase-3 and Bax mRNA expressions. These chemoprevention effects of PJ also alleviated the hepatic preneoplastic lesions induced by DENA. Although PCG treatment induced some modulation in DENA-treated rats, it did not show potent chemoprevention activity and induced some side effects.
Both of PJ and PCG downregulated miR-21 expression and triggered apoptosis. However, PJ was more effective than pure PCG in alleviating the hepatic antioxidant defense state and the inflammatory status. So, PJ was superior in prevention of DENA-induced hepatocellular carcinogenesis in rats than pure PCG.
Hepatocellular carcinoma (HCC) is the most common neoplasm among primary liver malignancies. It is the sixth most prevalent malignancy and the second-leading cause of cancer-related death worldwide, accounting for 70–85% of total liver cancer cases [1]. It usually occurs as a result of underlying liver disease and is frequently associated with liver fibrosis or cirrhosis arising from persistent liver injuries [2]. The lack of early diagnosis and high recurrence rates for HCC stages have drawn research attention toward studying the molecular mechanisms of this disease [3]. Disturbances in the hepatic antioxidant and oxidative stress and inflammatory systems have been implicated in the development and progression of cirrhosis to induce liver cancer [4, 5]. Additionally, overexpression of inflammatory cytokines linked to overproduction of reactive oxygen species (ROS) could inhibit apoptosis, presumably by activating the nuclear factor (NF)-κB-dependent pathway [6]. MicroRNAs (miRNAs), small noncoding single-strand RNA molecules (18–24 nucleotides), are involved in the regulation of gene expression at the post-transcriptional and translational levels [7]. The stability of miRNAs in the circulation from both healthy and diseased tissues makes them ideal biomarkers for different diseases. Successful miRNA-based therapy would certainly lead to improved efficacy [8]. MiR-21, one of the first identified mammalian miRNAs, has been identified as an oncogenic miRNA that is overexpressed in various human malignancies, including liver cancer [9]. MiR-21 upregulation has been reported in cirrhosis—the end-stage fibrosis—which is a prevalent preneoplastic condition linked to hepatocarcinogenesis [10]. Target-based therapy is widely thought to represent the future of cancer treatment, and so the development of inhibitors of HCC-signaling pathways and their upstream activators has generated considerable interest [11]. Thus, scientific interest in cancer management has been directed toward the utilization of natural products high in polyphenolic compounds due to their reported chemopreventive potential, and in an effort to prevent toxicities induced by chemicals, drugs, and carcinogenic xenobiotics. Furthermore, nearly 70% of all cancer medications used currently is derived from natural products [12]. Pomegranate (Punica granatum L.) fruit has become increasingly popular as a functional food of potential health benefits due to its ability to counter oxidative stress and reduce inflammatory mediators [13]. For cancer prevention and therapy, it is critical to maintain healthy physiological condition by balancing free radicals and antioxidants [14]. Among the bioactive ingredients in pomegranates is a group of polyphenols called punicalagins (PCGs), which are unique to the pomegranate. Pomegranate juice (PJ) has antioxidative, anti-inflammatory, anti-proliferative, and pro-apoptotic actions that exceed those observed with their isolated active components, suggesting that therapeutic strategies that do not rely on pure single agents could be developed [15, 16]. In this regard, this study evaluated and compared the chemoprevention effects of PJ and PCG in a hepatocellular carcinogenesis rat model. Moreover, this study tested a potential signaling pathway by surveying oxidative stress, inflammation, and apoptosis with particular focus on miR-21 expression. Diethylnitrosamine (DENA), an environmental and dietary hepatocarcinogen, is a DNA-alkylating agent widely used to induce liver cancer in animal models with a high success rate and with similarities to human HCC. Recently, DENA-induced rodent HCC models have been established to elucidate the pathogenesis, prevention, and treatment of liver cancer, including miRNA functions, antitumor effects of drugs and identification of biomarkers, and therapeutic targets [17].
Diethylnitrosamine (DENA) was purchased from Sigma-Aldrich (St. Louis, MO, USA). Fresh pomegranate fruits, free of obvious defects, were purchased from Dina farms (Cairo-Alexandria Desert Road, Egypt) and kindly were authenticated by our colleagues in the Botany Department.; fruits were manually washed and peeled, and separated arils were processed using a commercial blender to obtain PJ, and the juice was filtered and was provided orally to the animals within 1 week after squeezing. PCG (purity ≥ 80% HPLC) was purchased from Xi’an Rongsheng Biotechnology Co., Ltd. (82 Keji Road, Xi’an Hi-tech Zone, China).
Adult male Wistar albino rats (Rattus norvegicus; 200 ± 10 g) were purchased from the Nile Company for pharmaceuticals and chemical industries (Cairo, Egypt). The animals were housed in suitable cages and acclimatized to laboratory conditions for 1 week before the experiments began. Rats were provided with fresh tap water and standard rodent food pellets (Agriculture-Industrial Integration Company, Giza, Egypt). The animals were humanely treated in accordance with WHO guidelines for animal care, and the study design was approved by the Ain Shams University Research Ethics Committee (4/ 2018).
A hepatocellular carcinogenesis model was induced according to Cheng et al. [18] with some modifications, starting at the beginning of 3rd week; small doses of DENA (50 mg per kilogram body weight, once weekly) were injected intraperitoneally for 9 consecutive weeks. At the end, liver histopathology examination revealed early-stage HCC as demonstrated by dysplastic lesions, which have been described as possible progenitor lesions to HCC.
After 1 week of acclimatization, animals were randomly divided into six groups and treated for 11 consecutive weeks (77 days) as follows: Group I (control group): the animals received a basal diet and fresh water and housed in the same conditions as the treated groups. Group II (PJ group): 10 mL of PJ/kg bw, administered orally daily according to the previous studies of Ahmed et al. [19, 20]. Group III (PCG group): 18.5 mg of PCG/kg bw, administered orally daily according to our previous study El-Beih et al. [21]. Group IV (HCC control group): after induction of HCC, the animals served as a reference group for the DENA-treated groups. Group V (HCC + PJ group): the animals were injected with an intraperitoneal (i.p.) dose of DENA (50 mg/kg bw, weekly) beginning in the 3rd week until the end of the experiment and received 10 mL PJ/kg bw, administered orally daily, from the first day of the experiment until the end of the 11th week. Group VI (HCC + PCG group): the animals were injected with an i.p. dose of DENA (50 mg/kg bw, weekly) beginning in the 3rd week until the end of experiment and received 18.5 mg PCG/kg bw, administered orally daily, from the first day of the experiment until the end of the 11th week.
At the end of the experiment (day 78), the animals were subjected to light diethyl ether anesthesia before sacrificing. Part of the blood was collected into serum sep vacuette tubes and was then centrifuged in a cooling centrifuge (IEC centra-4R, International Equipment Co., Needham Heights, MA, USA) for 15 min at 4000 rpm and 4 °C to obtain serum. The serum samples were preserved at − 80 °C until they were used for biochemical analysis. Another part of the blood was collected into serum sep clot activator, left at room temperature (RT) for 30 min, and was then centrifuged at 10,000 rpm at 4 °C for 15 min. This part was used for detection and assessment of targeted miRNA. Immediately after sacrificing the animals, the liver was separated from the body, rinsed in 0.9% saline, weighted, and divided into three parts: (a) the first part was depressed in a 1-mL Qiazol solution to be used for the molecular assessments of different genes; (b) another part was stored at − 80 °C until it was used for biochemical assessment of oxidant/antioxidant markers; and (c) the last part was depressed in an appropriate fixative (10% formalin) for histopathological examination.
The activity of serum alanine aminotransferase (ALAT) and the level of serum total protein were estimated using commercial kits (Giza, Egypt).
Sandwich ELISA kits were used for quantitative estimation of serum interleukin-10 (IL-10) and TNF-α (Koma Biotech Inc., Seoul, Korea), as well as hepatic NF-κB-p65 (Elabscience, Biotech Inc., USA) concentrations, according to the manufacturers’ instructions.
Liver tissue homogenate was used for analysis of malondialdehyde [MDA; 22], nitric oxide [NO; 23], catalase [CAT; 24], superoxide dismutase [SOD; 25], and glutathione S-transferase [GST; 26].
Total ribonucleic acid (RNA) was extracted from liver tissues using Triazol extraction reagent (Bioflux Technology Co., China) following the manufacturer’s instructions. Reverse transcription of total RNA to first-strand complementary deoxyribonucleic acid (cDNA) was then carried out using Agilent Sure-Cycler 8800 (Agilent Technologies, Santa Clara, California) at 37 °C for 10 min. Gene expressions of Caspase-3, Bax, Bcl-2, and Bcl-XL were quantified by real-time polymerase chain reaction (qPCR), and the expression of hepatic glyceraldehyde-3-phosphate dehydrogenase (GAPDH, a house-keeping gene) was employed for normalizing the expressions of the tested genes. The qPCR was performed with specific primers obtained from Sigma-Aldrich for caspase-3 (sense, 5-CTGGACTGCGGTATTGAGAC-3; antisense, 5-CCGGGTGCGGTAGAGTAAGC-3), Bax (sense, 5-GACACCTGAGCTGACCTTGG-3; antisense, 5-GAGGAAGTCCAGTGTCCAGC-3), Bcl-2 (sense, 5-CAAGCCCGGGAGAACAGGGTA-3; antisense, 5-CCCACCGAACTCAAAGAAGGC-3) and Bcl-XL (sense, 5-TCAATGGCAACCCTTCCTGG-3; antisense, 5-ATCCGACTCACCAATACCTG-3), and GAPDH (sense, 5-ACCACAGTCCATGCCATCAC-3; antisense, 5-TCCACCACCCTGTTGCTGTA-3). All PCR reactions were performed using Maxima SYBR Green qPCR Master Mix (GenedireX, USA) according to the manufacturer’s instructions and were carried out using the Agilent Mx3005Pro qPCR system (Agilent Technologies Company, Santa Clara, CA, USA). The amplification conditions were one cycle for initial denaturation at 95 °C for 10 min, followed by 40 cycles of denaturation (15 s at 95 °C), annealing (60 s at 60 °C), and a final extension (20 s at 72 °C). Differences in gene expression between groups were calculated using the △△cycle threshold (Ct) method [27].
Total microRNA from the serum samples was isolated using the miRNeasy Mini Kit (Cat number #217,004; Qiagen, Hilden, Germany) in line with the manufacturer’s instructions. Reverse transcription of miRNA was carried out using Agilent SureCycler 8800 (Agilent Technologies, Santa Clara, California) with the MiScript II reverse transcription kit (Cat number # 218,160, Qiagen, USA). Finally, qPCR was performed using the MiScript primer assay (Cat number #218,300, Qiagen, USA), miRNA-21 (Hs_miR-21_2, MS00009079), and the reaction was tested using MiScript SYBR Green PCR kit (Cat number # 218,073; Qiagen, USA). Further, RNU-16 (Hs_RNU-2_11, MS00033740) was used as an endogenous control to normalize the expression level of the investigated miRNA. The relative expressions of miRNA were calculated according to Livak and Schmittgen [27].
Liver specimens previously fixed in 10% formalin were embedded in paraffin, sectioned at 5 µm, and stained with hematoxylin and eosin.
Statistical analysis was performed with one-way analysis of variance (ANOVA), followed by Tukey’s multiple comparison test, using GraphPad Prism version 4.03 for Windows (GraphPad Software, Inc., San Diego, CA, USA) for determining statistical significance. P values of < 0.05, < 0.01, and < 0.001 were considered statistically significant, highly significant, and very highly significant, respectively. Data are expressed as mean ± standard error of the mean (SEM).
The HCC control group showed a significant decrease (P < 0.05–0.001) in body weight, liver weight, and serum total protein levels and a significant increase (P < 0.05–0.001) in liver relative weight and the ALAT activity compared to the control group (Table 1). Although oral treatment of the DENA-treated groups with either PJ or PCG significantly increased (P < 0.001) body weight, only PJ treatment of the DENA-treated group led to a significant decrease in relative liver weight (P < 0.01). Treatment of the DENA-treated group with PCG non-significantly decreased (P > 0.05) relative liver weight as compared with the HCC control group. Despite the non-significant effect of PCG oral treatment on serum total protein levels in DENA-treated rats (P > 0.05), a significant increase was reported in total protein levels in the DENA-treated group that received PJ (P < 0.001) as compared to the HCC control group. While treatment of DENA-treated rats with PJ led to a significant decrease in serum ALAT (P < 0.001), treatment of DENA-treated rats with PCG did not significantly affect serum ALAT activity (P > 0.05) as compared with the HCC control group.
HCC control rats showed a significant increase (P < 0.05) in the activity of liver GST as compared with the control group. However, oral treatment of the DENA-treated rats with either PJ or PCG led to a significant decrease in the hepatic activity of GST (P < 0.05) as compared with the HCC control group. Liver CAT and SOD activities significantly decreased (P < 0.05–0.001) in the HCC control group as compared with the control group. However, oral treatment of the DENA-treated rats with PJ led to significant increases in the activity of liver CAT and SOD (P < 0.01–0.001), while DENA-treated rats that received PCG orally showed a significant increase in liver SOD activity (P < 0.001) accompanied with a non-significant increase (P > 0.05) in liver CAT activity as compared with the HCC control group. On the other hand, the HCC control rats showed a significant increase (P < 0.01) in liver MDA and NO levels as compared with the control group. However, these elevations showed significant decreases in the DENA-treated groups treated with either PJ (P < 0.01) or PCG (P < 0.001; Table 2).
HCC control group showed a significant increase (P < 0.001) in serum TNF-α levels and liver NF-κB-p65 levels as compared with the control group. Oral PJ treatment of DENA-treated rats led to a significant decrease (P < 0.05–0.001) in serum TNF-α and liver NF-κB-p65 levels as compared to HCC control rats. However, oral treatment of DENA-treated rats with PCG led to significantly increased serum TNF-α levels (P < 0.01) and did not significantly affect liver NF-κB-p65 levels (P > 0.05) as compared to the HCC control rats. On the contrary, the HCC control group showed a significant decrease (P < 0.05) in serum IL-10 levels as compared to the control group. This deterioration in serum IL-10 levels in the HCC control group showed increases with both PJ and PCG treatment. However, the increase was statistically significant (P < 0.05) in the PJ group and non-significant (P > 0.05) in the PCG group (Fig. 1).
The HCC control group showed a significant downregulation in hepatic caspase-3 and Bax mRNA expression (P < 0.05) accompanied by a significant upregulation in the Bcl-XL and Bcl-2 mRNA expression (P < 0.05 and P < 0.001, respectively). Moreover, the Bax/Bcl-2 ratio showed a significant decrease (P < 0.001) in the HCC control group as compared to the control group. However, oral treatment of the DENA-treated rats with either PJ or PCG improved these disturbances in apoptosis as shown by significant upregulation in caspase-3 and Bax mRNA expression (P < 0.001), significant downregulation in Bcl-XL and Bcl-2 mRNA expression (P < 0.001), and an increased Bax/Bcl-2 ratio as compared to the HCC control rats (Fig. 2A–E).
HCC control rats showed a significant upregulation in the expression of serum circulating miR-21 (P < 0.05) as compared to control rats. Oral treatment of DENA-treated rats with either PJ or PCG induced a significant downregulation in the expression of serum circulating miR-21 (P < 0.001) as compared with the HCC control rats (Fig. 2F).
Examination of the liver tissues of rats from the control, PJ-treated, and PCG-treated groups showed a normal pattern of liver architecture with no histopathological alteration. On the other hand, the hepatic tissues from the HCC control group showed distinct alterations, including nodular patterning of the hepatic tissue, dilated central vein, multiple scattered binucleated and multinucleated hepatocytes, vacuolated hepatocytes nuclei with multiple prominent nucleoli, dysplastic cells with increased nuclear cytoplasmic ratios, necrotic hepatocytes, and inflammatory cell infiltration. All of these reported alterations are defined as dysplastic nodules. These high-grade lesions of dysplasia are typically found in cirrhotic livers and have been described as possible progenitor lesions to HCC. In contrast, examination of liver tissue from the DENA-treated group that received PJ showed normal liver architecture with normal central veins and multiple scattered hepatic cells with clear vacuolated cytoplasm and dilated sinusoidal spaces. Liver tissues from the group treated with DENA that received PCG revealed nodular architecture of liver tissue (cirrhotic pattern) with dilated central veins, a few scattered hepatic cells with double nuclei, mild proliferation of interlobular connective tissue, moderate vacuolar degeneration, and mild bile duct hyperplasia (Fig. 3).
HCC is the predominant form of primary liver malignancy. Globally, it is the second-leading cause of cancer mortality, with over 500,000 new cases diagnosed each year [28]. One of the main goals of recent research has sought to determine cancer prevention strategies. To some extent, this can be accomplished by using naturally occurring or synthetic chemoprevention agents that can inhibit or prevent tumor development. Therefore, it is critical to identify agents, assess their efficacy, and elucidate their mechanisms of action. In this respect, it has been revealed that pomegranate and its components have cancer-inhibitory effects on many types of malignancies [29–31]. Accordingly, this study established a model of DENA-induced hepatocarcinogenesis in male albino rats to clarify the role of PJ and PCG in the prevention of HCC development. Experimental induction of liver cancer in rodents by DENA is one of the most well-characterized animal models of HCC, and it allows for screening of anticancer agents at different stages of neoplastic transformation [32]. DENA-induced rats developed various alterations in liver tissue, defined as dysplastic nodules, which are common in cirrhotic livers. According to an epidemiological survey, more than 80% of HCC cases are associated with advanced liver fibrosis or cirrhosis [33]. Considerable evidence has reported the nodules as precursors of hepatic cancer [34]. DENA-induced preneoplastic foci, as well as preneoplastic and neoplastic nodule formation in rodents, closely resemble the progression of HCC in humans. Cross-species comparison of gene expression revealed that DENA-induced liver tumors in rodents closely resemble a subclass of human HCC, allowing for the extrapolation of possible clinical chemopreventive effects of candidate medicines [32]. Here, the chemopreventive action of PJ in comparison to pure PCG on the development and progression of early preneoplastic lesions in the liver was established in a rat model of hepatocarcinogenesis induced with DENA. The results demonstrate that treatment of DENA-treated rats with PJ/PCG resulted in fewer animals with visible hepatocyte nodules and reduced nodular multiplicity compared to HCC control animals. In a previous literature data it was reported that pomegranate components could prevent malignancies of the skin, breast, lungs, and colon [35]. The present study reported that PJ had a potent chemopreventive effect greater than that of PCG via inhibition of hepatic nodule formation and suppression of nodule growth. These represent important steps for liver cancer chemoprevention. Liver cancer may produce complex metabolic disturbances resulting in rapid loss of body weight and tissue emaciation [36]. This study reported that the HCC control rats had a significant reduction in body and liver weight, with a significant increase in relative liver weight, as compared to the control group. This was likely attributable to liver carcinogenesis in the HCC control group. These findings converge with many previous studies [37–39]. Whereas treatment of DENA-treated rats with either PJ or PCG significantly increased body weight and decreased relative liver weight when compared to HCC control rats. This finding suggests that PJ and PCG altered the metabolism of energy. In this study, the HCC control rats showed a significant increase in serum ALAT enzyme activity and a significant decrease in total protein levels, indicating the toxic effect of DENA on liver tissue. These findings align with those of Assar et al. and Mokh et al. [37, 40], who found that ASAT, ALAT, and ALP activity significantly increased following nitroso compound treatment in rats owing to induced liver damage. The elevation of liver enzymes could be attributed to loss of cell membrane integrity, which allowed them to leak out of the damaged cell into extracellular space, indicating hepatic cellular injury. On the other hand, oral treatment of DENA-treated rats with PJ resulted in a significant decrease in ALAT activity—indicative of hepatic cell function recovery—suggesting that PJ may have a protective effect against DENA-induced liver damage. This hepatoprotective effect of PJ could emerge due to its antioxidant properties that lead to reduced ROS generation and, consequently, reduced membrane permeability and enzyme leakage into the blood [21, 41]. On the other hand, oral treatment of DENA-traeted rats with PCG did not affect serum ALAT activity. According to Lin et al. [42], despite the high antioxidant activity of PCG, it can induce liver damage when taken in high dosages. Although the PCG dose used in this study is less than the doses used in El-Beih et al. [21], we found that it was still not an effective dose. The decreased serum total protein levels in the HCC control group suggest that synthetic liver function declined and PJ treatment restored serum total protein and synthetic liver function, in line with Husain et al. [41]. This could be due to the antioxidant properties of PJ and could prevent oxidative modification of amino acid chains and ROS-mediated peptide changes caused by DENA. There is strong evidence that oxidative stress plays a role in the development and progression of cirrhosis, which can lead to liver cancer [4, 5]. ROS and RNS could trigger cellular damage upon exposure to carcinogens, and thus, intoxication of rats with DENA could initiate cell damage through the induction of lipid peroxidation formation, thereby causing cirrhosis and the appearance of preneoplastic lesions that characterize HCC [43]. Further, DENA biotransformation by cytochrome P450 in the rat liver produces ethyl diazonium ions that react with DNA, forming adducts recognized as the initial step in DENA-induced carcinogenesis [44]. Accordingly, HCC control rats showed a significant increase in hepatic levels of MDA and NO, as well as GST activity, which was accompanied by a significant decrease in SOD and CAT enzyme activity, suggesting that DENA injection causes an increase in free radical production, disrupts antioxidant defense systems, and increases ROS, consistent with findings from different cancer models [37, 40]. Interestingly, recent evidence has shown that overactive GSTs play a key role in tumor progression and cancer pathogenesis and are considered a common feature of various human cancers, as they actively participate in tumorigenesis processes such as cell survival, proliferation, and chemoresistance [45, 46]. This was clearly supported by this study, which showed a significant increase in hepatic GST activity in HCC control rats as compared with the control group. In contrast, this disturbance in the hepatic antioxidant defense system was attenuated in DENA-treated rats that received either PJ or PCG, as hepatic SOD and CAT enzyme activity showed significant increases in rats administered PJ. However, CAT activity improvement in the DENA-treated rats that received PCG was statistically non-significant. This improvement in hepatic antioxidants was coupled with a significant decrease in the hepatic levels of MDA and NO as compared to the HCC control group. Notably, the present data showed that treatment with either PJ or PCG led to a significant decrease in hepatic GST activity compared to the HCC control group. Thus, PJ or PCG could contribute to the development of GST inhibitors for cancer treatment. Pomegranate could reverse the progression of pathological lesions via its strong antioxidant capacity, as it has potent free-radical scavenging properties [47]. PJ may reduce the toxicity of heterocyclic aromatic amine and prevent the hepatocarcinogenic effect of nitrosamines. These findings support various studies documenting the hepatoprotective effects of pomegranate against chemically induced hepatocarcinogenesis and its ability to inhibit lipid peroxidation and oxidative damage [32]. In addition, Aloqbi et al. [48] reported that PCG contains 16 phenolic hydroxyls per molecule, whereas PJ contains higher concentrations of anthocyanins than tannin components, perhaps explaining why PJ has greater antioxidant capacity, as compared to PCG, in reducing stable radicals to a non-radical state. Active NF-κB is frequently implicated in various malignancies and in inflammation. Excessive generation of ROS may activate the NF-κB pathway, the most prominent pathway involved in the inflammation-fibrosis-cancer axis, and trigger release of IĸBs, leading to NF-κB nuclear translocation. Thus, it may enhance the release of pro-inflammatory mediators, thereby leading to inflammation. Inflammation could trigger disruption of the hepatic microenvironment, creating an adverse oncogenic field. Thus, inflammatory signaling pathways may be considered a target for cancer prevention [47, 49, 50]. A few recent studies have examined the molecular mechanisms that link inflammation to carcinogenesis. Considering that NF-κB-p65 has been reported to be related to HCC progression, Xu et al. [1] revealed that NF-κB-p65 was activated and that the translocation of its phosphorylated isoform into the nucleus was markedly increased after DENA and TNF-α treatment. Similarly, the data of this study showed that oxidative stress induced by DENA and initiated hepatocarcinogenesis in rats affected the inflammatory response of liver cells by triggering NF-κB-p65 protein and elevating serum TNF-α, in conjunction with reduced IL-10 levels, as compared to the levels observed in the control rats. This aligns with findings suggesting that NF-κB is the most important transcriptional factor, which directly controls pro-inflammatory cytokine cellular production [16]. TNF-α, which plays a major role in inflammation and has also been shown to be an accelerant of cell proliferation, is a powerful NF-κB-activating factor whose expression is regulated by NF-κB [51]. Driessler et al. [52] suggested that anti-inflammatory properties of IL-10 may act by suppressing the expression of pro-inflammatory cytokines via inhibiting NF-κB. However, it seems that the balance between TNF-ɑ and IL-10 is critical to the development of HCC [53]. Thus, elevated TNF-α associated with low levels of IL-10 were shown to be associated with increased risk of HCC development. In this study, DENA-treated rats that received PJ showed an improvement in the inflammatory state as demonstrated by the inhibitory effects of PJ on NF-κB-p65 production, which was associated with reduced serum TNF-α and increased IL-10 concentrations. While, DENA-treated rats that received PCG showed no effect on NF-κB-p65 and IL-10 levels. As NF-κB is an oxidant-sensitive transcription factor, PJ could inhibit the activation of NF-κB by suppressing oxidative stress. This is in alignment with the previous report showing that pomegranate extract inhibits nuclear translocation and activation of the NF-κB-p65 protein in human chondrocytes [54]. Further, in their review paper, Syed et al. [31] have presented literature data on therapeutic action of PJ via its ability to downregulate NF-κB expression by suppressing the IκBα kinase (IKK) activation, which stops cell growth and induces apoptosis. Moreover, PJ treatment reduced TNF-α level, which resulted in NF-κB inhibition and subsequent inhibition of cell proliferation. This reduction in TNF-α was associated with a significant elevation in IL-10 levels, indicating the potent anti-inflammatory effect of PJ in the initiation of HCC in DENA-treated rats. Oxidative stress and inflammation are thought to work as the primary initiators of apoptosis, which is controlled by either mitochondrial (intrinsic) or death receptor-mediated (extrinsic) signaling pathways. Anti-apoptotic proteins (Bcl-2 and Bcl-XL) and pro-apoptotic proteins (Bax and caspase-3) are essential components of mitochondrial-mediated apoptosis [55]. When Bcl-2 protein mRNA levels drop and Bax protein levels rise, mitochondrial membrane breakdown occurs, and this allows cytochrome C to leak into the cytosol. Then, a cascade of reactions occurs, including cytochrome binding to Apaf-1, resulting in activation of caspase-9 and caspase-3 [47]. Various studies suggest that DENA could trigger both apoptotic pathways initiated by oxidative stress in the liver [56, 57]. In contrast, our findings indicate upregulation in Bcl-2 and Bcl-XL and downregulation in Bax and caspase-3 mRNA levels in the livers of rats intoxicated with DENA. In rats, DENA mediated the anti-apoptotic effect via downregulation of caspase-3 mRNA levels and a decreased Bax/Bcl-2 ratio. Exposure to DENA is associated with hepatocellular accumulation of ROS [58], which in turn could trigger apoptosis in the liver [59, 60]. Chavda et al. [61] published literature evidence in their review work that the oxidative stress can cause cell apoptosis only at high concentrations. Thus, ROS levels play a critical role in either promoting tumorigenesis or causing apoptosis. The present results show that administering either PJ or PCG to DENA-treated rats reversed the progression of the pathological lesions through activation of the apoptosis process, as it significantly downregulated Bcl-2 and Bcl-XL mRNA and upregulated the expression of Bax and caspase-3 mRNA, in line with Ghani et al. [62], who found that pomegranate could trigger apoptosis via the intrinsic pathway through cytochrome C release and caspase-3 activation. PCG could render the hepatocytes more susceptible to TNF-α-induced (extrinsic) apoptosis, which is indirectly related to the depletion of hepatocytes GSH conjugates, resulting from decreased GST activity in the livers of DENA-treated rats that received PCG [46]. MiRNAs have been implicated in cancer development, and their expression in biological fluids offers considerable potential as nucleic acid markers in cancer diagnosis and prognosis. This study revealed that circulating oncomir miR-21 expression was significantly increased in HCC control rats as compared to the control group. This finding is in accordance with the previous work revealing miR-21 overexpression in DENA-infused rats and suggested that serum miR-21 could be employed as an early diagnostic molecular marker in hepatocarcinogenesis [63]. It was found that miR-21 usually has multiple targets and thus can modulate multiple molecular mechanisms of biological processes in HCC models [64]. Zhang et al. [65] found that the role of miR-21 in carcinogenesis might be linked to its effect on ROS levels via targeting SOD3 and TNFα. Moreover, miR-21 overexpression affects cell proliferation and inhibits apoptosis by targeting Bax downregulation and Bcl-2 upregulation [66]. In contrast, the current data demonstrate that oral administration of either PJ or PCG significantly downregulated miR-21 expression in comparison to the DENA-treated group, in line with studies reporting a downregulation effect of pomegranate on miR-21 expression [67, 68]. Given the current data, the downregulation effect of PJ or PCG on miR-21 expression may be attributable to their anti-inflammatory effect via the NF-κB-p65/TNF-α pathway, which can affect miR-21 expression (Fig. 4). This aligns with previous studies demonstrating that miR-21 is a type of NF-κB-dependent miRNA that shows increases in response to cytokines and inflammation, specifically via activation of NF-κB signaling, indicating the interplay between miR-21 and NF-κB in cancer [69, 70].
The present study revealed the chemopreventive role of PJ and PCG against hepatocarcinogenesis and elucidated their possible mechanisms of action. Both of PJ and PCG downregulated miR-21 expression and triggered apoptosis related genes expression. However; PJ was superior to pure PCG in improving the hepatic antioxidant defense state and the inflammatory status, which increases its chemoprevention effectiveness against DENA-induced hepatocellular carcinogenesis. So, further studies are required to provide the appropriate dose–response of PCG on HCC. | true | true | true |
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PMC9628169 | Baojun Zhao,Shengtao Gao,Mingyang Zhao,Hongyu Lv,Jingyu Song,Hao Wang,Qifan Zeng,Jing Liu | Mitochondrial genomic analyses provide new insights into the “missing” atp8 and adaptive evolution of Mytilidae | 02-11-2022 | Mitochondrial genome,Mytilidae,atp8,Molecular phylogeny,Positive selection | Background Mytilidae, also known as marine mussels, are widely distributed in the oceans worldwide. Members of Mytilidae show a tremendous range of ecological adaptions, from the species distributed in freshwater to those that inhabit in deep-sea. Mitochondria play an important role in energy metabolism, which might contribute to the adaptation of Mytilidae to different environments. In addition, some bivalve species are thought to lack the mitochondrial protein-coding gene ATP synthase F0 subunit 8. Increasing studies indicated that the absence of atp8 may be caused by annotation difficulties for atp8 gene is characterized by highly divergent, variable length. Results In this study, the complete mitochondrial genomes of three marine mussels (Xenostrobus securis, Bathymodiolus puteoserpentis, Gigantidas vrijenhoeki) were newly assembled, with the lengths of 14,972 bp, 20,482, and 17,786 bp, respectively. We annotated atp8 in the sequences that we assembled and the sequences lacking atp8. The newly annotated atp8 sequences all have one predicted transmembrane domain, a similar hydropathy profile, as well as the C-terminal region with positively charged amino acids. Furthermore, we reconstructed the phylogenetic trees and performed positive selection analysis. The results showed that the deep-sea bathymodiolines experienced more relaxed evolutionary constraints. And signatures of positive selection were detected in nad4 of Limnoperna fortunei, which may contribute to the survival and/or thriving of this species in freshwater. Conclusions Our analysis supported that atp8 may not be missing in the Mytilidae. And our results provided evidence that the mitochondrial genes may contribute to the adaptation of Mytilidae to different environments. Supplementary Information The online version contains supplementary material available at 10.1186/s12864-022-08940-8. | Mitochondrial genomic analyses provide new insights into the “missing” atp8 and adaptive evolution of Mytilidae
Mytilidae, also known as marine mussels, are widely distributed in the oceans worldwide. Members of Mytilidae show a tremendous range of ecological adaptions, from the species distributed in freshwater to those that inhabit in deep-sea. Mitochondria play an important role in energy metabolism, which might contribute to the adaptation of Mytilidae to different environments. In addition, some bivalve species are thought to lack the mitochondrial protein-coding gene ATP synthase F0 subunit 8. Increasing studies indicated that the absence of atp8 may be caused by annotation difficulties for atp8 gene is characterized by highly divergent, variable length.
In this study, the complete mitochondrial genomes of three marine mussels (Xenostrobus securis, Bathymodiolus puteoserpentis, Gigantidas vrijenhoeki) were newly assembled, with the lengths of 14,972 bp, 20,482, and 17,786 bp, respectively. We annotated atp8 in the sequences that we assembled and the sequences lacking atp8. The newly annotated atp8 sequences all have one predicted transmembrane domain, a similar hydropathy profile, as well as the C-terminal region with positively charged amino acids. Furthermore, we reconstructed the phylogenetic trees and performed positive selection analysis. The results showed that the deep-sea bathymodiolines experienced more relaxed evolutionary constraints. And signatures of positive selection were detected in nad4 of Limnoperna fortunei, which may contribute to the survival and/or thriving of this species in freshwater.
Our analysis supported that atp8 may not be missing in the Mytilidae. And our results provided evidence that the mitochondrial genes may contribute to the adaptation of Mytilidae to different environments.
The online version contains supplementary material available at 10.1186/s12864-022-08940-8.
Mitochondria are essential eukaryotic organelles, they play important role in ATP (the universal currency of biological energy) production through oxidative phosphorylation (OXPHOS) [1]. The typical mitochondrial genome of animals is a small (16 kb) circular molecule, which includes 13 OXPHOS-related genes, 22 transfer RNA (tRNA) genes and 2 ribosomal RNA (rRNA) genes [1, 2], and it usually follows a strictly maternal inheritance. In bivalves, some species of Mytilidae [2, 3], Donacidae [4] and etc. showed a unique Doubly Uniparental Inheritance (DUI) model. In this model, there are two highly divergent male (M-type) and female (F-type) mitochondrial genomes (M-type vs F-type DNA divergence exceeds 20%) [1, 5]. Females with DUI possess only F-type, and males possess two types, but transmit only M-type to their sons. The mitochondrial genomes of bivalve species are also characterized by extraordinary variability in gene arrangement, tRNA gene number, and genome size. And some bivalve species are thought to lack the mitochondrial protein-coding gene ATP synthase F0 subunit 8 (ATP8) [6–8]. The presence and absence of atp8 were mainly studied in Mytilidae, and atp8 gene has been identified and proved to be actively transcribed and translated in Mytilus spp. [6, 9, 10]. However, the atp8 gene of Limnoperna fortunei was presumed to be a pseudogene. Whether atp8 gene was actually “missing” in some species has become a concern for researchers [5]. Mytilidae, also known as marine mussels, are widely distributed in the oceans worldwide. Some mussels are important economic species, for instance, Mytilus chilensis, Mytilus. edulis, Mytilus coruscus, Perna viridis [11, 12]. According to the Fishery and Aquaculture Statistics 2018 reported by Food and Agriculture Organization, the total production of M. chilensis (major species) in 2018 was 365,595 tonnes. Members of Mytilidae show a tremendous range of ecological adaptions, from the species distributed in freshwater to those that inhabit in deep-sea. The deep-sea environment is one of the most extreme environments on Earth, with limited food, low oxygen, high hydrostatic pressure, toxic chemicals and extreme temperature [13]. The species of Mytilidae that invaded deep-sea environments are mainly in the subfamily Bathymodiolinae. The evolutionary stepping stone hypothesis believes that the ancestors of Bathymodiolinae progressively adapted to deep-sea environments by exploiting sunken wood and whale carcasses [14]. Bathymodioline species usually have reduced digestive systems [15] and rely instead on endosymbiotic bacteria, transmitted horizontally from the environment to gill tissues, which produce organic carbon with energy from hydrogen sulfide oxidation. [16]. L. fortunei, golden mussel, is a species of Mytilidae with freshwater independent colonization [6, 17]. In freshwater, the low levels of ionic concentration may force organisms to expend more energy regulating osmotic pressure [18]. Given the functional importance of OXPHOS, mutations of the mitochondrial genes can directly affect metabolic performance. Mounting evidence suggests that some non-neutral mutations in mitochondrial genes can contribute to the adaptation of animals to different environments [19–21]. Mitochondrial DNA has been one of the most useful tools that are widely used in species identification, phylogenetic studies [22], comparative genomics [23], and management of invasive alien species [24]. Xenostrobus securis, L. fortunei, and Mytilus galloprovincialis and etc., are regarded as notorious invasive species which have caused dramatic and devastating effects on ecosystems [25, 26]. However, the complete mitochondrial genome of X. securis is still unknown. In addition, more mitochondrial genomes may contribute to further understanding the differentiation and evolution of Mytilidae [27, 28]. The emergence of cost-efficient next-generation sequencing allows us to quickly obtain mitochondrial genomes from various data (genomic data, transcriptome data, and metagenomic data) [29, 30]. In the present study, the complete mitochondrial genomes of X. securis, and two deep-sea mussels (Bathymodiolus puteoserpentis, Gigantidas vrijenhoeki) were newly assembled. We re-annotated atp8 gene in Mytilidae, which is aim to answer whether atp8 is not missing in the whole family. Furthermore, we also performed positive selection analysis of 12 protein-coding genes. We aim to provide new insights into the molecular mechanisms of adaptive evolution (to different environments: deep-sea and freshwater) of Mytilidae.
The sequencing data were download from NCBI (X. secures SRR7751554, B. puteoserpentis ERR3959529, G. vrijenhoeki SRR10802050) and filtered by Trimmomatic 0.36 [31–33]. The mitochondrial genomes of those species were assembled with the NOVOPlasty software [30]. The MITOS web server (http://mitos2.bioinf.uni-leipzig.de/index.py) was used to annotate the mitochondrial genomes [34]. tRNA genes were also predicated by ARWEN v1.2.3 (http://130.235.244.92/ARWEN/) [35]. The AT and GC skews were calculated according to the following formulae: AT-skew = (A − T)/(A + T) and GC-skew = (G − C)/(G + C). Because of the small size and high variability of atp8, it is difficult for automatic annotation tools [5, 36]. The atp8 sequences were annotated by manually scanning the intergenic regions. ORFfinder (https://www.ncbi.nlm.nih.gov/orffinder/) was used to find the ORFs. The start codon of atp8 sequences was corrected according to the sequences of related species. TMHMM Server v.2.0 (http://www.cbs.dtu.dk/services/TMHMM/) was used to identify the transmembrane helices of atp8 sequences. The PROTSCALE tool of ExPASy (http://ca.expasy.org/tools/) was applied to calculate the hydrophobicity profiles. In addition, we also annotated the atp8 with HHblits v3.30 [37] referring to a previous study [38]. In brief, A Hidden Markov Model (HMM) was constructed for each ORF using HHblits with PDB70. An HMM for known atp8 genes was constructed with the latest Uniclust30 database. Then, the HMM-HMM alignment was run against ORFs with atp8.
In this article, only F-type was included in the analyses. The 12 protein-coding genes of 46 sequences were used to reconstruct the phylogenetic relationships [39]. The Crassostrea gigas (AF177226.1) and Atrina pectinata (KC153059.1) served as outgroups (Table 1). atp8 was excluded in the phylogenetic analysis as atp8 was highly variable in length and amino acid composition. The sequences were aligned with Muscle in MEGA7 [40]. The gap and ambiguously aligned sites were recognized and removed with Gblocks Version 0.91b [41]. ModelTest-NG was used to identify the best-fit models for each gene based on the Akaike Information Criterion (AIC) [42]. Bayesian phylogenetic inference was performed with Mrbayes 3.2.7 [43]. Two independent Markov chain Monte Carlo (MCMC) simulations were carried out with four chains (one cold, three hot) for 1,000,000 generations, sampling every 1000 generations. The initial 25% of sampled trees were discarded as burn-in. Maximum Likelihood (ML) inference was performed using RAxML-NG with 1000 bootstrap replicates [44]. The phylogenetic trees were visualized by Figtree. v1.4.4. The divergence time was estimated using the program MCMCtree in PAML4.9 [59]. Two nodes were used as calibrations, one of which was from the fossil recode data of Modiolinae (393–408 Mya) and the other was from previous studies [28, 60, 61], the time of divergence between B. themophilus and G. childressi was approximately 21.1–33.0 Mya.
Comparing the nonsynonymous/synonymous nucleotide substitution ratios (ω = dN/dS) has been widely used to evaluate the adaptive molecular evolution of protein-coding genes. The values of dN/dS mean changes in selective pressure, where the dN/dS < 1, = 1, > 1 correspond to negative purifying selection, neutral evolution and positive selection, respectively. The program CODEML in PAML4.9 was applied to calculate the values of dN/dS [59]. The phylogenetic tree of 12 protein-coding genes inferred with Mrbayes was used for selection analyses. The outgroups were not included in selection analyses. For branch model, One-ratio model (model = 0, NSsites = 0, icode = 4) and Three-ratios model (model = 2, NSsites = 0, icode = 4) were performed. The deep-sea branches (Bathymodiolinae) and freshwater branches (L. fortunei) were used as foreground branches (two foreground branches) and the remaining were used as background branches. In addition, the branch-site model (model = 2, NSsites = 2) was used to determine whether positive selection acted on specific sites on foreground branches. The sites under positive selection were identified with Bayes empirical Bayes posterior probabilities (> 0.95). The likelihood ratio tests were carried out to identify if the alternative model provided a significantly better fit than the null model. To explore the possible effects of positive selection sites on protein function, the three-dimensional structure of protein was predicted with phyre2 [62]. The protein structure of NuoM in Escherichia coli [63]was used as a template [21, 64]. The positive sites were marked using PyMOL.
We have successfully obtained the complete mitochondrial genomes of X securis, B. puteoserpentis, and G. vrijenhoeki, with lengths of 14,972 bp, 20,482, and 17,786 bp, respectively. The genomes we assembled showed high similarity with the known sequences of each species (100% for X. securis; 100% for B. puteoserpents; 99.42% for G. vrijenhoeki). It should be pointed that X. secures might be a cryptic species complex, and we cannot rule out the possibility that the mitochondrial genome of X. securis may belong to the M-type [65, 66]. The base composition analysis showed that three assembled genomes were biased toward A and T, with AT content of 59.08% in X securis, 63.55% in B. puteoserpentis, and 66.96% in G. vrijenhoeki. The assembled genomes are all characterized by negative AT skew and positive GC skew (Table 2). The base composition and skewness are consistent with most studies in bivalves [8, 67, 68]. For these three species, all genes encoded on the heavy strand (H-strand) except tRNA Gly in Light (L-strand). Each genome has 13 protein-coding genes and 2 ribosomal RNA genes (Fig. 1). However, the number of tRNAs is varied. Twenty-two typical tRNAs were identified in X securis. 27 tRNAs (four more tRNAHis and one more tRNALeu) and 23 tRNAs (one more tRNALeu) were identified in B. puteoserpentis and G. vrijenhoeki, respectively. The lengths of intergenic region between tRNAHis were 470 bp, 441 bp, 455 bp and 468 bp, respectively, which leads B. puteoserpentis to have the largest mitochondrial genome among Bathymodiolinae. In the assembled genomes of X securis, B. puteoserpentis, and G. vrijenhoeki, the total lengths of protein-coding genes were 11,060, 10,947, and 10,993, accounting for 73.87%, 53.45%, 61.81% of the whole genome, respectively. The protein-coding genes of X securis started with ATG and ATA, while both of B. puteoserpentis and G. vrijenhoeki started with ATG, ATA, ATT, and GTG. For these three species, the protein-coding genes mainly started with codon ATG. The stop codons of all species were either TAA or TAG except nad1 and cox3 of X. securis which had an incomplete stop codon of T. The presence of incomplete stop codons is a common feature of the mitochondrial genes among animals [5, 69, 70]. The incomplete stop codon is thought to be completed by polyadenylation of the transcript.
Some species are thought to lack atp8 gene that encodes a subunit of mitochondrial ATP synthase [6, 7]. Increasing studies indicated that the absence of atp8 may be caused by annotation difficulties for atp8 gene is characterized by highly divergent, variable length. Sometimes, atp8 gene could not be detected by automatic annotation software, the annotation of atp8 gene usually requires manual inspection and comparison to atp8 sequences from other species. In this study, we manually annotated atp8 in the sequences that we assembled and the sequences lacking atp8. Twelve atp8 sequences were manually annotated in the intergenic region (Table 3). The results of manual annotation were highly consistent with the results of HMM. However, HMM method was unable to detect atp8 in some species (e.g. L. fortunei, X. secures and Modiolinae,), probably due to the lack of atp8 sequences from related species and the low sequence similarity with known atp8 genes. For newly annotated atp8, start codons were ATG or GTG or ATC, and stop codons were either TAG or TAA. ATP8 usually has higher conservation of the secondary structure compared to the primary sequence [71]. The newly annotated atp8 sequences all have one predicted transmembrane domain, a similar hydropathy profile, as well as the C-terminal region with positively charged amino acids (R, H, and K). (Table 3, Figs. 2 and 3) [72]. In this study, all species of Mytilidae possessed an annotated atp8 gene, which allows us to further understand the features of atp8 gene in a family. The lengths of atp8 in Mytilidae were short and variable, ranging from 37 – 139 aa (Table 3 and Fig. 3). The longest atp8 was from Mytilaster solisianus (KM655841.1), and the shortest atp8 was from P. canaliculus. It should be noted that the annotation of the start codons and stop codons might be inaccurate in some species due to the lack of additional data. The atp8 sequence of M. solisianus was much longer than that of related species. We are not sure whether this sequence used an incomplete stop codon (TA or T), which caused the fact that the real length was shorter than the current length. The alignment of atp8 gene indicated that atp8 sequences were highly divergent that they showed similarity only in related species. The conserved ‘MPQL’ amino acid signature at the N-terminus, the typical characteristic for metazoan ATP8 proteins [71], was only found in L. fortunei (VPQL) (Fig. 3). However, the conserved ‘PQ’ amino acid signature was found in many species, for instance, Bathymodiolinae, Limnoperninae, Lithophaginae, P. viridis, P. canaliculus, Arcuatula senhousia and some species of Modiolinae [72]. Although not all species of Mytilidae have this feature, it still can contribute to identifying atp8 gene from ORFs in some species of Mytilidae. Given the characteristics of atp8 gene, it is not surprising that atp8 gene was once presumed to have lost in many species. Although atp8 gene of L. fortunei has the ‘MPQL’ amino acid signature at the N-terminus, it was still annotated as a pseudogene in an incorrect position [6]. In almost all lineages of animals, there has been strong selection to maintain a minimal set of 37 genes [5]. Researchers need to be cautious of assertions that a mitochondrial gene is missing [73]. Our results supported that atp8 gene may not be missing in the Mytilidae. Although we have no right to claim that whole Bivalvia class possesses an atp8 gene, we provided further evidence that a family possesses the atp8 gene. In the future, studies of transcriptional activity and function of these atp8 genes may be necessary. Moreover, we strongly encourage researchers to identify whether atp8 gene was not missing in other families.
To further examine the relationship among the Mytilidae species, the phylogenetic trees were reconstructed using Maximum Likelihood and Bayesian inference methods with a concatenated alignment. The tree topologies resulting from these two methods were consistent. The results supported that the Mytilidae is subdivided into two major clades [22]. The clade 1 contained the subfamilies Bathymodiolinae, Modiolinae, Limnoperninae, and Lithophaginae and the genus Xenostrobus (Arcuatulinae), and clade 2 included subfamilies Brachidontinae, Mytilinae, Crenellinae, Septiferinae, and genus Arcuatula (Arcuatulinae) (Fig. 4). The estimated divergence time between the two clades was around 399.37 Mya (95% HPD interval 392.74- 407.65 Mya), which is close to the estimated time in other analyses (Fig. 5) [22, 74]. The subfamily Bathymodiolinae was monophyletic, which is the same with previous studies [28, 60]. In this study, the Bathymodiolinae were divided into three separate clades, corresponding to the Gigantidas, Bathymodiolus, and “Bathymodiolus”. The Gigantidas was clustered with “Bathymodiolus” and then sister to Bathymodiolus, which is consistent with previous analysis [60], but different from zhang’s study [28]. It should be noted that although the Gigantidas clustered with “Bathymodiolus”, the node was not supported enough according to bootstrap value and posterior probability. Our results indicated that the subfamily Arcuatulinae was polyphyletic as genera Xenostrobus and Arcuatula were divided into the clade1 and clade2, respectively. In clade1, the genus Xenostrobus and (Modiolinae + Bathymodiolinae) were grouped in a subclade with high supporting values (100% BP and 1.00 BPP). The placement of Genus Xenostrobus was different between our results and a previous study based on 5 genes [74]. The tree of the previous study showed that Xenostrobus was clustered with Bathymodiolinae and then sister to Modiolinae. However, the gene order of 13 protein-coding genes and 2 rRNA (excepting tRNA) between Modiolinae and Bathymodiolinae was consistent, which supported our result (Fig. 1). Further increasing the sequences of Xenostrobus may contribute to resolving the phylogenetic relationship among Genus Xenostrobus, Modiolinae, and Bathymodiolinae. In clade 2, Brachidontinae were divided into three well-supported clades: [1] Geukensia [2] Brachidontes [3] Mytilisepta + Perumytilus + Semimytilus, which was similar to the results of nuclear genes18S and 28S [75]. However, the placement of Geukensia was inconsistent. Moreover, a previous study [22] and our result indicated that Perna perna (KM655841.1) had an unusual phylogenetic status, which showed high similarity with two Brachiodontes species rather than P. viridis and Perna canaliculus according to gene order and phylogenetic trees (Figs. 1 and 4) [22]. The sequence of P. perna (KM655841.1) showed 99.83% sequence identity with cox1 sequences of M. solisianus, which suggested that the sequence may belong to M. solisianus rather than P. perna.
Purifying selection has been widely recognized as the predominant force acting on the molecular evolution of mitochondrial genomes. However, some studies have demonstrated that relaxation of purifying selection or episodic positive selection on mitochondrial genomes may occur in species that have different types of locomotion [76] or species living in extreme environments [77–79]. The One-ratio model analysis the ω values of these 12 genes ranged from 0.0024 to 0.0435, where cox1-3 have lower ω values than other genes (Table 4). All the ω values were less than 1, indicating that the 12 genes of Mytilidae experienced constrained selection pressure to maintain their function. Members of Mytilidae show a tremendous range of ecological adaptions. To examine whether heterogeneous selective pressures act on the branches living in different environments (freshwater, deep-sea, and shallow sea), the Three-ratios model analysis was implemented. The likelihood ratio tests showed that the Three-ratios models have significantly better fit than the null models at cox1, atp6, cob, nad2, and nad5 (Table 4), suggesting divergence in selective pressure among the branches. In deep-sea branches, the ω values of those genes excepting cox1 are higher than those of other branches, suggesting those genes experienced relaxation of purifying selection. Relaxation of purifying selection in deep-sea branches has been found in many studies including deep-sea sea cucumbers and Boudemos sp. (Calamyzinae) [77, 80]. The relaxed purifying selection may be beneficial for deep-sea species to adapt to the reduction of oxygen levels and metabolic rates in extreme environments. In freshwater branches, only the ω value of atp6 was higher than that of shallow-sea branches, but still lower than the ω value of deep-sea branches. To identify whether positive selection acts on a few sites in freshwater branches or deep-sea branches, the branch-site model analysis was carried out. In deep-sea branches, although several sites of the genes (atp6, cob, nad2, nad4, nad5, and nad6) were recognized as positive sites according to BEB analysis (> 95%), the p-values of likelihood ratio tests were > 0.05 (Table S1). In freshwater branches, sites of nad2, nad4, and nad5 were identified as positive sites with BEB analysis (> 95%), however, only the p-value of nad4 was significant, which means nad4 may contribute to the adaptation of L. fortunei in freshwater (Table 5). Successful adaption to the freshwater environment may have required increased demand for energy involved in processes such as the osmotic balance [21]. NADH dehydrogenase, the largest and the most complicated enzyme of the respiratory chain, receives electrons from the oxidation of NADH and provides electrons for reduction of quinone to quinol [81]. nad4 together with nad2 and nad5 were considered to be the actual proton pumping devices as they showed homology with a class of Na + / H + antiporters [82]. Mutation in the members of NADH dehydrogenase would change the metabolic capacity which may further affect the fitness of an organism. To explore the possible effects of positive selection sites on nad4, the protein model was generated using the E. coli structure as a template. Most of the positive sites were directly located in the TMα7a which plays the most important role in the transportation of hydrogen ion (Fig. 6a). A positive site was found near the end of TMα9, which is adjacent to a positive site located in TMα7a. Intriguingly, both positive sites are polar amino acids, and these substitutions could change the environment between TMα7a and TMα9 (Fig. 6b) [21, 83]. This possible interaction was similar to a previous study of nad2 in freshwater dolphins [21]. We speculated that the mutations in NADH dehydrogenase may contribute to the survival and/or thriving of these species in freshwater.
Here, the mitochondrial genomes of three marine mussels (Xenostrobus securis, Bathymodiolus puteoserpentis, and Gigantidas vrijenhoeki) were assembled using the sequences deposited in NCBI. We annotated atp8 in the sequences that we assembled and the sequences lacking atp8. The newly annotated atp8 sequences all have one predicted transmembrane domain, a similar hydropathy profile, as well as the C-terminal region with positively charged amino acids. Our results supported that atp8 may not be missing in the Mytilidae. Furthermore, we reconstructed the phylogenetic trees of Mytilidae and carried out positive selection analysis. The results showed that the deep-sea bathymodiolines experienced more relaxed evolutionary constraints. And signatures of positive selection were detected in nad4 of Limnoperna fortunei, which may contribute to the survival and/or thriving of this species in freshwater.
Additional file 1: TableS1. Branch-site model analyses in deep sea branches | true | true | true |
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PMC9628239 | Jianwu Shi,Feng Qiao,Mei Ye,Ting Jiang,Jianni Liu,Mengya Zhang,Gangcai Xie,Kin Lam Fok,Xiaofeng Li,Hao Chen | CSE1L / CAS regulates cell proliferation through CDK signalling in mouse spermatogenesis | 13-09-2022 | CSE1L / CAS regulates cell proliferation through CDK signalling in mouse spermatogenesis
To the editor The cellular apoptosis susceptibility gene (CAS) (also named chromosomal segregation 1 like [CSE1L] and exportin‐2) has been found to play crucial roles in cell proliferation/apoptosis and progression of various cancers. While the functions of CAS in reproduction have not been well understood, previous studies in human trophoblast cells and seminoma showed that CAS is involved in cell proliferation and mitosis. , Our previous study demonstrated the expression of CAS in human testis and testicular cancers. We, therefore, speculate that CAS may exert its effect on spermatogenesis. The process of spermatogenesis is complicated and tightly regulated. The sequential differentiating germ cells were produced during the mouse testicular development. Undifferentiated spermatogonia begin to differentiate 4 days postpartum (dpp). The spermatocytes come up in 10dpp, and the meiosis was accomplished at 20–21dpp. The spermatozoa was found in the 35dpp testis. The mice are sexual maturation at 56dpp. Therefore, we collected several key time points of the spermatogonia, spermatocytes, round spermatids, and spermatozoa to illustrate the expression profile of CAS during mouse testicular development. qPCR results showed that CAS mRNA was expressed at all stages of testicular development from 7 to 56 days postpartum (dpp) in the development‐dependent manner (Figure 1A). Similarly, the protein expression of CAS was also increased during the mouse testicular development (Figure 1B). To further determine the locations of CAS in the testes, the immunofluorescent staining was carried on at the crucial mouse testicular development point. As shown in Figure 1C, the signals of CAS were detected in spermatogonia in day 7 testes. The stronger signal of CAS was observed in day 21 testes in the spermatocytes and enriched in the nuclear and cytoplasm after day 35 testes. Of note, the sperm were no obvious staining of CAS (Figure S1). Consistent with the results of Figure 1C, the CAS was highly expressed in spermatogonia, spermatocytes and round spermatid (Figure 1D,E) in the published scRNA‐seq dataset. Several studies have been demonstrated that CAS regulated the cell migration, proliferation and apoptosis in cancer cells. , However, the roles of CAS in germ cells proliferation are still unclear. To clarify this question, we employed a well‐established shRNA knockdown system used in our previous study , to knockdown the expression of CAS in spermatogonia cell line GC‐1 and spermatocyte cell line GC‐2 (Figure 1F and Figure S2A,B). The EdU incorporation assay revealed that knockdown of CAS resulted in a significant reduction of cell proliferation in GC‐1 and GC‐2 cells (Figure 1G,H). In addition, to elucidate the possible involvement of apoptosis elicited by interfering CAS, we determined the activity of caspase 3 and caspase 7 by the Caspase‐Glo 3/7 kit. As shown in Figure 1I, the activity of caspase 3/7 showed a remarkable increase in GC1‐shCAS and GC2‐shCAS cells compared with that of the GC1‐shNC and GC2‐shNC control group, suggesting that the knockdown of CAS was able to induce the cell apoptosis both in GC‐1 and GC‐2 cells. Our previous study has found the cell cycle arrest in the CAS knockdown breast cancer cells, with the observation that knockdown of CAS inhibited proliferation and induced apoptosis in GC‐1 and GC‐2 cells, we further explored the possible mechanisms underlying this phenomenon. Two markers of cell cycle CDK6 and CDK2, were significantly decreased after CAS knockdown in GC‐1 cells, while the CDK6, cyclin D3 and CDK2 were reduced after CAS knockdown in GC‐2 cells by western blot (Figure 1J and Figure S2C,D). Similar to the previous study, the migration ability of GC‐1 and GC‐2 was also blunted in the CAS knockdown group compared to the control group (Figure S3). In summary, the present study for the first time demonstrated the expression patterns of CAS during mouse testicular development. Knockdown of CAS resulted in the inhibition of proliferation and migration of immortalized spermatogonia and spermatocytes, GC‐1 and GC‐2 cells, suggesting its potential role during spermatogenesis. Cyclin‐dependent kinases (CDKs) are the central regulators in the cell cycle. Several studies including ourselves demonstrated that CAS was involved in the proliferation/apoptosis via regulating factors of cell cycle. , , Interestingly, CDK6 and CDK2, but not cyclin D1 and B1 in breast cancer cell, were significantly inhibited in the CAS knockdown GC‐2 cells. Of note, CDK2 was reported to be essential for the first meiotic division in germ cells, together with our results, indicating that the role of CAS in regulating cell proliferation and apoptosis is tissue/cell‐specific. It should be noted, however, that the mouse sperms showed weak immuno‐signal of CAS (Figure S1), indicating the turnover of CAS during spermiogenesis. In addition, the conditional knockout mouse models were warranted to be established due to the embryo lethal of knockout of CAS. Further investigation of these is undertaken in the authors' laboratory. Taken together, our results provide a novel role of CAS in spermatogenesis and a potential pathogenesis and diagnosis marker for male infertility.
Hao Chen conceived and supervised the project. Jianwu shi, Feng Qiao, Mei Ye, Jianni Liu, Ting Jiang, Mengya Zhang and Gangcai Xie performed the experiments and analysed the data. Jianwu Shi, Feng Qiao, Xiaofeng Li and Kin Lam Fok wrote the manuscript. Hao Chen, Kin Lam Fok and Xiaofeng Li revised the manuscript.
The authors declare that there are no conflicts of interest.
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PMC9628617 | The 48th Annual Meeting of the European Society for Blood and Marrow Transplantation: Physicians – Poster Session (P001 – P578) | 02-11-2022 | The 48th Annual Meeting of the European Society for Blood and Marrow Transplantation: Physicians – Poster Session (P001 – P578)
19 – 23 March, 2022 ● Virtual Meeting Copyright: Modified and published with permission from https://www.ebmt.org/annual-meeting Sponsorship Statement: Publication of this supplement is sponsored by the European Society for Blood and Marrow Transplantation. All content was reviewed and approved by the EBMT Committee, which held full responsibility for the abstract selections.
Background: Post-transplant cyclophosphamide (PTCy)-based anti-GVHD prophylaxis pioneered in haploidentical (Haplo) transplantation is being increasingly used in other transplantation settings. Both Haplo and mismatched unrelated donor (MMUD) transplantation are valid options in patients with ALL in the absence of a MRD. Methods: The study aim was to compare the outcomes of adult patients with ALL in CR who underwent Haplo versus 9/10 MMUD transplantation with PTCy in 2010-2020. Multivariate analysis adjusting for potential confounding factors was performed using a Cox’s proportional-hazards regression model for main outcomes. Results: This study included 781 pts: Haplo-678, MMUD-103. Median follow-up was 24 (range, 11.3-45.6) and 19 (5.3-39.0) months, respectively (p = 0.61). Median age was 38 (18-75) and 40 (19-73) years (p = 0.51) and 65% and 66% were male, respectively (p = 0.76). Year of transplant was 2018 vs 2017 (p = 0.68). 39% and 43% were Ph-, 35% and 38% were Ph + , and 26% and 19% had T ALL, respectively (p = 0.3). Disease status at transplant was CR1 in 67% and 64%, and CR2 in 33% and 36% of the patients, respectively (p = 0.54). Conditioning was myeloablative in 70% and 69%, (p = 0.19) in Haplo and MMUD, respectively. Fewer Haplo than MMUD patients received TBI (48% vs 59%, respectively, p = 0.038) while more Haplo compared with MMUD were performed with a BM graft (37% vs 16%, p < 0.0001), respectively. The most frequent immunosuppression agents added to PTCy were MMF/cyclosporine A, and MMF/tacrolimus. ATG was administered in 8% and 21% of the transplants, respectively (p < 0.0001). Engraftment (day 60) was 96.5% vs 97.1% (p = 0.77) in Haplo vs MMUD, respectively. Incidence of day 180 aGVHD grade II-IV and III-IV was 31.6% vs 21.4% (p = 0.034), and 11.1% vs 8.2% (p = 0. 34), respectively. 2-y cGVHD and extensive cGVHD were 33% vs 36% (p = 0.69), and 12% vs 10% (p = 0.55) in Haplo vs MMUD, respectively. Main causes of death were leukemia (42% vs 57%), infection (30% vs 13%), and GVHD (14% vs 7.7%). 2y relapse incidence (RI), NRM, LFS, OS and GRFS were 26% vs 31% (p = 0.18), 22% vs 13% (p = 0.1), 52% vs 55% (p = 0.45), 61% vs 70% (p = 0.45), and 42% vs 45% (p = 0.60), for Haplo and MMUD, respectively. On multivariate analysis, NRM and RI were similar between Haplo and MMUD, HR = 1.45 (95% CI 0.8-2.6, p = 0.21) and HR = 0.8 (95% CI 0.5-1.3, p = 0.38), respectively. LFS, OS and GRFS were comparable, HR = 1.0 (95% CI 0.7-1.5, p = 0.8), HR = 1.2 (95% CI 0.8-1.8, p = 0.46) and HR = 1.0 (95% CI 0.8-1.5, p = 0.7), respectively. aGVHD was significantly higher with Haplo, HR = 1.7 (95% CI 1-2.8, p = 0.023), while aGVHD grade III-IV and cGVHD did not differ significantly between the 2 transplant groups. Additional prognostic factors for HSCT outcomes were disease status for all parameters excluding GVHD; age for NRM, LFS and OS. Ph+ was a favorable predictive factor for RI and OS. RI was higher with reduced intensity conditioning while OS was lower with PB grafts. A female to male donor/recipient combination was associated with a higher incidence of cGVHD. Conclusions: Outcomes of Haplo and MMUD transplants for pts with ALL in CR are similar, apart from a higher incidence of aGVHD with Haplo transplants. Disclosure: Nothing to declare
Background: During the last decades, outcomes of patients with AML, receiving an alloSCT in complete remission (CR) have improved significantly, whereas the prognosis of patients transplanted with r/r AML remained dismal. Sequential conditioning regimens (e.g. FLAMSA-based or high-dose melphalan based) might improve outcomes of patients with r/r AML, particular in younger patients. A recent retrospective analysis showed promising survival data in younger patients conditioned with high dose melphalan, followed by 8 Gy TBI and fludarabine, while for elderly patients this intense therapy was associated with significant toxicities. Methods: In our retrospective study 99 patients, aged > 55 years, with r/r AML transplanted at the BMT-center of University Hospital of Muenster between 2014 and 2021, were included. Median age was 67 years (range 55-76), the median HCT-CI-score was 2 (range 0-10). Thirteen patients were initially diagnosed with favorable risk, 37 patients with intermediate risk, and 49 patients with adverse risk AML (ELN 2017). At transplant, 65 patients had primary refractory AML, 15 patients had AML refractory to salvage treatment and 19 patients had untreated relapsed AML. Median bone marrow (BM) blast count prior to start of conditioning was 30% (range 5-90%). 75 patients received melphalan on day -11 at a dose of 100 mg/m² and 24 patients at a dose of 140 mg/m² followed by fludarabine (4 x 30 mg/m²) and busulfan (cumulative dose on 2 days of 6,4 mg/kg body weight) on days -5 to -1 prior to transplant. One patient received a BM graft, all other patients received peripheral stem cell grafts from matched-related donors (MRD, 16 patients), from matched-unrelated donors (MUD, 10/10 HLA-matched, 62 patients) or from mismatched-unrelated donors (MMUD, 9/10 matched, 21 patients). Median follow-up of surviving patients was 749 days after transplant. Results: Overall survival (OS) rates at 1 and 2 years were 51% (95% CI, 41-61%) and 42% (95% CI, 32-53%), respectively. Cumulative incidences (C.I.) of relapse and non-relapse mortality (NRM) at 1 and 2 years were 14% (95% CI, 8-23%), 19% (95% CI, 12-30%) and 35% (95% CI, 26-46%), and 37% (95% CI, 29-49%), respectively. The 2-year-OS of patients with primary refractory AML, secondary refractory AML or untreated relapse showed no significant difference, with 46% (95% CI, 33-59%), 37% (95% CI, 9-65%) and 28% (95% CI, 7-50%), respectively. Patients with low disease burden (<20% BM blasts prior start of conditioning) had a C.I. of relapse of 6% (95% CI, 2-23%), compared to 28% (95% CI, 16-50%) and 32% (95% CI, 17-59%) for patients with 20-50% or >50% BM blasts (p .04). Furthermore, patients transplanted from a MMUD donor had a significantly higher risk of NRM of 70% (95% CI, 53-95%), as compared to patients transplanted from a MUD (30%, 95% CI, 21-45%) or MRD (24%, 95% CI, 9-63%) (p .001). Conclusions: Our data suggest that high dose melphalan-based sequential conditioning combined with busulfan and fludarabine followed by alloSCT is a feasible treatment option in elderly patients with r/r AML, which allows long term survival of >40% in this high-risk patient group. Disclosure: Nothing to declare.
Background: The best stem cell source for T-cell replete HLA-haploidentical transplantation with post-transplant cyclophosphamide (PTCy) remains to be determined. In patients with active acute myeloid leukemia (AML) at transplantation, one could speculate that the use of peripheral blood stem cells (PBSC) versus bone marrow (BM) could be associated with higher graft-versus-leukemia effects. These considerations prompted us to perform a retrospective study within the EBMT registry to assess this question. Methods: Inclusion criteria included adult AML patients with primary refractory or relapsed AML, first allogeneic transplantation with an HLA-haploidentical donor with PTCy as graft-versus-host disease (GVHD) prophylaxis, transplantation between 2010 and 2020, and no in vivo T-cell depleted grafts. Primary endpoint was leukemia-free survival (LFS). Results: A total of 668 patients (249 BM and 419 PBSC recipients) met the inclusion criteria. This include 380 patients with primary refractory AML, 229 in first relapse and 59 in second relapse at transplantation. Median follow-up was 36 months. Median age was 57 years (IQR, 45-64 years). There was a statistical interaction between patient age and stem cell source on LFS (P < 0.01 for age < or > 55 years). The analyses were thus performed separately for patients < or > 55 years of age. In multivariate Cox models, among patients < 55 years (n = 301, 114 BM and 187 PBSC), the use of PBSC versus BM resulted in comparable relapse incidence (HR = 0.85, P = 0.34), nonrelapse mortality (HR = 0.91, P = 0.8), LFS (HR = 0.82, P = 0.2; Figure 1) and overall survival (OS; HR = 0.81, P = 0.2) in multivariate Cox models. The use of PBSC was associated with higher incidence of grade II-IV acute GVHD (HR = 2.02, P = 0.01) and no significant difference in incidence of chronic GVHD (HR = 1.42, P = 0.3). In contrast, in patients > 55 years of age (n = 367, 135 BM and 232 PBSC), the use of PBSC versus BM was associated with higher nonrelapse mortality (HR = 1.64, P = 0.015), comparable relapse incidence (HR = 1.17, P = 0.43), lower LFS (HR = 1.39, P = 0.02, Figure 1) and lower OS (HR = 1.35, P = 0.03). Incidences of grade II-IV acute (HR = 1.55, P = 0.13) and chronic (HR = 1.2, P = 0.55) GVHD were comparable in the 2 groups of patients. Conclusions: Our data suggest that in patients > 55 years of age with active AML at HLA-haploidentical transplantation, the use of BM instead of PBSC as stem cell source results in lower nonrelapse mortality and better LFS and OS. In contrast among younger patients, the use of PBSC results in at least comparable LFS and OS. Disclosure: The authors have no COI to disclose
Background: The changes of genetic information play an important role in the pathogenesis and recurrence of acute lymphoblastic leukemia (ALL), but there is no consistent conclusion about the impact of molecular genetic changes on the diagnosis and prognosis of the disease. The purpose of our study was to investigate the frequency spectrum of gene mutation and its prognostic significance in combination with minimal residual disease (MRD) and hematopoietic stem cell transplantation (HSCT) in adolescents and adults ALL patients aged ≥15 years old. Methods: The basic characteristics, cytogenetics, molecular genetics, MRD, treatment regimen and survival outcome of ALL patients who were first diagnosed in Shandong Provincial Hospital and Yantai Yuhuangding Hospital from January 1, 2014 to May 1, 2021 were collected. Correlation analysis and survival analysis were performed by R and SPSS25 software, respectively. Results: A total of 353 patients were included in this study, of which 90.6% of the 128 patients with next-generation sequencing (NGS) results had at least one mutation, and 66.41% of the patients had polygenic (≥ 2) mutations. NOTCH1, PHF6, RUNX1, JAK3 and PTEN were the most common mutations in T-ALL, while FAT1, TET2, NARS, KMT2D, FLT3 and RELN were the most common in B-ALL. The incidence of NOTCH1, JAK3, PTEN, PHF6 and JAK1 in T-ALL is higher than that in B-ALL. Correlation analysis revealed common mutation patterns, which were significantly different between T-ALL and B-ALL. Then the prognostic factors of 92 patients with B-ALL were analyzed, including sex, age, white blood cell count, Ph chromosome status, HSCT, hepatitis B virus infection status, MRD level and bone marrow remission status after induction chemotherapy, and genes with mutation frequency ≥ 6. Univariate analysis showed that FLT3 mutation (P = 0.048), TP53 mutation (P = 0.088) and RELN mutation (P = 0.037) were adverse factors affecting the overall survival (OS) of B-ALL patients. Patients with negative MRD after induction therapy (P = 0.007) and receive HSCT (P = 0.003) had better OS. Multivariate analysis revealed that MRD ≥ 1% after induction chemotherapy (P = 0.007), RELN mutation (P = 0.022) and TP53 mutation (P = 0.012) were independent risk factors for OS. Patients who undergo HSCT tended to have better OS, but there was no statistical significance in multivariate analysis (P = 0.079). NOTCH1 mutation (P = 0.018) and recurrence (P = 0.000) were independent adverse prognostic factors of event-free survival. In addition, our study also found that among the 52 patients who achieved negative MRD (MRD < 0.01%) after the first induction chemotherapy, there was no significant difference in OS between the transplantation group (n = 26) and the non-transplantation group (n = 26), and the average age of the non-transplantation group was significantly higher than that of the transplantation group (41.16 vs 32.58 years old, P = 0.049). Conclusions: The distribution of gene mutations and the co-occurrence and repulsion of mutant genes in patients with ALL are closely related to the immunophenotype of patients. RELN and TP53 mutations are significantly associated with poor prognosis in patients with ALL. Patients with high sensitivity to chemotherapy do not seem to benefit from HSCT, which may be associated with high transplant-related complications and mortality and need to be confirmed in large prospective studies. Disclosure: The authors declare that they have no competing interests. Funding: This study was funded by the National Natural Science Foundation (grant nos. 82070203, 81770210, 81473486 and 81270598), the Key Research and Development Program of Shandong Province (grant no. 2018CXGC1213), the Technology Development Projects of Shandong Province (grant no. 2017GSF18189), the Translational Research Grant of NCRCH (grant nos. 2021WWB02 and 2020ZKMB01), the Technology Projects of Jinan (grant nos. 201704092 and 202019044), the Taishan Scholars Program of Shandong Province, Shandong Provincial Engineering Research Center of Lymphoma, and Academic Promotion Programme of Shandong First Medical University (grant no. 2019QL018).
Background: Stem cell transplantation (SCT) is a potentially curative post-remission therapy for intermediate-risk acute myeloid leukemia (AML) patients. For patients in first remission (CR1) with negative measurable residual disease (MRD) and without a HLA-matched donor, both autologous SCT (ASCT) and haploidentical donor SCT (haplo-SCT) were acceptable options, but it is controversial that which one is preferred. Methods: A retrospective study was conducted in 8 Chinese centers. The inclusion criteria were: 1) adult patients >18 years old; 2) diagnosis as AML with intermediate-risk according to ELN 2017; 3) ASCT or haplo-SCT underwent between 2010-2019; 4) in CR1 and MRD negative before transplant. The Primary endpoint was overall survival (OS). Secondary endpoints were progression-free survival (PFS), cumulative incidence of relapse (CIR), treatment-related mortality (TRM), and graft-versus-host disease-free and relapse-free survival (GRFS). Results: Totally 299 patients were enrolled in this study, including 97 recepients after ASCT and 202 recipients after haplo-SCT (Table 1). The median follow-up was 28 months in ASCT group versus 35 months in haplo-SCT group. Compared to haplo-SCT, patients after ASCT had increased 3-year CIR (27.0% ± 0.2% versus 13.5% ± 0.1%, p = 0.004) but reduced 3-year TRM (3.5% ± 0.0% versus 12.0% ± 0.0%, p = 0.013), which led to similar 3-year OS (80.8% ± 4.3% versus 79.2% ± 3.1%, p = 0.796) and PFS (69.5% ± 5.0% versus 73.7% ± 3.3%, p = 0.504). Moreover, the 3-year GRFS was remarkable better in ASCT group (69.5% ± 5.0% versus 55.9% ± 3.6%, p = 0.009) (Figure 1), which implied a survival with superior quality of life (QoL). In multivariate analysis, haplo-SCT independently related to an improved CIR, while increased TRM and reduced GRFS. Additionally, age more than 50 was associated with the worse OS, CIR and GRFS. Table 1 Characteristics of patients. Conclusions: We concluded that both ASCT and haplo-SCT were applicable for patients with intermediate-risk AML in MRD-negative CR1, but the absence of GVHD might potentially favor the QoL for patients receiving ASCT. Randomized trials are needed to confirm our conclusion. Disclosure: Nothing to disclose
Background: Transforming growth factor β1 (TGFβ1) is a pleiotropic regulatory cytokine secreted after hematopoietic stem cell transplantation (HSCT) by both donor T-cells and platelets and by recipient endothelial, connective and epithelial cells. TGFB1 − 1347C > T variant affects TGFB1 transcription and plasma levels and has been associated after HSCT with worse overall survival (OS) or graft versus host disease (GvHD).1,2 References: 1.-Arrieta-Bolaños E, et al. Polymorphism in TGFB1 is associated with worse non-relapse mortality and overall survival after stem cell transplantation with unrelated donors. Haematologica. 2016;101:382-90. 2.- Kövy P, et al. Investigation of TGFB1 -1347C > T variant as a biomarker after allogeneic hematopoietic stem cell transplantation. Bone Marrow Transplant. 2020;55:215-223. Methods: The study included 65 patients who underwent HSCT for acute myeloid leukemia (AML) disease. HSCT were performed between April 2013 and January 2021 at our single centre. OS was calculated from the day of HSCT until death for any cause or last follow up. Genomic DNA from donor and patient pairs was extracted from pre-transplant peripheral blood. TGFB1 − 1347C > T variant was determined by high resolution melting analysis on a LightCycler® 480 instrument (Roche Diagnostics) using an original primer design (Forward: 5′-CATGGGAGGTGCTCAGTAAA-3′; Reverse: 5′-AGGCTGGGAAACAAGGTAGG-3′). The effect of TGFB1 − 1347C > T variant was assessed in all three models: genotypic (CC vs. CT vs. TT), recessive (CC&CT vs. TT) and dominant (CC vs. CT&TT). The association of the clinical characteristics with the genetic findings was analyzed with the SPSS statistical program (v.20.0) and P values <0.05 were considered as statistically significant. Results: The median OS for patients after HSCT was 13 months (range, 0 – 78 months). Patient −1347C > T frequencies were CC: 24 (39%), CT: 27 (43%), TT: 11 (18%) [patient DNA was not available in 3 cases] and donor −1347C > T frequencies were CC: 31 (49%), CT: 25 (40%), TT: 7 (11%) [donor DNA was not available in 2 cases]. In the overall series, patients with TT donor showed compared to CT&CC genotypes a trend towards a higher incidence of acute GvHD grade III-IV (29% vs. 4%, P = 0.084). In addition, patients with TT genotype and TT donor had a significantly higher frequency of acute GvHD grade III-IV than the remaining patients (40% vs. 4%, P = 0.013). Interestingly, in the non-myeloablative subgroup (n = 47), patients with TT genotype had a lower OS compared to CC and CT genotypes (median, 20 vs. 30 vs. 55 months, P = 0.026; Fig. 1). On the other hand, in myeloablative subgroup (n = 18), patients with CC donor had a lower relapse-free survival compared to CT&TT genotypes (median, 25 vs. 50 months, P = 0.046). Conclusions: In our series of AML with HSCT analyzed, TGFB1 − 1347C > T variant was screened using a rapid and novel procedure and was associated with patients′ clinical outcome, especially in non-myeloablative conditioning. If these results were confirmed in a larger series, the analysis of that polymorphism could be useful in HSCT. Disclosure: Nothing to declare
Background: The association of GVHD and GvL after allogeneic SCT is well-established but has not been confirmed in recent years with the introduction of new transplantation techniques. Post-transplant cyclophosphamide (PTCy) has been used for GVHD prevention in haplo-identical transplantation and more recently also in HLA- matched SCT. We have shown that there was no association between GVHD and GVL in the haplo-identical setting with PTCy but it was not determined if this is related to the PTCy effect or to the haplo-identical setting itself. Methods: We assessed the impact of acute and chronic GVHD on SCT outcomes in patients with AML following SCT with conventional GVHD prophylaxis or PTCy by using Cox proportional-hazard analysis with acute and chronic GVHD as time- dependent variables. Results: 12,653 patients received a first allogeneic SCT between the years 2010-2019, from HLA- matched siblings (n = 6726, 53%) or 10/10 matched- unrelated donors (n = 5927, 47%), using standard GVHD prevention regimens. The median age was 52 years (range, 18-80). Status at SCT was CR1 (n = 10478, 83%) or CR2 (n = 2175, 17%). The conditioning regimen was RIC (n = 5711, 45%) or MAC (n = 6942, 55%). The incidence of acute GVHD grade II-IV and III-IV at 180 days was 23.8% and 7.5%, respectively. The incidence of chronic GVHD and extensive chronic GVHD at 2 years was 37% and 16.3%, respectively. Acute GVHD grade II was associated with lower relapse risk (HR 0.85, P = 0.002), higher risk of non-relapse mortality (NRM) (HR 1.5, P < 0.0001) and lower overall-survival (OS) (HR 1.49, P < 0.0001) in comparison with no or grade I GVHD. Similarly, acute GVHD grade III-IV was associated with lower relapse (HR 0.76, P = 0.003), higher NRM (HR 6.21, P < 0.0001) and lower OS (HR 6.1, P < 0.0001). Extensive chronic GVHD was associated with lower relapse (HR 0.69, P < 0.0001), higher NRM (HR 2.83, p < 0.0001) and lower OS (HR 2.74, P < 0.0001). Limited chronic GVHD was not associated with any of these outcomes.PTCy was given to 508 patients after SCT from HLA- matched siblings (n = 234, 46%) or 10/10 matched- unrelated donors (n = 274, 54%). The median age was 48.5 years (range, 18-72). Status at SCT was CR1 (n = 437, 86%) or CR2 (n = 71, 14%). The conditioning regimen was RIC (n = 215, 42%) or MAC (n = 293, 58%). The incidence of acute GVHD grade II-IV and III-IV at 180 days was 22.8% and 6.2%, respectively. The incidences of chronic GVHD and extensive chronic GVHD at 2 years was 35.5% and 17.7%, respectively.Acute GVHD II-IV was associated with a non-statistically different risk of relapse (HR 1.37, P = 0.15), higher risk of NRM (HR 3.34, P = 0.0005) and lower OS (HR 1.92, P = 0.001). Chronic GVHD was not associated with risks of relapse (HR 0.99, P = 0.98), NRM (HR 1.11, P = 0.83) or OS (HR 0.73, P = 0.19). The relatively low number of GVHD events in the PTCy setting did not allow differentiation between acute and chronic GVHD grading. Conclusions: GVHD and GVL are strongly associated in contemporary allogeneic SCT. However, in the PTCy setting these two effects can be separated and GVHD is not associated with reduced risk of post-transplant relapse. Disclosure: Nothing to declare
Background: Allogeneic hematopoietic stem cell transplantation (alloHSCT) from a haploidentical related donor (haploHSCT) is an important alternative for patients with acute myeloid leukemia (AML) in the absence of an HLA-matched donor. A substantial number of studies were published comparing matched unrelated donor (MUD) HSCT with conventional GVHD prophylaxis to haploHSCT with posttransplantation cyclophosphamide (PTCY). Several recent studies comparing both haplo and MUD HSCT with PTCY identified better outcomes after MUD HSCT. Our study aimed to compare the results of MUD and haploidentical alloHSCT both performed with PTCY prophylaxis. Methods: A total of 182 adult patients with de novo AML in CR1/CR2, who underwent alloHSCT at RM Gorbacheva Research Institute (CIC 725) between 2013-2021, were included. HaploHSCT was performed in 47 patients (haplo group), MUD alloHSCT was performed in 135 patients (MUD group). Median age was 31 (range 18-68) and 33 (range 18-59) years, 22 (46%) and 60 (44.4%) patients were male respectively. Most patients in both groups had intermediate ELN risk: 36 (76.6%) and 101 (74.8%) respectively. The ratio of patients in CR1 and CR2 prior to HSCT was comparable: 27 (57.4%) and 20 (42.6%) in haplo group, 94 (69,6%) and 41 (30.4%) in MUD group. Most patients received conditioning regimens with reduced intensity: 41 (87.2%) and 112 (83%) respectively. All patients received PTCY-based GVHD prophylaxis. Groups were statistically different only by graft source due to much more frequent use of bone marrow in haplo group: 23 (48.9%) vs 8 (5.93%) in MUD group (p < 0.001). Results: Median follow-up after alloHSCT in survivors was 26.6 months. Engraftment was achieved by 74,5% of patients in haplo group and 97% in MUD group (p < 0.001). Two-year OS was 55% and 82% (p < 0.001), EFS was 54% and 80% (p < 0.0001), GRFS was 38% and 53% (p = 0.021) in haplo and MUD groups respectively. The cumulative incidence of NRM at 2 years was 37% and 14% (p = 0.0014), RI was 19% and 7% (p = 0.014). Day-100 cumulative incidence of grade II-IV aGVHD was 27.7% and 11.1% (p = 0.013), with no significant difference for grade III-IV aGVHD (p = 0.427). In a more detailed study of the haplo group, it was found that the use of bone marrow as a graft source reduced EFS by 22% (p = 0.036) and increased RI by 27% (p = 0.049). In the multivariate analysis haploidentical donors had significantly reduced EFS (p = 0.026), status of CR2 significantly reduced GRFS (p = 0.029), bone marrow as a graft source did not significantly impact OS, EFS, GRFS. Conclusions: In this study we demonstrated the significant superiority of MUD HSCT over haploHSCT for de novo AML in adult patients when both are performed with PTCY prophylaxis. At the same time, haploHSCT remains an important and often the only option for patients without an available HLA-matched donor. Due to the revealed possible difference in EFS and RI between graft sources in the haplo group, further research is needed to identify the best graft source for replete haploHSCT. A significant advantage in GRFS has been demonstrated in patients in CR1, which allows for early consideration of the HSCT in patients with primary AML. Disclosure: Nothing to declare.
Background: Acute megakaryoblastic leukemia (AMKL) is a rare subtype of acute myeloid leukemia, arising from megakaryocytes. As survival rates are extremely poor due to higher rates of disease relapse (RI), consolidation treatment with an allogeneic hematopoietic cell transplantation (HCT) might offer the best chance of cure for patients in remission. Methods: We report on a retrospective analysis on adults affected by AMKL in first complete remission (CR1) that received a first allogeneic HCT, both from unrelated donors (UD) or HLA-matched sibling donors (MSD). Graft source was mainly peripheral blood (87%), or bone marrow (13%). All patients underwent transplantation between January 2000 and December 2020 and their data were reported to the ALWP of the EBMT. Results: A total of 201 patients (median age 48 years, 117 male and 84 female) were included in the analysis. Median follow-up for the entire population was 5.2 years. Donors were HLA-matched siblings (n = 98), 10/10 UD (n = 48), 9/10 UD (n = 17) and 38 were missing data on UD HLA-compatibility. Patients transplanted from UD were older (p < 0.01), more often presented the combination female donor to male recipient (p < 0.01) and more frequently received a reduced-intensity-conditioning (p < 0.01). Cytogenetic risk was intermediate in 54 patients, poor in 54 and not available in 93. Ninety-eight % of patients engrafted. The estimated 5-year rates of overall survival (OS) were 41.4% (95% CI 33.6 - 48.9), leukemia-free survival (LFS) 35.6% (95% CI 28.3-43), RI 40.5% (95% CI 33.2-47.7) and non-relapse mortality (NRM) 23.9% (95% CI 17.7-30.6) (Fig.1). Five-year GVHD-free, relapse-free survival (GRFS) was 26.8% (95% CI 20.3 - 33.7) overall. Global incidence of grade III-IV acute graft-versus host-disease (GVHD) at day-180 was 8.8% (95% CI 5.3-13.3), while 2-year extensive chronic GVHD incidence was 17.5% (95% CI 12.1-23.7). In multivariate model, patients transplanted from UD tended to have a trend towards higher rates of NRM (hazard ratio [HR] 1.90, 95% CI 0.99-3.63, p = .0053). Patient age (as incremental value, per 10 years), was associated with higher rates of NRM (HR 1.30, 95% CI 1.03-1.66, p = .03) and worse OS (HR 1.21, 95% CI 1.05-1.40, p = .01), as well as adverse cytogenetics with RI (HR 2.17, 95% CI 1.30-3.61, p = .003) and LFS (HR 1.58, 95% CI 1.04-2.40, p = .03), in multivariate analysis. Figure 1. Relapse incidence (RI), non-relapse mortality (NRM), leukemia-free survival (LFS) and overall survival (OS) for patients transplanted from HLA-matched siblings (blue line) and from UD (red line). Conclusions: Allogeneic HCT could be curative in a proportion of adult AMKL patients in CR after induction treatment. Patients transplanted from UD had increased rates of NRM. Disclosure: Nothing to declare
Background: For adult patients with acute myelogenous leukemia (AML), numerous patient and disease prognostic factors have been shown to impact the outcome following stem cell transplantation. In the past decade, several studies have shown the importance of the evaluation of minimal residual disease (MRD); undetectable MRD (uMRD) in particular at transplant, has been recognized as the most important predictor of favorable outcome, possibly erasing previously recognized poor prognostic factors. At the present time, there is no consensus on the consolidation therapy to offer to intermediate-risk patients in CR. The recent GIMEMA AML 1310 trial of risk adapted MRD directed therapy has shown equivalent outcome for AML patients autografted (ASCT) in uMRD-CR1 or allografted in the case of persisting MRD positivity. Methods: Using the EBMT registry, we collected data from 344 autografted patients and 200 patients who received a T-cell replete haploidentical (Haplo) transplant from January 2010 to December 2019. Results: The distribution of molecular markers was not even: for autografted patients, a NPM1 mutation was present in 63% and FLT3-ITD in 24% while it was 46% and 50% for Haplo (p < 0.0002 for each). The stem cell source was peripheral blood in 97% for ASCT and 64% for Haplo (p < 0.0001). In the autografted population the myeloablative conditioning (MAC) consisted essentially (69%) of the combination of busulfan + cyclophosphamide or busulfan + high-dose melphalan. For Haplo the conditioning regimen was MAC in 48% and reduced intensity in 52%. All Haplo recipients received posttransplant cyclophosphamide (PTCY) for GVHD prophylaxis. The outcome at 3 years posttransplant is indicated below (univariate p): Relapse NRM LFS OS Auto 50.5%[44.6-56.1] 7.1%[4.5-10.4] 50.3%[44.6-55.7] 67%[61.4-71.9] Haplo 14.5%[9.3-20.7] 23.6%[17.5-30.2] 65.2%[57.6-71.8] 72.6%[65.3-78.6] P value 0.001 0.001 0.005 0.5 Post Haplo, the rates of acute GVHD grades III-IV, chronic GVHD, and severe chronic GVHD at 3 years were 7%, 42%, and 17% respectively. The GVHD/relapse-free survival (GRFS) was 54%. 31% of autografted patients and 6% of Haplo received a second transplant. On multivariate analysis, Haplo was significantly associated with a higher NRM, a lower RI, and a higher LFS. Overall survival was not significantly different from ASCT. Other prognostic factors were patient age, and the presence of NPM1 and FLT3ITD mutations. No center effect was observed. Conclusions: For adult patients with AML with uMRD-CR1, Haplo with PTCY resulted in a superior LFS of 65% versus 50% at 3 years and a GRFS of 54%. By intention to treat there was no difference for OS. Disclosure: No conflict of interest
Background: Leukemic stem cells (LSCs), which play a crucial role in pediatric AML, are distinguished by a specific mRNA-expression signature. Recently, a six-gene leukemic stem cell score (pLCS6) value have been shown to be an effective prognostic marker independent of minimal residual disease (MRD) (Abdelrahman H. Elsayed et al., 2020). However, although initial high-pLCS6 status is associated with worse prognosis even in subsequent alloHSCT recipients, there is yet no data on pre-transplant pLCS6 measurement prognostic value. Methods: Gene expression profiling by RT-PCR was performed in 50 AML patients receiving alloHSCT in different disease status. Complete remission 1 (CR1) or CR2 was documented in 74% (n = 37) of children, 26% (n = 13) of patients had active disease before alloHSCT. The retrospective study cohort included 37 children with AML at CR1 or CR2 receiving alloHSCT from matched related (n = 3, 8%), matched unrelated (n = 15, 41%) or haploidentical donor (n = 19, 51%) in RM Gorbacheva Research Institute during 2014-2021 period. The median age was 6(1-18) years. In 28 (76%) cases myeloablative and in 9 (24%) non-myeloablative conditioning regimen was used. In 78% (n = 29) of cases the GVHD prophylaxis regimen included post-transplant cyclophosphamide. The gene expression profiling was performed via RT-PCR for DNMT3B, GPR56, CD34, SOCS2, SPINK2, IL2RA, FAM30A and ABL genes with subsequent pediatric six-gene leukemic stem cell score (pLSC6) calculation by previously described equation: (DNMT3b x 0.189) + (GPR56 х 0.054) + (СD34 х 0.0171) + (SOCS2 x 0.141) + (SPINK2 x 0.109) + (FAM30A x 0.0516).1 If an appropriate sample was available, the post-transplant pLSC6 value was also evaluated. After the median pLSC6 was determined all patients were divided into low-pLSC6 and high-pLSC6 groups. Results: A total of 18/37 (49%) patients had high-pLSC6 pre-transplant score value. Only 6/18 of high-pLSC6 patients with CR1-2 were MRD-positive. The post-transplant pLSC6 value was measured in 14 patients, in 85% cases the pre-transplant high-pLSC6 values converted to low-pLSC6. The linear regression analysis including patients with pre-transplant response as well as patients with active disease showed no association between blast count/MRD and pLSC6 values (OR 1.002; 95% CI: 0.979, 1.025). The 1-year RFS in CR patients was not significantly different between low-pLSC6 (78.9%) and high-pLSC6 (66.7%) patients (p = 0.62). However, while none of the clinical factors were significant in the multifactor analysis, the early relapse rate in CR patients was significantly higher in high-pLSC6 subgroup compared to low-pLSC6 (22% and 0%, accordingly; p = 0.03). Conclusions: Although current results do not support pLSC6 assay value as an indication for alloHSCT, there is still a tendency to worse prognosis in children with pre-transplant high-pLSC6 score in spite of the evidence of graft-versus-leukemia effect on LSCs. The pre-transplant high-pLSC6 status may, therefore, be a factor for pre-emptive post-transplant intervention in the future independent of blast count and MRD. As the effectiveness of this study is limited by its retrospective design, it warrants further research in a larger prospective cohort. Disclosure: The study was supported by Fund for promoting innovation «Fund-M», grant № 0059546.
Background: Early T-cell precursor lymphoblastic leukemia/lymphoma (ETP-ALL/LBL) is a hematological malignancy originating from immature T lymphoblastic cells with high rate of treatment resistance and poor long outcome. Hitherto, studies of survival outcomes in ETP-ALL/LBL were still controversial. Data of large cohort in ETP-ALL/LBL are still lacking. Methods: In this retrospective analysis, we performed a real-world multicenter study to explore the clinical characteristics and prognosis of adolescent and young adults (AYA) and older adult ETP leukemia/lymphoma. A total of 103 patients with ETP-ALL/LBL in five centers in China between January 2016 and February 2021 were included in this study. Outcomes were assessed in terms of overall survival (OS) and relapse-free survival (RFS). Results: The median age was 29 years (range, 15–70 years). Next-generation sequencing was performed in 94 patients and revealed that NOTCH1 (35.1%, 33 cases) was the most frequently mutated gene, followed by JAK3 (16.0%, 15 cases), PHF6 (13.80%, 13 cases) and EZH2 (11.70%, 11 cases). Complete remission (CR) was obtained in 74.2% (72/97) of patients, and 6 relapsed/refractory patients received a decitabine combined with AAG priming regimen as reinduction therapy with a CR rate of 50%. With a median follow-up of 18 months (0.5–60 months), the 2-year overall survival (OS) and relapse-free survival (RFS) rates for the entire cohort were 54% and 57.7%, respectively. Allogeneic stem cell transplantation (allo-SCT) was performed in 59.8% (58/97) of patients. Patients who experienced transplantation in CR had better OS than transplantation in NR (P = 0.029, HR: 0.3625, 95%CI 0.1005 to 1.308), while the RFS was no statistical significance. (P = 0.078, HR:0.4431, 95% CI 0.1340 to 1.465). Patients who were MRD negative at transplantation had better OS and RFS than those who were MRD positive (OS: P = 0.032, HR: 0.3306, 95%CI 0.09829 to 1.112; RFS: P = 0.0035, HR: 0.2492, 95%CI 0.07993 to 0.7770). Additionally, there was no statistical significance in the OS and RFS of HLA-matched and HLA-mismatched patients (OS: P = 0.63, HR: 0.7996, 95% CI 0.3144 to 2.034, RFS: P = 0.97, HR: 0.9844, 95% CI 0.3982 to 2.433). The 2-year OS of patients was 68% in the allo-SCT group and 26% in the chemotherapy group (p < 0.001, HR: 0,2567, 95% CI 0.1336 to 0.4932). A multivariate analysis suggested that allo-SCT and CR after the first course induction were independent prognostic factors for OS. Conclusions: Collectively, we reported the largest cohort study with AYA and older adult ETP-ALL/LBL, and we found that ETP-ALL/LBL was highly invasive and had a poor long-term prognosis. Allo-SCT could significantly improve ETP-ALL/LBL patient survival. Disclosure: Nothing to declare.
Background: In acute myeloid leukemia (AML) current risk stratification (e.g. by the European LeukemiaNet (ELN) 2017) relies on diagnostic genetic aberrations and help to inform treatment decisions, including those for an allogeneic stem cell transplantation (HSCT) in first remission. Recently Gerstung et al. (Nature Genetics, 2017) developed a knowledge bank (KB)-based algorithm based on demographic, clinical, and genetic data to predict individual outcomes. Two studies validated the feasibility of KB prediction in AML patients consolidated with chemotherapy. However, a validation in a HSCT-treated cohort, crucial with respect to informed decisions towards HSCT, is missing. Methods: We analyzed 545 AML patients (median age at diagnosis 62, range 21-77 years) receiving a non-myeloablative (77%) or reduced-intensity (23%) HSCT (60% were in first remission). Clinical variables included in the KB were available for our cohort, while our gene mutation panel did not include 17/58 of genes included in the KB. KB predictions 3 years after diagnosis were calculated by using the adapted transplant strategy and compared to the observed outcomes using receiver operating characteristics (ROC) curves. In addition, the measurable residual disease (MRD) status at HSCT was evaluated in patients with material available and based on NPM1 mutation and BAALC, MN1, and WT1expression. Results: The KB approach had an area under the curve (AUC) to predict 3-year OS of 0.69 (95% CI 0.62-0.72) which was not significantly different compared to the AUC of the ELN2017 risk stratification (0.66 [95% CI 0.57-0.71], P = 0.23), and worse compared to the published results in patients receiving chemotherapy (AUCKB = 0.80, Bill et al. 2021). However, in a multivariate analysis the KB prediction for 3-year OS significantly impacted OS (OR 6.25, CI 2.9-13.2) after adjustment for the MRD-corrected remission status at HSCT and Aikaike Information criterion (AIC) comparison with a model including the ELN2017 classification demonstrated the model containing the KB prediction as preferable. When introducing arbitrary cut-offs according to the KB prediction for OS at 3 years, we observed a clear separation of OS curves according to a KB value of <20, 20-39, and ≥ 40 (with higher values indicating a higher likelihood for OS, P < 0.001). Regarding other endpoints for which the KB algorithm provides outcome prediction, we observed the highest probability to correctly predict non-remission death (AUCKB = 0.75), restricted prediction for death in first remission (AUCKB = 0.61) or after relapse (AUCKB = 0.63), but good outcome prediction for being alive after relapse (AUCKB = 0.77) or being alive in first remission (AUCKB = 0.69). Conclusions: For HSCT treated AML patients the KB-based outcome prediction for HSCT treated patients was inferior compared to previous studies of patients receiving chemotherapy. The likely reason for the inferiority is the introduction of confounders (e.g. donor selection, graft-versus-host-disease), especially for treatment-related mortality, not integrated in and not predictable by the current KB algorithm. Inclusion of these additional factors might allow for a more precise outcome prediction for AML patients receiving HSCT. Disclosure: Nothing to disclose
Background: Introduction: FLT3-ITD mutation is associated with adverse prognosis (ELN2017, Dohner et al, Blood 2017) and SCT has been the standard of treatment for FLT3 mutated AML for decades. Midostaurin (Midos) has been approved in combination with intensive chemotherapy (IC) for FLT3-mutated AML, midostaurin maintenance was part of the treatment schedule in the RATIFY trial but not randomly explored (Stone et al, N Engl J Med 2017). Methods: Aims: The aims of this study are to analyze safety and effectiveness of midostaurin maintenance in FLT3 AML in a “real-world” setting and to evaluate maintenance versus alloSC versus W&W and the impact of risk factors. Methods: We carried out a retrospective multicenter study (MDA-AML-2018-06) in 27 Spanish centers. Inclusion criteria: age >18 years, FLT3-mutated AML diagnosis according to WHO criteria and start of treatment with midostaurin in combination with IC between June 2016 and December 2020. We evaluated the response according to 2017 ELN criteria, toxicity according to CTCAE v4.0 and overall survival (OS) by Kaplan-Meier. Statistical analysis was performed using SPSS program version 20.0. Results: Results: A total of 175 (93 female) patients were included, median age 53 (18-76) median OS for the whole population not reached, 24months OS 68%. Of those who achieved CR after Induction1or2 144 (81.4%) patients, 24p received maintenance, 76p were consolidated with alloSCT and 41p proceed to W&W (table 1). Safety: AE during maintenance were one case of QT prolongation which required Midos discontinuation. No cases of febrile neutropenia and no cases of deaths related to Midos. Regarding OS the ELN2017 classification resulted in a trend of differences in all groups maintenance, alloSCT and W&W. We observed significant differences for maintenance versus W&W (p0.001) in low and intermediate risk patients. Comparing maintenance vs alloSCT we observe no differences in the Int ELN2017 group. Table 1 Conclusions: Conclusions: Our experience confirms safety of maintenance therapy in AML FLT3 patients after intensive chemotherapy. We also observed a benefit for maintenance versus W&W in low and intermediate risk population. Clinical Trial Registry: Not registered at Clinical Trials Approved by National Ethics Comitie Disclosure: Study founded by Novartis
Background: Using EBMT registry data, the DRSS has been proposed to predict relapse risk after allogeneic hematopoietic cell transplantation (HCT) across disease subtypes and remission states ordered in 55 categories and 5 risk levels (Shouval R et al. Lancet Haematol. 2021). For acute myeloid leukemia (AML) the DRSS combines ELN risk group, remission rank, and de novo vs. secondary AML in 19 categories. We sought to determine its reproducibility in a cohort of subjects transplanted for AML. Methods: Data from a single-centre cohort of adult AML patients transplanted between 01/07/2015 and 30/06/2020 was analysed retrospectively. Baseline characteristics and outcomes were extracted, and Fine-Gray regression was used to determine the association between cumulative incidence of relapse (CIR) and patient, disease, and transplant characteristics, as well as the influence of graft-versus-host disease (GVHD) as a time-dependent covariate. Model selection techniques were used to select the least number of significant predictors of CIR. Results: In this cohort of 89 patients, median follow-up was 2.7 years (interquartile range: 0.9-3.2) and CIR was 29% at 5 years (95% confidence interval: 19-40). The study of the association between CIR and patient age >60 years, donor type (related matched, haploidentical, matched unrelated, cord blood), DRSS category, chronic (c) GVHD yielded a model using two covariates: DRSS and cGVHD, to predict CIR (hazard ratio (HR) 0.38, p = 0.03 and HR 0.43, p = 0.12, respectively). Univariate graphic representation of CIR according to DRSS is shown in Figure 1. Conclusions: In adults with AML, cumulative incidence of relapse after allogeneic HCT can be predicted by DRSS across all donor types and age groups. Disclosure: Nothing to declare.
Background: Protein tyrosine phosphatase non-receptor type 21 (PTPN21) gene mutations and its elevated expression level have been reported in different type of tumors. We have also found that PTPN21 gene mutations are associated with disease relapse in B-cell acute lymphoblastic leukemia (B-ALL) patients. However, the structure and molecular mechanism of PTPN21 wild type and mutations remain to be determined. Methods: We use protein crystallization and X-ray diffraction to determine the structure of protein PTPN21. We further used miniTurbo-mass spectrometry analysis to study the difference of protein interaction networks between PTPN21 wild-type protein and PTPN21 mutant proteins in living cells. Results: We have determined the structure of FERM domain and PTP domain of PTPN21. We also found that compared with PTPN21 wild-type protein, three PTPN21 mutant proteins, which were involved in the pathogenetic process of relapse of B–ALL, significantly reduce the binding affinity to 202 proteins and increase the interaction with 119 proteins in cells. The KEGG pathway analysis showed involvement in the vesicle docking, extra-cellular matrix(ECM) receptor interaction, MAPK pathway and so on. Notably, the most remarkably decreased interacting proteins of all three PTPN21 mutations are centrosome associated protein HP5, the Hippo pathway component WWC2, the trafficking protein TMED10, actin binding protein FSCN1 and a newly emerged cell fate regulators as well as the Hippo pathway kinase LATS1. Conclusions: ALL-associated PTPN21 mutant proteins may promote cell-matrix interaction, MAPK signaling, the Hippo pathway and the expansion of centrosomal amplification to assist B-ALL cells to survive chemotherapy and disease relapse. Disclosure: Nothing to declare
Background: For acute lymphoblastic leukemia (ALL) patients, total body irradiation (TBI) based conditioning regimens are often the first choice, considering their positive impact on relapse incidence. However, TBI is associated with toxicity and long-term morbidity, and its accessibility can be a major issue for many hematological centers. Several studies have shown an equivalence in clinical outcomes with chemotherapy-based conditioning, notably with the use of thiotepa. We performed a retrospective bicenter study to evaluate the outcome of adult ALL patients who had received, before allogeneic hematopoietic stem cell transplantation (allo-HCT), a thiotepa-busulfan-fludarabine (TBF) myeloablative conditioning regimen with reduced toxicity. Methods: Fifty-five patients (not eligible to high dose TBI) from Saint-Antoine Hospital (Paris, France) or the American University of Beirut Medical center (Beirut, Lebanon), received a TBF regimen consisting of 1-2 days of thiotepa (5mg/kg/day), 2-3 days of busulfan (130 mg/m2), and 5 days of fludarabine (30 mg/m2). The repartition of conditioning-regimen was 34.5% of T2B3F, 32.7% of T1B3F, 30.9% of T1B2F, and 1.8% of T2B2F. The median age of the patients was 51 years (range 17 to 72.4). Twenty-eight (50.9%) were male. Most patients had a diagnosis of B-ALL (93%) and 7% of T-ALL. Forty-two (76.4%) patients had a high-risk cytogenetic ALL. At the time of transplant 52 (94.5%) patients were in complete remission, 2 patients had a positive minimal residual disease (MRD) and 1 patient was refractory. For assessment of minimal residual disease (MRD): 27 (50%) patients had a Philadelphia chromosome, 8 (14.8%) had an immunoglobulin (Ig) and/or T-cell receptor (TCR) rearrangement, and 4 (7.4%) had an MLL rearrangement. The remaining MRD assessments were carried out using multiparameter flow cytometry. Peripheral blood stem cells were the main stem cell source (90.9%). Twenty-seven (49.1%) patients were transplanted from a matched sibling donor, 12 (21.8%) from a matched unrelated donor, and 16 (29.1%) from a haploidentical donor. The graft-versus-host disease (GVHD) prophylaxis was cyclosporine A (CsA) alone (32.7%), or CsA with mycophenolate mofetil. In addition, antithymocyte globulin (ATG) was used for a median of 2 days, and patients with a haploidentical donor received low-dose ATG and post-transplant cyclophosphamide (PT-Cy). All patients engrafted at a median time of 15 days (range, 5-27). Results: With a median follow-up of 43 months, 2- and 5-year overall survival (OS) was 73.2 % (95% CI: 58.9 - 83.2) and 64% (95% CI: 48.8 - 75.7), respectively. At 2 years, leukemia-free survival (LFS) and GVHD-free, relapse-free survival (GRFS) were 59.5% and 57.6%, and at 5 years, 53.4% and 51.8%, respectively. The 5-year non-relapse mortality (NRM) was 15%. The day 180 cumulative incidence (CI) of grade II-IV acute GVHD and grade III-IV acute GVHD were 44.7% and 6.4%, respectively. At 2 years, the CI of chronic GVHD and extensive chronic GVHD was 16.9% and only 1.9%, respectively. Conclusions: Our retrospective study does suggest that using TBF as the conditioning regimen in adult ALL patients is a promising option with acceptable toxicity. Disclosure: Nothing to declare
Background: With improvement in transplant related mortality (TRM), relapse is the major factor affecting survival post HSCT. Prognosis of patients with acute leukemia (AL) relapsing after HSCT is dismal. We analysed incidence and patterns of relapse, factors predicting relapse and determinants of post relapse OS (PROS). Methods: This is a single centre retrospective analysis of AL patients who underwent 10/10 or 9/10 matched related or unrelated ASCT from January 2008 to December 2019. AL included lymphoid (ALL), myeloid (AML) and mixed phenotypic (MPAL) leukemias. Nineteen patients with AML had active disease; rest were in complete remission at ASCT. Conditioning included either full intensity regimens [TBI-cyclophosphamide (Cy), busulfan (Bu) - Cy or Fludarabine (Flu) – Bu] or reduced intensity regimens (Flu based). GVHD prophylaxis consisted of calcineurin inhibitor (CNI) with either methotrexate or MMF or PTCY with CNI and MMF. Factors studied as predictors of relapse were DRI, EBMT risk score, HCT-CI, transplant conditioning intensity (TCI), mixed chimerism (MC) at day 30, 90, 180 and 360, grade II-IV aGVHD and cGVHD. Treatment after diagnosis of relapse was at the discretion of treating physician. Ongoing immunosuppresion (if any) was stopped. In general, patients received 1 or more DLI and/or immunomodulatory or targeted agents (lenalidomide, dasatinib or imatinib, nivolumab, palbociclib, etc). Patients with morphological relapse were treated with chemotherapy with or without DLI. Some patients with extramedullary relapse (EMR) received radiotherapy. Factors analysed for effect on PROS were time to relapse (TTR), TCI, DRI and prior aGVHD or cGVHD. Cumulative incidence of relapse (CIR) was calculated using competing risk regression with TRM being the competing event. Results: Total of 162 AL patients underwent HSCT. Sixty four (39%) relapsed. Of these, 52 (81%) had medullary relapse (MR), 8 had EMR and 4 had combined relapse. Among isolated MR, 41 (78.8%) were morphological, 3 were cytogenetic, 3 were flowcytometric, and 5 were molecular relapse. CIR at 1 yr and 2 yr was 24% (95%CI; 18%-31%) and 33% respectively (95% CI; 26%-40%).Significant factors associated with relapse were MC at day 90 (p = 0.004) and day 180 (p = 0.001) and absence of aGVHD (p = 0.01) and cGVHD (p = 0.00). However, cGVHD was present in 5/8 with EMR as compared to 12/56 with MR or combined relapse (p = 0.026). Similarly, more patients with MR and combined relapse had MC at relapse (28/51, 54.9%) compared to none with EMR (p = 0.005). Median PROS was 4.2 months while 1 yr PROS was 31.1%. Factors associated with better PROS included low - intermediate DRI (5.3 vs 1.1 months with high DRI, p = 0.004) and receipt of targeted therapy (median PROS 16.5 vs 6.4 months with conventional chemotherapy alone, p = 0.016). Conclusions: About 40% of AL patients experienced relapse post ASCT. Patients with MC (at any time through day +180) and those without acute or chronic GVHD were more likely to have MR or combined relapse. Presence of aGVHD or cGVHD and full donor chimerism did not protect against EMR. Survival after relapse was poor, however those with low and intermediate DRI, and those who received targeted therapies post relapse fared better. Disclosure: Nothing to declare
Background: Anti-thymocyte globulin(ATG) is widely used to prevent graft-versus-host disease(GVHD) after allogeneic peripheral blood stem cell transplantation(HSCT). High dose cyclophosphamide post-transplant is used as a potent agent for GVHD prophylaxis in matched related(MRD), matched unrelated (MUD), mismatched unrelated(MMUD) and haploidentical HSCT. In this retrospective analysis, we compared the use of ATG to post-transplant cyclophosphamide(PT-CY) on leucocyte engraftment, acute and chronic GVHD, overall survival(OS), disease free survival(DFS), non-relapse mortality(NRM) relapse incidence(RI). Methods: A total of 57 patients with ALL in CR1 were treated with a preparative regimen of TBI 12Gy in combination with cyclophosphamide or fludarabine between 2002 and 2021. Of the 57, 38.6% patients received ATG and 61.4% PT-CY with calcineurin inhibitors±mycophenolate mofetil. Median age was 37(11-56) years and the majority were male(52.6%). ALL patients(35.1% bcr/abl+) were treated according to GMALL protocols including prophylactic cerebral irradiation in 49.1%. MRD positivity was observed in 45.6% and negative in 43.9% of patients. Patients were positive for CMV in 57.9%. Karnofsky score was median 80(60-100)%. Donors were median 32(5-57) years old. More male(66.7%) than female donors and more unrelated(71.9%) than related(28.1%) were used. Included in this analyses were, for the PT-CY group MRD(n = 9), MUD(n = 17), MMUD(n = 4) and haploidentical(n = 5) and for the ATG group MRD(n = 2), MUD(n = 14), MMUD(n = 5). Most of the donors were CMV positive(59.6%) and more PBSC 89.5% than bone marrow were given. Of the six patients receiving bone marrow four received PT-CY and two received ATG, two were related and four were unrelated. Results: Patients characteristics were equally distributed in both treatment groups regarding patient age, gender and bcr/abl+(Table 1), but statistically significant differences were noted in pre-transplant radiation therapy(p = 0.02) and related/unrelated donors (p = 0.01). MRD status was not different between the groups. Engraftment was observed in all patients except two in the ATG group. Recovery of WBC was faster in PT-CY as in ATG group. No difference in acute and chronic GVHD was observed between the two groups. After a median follow-up of 2.77 (range 0.05-13.14) years, OS of all patients was 76.7% at 3 years, 72.0 ± 9% in the PT-CY group and 81.8%±8 in the ATG group. DFS of all patients resulted in 72.8% at 3 years, 66.7 ± 8.7% in PT-CY and 81.0 ± 8.6% in ATG treated patients. Only bone marrow source (p = 0.03) remained an independent factor for OS after accounting for type of SCT, ATG and prophylactic cerebral irradiation. For DFS type of SCT remained as a trend (p = 0.07). RI for PT-CY was 9.1 ± 5.1% and for ATG 4.5 ± 4.6%, while NRM was 21.2 ± 7.3 und für 13.6 ± 7.5% for PT-CY and ATG treated patients, respectively. When analysing only patients receiving peripheral blood, there only was a difference between the related and unrelated(p = 0.03) and no difference in outcome and GVHD. Conclusions: The use of PT-CY for GVHD-prophylaxis resulted in faster leucocyte engraftment, but similar acute and chronic GVHD incidence, OS and DFS. Bone marrow source was the only independent risk factor for OS. This finding needs to be confirmed in a larger more homogenous cohort. Disclosure: Nothing to disclose
Background: Allogeneic hematopoietic stem cell transplantation (allo-HSCT) remains an efficient therapy for hematologic malignancies. Relapse of acute leukemia following allo-HSCT usually represents return of an original disease clone. The development of de novo hematological malignancies in cells of donor origin is a rare but severe complication, known as donor cell leukemia (DCL). To date, the reported patients with DCL are mainly following HLA-matched sibling or unrelated donor HSCT. We present here a rare case of late-onset DCL that developed in HSCT recipient and not in his HLA haploidentical father donor. To reveal the precise etiologic mechanisms of such DCL, we integrated genomic and single-cell molecular analyses. Methods: We performed short tandem repeat (STR) analysis on bone marrow (BM) samples obtained from patient pre-HSCT and relapse post-HSCT, and donor PBSC sample. Genomic DNA was isolated and subjected to whole-exome sequencing (WES) from specimens of BM sample at relapse post-HSCT, and buccal mucosal specimen from the patient, as well as BM cells and the buccal mucosal specimen from the donor. 10x Genomics single-cell RNA sequencing was performed on patient’s BM sample at relapse post-HSCT and donor’s BM sample. Results: STR analysis confirmed that leukemia cells originated from the donor. We compared data sets from donor and DCL samples in order to detect variants that expandedå 3-fold or de novo in DCL with WES. The mutation affected C17orf97 (chr17: p.Asp220_Pro229del) has been present in donor BM cells, while at a 7-fold higher frequency in the DCL sample, with variant allele frequency (VAF) from 3.85% to 30%. The evolution of DCL was further associated with the acquisition of mutations in IDH1 (chr2: p.Arg119Trp; VAF = 6.98%) and NSD1 (chr5: p.His2205Asp; VAF = 10%). In addition, we also found ATF7IP (chr12: p.Gly1122AspfsTer53) mutation with VAF = 5.41% and ZNF33A (chr10: p.Cys607Tyr) mutation with 9.64%, which have not been reported in leukemia. To better reveal the hematopoietic hierarchy and leukemic transformation involved in DCL progression, 10X Genomics single-cell RNA sequencing was performed. We deciphered an atlas covering 22399 cells and 12 major cell types (20 clusters) according to the established markers of hematopoietic populations. We observed an increase in the fraction of granulocyte-monocyte progenitors, while decreased proportions of mature monocytes and neutrophils within DCL cells. We next performed differential gene expression analyses between healthy donor and DCL hematopoietic stem and progenitor cells (CD34+ckit+ cells), 245 differentially expressed genes (p <0.05, log2FC > 2) was found. KEGG analysis revealed the top2 enrichment of pathways related to metabolic pathways and pathways in cancer. To understand the specific HSPC subpopulations involved in DCL, 4 transcriptionally distinct clusters were identified. HSPCs from donor and DCL harbour distinct transcriptional programs, and GO analysis in cluster2 (from DCL) showed enrichment of the pathway related to myeloid cell differentiation. CRIP1 and LGALS1, recently reported associated with AML, are the top2 significantly differentially expressed genes in cluster2. Conclusions: Our study distinguishes HSPCs from healthy haploidentical donor and DCL at a single-cell resolution. With the help of integrated genomic and single-cell molecular analyses, we provide more comprehensive mechanisms of DCL after allo-HSCT. Disclosure: Nothing to declare
Background: Allogeneic hematopoietic stem cell transplantation (Allo-HSCT) as post-remission treatment in cytogenetically defined intermediate-risk Acute Myeloid Leukemia (AML) in first complete remision (CR1) is effective, but has been associated with high transplant-related mortality (TRM). The aim of this study is to analyze the outcome of this procedure using HLA-identical sibling, unrelated or alternative donors. Methods: We conducted an observational retrospective study in all patients with intermediate-risk cytogenetically defined (Upon The European LeukemiaNet 2017 criteria) AML in CR1 who underwent an allo-HSCT in our center between 2010 and 2020. The primary endpoint was OS, secondary outcomes were relapse-free survival (RFS), cumulative incidence of relapse and TRM. Minimal residual disease (MRD) was measured by flow cytometry (using a cut off of 0,01%) and/or RT-PCR (NPM1). A multivariate Cox regression model was performed. Results: We analyzed 58 patients with a median age of 58 (R 19-82) years. Ten (17,2%) patients had negative MRD and 29 (50%) had an unrelated donor (5 had a 9/10 HLA allelic match) at the moment of transplant. Forthy three (74,1%) patients received myeloablative conditioning regimens (Table 1). The median time from diagnosis to transplant was 5 (IR 4-7) months. With a median follow up of 40 months, the OS at 1, 2 and 3 years was 88% (95% CI 77-98%), 84% (95% CI 72-96%) and 78% (95% CI 64-92%) respectively (Figure 1). The RFS at 1 and 3 years was 88% (95% CI 77-99%) and 73% (95% CI 58-88%), the cumulative incidence of relapse at 1 and 3 years was 7% (95% CI 2-15%) and 14% (95% CI 7-25%) and the TRM at 1, 2 and 3 years was 7% (95% CI 2-15%), 11% (95% CI 4-20%) and 15% (95% CI 7-26%) respectively. Among all the clinical features which could be related to OS: group of age, gender, conditioning, MRD and type of donor were included in the multivariate analysis. A non-myeloablative conditioning (HR 15 p = 0,01) an haplo-identical donor (HR 6 p = 0,03) and an unknown/undefine MRD (HR 13 p = 0,03) were associated with a significant higher risk of death. Age and gender showed no differences. Conclusions: Allo-HSCT in patients with an intermediate-risk LMA in CR1 is the standard consolidation therapy at our institution mainly when a HLA identical sibling or unrelated donor is available, and especially when using a myeloablative conditioning, as it is associated with low TRM and a high RFS. Disclosure: Nothing to declare
Background: Nearly 30% of AML patients harbour a FMS-like tyrosin-kinase 3-gene alteration (FLT3) driver mutation. The increased relapse risk associated with internal tandem duplications (ITD) may be counteracted by allogeneic hematopoietic stem cell transplantation (allogeneic HSCT) followed by FLT3 inhibitor (FLT3i) maintenance. We here describe risk factors for post-HSCT central nervous system (CNS) relapse, an uncommon but prognostically extremely unfavourable event in adult FLT3-mutated AML. Methods: We retrospectively analysed data of 39 patients with FLT3-ITD (n = 34) and/or TKD-mutated AML (n = 5) who were transplanted 2017-2020 at our institution. Minimal residual disease (MRD) was determined prior to and 60-100 days after allogeneic HSCT by qRT-PCR (NPM1mut, n = 26; KMT2A-PTD, n = 2; JAK2mut, RUNX1/RUNX1T1, NUP98-NSD1, n = 1 each) or multicolour flow cytometry, (n = 8). Cumulative incidence of relapse (CIR) was calculated with non-relapse mortality as competing risk and survival probabilities were compared by log-rank test. Results: At the time of allogeneic HSCT, 6 patients (15%) were in MRD-negative CR (molCR), 24(61%) in MRD-positive CR and 9(23%) had active disease. All patients achieved CR with 30(77%) molCR and FLT3i was started prophylactically (n = 11) or pre-emptively (n = 11) at a median of 54(range, 41-713) days post-HSCT for a median of 12.6 months. Reasons for no FLT3i were allogeneic HSCT before 2019 (n = 12), renal insufficiency (n = 1), FLT3-TKD only (n = 3) and early relapse (n = 1). With a median follow-up of 27 months in surviving patients, probability of OS was 67.9% at 3 years. Ten patients relapsed at a median of 9.3(range, 3.3-33.6) months post-HSCT, the CIR was 32%. Survival was significantly longer in patients with molCR vs no molCR post-HSCT (OS: not reached vs. 30.4 months, p = 0.004; relapse-free survival: not reached vs. 9.1 months, p < 0.001), while other factors, such as cytogenetic risk, total body irradiation-based conditioning, RIC vs MAC, use of FLT3i pre-HSCT or post-HSCT had no significant impact on survival. Meningeal leukemia (n = 5) or CNS chloroma (n = 1) were observed as late events at a median of 16.3 months post-HSCT and occurred on Sorafenib (n = 2), Gilteritinib (n = 1) or decitabine-based salvage therapy after failure of FLT3i (n = 3). All patients with CNS relapse had active disease at allogeneic HSCT (median: 14% bone marrow blasts) and no molCR post-HSCT. CNS relapse was preceded by hematologic relapse in 5/6 patients by a median of 2.7 months. Conclusions: Patients with FLT3-mutated AML and active disease at the time of allogeneic HSCT, combined with failure to achieve MRD-negativity after allogeneic HSCT have a high risk of CNS-relapse and leukemic death. This was not abrogated by pre-emptive or salvage-therapy with FLT3i or HMA and prophylactic intrathecal therapy may be considered to avoid CNS relapse. Disclosure: Gesine Bug, Honoraria: Jazz, Celgene, Gilead, Novartis, Hexal, Pfizer, Eurocept
Background: Despite progress in therapies, allogeneic hematopoietic stem cell transplantation (alloHCT) still plays a pivotal role in the treatment of adult Acute Lymphoblastic Leukemia (ALL). Nevertheless, relapse and non-relapse mortality remain a significant concern. The aim of this retrospective real-life study was to evaluate overall survival (OS), disease-free survival (DFS) and non-relapse mortality in a multicenter series of adult ALL patients undergoing alloHSCT. Methods: The study included adult patients, affected by either B-ALL (Ph negative or positive) or T-ALL, who underwent alloHSCT in three Italian Bone Marrow Transplant Centers between July 2003 and July 2020. Patient outcomes were evaluated by univariate analysis based on variables defined as pre-alloHSCT [i.e., ALL phenotype, white blood cell count (WBC) at diagnosis, number of complete remissions (CR)], alloHSCT-related [i.e., EBMT risk score, donor type], and post-alloHSCT [Graft versus Host Disease (GvHD) and minimal residual disease (MRD)]. The observation period ended on October 2020. Results: 133 subjects with a median age of 40 (range: 18-70) years were enrolled. Patients were affected by Ph- (66) or Ph + (33) B-ALL (33), and T-ALL (34). With a median follow-up of 18.5 (range: 0.9-187.5) months, OS was 47.4% (median survival: 37.4 (range: 0.9-187.5) months) and DFS 67.7% (median survival not reached). Relapse mortality accounted for 24% and non- relapse mortality for 28.6% of deaths. According to univariate analysis, DFS had a better trend in patients with T as compared to B phenotype (p = 0.09) (A) and in those with negative MRD (immunophenotypic detection) before alloHSCT (p = 0.08). The number of pre-alloHSCT complete remissions (CR1 vs CR > 1) did not affect DFS (p = 0.08). Patients with matched unrelated donors had a better DFS as compared to those with matched related (p = 0.07) (B). DFS was significantly higher in patients with GVHD (p = 0.002). Among B-ALL patients, a WBC count at diagnosis > 30.000/mmc had a negative impact (p = 0.09). Ph- B-ALL and T-ALL patients with a negative immunophenotypic MRD at day +90 after alloHSCT showed a better DFS (p = 0.005) (C), Ph+ B-ALL patients with a major molecoular response at day +21 had better DFS (p = 0.003) (D). OS and non-relapse mortality were lower in patients with EBMT score ≥ 4 (p = 0.01) Conclusions: This real-life study confirms that in adult ALL patients alloHSCT is effective, though associated to a significant transplant-related mortality that can be predicted by the EBMT score. A negative post-alloHSCT immunophenotypic MRD (day + 90) and a major molecular response (day + 21) are predictive for a favorable outcome in Ph- B- and T-ALL, and Ph+ B-ALL, respectively. Disclosure: Nothing to declare
Background: Although allogeneic transplantation in high risk myeloid malignancies is the best way of consolidation, relapse of the original disease remains the major cause of transplant failure, the treatment is difficult and until now there is no definite way how to deal with it. Methods: We retrospectively evaluated 186 AML and 51 MDS patients transplanted between years 2001 and 2021 who relapsed after transplantation. The therapeutic approach was individual considering the status of the patient, risk of the disease assessment and the patient’s will. For the evaluation the patients were divided into three groups. 1) no chemotherapy approach (including immunosuppression tapering and DLI only), 2) low dose chemotherapy considering of either low dose ARA-C or 5-azacytidine or both, including five patients treated with addition of gilteritinib, 3) high dose chemotherapy considering of antracyclin and ARA-C base regimens, or/and second transplant. The majority of the patients were in parallel additionally treated with DLI. Results: In AML: the survival in 1, 2 and 5 years was 7%, 3% and 0% in no chemotherapy gr., 38%, 31% and 19% in low dose gr.,and 42%, 32%, and 20% in high dose gr. In MDS 7% in no chemotherapy gr., 15%, 13% and 12% in low dose gr., 51% 36% and 32% in high dose group. No statistical difference in OS was found between treated groups. Time of relapse from transplant significantly influenced the overall survival in all tested time points (3, 6 and 12 months) OS in 2 years 25%, 22% and 32%. Not surprisingly the patients who achieved remission with any type of chemotherapy had OS better in 2 years comparing to those, who did not (60% vs 5%). Anyway 43% of patients suffered from subsequent relapse. 21% of patients who achieved remission died from treatment related complications (toxicity or GVHD). There was no substantial difference in the outcome between AML and MDS patients. Conclusions: Although the outcome of the AML/MDS patients relapsed after transplantation is very poor, many of them can profit from additional treatment and some of them achieve further remission. Immunomodulation with DLI is considered to play substantial role and its prophylactic and preemptive use is promising. New drugs such as venetoclax or gilteritinib need to be introduced and evaluated. Disclosure: I have no conflict of interest.
Background: There are virtually no treatment options for therapy-refractory or relapsed AML/MDS and high rates of relapse in successfully treated patients. Methods: The combination of the (clinically approved) immune-modulatory compounds GM-CSF + Prostaglandine 1 (PGE1; the combination referred to as KIT M) converts myeloid blasts into dendritic cells of leukemic origin (DCleu). After stimulation with DCleu, antileukemic (T) cells are activated. Kit M treatment may be an attractive tool for immunotherapy in myeloid leukemia. Results: 1. ex vivo: Treatment of 65 leukemic WB samples with KIT M does not induce blast proliferation, but triggers generation of mature DC/DCleu and reduces tolerogenic DC. Kit treated WB activates the adaptive and innate immune system after MLC (T cell proliferation, antitumor-supportive T cells (TCRgd,Tb7), memory cells (Tcm,Tb7cm) and downregulates immune suppressive T cells (Treg4 and 8). Moreover leukemia specific (interferon g (g) and/or degranulating (deg)) adaptive (g-degT4,T8,TCRgd,Tb7,Tcm) and innate cells (g-degNK,NKb7,CIKb7) are increased and regulatory cells (g-degTreg4) downregulated. In addition, blast lysis is increased vs control. Ex vivo achieved blast lysis correlates positively with frequences of mature DC/DCleu, leukemia specific T3,T4,T8,TCRgd,Tb7 and NK cells and negatively with Treg4 and 8. Blast lysis does not correlate with age, sex, ELN risktype, blast counts, or response to chemotherapy. 2. In vivo - rats: Kit M treatment of 3 leukemically diseased (vs 3 control) rats (followed by sacrification after treatment) leads to reduced blasts and Tregs in blood and spleen and increased DCleu and memory-like T cells. 3. In vivo - human: Kit M therapy was offered to a 72 year old pancytopenic male as an individual salvage attempt (applied as continuous infusion), after discussion with the ethical commitee, the patient’s information about the experimental nature of the treatment and his written consent. The treatment was well tolerated and the patient improved clinically. Neutrophils in WBC increased from 10% to 50%, thrombocytes reached 100 G/l after 24 days. Immune monitoring showed a continuous increase of proliferating and non-naïve Tcells, NK, CIK- and NKT-, TH17 cells, Bmem-cells and DC in PB. The production of IFNg producing T-, CIK and NKT-cells was demonstrated, suggesting an in vivo production/activation of (potentially leukemia-specific) cells. Immune stimulatory effects decreased after discontinuation of therapy. After 4 weeks of treatment, the patient was discharged in good clinical condition. Unfortunately, at two weeks from discharge, AML progressed and the patient died few days later. Conclusions: Treatment of WB ex vivo with Kit M leads to activation of adaptive and innate (leukemia specific) immune reactive cells (and downregulated suppressive mechanisms) via a DC/DCleu triggered mechanism – resulting in significantly improved blast lysis compared to controls (independent of patients‘ risk classification, MHC, age or sex). In vivo treatment of leukemically diseased rats or humans was well tolerated, led to an increase of platelets and granulocytes and stable (low) blast counts in PB – probably mediated by a (leukemia specifically) DC/DCleu activated immune system. A dose defining clinical trial in carefully selected patients to confirm clinical safety and underscore clinical efficacy is being prepared. Disclosure: Helga Schmetzer is associated with Modiblast Pharma GmbH
Background: The second allogeneic hematopoietic stem cell transplantation (alloHSCT) is the most effective treatment option for patients (pts) with acute myeloid leukemia (AML) who relapsed after the first alloHSCT. The strategy of this procedure, especially optimal reinduction, choice of donor and type of conditioning remain unknown. Methods: We retrospectively analyzed the outcome of the second alloHSCT in 40 pts (21 women, 19 men) with AML, transplanted in one center between 2005 and 2020. At the first alloHSCT most pts (36) were transplanted in complete remission (CR) – 26 pts in CR1, 8 pts in >CR1, 4 pts were transplanted with active disease (nonCR). Most pts (32) received myeloablative first conditioning. Donor at first alloHSCT was sibling (16), unrelated (23) or haploidentical (1). The median time between first alloHSCT and relapse of disease was 10 (3-120) months; 13 pts relapsed within 6 months, 6 pts later than 2 years, one after 10 years. Only seven pts presented graft versus host disease (GvHD) symptoms after first alloHSCT. At the time of the second transplant median age was 41 (20-69) years. All but two pts received reinduction chemotherapy (based on Flag or Clag – 28 pts, HDAraC- 9 pts) before the second alloHSCT. At the time of the second transplant 28 pts were in CR, 12 were transplanted in nonCR. Before the second alloHSCT pts received myeloablative (n = 14) or reduced-intensity (n = 26) conditioning regimen and peripheral blood stem cells (PBSC) (n = 39) or bone marrow cells (n = 1) from matched sibling donors (n = 11), matched/mismatch unrelated donors (n = 13) or haploidentical donors (n = 16). 22 pts received second alloHSCT from the same donor as for the first transplant, 18 from different ones. Results: Neutrophil engraftment was achieved in 35 patient, with median time 22 days, range 10-47. Eight pts (20%) died up to 100 days due to transplant related reason (infection, MOF). GVHD was seen in 7 pts only – acute in 5 pts, chronic in 3. Relapse occurred in 17 (42%) pts and was the cause of death in 15 of them. The median time between the second alloHSCT and relapse was 7 (2-30) months. After the median follow-up of 40 months, 15 (37%) pts remained alive with 14 in remission of disease. Median overall survival (OS) was 16 months. The one-year and five-year OS was 63% and 35%, respectively. In Cox-model-based tests only disease status at time of second alloHSCT (CR vs nonCR) significantly improve OS – one-year and five-year OS was 76% and 48% vs 49% and 15% respectively (HR 95% CI 0.39 (0.17-0.89); p = 0,02), with no influence of time to relapse, conditioning, donor type and GvHD status. Of the patients who have been transplanted in CR 14 (50%) remain alive, while of those who were not in CR only one is alive. Conclusions: The second alloHSCT remains a curative option for patients with AML relapsing after first alloHSCT, however achievement of complete remission before transplantation is required for successful treatment. The rate of transplant related mortality is high. Most patients died due to relapse of disease. Disclosure: Nothing to declare
Background: Endothelial Activation and Stress Index (EASIX)—lactate dehydrogenase (U/L)×creatinine (mg/dL)/thrombocytes (109/L)—was reported to be useful in predicting outcomes after allogeneic hematopoietic stem cell transplantation (allo-HSCT). However, due to controversies regarding the validity and usefulness of this simple predictive marker, EASIX needs to be verified in various cohorts. Thus, we investigated whether the EASIX measured before allo-HSCT conditioning (EASIX-pre) correlates with transplant-related outcomes in acute myeloid leukemia (AML) patients who underwent allo-HSCT at a single center in Korea, and compared its predictive ability with the established prognostic indices. Methods: We conducted a pilot study with AML patients who received allo-HSCT in 2017 among a prospective observation cohort for acute leukemia, in the Catholic Hematology Hospital, Korea (CRIS# KCT0002261). Each patient’s EASIX-pre was calculated one day before allo-HSCT conditioning. We evaluated the association between EASIX-pre and overall survival (OS) or failure (relapse or non-relapse mortality)-free survival (FFS) after allo-HSCT using the Kaplan-Meier estimates and the Cox model. We inspected whether EASIX-pre correlates to cumulative incidences of non-relapse mortality (NRM), relapse, acute graft versus host disease (aGVHD), transplant-associated thrombotic microangiopathy (TA-TMA) and sinusoidal obstruction syndrome (SOS), using cumulative incidence estimates and the Fine-Gray model. The usefulness of EASIX-pre in predicting death in 2 years after allo-HSCT was compared with other known predictive indices—The European Group for Blood and Marrow Transplantation (EBMT) risk score, Hematopoietic Cell Transplantation-specific Comorbidity Index (HCT-CI), Pretransplant Assessment of Mortality Score (PAM score)—using receiver operating characteristic (ROC) curves and their area under curve (AUC). Results: A total of 117 patients were included in this study. In our analyses, EASIX-pre showed strong association with OS and FFS. The hazard ratios for OS and FFS per 1 increment in EASIX (log 2 scale) were 1.37 (95% confidence interval (CI): 1.20–1.55, p < 0.001) and 1.36 (95% CI: 1.19–1.54, p < 0.001), respectively. EASIX-pre correlated with OS and FFS in each subgroup stratified according to their pre-HSCT disease status, cytogenetic risk, HSCT donor, and conditioning intensity. Also, EASIX-pre showed significant relationship with the cumulative incidence of NRM (coefficient of Fine-Gray subdistribution hazard model: 1.38, 95% CI: 1.14–1.66, p < 0.001), rather than those of relapse (coefficient: 1.17, 95% CI: 0.97–1.37, p = 0.055). There were no significant associations of EASIX-pre with the cumulative incidence of aGVHD (p = 0.756), TA-TMA (p = 0.893), and SOS (p = 0.923) after allo-HSCT. In predicting death in 2 years after allo-HSCT, EASIX-pre showed high predictability, of which AUC of ROC curve was 0.7534, which was comparable to other established prognostic indices (Figure 1). EASIX-pre cut-off of highest accuracy was 2.1 (log 2 scale). Conclusions: This study is the first to show the validity of EASIX-pre as a prognostic index in a cohort of Asian patients with AML. EASIX-pre significantly associated with the patient’s OS, FFS, and NRM. EASIX-pre had comparable predictive capacity with established prognostic scores. Although EASIX is known to be a marker that reflects endothelial stress, we did not find a significant association with vascular stress-related complications, such as GVHD, TA-TMA, and SOS. This pilot analysis warrants further validation with larger prospective cohorts. Clinical Trial Registry: CRIS# KCT0002261 https://cris.nih.go.kr/cris/search/detailSearch.do?seq=8588&search_page=L&search_lang=E&lang=E Disclosure: Nothing to declare
Background: The prognosis of patients with nucleophosmin 1 gene mutated (NPM1mut) acute myeloid leukemia (AML) depends on the presence of concomitant chromosomal aberrations and mutations. Although, patients with isolated NPM1 mutation are considered to convey a favorable prognosis, relapse still remains the most common cause of treatment failure and allogeneic hematopoietic blood stem cell transplantation (alloHSCT) as second line therapy is required to achieve durable molecular remissions. Patients who relapse molecularly or morphologically after or during conservative chemotherapy require immediate therapy to achieve a molecular or at least hematological remission again prior transplant. Patients with ratio > 0.5 of FMS-like tyrosine kinase 3 internal tandem duplication (FLT3-ITDhigh) or adverse chromosomal aberrations have a poorer outcome and are therefore considered as candidates for first line alloHSCT in first remission. Methods: Here we demonstrate our single center experience concerning alloHSCT and peri-transplant strategies in 73 patients with relapsed/refractory good or intermediate/high risk NPM1mut AML out of whom 55 patients received an alloHSCT at our center since 2008. Results: After a median follow-up of 2.7 years from alloHSCT, patients with hematologic complete remission (CR) compared to patients with morphological active disease (HR + ) had an estimated 2-y-OS of 73% vs. 62% (ns) and an estimated 2-y-RFS of 63% vs. 20% (P = 0.0070). Focusing on patients with CR before alloSCT, those with no detection of minimal residual disease (MRD-) showed a trend towards higher RFS compared to MRD + patients with a 2-y-RFS of 79% versus 47% (ns). This was not reflected in a different 2-y-OS (78% vs 75%, ns). Patients with FLT3-ITDhigh and MRD + CR before alloHSCT showed a trend towards a lower 2-y-RFS compared to patients without FLT3-ITDhigh (29% vs 62%, ns). Focusing on patients with second line indication for alloHSCT because of inadequate response or relapse post or during therapy we analyzed status of high dose cytarabine based salvage chemotherapy (S-CT). Thirteen patients got s-CT with 8 patients achieving CR (MRD- n = 6, MRD + n = 2). Sequential conditioning with FLAMSA without previously performed S-CT showed similarly good OS but a high risk of relapse. Patients with HR + showed a 2-y-OS of 52% and a 2-y-RFS of 24%, whereas patients with MRD + CR and MRD- CR showed quite similar 2-y-OS of 80% and 83% but a different 2-y-RFS of 62% and 83%, suggesting effective relapse strategies like hypomethylating agents (HMA) and donor lymphocyte infusion (DLI), especially in case of late and molecular relapse, detected by intensive MRD monitoring. Conclusions: Patients with second line indication for alloHSCT have an excellent chance of long-term remission if they are MRD- before alloHSCT. Patients with MRD + CR also have a good chance of long-term remission if no FLT3-ITDhigh mutation is present. To achieve a second remission S-CT is effective. Only the group of patients with HR + during therapy present a clinical challenge. For all groups, sufficient relapse strategies after alloHSCT such as HMA and DLI exist, especially if the relapse occurs late and was diagnosed early by intensive MRD monitoring. Disclosure: Thomas Schroeder JAZZ, Pfizer, BMS advisory boards, lecture fees JAZZ, BMS research funding
Background: Acute myeloid leukemia (AML) is an aggressive malignancy and is the most common and second most common form of acute leukemia in adults and children, respectively. The combination of the highly selective BCL-2 inhibitor venetoclax and the hypomethylating agent azacitidine was shown to be safe and effective in clinical trials (DiNardo et al. Blood. 2019;133:7-17; DiNardo et al. N Engl J Med. 2020;383:617-629) and is approved by the United States Food and Drug Administration and European Medicines Agency for the treatment of patients with AML who are not eligible to receive intensive chemotherapy. Following allogeneic stem cell transplantation (alloSCT), most patients do not receive antileukemic therapy; however, an unmet need remains as disease relapse and graft-versus-host disease (GvHD) commonly occur posttransplant. In addition to the antileukemic effect of venetoclax shown in clinical studies, preclinical studies suggest venetoclax may mitigate the risk of GvHD. VIALE-T is a Phase 3, randomized, open-label trial in progress (NCT04161885) evaluating the safety and efficacy of venetoclax in combination with azacitidine versus best supportive care (BSC) as maintenance therapy following alloSCT in patients with AML. Methods: This Phase 3 study consists of 2 parts (Figure). Key inclusion criteria include diagnosis of AML; plans to receive alloSCT or have received alloSCT within the past 30 days; bone marrow blasts <10% before pretransplant conditioning and <5% posttransplant; have received myeloablative, or reduced intensity, or nonmyeloablative pretransplant conditioning protocols. Grafts are allowed from various sources (bone marrow, peripheral blood stem cells, cord blood cells). Patients must be ≥18 years old for Part 1 and ≥12 years old for Part 2. Additionally, patients must meet key laboratory values for absolute neutrophil count (Part 1, ≥1500/µL; Part 2, ≥1000/µL), platelet count (Part 1, ≥80,000/µL; Part 2, ≥50,000/µL), bilirubin ≤3 times the upper limit of normal, and creatinine clearance >30 mL/min. Patients who have received venetoclax and had no history of disease progression while receiving venetoclax are eligible. Part 1 evaluates dose levels of venetoclax combined with azacitidine to determine the recommended Phase 3 dose (RP3D), which will be confirmed in approximately 12 additional patients enrolled in the Safety Expansion Cohort. Part 2 will be a randomized, open-label evaluation of the RP3D of venetoclax combined with azacitidine and BSC versus BSC only in adults and children aged 12 years or older. All venetoclax-treated patients will receive antibiotic prophylaxis during Cycle 1. The primary endpoint for Part 1 is the frequency of dose-limiting toxicities. The primary endpoint for Part 2 is relapse-free survival as assessed by an independent review committee. Key secondary endpoints for Part 2 include overall survival, GvHD-free relapse-free survival, and the rate of patients without higher grade GvHD at 90 days after randomization. Enrollment into the Safety Expansion Cohort will be completed in 2021. Part 2 will enroll approximately 400 patients across approximately 175 participating study sites in 17 countries, with recruitment beginning in 2022. Results: N/A Conclusions: N/A Clinical Trial Registry: NCT04161885 https://clinicaltrials.gov/ Disclosure: CC has served as a consultant for AbbVie and Bristol Myers Squibb; has participated in speakers’ bureaus for AbbVie and Bristol Myers Squibb; and has received research support from Bristol Myers Squibb. UP has received honoraria from AbbVie and Bristol Myers Squibb. MH has served as a consultant for AbbVie, Agios, Bristol Myers Squibb, Daiichi Sankyo, Jazz Pharmaceuticals, Kura Oncology, Novartis, Pfizer, PinotBio, Roche, and TOLREMO; has received honoraria from AbbVie, Eurocept Pharmaceuticals, Jazz Pharmaceuticals, Janssen, Novartis, and Takeda; and has received research support from Astellas, Bayer AG, BerGenBio, Daiichi Sankyo, Jazz Pharmaceuticals, Karyopharm, Novartis, Pfizer, and Roche. VP has served as a consultant and an advisory board member for AbbVie, has participated in speakers’ bureaus for AbbVie, and has received honoraria from AbbVie. SC has participated in advisory boards for bluebird bio, Viacord, and AbbVie; and has received research support from Bristol Myers Squibb and Karius. DW reports no conflict of interest. SA, BC, QJ, PL, and JW are employees of AbbVie and may hold stock or stock options in AbbVie. Venetoclax is being developed in collaboration between AbbVie and Genentech. AbbVie funded this study and participated in the study design, research, analysis, data collection, interpretation of data, reviewing, and approval of the publication. All authors had access to relevant data and participated in the drafting, review, and approval of this publication. No honoraria or payments were made for authorship. Medical writing support was provided by Laura Ruhge, PhD of Bio Connections, LLC, funded by AbbVie.
Background: Acute myeloid leukemia (AML) is a hematological, clonal malignancy of the myeloid stem cell precursors, which is proliferative and is characterized by clonal evolution and genetic heterogeneity. For adverse risk acute myeloid leukemias, allogeneic stem cell transplantation (allo HCT) is one of the most potent curative options. Unfortunately, disease relapse is still around 40% in the first year after HSCT. In allo HCT, fludarabine is a frequently used agent, mainly in reduced intensity conditioning regimes. It is often combined with busulfan for which dose individualization based on pharmacokinetics with area under the curve (AUC) determination is used successfully for many years. It was already shown that fludarabine exposure might be predictive for the survival in allo HCT and it is suggested that individualized dosing can improve the survival after transplantation within the first year. Actually for the fludarabine dose calculation in adults, only the body surface area is used, which might either result in too high exposition causing increased toxicity or with too low exposure and increased relapse incidence (RI). With the aim to reduce RI we established pharmacokinetic measurements for fludarabine in addition to the established ones for busulfan. Methods: Fludarabine was measured with a validated LC-MS/MS method. The exposure as AUC was calculated for each patient using a three-compartmental model (adapted from Langenhorst et al.) in n = 13 consecutive patients receiving a conditioning regime with fludarabine being diagnosed with the acute myeloid leukemia. Fludarabine was given on days -7 to -2 with a dose calculation based on 30mg/m2 with an infusion rate of 30 minutes. The time points of analysis were 30 minutes, 4h, 6h, and 7h after the end of infusion. In addition patients received peroral busulfan 4mg/kg bw on days -3 and -2 and ATG 10mg/kg bw (Grafalon®) on days -4 to -1. Results: So far, there is no dose individualization based on pharmacokinetic parameters for fludarabine specifically suggested in AML patients. The general study published by Langenhorst et al. that mixed up benign and malignant hematological malignancies, suggests lower fludarabine AUC than we observed in our patients. We have seen that the fludarabine AUC were higher within our patients. In the analysis of Langenhorst et al. the optimal AUC was postulated to be 20 mg*h/l. In our (n = 19) AML patients the median of the AUC was 42.97 (+/− 11.5) mg*h/l, twice as high than the suggested level for an optimal toxicity profile. However, none of our patients experienced any acute toxicity (within 30 days after transplant). Moreover, there was also no major toxicity seen at day 100. So far, we did not see a reduced RI, despite the AUC was much higher than the suggested optimal levels. Conclusions: In a regimen with more (6 vs 4), but lower single fludarabine dosage (30mg/m2 vs 40mg/m2) and a shorter infusion duration (30min vs 60min) resulting in higher AUC (40mg*h/l vs 20 mg*h/l) than previously published, we did not observe an increase in transplant-related toxicity or a lower RI in AML patients. Disclosure: Tharshika Thavayogarajah - Research grant from the Kurt-and Senta Herrmann Foundation Nothing further to declare.
Background: In HSCT, vitamin D deficiency has been associated with increased complications, primarily chronic GvHD, with a potential impact on survival. Results from various studies however, have not been consistent. At our center, vitamin D levels are done in all patients as part of pre-transplant assessment. This analysis was conducted to study the incidence of vitamin D deficiency, its correctability following oral replacement and the impact of vitamin D levels on transplant outcomes. Methods: This was a single center retrospective study. Patients of Acute leukaemia (AL) who underwent fully matched or 9/10 transplants (related/unrelated) between 2008 and 2019 were included. In all patients, vitamin D levels were measured at the time of referral to the transplant unit for HSCT counselling (baseline vitamin D). Vitamin D deficiency was defined as 25-hydroxy vitamin D3 level less than 20 ng/mL. Prior to January 2012, patients with vitamin D deficiency did not receive correction. From January 2012 onwards, those with deficiency received replacement with oral vitamin D (60,000 IU weekly for 8 weeks followed by maintenance with 800 IU/day). For patients who received correction, vitamin D level was repeated after 4 months. Based on vitamin D level within 120 days of transplant (peri transplant vitamin D), patients were categorised as either vitamin D replete (>20 ng/ml) or deplete (≤20 ng/ml). Transplant outcomes were compared between these two groups. Results: One hundred and sixty two patients of AL underwent HLA matched transplants during the study period. Baseline vitamin D levels were available for 145 patients. Of these, 126 (86.9%) were deficient at baseline. One hundred and six out of these 126 patients with vitamin D deficiency (84.1%) received correction. Eighty three patients (78.3%) achieved levels above 20 ng/ml and 11 remained deplete. For 12 patients, repeat levels were not available and these were excluded from subsequent analysis. In all, 31 patients were deplete and 102 were replete in the peri-transplant period. The median peri-transplant serum vitamin D level was 34 ng/ml (range 20.4-102.4 ng/ml) in the replete group and 15.0 ng/ml (7.7-19.8 ng/ml) in the deplete group. Both groups were matched for age, diagnosis, EBMT score and disease risk index (DRI). Between the deplete and replete groups, there were no significant differences in time to neutrophil or platelet engraftment, CMV reactivation, day 100 absolute lymphocyte count, aGVHD, cGVHD and TRM. PFS and OS (figure 1) were also comparable between the two groups. Figure 1: Overall survival in vitamin D replete and deplete groups Conclusions: The incidence of vitamin D deficiency was high in our patients. In a majority of patients, the levels normalized following adequate oral supplementation. Patients who were vitamin D deficient in the peri-transplant period did not have inferior outcomes, suggesting a limited role of vitamin D in influencing transplant outcomes in our patient cohort. Clinical Trial Registry: Not applicable. Disclosure: Nothing to declare.
Background: Relapse remains the most frequent type of treatment failure after allogeneic hematopoietic stem cell transplantation (allo-HSCT). Particularly patients with FLT3 mutations have an increased risk of relapse and thus a lower chance of cure. Hence, there is further medical need to evaluate different maintenance therapies strategies after allo-HSCT regarding their impact on outcome including the classic one with application of prophylactic donor lymphocyte infusion (pDLI) only. Methods: We performed a retrospective study of 132 FLT3-positive patients who underwent allo-HSCT at our center from 2005-2021. Patients in complete remission (CR) were put under maintenance therapy if lacking any signs of higher grade GvHD, severe infection or organ toxicity. Since 2005 pDLIs were used applied in up to three escalating doses with respect to the donor type. Sorafenib was regularly administered from 2018 onwards, starting with 200mg up to 800mg daily for an expected duration of 24 months. Overall survival (OS), progression-free survival (PFS), non-relapse mortality (NRM) as well as adverse events (AEs) were evaluated, retrospectively. Results: We identified 19 patients (15%) from our underlying cohort who were considered for maintenance therapy and of whom eleven were treated with Sorafenib and eight underwent pDLI only. Of the entire cohort, 21 died from early death, 35 suffered from GvHD requiring steroid treatment and 14 were lost to follow up while the rest of the patients was not eligible for other reasons including corresponding comorbidities. Independent of the chosen post transplantation treatment strategy the estimated one- and two-year OS for the entire cohort was 78/70% with a median follow up of 2.1 years (range 7 months-6.3 years). One- and two-year PFS was 72/64%, respectively. Our preliminary analysis points towards an increased therapy efficacy in the Sorafenib group compared to the DLI group. 2 out of 8 patients in the DLI group died, one due to relapse. Up to now, no relapse or death was recorded in the sorafenib group. Further, we did not observe major differences in both groups when regarding AEs leading to similarly therapy interruption in both groups (Sorafenib n = 3, DLI n = 3). Conclusions: Maintenance therapy after allo-HSCT has gained increasing importance in recent years. Our data indicate that Sorafenib and pDLI only are both well tolerated after transplantation. However, patients treated with Sorafenib might have a more prominent survival benefit. In our cohort only a minority of patients was eligible for a maintenance therapy while median follow up differed significantly among the groups. Further studies might elucidate whether more patients are applicable when inclusion criteria were extended. Disclosure: Nothing to declare
Background: Leukemia in infants is rare, and one of the most challenging diseases in pediatric hematology/oncology. Survival rates are poor compared with leukemia in older children despite the use of maximally intensified standard therapies, and the indication for hematopoietic stem cell transplantation (HSCT) is restricted to specific subgroups with poor-risk factors. Methods: We reported a retrospective analysis of a cohort of 39 patients diagnosed with infant leukemia during the period 1990-2020 who received treatment at the Pediatric Hemato-Oncology Service of a tertiary hospital in Madrid, Spain. Results: Within our study period, we diagnosed 39 cases with infant leukemia out of 588 cases of childhood leukemia (incidence of 6.6%). 61.5% were females and 38.5% were males. The median age at diagnosis was 5.8 months (IQR 5.4) and 20 (51.2%) patients were under 6 months of age. A total of 27 (69%) patients were affected by acute lymphoblastic leukemia (ALL), 11 (28%) patients by acute myeloblastic leukemia (AML), and 1 (3%) patient by mixed phenotype acute leukemia. At diagnosis, 12 (30.8%) had leukocytosis >300.000/mm3, 22 (59.5%) presented MLL gene rearrangements and 2 (5.1%) suffered from CNS involvement. Induction failure and relapse occur in 8 (20.5%) and 14 patients (35.9%) respectively. The median duration of first remission before relapse was 4 months (IQR: 6.7). A total of 26 patients (66.6%) received HSCT, 24 (92.3%) in first complete remission. With a median follow-up period of 15 months, the 5-year event-free survival and 5-year overall survival at 5 years was 56.7% (SD 4.6) and 44.9% (SD 4.2) respectively. In a multivariate analysis, younger age at diagnosis was associated with poor outcome (p = 0.027). HSCT reduced the risk of mortality by 81.8% (p = 0.001), with transplant patients in first complete remission having a longer survival compared to the rest (p = 0.002). Transplanted related mortality was 31.6% and relapse after HSCT occurs in 7 patients (17.9%). Conclusions: Most of the infant leukemias diagnosed in our center were ALL. The main risk factors (hyperleukocytosis, MLL) and relapse were found more frequently in the group of patients with ALL, although this difference was not statistically significant. Most of the patients received HSCT, with a favorable impact on overall survival, especially when it was performed in first complete remission. Our results suggest that HSCT seems to be a good and efficient choice of treatment for selected patients. However, there is still a big issue to decide which patient should undergo transplantation and more studies are needed to reevaluate the eligibility criteria for HSCT in this group of patients. Disclosure: nothing to declare
Background: Disease relapse is still the major cause of transplant failure and it is associated with a dismal outcome. In the last years, a better characterization of genomic profile of acute myeloid leukemia made post-transplant target therapies available in cases deemed at high risk of relapse, or frankly relapsed. We report our experience in the use of innovative target therapies (FLT3 inhibitors, venetoclax with or without hypomethylating agents or low dose cytarabine) as pre-emptive therapy in high-risk disease or in cases of molecular or hematological relapse. Methods: We analyzed 24 patients transplanted in our Centre from 2015 to 2021, with a median follow-up of 10 months (range 2.5-70.5). Our study population comprises acute myeloid leukemia cases, the majority of which (18/24) is considered at high risk, either according to ELN 2017 classification (6/24) or due to high-risk clinical features (12/24, hyperleukocytosis, s-AML, advanced disease). Donor was matched unrelated in 79% of patients and peripheral blood stem cells was the graft source in 91.7% of cases. Conditioning regimen was myeloablative in 54.2% of cases. Only 12.5% of patients achieved a complete remission disease status at transplant; the rest of the study group had pre-transplant active disease (41.7%) or minimal residual disease (MRD) positivity (45.8%). Results: Our study population started a post-transplant therapy either for hematological (12/24) or molecular relapse/persistent disease (12/24) and median time to treatment start was 2.4 months. 54.2% of patients was started on FLT3-inhibitors (gilteritinib 3/24, sorafenib 10/24) and 45.8% was started on venetoclax with or without a second drug (venetoclax and hypomethylating agents in 5/24, venetoclax and cytarabine in 3/24, venetoclax single agent in 3/24). We performed a univariate analysis on overall survival (OS) and leukemia free survival (LFS). OS for our study population was 39%, as shown in Figure 1. We observed no significant difference regarding the type of conditioning regimen, the donor type and the graft source. Similarly no differences were seen if, at conditioning before transplant, patients had an active hematological disease or an MRD positivity. On the contrary, a timely beginning of the post-transplant therapy to treat minimal residual disease rather than an overt hematological relapse was significantly associated to a better LFS (P 0.0274). Finally, patients with FLT3 mutated patients are those benefit the most of post-transplant treatment. Figure 1 Conclusions: Post-transplant therapy is feasible and induces a significant improvement in LFS in a very high-risk AML patient population. The clinical benefit seems more pronounced for patients treated with FLT3 inhibitors, especially when therapy is promptly started before an overt relapse. Disclosure: Nothing to declare
Background: Recently, Venetoclax-hypomethylating agents (HMA) combination improved outcomes of untreated and relapsed/refractory acute myeloid leukemia (AML). We hereby report our experience of venetoclax + HMA +/− DLI for post-transplant AML at our center. Methods: We conducted an observational retrospective study to report response rate and safety of venetoclax + HMA in a cohort of AML patients relapsed after HSCT. Venetoclax was administered off-label in association with or added to ongoing HMA therapy. Venetoclax was given after a short ramp-up continuously until response and then in 21 out of 28 days cycles. Dosage was reduced in case of concomitant CYP3A4 inhibitors. Venetoclax was temporarily discontinued in case of grade 4 cytopenia or infection. In case of multiple neutropenic febrile episodes, grade >3 infection or worsening of ECOG > 2, venetoclax was permanently discontinued. Results: From September 2016 to March 2021, 11 patients were treated with venetoclax + HMA for post HSCT AML relapse. Their characteristic are summarized in Table 1. One patient was treated for molecular relapse, all others for hematological relapse. Venetoclax was added to HMA after a median of 2 cycles of HMA (0-10); three patients started directly with the combination therapy; all had active disease at start of venetoclax. Five (45%) patients received DLI, with one infusion per cycle at incremental doses. Reasons for not giving DLI were active GvHD, no availability and previous use. Nine (82%) patients experienced grade ≥3 toxicity (8 hematological, 3 infectious, 1 gastroenteric). Five (45%) patients had to stop venetoclax prematurely after a median time of 35 days (25-46); two of them still proved to have benefited from therapy (1 CRi and 1 MRD negativity after molecular relapse). Both had received DLI, with a median response duration of 1,5 month. Six patients did not permanently discontinue venetoclax, but at least one temporary suspension was required in four of them. Median duration of therapy was 90 days (37-341). Among these six patients, four had NR (3 DLI), one had PR and one achieved CR with MRD negativity, lasting 12 months. Median OS was 122 days from venetoclax start. Conclusions: Venetoclax-HMA combination induced CR in 27% of these heavily pre-treated patients. The major challange lies in optimizing venetoclax therapy due to higher toxicity rates compared to historical pre-transplant cohorts. At end of study, all patients eventually relapsed, suggesting long-term control is difficult without a second HSCT. Further studies focusing on appropriate patient selection and treatment schedule are warranted in this setting. Disclosure: No disclosure
Background: Despite significant therapeutic progress in recent years, acute leukemia that appears early in the first two years of life still have a poor prognosis with high mortality rate as well as a considerable risk of relapse. The bone marrow transplant improved the prognosis especially by integrating Haplo-identical hematopoietic stem cell transplantation (haplo-HSCT). This study, aimed to describe the outcome of a single center experience with HSCT in early childhood acute leukemia in Tunisia. Methods: This was a retrospective study carried out in immune-hematology pediatric department of Tunisia between 2010 and 2020 and including children aged under 2 years old who underwent HSCT for acute leukemia. Results: Among 21 patients included, 11 had acute myeloid leukemia, 9 had acute lymphoid leukemia and one patient had an ambiguous Lineage leukemia. Most patients (n = 20) were in their first complete remission (CR1), only one was in his second remission (n = 1) (CR2). Ten patients (48%) underwent geno-identical HSCT, and 11 (52%) underwent an haplo- HSCT. The median age of the patients was 14 months old with a sex ratio= 0.3. Ten of these patients had a positive MRD before HSCT. Most patients had Thiotepa-busulfan-fludarabine as a conditioning regimen. The graft-versus-host disease prophylaxis was based ciclosporin (n = 10) and post transplant cyclophosphamide + MMF + ciclosporin (n = 11). Four patients presented CMV reactivation. Three patients had a grade III/IV acute graft-versus-host disease (GVHD). Four patients died after relapse following HSCT. One patient died of transplant-related causes. The 2-years overall survival and disease-free survival (DFS) are 79% and 73%, respectively. The haplo-HSCT and geno-identical HSCT offered similar outcome. Conclusions: These results support the fact that HSCT is a great option in the treatment of the early childhood acute leukemias, especially when used in CR1. Further studies should be performed to determine the long-term effects of the HSCT. Disclosure: we confirm that there are no competing interests to declare and no relevant financial or non financial interests to report.
Background: Ph-like ALL is a subtype of ALL with poor prognosis and undefined prevalence in Russian population of adult patients mainly due to diagnostic challenges. Simultaneously, there is no standard approach to the diagnosis and most of the methods used are not financially and technically available in clinical laboratories. Here we present the screening diagnostic algorithm and description of several clinical cases. Methods: The study included 26 patients with B-ALL, median age 31 (19-78) years, who were diagnostically screened for Ph-like ALL from 2020 to 2021. It is cost and time consuming to screen for Ph-like ALL by searching directly for specific translocations with the particular gene-partners and mutations. Due to this reason the screening algorithm used in our laboratory is based on the determination of the signaling pathways involved in a particular case in order to choose targeted therapy (TKI/JAK-inhibitor). Algorithm is based on measurement of B-lymphoblasts’ cell-surface TSLP levels with further screening for rearrangements in genes by DNA-specific FISH probes (ABL1, ABL2, CSF1R, PDGFRB, JAK2, CRLF2) or qPCR (CRLF2) (Figure 1). Results: According to the diagnostic algorithm the Ph-like incidence was 8 of 26 cases (30,7%). The most frequent finding was CRLF2 gene rearrangement (n = 4, 15,4%) (Table 1). Two patients were diagnosed with Ph-like during primary diagnosis of ALL, while others in 1st or subsequent relapses. Five patients proceed to alloHSCT, 4 of them are still in CR at the moment of last follow-up. Two patients received dasatinib after alloHSCT: patient #1 with the prophylactic aim, patient #2 in combination with chemotherapy in 3rdrelapse. Despite early dasatinib administration, patient #2 demonstrated r/r course of the disease after alloHSCT. Patient #3 with both EPOR and CRLF2 rearrangements died in the progression without response to the combination therapy with Inotuzamab ozogamicin and ruxolitinib. At the same time, patients #5 and #8 with CRLF2 rearrangement and extramedullary disease did not receive JAK2-inhibitors and still in CR after alloHSCT for 12 and 45 months, respectively. Conclusions: Diagnosis of Ph-like ALL may not rely on the definite gene expression phenotype, but rather on the identification of a genetic aberration in the signaling related genes. A panel of FISH probes covering the most common translocations can be employed as a screening tool. Our limited clinical experience demonstrated potential resistance even to combination therapies with TKI and JAK inhibitors. Apparently, these patients are likely to benefit from alloHSCT as soon as possible in CR, but optimal strategies are yet to be determined. Disclosure: Nothing to declare
Background: The impact of genetic risk profiles on the outcomes of patients with acute myeloid leukemia (AML) following allogeneic hematopoietic stem cell transplantation (HCT) has not yet been fully established. The objective of this work is to study whether the application of European LeukemiaNet (ELN) 2017 scale improves prognostic risk stratification with respect to ELN2010 and Medical Research Council (MRC). Methods: We included 37 adult AML patients treated with PETHEMA AML chemotherapy protocols and who underwent allogeneic HCT at our institution between June 2017 and November 2021 and were studied at diagnostic using the Spanish PLATAFOLMA PETHEMA. NPM1 and FLT3-ITD were determined by melting curve analysis and standard PCR-EC technique according to Thiede et al (Blood 2002) in ABI 3130 Analyzer (Thermofisher). For NGS, the commercial panel Myeloid SolutionTM (Sophia Genetics) KAPA Kit amplification libraries and sequencing on ILUMINA Myseq platform were used. Variant analysis was performed using DDM software (Sophia Genetics). Baseline demographic, disease characteristics, transplant procedures and mutations by functional groupsare summarized in Table 1. The prognostic risk was established according to MRC, ELN2010 and ELN2017 classification. Results: The majority of the patients (91.6%) had at least one mutation at diagnosis. The median number of mutations was 3 (0-6). Grouped by functional groups, the most frequent were those related to DNA methylation (54.1%) and signaling/kinase pathways (51.4%). The most prevalent were FLT3 (45.9%), IDH1/IDH2 (24.3%), TET2 and NPM1 (18.9%) followed by N-RAS and DMT3A (16.2%). The overall survival (OS) analysis according to ELN17, although not showing statistically significant differences between the 3 groups (LongRank p = 0.219) with a 2-year OS estimate of 100%, 65.8% and 50.2% in the favorable, intermediate and unfavorable group; respectively, establishes greater differences between intermediate and unfavorable group than the risk stratification according to ELN 2010 and MRC (p = 0.702 and p = 0.614, respectively). Progression-free survival (PFS) according to ELN2017 did not reach statistical significance, possibly due to the small number of patients included (p = 0.465) with an estimated 2-year PFS of 50%, 30.8% and 45.8% in the favorable, intermediate and unfavorable group, respectively. Our data show a higher OS in patients with mutations in genes involved in signaling pathways (including FLT3) compared to non-mutated patients with a 2-year OS estimate 71.8 % vs 47.8 %; reaching statistical significance (p = 0.037). In addition, FLT3 mutated patients showed a lower risk of relapse compared to non-mutated patients with a cumulative incidence of relapseat two years of 32.6% vs. 65.1% (p = 0.034). No other individual mutations showed differences in survival or cumulative incidence of relapse. Conclusions: In our series, ELN 2017 shows better prognostic stratification between intermediate and unfavorable groups, although without statistically significant differences with respect to ELN 2010 and MRC. FLT3 mutated patients showed a strikingly lowrisk of relapse, related in part to treatment with FLT3 inhibitors prior and/or after transplant. New studies including a larger number of patients are needed to corroborate our results. Disclosure: Nothing to declare
Background: Acute lymphoblastic leukemia (ALL) is a malignant disease that arises from several cooperative genetic mutations in a single B or T lymphoid progenitor. The collaborative work of the last 60 years has led ALL to be a fatal disease in all of the cases, to a curable disease in more than 90% of cases in developed countries, however in Mexico a high percentage of children with ALL have high-risk relapses and the prognosis is less than 16% with conventional treatments only with chemotherapy, the objective of the present study was to determine the survival of patients treated with bone marrow transplant patients with high-risk relapsed ALL Methods: An observational, analytical and retrospective study was carried out INP patients of either sex from 0 to 18 years old who had received hematopoietic progenitor cell transplantation with a diagnosis of "ALL" and high-risk relapse were included. The patients received an allogeneic transplant with conditioning based on TBI/CFM/VP16: With the statistical program SPSS version 25, descriptive statistics were obtained according to the type of variable. A Kolmogórov-Smirnov test will be performed to fit a normal distribution. Of the qualitative variables, absolute frequencies and percentages were obtained. Survival curves for global and disease-free survival were performed using the Kaplan Meier method and the influence of risk factors will be evaluated using the log-Rank method. Results: A total of 47 patients undergoing stem cell transplantation with a diagnosis of high-risk relapsed ALL were included, with a predominance of males with a male: female ratio of 3: 1, the minimum age of the patients was 3 years and a maximum of 19 years, in 47% of the cases the transplant was performed after the first relapse, in 89% of the cases the leukemias were precursors B. The overall survival was 79 and the event-free survival was 71 with a 5-year follow-up, among the variables that were analyzed as: Initial risk assigned (0.112),Hypodiploidy (0.133), Development of EICHa (0.242), ALL Ph + (0.312), Year in which the TCPH was carried out (0.348), source cell (0.570), Remission number (0.598), CNS + (0.694), 100% compatibility (0.738), Immunophenotype of ALL at Dx (0.825), Relapse rate (0.911). No statistical significance was found. Figure 1. Among the 47 patients, a follow-up of between 1 and 114 months was achieved. For the purposes of the survival study, the Kaplan-Meier method established a maximum limit to the follow-up time of 60 months (5 years), obtaining a probability of that patients survive 5 years after HSCT of 0.71. Conclusions: The survival of children with high-risk relapse ALL in the study was 71% at 5 years compared with the survival of less than 16% of children who received 2nd-line chemotherapy treatment reported by other groups in Mexico. Disclosure: All authors declare that they have no conflict of interest with the publication of this study. Cesar Galván-Diaz, Haydee Salazar-Rosales Gerardo Lopez-Hernandez, Nideshda Ramírez-Uribe, Ángeles Del Campo-Martínez, Yadira Melchor-Vidal, Roberto Rivera-Luna, Alberto Olaya-Nieto, Alberto Olaya-Vargas.
Background: Median age of acute myeloid leukemia (AML) onset is around 68 years old. Allogeneic hematopoietic stem cell transplantation (allo-SCT), the curative landmark for high-risk AML, is generally not recommended to patients older than 70 years due to higher transplant-related morbidity and mortality. However, with recent improvements in transplant procedures, allo-SCT could be offered to thoroughly selected older patients. Here we report favorable outcome in a series of patients aged ≥70 transplanted from HLA-haploidentical donors following treosulfan-based myeloablative conditioning (MAC). Methods: We retrospectively collected data of 4 patients older than 70 consecutively transplanted from 11/2019 to 10/2021 at our Centre. The conditioning regimen (TTF) included treosulfan 10 or 12 g/mq from day -6 to day -4, fludarabine 30 mg/mq from day -6 to day -2 and a single dose of thiotepa 5 mg/kg on day -3. Patients underwent allo-SCT from haploidentical donor with peripheral blood as a stem cell source; graft versus host disease (GvHD) prophylaxis included ATG 1.25 mg/kg on days -3 and -2, post-transplant cyclophosphamide (PTCy) 50 mg/mq on days +3 and +4, mycophenolate mofetil (MMF) and cyclosporine (CsA) from day +5. CMV-positive patients underwent prophylaxis with letermovir until day +100. Results: Patient and transplant characteristics are shown in Table 1. Median time from diagnosis to allo-SCT was 219 days (175-640). Treosulfan dose was 10 g/mq in the 3 patients aged 74-yo and 12 g/mq in the patient aged 71-yo. Median number of days to neutrophil and platelet engraftment was 18.5 (16-22) and 13 (11-23), respectively. Early complications included neutropenic fever (highest grade III), atrial fibrillation (highest grade III) and mucositis (grade II). Two patients developed grade I acute GvHD involving only the skin, fully recovered with low-dose prednisone. One patient developed mild chronic cutaneous GvHD with no indication to treatment. Worsening of cardiac ejection fraction from 52% at baseline to 45% was documented in one patient 400 days post allo-SCT. After a median follow-up of 361 days (51-539), all patients were alive in complete remission, with full donor chimerism. Conclusions: Positive outcome of this case series confirms the feasibility of haploidentical myeloablative allo-SCT for AML patients older than 70 years, with no major complications. The TTF conditioning regimen with single administration of thiotepa was very well tolerated. Low dose ATG associated to the classic PTCy/CsA/MMF platform seems to be very effective in preventing both acute and chronic GvHD. Altogether, our experience support the inclusion of fit patients older than 70 years in MAC allo-SCT programs. Disclosure: Nothing to declare
Background: The incidence of most hematologic malignancies increases with age. Aging is related with a greater prevalence of impaired functional status and comorbidities. Although cure of malignant and non-malignant hematological diseases is potentially possible with alloHSCT, it could lead to significant transplant-related mortality. Decision making about referral to allo-HSCT in older adults is a challenging task. In this study we aim to present our geriatric allo-HSCTs. Methods: From 2004 to 2021, 31 patients (age ≥ 60) underwent allo-HSCT in our center included to this retrospective study. Pre-transplant status as well as posttransplant toxicities, complications and outcomes were determined. Results: Patient characteristics and transplant results are summarized in Table-1. Twenty-three patients were between the ages of 60-65, and 8 patients were over 65 years of age. Neutrophil engraftment (>0.5x109 /L) occurred in a median of 19 days (range: 11-38) and platelet engraftment (20x109 /L) in 24 days (range: 15-36). Post-transplant complications are detailed in the table. Acute graft versus host disease (aGvHd) was occured in 9 patients (29%) and chronic graft versus host disease (cGvHD) seven patients (22%) were diagnosed with a relapse and 1 year relapse-free survival was 45%. The 1-year and 2-year OS were detected as 45% and 19%. At the end of the long-term follow-up of the patients, 20 patients died, the most common cause ofdeath was relapse of the primary disease. Conclusions: Since increasing number of older patients being diagnosed with hematologic malignancies, this trend of increasing number of allo-HSCT will continue. Tolerability and effectiveness are lesser, toxicity is higher in older adults. Although study population is relatively small, reduced-intensity conditioning and pre-transplant remission status may be related to beter survival. Comprehensive geriatric assessment may be considered prior to alloHSCT for global evaluation. Disclosure: Nothing to declare.
Background: Allogeneic hematopoietic stem cell transplantation (allo-HSCT) in pediatric acute myeloid leukemia (AML) patients (pts) is widely recommended with an HLA identical donor. This retrospective study describes the long-term results of this procedure in a single-center series, among pts whose age is less than 18 years. Methods: Between September 1998 and December 2020, 161 pts younger than 18 years with AML underwent allo-HSCT. Median age was 11 years (4-17) and sex-ratio (M/F):1. The average time from diagnosis to transplant was 9 months (3-73). At the time of the transplant, 132 pts were in first complete remission (CR1), 21 pts in second CR and 8 pts in active disease. Myeloablative conditioning regimens based on Busulfan were used in 155 pts with Cyclosporine-Methotrexate as prophylaxis of GVHD. Six pts were transplanted from a haploidentical donor with addition of mycophelonate as prophylaxis of GVHD. The grafts used are peripheral blood stem cells in 147 pts with an average rate of CD34 cells:8,07.107/kg (1,5-23,9), bone marrow in 13 pts with an average rate of nucleated cells: 3,33.108/kg, and cord blood in 2 pts with a rate of NC: 4,4.108/kg. At September 2021, the minimal follow-up (FU) was 11 months and maximal FU was 276 months. Results: Aplasia was observed in all pts with median day of neutrophils engraftment was 15 days (13-29). Eighty thirteen pts (60%) required RBC transfusions (1,4 unit/pt) and 150 pts (97%) needed platelet transfusions (1,7 units/pt). Three pts presented a moderate veino-occlusive disease. Acute GVHD occurred in 44 pts (28%) including 34 (22%) grade II-IV. Chronic GVHD was seen in 53 pts (38%) with extensive form in 39 pts. Twenty-one pts (13%) showed CMV reactivation on average at day 72 (19-237). Forty pts (26%) relapsed (26 pts in CR1, 8 pts in CR2 and 6 pts with blast crisis at the time of the transplant). Disease status before transplant (CR1 vs CR2) affect the incidence of relapse (p:0,05). After follow-up of 103 months (11-276), 91 pts (56,5%) are alive in CR and 70 pts (43,5%) died within 30 pts (18,6%) from TRM (acute GVHD:16, severe infection:9, early rejection :2, capillary leak syndrome:1, kidney failure:2) and 40 from relapse (24,8%). The overall survival (OS) and disease free survival (DFS) are 54% and 53% respectively. CR1 is associated with better EFS in univariate analysis (60% vs 29%, p:0,05). Conclusions: In our practice, on the absence of cytogenetics and molecular genotyping analysis, allo-HSCT from an HLA matched family donor became the standard of care for treatment of childhood AML. This retrospective study with long-term follow-up shows interesting results; however, monitoring for long-term complications after HSCT needs special consideration. Disclosure: Nothing to declare
Background: Routine blood counts and donor chimerism studies are monitored regularly after engraftment in patients with severe aplastic anemia (SAA) following HSCT. Mixed chimerism (MC) may be associated with cytopenia, graft rejection/failure or poor graft function Methods: This is a retrospective chart review of patients with SAA who underwent allogeneic HSCT at Nationwide Children’s hospital between 2007-2021. Whole blood and sorted chimerism (CD3 and CD33) were performed using PCR-based testing. Mixed chimerism was defined as donor chimerism of 5-95%. Data on patient demographics, transplant details and outcomes were collected in a secure database and results were analyzed using descriptive statistics. Results: Eighteen patients (10 males) with SAA underwent HSCT, with 14 patients having sufficient chimerism and outcome data to be included in this study. Median age at HSCT was 9 years (range: 4-17); 7 received HSCT from MSD, 5 from MUD and 2 from haploidentical donors. Cyclophosphamide with or without fludarabine, and alemtuzumab or rabbit ATG (n = 10) were the common conditioning regimens with low dose TBI used in 4 patients. At day +30, mean whole blood (WB), CD3 and CD33 donor chimerism were 97%, 90%, and 98%, respectively. While the mean CD33 donor chimerism, stayed >95% at all time points studied, the mean CD3 donor rapidly fell and reached a nadir of 46% (range:11-99%) by day +100 (Fig 1). With continued immunosuppression, the CD3 chimerism gradually increased to 74% (range:48-100%) by day +180 and then stabilized around a mean of 85% (range:62-100%) on follow-up. A similar pattern was observed for patients given either Alemtuzumab or rATG. Overall, mixed chimerism were seen in 7/14 patients (50%) in whole blood, 11/14 (79%) in the CD3 fraction and 2/14 (14%) in CD33 fraction on at least one timepoint post-HSCT. One patient with Down syndrome, died from pneumococcal septicemia but had WB, CD3, and CD33 chimerism of 98, 98 and 99% respectively at 4 years after HSCT. Two patients developed recurrence of SAA at 45 and 21 months after MSD HSCT, had donor chimerism of 79 and 91%, 74 and 90%, and 89% and 100% in WB, CD3 and CD33 fractions respectively at the time of recurrence of SAA. One underwent a successful second HSCT while the other is in planning stages for a second HSCT. Cytopenia was noted in 3/14 (21.4%) patients (excluding the 2 with recurrence of SAA) at a mean of 107 days (range 89-130) after HSCT but fully recovered subsequently. Thirteen patients are alive at the time of this report with a mean follow up of 4 years (range 0.5 to 10). Conclusions: After HSCT, patients with SAA showed sustained and stable mean CD33 donor chimerism of ≥95% at all time points while the CD3 chimerism reached a nadir of 46% around day +100, before slowly recovering in all patients. More importantly, there was no correlation between CD3 chimerism at any time point after HSCT with conditioning regimen, serotherapy, development of cytopenia, overall survival, graft failure or recurrence of SAA. Disclosure: All authors have nothing to declare
Background: Bone marrow failure (BMF) is the main cause of morbidity and mortality in Fanconi Anemia (FA) patients. Hematopoietic Stem Cell Transplantation (HSCT) is the only therapeutic option capable to durably correct hematopoiesis. However, HSCT also negatively affect development of post-transplant solid tumors namely especially head and neck or lower gynecological squamous-cell carcinoma (SCC). A previous analysis of 97 FA patients from the Italian national database (Svahn J et al Am J Hematol 2016), demonstrated that the majority of patients developed cytopenia during the follow-up but 32 subjects maintained a mild/moderate cytopenia without need for HSCT. Overall survival (OS) of the whole original cohort was 74.2%. Methods: This is an extension study in which we evaluate, over a longer follow-up (ended on 31/05/2021), the cohort of 32 FA patients who had a marginally compromised hematopoiesis in the original study. Demographic data, presence, grade and trend of cytopenia, defined according to international guidelines, HSCT characteristics and the development of secondary cancer were recorded. Results: Of the 32 patients evaluated cytopenia was present in 90.6% at diagnosis. At the time of the first pathological blood count, 53.1% had mild cytopenia, 40.6% moderate and 6.3% severe; at the end of the follow up of the original study 9.4% had no cytopenia, 43.8% mild, 31.2% moderate and 15.6% severe. Among 32 patients only 26 were evaluable with a median follow-up of 9.5 years (IQR 6.25-12.75, range 0-27.0). 38.5% (10/26) maintained stable cytopenia (1 have no cytopenia, 6 mild, 2 moderate, 1 severe), in 11.5% (3/26) cytopenia worsened, while 50% (13/26) underwent HSCT. Indications for HSCT were: progression of BMF (10/26, 76.9%) clonal evolution (2/26), unknown (1/26). Donor type was: matched-sibling (MSD) (46.2%), alternative (AD) (23.1%) or haplo (15.4%). The source of cells was bone marrow (38.5%), peripheral blood (30.8%) and bone marrow + cord blood from the same donor (15.4%). One patient developed a uterine cervix SCC and two women a precancerous lesion (in both cases HPV-related low-grade squamous intraepithelial lesions of uterine cervix). They were all FANCA mutated and none underwent HSCT. The 5.8 years OS of this cohort was 92.3% (2/26 died for transplant-related mortality). Conclusions: This extension study confirms that in FA cytopenia tends to progress overtime but that a proportion of patients maintain a stable level of hematopoiesis. These data outline the need for a close and prolonged follow-up to identify the more suitable moment to transplant FA patients, considering the different variables, like the available donor, the HLA matching and the patient’s comorbidities. Disclosure: Nothing to declare
Background: Aplastic Anemia is a benign disease associated with significant morbidity and mortality in severe forms. For those patients without an HLA-identical donor or not responding to immunosuppression treatment, a related HLA-haploidentical donor is an immediately available donor source. Methods: Retrospective study of patients with a diagnosis of severe or very severe aplastic anemia who underwent haploidentical stem cell transplantation (Haplo-SCT) using post-transplant cyclophosphamide (PT-Cy) between 2016 and 2021 from Argentine centers affiliated to GATMO-TC. We analyzed overall survival, the cumulative incidence of acute and chronic graft versus host disease (aGVHD and cGVHD), and graft failure. Results: A total of 20 patients were reported (median age 19; IQR 12-27; 65% below 21 years), without response to immunosuppression treatment (n = 18). The median interval from diagnosis to transplant was 9.72 months (IQR 5.5 – 14.9) and the median pre-transplant serum ferritin level was 2956 ng/ml (IQR 1532 - 3355). Donors (median age 32.8 years) were: parents (n = 12; 60%), siblings (n = 7; 35%) and children (n = 1; 5%). Stem cell source was peripheral blood (n = 11; 55%) and bone marrow (n = 9; 45%). The conditioning regimen was predominantly a combination of fludarabine, Cy, and low dose total body irradiation (TBI) (n = 15; 75%). All patients received PT-Cy, antithymocyte globulin was used in 11 patients (55%), and all patients received either tacrolimus (n = 12; 60%) or cyclosporine (n = 8; 40%). The cumulative incidence at day 28 of neutrophil recovery was 95% (median 16 days), and day-28 platelet recovery of more than 20,000/microliters and 50,000/microliters was 80% (median 18.5 days) and 65% (median 24.5 days), respectively. Two patients developed primary graft failure and secondary graft failure, respectively; both received bone marrow as a stem cell source. The cumulative incidence of aGVHD at day 100 was 42.3% (Grade I: 12.5%; Grade II: 75%; y Grade III: 12.5%); with a trend to a higher rate with peripheral blood (54.5% vs 27.5%; p = 0.074). One-year cumulative incidence of cGVHD was 26.1% (Mild: 57.6%; Moderate: 11.1%; and Severe: 33.3%). Two-year overall survival was 84.4% (CI95% 59-95). Variables associated with better survival were: age below 21 years (100% vs 57%; p = 0.020), tacrolimus-based prophylaxis (100% vs 62%; p = 0.041) and conditioning regimen Baltimore-type vs others (93% vs 60%; p = 0.079). Death causes were: refractory GVHD (n = 1), primary graft failure and sepsis (n = 1). Conclusions: Haplo-SCT with PT-Cy is an option for patients without response to immunosuppression treatment and without an HLA-identical donor. Advantages such as the widespread availability and a lower cost of graft acquisition compared with other alternatives sources make Haplo-SCT the best choice. Some key factors such as conditioning regimen and GVHD prophylaxis might have an impact on the results of the procedure. Disclosure: Nothing to declare.
Background: Pure red cell aplastic anemia (PRCA) is a heterogeneous syndrome characterized by a simple erythroid hematopoietic disorder. The main clinical symptom is anemia. Most patients become transfusion dependent, and are at considerable risk for infection and of the syndrome to evolve into leukemia. At present, about 50% of patients can achieve remission,mainly through immunosuppressive therapy. Hematopoietic stem cell transplantation (HSCT) has also been used in patients who are unresponsive to glucocorticoids, but data on outcomes on these patients are scarce in China. Methods: We retrospectively analyzed the clinical characteristics of 6 patients with PRCA who underwent HSCT in our hospital from May 2019 to November 2021. Two patients had a congenital form of PRCA and 4 patients had acquired PRCA. All patients were unresponsive to glucocorticoids prior to HSCT. The male to female ratio was 2:4. The median age was 30.5 months (range: 17-42 months). The median disease course from diagnosis to transplant was 29 months (range: 13-42). Four patients received umbilical cord blood-HSCT. The conditioning regimen was fludarabine (40mg/m2/d×5days IV) + Ara-C (2g/m2/d×5days IV) + Bu(0.8mg-1.2mg/Kg Q6h×4days) + cyclophosphamide (1.8g/m2/d×2days) +ATG (thymoglobuline, Sanofi, 1.25mg/Kg/d×2days). One patientunderwent a matched unrelated donor (MUD) HSCT. Another patientunderwent a haplo-HSCT with the following conditioning regimen: fludarabine (40mg/m2/d×3days IV) + Ara-C (2g/m2/d × 3days IV) + Bu(0.8mg-1.2mg/Kg Q6h×4days) + cyclophosphamide (1.5g/m2/d×2days) +ATG (thymoglobuline, Sanofi, 1.25mg/Kg/d ×4days). For GVHD prophylaxis, we used CsA/tacrolimus plus sMTX for all of the patient. Results: All 6 patients tolerated the pre-treatment well. The total implantation rate was 100%. The median time of neutrophil engraftment was 12.5 days (range: 11-21 days). The median time to platelet engraftment was 18.5 days (range: 11-75 days). During the median follow-up period of 25.5months (range: 6-30 months), all patients had complete donor chimerism and became transfusion-independent by subsequent normalization of hemoglobin levels. The incidence of CMV was 50% (3 of 6 patients). Following transplantation, EBV DNA load was undetectable among all of patients who were infected with EBV. Grade II aGVHD occurred in 1 patients (16.7% incidence) . One patients had cGVHD. All patients survived, with an overall survival rate of 100%. Conclusions: In our small cohort of 6 PRCA patients, we demonstrate that HSCT has a high implantation rate and overall survival rate, and may become a viable therapeutic option for these patients. Nevertheless, because of the risk of transplant-related death, careful selection of HSCT candidates and the optimal timing of HSCT is necessary. Future studies need to examine the PRCA patient characteristics that are likely to result in a successful HSCT as well as the optimal time for a patient to undergo transplantation. Disclosure: Nothing to declare
Background: Autologous haematopoietic stem cell transplantation (AHSCT) is a well-established therapeutic option for severe systemic sclerosis (SSc), however application is limited by treatment-induced cardiotoxicity apparent during conditioning, particularly in the setting of established cardiac disease. In this study we examined the impact of cyclophosphamide conditioning therapy on cardiac enzymes over the transplantation period. Methods: The records of all patients from 2017 who underwent AHSCT for SSc at our hospital were retrospectively reviewed to identify those with cardiac enzyme measurement pre- and during conditioning therapy. All patients who received treatment were enrolled in AHSCT clinic trials approved by the St Vincent’s Hospital ethics committee. Statistical analysis was performed by one way analysis of variance (ANOVA) or Mann-Whitney U test. Results: Chart review identified 22 patients with measurement of proBNP and/or troponin I during conditioning. Four patients underwent a repeat AHSCT procedure, giving a total of 26 conditioning episodes. All patients received cyclophosphamide (Cy) conditioning at doses between 50-200mg/kg divided over 4 days (D-5 to D-2); patients who received <200mg/kg had either pre-existing cardiac disease or other significant risk factors. Patients who received lower doses of Cy conditioning had significantly higher NTproBNP at baseline compared to the standard dose group (265 vs 67.5 ng/L, p < 0.01). In all patients, NTproBNP was significantly increased from D-5 to D-1 compared to baseline (p < 0.01) with a peak at D-1. Peak NTproBNP was higher in the lower dose group compared to the standard dose group, however this was not statistically significant. The relative increase of NTproBNP was higher in the standard dose group (1329% vs 271%), however this did not reach statistical significance (p = 0.09). Following transplantation, the median NTproBNP returned to baseline. There was no significant changes in troponin I during Cy conditioning. Conclusions: In summary, these data suggests that Cy conditioning induces markers of cardiac stress in an already high-risk population. Further data are needed to explore the relationship between relative NTproBNP increase during conditioning and Cy dose. Measures to reduce cardiovascular risk during the conditioning period are warranted. Clinical Trial Registry: ACTRN12617000216314 https://www.anzctr.org.au/Trial/Registration/TrialReview.aspx?id=371990. Disclosure: Nothing to declare
Background: RAS-associated lymphoproliferative syndrome (RALS) is a relatively new entity causing defects in intrinsic pathways of apoptosis. Although management has been mainly with immunosuppression, we report haematopoietic stem cell transplant for refractory autoimmunity and for lupus-associated nephritis. We report persistent lupus nephritis despite achievement of fully donor myeloid and B-lymphoid engraftment and speculate that residual recipient T-cells are responsible for persistent disease manifestations. Methods: Retrospective review of the patient’s case records and laboratory investigations was undertaken. Results: An 18 months old boy presented with autoimmune haemolytic anaemia and immune thrombocytopenia with leucocytosis and monocytosis. He had a gross splenomegaly, a leucoerythroblastic blood picture and raised fetal haemoglobin raising concerns of underlying Juvenile myelomonocytic leukemia (JMML). Further investigations revealed a high titre of anti-double stranded DNA antibodies and antinuclear antibodies. Investigations for Autoimmune lymphoproliferative syndrome (ALPS) were negative. Bone marrow assessment revealed hypercellular marrow with no increase in blast population with normal cytogenetic studies. Next generation sequencing from peripheral blood revealed a somatic gain of function mutation of NRAS leading to a diagnosis of RALS. The immune cytopenias were refractory to treatment with steroids and multiple immunosuppressants. In view of refractory autoimmunity, he was treated with a matched sibling donor(MSD) bone marrow transplant with Fludarabine, treosulfan and thiotepa conditioning at 30 months of age. He engrafted at 22 days after transplant with a donor chimerism of 90%. However, there was a rapid loss of donor chimerism with concurrent autologous haematopoietic reconstitution. Consequently, he had recurrence of primary disease manifesting as monocytosis with splenomegaly alongside lupus nephritis.On account of refractory and severe, biopsy-proven lupus nephritis induced by the underlying RALD, he had a second MSD bone marrow transplant from a different sibling at 38 months of age with Fludarabine, busulfan and alemtuzumab conditioning. He remains engrafted with 100% donor myeloid and B-lymphoid chimerism, but with persistent rising mixed T cell chimerism. He has relapsed, persistent lupus nephritis and a recurrently raised titre of anti-DS-DNA antibodies, despite treatment with steroids, immunosuppresants, Rituximab, Bortezomib. After a diligent multidisciplinary input, it is decided to use Daratumomab, a CD38 directed monoclonal antibody in an attempt to eradicate the recipient T cells, which is inferred to be the principal cause of the underlying lupus nephritis. Conclusions: HCT for treatment of autoimmunity in RALS has not been documented in literature to the best of our knowledge. Further data is needed to ascertain the unique complications associated with HSCT in RALS.While mixed T cell chimerism does not affect outcome in non malignant transplants in general, it is proposed that residual inflammatory activated autologous cells here drive persistent disease manifestations, and that full donor T cell chimersim is required to fully control disease. Disclosure: Nothing to declare.
Background: Hematopoietic stem cell transplantation (HCT) recipients have a high risk of mortality with COVID-19 because of severe immune dysregulation1. Several vaccines have been developed whose safety has been proven, however, infrequent complications include neurological complications2 or autoimmune phenomena3. Moreover, there are still scarce data on its safety and tolerability in recipients of allogenic hematopoietic stem cell transplant4. Methods: We report a case of acute encephalopathy and subsequent thrombotic microangiopathy within two weeks of receiving the first dose of mRNa SARS-COV2 vaccine (Moderna). Results: 68-year-old male recipient of allogenic matched unrelated donor transplantation in 2009 because of AML. Since then in complete remission. As postransplant complications he presented extensive severe chronic sclerotic cutaneous, ocular and pulmonary chronic graft-versus-host disease (cGVHD) that required immunosuppressive treatment with systemic steroids and photopheresis for several years. In addition to chronic kidney disease and adrenal insufficiency for which he was under treatment with steroids until now. He presented to the emergency department with motor aphasia, generalized tremor and fever. The patient had received the first dose of mRNa SARS-COV2 vaccine (Moderna) 20 days earlier. In laboratory tests, hemoglobin 15 g/dL, platelets 212,000/mm3 and leukocytosis of 19,920/mm3 with neutrophilia and lymphocytosis. CT brain scan and lumbar puncture were performed whitout significant alterations. We started antibiotic therapy with Ceftazdime, Ampicillin, Vancomycin and Acyclovir. The study was extended with brain MRI and electroencephalogram, which ruled out ischemic phenomena. Negative onconeurolane and PF4–heparin antibodies. Everything suggested that the most likely cause was acute post-vaccinal encephalitis. We started treatment with high dose steroids and plasma replacements with symptom improvement 48 hours later. All microbiological cultures were negative. One week after admission, anemia and thrombopenia were observed, reaching a platelets 40,000/mm3 with worsening renal function, increase LDH up to 415U/L, uncontrolled hypertension, 4% of schistocytes in blood smears and ADAMTS13 within normal range. Bone marrow study was performed and was in complete remission. Steroids 1 mg/kg/12h and immunoglobulins were administrated with initial response the first week with subsequent worsening after 10 days. Plasma exchange was restarted with clinical and analytical response after two weeks: recovery of thrombopenia, optimal blood presure control and disappearance of schistocytes. All this established a possible diagnosis of thrombotic microangiopathy secondary to post-vaccinal autoimmune phenomena. Conclusions: Hematopoietic transplant recipients who developed COVID-19 have worse prognosis. Vaccination of these patients is one of the best preventive strategies. However, the efficacy and safety of these vaccines in transplant recipients is currently lacking4,5 The most frequently hematological adverse effects are cytopenias and worsening or establishment of GVHD6. This is a patient with severe cGVHD who, after first dose of vaccine, presented with encephalopathy without infectious or ischemic cause, with favourable outcome after immunosuppressive treatment and plasma exchanges. One week later, he presented thrombopenia, anemia, impaired renal function and schistocytosis; test to rule out vaccine induced immune thrombotic thrombocytopenia (VITT) was negative. So we conclude that it was an autoimmune thrombotic microangiopathy, reflecting a complex inumnologic process in patient with cGVHD with the vaccine as the only identified trigger factor. Disclosure: Nothing to declare
Background: CD19-directed CART therapy has become a standard of care in various B-cell malignancies. However, application of CD19 CARTs in CLL has been hampered by the disease-inherent T-cell dysfunction. Here, we show preliminary results obtained with HD-CAR-1, a 3rd generation CD19-directed CART, in patients with high-risk r/r CLL. Methods: HD-CAR-1 is an investigator-initiated trial evaluating efficacy and safety of escalating doses of CD19-directed CARTs comprising CD28 and 4-1BB as costimulatory molecules in patients with advanced B-cell malignancies after fludarabine/cyclophosphamide lymphodepletion. Leukapheresis, manufacturing, administration, patient monitoring and follow-up were all conducted in-house at the Heidelberg University Hospital. Patients with CLL were eligible if they had failed chemoimmunotherapy and at least one pathway inhibitor and/or alloHCT. Results: Between Oct 2018 and Nov 2021, 32 patients were enrolled of whom seven had CLL. Patients with CLL had a median age of 62 years and had received two to ten prior treatment lines. All patients were r/r to therapy with at least one pathway inhibitor, and three patients were in addition r/r to alloHCT. TP53 abnormalities were present in six of seven patients. Disease status at lymphodepletion was CR in three patients, PR in two patients, SD in one patient and PD in one patient. Despite heavy pretreatment, leukapheresis yielded sufficient T-cell numbers for production in all instances. CART manufacturing was successful for all seven patients. Dose levels administered were I ( = 0.1x107 CARTs/m2) in one patient, II ( = 0.5x107 CARTs/m2) in one patient, and V ( = 10x107 CARTs/m2) in five patients. Rapid CART expansion was observed in four of five patients evaluable so far. Peak levels ranged between 37,792 and 369,756 copy numbers/µg PBMC DNA and correlated with administered CART dose level. Toxicity was moderate with a single case of CRS > G2 and no severe neurotoxicity. However, prolonged G4 neutropenia occurred in one of five patients with ANC recovery on day +32. Responses were observed in all five patients evaluable for response with CRs in three patients treated at dose level V. Conclusions: Homebrewed 3rd generation HD-CAR-1 CART could be successfully generated for heavily pretreated patients with high-risk CLL and exerted a promising safety and efficacy profile. Clinical Trial Registry: ClinicalTrials.gov Identifier: NCT03676504 Disclosure: P.De.: received an honorarium for a scientific presentation by MSD. M.S.: funding for collaborative research from Apogenix, Hexal and Novartis, travel grants from Hexal and Kite, financial support for educational activities and conferences from bluebird bio, Kite and Novartis, board member for MSD and (co-)PI of clinical trials of MSD, GSK, Kite and BMS, as well as co-Founder and shareholder of TolerogenixX Ltd. P.Dr.: consultancy for AbbVie, AstraZeneca, Gilead, Janssen, Novartis, Riemser, Roche; speakers bureau for AbbVie, Gilead, Novartis, Riemser, Roche; research support from Neovii and Riemser. C.M.-T.: consultancy Advisory Board for Pfizer and Janssen, and has received grants and research support from Pfizer, Daiichi Sankyo, BiolineRx and Bayer AG. A.S.: travel grants from Hexal and Jazz Pharmaceuticals, research grant from Therakos/Mallinckrodt and co-founder of TolerogenixX LtD. M.-L.S.: consultancy for Gilead. All other authors report no potential conflicts of interest.
Background: ZUMA-7 (NCT03391466) is a global, randomized, Phase 3 trial of axicabtagene ciloleucel (axi-cel), an autologous anti-CD19 chimeric antigen receptor T-cell (CAR T) therapy, vs. standard of care (SoC: salvage chemoimmunotherapy followed by high-dose therapy with autologous stem cell rescue [auto-SCT] for responders) in second-line large B-cell lymphoma (2L LBCL). Axi-cel demonstrated a statistically significant and clinically meaningful improvement in event-free survival, and despite 56% of the SoC arm receiving third-line (3L) CAR T therapy, a trend toward improved OS was observed. This study estimated the cost-effectiveness of axi-cel versus SoC from a third-party US payer perspective in the 2L setting. Methods: We developed a three-state partitioned-survival model to estimate lifetime cost and benefits. Health care resource use, adverse event (AE) rates, and survival data were taken from the ZUMA-7 trial where possible, costs (including acquisition, administration, monitoring, auto-SCT, 3L treatment [including 3L CAR T after SoC treatment], and AEs) from published sources (in 2021 USD), and utilities from the literature. Long-term EFS and OS were estimated using mixture cure modelling methods. A 3% discount rate was applied to costs and health effects. The model estimated expected life years (LYs), quality-adjusted life years (QALYs), total costs, and the incremental cost-effectiveness ratio (ICER). Additional model outputs included health state occupancy, spending on 2L/3L treatments and granular costs. One-way and probabilistic sensitivity analyses were performed to test model robustness. Results: The model projected median OS was 59 months for the axi-cel arm and 25 months for the SoC arm (Figure 1). Incremental LY and QALY gains for axi-cel versus SoC were 1.34 and 1.37, respectively. The discounted incremental cost for axi-cel versus SoC was $119,055. Despite the higher upfront treatment costs with axi-cel, the high cost of 3L treatment in the SoC arm due to CAR T use was one key cost category that reduced the difference in cost between the two arms (axi-cel: $144,281; SoC: $373,162; difference: -$228,341). Incremental costs and QALY differences resulted in an ICER of $87,026/QALY versus SoC. At a willingness-to-pay threshold of $150,000/QALY, probabilistic sensitivity analysis demonstrated that axi-cel has a 72% probability of being cost-effective versus SoC. Figure 1. Projected 5-Year Survival Outcomes for Axi-Cel and SoC Table 1. Discounted Cost-Effectiveness Results Abbreviations: Axi-cel: axicabtagene ciloleucel; SoC, standard of care; LYs, life-years; QALYs, quality-adjusted life-years Conclusions: Findings from this study suggest a sizable improvement in quality and length of life after axi-cel compared to SoC. While incremental costs are higher with 2L CAR T use, the offsets in 3L CAR T use lead to a limited incremental cost difference resulting in a highly cost-effective ICER by US standards. In addition to meaningfully improving key clinical endpoints, axi-cel is a cost-effective treatment option that can address an important unmet need. Clinical Trial Registry: Not applicable Disclosure: JTS, SV, and AP are employees and shareholders of Kite Pharmaceuticals. FE, RB, and NS received consulting fees from Kite Pharmaceuticals. MP: employment with Memorial Sloan Kettering Cancer Center; honoraria from AbbVie, Astellas, Bellicum, Celgene, Bristol Myers Squibb, Incyte, Karyopharm, Kite, a Gilead Company, Miltenyi Biotech, MorphoSys, Nektar Therapeutics, Novartis, Takeda, VectivBio AG and Vor Biopharma; consultancy or advisory role for Merck, Omeros, OrcaBio; research funding from Incyte, Kite, a Gilead Company, and Miltenyi; and other relationships with DSMB, Cidara Therapeutics, Medigene, Sellas Life Sciences and Servier. JK: honoraria from Amgen, AstraZeneca, Bristol Myers Squibb, Celgene, Gilead, Janssen, Karyopharm, Merck, Novartis, Roche, and Seattle Genetics; consultancy or advisory role for AbbVie, Bristol Myers Squibb, Gilead, Karyopharm, Merck, Roche, and Seattle Genetics; and research funding from Roche and Janssen. PJ: nothing to disclose.
Background: Prediction and management of CAR-T cells specific toxicities, mainly cytokine release syndrome (CRS) and immune effector cell–associated neurotoxicity syndrome (ICANS), represent a challenge for physicians. An emerging role is being assigned to the interaction between inflammation and endothelium. In the setting of allogeneic stem cells transplantation, Endothelial Activation and Stress Index (EASIX) can predict endothelial impairment and patients outcomes. We aimed to assess the predictive value of a modified EASIX (mEASIX) towards occurrence of CRS. Methods: m-EASIX was calculated as described by Pennisi et al (BloodAdv 2021) as [CRP (mg/dL) x LDH (mg/dL)/ platelets count (109 cells/L)]; we applied log transformation using base 2 (log2) in order to reduce skewness. We retrospectively analyzed 33 patients with DLBCL/PMBL treated in the two adults CAR-T centers in Rome from 2019 to 2021 both with tisa-cel and axi-cel. mEASIX was assessed at baseline, and 2 and 4 days after CAR-T cells infusion. We then explored the role of procalcitonin and the ability of mEASIX to predict outcomes. Results: Seven patients had no CRS, seven had a grade 1 CRS, while 19/33 (56%) patients had grade 2-3 CRS. Patients with grade 2-4 CRS had higher mEASIX at baseline (median -0.9 vs 4.5, p > 0.001) and at day 0, 2 and 4. In order to assess if mEASIX calculated on day 2 or 4 after CAR-T cells infusion could predict the development of a grade 2-4 CRS on the same day, we performed a ROC analysis and identified that mEASIX of 6.4 had 100% sensitivity and 71% specificity in predicting grade 2-4 CRS. An mEASIX of 6.4 or more at day 2 or 4 had associated with grade 2-4 CRS in 27% of cases during the same day (vs 2% among patients with mEASIX lower than 6.4, p = 0.002). Moreover, higher mEASIX during day 2 and 4 also predicted CRS 2-4 anytime during hospitalization. Procalcitonin (PCT) was not different in patients with CRS graded 0-1 vs 2-4 (0.16 vs 0.21 ng/ml, p = 0.11). However, patients with PCT > 0.5 ng/ml had higher mEASIX (median, 4,6 vs 7,8, p = 0,001).Patients with higher mEASIX (>6.4) were more likely to develop grade 3-4 cytopenia between day 30 and 90. Progression free survival was similar both for patients who experienced grade 0-1 vs 2-4 CRS (p = 0.58) and for patients with mEASIX higher or lower than 6.4 at any timepoint. Conclusions: In this analysis, anyway, we confirmed that baseline mEASIX correlated with higher risk for developing grade 2-4 CRS. Nevertheless, patients with higher mEASIX had also higher procalcitonin, thus infectious etiology of hyperpyrexia still needs to be carefully investigated when CRS is suspected. With the limitations of our study, we were not able to confirm the correlation between mEASIX and ICANS and PFS as reported elsewhere. We identified a daily cut-off of mEASIX as able in forecasting the development of a CRS of grade 2-4; this information could alarm the physician and lead to intensified patients monitoring before severe CRS occurrence, possibly facilitating an immediate intervention. Disclosure: Nothing to declare.
Background: Axi-Axi-cel, an autologous anti-CD19 chimeric antigen receptor (CAR) T-cell therapy, is approved for treatment of patients with relapsed/refractory LBCL with ≥2 prior therapies. In the 2-year analysis of ZUMA-1 (NCT02348216), the objective response rate (ORR) was 83%, with a 58% complete response (CR) rate (Locke et al. Lancet Oncol. 2019). After ≥4 years of follow-up, median OS was 25.8 months with a 4-year OS rate of 44% (Jacobson C, et al. ASH 2020. #1187). Event-free survival (EFS) has emerged as a robust surrogate of OS in hematologic cancers. Here we report updated survival results from phase 2 of ZUMA-1 after ≥5 years of follow-up, including an exploratory evaluation of the association of 5-year OS and EFS at 12 and 24 months.cel, an autologous anti-CD19 chimeric antigen receptor (CAR) T-cell therapy, is approved for treatment of patients with relapsed/refractory LBCL with ≥2 prior therapies. In the 2-year analysis of ZUMA-1 (NCT02348216), the objective response rate (ORR) was 83%, with a 58% complete response (CR) rate (Locke et al. Lancet Oncol. 2019). After ≥4 years of follow-up, median OS was 25.8 months with a 4-year OS rate of 44% (Jacobson C, et al. ASH 2020. #1187). Event-free survival (EFS) has emerged as a robust surrogate of OS in hematologic cancers. Here we report updated survival results from phase 2 of ZUMA-1 after ≥5 years of follow-up, including an exploratory evaluation of the association of 5-year OS and EFS at 12 and 24 months. Methods: After leukapheresis, eligible patients received conditioning chemotherapy followed by a target dose of 2×106 anti-CD19 CAR T cells/kg. The primary endpoint was ORR. Comparisons of OS by EFS, defined as the time from axi-cel infusion until progressive disease (PD), initiation of new lymphoma therapy (excluding stem cell transplant [SCT]), or death by any cause, were analyzed via Kaplan-Meier estimates. Results: As of August 11, 2021, 101 patients received axi-cel with a median follow-up of 63.1 months. With ≥5 years of follow-up, median OS was 25.8 months with a 5-year OS rate of 42.6% (95% CI, 32.8-51.9). The median OS among complete responders was not reached (5-year OS rate, 64.4% [95% CI, 50.8-75.1]). Since the 4-year analysis, 1 death and 1 PD were observed. Median EFS was 5.7 months, with a 12-mo EFS rate of 42.8% (95% CI, 33.0-52.3) and a 24-mo EFS rate of 37.7% (95% CI, 28.3-47.2), respectively. In patients with an EFS event by Month 12 (EFS12; n = 57) versus without EFS12 (n = 44), 5-year OS rates were 5.3% (95% CI, 1.4-13.2) versus 90.9% (95% CI, 77.6-96.5), respectively. In patients with an EFS event by Month 24 (EFS24; n = 62) versus without EFS24 (n = 39), 5-year OS rates were 11.3% (95% CI, 5.0-20.5) versus 92.3% (95% CI, 78.0-97.5), respectively. Since the 4-year analysis, there have been no new safety signals. Overall, 34 patients (34%) were still alive and received no subsequent therapy (excluding SCT) or axi-cel retreatment; median time to next therapy was 8.7 months, as previously reported. Of treated patients, 59 (58%) have died, primarily due to PD (45%; n = 45), followed by other reasons (9%; n = 9), adverse events (4%; n = 4), and secondary malignancy unrelated to axi-cel (1%; n = 1). Median peak CAR T-cell levels were numerically higher in patients with ongoing response at Month 60 and were considerably lower in patients who relapsed and nonresponders. A similar trend was observed with CAR T-cell expansion by area under the curve from Day 0 to 28. Conclusions: In this long-term survival analysis of ZUMA-1 with ≥5 years of follow-up, axi-cel induced long-term OS with no new safety signals. Axi-cel demonstrated longer OS in patients without EFS12 and EFS24 versus patients with events at these timepoints. These data potentially support EFS as a surrogate endpoint for long-term OS in refractory LBCL. Clinical Trial Registry: Clinical Trial Registry: NCT02348216 https://clinicaltrials.gov/ct2/show/NCT02348216 Disclosure: Caron A. Jacobson: honoraria from Kite, a Gilead Company, Celgene, Novartis, Bluebird bio, Epizyme, Humanigen, Pfizer, Precision BioSciences, Nkarta, Lonza, and AbbVie; consultancy or advisory role for Kite, a Gilead Company, Celgene, Novartis, Pfizer, Humanigen, Precision BioSciences, Nkarta, Bluebird bio, Lonza, Pfizer, Ispen and AbbVie; speakers’ bureau participation for Axis and Clinical Care Options; research funding from Pfizer; and travel support from Kite, a Gilead Company, Celgene, Novartis, Precision Biosciences, Lonza, Pfizer, and Humanigen.
Background: CAR-T therapy has improved the outcome of pediatric patients with R/R B-ALL. However, about half of them (41-51%) relapse. There is scarce information about treatment and prognosis in patients relapsing after CAR-T. The aim of the study is to describe the type of relapse, management and outcome in pediatric and young adults patients with R/R B-ALL relapsing CD19 CAR T-cells. Methods: We analyzed a cohort of consecutive patients under 25 years relapsing after CAR19 T-cells in a single center from Jan-2016 to Nov-2021. Results: Fifty-six patients were infused (Tisagenlecleucel: n = 39, ARI_0001-cells: n = 17) and 51 (91%) achieved complete remission (CR) with negative measurable residual disease. Twenty-one (42%) relapsed at a median of 8.9mo (range:2.2-28.4), 5 beyond 1 year. Thirteen patients (62%) had CD19- relapses, all in bone marrow (BM); median 4.9mo (range:2.2-18.6). Eight patients had CD19 + relapses, median 15.1mo (range:3.0-28.4); 3/8 in BM, 2 combined (BM and CNS) and 3 isolated extramedullary disease (EMD) (testes, breast, subcutaneous). All patients with CD19- relapses had persistent B-cell aplasia (BCA) whereas among CD19 + relapses all except two with isolated EMD had lost BCA before relapse. With a median follow-up of 28.6mo, the cumulative incidence of relapse at 24mo was 30.7%(19.5-46.4) Regarding treatment and outcome of patients relapsing after CAR T-cells (Table1), 3 patients relapsing <1mo from data cut-off were excluded. Among 18 relapsed patients, 8 died from progressive disease (PD). Ten patients achieved a subsequent CR (5 with inotuzumab, 3 with chemotherapy and 2 with blinatumomab). Two died from PD; 7/10 were bridged to HSCT. Three died from transplant-related mortality (TRM). Only 5 patients remained in CR at the data cut-off, 4/5 bridged to transplant. Of these, 2 are in remission >1 year after HSCT and 2 patients are <3 months since HSCT. Reinfusion with CAR19 T-cells was performed in 3 patients (CD19 + relapses) and was unsuccessful. Two achieved CR with blinatumomab after reinfusion failure. Five patients are alive with disease: 2 patients with CD19- relapse with a follow-up since relapse <1 month and 3 CD19 + with 1, 2, and 11mo of follow-up, respectively. ª1 patient discontinued inotuzumab and died from PD, 5 achieved CR, 4 were bridged to HSCT, one died from TRM and 3 are alive in CR (28, 3 and 0.5 months after HSCT). bBoth achieved CR, one was bridged to 2nd HSCT and died from TRM, in the other a 2nd HSCT is planned. cReinfusion was unsuccessful in the 3 patients.dOne achieved CR and was bridged to HSCT (<1 mo after SCT at data cut-off). eBoth achieved CR, 1 died from TRM, 1 is alive in CR 27months after HSCT. Conclusions: The outcome of patients who relapse after CART19 therapy is very poor and therapeutic options are scarce. Although CR can be achieved in some patients with inotuzumab or blinatumomab (CD22 + and CD19 + disease, respectively) consolidation with HSCT treatment is needed and long-term remission is very rare. Clinical Trial Registry: CTL019: EudraCT 2013-003205-25/EudraCT 2016-001991-31 and approved tisagenlecleucel; ARI-0001 cells EudraCT 2016-002972-29 Disclosure: A.A.S: consultant or advisory role (Novartis), travel grants (Novartis), S.R.: consultant or advisory role (Novartis, Jazz-Pharma, Shire/Servier, Amgen, Cellectis, Celgene/Bristol Myers Squibb), travel grants (Novartis, Jazz-Pharma, Shire/Servier, Amgen, Celge), honoraria (Novartis, Jazz-Pharma, Celgene, Shire/Servier, Amgen), A.C.: consultant or advisory role (Novartis, Celgene), travel grants (Novartis, Celgene), honoraria (Novartis,Celgene). M-T: consultant or advisory role (Novartis), travel grants (Novartis), honoraria (Shire/Servier), M.J.: Consultant role at Grifols in 2018.
Background: CAR-T cell therapy has been one of the most arresting immunotherapies in the treatment of cancers. With its widening application, a number of characteristic toxicities have also been identified, and amongst them is an increasingly recognized severe toxicity resembling hemophagocytic lymphohistiocytosis (carHLH). Methods: We retrospectively identified and profiled patients who developed carHLH following BCMA CAR-T therapy in our center. For this work, the diagnostic criteria of carHLH proposed by Neelapu et al were adopted. Patients received CAR-T therapy were then divided into 3 groups: patients developed CRS and carHLH (carHLH group), patients developed grade ≥ 3 CRS alone (severe CRS group), and patients developed grade ≤ 2 CRS or no CRS (mild/no CRS group). And factors associated with or predictive of carHLH were explored. Results: Amongst 99 patients treated with BCMA CAR-T cells, a considerable portion (20.2%) have developed carHLH. In the context of CRS (96 patients), we report a carHLH rate of 20.8% (21 patients). Preliminary cytokine profiling revealed a cytokine storm partially resembles that of severe CRS (e.g., elevated levels of IL-4 and IL-6), but significant elevation in the peak levels of IFN-γ and IL-10 were found in carHLH group in comparison to severe CRS group, indicating a different cytokine network lying under the development of carHLH. Thus, further investigation using a broadened panel is being conducted, and the results are still incomplete. Coagulopathy has been proposed to be one of the manifestations of carHLH, but our analysis of related biomarkers showed only a decrease in fibrinogen with a potential risk to bleed, which might alternatively relate to liver injury.Regarding baseline characteristics, disease burden is a prominent factor associated with the development of carHLH (median: 11.04% in carHLH group vs. 1.495% in severe CRS and mild/no CRS groups together). In addition, we found that baseline T:NK ratio in peripheral blood was negatively related to the development of carHLH, the patients who experienced carHLH had a lower T:NK ratio at the baseline (median: 2.8422 in carHLH group vs. 5.894 in severe CRS and mild/no CRS groups together), which is the opposite of one of the previous carHLH study in the context of anti-CD22 CAR-T. Whether this was a coincidence or this represented different features of carHLH following CAR-T therapies of different targets need more investigations to elucidate. Conclusions: Although for now, we generally believe that carHLH develops on the basis of CRS, but additional pathophysiological mechanisms, apart from the shared ones, definitely play a role in the evolution between these inflammatory syndromes. And in view of the life-threatening complications which CarHLH can be associated with, it’s imperative to establish precise recognition and proper management of carHLH. Both require more detailed description of carHLH in different contexts. Clinical Trial Registry: NCT03716856 Disclosure: The research was funded by the National Natural Science Foundation of China (grant No. 81730008, 81770201, He Huang), Key Project of Science and Technology Department of Zhejiang Province (grant No. 2019C03016, 2018C03016-2, He Huang), and Key R&D Project of Zhejiang Science and Technology Department (grant No. 2020C03G201358, He Huang). The authors declare no other potential conflict of interest.
Background: Cytokine detection is an important basis for evaluating Cytokine Release Syndrome (CRS). To study the role of cytokines in CRS evaluation and to make a rapid and simple quantitative scoring system, we conducted this study. Methods: From March 2020 to February 2021, 111 patients with refractory relapsed B-ALL were treated with CD19-CAR T at Hebei Yanda Lu Daopei Hospital. Blood samples were collected after CART treatment D0, D4, D7, D14, D21 and D28.the concentrations of 24 serum cytokines were detected by flow cytometry: IFNγ, IL-1β, IL-2, IL-6, IL-10, IL-12p70, TNFα, TNFβ, IL-4, IL-5, IL-8, IL-17A, IL-17F, IL-22, IL-2RA, MCP-1, MIP-1α, GM-CSF, IL-15, GranzymeB, REG3a, ST-2, TNFRI, and Elafin. The integral formula was made according to the test values of 34 cases, and the test values of the other 77 cases were calculated by the formula. Results: The changes of IL-1β, IL-2, IL-6, IL-10, IL-17F, GM-CSF, ST-2, TNFRI, REG3a and IFN-γ were statistically different at different time points (all P < 0.05). D7-d10 was the peak, and D28 basically returned to normal, which was consistent with the clinical manifestations of the patients, the proportion and cell number of CAR T cells, CD3 + T cells and CD8 + T cells in peripheral blood. It can also distinguish different levels of CRS. The scoring formula = ∑βi*Si, where βi refers to the weight of the ith cytokine; Si refers to the score of the ith cytokine. The score of the ith cytokine is determined according to whether the peak level of the ith cytokine exceeds 3 times of the base value. The 34 patients with different CRS grades were randomly selected, and the weight of each cytokine was found to be: IFN-γ, IL-2, IL-6, IL-10, ST-2, IL-8, and GM-CSF was 2 points. The weight of IL-2RA, IL-17F, REG3a, IL-1β, McP-1 and TNFRI was 1 point. The weight of IL-4, IL-5, IL-22, IL-15 and IL-12P70 was 0.5 points; The weight of Elafin, TNFα and Granzyme B was -1 point. If the score is ≤8, belongs to CRS 0-1; if > 8, < 18, CRS 2; if ≥18, CRS 3-4. Tested in the other 77 patients, the sensitivity was 89.61%, the specificity was 96%, the positive predictive value was 95.83%, and the negative predictive value was 90%. In particular, the sensitivity was 91.55%, the specificity was 99%, the positive predictive value was 98.48%, and the negative predictive value was 92% in the identification of patients with grade 0-1. Conclusions: The evaluation method of immune status after treatment of CART obtained in this study provides an effective tool for simple, scientific and rapid evaluation of high information data, especially for objective judgment and early detection of CRS. Disclosure: Nothing to declare
Background: CD19-directed chimeric antigen receptor (CAR) T-cell has revolutionized the treatment paradigm of relapsed/refractory B-cell Non-Hokdgkin’s lymphoma (B-NHL). Bacterial and fungal infections have been well described, but many questions remain unanswered regarding the role of cytomegalovirus (CMV) in this setting. We studied the incidence and risk factors associated to CMV reactivation and CMV disease. Methods: We retrospectively reviewed the consecutive CMV viral load determinations of B-NHL treated with CAR T-cells from July 2018 to September 2021. CMV-seronegative patients, and previous allogeneic stem cell transplant recipients were excluded. Significant CMV viral load was defined as higher than 1000 IU/mL. Results: Overall, 98 patients met the inclusion criteria. Median age was 61 years (IQR 53-68), and 63 were men (64.3%). Median number of previous therapeutic lines was 2 (IQR 2-3). All patients had a clinical follow-up of 2 months, and 83 patients had at least 3 CMV viral load determinations during the first 2 months after CAR-T treatment. Among patients who fulfilled the inclusion criteria, 44 (44.9%) had at least one positive CMV determination in peripheral blood. Of them, 24 patients (24.5%) had a significant CMV viral load (CMV > 1000), and in 7 patients it was greater than 10000 UI/mL (Log > 4). Six patients were excluded from the analysis for presenting CMV reactivation before receiving CAR-T therapy. Median time from treatment to reactivation was 18 days (IQR 7-15.5), and the median duration of viremia was 8 days (IQR 5-24). Baseline variables as age, median number of previous lines, median number of CD3 cell count, ferritin levels, or D-dimer levels before lymphodepleting chemotherapy were not related with CMV > 1000. The only independent risk factor associated with a higher risk of CMV > 1000 was having received dexamethasone and/or tocilizumab within the first month (72.2% vs. 27.8%; adjusted OR 3.07 [IC 95% (1.2-8.0) p = 0.021]. No relationship was observed with hypogammaglobulinemia, or the product administered. The median lymphocyte count in the moment of reactivation was 0.5 x 109/L (IQR 0.2-0.6 x 109/L). No patient had evidence of CMV disease, and only the patients with viral load superior to 10000 UI/mL received treatment with ganciclovir or valganciclovir. Of these, one presented valganciclovir-induced neutropenia. Conclusions: CMV monitoring can be useful during the first 2 months after CAR T-cell therapy, especially in those receiving dexamethasone and/or tocilizumab. CMV replication has doubtful clinical significance in this setting, so treatment should be carefully individualized assessing risk-benefit in terms of toxicity. Disclosure: We declare no conflict of interest. This study was not funded.
Background: CAR-T cells therapy is an highly effective third line salvage treatment in relapsed/refractory diffuse large B-cell lymphomas (DLBCL), primary mediastinal B-cell lymphomas (PMBCL) and Acute Lymphoblastic Leukemia (ALL), but up to 60% of patients still relapse. CAR-T cells generated from “exhausted” T-lymphocyte (Ly) have been correlate with a worse response. In this prospective study, we assessed the fitness of pre-apheresis Ly for evaluating the impact of previous treatments on the T-cells repertoire. Methods: From January 2021, Ly-subsets were evaluated at the time of leukapheresis in 13 patients: 11 DLBCL, 1 PMBCL and 1 ALL. Since cryopreservation of the apheresed Ly is allowed for Kymriah, 8 DLBCL patients were enrolled in a “pre-emptive” Ly-apheresis program, scheduling leukapheresis as soon as possible in patients selected for the following poor prognostic risk factors: refractory to first line of treatment; PET positivity before ASCT; first complete response less than 12 months. Combinations of monoclonal antibodies directed against CD45RA, CCR7, CD3, CD4, and CD8 were used by Flow Cytometry to evaluate the following CD4 + /CD8 + T-ly subsets: T-naïve (CD45RA + CCR7 + ); T-central memory (CD45RA-CCR7 + ); T-effector memory (CD45RA-CCR7-); T-terminally differentiated (CD45RA + CCR7-). All stained samples were acquired on a Canto II (BD Bioscience) flow cytometer and analyzed using DIVA software version 8.0.2. Continuous variables were compared using Mann-Whitney test. Results: Table1 shows the main patients’ clinical characteristics. Mainly, 8/11 High-Risk DLCBL patients received one line treatment (R-DAEPOCH or R-CHOP), including ASCT according to the Centre policy. Only ASCT had a significative impact on Ly-subsets distribution. In particular, in patients who had not previously received ASCT, CD4/CD8 ratio, percentage of CD4-naïve and CD8-naïve were significantly higher (p = 0,04; p = 0.006 and p = 0.012, respectively). The effects of ASCT on Ly-subsets distribution were detected even after one year from ASCT. No other significant differences were found between patients who received previously ASCT or not, except for age (66 [57-70] vs. 49 [29-59], p = 0.01). Patients with ALL or PMBCL had few circulating naïve T-cells, similarly to the patients who underwent ASCT before leukapheresis. Table1 Conclusions: Previous ASCT in DLBCL patients can result in a significant more percentage of “exhausted” T-Ly at the time of leukapheresis. This may lead to an higher percentage of “exhausted” CAR-T cells and, potentially, to their reduced efficacy. In patients at higher risk of relapse after ASCT a “pre-emptive” leukapheresis should be considered a timely clinical option to cryopreserve more “fit” T-Ly to be sent for manufacturing in case of relapse. Disclosure: Nothing to declare
Background: Chimeric antigen receptor (CAR) T cells targeting CD19 antigen provide a highly effective immunotherapy for B cell malignancies. The anti-tumor effect depends on high proliferative and cytotoxic activity of CAR T cells, hence the importance of proper in vitro assessment of the CAR T cell functionalities. This study compares functional properties of CD19 CAR T cells obtained from patients without a history of HSCT vs. HSCT recipients vs. healthy donors, and CAR-T manufactured with either a short (7 day) or long (11 day) protocol. Methods: The study enrolled patients with relapsed/refractory B cell acute lymphoblastic leukemia who met inclusion criteria for the clinical protocol at the Dmitry Rogachev National Medical Research Center of pediatric hematology, oncology and immunology. A total of 78 cell products were derived from patients who never received HSCT (‘auto’, n = 50), patients who received HSCT within 4 to 24 months before (‘pseudo-auto’, n = 19), or haploidentical healthy donors (‘haplo’, n = 9). The production was performed based on the automated CliniMACS Prodigy® system with the full cycle lasting 11 days (n = 50) or 7 days (n = 28). The lentiviral vector transduction efficiency was determined for all CAR T cell products by direct staining. Functional testing of the final CAR T cell products included degranulation assay (measuring CD107a externalization by CD4 + and CD8 + lymphocytes, n = 33) and TNFα and IFNγ secretion assay upon incubation with the target cell line JeKo-1 (n = 41). The flow cytometry measurements were based on the customary surface and intracellular staining protocols with commercially available antibodies. Cytotoxicity of CAR T cells towards the target cell line JeKo-1 was assessed using the Incucyte® Live-Cell Analysis system. Results: T lymphocytes from pseudo-autologous donors were transduced with lentiviral vectors more efficiently than the cells from autologous donors. Besides, the longer 11-day manufacturing afforded significantly higher transduction efficiency than the shorter 7-day cycles. On the other hand, CAR T cells manufactured over 7-day process showed higher rates of degranulation than the 11-day cycle products. CD 4+ CAR T cells from haploidentical donors showed a higher level of degranulation compared to autologous donors. The rates of cytokine production and cytotoxic activity of CAR T cells were similar in all groups (p > 0.05). Conclusions: The established protocol affords a stable cell product with high anti-tumor activity and functional properties largely independent of clinical history of the donor. Moreover, CAR T cells produced in an expedite time-frame of 7 days exhibit the same (and in certain aspects even higher) anti-tumor activity as the cells obtained in 11-day production cycles. For patients with rapid progression, reduction of the waiting time between apheresis and CAR T cell infusion to 7 days may be vital. Disclosure: Nothing to declare
Background: CAR-T cell therapy is approved for adult patients with relapsed/refractory diffuse large B-cell lymphoma (DLBCL) or primary mediastinal lymphoma (PML) after two or more lines of therapy. Prolonged cytopenias have been reported in 30-60% of patients undergoing CAR-T cell therapy, but information about its dynamics and etiology is scarce. Methods: We conducted a retrospective study of consecutive patients with a DLBCL or PML undergoing commercial CAR-T cell therapy in our center between June 2019 and September 2021. We analyzed blood cell counts pre-lymphodepletion and during hospitalization. This information was correlated with clinical and analytical parameters. Results: 54 patients were included. Median age was 58 years (range 22-79). 34 patients (63%) were treated with Axicabtagene ciloleucel (Axi-cel).Patients had received a median of 2 prior lines of therapy (range 2-6) and 35.2% had undergone a previous HSCT. 47 patients (87%) received bridging therapy (Table 1). TABLE 1. PATIENTS GENERAL CHARACTERISTICS Median hospitalization was 23 days (range 11-87) and 47 patients (87%) showed profound neutropenia (ANC ≤ 500/μL), lasting >7 days in 42 patients (77.8%). 28 patients (51.9%) showed profound thrombocytopenia (≤50000/ μL) and 31 (57.4%) anemia (≤8g/dL). Cytopenias during hospitalization were more frequent with Axi-cel, with higher rates of neutropenia (97% vs 70%; p = 0.004), thrombocytopenia (61.7% vs 35%; p = 0.05) and prolonged cytopenias (90.9% vs 60%; p = 0.007, Figure 1). FIGURE 1. CYTOPENIAS DURING HOSPITALIZATION On day +28 post CAR-T, 28 patients (56%) showed persistent cytopenias: 3 (6%) neutropenia, 9 (18%) thrombocytopenia and 16 (32%) both, with higher rates of cytopenias at day +28 with Axi-cel (36.8% vs 67.7%; p = 0.03). Cytopenias during hospitalization were related to treatment with Axi-cel (p = 0.004) and bridging therapy (p < 0.001), while its prolonged duration was associated with Axi-cel (p = 0.007), bridging therapy (p = 0.011) and progressive disease (p = 0.05). Cytopenias on day +28 were related to Axi-cel (p = 0.03), bridging therapy (p = 0.03), higher ferritin levels (p = 0.01), CRS (p = 0.003) and ICANS (p = 0.05). A multivariate analysis using logistic regression showed that bridging therapy (OR 25.4; 95% CI 2.2-29.1; p = 0.009) and treatment with Axi-cel (OR 17.9; 95% CI 1.3-24.7; p = 0.03) were independent predictors for profound cytopenias during hospitalization, while no independent predictor was found for its duration nor for day +28 cytopenias. Conclusions: Profound cytopenias were frequent in our cohort, with up to 87% of cases during hospitalization and 56% on day +28, being bridging therapy and use of Axi-cel independent predictors for its development. Improving the knowledge on these cytopenias can contribute to a better outpatient management. Disclosure: Nothing to declare
Background: Chimeric antigen receptor (CAR)-T cell therapy has revolutionized the treatment paradigm for children with relapsed/refractory (R/R) B-cell acute lymphoblastic leukemia (B-ALL). Effective treatment of extramedullary central nervous system (CNS) B-ALL with CAR-T cells has been reported, however little is known about the effectiveness of CAR-T cells for bulky CNS disease. While CNS chloromas are an extremely rare presentation of R/R B-ALL, they have been reported, are difficult to treat in heavily pre-treated patients, and are associated with poor outcomes. Herein, we report the effectiveness and safety of CD19-directed CAR-T cell consolidation with tisagenlecleucel in two pediatric patients with B-ALL CNS chloromas. Methods: Medical records of 2 pediatric patients with R/R pre-B-ALL treated with tisagenlecleucel for CNS chloromas at MSKCC were reviewed. Clinical course, treatment, and outcomes are discussed here. Additionally, all pediatric patients who received tisagenlecleucel from 2018-2021 at MSKCC were evaluated based on CNS disease (spinal fluid flow cytometry/cytology and/or presence of chloroma) at time of treatment to evaluate toxicities and clinical outcomes. Results: Patient 1 was diagnosed with standard-risk pre-B-ALL (CNS1) at age 5. She had 2 isolated CNS relapses followed by a concurrent medullary and CNS relapse, which was treated with allogeneic hematopoietic stem cell transplantation (allo-HSCT) and post-transplant intrathecal chemotherapy for CNS prophylaxis. Sixteen months after allo-HSCT, she was found to have a CNS chloroma with a concurrent medullary relapse. She underwent subtotal surgical resection of the chloroma, focal proton radiation (2400cGy), and bridging chemotherapy (vincristine/mercaptopurine) followed by tisagenlecleucel. She experienced minimal toxicities: grade I cytokine release syndrome (CRS) and grade II immune effector cell-associated neurotoxicity syndrome (ICANS) requiring 2 doses of dexamethasone. She achieved a complete response (CR) at 1-month after tisagenlecleucel with a negative brain MRI, and she maintained disease control for 6 months. She developed an isolated CNS2 relapse 6 months post-treatment, responded to a second infusion of tisagenlecleucel with no toxicities, and is now undergoing a second allo-HSCT. Patient 2 was diagnosed with standard-risk pre-B-ALL (CNS1) at age 3. Fourteen years after initial diagnosis, he had a late isolated CNS relapse. He subsequently had a second isolated CNS relapse 3.5 years later followed 2 years later by a third isolated CNS relapse. Fourteen months after his third relapse, brain MRI revealed a CNS chloroma, which was treated with surgical resection, focal proton radiation (2400cGy), and then tisagenlecleucel. He experienced minimal toxicities: grade I CRS and no ICANS. He achieved a CR at 1-month after CAR-T cell treatment. Thirteen months later, he has maintained no evidence of disease. Upon further review of all pediatric patients treated with tisagenlecleucel at MSKCC (n = 20), 30% (n = 6) had CNS disease at time of tisagenlecleucel treatment, of which 33% (n = 2) developed ICANS (both grade II), 33% (n = 2) relapsed after treatment, and 83% (n = 5) remain alive to date. Conclusions: Two pediatric patients with R/R B-ALL with CNS chloromas were successfully treated with tisagenlecleucel, both achieving a CR at 1-month post-treatment. Both patients had minimal treatment-associated CNS toxicities. Our experience highlights consideration of CD19-directed CAR-T cell therapy in pediatric patients with CNS chloromas. Disclosure: Boelens, Jaap J. discloses consulting or advisory roles (Avrobio; Advanced Clinical; Bluerock; Omeros; Race Oncology; Sanofi; Equillium; Medexus; Sobi). Curran, Kevin J. discloses consulting or advisory role (Novartis; Mesoblast) and research funding (Juno Therapeutics; Novartis; Celegene; Cellectis). The remaining authors have no conflicts of interest to declare relevant to this abstract.
Background: The majority of patients with diffuse large B cell lymphoma (DLBCL) undergoing adoptive transfer of CD19-directed CAR T cells have been exposed to multiple rounds of cytotoxic therapies. It has been noted that the quality of the collected T cells is a significant factor determining their toxicity and efficacy. In order to explore the influence of early T cell collection, we have conducted a study comparing T cells parameters of patients who underwent lymphocyte collection after failure of first line therapy for DLBCL (early apheresis) with patients who underwent lymphocyte collection after second line therapy or later (late apheresis). Methods: Patients were assigned to groups according to referral time: early versus late. Blood samples were collected at the day of apheresis, representing the starting material for manufacturing. Immune phenotyping (FACS) was performed for T cell subpopulations, differentiation and exhaustion markers (CD3, CD4, CD8, CD45RA, CCR7, CD27, CD28, TIM-3, LAG-3). T cell activation and proliferation were analyzed using PBMCs labelled with carboxyfluorescein succinimidyl ester (CFSE) and anti-CD3/ CD28 or (phytohemagglutinin) PHA. Results: Thirty-six patients were enrolled: 15 to the early group and 21 to the late group. The mean percentage of circulating CD3 + lymphocytes was 48.9 ± 3.5 and 58.5 ± 1 in the late and early group, respectively (p < 0.05) (Fig.). The early group had a significantly higher proportion of CD8 + lymphocytes (p < 0.05) and a lower proportion of CD4 + lymphocytes (p < 0.01) compared to the late group (Fig.). Among CD4 + and CD8 + T cells, the early samples showed increase of both naïve (p = 0.07, p < 0.005) and central memory T cells (TCM) (p < 0.0005, p < 0.05), and a reduction of effector memory (TEM) (p < 0.05, p < 0.005) and effector T cells (TEF) (p < 0.05, ns), respectively. (Fig.) Both CD4 + and CD8 + T lymphocytes showed higher expression of the exhaustion markers, TIM-3 and LAG-3 in the late apheresis group (p < 0.0001). CD8 + cells analysis revealed a significant increase of senescent CD27−/CD28− T-cells, in the late group compared to the early group (p < 0.05). In the early samples there was a significant increase in T-cell proliferation, in response to either stimulus: anti CD3/CD28 or PHA in both CD4 (p < 0.001) and CD8 (p < 0.05) subsets. Conclusions: Early apheresis, after first line of chemotherapy, shows abundance of T lymphocytes, mainly CD8 + cells, with an improved quality of the starting material, represented by a higher percentage of naïve, less exhausted and senescent cells. Furthermore, early apheresis shows a higher T cell proliferation potential. These improved parameters in the initial collected product may have an influence on the manufacturing process and eventually on the efficacy of the infused CAR T cells. Disclosure: "Nothing to declare"
Background: The program of cellular therapy with chimeric antigen receptor T-cells (CAR-T) has been established in the Czech Republic in 2019. Since then, five centers have been certified for tisagenlecleucel (tisa-cel), axicabtagene ciloleucel (axi-cel), or both. This report summarizes all the treatment that has been administered so far. Methods: All patients treated with commercial tisa-cel or axi-cel in the Czech Republic until August 2021 were included into this retrospective analysis. The data were analysed for overall response rate (ORR) and complete remission (CR) rate, incidence and severity of cytokine release syndrome (CRS) and immune effector cell-associated neurotoxicity syndrome (ICANS), and progression-free (PFS) and overall survival (OS). CRS and ICANS were graded according to ASTCT consensus criteria. Results: A total number of 66 patients were included into this analysis, 51 treated with tisa-cel and 15 with axi-cel. Tisa-cel was infused in 43 patients with B-cell non-Hodgkin lymphomas (B-NHL). ORR was observed in 47% patients, while CR was reached in 35%. CRS occurred in 74% of cases (grade 3 or higher in 16%), and ICANS in 23% (grade 3 or higher in 2%). Six-month PFS and OS estimates were 37% and 52%, respectively. Better PFS was observed in patients with pre-treatment CRP below median (p = 0.02), and in patients who developed CRS (p = 0.03). Tisa-cel was infused in 8 patients with B-cell precursor acute lymphoblastic leukemia (B-ALL), of whom 5 were children and young adults under the age of 18. All patients but one (88%) achieved a CR. CRS occurred in 63% of cases (none grade 3 or higher), and ICANS in 25% (one grade 4). Six-month EFS and OS estimates were 50% and 88%, respectively. Axi-cel was infused in 15 B-NHL patients (including primary mediastinal B-cell lymphoma, PMBCL). ORR was observed in 69% of cases, CR in 46%. CRS occurred in 80% of cases (none of them grade 3 or higher), and ICANS in 33% (grade 3 or higher in 3 patients). Six-month PFS and OS estimates were 69% and 74%, respectively. Better survival was observed in patients with PMBCL but the difference was not statistically significant. Conclusions: Cellular therapy with commercial CAR-T products is a well-established treatment modality for patients with relapsed/refractory B-NHL and B-ALL. Real world data observed in our nation-wide cohort are comparable to already published results of larger patient populations. Disclosure: Nothing to declare
Background: Prolonged cytopaenias are an under-recognised toxicity of Chimeric Antigen Receptor-T cell(CART) therapy for B-cell malignancies(53% of B-ALL patients in the ELIANA study had grade 3-4 neutropenia persisting beyond day 28). Persistent cytopaenias post-CART have been associated with baseline cytopaenias and pro-inflammatory milieu(Rejeski et al, Blood, 2021). Cytopaenias generally resolve spontaneously over 2-3 months post-CART therapy but some patients develop prolonged cytopaenias associated with susceptibility to infection(s) and/or transfusion dependence and a hypoplastic bone marrow(BM). Cytopaenias can be treated with an unconditioned CD34 stem-cell boost in those who have undergone prior stem cell transplant(SCT). We reviewed our institutional data on outcomes with this approach. Methods: Data was retrospectively analysed from paediatric and young adults with relapsed/refractory(r/r) B-ALL treated with CART therapy and who received CD34 stem-cell boost from their SCT donor between May 2016 and December 2021 at 2 centres in the UK. Demographic details, disease status and cytopaenias pre-CD34 stem-cell boost, toxicity and outcomes were collated. Results: Over 5 years, 103 patients received CART therapy for r/r B-ALL. Seven (6.7%) of these patients received an unconditioned CD34 stem-cell boost from their SCT donor due to severe grade 3-4 cytopaenia (pancytopenia in 6/7, bicytopenia in 1/7) beyond 1 month after CART. CD34 stem-cell boost was infused at median of 2.6 months (range 2-16.5m) after CART therapy. All 7 demonstrated BM hypoplasia for age without a clear drug or viral etiology and were MRD negative. Three(43%) had ongoing invasive fungal infections at CD34 stem-cell infusion. Median age at CD34 stem-cell boost was 16 years(range 11-27y). All patients were heavily pre-treated and had undergone previous SCT and 6/7 received CART in > 2 relapse. Four(3 CD19, 1 CD19/CD22 directed) received CAR as per CARPALL study(NCT02443831), 1 received CD19/CD22 directed CAR as per AMELIA study(NCT03289455), 1 received licensed product Tisagenlecleucel and 1 received allogenic CD19 directed CAR as per CARD study(NCT02893189). Median CD34 and CD3 doses in the infused product were 6.75 x106/kg(range 2.5-11.2x106/kg) and 0.19x104/kg(range 0.07-1.22x104/kg) respectively. CD34 boost was well tolerated:1 patient developed grade 2 cytokine release syndrome(CRS) at day 10 but no acute or chronic GVHD was observed. Two patients were not evaluable for response to CD34 stem-cell boost:1 died due to gastro-intestinal haemorrhage caused by disseminated Mucormycosis on day 24 and 1 relapsed at day 38. Of the 5 evaluable patients one had transient recovery of the bicytopenia followed by ongoing cytopenias until demise while 4 patients recovered neutrophils >1x109/L without GCSF, were blood and platelet transfusion independent by a median of day 42 (range 11-192 days), day 33 (range 4-106 days) and day 33 (range 7-73 days) respectively. At a median follow-up of 9 months(range 24 days-2.5 years) from CD34 stem-cell boost, 5 died (2:relapse, 2:infections, 1: further therapy related complications) and 2 were alive, in CR with normal blood counts at last follow up. Conclusions: Unconditioned CD34 stem-cell boost is well tolerated and can ameliorate prolonged cytopaenias post-CART therapy. However, CRS is a potential complication after the infusion due to the presence of CD19+ progenitors within the CD34 selected product. Disclosure: None
Background: Low tumour burden prior to CART-cell infusion for B-ALL is associated with less CRS toxicity and improved survival1,2. The choice of bridging chemotherapy (BCT) is crucial to achieve adequate disease control and avoid toxicity-related infusion delays; however, there is no consensus as to which is the optimal strategy. We have analysed the use of BCT in two of the UK’s largest CART-cell centres in terms of both feasibility to deliver CART-infusion on time and efficacy in reducing large/bulky tumour burden. Methods: We performed a descriptive analysis on restrospectively collected data. BCT was defined as systemic anti-leukaemic treatment given between leukapheresis and lymphodepletion. A BCT course was defined as a particular chemotherapy schedule given with the intent to control disease. Disease burden was measured by flow cytometry or molecular MRD for patients with <5% disease and confirmed by a local consultant in each case. Results: Data was collected from 49 paediatric patients who underwent 70 BCT courses (median number of courses per patient was 1, range 0-3). Patient characteristics are shown in Table 1. Thirty-two patients (65.3%) received their CART-cell infusion on the expected date, whereas 15 patients (30.6%) suffered a delay, 1 patient (2%) died prior to infusion. A total of 20 patients (40.6%) experienced some kind of toxicity, leading to an infusion delay in 15 (75%) of them. The majority (n = 11) of these suffered from an infection or likely infection. Other non-infectious reasons for delay included leukoencephalopathy, small bowel obstruction and difficulties in disease control (n = 3, 20%). BCT regimens were heterogeneous; we have grouped them in 5 categories in decreasing order of popularity: Capizzi-like (n = 28; MTX + VCR as per UKALL-2011 - up to 9 variations were recorded in this cohort), Maintenance (n = 12; oral 6MP + MTX), Inotuzumab (n = 11; single 0.5-0.8 mg/m2 doses to avoid toxicity), HiDAC (n = 7; AraC in doses 9-12 g/m2), and Other (n = 12; including exclusive intrathecal therapy, TKI and other miscellaneous schedules). Maintenance was usually given to hold/maintain low-level disease. Inotuzumab was not given as upfront BCT. Both Inotuzumab and Capizzi regimens seemed effective in debulking disease. Table 1. Patient characteristics. Conclusions: The ideal BCT schedule should achieve adequate disease control with minimal toxicity facilitating timely CART-cell infusion. Capizzi-like and Inotuzumab regimens may be suitable agents to debulk disease. Infection is the most common reason for delays in infusion. Disclosure: No conflict of interest of any of the authors.
Background: COVID-19 disease is caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2); its course can be severe, the overall mortality approaching 1.5%. Patients with malignant diseases and an impaired immune system, especially patients after hematopoietic stem cell transplantation (HSCT) and immune cell therapy, have an increased risk of an aggravated course of the disease or death and despite the expectation of less than optimal vaccine responsiveness were granted prioritized access. Inherently, patients with anti-CD19 CAR-T-mediated B-cell aplasia would be incapable of generating humoral responses, so that assessment of the vaccine-induced cellular immunity was all the more important to gauge whether the vaccine can induce meaningful protection. Methods: The prospective study included 8 patients aged >12 years (and hence, eligible for SARS-CoV-2 vaccination) diagnosed with multiply relapsed B-cell precursor acute lymphoblastic leukaemia (ALL) and treated with anti-CD19 chimeric antigen receptor T-cell (CAR-T19) therapy between 2016 and 2021. The primary endpoint was the detection of humoral and cell-mediated response to vaccine. Secondary endpoints included the incidence of grade 3 or 4 adverse events and GVHD exacerbation and the influence of the vaccine to CAR T cells and lymphocyte subset. Results: Even though half the patients exhibited sub-normal lymphocyte counts and marginal CD4/CD8 ratios, after two vaccinations all showed brisk T-cell responsiveness to spike protein, predominantly in the CD4 compartment which was qualitatively well within the range of healthy controls. In none of the patients severe vaccine-associated (grade 3 or 4) adverse events were observed. None of the eight patients developed a cytopenia post vaccination. When looking at the differentiation of the CD4 + and CD8 + T cells into naïve, central-, effector memory and EMRA T cells, also no significant changes in composition could be observed. The slight changes which could be seen in an increase of the naïve T4 and T8 cell subpopulation is probably related to the restoration of the naïve T-cell pool in the course of immune regeneration after stem cell transplantation. The effectory compartment (TEM and TEMRA) showed no significant changes with respect to expansion of these cells. Conclusions: We posit that SARS-CoV-2 mRNA vaccines induce meaningful cellular immunity in patients with isolated B-cell deficiency due to anti-CD19 CAR-T therapy. Clinical Trial Registry: The study was approved by Goethe University Medical Center’s ethics committee (case number 2021-180). Disclosure: "Nothing to declare"
Background: Apheresis and cell processing facilities, although experienced in HPC collection, could face challenges when introducing the collection and storage of cells intended for manufacturing of cellular therapy products, because the validation of collection and cryopreservation of other cell types is needed. The aim is to present results of validation of collection and cryopreservation of autologous mononuclear cells (MNC) and lymphocytes subpopulation intended for CAR-T cells production. Methods: Collection and cryopreservation of lymphocytes for production of autologous T cell immunotherapy tisagenlekleucel (Kymriah, Novartis) in University Hospital Centre Zagreb started at the end of 2019. Leukapheresis were performed on Spectra Optia system (Terumo BCT) using CMNC procedure and acid citrate dextrose A (ACD-A) anticoagulant. All patients received prophylactic continuous intravenous calcium gluconate. Leukapheresis product was considered eligible for CAR-T product manufacturing if specification requirements were met: total nucleated cells (TNC) ≥ 2x109, CD3 + lymphocytes ≥1x109, and ≥3% CD3 + lymphocytes. Collection efficiency (CE1) for MNC and CD3 + cells was calculated based on pre- and postapheresis MNC and CD3 + cells peripheral blood counts. Cells were cryopreserved with DMSO cryoprotectant and controlled-rate freezing. The effect of cryopreservation on the recovery of lymphocyte subpopulations was evaluated in cryovials representative of cryopreserved products using flow cytometer BD FACSCanto II. Results: From December 2019 to November 2021, two children and two young adult patients with ALL, and 23 adults with DLBCL underwent leukapheresis. The median age was 57 years (range 5-71), and median body weight was 75 kg (range 21-109). 15 patients required central venous catheter, while in 12 patients peripheral veins were used. In all patients, except in one child, the target number of cells was obtained with one leukapheresis. The median of total blood volume (TBV) processed was 2,1 (range 1,4-2,8) during 240 min (range 160-300). In two procedures heparin was added to resolve clotting. In only one patient mild symptoms of hypocalcaemia were observed. Median of collected TNC was 8,9x10E9 (range 0,7-27,7), and CD3 + cells 4,1x10E9 (range 0,7-11,1). Median of CE1 for MNC was 24% (range 2,4-32), and for CD3 + cells 43,4% (range 18,9-56,8). The number of CD3 + cells in peripheral blood significantly correlated with CD3 + cell yields (correlation coefficient r = 0.680, p < 0,0001). All leukapheresis products were cryopreserved on the day of apheresis. The median recovery of CD3 + cells after cryopreservation was 79% (range 34,8-106,4), CD4 + cells 83,5% (range 15,1-136,9), CD8 + cells 84,8% (range 34,1-145,7), CD19 + cells 96,3% (range 41,8-107,7) and CD56 + cells 74,7% (range 8,3-121,1). Conclusions: Leukapheresis is an efficient and safe procedure for the collection of cells for cellular therapy production. CE1 calculated for CD3 + lymphocytes exhibited a relatively wide range, therefore further evaluation of factors affecting apheresis performance is necessary. Results of CE1 validation for CD3 + cells allow calculation of required TBV processing based on patient’s pre CD3 + count and optimization of the apheresis procedure. The recovery of different lymphocyte populations after thawing was lower compared to the results of our previous validation of CD34 + cell recovery, and further clarification of the different tolerance to cryopreservation of cell subpopulations in apheresis products is needed. Disclosure: Nothing to declare.
Background: Treatment based on target CD19 protein by a chimeric antigenic receptor expressed on T lymphocytes (antiCD19 CAR-T) is a novel immunotherapy that has led to a revolution in the management and treatment of relapse and refractory (r/r) B cell acute lymphoblastic leukemia (B-ALL) even after an allogenic hematopoietic stem cell transplant (HSCT) relapse. After antiCD19 CART therapy, the successful functionally effective of the CAR-T is monitored by bone marrow negative minimal residual disease but also by the absence of peripheral CD19+ B lymphocytes (B cell aplasia). In addition, continuous chimerism monitoring after HSCT is a routinely well-established method which is critical for early therapeutic interventions. So, in patients who have received an allogenic-HSCT prior to treatment with antiCD19 CAR-T, monitoring of chimerism and lineage-specific chimerism on CD19+ and CD3+ cells could be a helpful complementary tool to early evaluate the risk of relapse and it could also help to propose early treatment. Methods: We describe four B-ALL pediatric patients who received anti-CD19 CART therapy in the setting of a relapse after an allogeneic HSCT. They all received fludarabine and cyclophosphamide (FluCy) lymphodepleting regimen. Lineage chimerism in peripheral blood (PB) and bone marrow (BM) was done in all of them periodically after the CART infusion. Results: In patient 1, mixed chimerism was observed specifically in the CD3+ lineage, and it reverted with the administration of serial donor lymphocyte infusion (DLI). In the case of patients 2 and 4, a loss of chimerism was observed in the CD19+ lymphocytes subset without being accompanied by a loss of B cell aplasia. In these patients, treatment with DLI, although it could turn back to complete chimerism in patient 2, did not prevent the later progression to relapse in both. In patient 3, mixed chimerism in CD19+ subset was associated with very early loss of B cell aplasia. Given that after these findings an allogenic HSCT was indicated, he did not receive DLI. Conclusions: The CD19+ lineage mixed chimerism but not CD3+ lineage mixed chimerism monitored by single tandem repeat (STR) techniques could anticipate earlier than B cell aplasia determined by flow cytometry, the antiCD19 CAR-T lack of persisting functionally effective and leukemia relapse. Treatment with DLI would not avoid relapse but could recover CD3+ full donor chimerism. We suggest that continuous lineage chimerism analysis should be a routinely tool for monitoring in patients who received anti-CD19 CART after an allogeneic HSCT and achieved complete remission, because it could help to propose early treatment. However, the role of DLI in this setting looks useless, although prospective studies should be proposed. Disclosure: Nothing to declare.
Background: CD19-targeted chimeric antigen receptor modified T-cell (CAR-T) infusion is an established treatment in relapsed or refractory diffuse large B-cell lymphomas (DLBCL). While early adverse events like cytokine release syndrome (CRS) and neurotoxicity are well investigated, in late occurring complications like prolonged cytopenias the underlying mechanisms are still unclear. Grade 3-4 neutropenia, anemia and thrombocytopenia at day 30 have been reported in 48% of patients given axicabtagene-ciloleucel (axi-cel) infusion in the ZUMA-1 and ZUMA-9 trials. We present two patients who received an autologous peripheral blood stem cell (PBSC) boost for prolonged severe cytopenias. Methods: Patient A, a 65 year old man, received an autologous PBSC transplantation for DLBCL in partial remission, but relapsed 106 days later. Patient B, a 69 year old woman had an early relapse of a DLBCL and was not eligible for autologous PBSC transplantation due to progression under salvage treatment. Both patients fulfilled the criteria of the Austrian CAR-T-cell platform and thus, were eligible for anti-CD19 CAR-T-cell infusion with axi-cel (Yescarta ®) after lympho-depleting chemotherapy with 3 doses of cyclophosphamide 500mg/m² and fludarabine 30mg/m² body surface area. While patient A did not reveal any acute adverse events after CAR-T-cell infusion, patient B developed CRS grade I and obtained consecutively tocilizumab on days +9 and +10. Both individuals suffered from prolonged WHO grade 4 neutropenia, anemia and thrombocytopenia beyond day 30 including G-CSF refractory neutropenia. Therefore, bone marrow examination was performed at days 40 and 33 after CAR-T-cell infusion revealing no signs of DLBCL or myelodysplastic syndrome (MDS) but severely hypoplastic marrow involving all three cell lines. Therefore, we administered stored autologous PBSCs of 2.99 and 2.86 x106 CD34+/kg body weight 48 and 33 days after CAR-T-cell infusion. Results: Leukocyte engraftment (ANC > 500/μL) supported by G-CSF stimulation occurred 3 and 11 days after autologous PBSC boost. Platelet engraftment with platelets over 20.000/μL was observed in patient A 40 days after autologous PBSC boost. In patient B no platelet engraftment could be achieved within an observation period of 52 days after autologous PBSC boost. No signs of clinical infection or bleeding were observed both patients showed persistent complete metabolic remission of their malignancies at days +158 and +63 after CAR-T-cell infusion. Conclusions: Autologous PBSC boost seems to be a rescue therapy for prolonged severe cytopenia after CAR-T-cell therapy. Reducing the duration of late cytopenia could lower risk of infectious complications and improve outcome of patients given CAR-T-cell therapy. Disclosure: Nothing to declare
Background: CD19-Chimeric Antigen Receptors T cell(CAR-T) has made a very big progress in treatment of B acute lymphoblastic leukemia(B-ALL) and lymphoma. CD19 is a very good target for CAR-T treatment and pan- B marker for Flow Cytometry(FCM) detection for it’s high coverage on B malignancies. However, 13%-60% cases were CD19 dim or negative when relapsed after CD19-CAR-T, so it’s urgent to find a substitute for CD19 not only as a new target of CAR-T but pan-B marker for FCM detection. Methods: From October 2020 to August 2021, 315 newly diagnosed patients with hematological tumor in Hebei Yanda Lu Daopei Hospital were investigated for immunophenotyping. 350 bone marrow minimal residual diseases(MRD) tests were performed in 200 B-ALL patients. 3 Laser 10-color FACS Canto Plus was used for FCM detection, and Diva and kaluza softwares were used to analyze data. Combining rough B gates were set as CD24 or CD19 positive and CD72, cCD79a or CD19 positive, and MRD were detected by multi dimensional radar photos for quick and intuitive observation. Results: 1. In 315 patients with hematological tumors, the positive rate of CD72 was 97.73%(129/132) in ALL-B, and 100%(32/32) in lymphoma, which includes 3 Burkitt lymphoma, 5 chronic lymphocytic leukemia (CLL), 8 diffuse large B-cell lymphoma (DLBCL), 2 follicular lymphoma (FL), 1 hair cell leukemia (HCL), 1 lymphoplasmacytic lymphoma (LPL), 4 mantle cell lymphoma (MCL), 7 marginal zone lymphoma (MZL), and 1 small cell lymphoma (SLL). CD72 positive rate was 10.27%(17/151) in non-B-cell tumors, including 10.85%(14/129) in AML, 40%(2/5) in mixed phenotype acute leukemia(MPAL), 14.29%(1/7) in T-ALL/LBL, and 0%(0/10) in multiple myeloma(MM). These results suggest that CD72 is a marker comparable to CD19 for its coverage, specificity and expression intensity are comparable to that of CD19, it can be used as a rough B gating marker for FCM MRD of post-targeted therapy and a highly effective target for further CAR-T therapy of CD19-negative relapsed cases. 2. In MRD detection, CD72 combined with CD19 and cCD79a can cover all B cells, with sensitivity and specificity as high as 100%. 3. different from old analysis methods in which many gate strategies and dot plots were used to analyze data, a multi-parameter and multi-dimention radar photo could offer a quick and high efficient method. Conclusions: CD72 is highly expressed in B-cell tumors and may be an effective B cell marker after CD19. It can be used not only as a gating marker of B-ALL MRD, but also as a highly promising biomarker for further targeted therapy of CD19 negative relapsed cases. Disclosure: Nothing to declare
Background: There is limited evidence regarding the role of nutrition during a CAR-based cellular therapy transplant. However, patients experience similar nutritional challenges to a HSCT. Patients will have increased nutritional requirements and experience side effects of their underlying condition including side effects of the transplant, which affects nutritional intake. Malnourished patients have poorer outcomes than well-nourished patients with 10% weight loss being regarded as a clinical prognostic factor. Nutrition & Dietetics at the University Hospital Wales has set up a service for CAR-T patients. Methods: The dietitian benchmarked the service, developed the Dietetic nutrition pathway, worked as part of the CART MDT, developed patient nutritional resources and developed nutritional guidelines. Patients had a consultation with the dietitian during or just after apheresis for nutritional assessment and information sharing. The patients were supported with nutritional pre-habilitation until admission, for those who required it. Nutrition support and advice was provided by the dietitian during their inpatient stay for their CART cell therapy which continued until 30 days post- CART. Following this, patients who had ongoing nutritional challenges continued to have dietetic support regularly. All patients continued to have nutritional assessment, advice and support at 3 month and 6 month clinic appointments. Data collected for outcome measures included weight and patients reported experience measures (PREMs) using a questionnaire. Results: 14 patients received their CART and experienced the dietetic service. All patient’s received nutritional support dietary advice in the pre-hab phase. 21% (n = 14) of patients required more aggressive nutritional pre-hab including nutritional supplementation. 100% (n = 14) of patients received nutrition support dietary advice and nutritional supplements during their inpatient stay with 14.3% (n = 14) of patients requiring nasogastric feeding during their CART. Mean weight loss in the pre-hab phase was 3.4% (range 0-12.5% median 2.3%) and mean weight loss from pre-hab to discharge at 30 days post-transplant was 6.6%.(range 0-23.2%, median 6.78%). Regarding PREMs, 100% (=14) of patients reported they strongly agreed that the dietetic input was clear, concise, easy to understand and empowered them to make dietary changes. 100% (n = 14) also reported they strongly agreed that they benefited from the dietetic service and patient information. Conclusions: 21% (n = 14) of patients experienced weight loss above the 10% clinical prognostic factor. Patients experience of the dietetics service was very positive. The dietetic service to the CART patients will continue. Future development of this service will also include outcomes including bioelectrical impedance analysis (BIA) and hand grip strength. Disclosure: Nothing to declare
Background: CAR-T is an innovative anti-cancer therapy, treating B-cell ALL in children and young adults up to age 25. Despite a significantly increased survival in patients with ALL in the last decades, 2-3% of patients show a disease refractory to chemotherapy treatments and about 10-15% eventually relapse. Methods: The first pediatric patient candidate to CAR-T therapy at the Children’s Hospital of Brescia was a 12-years-old girl diagnosed in 2015 with Non-Hodgkin B Lymphoma, stage III involving bone, CNS with BOM negative. She underwent therapy according to the LNH-97 protocol and achieved stop therapy in July 2017. In January 2021, relapse occurred with a common B-ALL phenotype, CNS negative, AIEOP LLA REC 2003 protocol was started. Over the end of the therapy, persistence of blasts was present with strongly positive MRD. Therefore InterALL-HR-2010 R3 + Bortezomib protocol was given with, nevertheless, persistence of disease at the end of the treatment, with a marrow infiltrate of 12%. In March 2021, she was enrolled in CAR-T program and autologous T lymphocytes were collected. Results: In May 2021, after lymphodepleting therapy, 1.6 x 108 CD3 + CAR-T cells were infused (Kymriah). On post infusion day+4, patient presented fever, hypotension, hypoxemia attributable to CRS grade II associated with an increase of IL-6 (peak at day+7, 266 ng/L, range<7 ng/L). She was treated with a single dose of Tocilizumab with benefit and resolution of symptoms. On day+17,+ 30,+ 60 MRD was negative. At days+1,+3,+7,+10,+14,+16,+30,+86,+115,+120,+146 the presence of CAR-T was analyzed by flow cytometry and by a highly sensitive technique: digital droplet PCR(ddPCR), CAR-T were detected already on day+1 in ddPCR with a peak at day+10. Five months after CAR-T infusion, patient lost B cell aplasia and MRD resulted positive with a rapid loss of CAR T cells. Soon after she received HSCT from MUD after conditioning therapy with total body irradiation, Fludarabine and Thymoglobulin. HSCT was performed with a positive selection of CD34 + (10x106 CD34 + /Kg infused) cells and T controlled addback (30x106 CD3 + /Kg infused) from PBSC. After HSCT, patient was monitored and MRD became negative in presence of cutaneous GvHD stage II. Donor chimerism performed on CD34 + cells from BM was 97.3%. Analysis of BM, moreover, showed persistence of CAR-T cells. The surprisingly persistence of CAR-T after HSCT can be explained by the results of the study of donor chimerism performed with ddPCR, that show 12% of autologous cells. Conclusions: Monitoring of CAR-T cells with a very highly sensitive techniques, such as ddPCR, is of critical importance because it has been demonstrated that even low concentrations of CAR-T could have a therapeutic function. However, in this case, the sudden decrease of CAR-T circulating cells and the appearance even of a very low number of B cells, suggested to immediately proceed to HSCT. In fact, MRD short after became slightly positive. CAR-T is an innovative therapy, several studies nevertheless, are ongoing to understand if it is a definitive therapy or a bridge to transplant. In our case it was the latter. It’s no clear if persistence of CAR-T after allogeneic HSCT could have a role of relapse prevention. Disclosure: Nothing to declare
Background: Peripheral blood autologous stem cells transplantation (PBSCT) has played pivotal role as consolidation for relapsed or high risk lymphomas, while its role is discussed in CAR-T cells era. Lower disease burden has been associated with better outcome after treatment with CAR-T cells; the intensity and role of bridging therapy has not been clearly stated. In this study, we aimed to assess the feasibility of PBSCT as bridging therapy before treatment with CAR-T cells in the setting of relapsed/ refractory B cell lymphomas. Methods: We retrospectively analyzed six patients who had received FEAM-conditioned PBSCT as a bridging to CAR-T: we focused on cytopenias, specific CAR-T toxicities and outcomes. Results: At the time of eligibility for CAR-T, all patients had ECOG 0-1, three had elevated LDH and five out of six had an IPI score of 3 or more. All patients had either refractory or early relapsed NHL. After lymphocyte collection was completed, patients were hospitalized for receiving PBSCT. Median time from lymphocyte collection to the beginning of bridging with PBSCT therapy was 6 days (range 1-44 days). Main toxicities related to PBSCT were grade 3 infections and grade 4 cytopenias. No unexpected toxicities were observed. Patients were discharged at a median of 12 days (range 11-15) after PBSCT. CAR-T manufacturing process took a median of 31 days. At the time of arrival of the product in our center, five of the six patients had been already discharged from the hospital after PBSCT. Prior to CAR-T cell conditioning, disease response to bridging with PBSCT was evaluated using PET-CT. Three patients obtained complete (CR) or partial (PR) response, two were in stable disease (SD) and one in progressive disease (PD) (Figure 1A). Before the start of lymphodepletion, complete blood counts (CBC) had recovered from PBSCT with no grade 4 cytopenia. Median time from arrival of frozen CAR-T cells at our center and start of lymphodepletion was 22 days (12-46). Median time from PBSCT to CAR-T cells infusion was 50 days (48-74). All patients experienced CRS, with only one grade 3 CRS, and no ICANS was documented. In terms of anti-lymphoma efficacy of PBSCT bridging, overall response at one month from CAR-T cell infusion was obtained in five out of six patients (83%), with three (50%) CR and two PR; responses were stable with those five patients still being alive without disease progression at median follow-up of 6 months after CAR-T. Conclusions: We conclude that PBSCT is a feasible option as bridging therapy prior to CAR-T, with reasonable toxicities and efficient action on disease bulk. We than provide a review of literature on this topic. Disclosure: Nothing to declare.
Background: Tisagenlecleucel (Kymriah), an autologous CD19-directed CAR-T-cell therapy, has been approved by the European Medicines Agency in Aug 2018 for the treatment of children and young adults (aged up to and including 25 years) with relapsed/refractory (r/r) B-cell acute lymphoblastic leukemia and adults with r/r diffuse large B-cell lymphoma. Here, we discuss >3-year commercial tisagenlecleucel manufacturing experience across the four manufacturing sites in Europe and US (Les Ulis, France; Stein, Switzerland; Fraunhofer, Germany; and Morris Plains, US), for patients in Europe. Methods: Tisagenlecleucel manufacturing process involves leukapheresis to collect patients’ peripheral blood mononuclear cells, which are cryopreserved and shipped to the manufacturing site. This is followed by enrichment and activation of T cells, transduction of the lentiviral vector containing the anti-CD19 CAR transgene, activation with anti-CD3/CD28 antibody-coated beads, expansion in cell culture, washing, and formulation of the viable cells into a cryopreservation medium, and shipping back for infusion (Tyagarajan, 2020). Manufacturing success rate (MSR) was defined as the proportion of patients for whom the manufactured product met the commercial release criteria of the total number of patients leukapheresed. Shipment success rate (SSR) was defined as the proportion of patients for whom the manufactured product was shipped for infusion. Results: Novartis has a significant global commercial manufacturing footprint with six sites located across the globe (US, France, Switzerland, Germany, Japan, and Australia) and a treatment network of >340 certified centers worldwide, including 164 centers in Europe. This allows for immediate manufacturing availability, thereby meeting the needs of patients. As of Aug-2021, tisagenlecleucel has been manufactured for more than 5300 patients worldwide. For patients in Europe, the SSR was consistently high at 96%, and the MSR increased progressively from 84% in 2018 to 93% in 2021 (Figure). The corresponding out-of-specification (OOS) rates decreased considerably between 2018 and 2021 from 13% to 5%, with the viability OOS rates decreasing from 9% to 0%. The median turnaround time (apheresis pickup to delivery back to center) has improved from 33 days at launch in 2018 to 26 days currently. Since the approval of tisagenlecleucel in 2018, a key goal has been to upscale and continuously improve manufacturing success, decrease OOS rate, and minimize the turnaround time in the commercial settings to meet the needs of a global patient population. In July 2021, two key process and analytical improvements have been introduced. Firstly, an alternate serum source (5% plasma-derived human AB serum) which further improves process robustness with a trend towards improved growth and higher peak cell counts. Secondly, a simplified sample preparation procedure for final product cell count and viability measurement, which is more reflective of final product at infusion. Conclusions: Tisagenlecleucel’s current global commercial manufacturing footprint and treatment network are well-positioned to meet the anticipated increase in demand for CAR-T therapies. Over the last 3 years, SSR remained high at 96% and MSR improved to 93%. Continuous investment in process improvements has helped improve manufacturing capacity, robustness of manufacturing and testing processes, as well as speed and reliability to deliver tisagenlecleucel to patients in need of treatment. Disclosure: All authors are employees of Novartis.
Background: Patients with acute myeloid leukemia (AML) relapsed after allogenic hematopoietic stem cell transplantation (allo-HSCT) have poor prognosis although some therapeutic options including hypomethylating agents, chemotherapy, donor lymphocyte infusion (DLI), or a second allo-SCT could be adopted. The anti-CD123 chimeric antigen receptor-γδT (CAR-γδT), which is a promising therapeutic target for AML, might be a new way for relapsed AML after allo-HSCT. Methods: A 43-year-old male patient with AML transformed from myelodysplastic syndrome underwent haploidentical-SCT in June 2019. Before transplantation, the percentage of primordial monocytes in bone marrow was 22%. Flow cytometry showed: primordial monocytes expressed CD34, CD123, HLA-DR. He received a myeloablative conditioning regimen and a successful hematopoietic reconstitution. Bone marrow examination showed complete remission with minimal residual disease (MRD) negative. Segment tandem repeat (STR) showed complete donor chimerism. To prevent relapse, the patient received 6 cycles of Azacytidine (50mg/m2/d*5d, once a month) since 4 months after haplo-SCT. He suffered MRD positive (1.47%) relapse in July 2020. One cycle of Azacytidine combined with low-dose Cytarabine (20mg/m2/d*7) and 2 courses of Venetoclax (400mg/d*28) were administered successively. After above treatment, he experienced extensive chronic graft versus host disease (cGVHD) involving skin, eyes and mouth. Unfortunately, in December 2020, 1.5 years after haplo-SCT, he suffered hematological relapse. The percentage of primordial monocytes was 9.5% and STR dropped to 78.83%. Universal anti-CD123 CAR-γδT cells were prepared from a third party umbilical cord blood. In brief, the γδT cells were selected using TCRg/d + T Cell Isolation Kit (Miltenyi) and stimulated with an anti-gd TCR antibody. Activated gd T cells were transduced with an anti-CD123 CAR-carrying lentivirus. Cell product were manufactured by Senlangbio company. Pre-treatment was Fludarabine 50mg/d*5 and Melphalan 50mg/d *2. The patient received universal anti-CD123 CAR-γδT (2.5 × 106/kg) and 7-days later, G-CSF mobilized and cryopreserved donor mononuclear cells (1.98 × 108/kg) were infused including 0.693 × 108/kg CD34 + stem cell and 1.67 × 107/kg CD3 + T cell. Sirolimus(0.5mg/d) was started after donor cells infusion for GVHD-prophylaxis. Results: After pre-treatment, MRD was 3.69% and STR was dropped to 61.12%. The universal anti-CD123 CAR-γδT proliferated rapidly from day+3 to day+7 and then decreased. The patient experienced grade 1 cytokines release syndrome including fatigure, nausia, headache and without neurotoxic syndrome. Seven days after CAR-γδT infusion, the bone marrow test showed: AML-MRD was negative and STR was increased to 90.24% and then donor mononuclear cells were infused. On day17 after CAR-γδT infusion, he was in agranulocytosis stage and suffered bacteremia and received antibiotic treatment. The patient achieved MRD negative remission and complete donor chimerism 21-days after CAR-γδT infusion. He did not develop acute GVHD or new onset cGVHD and sirolimus was stopped 1 month after donor lymphocyte infusion. There was an interesting finding that the number of universal anti-CD123 CAR-γδT increased again after donor cells infusion which might be due to the CD123 antigen expressed on donor cells (Figure-A,B). Six months later, the patient suffered hematological relapse again and received supportive treatment. Conclusions: Universal anti-CD123 CAR-γδT combined with donor lymphocytes infusion seems to be an effective and safe treatment for AML relapsed after allo-HSCT. Disclosure: no conflict of interest statement
Background: CAR-T cell is an effective treatment in B cell malignancies. Cytokine Release Syndrome and CNS toxicity are the most frequent adverse reactions following CAR-T cell infusion. Although hemolytic anemia has been reported following hematopoietic stem cell translantation, cold aglutinin mediated hemolytic anemia after CAR-T cell infusion has not been reported. Anti CD20 therapies are the preferred first line therapy in cold agglutinin-mediated hemolytic anemia but response is usually not sustainable. Daratumumab, a novel anti CD38 monoconal antibody which has been approved for treatment of plasma cell dyscrasias is a logical option in refractory immune hemolytic anemia when conventional therapies fail as it targets autoantibody-producing plasma cells. Methods: A 22 years old male patient received anti CD-19 directed academic CAR-T cell (ISIKOK-19) infusion due to relapsed acute lymphoblastic leukemia following haploidentical stem cell transplantation from his mother. He presented with post CAR-T cell refractory autoimmune hemolytic anemia. The case report presents treatment approach with Daratumumab for this patient. Results: Patient with relapsed acute lymphoblastic leukemia following haploidentical stem cell transplantation was discharged on day +43 of CAR-T cell infusion but had to be re-admitted with acute Coombs positive hemolytic anemia on day +87. Bone marrow analysis was consistent with morphological and flow cytometric remission with full donor chimerism. Peripheral blood CAR-T cell level was adequate. Initial work-up excluded infectious and post-transplant lymphoproliferative etiologies. Low dose methylprednisolone of 20mg was chosen as first line treatment in order to avoid CAR-T cell apoptosis by steroid treatment. When there was no response IVIG was administered. Again no response was obtained. Autoantibodies were determined to be cold agglutinin in nature and weekly rituximab was commenced on day +92 . Anemia requiring massive transfusion persisted with severe hyperbilirubinemia reaching 30 mg/dL. Patient was administered a total of 97 units of RBC transfusion from beginning of hemolytic anemia and considering patient’s severe condition and lack of rituximab response, Daratumumab 16 mg/kg per week was commenced on day + 137. Following the first dose patient’s hemoglobin level continued to fall and blood transfusion and pulse steroid treatment at 250mg of prednisolone for 3 days had to be performed. Second, third and fourth doses of Daratumumab were administered weekly as scheduled. Transfusion requirement decreased from the second dose of Daratumumab, but patient received a total of 18 units of RBC transfusion whilst on 2 weeks of Daratumumab therapy. Steroid treatment was weaned down and stopped while consecutive weekly doses of Daratumumab continued. Patient reached a stable hemoglobin level following the third dose of daratumumab and remains steroid and transfusion free following the 4th and final dose of Daratumumab. Conclusions: Cold agglutinin mediated hemolytic anemia post CAR-T cell treatment is a complex entity with lack of evidence based treatment . Anti CD38 monoclonal antibody daratumumab induces rapidly depletion of antibody producing plasma cells and can be an option in steroid, IVIG and Rituximab refractory cold agglutinin mediated autoimmune hemolytic anemia in CAR-T cell patients. Disclosure: No conflict of interest
Background: Chimeric antigen receptor T (CAR-T) cells mediate impressive anti-tumor effects in B cell malignancies, but fewer than 50% of patients experienced long-term disease control due to T cell exhaustion, which is a state of antigen-specific T cell dysfunction and subsequent physical deletion. During exhaustion, T cells can upregulate various inhibitory receptors, including PD-1, Lag-3, Tim-3, and progressively loss their effector function and proliferative capacity. The transcription factor T cell factor 1 (TCF-1) plays an important role in T cell development and maturation. T cells expressing a high level of TCF-1 exhibit a stem-cell-like phenotype and maintain a better proliferative capacity. To investigate the role of TCF-1 in CD19 CAR-T cells, we performed this study. Methods: CD19 CAR-T cells were manufactured using the third-generation retroviral CAR vector (SFG.CD19.CD28/4-1BB/ζ). Double transduced T (DT-T) cells were generated using the same CD19 CAR vector with an extra TCF-1 vector (SFG.CD70_1F6.CH3-IgG4h-CH2-IgG4h.Tcf7.NGFR). The expansion of CAR-T cells during the manufacturing was determined by cell counting. The apoptotic state of CAR-T cells at the end of generation was evaluated by western blot using anti-PARP, anti-caspase-3, and anti-cleaved caspase-3 antibodies. The cytokine release capacity of CAR-T cells was analyzed by an intracellular cytokine staining. Moreover, a co-culture system was applied to determine the long-term function of CAR-T cells, where effector cells and target cells were plated at a 1:1 or 1:2 ratio, followed by a repetitive tumor challenge. The proliferative capacity of CAR-T cells, the number of challenging procedures, the number of residual tumor cells, and the exhaustion status of CAR-T cells were investigated in a co-culture assay using flow cytometry. Results: Both CD19 CAR-T cells and DT-T cells were successfully generated with a stable transduction efficiency (CD19 CAR-T cells: 80.2%±5.21, DT-T cells: 55.1% ± 5.91) as well as a potent and specific killing capacity. Of note, DT-T cells at the end of generation showed downregulated expression of PARP and cleaved caspase 3, suggesting a lower tendency to apoptosis than CD19 CAR-T cells. The array resulted in a greater cell number of DT-T cells than CD19 CAR-T cells. Although DT-T cells showed a lower killing efficiency in a 4-hour killing assay due to the reduction of cytokines (CD19 CART group: TNF-α 67.68% ± 4.57, IFN-γ 42.6% ± 10.1, CD107a 82.4% ± 4.2, DT group: TNF-α 52.3 ± 3.9, IFN-γ 29.6%±8.1, CD107a 61.8%±8.1), they exhibited a superior functionality than CD19 CAR-T cells in the long-term killing assay. An improved proliferative capability was observed in DT-T cells throughout the co-culture, showing a great expansion(Day11, NT 41.97E7 ± 15.37E6, CD19 CART 28.53E6 ± 9.6E6, DT46.61E6 ± 10.69E6). The inhibitory receptors (PD-1, Tim-3, and Lag3) were reduced on DT-T cells when compared with CD19 CAR-T cells. The long-term killing ability of DT-T cells was dramatically improved, which was evidenced by an increased number of challenging procedures and a powerful reduction of residual tumor cell number. Conclusions: Overexpression of TCF-1 might constitute a novel way to improve the functionality of CAR-T cells through the reduction of apoptosis, the improvement of proliferation, and the enhancement of the resistance to exhaustion. Disclosure: Nothing to declare
Background: CD19-specific chimeric antigen receptor (CAR) T-cells (CD19CARTs) have significantly improved the outcome of patients with relapsed B-cell malignancies such as acute lymphoblastic leukemia (ALL) and certain type of non-Hodgkin’s lymphoma (NHL). However, despite treatment with CD19CARTs, a significant proportion of patients eventually relapse, highlighting the need to further improve the functionality of CD19-CAR T-cell products. CIN85 protein, also known as SH3 domain-containing kinase-binding protein1 (SH3KBP1), has been shown to inhibit T-cell activation through its interaction with phosphatase suppressor of TCR signaling-2 (Sts-2) within the T-cell receptor (TCR) complex. As the CAR-mediated activation of T-cells depends on pivotal components of the TCR, we hypothesized that the disruption of CIN85 may lead to the enhancement of CAR-mediated T cell activation, hereby improving the proliferation and anti-tumor efficacy of CD19 CAR T-cells. Methods: Two days after activation using CD3 and CD28-specific antibodies, T-cells from healthy donors were retrovirally transduced with a third generation CD19 CAR construct containing the CD28 and 4-1BB co-stimulatory domain followed by a CRISPR/Cas9-mediated disruption of the CIN85 gene using ribonucleoprotein (RNP) complexes on day 6 (CIN85KO-CD19CARTs). Successful CAR transduction and CIN85 gene disruption were confirmed by flow cytometry (FACS) and western blot (WB) respectively. Subsequently, we performed in vitro studies to evaluate proliferation capacity, viability/apoptosis, and potential changes in the T-cell phenotype of CIN85KO-CD19CARTs. In addition, cytokine production upon antigen stimulation was determined by intracellular cytokine staining (ICS) and cytotoxicity was tested using a standard chromium (Cr51) release assay. Results: During ex-vivo expansion, CIN85KO promoted enhanced proliferation of CD19CARTs compared to non-modified CD19CARTs leading to a higher expression of the proliferation marker Ki67 as detected by flow cytometry. In addition, we noticed a higher percentage of CAR expressing T cells in the CIN85-KO-CD19CART product compared to the control CAR T-cells. CIN85KO-CD19CARTs showed a higher surface expression of markers associated with T-cell activation such as CD69, CD25, HLA-DR, Tim-3, PD-1 and LAG-3 than CD19CARTs whereas CIN85 disruption did not affect the CD4/CD8 composition of the CAR T-cell product. Importantly, higher activation status did not lead to a higher rate of apoptosis and cell death on CIN85KO CD19 CAR T-cells. In both, the CD4 positive and CD8 positive subpopulations, a higher percentage of CIN85-CD19CARTs expressed surface markers associated with a central memory and effector memory phenotype whereas a lower percentage of CIN85-CD19CARTs exhibited a terminally differentiated T cell phenotype compared to non-modified CD19-CARTs. Functionally, CIN85-CD19CARTs secreted a significantly higher amount of activating cytokines such as IFN-γ, TNF-α and IL-2 and exhibited a stronger cytotoxic activity in the 51Cr release assay upon stimulation with CD19-positive tumor cells than CD19CARTs. Conclusions: Disruption of CIN85 enhances proliferation, activation, and cytotoxic activity of CD19CARTs, while at the same time skewing the cells to a more favorable T-cell phenotype. Our results warrant further in-vitro and in-vivo studies to determine the potential of this approach to improve the functionality of CAR T-cells products . Disclosure: Nothing to declare
Background: Fetal bovine serum (FBS) or human serum (HS) is widely used in the production of chimeric antigen receptor T cells (CARTs). To overcome a lot-to-lot inconsistency and a risk of contamination the use of chemically-defined, animal-component free medium would be desirable. In this study, we compared three serum-free media to CART medium containing FBS. Methods: After 12 days of CD19.CART culture, we assessed expansion, viability, transduction efficiency and phenotype by flow cytometry. Functionality of CARTs was tested by intracellular staining, chromium release assay and long-term co-culture assay: CARTs co-cultured with tumor cells for the period of 30 day were subjected to multiparametric flow cytometry. Results: CARTs were cultured in different media such as Fujifilm™ Prime-XV™ T Cell CDM (FF), Takara Bio™ LymphoONE™ T-Cell Expansion Xeno-Free Medium (TB) or CellGenix™ TCM GMP-Prototype (CG). These CART cultures did not vary in terms of expansion and viability when compared to FBS-containing medium. Transduction efficiency and CD4 + /CD8 + ratio of CARTs were significantly lower for CARTs cultured in TB (64.5% vs. 86.5%, P = 0.0167; 1.4 vs. 2.8, P = 0.0319) and CG (65.8% vs. 86.5%, P = 0.0358; 1.5 vs. 2.8, P = 0.0232) compared to CARTs of serum-containing medium. The functionality of CARTs was tested by intracellular staining and chromium release assay. CARTs of CG had the highest frequency of IFNγ + and IFNγ + TNF-α + CARTs compared to CARTs cultured with serum (22.5% vs. 7.6%, P = 0.0194; 15.3% vs. 6.2%, P = 0.0399). IFNγ-expression of CARTs cultured in TB was also significantly higher (16.9% vs. 7.6%, P = 0.0336). These findings corresponded to the results of chromium release assay. On average of four effector-to-target cell ratios CARTs of CG showed the highest cytotoxicity (P = 0.0182), CARTs of FF showed a similar high effectiveness (P = 0.0482) and CARTs of TB had also a higher rate of killed tumor cells (P = 0.0428) than CARTs cultured with FBS. Phenotyping on day 12 of CART production did not show a significant difference in expression of exhaustion markers PD-1, LAG-3 and TIM-3. CARTs cultured in FF had a higher percentage of central memory CARTs (40.0% vs. 14.3%, P = 0.0470) than CARTs cultured with FBS, whereas CARTs of CG (9.8% vs. 14.3%, P = 0.0092) and TB (6.1% vs. 14.3%, P = 0.0210) had a significantly lower frequency. In contrast, CARTs of FF (6.2% vs. 24.2%, P = 0.0029) and CG (11.0% vs. 24.2%, P = 0.0468) had a lower frequency of naïve CARTs. Long-term cytotoxicity was tested by co-culture assay. Cells cultured with FBS showed the highest CART expansion and lowest expansion of target cells indicating the best long-term cytotoxicity of CARTs. On day 30 of co-culture CARTs cultured in FF, TB and CG had a higher expression of LAG-3 (non-significant; 91.7% vs. 41.1%, P = 0.0306; 86.8% vs. 41.1%, P = 0.0205) and TIM-3 (77.3% vs. 32.5%, P = 0.0159; 90.8% vs. 32.5%, P = 0.0034; 68.1% vs. 32.5%, P = 0.0294) compared to CARTs cultured with FBS. Conclusions: We could demonstrate that functionality and expansion of CARTs are maintained in serum-free media. Given the advantages of freedom from bovine material and consistent quality, serum-free media keep promise for the future development of the field of GMP manufacturing of CARTs. Disclosure: A.S.: Hexal and Jazz Pharmaceuticals (travel grants), Therakos/Mallinckrodt (research grant), TolerogenixX Ltd. (co-founder and part-time employee). C.M.-T.: Bayer AG (research support), Pfizer and Janssen-Cilag GmbH (advisory board member), Pfizer, Daiichi Sankyo, BiolineRx (grants and/or provision of investigational medical products), Pfizer (clinical trial financial support). M.S.: Apogenix, Hexal and Novartis (research support), Hexal and Kite (travel grants), bluebird bio, Kite and Novartis (financial support for educational activities and conferences), MSD (advisory board member), MSD, GSK, Kite and BMS ((co-)PI of clinical trials), TolerogenixX Ltd. (co-founder and shareholder). The other authors have no COI.
Background: The generation and administration of Chimeric Antigen Receptor (CAR)-T cells represents a therapeutic approach that is approved for the treatment of distinct hematological malignancies and evaluated intensively for the extension to other tumor entities including solid tumors. CAR-T cell therapy relies on autologous lymphocytes, which are transduced to express a tumor antigen-specific CAR and transferred back into the patient, where they exhibit potent anti-tumor activity. However, CAR-T cells also cause adverse side effects such as the potentially life-threatening cytokine release syndrome. Moreover, CD19 CAR-T cell therapy does not distinguish between healthy and malignant B cells, indicating the necessity to develop CAR-T cells against more suitable target molecules. Nearly 80% of carcinomas and leukemia are positive for glycan structures from the Thomsen-Friedenreich (TF) antigen family, one of whose members is CD176 (Galβ1-3GalNAcα1-R). Due to different modifications (e.g. sialylation or fucosylation), CD176 is not accessible for ligand binding on healthy cells, but exposed on several carcinomas including hepatocellular, breast, colorectal and lung carcinomas as well as various leukemic cells. Thus, CD176 is a potential target for immunotherapy of multiple carcinomas. Methods: We designed a 2nd generation CAR to direct T cells against the carbohydrate epitope CD176. The ability of this CD176 CAR to initiate T cell signaling upon distinct target recognition of different carcinomas was tested in a reporter assay using a variety of tumor cell lines with different levels of CD176 expression as target cells. These included healthy CD176-negative cells, CD176-positive lung, breast and pancreatic cancer, as well as acute myeloid leukemia cell lines. Furthermore, primary CD176 CAR-T cells were generated and their functionality assessed in co-cultures with the same target cells evaluating the potential treatment of different tumor entities. Results: A reporter assay revealed that T cell activation initiated by the CD176 CAR constructs upon recognition of cell lines derived from different carcinomas was specific to the presence of CD176. Upon co-cultivation of primary CD176 CAR-T cells with the same CD176-positive cells, the expression of activation markers (e.g. CD69) and the release of pro-inflammatory cytokines (e.g. IFN-γ, TNF-α) was equally upregulated in a target-specific manner. Moreover, the engineered T cells released cytotoxic mediators (e.g. granzyme B, granulysin) and exhibited cytotoxicity towards different cancer cell lines determined by 7-AAD staining in flow cytometry and confirmed by real-time impedance measurements (xCELLigence). Taken together, CD176-specific CAR-T cells were generated and showed a CD176-specific cytotoxicity towards a variety of different cancer cell lines. Conclusions: Due to its differential modification – being accessible for ligand or antibody binding on malignant cells, but not accessible on healthy tissue – the carbohydrate antigen CD176 is a promising target for cancer immunotherapy. Our results demonstrate that CD176-specific CAR-T cells specifically recognize and react towards target cells from different tumor entities. Generating CAR-T cells targeting a structure present on a variety of tumor entities might allow for the simultaneous targeting of multiple carcinomas in the future. Disclosure: Nothing to declare.
Background: Acute Myeloid Leukemia (AML) is a hematological malignancy still incurable for almost all patients. Chimeric Antigen Receptor (CAR) therapy is showing astonishing results in other hematological disorders but remains challenging in AML since no specific antigens have been described yet. Using NKG2D as a CAR, a natural NK receptor with 8 ligands (NKG2DLs) overexpressed in several tumors, could surmount AML targeting limitations. T cells are considered the gold standard immune effector cells for CAR therapy, but they show some toxicities that could be overcome using other cells such as activated and expanded natural killer cells (NKAE), that can be used in an allogeneic context with no GvHD, have natural anti-tumor properties and have demonstrated to be clinically safe. In this project we analyze the anti-leukemia activity and safety of peripheral blood NKAE cells lentivirally transduced with an NKG2D-41BB-CD3z CAR. Methods: Peripheral blood mononuclear cells (PBMCs) from healthy donors (HD) and/or AML patients were cocultured during 7 days with the irradiated cell line k562-mb21-41BBL, when NKAEs were isolated by magnetic immunodepletion and lentivirally transduced with an NKG2D-41BB-CD3z CAR. CAR expression was measured up to 13 days post-transduction by flow cytometry. Cytotoxic activity against AML cell lines was performed by Propidium Iodide and Annexin V staining. Toxicity was evaluated by Europium-TDA assays. Effector cells were characterized by flow cytometry and Cytometric Bead Arrays were done to study cytokine release profile. Results: AML can be targeted with an NKG2D CAR since all patients studied express at least one NKG2DL. Primary NK cells can be lentivirally transduced with an NKG2D CAR, showing a modest but stable CAR expression up to 13 days after transduction (23% ±4,7% NKG2D + , 33,35% ±4,25% GFP + ). CAR-NKAE cells perform robust cytotoxicity towards MOLM-13 AML cell line after 24h of coculture at an effector:target ratio of 1:1, exerting a quasi-total lysis of AML cells. This represents a significant increase in anti-leukemia effect compared to untransduced NKAE (92,6% ±0,3% vs 72,2% ±10%, p = 0,0138). Surface expression of relevant molecules for NK activity showed that NKp30, NKp44 (natural cytotoxicity receptors), CD69 (early activation marker), CD25 (IL2R alpha chain), FasL (mediates FasL-mediated cytotoxicity), NKp80 (C-type lectin-like surface-activating receptor) and TRAIL (TNF-Related Apoptosis Inducing Ligand) were more expressed by transduced NKAE. Cytokine release profile revealed a higher production of IL-6, IL-17A, sFasL and IFNg by CAR-NKAE. These results suggest that the studied mechanisms could be underlying the increased anti-tumor activity shown by CAR-NKAE. We did not observe relevant toxic effect of none of the cells against PBMCs from HD. Low toxicity was found against NL-20 lung cell line, but there were no differences between NKAE and CAR-NKAE. Conclusions: Our preliminary results show that primary NK cells from HD and/or AML patients can be lentivirally transduced with a second generation NKG2D CAR, exerting a robust anti-leukemia activity towards AML cell lines and a safe profile over healthy tissues. Therefore, NKG2D CAR NKAE could be considered a promising approach to treat AML. Disclosure: DJPJ holds patents in CAR-T-cell therapy field. DAL declares an equity interest, advisory role, and intellectual property licensing to CytoSen Therapeutics and Kiadis Pharma, and advisory role with Caribou BioSciences and Courier Biosciences. PR has licensed medicinal products and receives research funding and equity from Rocket Pharmaceuticals, Inc., Patents & Royalties, Research Funding. The remaining authors declare no competing interests.
Background: Since CD33 is expressed on over 90% of acute myeloid leukemia (AML) blasts and leukemic stem cells and is indispensable for cell survival, it is a promising target for immunotherapy in AML, such as CAR-T cells or antibody drug conjugates. Methods: To compare the functionalities of second generation CARs and third generation CARs we generated two second generation anti-CD33 CAR (CAR33) with either 4-1BB or CD28 as costimulatory domains and a third generation CAR33. We evaluated the proliferation and cell viability of second and third generation CAR33-T cells. The cytolytic capacity of the CAR33-T cells against AML cell lines, primary AML blasts and human stem and progenitor cells (HSPCs), was determined by Chromium51 release assay and the antigen-specific response by cytokine secretion assay. The long-term killing capacity was assessed by coculturing CAR-T cells with target cells for 10 days with rechallenging of the CAR-T cells on every second day. To prove the antigen specificity of CAR33-T cells, functional assays were performed against CD33 positive and negative target cells. Results: First, the cytotoxicities of CAR33-T cells are antigen-specific and antigen-dependent. Only CD33-positive, but not CD33-negative cells were killed by CAR33-T cells and could stimulate CAR33-T cells to secrete cytokines. Second, we found that the second generation CAR33-T cells with CD28 as costimulatory domain (2G.CD28.CAR33-T) had comparable viability but reduced proliferation capacity compared to the third generation CAR33-T cells (3G.CAR33-T), whereas the second generation CAR33-T cells with costimulatory domain 4-1BB (2G.4-1BB.CAR33-T) expanded less with lower viability. In terms of short- and long-term killing capacity and levels of cytokine release 2G.CD28.CAR33-T cells and 3G.CAR33-T cells showed similar abilities, while 2G.4-1BB.CAR33-T cells exhibited the lowest potential. Conclusions: In summary, third generation CAR33-T cells exhibited improved properties in terms of viability and proliferation as well as short- and long-term anti-tumor activity when compared to second generation CAR33-T cells. Disclosure: Maximilian Felix, Blank: Molecular Health GmbH (consultant) Maria-Luisa, Schubert: Kite/Gilead, Takeda (consultant). Michael, Schmitt: Apogenix, Hexal and Novartis (research support). Hexal, Kite/Gilead (travel grants). Bluebird bio, Kite, Novartis (financial support for educational activities and conferences). MSD (advisory board member). MSD, GSK, Kite, BMS ((co-)PI of clinical trials). TolerogenixX Ltd. (co-founder and shareholder). Carsten, Müller-Tidow: Bayer AG (research support). Pfizer, Janssen-Cilag GmbH (advisory board member). Pfizer, Daiichi Sankyo, BiolineRx (grants and/or provision of investigational medicinal products). Christian, Kleist: TolerogenixX Ltd. (co-founder and shareholder). Tim, Sauer: Pfizer, Gilead, Amgen, Takeda, Astellas, BMS (advisory board member), AbbVie, Pfizer (financial support for educational activities and conferences), Matterhorn Biosciences, Ridgelien Discovery (consultant).
Background: Relapse and graft-versus-host disease (GvHD) are the main causes of morbidity and mortality after allogeneic hematopoietic cell transplantation (HCT). We recently showed that human iNKT cells inhibit alloreactive donor T cells and promote graft-versus-leukemia (GvL) effects. Now, we aim to further enhance the antileukemic potential of iNKT cells by introducing a chimeric antigen receptor (CAR) while maintaining their tolerogenic properties making them an ideal cytotherapeutic candidate to treat relapse after allogeneic HCT. Methods: CD19-CAR-iNKT cells were co-cultured with acute lymphoblastic leukemia (ALL) and Burkitt lymphoma (BL) cells. Cytotoxicity was analyzed by subsequent multiparametric flow cytometry and multiplex analysis. To assess the immunomodulatory properties of CD19-CAR-iNKT cells, T-cell activation (flow cytometry) and proliferation (CFSE dilution) of conventional T cells were analyzed after co-culture with allogeneic mo-DCs in presence or absence of CD19-CAR-iNKT cells. Results: CD19-CAR-iNKT cells showed robust cytotoxic activity against ALL and BL cell lines and primary patient cells. Multiplex analysis revealed the release of inflammatory cytokines such as IFN-γ and TNF as well as cytotoxic effector molecules like granzyme A, granzyme B and perforin. Interestingly, PD-1 expression was increased on CD19-CAR-iNKT cells after being challenged with lymphoma cells. Consequently, adding the checkpoint inhibitor nivolumab further increased the activity of CD19-CAR-iNKT cells. Regarding their tolerogenic properties, CD19-CAR-iNKT cells retained their ability to induce apoptosis of mo-DCs through CD1d signaling independent of the CAR resulting in reduced activation and proliferation of alloreactive T cells. Conclusions: We demonstrate that CD19-CAR-iNKT cells efficiently lyse CD19 + leukemia and lymphoma cells through their CAR while preventing alloreactive T cell responses interacting with dendritic cells. Checkpoint inhibition may further increase their cytotoxic activity without exacerbating the risk of GVHD after allogeneic HCT making them an ideal cytotherapeutic to treat relapse in this challenging clinical setting. Disclosure: Nothing to declare
Background: Multiple myeloma is a hematological disease characterized by an uncontrolled proliferation of plasma cells. New therapeutical agents developed survival rates but the outcome of the disease remains to be improved. The search for target antigens for CAR-T cell therapy against multiple myeloma yielded with the B-cell maturation antigen (BCMA) a possible candidate. Several studies of BCMA-directed CAR-T-cell therapy showed promising results. Methods: Second generation BCMA-CAR-T cells were manufactured at GMP standard by using the CliniMACS Prodigy® device. Cytokine-release in BCMA-CAR-T cells after stimulation with BCMA positive versus negative myeloma cell lines, U266/HL60, was determined by intracellular staining and flow cytometry. The short-term cytotoxic potency of CAR-T cells was evaluated by chromium-51 release, while the long-term potency used co-culture (3 days/round) at the effector:target cell ratio of 1:1 and 1:4. To evaluate the activation and exhaustion of CAR-T cells, exhaustion markers were assessed by flow cytometry. Stability was tested by comparison of these evaluation on different timepoints: d0 as well as d + 14 and d + 90 of cryopreservation. Results: BCMA-CAR-T cells can release much more cytokines upon stimulation with U266 cells in all donors (20.29 ± 4.868 and 1.193 ± 0.560, P = 0.0199; 20.16 ± 5.122 and 0.764 ± 0.597, P = 0.0194; 62.21 ± 2.029 and 1.348 ± 0.134, P = 0.0004) but not HL60 cells (1.820 ± 0.866 and 1,193 ± 0.560, P = 0.3102; 1.533 ± 0.627 and 0.764 ± 0.597, P = 0.0921; 2.922 ± 0.667 and 1.348 ± 0.134, P = 0.0602). Interestingly TNFa release was higher than IFNg on every timepoint (58.80 ± 8.890 and 18.92 ± 4.604, P = 0.0188; 59.35 ± 4.000 and 20.68 ± 5.198, P = 0.0179; 72.19 ± 2.379 and 63.32 ± 0.723, P = 0.0266). For CD8 + BCMA-CAR-T cells, TNFa and IFNg had the same level of cytokine-release (47.61 ± 10.07 and 30.00 ± 1.143, P = 0.076; 40.99 ± 10.42 and 33.88 ± 8.733, P = 0.1922; 72.85 ± 4.142 and 68.48 ± 4.195, P = 0.4590). However, CD4 + BCMA-CAR-T cells had lower level of IFNg than TNFa (10.40 ± 3.332 and 61.86 ± 10.57, P = 0.0065; 63.99 ± 6.929 and 13.50 ± 5.956, P = 0.0134; 49.82 ± 4.682 and 76.72 ± 4.737, P = 0.0345). There was no significant difference in cytokine-release after cryopreservation: neither double positive CAR-T cells (P = 0.9350) nor single positive (TNFa/IFNg) CAR-T Cells (P = 0.2786, P = 0.2489). Killing efficiency of U266 cells correlated with the dose of CAR-T cells in a classical 4-hour chromium-release assay. There was no significant difference after cryopreservation on timepoints d + 14 or d + 90 (P = 0.1300, P = 0.9602). As for long-term potency, after 3-rounds co-culture, BCMA-CAR-T cells reached to dominant position while U266 cells nearly disappeared at both ratios. As for endurance of BCMA CAR-T cells function, BCMA CAR-T cells kept their ability to kill all U266 cells over six rounds. Exhaustion markers were detected to evaluate CAR-T cells: LAG3 declined when CAR-T cells were activated and proliferated but decreased when they failed to kill; PD1 showed a similar trend as LAG3 but TIM3 had a curve trend. Conclusions: BCMA-CAR-T cells manufatured under GMP conditions possessed the ability for robust and specific killing of target tumor cells with a higher release of cytokines. Even after 14 or 90 days of cryopreservation, their cytotoxic functions were maintained at the same level. This give clinicians enough time to schedule the timepoint of BCMA CAR-T cell application to the patient. Disclosure: Nothing to declare
Background: Allogeneic hematopoietic stem cell transplant (allo-HSCT) represents an effective curative option for several malignant diseases; relapse remains the main cause of treatment failure. ab-T and B-cell depleted haploidentical HSCT offers a unique platform to evaluate the effect of post-HSCT Donor Lymphocyte Infusion (Haplo-DLI) in high-risk patients. Methods: Between August 2016 and July 2021, 32 high-risk patients received unmanipulated haplo-DLI after an ab-T and B-cell depleted haploidentical-HSCT (tab.1). Haplo-DLI were prophylactic in 19 cases due to high-risk disease [very high risk at diagnosis, previous allo-HSCT and positive minimal residual disease (MRD) at HSCT] and pre-emptive in 13 cases due to early post-transplant positive MRD. Results: Haplo-DLI were infused at median time of 4,2 months after transplant (range 1-43). Median CD3 + , CD4 + and CD8 + infused cells were 0,473x10^6/kg (range 0,059-3,044), 0,289x10^6/kg (range 0,022-2,140) and 0,160x10^6/kg (range 0,012-0,069), respectively. Fifteen patients received more than one infusion (table 1). Six patients developed grade III-IV acute GvHD at a median of 43 days after Haplo-DLI (range 34-76), the cumulative incidence of this complication being 19% (95% CI 9-38%). Seven out of 13 patients (54%) who received pre-emptive Haplo-DLI remained disease-free over time, while response rate for prophylactic haplo-DLI was 74% (14/19). With a median follow-up of 24 months, global progression free survival (PFS) and overall survival (OS) were 64% (95% CI 45-78%) and 71% (95% CI 50-84%), respectively; non relapse mortality (NRM) was 13% (95% CI 5-13%). Table 1. Patients characteristics. ALL, acute lymphoblastic leukemia; AML, acute myeloid leukemia; CML, chronic myeloid leukemia; DLI, donor lymphocyte infusion; GvHD, graft versus hosti disease; HL, Hodgkin lymphoma, HSCT, hematopoietic stem cell transplant; MAC, myeloablative conditioning; MDS, myelodysplastic syndrome; MRD, minimal residual disease; RIC, reduced intensity conditioning. Conclusions: Unmanipulated haplo-DLI after ab-T and B-cell depleted haploidentical-HSCT were feasible and safe, with encouraging result in terms of PFS and clearance of MRD. Disclosure: Nothing to declare
Background: Mesenchymal Stromal Cells (MSC) are multipotent non-hematopoietic cells with immunomodulatory and regenerative properties that are being used as a treatment for allogeneic HSCT complications. Access to advanced-therapy medicinal products on a named-patient compassionate-use basis is available in special situations and requires pharmacovigilance and subsequent audit and investigation of their safety and efficacy. Methods: Retrospective analysis of the use of MSC in our center in a compassionate-use basis for complications of allogeneic HSCT (2006 to 2021). Use of MSC in clinical trials are excluded. We use an AEMPS approved investigational MSC product derived from bone marrow of healthy donors and expanded under GMP conditions (PEI 10-146). Results: Sixty-two allogeneic HSCT recipients (37 male, 60%; median age 48 years, range 17-73; 29 AML/MDS, 10 ALL, 8 NHL/CLL, 4 MM, 4 SAA, 7 other indications; 27 matched related, 15 matched unrelated, 14 cords and 6 haploidentical donors) received a total of 248 infusions (median 4, range 1-16) of approximately 1x106 MSC cells/kg for the following transplant complications: 50 GVHD (81%), 8 hemorrhagic cystitis (HC; 13%), 3 cytopenia (5%) and 1 transplant-associated thrombotic microangiopathy (2%). GVHD target organ involvement included 39 gastrointestinal (78%), 34 cutaneous (68%) and 18 hepatic (36%), with severity score 2, 3 and 4 in 20 (40%), 17 (34%) and 13 (26%), respectively. One GVHD case with concomitant CMV infection received MSC as first-line to spare further immunosuppression with corticosteroids, but all others had MSC as second-line after failure of corticosteroids, alone in 14 cases (29%) and in association with other drugs (etanercept, ruxolitinib, alemtuzumab, basiliximab and vedolizumab) in the rest. Thirty-seven patients (74%) achieved an overall response at day +28 from MSC treatment, including 17 (34%) with complete response and 20 with partial response (40%). All-cause mortality was 26% (13/50) at 90 days from treatment. Clinical response to MSC was associated with survival at day 90 (p = 0.003). Eight cases with grade 3 (3, 37%) or 4 (5, 63%) HC refractory to first-line therapy (most commonly continuous saline irrigation and hyaluronic acid) received MSC as a named-patient compassionate-use basis, achieving 3 complete responses, 2 partial responses and 3 refractory cases by day +28 from treatment. By day +90, four patients died and the other four were in complete response from their HC. Overall, in our named-patient compassionate-use series in the various indications, overall response rate at day +28 was 69%, complete response rate 32%, and 44 patients (71%) were alive 90 days after the first MSC infusion. Conclusions: In the absence of formal regulatory approval, access to MSC in special situations including named-patient compassionate-use is available for life-threatening transplant complications which cannot be treated satisfactorily with authorized alternatives. This study audits our practice with MSC in transplant complications and suggests that their use is safe and effective in this setting. Disclosure: Nothing to declare
Background: Relapse after allogeneic hematopoietic cell transplantation (HCT) is the leading cause of mortality in patients with acute myeloid leukemia (AML). Increased recovery of γδ T lymphocytes after HCT is associated with better disease-free survival. The γδ T cells have MHC-independent, potent cytotoxic activity against AML and other neoplastic cells. Thus, donor γδ T cells can play an important role in regulating AML relapse after HCT. We assessed if γδ T cell clonality, distinct clones or specific receptor expression can predict AML relapse prevention after HCT. Methods: We used previously frozen banked bone marrow biopsy samples containing viable mononuclear cells, obtained at AML diagnosis, at 3 months and at relapse after HCT. We performed flow cytometry sorting of γδ T cells, extraction of genomic DNA and γδ T cell clonality assessment by T cell receptor γ locus (TRG) sequencing (ImmunoSEQ platform). Multiparametric flow cytometry was used to assess the expression of γδ T cell receptors and AML blast ligands. Results: We identified 24 adult (18 years and older) patients with available bone marrow samples who received their first HCT for AML in complete remission: 12 patients did not relapse, and 12 patients relapsed after HCT. Non-relapsed patients had higher TRG clonotype diversity at 3 months post-HCT as compared to the relapsed patients. Non-relapsed vs. relapsed patients had higher expression of TIGIT (46.5% vs. 34.2%, p = 0.03) and CTLA4 (72.8% vs. 34.6%, p = 0.05) receptors on γδ T cells (CD3 + /γδ TCR + ) but similar expression of NKG2D/CD314 (60.2% vs. 79.2%, p = 0.15). The expression of corresponding ligands on AML blasts at diagnosis for CD155 (TIGIT ligand) was 11.4% vs. 17.9% (p = 0.85), for CD86 (CTLA-4 ligand) was 69.3% vs. 64.7% (p = 0.88) and for MICA/B (NKG2D ligand) was 3.2% vs. 0.26% (p = 0.1) in non-relapsed vs. relapsed patients, respectively. Comparing 3-month and relapsed samples in patients relapsing after HCT, there was decreased TRGV9-1, TRGV7-1 and TRGJP-1 repertoires from 3 months to relapse. Conversely, the usage of TRGV5-1 gene was increased at the time of relapse. Conclusions: We found that higher diversity of donor TRG clonotypes are associated with AML relapse prevention after HCT. In addition, decreased usage of TRGV9-1 and TRGV7-1 genes influences higher relapse risk after HCT. This study findings can help to implement γδ T cell immunotherapy strategies against AML. Disclosure: Funding Source: ORIEN NOVA Award M2Gen COI: Nelli Bejanyan serves on advisory board for Medexus pharma, Magenta therapeutics and CTI Biopharma. Marco Davila has equity in Adaptive Biotechnologies and Precision Biosciences, and has received licensing fees and research support from Atara Biotherapeutics and CRISPR.
Background: Sequential therapy as conditioning in allogeneic stem cell transplantation (SCT) is frequently used in higher risk MDS, but comparisons with another regimen are rare. Methods: In this retrospective study conducted at the University Medical Center Hamburg/Germany we compared the impact of FLAMSA-Busulfan-Fludarabine (n = 81) with Treosulfan-Fludarabine (n = 95) conditioning on post-transplant outcomes in MDS. FLAMSA-FB regimen consists of fludarabine (30 mg/m2; total dose 120 mg/m2), amsacrine (100 mg/m2; total dose 400 mg/m2), and cytarabine (1 g/m2; total dose 4 g/m2) therapy from days -11 to -8, followed by a three-day interval without therapy and busulfan from day -4 to -3 with a total dose of 6.4mg/Kg and fludarabine on day -4 and -3 (30 mg/m2, total dose 60mg/m2). Treo-Flu regimen consisted of Treosulfan (12 g/m2, total dose 36 mg/m2) on days -6 to -4 and fludarabine (30 mg/m2; total dose 150 mg/m2) on days -6 to -2. Results: In the FLAMSA-FB group 11 patients (13%) had related donor (MRD 11% MMRD 2%) and 70 patients (86%) had unrelated donors (MUD 54% MMUD 32%), compared to 17 patients from MRD (18%) and 78 patients from unrelated donor (MUD 61% MMUD 21%) in the Treo-Flu group (p = 0.1). ATG was the major GvHD prophylaxis in both arms. Second allograft was seen only in the Treo-Flu group (n = 9) and the median number of blasts in bone marrow at transplantation were higher in the FLAMSA-FB than in the Treo-Flu group (9vs 2%, p < 0001) IPSS- low, intermediate 1 and 2 and high risk was 3%,15%,57% and 26% in the FLAMSA-FB arm and 13%, 53%, 25% and 9% in the Treo-Flu arm, respectively. Median platelet and neutrophil engraftment were significantly delayed in the Treo-Flu group when compared to the FLAMSA-FB group: 15 vs 12 days(p = 0.02) and 13 vs 12 days (p = 0.009). The cumulative incidences of aGVHD grade II-IV, III-IV and cGvHD were similar between the two groups: 37%, 9% and 49% in the FLAMSA-FB group and 37%, 16% and 47% in the Treo-Flu group (p = 0.2; p = 0.7 and p = 0.9, respectively). The cumulative incidence of non-relapse mortality at 5 years and relapse was 23% and 35% in the FLAMSA-FB and 14% and 23% in the Treo-Flu group (p = 0.07 and p = 0.06, respectively). After a median follow-up of 30 months (range, 1-218), the 3-year overall Survival (OS) and progression free survival (PFS) were in a univariate analysis higher in the Treo-Flu group: 72 vs 51% (p = 0.001) and 58 vs 45% (p = 0.04), respectively. In a multivariable analysis for OS only low IPSS (low-intermediate I vs Intermediate II-high risk) (HR = 0.2, 95%CI 0.2-0.95, p = 0.037) was associated with improved OS. Conditioning regimen, patient age (≤61 vs >61) donor age ((≤34 vs >34), type of Donor (MRD vs MUD vs MMRD vs MMUD), status at transplant (progressive/refractory disease vs others) and blasts in bone marrow (≤3.5 vs >3.5) had no significant impact on OS. Conclusions: Acknowledging the retrospective nature of our study, our results suggest that sequential conditioning with FLAMSA-FB does not improve survival in MDS-patients undergoing allo-SCT. Disclosure: nothing to decalre
Background: Natural Killer (NK) cells are known for their high intrinsic cytotoxic capacity and the possibility to be applied as ‘off-the-shelf’ third party donor cell therapy. In cancer patients suffering from multiple myeloma (MM), an elevated number of NK cells has been correlated with a higher overall-survival rate. However, NK cell function can be impaired by upregulation of inhibitory receptors, such as immune checkpoint NKG2A (natural killer group 2A). With the aim to overcome suppression of anti-tumor NK cell function, we used the CRISPR-Cas9 nuclease to knockout (KO) the killer cell lectin like receptor C1 (KLRC1) locus encoding NKG2A in primary NK cells, which led to significant increase in NK-cell mediated cytotoxicity against both MM cell lines and patient-derived MM cells. Methods: Primary NK cells were isolated from PBMCs from healthy donors. Upon transfer of KLRC1-targeting CRISPR-Cas9 nuclease, KLRC1 KO-NK cells were expanded using IL-15 cytokine under feeder-cell free conditions. KLRC1 KO was analyzed using Tracking of Indels by Decomposition (TIDE), T7 endonuclease I (T7E1) assay and next-generation sequen cing (NGS). NKG2A expression of KO-NK cells was compared to non-edited NK cells (NT-NK cells) by flow cytometry. Cytotoxicity of NK cells was analyzed against MM-tumor cell lines and allogenic MM patient-derived tumor cells. Results: The chosen CRISPR-Cas9 nuclease disrupted 70-86% of KLRC1 alleles, as evaluated by T7E1 (70%), TIDE (75%), or NGS (86%). KLRC1 KO significantly reduced NKG2A expression on gene-edited NK cells analyzed after three weeks of cultivation compared to NT-NK cell population (KO-NK cells 43.5% vs NT-NK cells 90%; n = 10, p < 0.05). Inhibition of NKG2A-expressing NK cells is mainly related to HLA-E ligand expression, which is often over expressed in anti-tumor response and can be particularly upregulated by IFN-γ. After 24h co-culture of IFN-γ pre-stimulated U266 tumor cells, they showed increased lyses induced by NKG2A KO-NK cells compared to NT-NK cells at different effector:target (E:T) ratio (E:T 2.5:1, 40% vs 15.5%, 1:1, 42.2% vs 13.4%, 0.5:1, 12.3% vs 5.6%; n = 4, p < 0.05). Additionally, after co-culture of NK cells with non-stimulated MM1.S, significantly higher lyses for NKG2A KO-NK cells compared to NT-NK killing capacity could be shown (E:T 2.5:1, 82% vs 70%; 1:1, 82.2% vs 61%; 0.5:1, 72.3% vs 38.9%; n = 5, p < 0.5). To address the increased killing capacity in a preclinical setting, we isolated primary tumor cells from bone marrow aspirates of differently treated MM patients. Allogenic NKG2A KO-NK cells showed significantly higher primary MM tumor cells lyses as compared to allogenic NT-NK cells (E:T 2.5:1, 40.7% vs 35.5%, 1:1, 36.7% vs 30.8%, 0.5:1, 29.7% vs 25.6%; n = 10 p < 0.05). Conclusions: Taken together, the deletion of inhibitory KLRC1-NK cell receptor resulted in a significantly increased NK cell-mediated cytotoxicity against allogenic patient-derived MM cells. Our protocol for gene editing of NK cells provides a robust platform to perform variety of further modulations to design NK cell-based therapeutic approaches to overcome immune checkpoint inhibition against different tumor entities. Disclosure: E.U. has a sponsored research project with Gilead and BMS. T.C. has a sponsored research collaboration with Cellectis and is a scientific advisor to Excision BioTherapeutics. The other authors declare no competing financial interest.
Background: Progressive multifocal leukoencephalopathy (PML) is a serious opportunistic viral disease of the brain caused by the human polyomavirus 2 (HPyV-2) (previously known as: JC polyomavirus). It usually affects patients with significantly impaired cellular immune defenses. The majority of patients suffer from an underlying malignant hematological disease. The natural course of PML is usually fatal, especially in this group of patients; about 90% die within a few months. To date, there is no approved therapy for PML and all antiviral strategies have failed in trials. Since 2018 several case reports and case series reporting successful treatment with allogeneic virus-specific T cells have been published. Methods: Sixteen PML patients have received at least one infusion of virus-specific T cells at the Department of Neurology at Hannover Medical School on a compassionate use basis. T cells were manufactured from HLA-partially matched healthy donors using cytokine capture system and immunomagnetic selection. According to current publications, this is the largest cohort worldwide as far as we know. Enriched human polyomavirus 1 (HPyV-1) specific T cells, a close relative of HPyV-2, were used in all of the cases. All patients were closely monitored by clinical examination, MRI imaging and laboratory analysis. CSF analysis included cell count, albumin quotient, detection of oligoclonal bands, determination of HPyV-2 viral load, analysis of HPyV-2 antibody specificity index, and measurement of neurofilaments (phosphorylated heavy chain, pNfH). Results: Therapy has been completed in 16 patients, who received between one and five T-cell infusions with an interval of usually two or four weeks between doses. As underlying diseases they suffered from lymphoproliferative disorders (n = 9), autoimmune diseases (n = 3), lymphopenia (n = 2), acquired immune deficiency syndrome (n = 1), and breast cancer (n = 1). Median observation time between first T-cell administration and last follow-up was 3 months (1-12 months). Improvement in neurological symptoms occurred in 10 cases (62.5%), and symptoms remained stable in two cases (12.5%). A total of four patients (25%) experienced worsening of symptoms during therapy, of which three patients (19%) died of PML. The median cell count at baseline lumbar puncture prior to the first infusion was 1/µl (1-16/µl), the median albumin quotient was 7.4 (3.3-11.6), and the proportion of patients with positive oligoclonal bands as evidence of intrathecal immunoglobulin G synthesis was 71% at the first CSF analysis and as high as 80% at the second. Comparison of first and last CSF analysis showed a decrease in CSF pNfH in patients with improvement or stabilization of symptoms, while the values increased in patients with poor outcome (p = 0.0040). Furthermore, a decrease in HPyV-2 viral load between first and last CSF analysis correlated with a positive outcome (p = 0.0013). Conclusions: Therapy with allogeneic virus-specific T cells leads to an improvement or stabilization of neurological symptoms in the majority of patients. Close monitoring of patients is crucial, and a reduction in viral load as well as a decrease in pNfH in the CSF indicate to be positive prognostic markers. However, controlled clinical trials are needed to better assess the efficacy of the therapy and to identify further prognostic factors. Disclosure: Nothing to declare.
Background: Epstein-Barr virus (EBV) causes significant morbidity and mortality in immunocompromised patients. Functional EBV-specific cellular immunity can be restored by adoptive T-cell transfer in patients with EBV-associated complications following transplantation or immunosuppression. The current study explores results of a personalized T-cell manufacturing program evaluating donor, patient, T-cell product and outcome data. Methods: Patient-tailored clinical-grade EBV-specific T cells from stem cell, haplo-identical family or third-party donors were manufactured by stimulation and immunomagnetic selection using the CliniMACS Plus or Prodigy device and PepTivator EBV EBNA-1 and Select. T-cell donors were selected by best HLA-matching (minimum 3/6 matches in HLA-A, -B, -DR) and EBV-specific T-cell precursor frequency in peripheral blood. Consecutive manufacturing processes were evaluated and patient outcome and side effects were retrieved by retrospective chart analysis. In a subset of 18 patients, EBV-specific T cell frequencies were monitored in peripheral blood by interferon-γ ELISpot assay following stimulation with peptide pools. Results: Forty clinical-grade EBV-specific T-cell products from stem cell (n = 13), family (n = 9) or unrelated third-party donors (n = 18) were generated between 2015 and 2019 for n = 37 patients with hematopoietic stem cell (HSCT, n = 27), solid organ (SOT, n = 5) or no (n = 5) transplantation history. Median time from initiation of third party donor search to CTL manufacturing start was 10.1 days. There was no significant difference in terms of cell yield and purity in the final T-cell products from stem cell, family or third party registry donors. Three products have not been infused due to prior death or cure. Thirty-four patients received 1-12 (median 2.0) EBV-CTL products (fresh and cryopreserved); the median number of transferred cells for the first transfer was 2.7x104 CD3+ cells/kg body weight (bw) in stem cell donor products and 1.4x104 CD3+ cells/kg bw when derived from third party donors. EBV-CTL led to complete clinical response in 19 of 31 patients, who survived at least three weeks after transfer. Complete viral clearance was documented in 13 of 20 HSCT patients, for whom data were available. Responses did not correlate with transferred CTL numbers. While no infusion-related toxicities were reported, two HSCT patients developed de novo GvHD (skin °I, n = 1; liver °IV, n = 1) after T-cell transfer, both had received EBV-CTL from the stem cell donor. These two patients had received CTL numbers above median (3.04x104 and 5.0x104 CD3+ cells/kg bw, respectively). EBV-specific T cells could be detected in 15 of 18 monitored patients (83.3 %) after transfer and detection of antiviral T-cell responses correlated with a favorable clinical response in these patients. Conclusions: Personalized clinical-grade manufacturing of EBV-CTL from stem cell, family or third-party donors is fast and feasible. Adoptive transfer of manufactured EBV-CTL is effective and safe regardless of EBV-CTL donor origin. Timely production of EBV-CTL from pre-characterized registry donors is a valuable alternative to cryopreserved CTL lines for patients lacking an EBV-positive stem cell donor. Transfer of EBV-CTL to patients being immunocompromised for other reasons than in a transplantation context provides an attractive new option, which should be further explored in clinical trials. Disclosure: Nothing to declare.
Background: Donor lymphocyte infusions (DLI) hold the potential to re-induce remission in patients suffering from relapsed myeloid malignancies after allogeneic hematopoietic stem cell transplantation (HSCT). Analysis of the cell product with regard to prediction of response to DLI is largely missing. Here, we perform in-depth investigation of surface molecules present on donor T-cells in respect of clinical outcome. Methods: In this prospective clinical study, we enrolled 14 patients with relapsed myeloid malignancies (Table1) after HSCT, who were treated with unmodified DLI. The median follow-up was 22 months post first DLI. An aliquot of the infused DLI product was collected and 30 color-spectral flow cytometry for extensive immunophenotyping of T-cells was performed. Functional markers, such as 4-1BB, LAG3, NRP1, PD1, TIGIT, TIM3, VISTA were included in the staining panel. Results were evaluated both, via conventional 2D-gating and complementary unsupervised cluster analysis by Uniform Manifold Approximation and Projection (UMAP). Statistical analyses were performed with unpaired t-test or Mann-Whitney-Wilcoxon test. Results: After DLI treatment, 5 patients had a durable remission, whereas 9 patients developed a subsequent relapse (Table1). Patients without relapse after DLI had received significantly lower numbers of CD4+conventional (Tconv) effector-memory (CD45RA-CCR7+/−, EM) cells compared to relapsing patients (1.4 × 106/kg BW (range 4.6 × 105-1.4 × 106) vs. 3.6 × 106/kg BW (1.4-6.3 × 106), P = .03). Also, the absolute numbers of EM-like regulatory T-cells was lower in patients without relapse (0.6 × 105/kg BW (4.5-8.9 × 104) vs. 1.6 × 105/kg BW (3.7 × 104-2.3×106), P = .02). Additionally, frequencies of CD4+ naïve (CD45RA+CCR7+) Tconv were higher in patients without relapse (53.75% (31.34-84.77%) vs. 32.65% (14.96-53.59%), P = .01). UMAP analysis of DLI cell products revealed a subpopulation of CD4+EM-like cells, which had lower frequencies in patients without relapse. Moreover, lower frequencies of CD8+EM-like cells were seen in this group. Of note, CCR5+ expression within this CD8+EM-like population was significantly lower in patients without relapse (1.34% (0.23-2.98%) vs. 5.04% (1.74-13.33%), P = .002). Next, functional markers were analysed based on their mean fluorescence intensity (MFI). Patients without relapse showed lower MFI values of VISTA on CD4+naïve and EM-like cells. Along the same lines, we observed lower PD1 expression on CD8+naïve-like and CD4+EM-like cells in the same group . With regard to GVHD post DLI, UMAP analysis of functional markers revealed lower PD1 expression on CD8+effector-like cells in patients developing GVHD when compared to patients without GVHD. Conclusions: In conclusion, we identified phenotypical differences of T-cells within the DLI cell product comparing patients with and without relapse after DLI. Therefore, deep phenotyping of the DLI product might be useful for prediction of response to therapy, including GVHD. However, this dataset is limited by the small cohort size. Further validation in a larger cohort is underway. Disclosure: Nothing to declare.
Background: Effector γδ T cells immediately recognize and kill malignant cells in a broad-based non-MHC restricted manner. Increases in circulating donor-derived γδ T cells during post-bone marrow transplant (BMT) recovery have been significantly associated with improved disease-free survival (DFS). Relapse post-Haplo/Cy BMT occurs in approximately 45% of patients. We sought to mitigate relapse in this context by expanding, activating, and infusing donor-derived haploidentical γδ T cells. We now report preliminary clinical and biologic correlative findings from the first cohort of patients who have been treated with ex vivo expanded and activated donor γδ T cells (EAGD). This single-center Phase I clinical trial represents the first systemic infusion of allogeneic EAGD cells in the post-BMT setting Methods: Standard of care reduced-intensity flu/cy/TBI conditioning was followed by an unmanipulated bone marrow graft and 50mg/m2 Cy on days +3 and +4 post-transplant. EAGD were manufactured using the Miltenyi Prodigy® bioreactor and cryopreserved. The product was infused intravenously within 5 days of neutrophil engraftment (ANC > 500/µL X 3d). Peripheral blood was collected at EAGD infusion and monthly thereafter through day +90, with additional collections every 6 months through 1 year. Biologic parameters included multiparameter flow cytometric immunophenotyping and single cell cytokine analysis of the EAGD graft. Peripheral blood analysis includes leukocyte count and differential, immunophenotyping, and serum Th1/Th2/Th17 cytokine analysis. Primary endpoints include dose-limiting toxicities (DLT) and grade 3-4 adverse events while secondary endpoints include incidence of acute and chronic GvHD, relapse, and overall survival. Results: Three patients have received the first dose level of 1 x 106 EAGD/kg. All three patients remain in morphologic complete remission at 20.1, 17.8, and 6.1 months post-BMT. One patient is receiving ongoing hypomethylating therapy for the occurrence of recipient chimerism. Grade 1-2 toxicities include constipation, CMV reactivation, emesis, fatigue, and hypomagnesaemia. Steroid-responsive cutaneous acute Grade I-II GVHD has been observed in all patients with one patient experiencing Grade II intestinal GvHD. No chronic GVHD, DLTs, treatment-related ≥ grade 3 adverse events, or cytokine release syndrome has occurred. EAGD grafts contained 88.7%-99.2% Vγ9Vδ2 + γδ T cells with small populations of NK cells and <1.0 x 105 αβ T cells/kg. EAGD principally expressed Granzyme B, MIP1α, MIP1β, and IL-2. Significant peripheral lymphodepletion persisted through the first 100 days post-BMT followed by slow recovery of CD4 + , CD8 + , γδ T and B cells. NK cells remained within the low normal range throughout. T cells transitioned from a CD45 + CD27- effector phenotype to CD45RA-CD27+/− central to effector memory phenotype as recovery progressed. CD3+CD4+CD25hiFoxP3+ Treg cells remained <3% of circulating T cells. Preliminary serum cytokine analysis revealed an initial inflammatory environment with predominant expression of IFNγ, and TNFα and T cell expression of Granzyme B, MIP1α, IFNα, and TNFα that gradually decreased as recovery progressed. Conclusions: Early indications suggest that EAGD transfusion with the initial dose level of 1 x 106 EAGD/kg has manageable toxicity and an appropriate immune recovery profile with 3 of 3 patients alive and progression-free. Clinical Trial Registry: https://clinicaltrials.gov/ct2/show/NCT03533816 Disclosure: The clinical trial is conducted as an Investigator-Initiated Trial with the Kansas University Cancer Center as the sponsor and Dr. Joseph P. McGuirk as the Principal Investigator. The trial is funded by IN8Bio, a US public biopharmaceutical company. Dr. McGuirk also received funding through the Kansas University Cancer Center from Kite, Novartis, Bristol Myers Squibb, and Allovir as a site investigator for ongoing clinical trials. Dr. Lawrence Lamb, Mariska ter Haak, and Samantha Youngblood are employees of IN8Bio.
Background: Allogeneic T-memory cells (Tm) co-infused with T-cell depleted (TCD) peripheral blood stem cells (PBSC) is known to protect against post-transplant infections. However, the effective Tm dose for infections protection relative to engraftment/ cytokine release syndrome (ES/ CRS) and graft-versus-host disease (GVHD) risks is unknown. Many programs, including ours, set Tm doses arbitrarily because data guiding Tm dosing is lacking. Between 2014 and 2019, we systematically reduced Tm doses in 54 paediatric patients transplanted consecutively using published experiences and our patients’ outcomes as guidance. Methods: We retrospectively reviewed transplant outcomes, including infections, graft failure (GF), ES/ CRS and GVHD rates in patients given Tm (denoted by CD45RO + ) doses categorised in 3 strata (S): S1: > 3 to 11 x 106/kg (N = 17); S2: > 11 to 31 x 106/kg (N = 20), and S3: > 31 to 330 x 106/kg (N = 17). Transplant indications included cancers (N = 42) and non-cancers (N = 12) diseases. PBSC were T-cell depleted as the main form of GVHD prophylaxis with: CD34 + selection (N = 4), CD3 + depletion (N = 48) or alpha-beta T-cells depletion (N = 2). The majority of patients (N = 34) received non-radiation based preparative regimens. Anti-microbial prophylaxis included echinocandins and ganciclovir. ES/CRS and GVHD were pre-emptively treated. Results: The median age of patients in the 3 strata were: 94 (range, 9 to 231); 84 (range, 10 to 192); 48 (range, 6 to 183) months; and their donors: 38 (range, 14 to 56); 37 (range, 21 to 52); 38 (range, 31 to 52) years, respectively. The CD34 + cell doses co-infused with Tm in the 3 strata were: 18 (range, 14 to 32); 18 (range, 7 to 45); and 20 (range, 13 to 57) x 106/kg, respectively. The CD3 + cell doses in the stem cell products averaged 1.72 (range, 0.0 to 4.97) x 104/kg. Transplant outcomes including cytomegalovirus (CMV), adenovirus (ADV), BK virus (BKV), and fungal infections, GF, ES/ CRS, acute and chronic GVHD rates in the 3 strata are summarised in Table 1. Infections, GF and chronic GVHD rates were not statistically different among patients in the 3 strata. However, ES/CRS and acute GVHD rates were statistically different in patients receiving different Tm doses. At a median follow-up of 452.5 (range, 18 – 1965) days, the 3-year overall survival was not statistically different among patients in the 3 strata (Figure 1): (S1) 69.6% vs. (S2) 69.2% vs. (S3) 80.2% (p = 0.692). Figure 1: Overall Survival of 54 patients Given Different Tm Doses Conclusions: There appears to be no infection protective advantage with higher Tm doses. However, ES/CRS and acute GVHD rates were significantly different with different Tm doses. This preliminary data supported the Tm dosing strategy used in our program. Disclosure: Nothing to declare.
Background: The total blood volume (TBV) to process during extracorporeal photopheresis (ECP) and sufficient number of collected mononuclear cells (MNCs) is not clearly defined. Cell yields vary between apheresis devices. The duration of ECP may affect the clinical benefit but also pose a threat to a patient’s safety. In this study we have made an attempt to determine if the blood volume processed can be minimized to increase the patient’s safety, concurrently preserving the ECP therapeutic effect. Methods: 23 patients (F/M – 12/11, median age 52 (17-66)) who have underwent allogeneic hematopoietic cells transplantation complicated by steroid resistant chronic graft-versus-host disease were enrolled in the study. A median number of 10 (3-30) procedures was performed between January 2019 and October 2021 with 1 or 2 TBV being processed. ECP was achieved in an offline manner. MNCs were collected with the Spectra Optia device (Terumo BCT) continuous mononuclear cell collection (cMNC) protocol. The final product was irradiated with UVA-PIT (PIT Medical Systems GmbH). Results: 100 ECP procedures with 1TBV processed and 79ECP procedures with 2TVB processed procedures were performed. No statistical difference in patient age, body weight and TBV was observed between the compared sets of data. The procedures where 1TBV was processed resulted in shorter duration than 2TVB procedure: 114 min (88-189) vs 224 min (158-253), p < 0,001; lower product volume 104 mL (65-200) vs 210 mL (139-237), p < 0,001, lower MNC content 4,42 × 1012 (0,08-20,84) vs 11,3 (2,4-30,1), p < 0,001 and lower MNC content per kg body weight 159,8 ×106/kg body weight (24,4-373,1) vs 75,7 (2,1-235,4), p < 0,001. The MNC CE1 collection efficiency was 37,3% (4,1-99,9) for 1TBV and 44,9% (14,3-81,6) for 2TBV, p = 0,004. Parameters characterizing the patient safety i. e. platelet drop and ACD(A) infused per patient were lower for the procedures with 1TBV processed: 18,1% (-71,2-63,4) vs 34,2% (-15,0-48,1), p < 0,001 and 449 mL (161-694) vs 752 mL (439-1101), p = 0,004 respectively. The clinical response rate was 77,3% for the skin (n = 22), 100,0% for the liver (n = 5), 0,0% for the gastrointestinal (GI) tract (n = 5), 42,9% for ocular GVHD (n = 7), 55,6 for oral GVHD (n = 9), 50,0% for the musculoskeletal system (n = 2), and 100,0% for bronchiolitis obliterans (n = 2). Conclusions: Although there is no consensus on the MNC cell dose to be irradiated in ECP, Worel et al. has indicated a cut-off MNC level 13,9×106 /kg body weight which predicts 75% overall response. Except for one patient who was leukopenic, all of our patients have reached (and often significantly exceeded) the cut-off MNC /kg body weight level. The MNC count collected during 1TVB cycle was comparable or higher to those reported by other authors who have also concluded an efficacious ECP therapeutic effect. This preliminary analysis shows that it is possible to collect sufficient number of MNC through processing of one total blood volume. Moreover, this approach increases patient’s safety by lowering the ACD(A) volume infused, lowering platelet loss and improving patient comfort by lowering the time of the procedure. The presented data and response ratio support processing 1TBV with Spectra Optia cMNC protocol for UV irradiation. Disclosure: Nothing to declare.
Background: Viral infections is a major cause of post-HSCT complications and transplant-related mortality. Delayed post-HSCT immune reconstitution fails to support the resistance to common infections with CMV, AdV, EBV and other opportunistic agents. In the context of other post-HSCT complications such as graft-vs-host disease and graft hypofunction, this failure may lead to longer-term immune deficiency with severe consequences. The CliniMACS Prodigy® platform (Miltenyi Biotec) allows obtaining IFNγ-secreting virus-specific lymphocyte-enriched cell products by immunomagnetic separation (IMS). Here we present clinical experience of using such products in patients with post-HSCT viral infections. Methods: The study enrolled 5 patients with post-HSCT viral complications, receiving infusions of virus-specific cell products. Magnetic separation of IFNγ + lymphocytes obtained from a haploidentical donor by leukapheresis was performed using the CliniMACS Prodigy platform in accordance with the recommended protocol. In vitro stimulation was performed with PepTivator® peptide pools (Miltenyi Biotec) in combinations corresponding to the current condition of the patient and possibly preventing related complications (see the Table). Subpopulation composition of cell product was assessed by flow cytometry using routine surface staining for CD3, CD4, CD8 and IFNγ. The median content of viable T lymphocytes in the cell product was 35%. The median content of virus-specific interferon-expressing T cells in the graft was 90%. The median infusion dose was 39.27 ∙ 103 of viable CD3 + cells per kg weight (min 11 ∙ 103/kg, max 432 ∙ 103/kg). The median time after HSCT at the moment of infusion was 125 days (min 30 days, max 1.5 years). Viremia was monitored by PCR. Detection and monitoring of virus-specific donor T cells was performed by IFNγ ELISpot assay. Results: Robust response to the treatment correlating with the ELISpot data was observed in 4 of 5 patients. Clinical and laboratory indicators for the patients are given in the Table: Conclusions: Infusions of multivirus-specific lymphocytes obtained by IFNγ-directed IMS provide effective treatment of viral complications that arise during post-HSCT immune reconstitution. Polyspecific antigenic stimulation performed in advance may facilitate prevention of viral complications during post-HSCT immune reconstitution. Disclosure: Nothing to declare
Background: Subarachnoid placement of bone marrow (BM)-derived total nucleated cells (TNCs) has been reported to be safe and relatively easy to perform in children with cerebral palsy (CP) and autism spectrum disorder (ASD). Methods: This was a retrospective, open-label trial to assess the side effects, safety, and tolerability of a single subarachnoid BM-derived TNC injection in patients with CP and ASD. Patients aged between 1 and 18 years were included in this study. The outpatient-based autologous BM stimulation consisted of 10 mg/kg/day G-CSF subcutaneously for 3 days. The procedure was performed under sedation and local anesthesia. We harvested 8 mL/kg of body weight of BM, filtered on a laminar flow cabiet, centrifuged, and enumerated using CD34 + and CD45 + flow cytometry. Caretakers were instructed to contact the research team if they developed symptoms. Results: Between May 2009 and December 2021, 640 patients were treated with BM-derived TNCs, 303 patients with ASD (47.3%) and 337 patients with CP (52.7%). The median age was 6 years (range,1 month to 18 years). Males comprised 71.6% of the study population, and 28.4% were women. Almost half of the patients (n = 309,48.3%) presented with any symptoms after the procedure. The characteristics of the study population are summarized in Table 1. Among the patients with ASD, the most common symptom was vomit/nausea in 63 patients (20.5%). Headache/irritability affected children over 60 months of age (p = 0.038). There was an association in patients reporting vomiting and nausea with a larger TNC volume infused (median 6.2 ml) (p = 0.003) and with fewer absolute neutrophil count (ANC) infused (p = 0.026). In children diagnosed with CP, symptoms were present in 160 patients (51.8%). We found an association between age >60 months and headache/irritability (p = 0.004). Conclusions: There was no association between symptomatic patients and leukocyte count or CD34 + x106/kg infused. The study reported an incidence of up to 309 (48.3%) symptoms after subarachnoid TNC administration. These secondary effects were not related to the laboratory parameters of the cells (leukocyte count or CD34 + x106/kg infused), but only the volume infused was associated with nausea and vomiting in children with autism older than five years of age. In most cases, these symptoms can be satisfactorily controlled without hospital admission, so we can consider it a safe procedure and possibly improve the quality of life of patients. Disclosure: Nothing to declare
Background: Progressive multifocal leukoencephalopathy represents an opportunistic viral infection of the brain with potential fatal outcome. Triggered by an immunosuppressive constitution, e.g. oncological diseases, chronic viral infections or immunosuppressive therapy in autoimmune disease/transplantation, affected individuals suffer reactivation of latently existing infection by human polyomavirus 2 (HPyV-2, former: JCV), which leads to lytic destruction of the brain parenchyma. The diagnosis is based on a triade of suitable clinical symptoms, typical MR-imaging findings and detection of HPyV-2 in cerebrospinal fluid (CSF)/brain biopsy. No approved effective therapy exists to date, but adoptive transfer of allogenic human polyomavirus 1 (HPyV-1) specific T cell proves to be a promising approach - basing on induction of immune reaction by cross-reaction due to partial equal epitopes of HPyV-1 and HPyV-2. Methods: Since March 2020, patients referred to our clinic suffering from defined, progressive PML were analyzed regarding endogenous amount of HPyV-1/ HPyV-2-virus specific T cells. If examination presented insufficient amount, adoptive HPyV-1-specific T cell transfer, extracted from partially human leukocyte antigen compatible donors, was initiated. Dosage varied between 2.0 × 10e4 and 1.0 × 10e4 CD3 + T cells per kg body weight. Targeted therapy regime included at least two doses of allogenic T cells. Follow up investigations included routine neurological examination which partially included examination of 55 m walking distance and Montreal cognitive assessment scale, CSF analysis to investigate HPyV-2 level, virus specific T cells within blood and magnetic resonance imaging. Results: Sixteen PML-patients received at least one dose of allogenic T cells and were followed up > six weeks after therapy initiation, fifteen patients completed aimed application of two doses. Follow up examination time varied between 42 and 379 days. The majority, eleven patients, suffered from oncological disease, two patients had a history of immunosuppressive disease, two patients suffered from idiopathic lymphopenia and one patient had acquired immune deficiency syndrome but developed PML despite sufficient antiretroviral therapy. In total, ten of sixteen patients showed improvement of symptoms, two patients presented with clinical stabilization and four patients suffered from progression of disease. Of ten patients course-controlled by walking distance/Montreal cognitive assessment scale, two patients showed objective improvement within walking distance and cognition with one further patient exhibiting improvement within cognition alone. Other investigated patients suffered from severe paresis or aphasia, so that examination tools did not fit to exhibit improvement demonstrated by elsewhere diagnostic. Conclusions: Allogenic HPyV-1-virus specific T cell therapy shows promising therapeutic approach in patients suffering from PML leading to an improvement or stabilization within the majority of treated patients, but examination of walking distance and cognition by Montreal cognitive assessment scale alone appears to be inadequate to show full therapy response. Despite the need of large controlled clinical trials to better assess the efficacy of therapy, further investigations regarding specific clinical scores are needed. Disclosure: Nothing to declare.
Background: Cytomegalovirus (CMV) reactivation is common after allogeneic hematopoietic cell transplantation (HCT) and may result in fatal CMV disease. Invariant natural killer T (iNKT) cells are potent modulators of the immune system preventing graft-versus-host disease (GVHD) while promoting graft-versus-leukemia (GVL) effects. It is thought that iNKT cells selectively influence mediators of both innate and adaptive immunity. Here, we investigated the impact of iNKT cells on virus control after allogeneic HCT. Methods: We report a single-center prospective observational study designed to investigate the impact of graft iNKT cells on early CMV reactivation in peripheral blood measured by weekly PCR from patient plasma. The primary endpoint was defined as detection of CMV DNA within 100 days following allogeneic HCT. Secondary endpoints were incidence of GVHD, non-relapse mortality (NRM), event-free survival (EFS) and overall survival (OS). The graft composition was studied by flow cytometry. Results: Median age of patients (n = 50) was 57 years (range 25-76). Two thirds of patients were CMV IgG seropositive and about half of donors were latently infected with CMV. Reactivation of CMV was noted in 23 (46%) patients after a median of 39 days (range 11-59). iNKT-cell numbers were significantly decreased (0.1% vs. 0.3%, p = 0.0001) in patients with early cytomegaloviremia. We also found a significantly reduced cumulative incidence of CMV reactivation after 100 days in patients with higher numbers of iNKT cells in their allograft (24% vs. 68%; p = 0.002). Acute GVHD °II-IV was observed in 5 (10%) patients. Also, extensive chronic GVHD occurred in 5 (10%) patients. Cumulative incidence of relapse or progression and NRM as competing risks at 2 years were 26% and 28%, respectively. 38% of all patients died during a median follow-up of 27 months resulting in a 2-year EFS of 47% and a 2-year OS of 61%. Conclusions: This study provides evidence that graft iNKT cells improve post-transplant immunity towards reactivation of latent virus infections. Therefore, iNKT-cell enriched grafts or adoptive transfer of iNKT cells are compelling cytotherapeutic strategies to improve outcomes after allogeneic HCT. Disclosure: Nothing to declare.
Background: Peripheral T-cell lymphomas (PTCL) and NK/T-cell lymphoma (NKTCL) share common characteristics of high chemotherapy resistance with frequent relapses and rapid disease progression. Efforts to improve outcome have incorporated autologous (auto-SCT) and allogeneic stem-cell transplantation (Allo-SCT). Allo-SCT has been to show a plateau of survival in responding patients, and even complete responses in patients who relapsed after various chemotherapy regimens. Although attempts to apply Allo-SCT in adult PTCL and NKTCL are steadily increasing, cases are still scarce so that there are very few prospective trials. Even though Allo-SCT could improve survival in relapsed and refractory patients who would otherwise have grave prognosis, there are several unsolved problems: suitable patient populations, HSCT timing (first relapse versus beyond first-relapse) and proper conditioning intensity and regimens (myeloablative conditioning vs. reduced-intensity conditioning). Methods: Peripheral T-cell lymphomas (PTCL) and NK/T-cell lymphoma (NKTCL) share common characteristics of high chemotherapy resistance with frequent relapses and rapid disease progression. Efforts to improve outcome have incorporated autologous (auto-SCT) and allogeneic stem-cell transplantation (Allo-SCT). Allo-SCT has been to show a plateau of survival in responding patients, and even complete responses in patients who relapsed after various chemotherapy regimens. Although attempts to apply Allo-SCT in adult PTCL and NKTCL are steadily increasing, cases are still scarce so that there are very few prospective trials. Even though Allo-SCT could improve survival in relapsed and refractory patients who would otherwise have grave prognosis, there are several unsolved problems: suitable patient populations, HSCT timing (first relapse versus beyond first-relapse) and proper conditioning intensity and regimens (myeloablative conditioning vs. reduced-intensity conditioning). Results: Fifteen patients received Allo-SCT with Bu3Flu6 conditioning regimen for relapsed and refractory T- and NK/T-cell lymphomas. Median age was 54 years (range, 33-65 years) and median previous lines of therapies was 2 (range, 1-3). 53.3% of the patients had received auto-SCT. Stem cell source were PB and CB in 14 patients and 1 patient, respectively; stem cell donor type were full-matched sibling and unrelated donor in 46.7% and 40% of the patients, respectively. After a median 3 cycles of salvage chemotherapies, 66.7 % and 33.3% of the patients were in CR and PR, respectively, before enrollment to the study, and for 5 patients who were in PR before Allo-SCT, 3 patients further achieved CR after Allo-SCT with Bu3Flu6. After a median follow-up duration of 17.7 months (range, 2.13-51.47 months), 2-year PFS and OS were 77.8% (95% CI, 45.5-92.3%), and 68.4% (95% CI, 35.9-86.8%), respectively. All patients engrafted neutrophils and platelets rapidly with a median of 12 and 12 days, respectively. There were no unexpected regimen-related toxicities including sinusoidal obstruction syndrome, hemorrhagic cystitis, and sepsis. Grade 3-4 acute graft-versus-host disease (GVHD) and moderate-to-severe chronic GVHD occurred in 40% and 60% patients, of which chronic GVHD combined with infection lead to death in 2 patients. Conclusions: 3-days Bu and 6-days Flu combination as a conditioning regimen is effective with tolerable safety profile for relapsed or refractory T- and NK/T-cell lymphoma patients who are undergoing Allo-SCT. Clinical Trial Registry: NCT02859402 Disclosure: This work was supported by Korea Otsuka Pharmaceutical Co., LTd. This study would not have been possible without the cooperation of the Korean lymphoma transplantation group (KLTG) and the Consortium for Improving Survival of Lymphoma (CISL). The authors have no conflicts of interest to declare.
Background: The collection of haematopoietic progenitor stem cells (HPSC) using an apheresis method for the treatment of various haematological malignancies is a standard procedure performed by the South African National Blood Service. Mesenchymal stromal cells (MSC) are CD45 negative fibroblast-like cells that have the potential to regulate immune and inflammatory responses, including possibly preventing and treating engraftment failure and graft-versus-host disease. This study investigated the possibility of the presence of MSCs in the HPSCs collection fraction. This would allow for using the same collection to obtain both HPSC and MSCs, thereby not only treating the malignancy, but also the potential side effects of the treatment. Methods: The HPSC collections were performed in mobilized donors as per standard apheresis protocols. Signed consent was obtained to use excess HPSC that were not required for the patients for research purposes. The HPSC collections were de-identified prior to being sent to the research laboratory. The mononuclear cells were isolated using a density gradient, followed by a CD45 bead isolation. The CD45 negative mononuclear cells were incubated in Dulbecco’s Modified Eagle Medium (DMEM) in the presence of 10% foetal bovine serum (FBS), or 5% human platelet lysate (HPL - produced by SANBS) and 2% penicillin-streptomycin, in a humidified 37°C CO2 incubator. Cell culture media was changed every 2-3 days, with trypsonising and splitting of cell numbers when 80% confluence was reached. The presence of fibroblast-like cells was visually evaluated using a phase-contrast microscope. Results: A total of three donations were obtained and the cells processed. The cells for each donation were grown in both FBS and HPL. The presence of fibroblast-like cells was seen in both culture conditions; Figure 1 shows the unstained fibroblast-like cells grown in the presence of HPL. Figure 1: Fibroblast-like cells grown in HPL (unstained) from HPSC apheresis collection Conclusions: The presence of fibroblast-like cells in three HPSC apheresis donations shows promise for the use of this fraction as a potential source of MSCs that can regulate immune and inflammatory responses. Future research will include the culturing of the fibroblast-like cells to larger numbers to facilitate the identification of these cells and confirm if they are indeed MSCs. Clinical Trial Registry: N/A Disclosure: Nothing to declare
Background: Allogeneic hematopoietic stem cell transplantation (HSCT), provides curative treatment chance for many patients with hematologic malignancy. But the complications such as infections, mucositis, and graft versus host disease are the main obstacles to be overcome in the post-transplantation period. Moreover, each of these complications can provoke the others. Granulocyte transfusion can be a reasonable option to support this critical neutropenic period, however, there are not enough randomized controlled trials, and the utility of this approach is controversial. This study aimed to evaluate the efficacy of granulocyte transfusion in allogeneic HSC recipients. Methods: We retrospectively examined the data of patients who underwent allogeneic HSCT because of acute leukemia, in Erciyes University Hospital, Bone Marrow Transplantation Center, between 2019 and2020. Thirty-one patients who have severe neutropenia lasting more than 15 days (absolute neutrophil count ≤0.5x 109/L) or severe infection or mucositis (grade 2-4) were considered eligible for granulocyte transfusion. Eleven patients who have an appropriate donor received granulocyte transfusion. The remaining 20 patients were considered as a control group and received conventional treatment for mucositis and infection. The clinical course was considered as ‘favorable’ if neutrophile recovery was achieved or the clinical symptoms and signs improved proceeding one week of treatment. Granulocyte concentrates were collected using apheresis from donors stimulated with corticosteroid and GCSF. Results: The number of patients suffering from mucositis was 6 (54.5%) in the granulocyte receiving group and only two of them responded to granulocyte transfusion favorably. Fourteen (70.0%) patients had mucositis in the control group and 5 of them were responsive to conventional treatment. Engraftment failure was seen in 3 patients in the granulocyte receiving group and there was no engraftment failure in the control group. Engraftment times for neutrophil and platelet were not significantly different between the two groups. The duration of hospitalization after transplantation was significantly longer in the granulocyte receiving group (p < 0.05). There was no adverse event related to granulocyte transfusion. Conclusions: In the present study, granulocyte transfusion did not provide any clinical benefit in the treatment of mucositis and in neutrophile recovery. Given the small size of the population, more comprehensive studies are needed. Disclosure: Nothing to declare
Background: Primary myelofibrosis (PMF) is a clonal myeloproliferative neoplasm and is associated with marrow fibrosis, extramedullary hematopoiesis, and the propensity of leukemia transformation. Allogeneic hematopoietic stem cell transplantation (allo-HSCT) is the only curative option, but the reports regarding outcomes of patients with PMF receiving allo-HSCT the literature were heterogeneous. In this study, we aimed to evaluate the efficacy and safety of allo-HSCT in patients with PMF. Methods: From 1996 to 2020, we consecutively enrolled 35 PMF patients who received allo-HSCT at our institute. Next-generation sequencing focusing on 54 myeloid disease-related gene mutations was performed in 29 patients who had enough specimens. The survival was calculated from the date of allo-HSCT. Results: The median age at allo-HSCT was 57.6 years. According to the Dynamic International Prognostic Scoring System plus (DIPSS-plus) risk stratification right before the HSCTs, 8.6% of patients were categorized in the intermediate-1; 40%, intermediate-2; and 51.4%, high-risk group. Nineteen (54.3%) patients had JAK2 V617F mutation, 2 (5.7%) MPL mutation, 5 (14.3%) CALR mutation, 1 triple-negative, and 6 (17%) unknown because there was no specimen for retrospective analysis (mostly diagnosed before 2006). Eleven (31%) patients had high molecular risk mutations (HMR), defined as ASXL1, EZH2, SRSF2, and IDH1/2 mutations. Twenty-five (71.4%) patients received reduced-intensity conditioning, which is associated with better NRM compared to myeloablative conditioning (MAC) (not reached vs. 7.9 months, P = 0.05), and earlier leukocyte engraftment (12 days vs. 15 days, P = 0.02). The median duration from diagnosis to allo-HSCT was 13.5 months (range 1.2-279.9 months). Eighteen (51.4%) patients received spleen management before allo-HSCT, including 5 with splenectomy and 13 with splenic irradiation. With the median follow-up of 54.9 months, the median overall survival (OS) was 20.0 months and the 5-year survival rate was 35.5% (Figure 1). The 1-year cumulative incidence of relapse (CIR) was 26.6% and 1-year non-relapse mortality (NRM) was 25.2%. Of the 18 mortality cases, 9 patients died of infection (including 1 graft failure), 4 died of the disease, and 4 died of graft-versus-host disease (GvHD). The 100-day cumulative incidence of grade 2-4 acute GvHD was 47%. Intriguingly, age or donor source has no prognostic impact on OS, NRM, or CIR. Similarly, there was no difference in terms of CIR, NRM, and OS among patients with various DIPSS-plus risks. Intriguingly, patients with spleen management had a trend of lower NRM but similar CIR and OS compared with those without. Furthermore, the patients with HMR share similar outcomes with those without HMRs, suggesting that HSCT may alleviate the negative prognostic impact of HMR mutations. Conclusions: Allo-HSCT has the potential to cure some PMF patients. However, it remains to be a challenging task. Finding strategies to reduce CIR and NRM and improve the outcome is warranted. The role of spleen management before allo-HSCT needs to be further clarified in larger cohorts. Disclosure: Nothing to declare
Background: Despite the widespread use of 2nd and 3rd generation tyrosine kinase inhibitors (TKIs), patients with advanced phase CML, blast crisis (BC) or accelerated phase (AP), still have poor prognosis. This study compares the results of conservative therapy and allo-HSCT in patients with advanced CML Methods: This retrospective study includes 162 patients with CML BC/AP. All patients received TKIs, in some cases (n = 62/20) followed by allo-HSCT. All patients received allo-HSCT with a reduced dose intensity conditioning regimen (fludarabine 180 mg/m2, busulfan 8-14 mg/kg or melphalan 140 mg/m2). In post-transplant period TKIs, mostly dasatinib (n = 36), were reinitiated in 42 cases. Non-transplant group consisted of patients with disease progression, late transplant center referral, or patients refusing the procedure. In these cases TKIs (2nd or 3rd generation in most cases) were continued as monotherapy (n = 60) or in combination with chemotherapy (n = 20). Allo-HSCT and TKI groups did not differ in age, sex, comorbidity, disease phase or presence of additional chromosomal aberrations (ACAs) (Tab.1). OS and EFS were defined as the time from treatment initiation (allo-HSCT/TKI) to death and/or loss of response/post-transplant relapse. The response was assessed in accordance with the recommendations of the European Leukemia Net. Results: A total of 71 (86%) patients engrafted. Gr2-4 aGVHD developed in 21(29%), Gr3-4 aGVHD in 14(20%), and cGVHD in 18(27%) of cases (severe cGVHD in 4 cases). Within 100 days past allo-HSCT the cumulative risk of relapse and NRM were 10% and 18%, accordingly. With a median follow-up of 44(1–344) months the cumulative relapse rate was 39% with 26 patients receiving subsequent DLIs and TKIs achieving complete molecular response (CMR) in 9, and progressing in 19 cases, accordingly. In TKIs group 71 patients were available for follow-up with 36(59%) progressing on therapy, and 25 achieving complete hematologic (CHR, n = 22), cytogenetic (CHR, n = 1) or molecular (CMR, n = 2) response, accordingly. Among 10 patients without history of BC one did not respond to therapy, while 9 achieved CHR (т=5), CCR (n = 2) or CMR (n = 2). Sixty-nine patients died due to disease progression. The allo-HSCT effect on OS of patients with AP or BC was also assessed by landmark analysis for 2 and 3 years with maximal phase onset chosen as a starting point. In 2 years the allo-HSCT significantly improved OS in patients with history of BC (71%) compared to TKI recipients (28%; p < 0.0001). In Cox’s regression model allo-HSCT was also associated with higher OS compared to TKIs (HR 0.37; 95%CI 0.15-0.89; p = 0.026). Three-year landmark analysis have also demonstrated allo-HSCT advantage with 82% OS compared with 32% in TKIs recipients (p < 0.0001; Fig 1) with this advantage retained in Cox regression model with allo-HSCT being a positive (RR 0.22; 95% CI 0.07-0.74; p = 0.014) and history of BC a negative (RR 20.5; 95% CI 2.77-151.45; p = 0.003) influence on 5-year OS. Conclusions: Despite of TKIs being the mainstay of therapy in CP CML, allo-HSCT still remains the only curative option for poor-prognosis patients. A timely referral to transplant center may salvage a patient in AP or BC. Disclosure: Nothing to declare
Background: Hematopoietic Stem Cell Transplantation (HSCT) in Chronic Myelomonocytic Leukemia (CMML) has a very important role, for being the only curative procedure in high-risk patients. Despite this statement, there is a small number of transplants in this pathology in Latin America. OBJECTIVE to evaluate the HSCT scenario in Latin America. Methods: Data from 29 patients with LMMC from 32 centers of the Latin American Registry of Transplantation in MDS, from April/1988 to December/2020, were analyzed. Statistical analysis was performed using the R program. Survival was analyzed using the Kaplan Meier curve and the prognostic factors, by Cox proportional risk. Results: The mean age was 56, 52 years, with a predominance of males (79,31%, n = 23) and Caucasian (89,66%, n = 26). According to R-IPSS stratification patients were Very Low risk (3,45%,n = 1), Low risk (10,34%, n = 3), Intermediate (17,24%, n-5), High Risk/ Very High Risk (13,80%, n = 4). About 55,17% had not stratification. A total of 24,14% of patients received more than 20 unities of red blood cells and 27,59% received more than 15 unities of platelets. A total of 23 (79,31%) of patients underwent treatment before BMT, in which 47,82% took Hypomethylating. The Myeloablative regimen was the most frequent (62,07%, n = 18), followed by the Reduced Intensity (20,69%, n = 6) and Non-myeloablative (17,24%, n = 5). In 72,41% of cases, the donors were related, and of these, 10.34% were haploidentical; 17.24% not related. The main sources of cells used were peripheral blood (62,07%, n = 18) and bone marrow (37,93%, n = 11). Post-transplant complications were observed in 72.41% (n = 21. The most frequent was infection (57.14%), mainly by CMV (45.45%); acute GVHD (42.86%), chronic GVHD (33.33%) and veno-occlusive disease (9.52%). Recurrence occurred in 30% of cases. The frequency of deaths was 37.93% (n = 11). The survival probability of transplanted patients was 47.40% in 5 years. In the Cox regression model, the risk of death was 6.72 times greater in ≥ 65 years patients (p = 0.015) (CI95%: 1,44 - 31,40). Cox’s model was evaluated using the proportional hazards hypothesis. The Global and individual Schoenfeld test was performed and the adequacy of the model was demonstrated. Patients were also stratified according to Bournemouth scores: 55,1% (n = 16) were high risk and 44,9%, (n = 13)were low risk. For MDAPS score patients were classified as: Low (13,7%, n = 4); Intermediate 1 (3,44%, n = 1), Intermediate 2 (20,6%, n = 6) and high (58,6%, n = 18). Regarding Mayo score, patients were stratified as: low (17,24%, n = 5); intermediate (17,24%, n = 5); high (65,51, n = 19%). Conclusions: This is the first study of HSCT in CMML performed in Latin American. It presents the difficulties of correct diagnosis and possibilities of HSCT and reflects the efforts of different centers for conducting patients to the correct diagnosis therapeutic strategies, including the management of pre and post HSCT. Age at HSCT was the only factor that influenced in OS. The advances in technology around molecular features of this disease are gradually being incorporated into clinical protocols and has been show a powerful tool for better predicting outcomes. However, in some centers the molecular approach is still a challenge. Disclosure: Nothing to declare
Background: Total body irradiation (TBI) at myeloablative doses is superior to chemotherapy-based regimens in young patients with acute lymphoblastic leukemia (ALL) undergoing allogeneic hematopoietic stem cell transplantation (allo-HSCT). However, in elderly and unfit patients, where reduced-intensity conditioning (RIC) regimens are preferred, whether a TBI- or chemotherapy-based approach is better is an unexplored issue. Thiotepa is an alkylating agent with radiomimetic activity and capability to cross the blood-brain barrier, that is used as part of ALL conditioning regimens. The aim of the current study is to compare transplant outcomes after RIC with TBI- or thiotepa-based regimens in ALL. Methods: Included were patients aged ≥40 years undergoing allo-HSCT for ALL in first complete remission between 2000-2020, receiving a RIC regimen containing either TBI- (4-6 Grays, Gy) or thiotepa-based regimen. Results: We identified a total of 265 patients, including 117 receiving TBI- (4 Gy, n = 65; 6 Gy, n = 52) and 148 receiving a thiotepa-based RIC regimen. Median age was 56 (range 40-72) versus 59 (range 40-75) years for TBI and thiotepa, respectively (p = 0.32). Thiotepa was more frequently associated to busulfan and fludarabine (n = 88) while TBI was more frequently associated to cyclophosphamide and fludarabine (n = 52), fludarabine alone (n = 27) or cyclophosphamide alone (n = 17). Most patients were diagnosed with Philadelphia positive ALL in both groups (59% for TBI and 55.4% for thiotepa, p = 0.14); T-ALL was diagnosed in 26 and 24 patients receiving TBI or thiotepa, respectively. HLA-identical and mismatched sibling donors were more frequent with thiotepa (35% versus 31% for matched and 21% versus 11% for mismatched siblings) while unrelated donors were more frequent in the TBI group (58% versus 44%) (p = 0.03). A longer interval from diagnosis to transplant was observed with thiotepa (6.7 versus 5.5 months, p < 0.01). Stem cell source was predominantly peripheral blood (94% for TBI and 81% for thiotepa, p < 0.01). The mainly used graft-versus-host disease (GVHD) prophylaxis was cyclosporine with either methotrexate or mycophenolate mofetil in both groups. In vivo T-cell depletion was more frequently used in the TBI group (54% versus 40%, p = 0.02). No imbalances for Karnofsky score (<90 in in 22% and 23% for TBI and thiotepa, p = 0.81) were observed. A Sorror score of 1-2 or ≥3 was observed in 19% and 20% of patients receiving TBI and 26% and 27% of those receiving thiotepa, respectively (p = 0.19). In univariate analysis, no differences were observed in transplant outcomes (for TBI vs thiotepa: relapse 23% versus 28%, p = 0.24; non-relapse mortality, 20% versus 26%, p = 0.61; leukemia-free survival, 57% versus 46%, p = 0.12; overall survival, 67% versus 56%, p = 0.18; GVHD/relapse-free survival, 45% versus 38%, p = 0.21; grade II-IV acute GVHD, 30% in both groups, p = 0.84; grade III-IV acute GVHD, 9% versus 10%, p = 0.89) except for chronic GVHD that was higher for TBI-based regimens (43% versus 29%, p = 0.03). However, in multivariate analysis we observed no differences in transplant outcomes according to the conditioning regimen used. Conclusions: In patients aged more than 40 years receiving a RIC regimen, use of thiotepa-based regimen may represent a valid alternative to TBI-based regimens due to no differences in the main transplant outcomes. Clinical Trial Registry: In patients aged more than 40 years receiving a RIC regimen, use of thiotepa-based regimen may represent a valid alternative to TBI-based regimens due to no differences in the main transplant outcomes. Disclosure: No COI to disclose
Background: Allogeneic haematopoietic stem cell transplant (HSCT) is considered a curative strategy for acute myeloid leukaemia (AML) and myelodysplastic syndrome (MDS) with excess of blasts or complex/adverse cytogenetic. The evaluation of comorbidities with HCT-CI score and the upper limit of 55 years for administering myeloablative conditioning (MAC) are common strategies to minimise HSCT non-relapse mortality (NRM). Despite multiple studies performed previously, this remains an area of uncertainty and precise data guiding MAC selection are still needed. Herein we report the outcome of patients affected with AML and MDS conditioned with MAC. Methods: HSCT was performed with GCSF mobilised peripheral blood stem cells. Conditioning protocol was with fludarabine 30 mg/m2 days -7, -6, -5, -4, -3 busulfan 3.2 mg/Kg days -6, -5, -4, -3 (FB4); graft versus host disease (GVHD) prophylaxis consisted of thymoglobulin (ATG 5 mg/Kg) or Campath 60 mg (27 and 94 patients, respectively) and single-agent ciclosporin 3 mg/Kg (therapeutic level of 150-200) until d + 56 and then tapered in absence of GVHD. Results: Between January 2016 and November 2020, 121 patients (77 AML, 44 MDS) with a median age of 56 (19-73) had FB4 conditioning. A median of 5.5x106 CD34 + /Kg was infused (3.1 – 8). Donors were: 21 full matched siblings, 76 full matched unrelated donors, 24 mismatched unrelated donors. Patients aged > 55 were 64 (53%). HCT-CI score <2 and ≥2 was present in 48 and 73 patients, respectively. Two years overall survival (OS) was 55% with a median OS of 42 months. No septic death before engraftment or primary graft failure were noted. Median time to neutrophils ≥1000/mL was 12 days (10-18), and 10 days (8-48) to platelets ≥ 20.000/mL. Median CD3 and CD15 chimerism at day 365 were 98% and 100%. Incidence of acute GVHD was 60% (grade III-IV 9%); overall chronic GVHD rate was 33% (moderate 14%, severe 7%). Incidence of venous occlusive disease (VOD) was 7%, no VOD-deaths were recorded. Cumulative incidence of relapse was 19%. Flow cytometry minimal residual disease (MRD) was positive in 28 patients at the time of HSCT and didn’t affect the OS. There was no significant difference in OS when patients were stratified according to age even if there is a non-significant trend for patients younger than 55. Age at HSCT did not influence NRM but was higher in patients with higher HCT-CI: 10% versus 43% if HCT-CI was <2 and ≥2, respectively (P 0.04). Two years OS for patients aged ≥ 55 and with HCTI-CI < 2 and for those with HCTI-CI ≥ 2 were 63% (median OS not reached in this group) and 45%, respectively. Three years OS was 57% and 42%, respectively. Conclusions: This analysis supports the feasibility of FB4 conditioning in patients affected with AML and MDS regardless of age. The decision for myeloablation should rely on comorbidities and disease characteristics rather than chronological age, especially for those with positive MRD at the time of HSCT. Disclosure: Nothing to declare
Background: Conditioning protocols for patients undergoing allogeneic hematopoietic cell transplantation (allo-HCT) are developing continuously to improve their anti-leukemic efficacy and to reduce their toxicity. A recent developed score (transplantation conditioning intensity or TCI) considers the intensity of conditioning as a continuum and not as “classical” defined myeloablative or reduced intensity conditioning. In previous studies, we compared two of the most used conditioning protocols from the intermediate TCI score based on one alkylating agent as fludarabine/melphalan (FluMel) vs. fludarabine/treosulfan (FluTreo) using the EBMT ALWP registry, which serve as basis for the comparison of the present study. Methods: In the present study, we compared the conditioning protocol FluMel with conditioning protocols based on FluMel (fludarabine 150 mg/m2, melphalan 140mg/m2) or with the addition of a second alkylating agent as FBM (fludarabine, mean 150mg/m2, carmustine 300-400mg/m2 and melphalan, mean 110 mg/m2) or FTM (fludarabine, mean 150mg/m2, thiotepa 5-10mg/kg and melphalan, mean 110 mg/m2). FBM and FTM have been shown to be equivalent at the dosages used in this study. We used following inclusion criteria: first allo-HCT from a matched sibling donor (MSD) or unrelated donor (UD) for patients with AML in complete remission (CR), (3) transplantation date between January 1st, 2009 and December 31st, 2020, (4) with an unmanipulated peripheral blood graft. Results: We included 3417 adult patients with acute myeloid leukemia (AML) in complete remission (CR) from the registry of the EBMT Acute Leukemia Working Party, 2567 patients were conditioned with FluMel and 850 patients in FBM/FTM. The median follow-up was 4.0 years in FluMel and 3.0 years in FBM/FTM cohorts. Patients in the FBM/FTM group were older (59.9 years vs. 59.0 years, p < 0.001) and had a worse Karnofsky performance score (KPS < 90, 27.1% vs. 20.1%, p < 0.001). Additional transplant characteristics as female donor (FBM/FTM: 28.2% vs. 32.4%, p = 0.02), CR1 status at allo-HCT (FBM/FTM: 82% vs 78.1%, p = 0.02), matched sibling donor (FBM/FTM: 21.1 vs. 31.7%, p < 0.0001) and in vivo T-cell depletion (anti-thymocyte-globuline in 75.8% of FBM/FTM patients, alemtuzumab more used in 66% of FluMel patients) were different among cohorts. In univariate analysis, patients in FBM/FTM group showed a better overall survival at 2 years (65.9% vs. 58.7%, p = 0.03) but a higher incidence of aGvHD II-IV at day 100 (25.8% vs. 16.8%, p < 0.0001) compared to patients treated with FluMel. In multivariate analysis, patients treated with FBM/FTM showed a trend for improved overall survival (FluMel with HR 1.17, 95%CI 1-1.38, p = 0.057) and leukemia-free survival (HR 1.14, 95%CI 1-1.3, p = 0.059) compared to FluMel treated patients. No significant differences were observed in relapse incidence (HR 1.12, 95%CI 0.94-1.33, p-value 0.21) and non-relapse mortality (HR 1.14, 95% CI 0.88-1.49, p-value 0.32). Conclusions: In conclusion, the addition of a second alkylating agent (BCNU/carmustine or thiotepa) to FluMel as FBM/FTM conditioning seems to improve overall survival while maintaining similar toxicity in AML patients in CR undergoing allo-HCT. Due to several limitations of the study including the retrospective nature of the study and unbalanced patient characteristics, these data should be interpreted with caution. Disclosure: Nothing to declare
Background: Remarkable improvements in haplo-SCT outcomes are made since the introduction of posttransplant cyclophosphamide (PTCy)-based GVHD prophylaxis. There remains, however, no consensus regarding best conditioning platform; choice of regimen is mostly dependent on center experience. Fludarabine (total 160 mg/m2)/Melphalan (100-140 mg/m2) combined with thiotepa (5 mg/kg) (FMT) vs 2 Gy total body irradiation (TBI) are two reduced-intensity regimens commonly used at our center. We present here the largest single center study to compare the transplant outcomes in patients with acute leukemia and MDS who underwent haplo-SCT using FMT vs FM/TBI. Methods: We included all consecutive AML/MDS and ALL patients who underwent haplo-SCT between 01/2012 and 12/2019 and received FMT or FM/TBI with PTCy/Tacrolimus/MMF GVHD prophylaxis. Primary objectives were to compare PFS and OS by conditioning regimen. Secondary objectives included cumulative incidence (CI) of NRM, CIR, and GVHD. Results: 134 patients with a median age of 52 (IQR 35-60) years were identified, 64 (48%) received FMT and 70 (52%) received FM/TBI. Table 1 summarizes baseline characteristics. At a median follow up of 3 years, the 3-year PFS/OS rates were 33%/47% and 37%/53% in the FMT and FM/TBI, respectively (p = 0.127 for PFS; p = 0.107 for OS). One-year NRM rates for FMT and FM/TBI were 37% and 23% (HR 0.585, 95% 0.325-1.050; p = 0.072). The CIR at 1/3 years were 19%/ 25% for FMT and 23%/27% for FM/TBI (p = 0.690). In UVA, age ≥ 55, high/very-high DRI, HCT-CI > 3, and reduced-dose melphalan 100 mg/m2 were significantly associated with inferior PFS and OS. In MVA, high/very high DRI (HR 1.950, 95%CI 1.248-3.046; p = 0.003) and HCT-CI > 3 (HR 1.586, 95%CI 1.009-2.493; p = 0.046) were associated with worse PFS. In MVA for OS, age≥ 55 (HR 1.997, 95%CI 1.093-3.650; p = 0.024), high/very-high DRI (HR 2.020, 95%CI 1.263-3.231; p = 0.003) and HCT-CI > 3 (HR 1.747, 95%CI 1.090-2.798; p = 0.020) were associated with inferior survival. In MVA for NRM, age≥ 55 (HR 2.646, 95%CI 1.453-4.816; p = 0.001) was associated increased NRM, with a trend for lower NRM with FM/TBI (HR 0.617, 95%CI 0.341-1.115; p = 0.110). Grades 3-4 acute GvHD at day 100 were 5% and 6% for FMT and FM/TBI, respectively (p = 0.8). The CI rates of chronic GvHD at 3 years for FMT and FM/TBI were 19% and 12%, respectively (p = 0.2) Conclusions: FMT and FM/TBI conditioning with PTCy/Tacrolimus/MMF GVHD prophylaxis showed comparable survival outcomes in haplo-SCT. There was a trend for better outcomes with FM/TBI related to decreased NRM. Prospective controlled studies to optimize conditioning regimen for haplo-SCT are needed. Disclosure: Nothing to declare
Background: Myeloablative conditioning schemes are the gold standard conditioning therapy in patients with acute myeloid leukemia (AML) or myelodysplastic syndrome (MDS) that underwent allogeneic haemopoietic stem cell transplantation. Reduced toxicity conditioning regimens, such as treosulfan-fludarabine (Treo-flu), have been increasingly used for treating comorbid and elderly patients, and they could be as effective as standard myeloablative, with fewer toxicity and mortality rates in relation with the procedure. Our primary objective was to compare overall survival (OS) and relapse free survival (RFS) in patients as contrast with standard myeloablative regimens. Our secondary objectives were evaluating toxicity and GVHD incidence. Methods: We retrospectively studied 97 patients, diagnosed with AML (n = 71) and MDS (n = 26), that underwent allogeneic stem cell transplantation at our institution between the years 2012-2020. Control myeloablative conditioning regimen was Bu-Flu (n = 67)[Busulfan total dose: 9,6-12,8mgr/Kg, fludarabine 160mg/m2]. Treo-Flu was used in 30 patients [Treosulfan total dose: 30-42 g/m2 (in 5 and 25 patients), fludarabine 150mg/m2]. Busulfan levels were not extracted as a rule. Results: Patient’s characteristics of both conditioning regimens were similar, excluding age (62 vs 54 p = 0,003) and HCT-CI score >3 (66,7% vs 44,8% p = 0,037) that were higher in Treo-Flu patients, as shown in table 1. Treosulfan group had more patients with uncontrolled disease (33% vs 16% p = 0,057). With a median follow up of 50,5 months (11,7-109,4 months), there weren’t significant differences between treosulfan and busulfan in 3-year overall survival (64% vs 73% p = 0,101), relapse free survival (64% vs 65% p = 0,385) and relapse-free mortality (21% vs 14%; p = 0,164) . The presence of grade >2 toxicity with treosulfan (40%) was lower than with busulfan (59,7%; p = 0,057). These results rely mostly on the presence of mucositis, which was significantly lower in treosulfan group (13,3% vs 49,3%; p = 0,001). There weren’t significant differences on GI, hepatic, pulmonary, renal or cardiac toxicity between both groups. There were no differences between CMV or fungal infections. There weren’t significant differences between both conditioning regimens neither in 100-days cumulative incidence of severe aGVHD (7% vs 9%; p = 0,766) nor 3-year cumulative incidence of moderate-severe cGVHD (32% vs 34%; p = 0,591). Conclusions: Considering patient selection and retrospective study limitations, in our experience treosulfan-fludarabine as allogeneic transplant conditioning regimen, regarding OS and RFS, offers similar results as myeloablative busulfan-based, despite patients were older with more comorbidity and poor disease status at transplantation. Disclosure: Nothing to declare
Background: This study investigates the incidence and risk factors for hemorrhagic cystitis (HC) in a large cohort of adults undergoing allogeneic hematopoietic stem cell transplantation (alloHSCT) from a single Institution. Methods: Between January 2015 and June 2021, 960 adults underwent first alloHSCT at our Institution and included in the study. Data was collected retrospectively and updated in October 2021. Results: Overall, the median age was 58 years, 45.7% of patients underwent MUD alloHSCT, and 252 received MAC regimens. Of the 252 patients transplanted using MAC regimens, 81.4% received high doses of intravenous busulfan (HD BU). PTCY was given to 72.4% patients, and among them, 91.4% received dual T-cell depletion with ATG. Overall, the cumulative incidences of grade 2-4 and grade 3-4 HC at day +180 were 13.2% and 5.8%, respectively, and the median of days to grade 2-4 and 3-4 HC were 39 and 44 days. BK virus was analyzed on 95% of cases and only 60% with HC grade 2-4 were positive. Additionally, the level of BK did not correlate with severity. Those patients receiving HD BU (Day + 180 23.1% vs 10.5%) had higher incidences of grade 2-4 HC than those that did not. Patients receiving PTCY (the majority of them in combination with ATG-CsA) had comparable incidences of grade 2-4 HC (Day + 180 14.6% vs 10.5%, P = 0.12). Additionally, patients with blood group O had higher incidence of grade 3-4 HC than patients with other blood groups (7.7% vs 3.9%, P = 0.002). A multivariate analysis exploring risk factors for grade 2-4 and 3-4 HC was calculated including conditioning regimen, HCT-CI, donor type, blood group, and GVHD prophylaxis. The administration of HD BU (HR 3.06, P < 0.001) and the use of PTCY (HR 1.69, P < 0.001) were found to be risk factors for being diagnosed with grade 2-4 HC. Secondary to the results obtained in the cumulative incidence and MVA analyses, the effect of HD BU and PTCY was explored in detail. The 545 patients receiving HD BU without PTCY were 1.9 times more likely to have grade 2-4 HC compared with patients that did not received any of these drugs (P = 0.038). The 90 patients that received HD BU with PTCY-based GVHD prophylaxis were 4.6 times more likely to present grade 2-4 HC compared with patients that did not received any of these drugs (P < 0.001). The use of PTCY, without HD BU did not increase the probability of grade 2-4 HC in our analysis (HR 1.4, P = 0.20). Conclusions: The incidence of grade 2-4 HC at our Institution was 13.2%. HD intravenous BU was found to be an independent predictor for grade 2-4 HC; and when combined with PTCY the risk increased x2.38 times. PTCY-based GVHD prophylaxis, alone, did not increase the probability of HC in our study. Patients receiving MAC alloHSCT with HD intravenous BU combined with PTCY-based GVHD prophylaxis may need change in supportive care with forced diuresis and increased dose of MESNA. Disclosure: Nothing to declare
Background: The pre-transplant minimal residual disease (MRD) has a negative impact on post-transplant survival in AML patients due to increased relapse risk. An increased busulfan dosage may lead to lower relapses, however it may be also associated with increased NRM. Due to its narrow therapeutic index, the therapeutic drug monitoring approach based on the calculation of area under the curve (AUC) was developed to optimize the busulfan exposition. In this study, we compared post-transplant outcomes after the administration of personalized or fixed busulfan dosage in patients with intermediate risk AML focusing on pre-transplant MRD status. Methods: 86 patients (male = 48; median age 56 years, 21-73) with intermediate risk AML and available pre-transplant MRD data (multicolored flow cytometry, “different from normal” approach, according to ELN guidelines), who received allografts (matched, n = 61; mismatched, n = 25) during 2015-2020 years at the University Cancer Centre Hamburg-Eppendorf were included. 33 patients received personalized busulfan dosage (AUC) after model-based AUC-calculation and 53 fixed busulfan dosage (12.8 mg/kg bw iv, n = 30; 9.6 mg/kg bw iv, n = 15, 6.4 mg/kg bw iv, n = 8). There were more females in the AUC group (67% vs 30%, p = 0.01). The myeloablative busulfan/fludarabine regimen was the most used in the both groups (82% and 58%, respectively). Patients from non-AUC group received more post-transplant cyclophosphamide than ATG as GvHD prophylaxis (25% vs 6%, p = 0.022). Results: The median follow up was 27 months (1-61). The relapses were lower in pre-transplant MRDneg patients (11%, 5-25% vs 35%, 22-51%, p = 0.008) and in those who received MAC (19%, 11-31%) vs RIC (65%, 20-93%, p = 0.04). The non-AUC led to higher relapses at 3 years (35%, 23-49% vs 6%, 2-19%, p = 0.02) resulting in lower 3-year LFS (55%, 40-70% vs 78%, 54-91%, p = 0.009) and OS (69%, 54-81% vs 82%, 60-93%, p = 0.05) comparing to AUC. The NRM at 3 years was not different (AUC: 7%, 2-19% vs non-AUC: 10%, 5-21%, p = 0.48). The aGvHD at 1 year (AUC: 21%, 11-37% vs non-AUC: 14%, 7-27%, p = 0.41) and cGvHD at 3 years (AUC: 57%, 40-73% vs non-AUC: 45%, 31-60%, p = 0.30) were not significantly different. Of the pre-transplant MRDpos patients, those from AUC group (n = 13) showed lower relapses at 3 years (8%, 1-36% vs 49%, 31-67%, p = 0.07) resulting in higher 3-year LFS (92%, 69-98% vs 41% 24-61%, p = 0.023) and OS (100% vs 58%, 39-75%, p = 0.032) compared with non-AUC group (n = 31). The NRM at 3 years was not significantly different (AUC: 0%, non-AUC: 10%, 3-28%, p = 0.24). Of the pre-transplant MRDneg patients, there were no significant differences concerning relapses at 3 years (5%, 1-24% vs 17%, 6-40%, p = 0.32), NRM at 3 years (6%, 1-28% vs 9%, 3-27%, p = 0.56), 3-year LFS (84%, 61-95% vs 74%, 52-88%, p = 0.43) and OS (84%, 61-95% vs 84%, 61-95%, p = 0.92) between AUC (n = 19) and non-AUC (n = 23) groups, respectively. Conclusions: The personalized, AUC-based, busulfan administration as part of conditioning seems to overcome the negative impact of pre-transplant MRD positivity with acceptable NRM in patients with intermediate risk AML undergoing allo-HSCT. Disclosure: Nothing to declare
Background: Alemtuzumab is a humanised monoclonal antibody specific for CD52 that depletes T cells in vivo and reduces acute and chronic graft versus host disease (GVHD). Alemtuzumab is highly efficient at preventing acute and chronic GVHD in Fludarabine and Melphalan (FM) conditioning but at the cost of prolonged immunosuppression, increased infections, and relapse. The optimal dose and scheduling of Alemtuzumab is not defined, particularly in the matched unrelated donor (MUD) setting where empiric flat-dosing, unsupported by pharmacokinetics, is the rule. Methods: In this retrospective single centre study we compared two empiric dose reductions from the original 100mg dose regimen (Kottaridis et al., 2000). These were introduced as policy changes in an attempt to minimise excessive T cell depletion. From 2015-2018, 48 patients received 60mg (30mg on days -4 and -2) (FMA60) and from 2019-2021,40 patients received 30mg delivered on day -1 (FMA30). Transplant serum samples were available from 21 FMA60 and 16 from FMA30 at days 0, +7 and +14. Alemtuzumab levels were measured by ELISA assay. All Patients were transplanted with fludarabine 150mg/m2 and melphalan 140mg/m2 conditioning chemotherapy at the Northern Centre for Bone Marrow Transplantation (Newcastle upon Tyne Hospitals UK). We compared the overall incidence and severity of acute GVHD in the two cohorts. Overall survival (OS) and relapse free survival (RFS) were analysed by Kaplan Meier compared with log-rank tests. Results: On all days, the mean (SD) of Alemtuzumab concentration were significantly different between FMA60 and FMA30 cohorts. Respectively, Alemtuzumab on day 0 was 6.22 (2.09) ug/ml and 3.33 (0.97) ug/ml (p < 0.0002); on day+7: 2.385 (1.456) ug/ml and 0.8423 (0.4431) ug/ml (p = 0.0003), on day +14: 1.124 (0.8435) ug/ml and 0.4768 (0.4907) ug/ml (p = 0.0410). No differences were observed in the incidence and severity of acute GVHD between the two cohorts (Chi-square test p = 0.6801). In FMA60, GVHD was 43.75%, 8.33% and 2.08% compared with 50%, 2.5% and 2.5% in FMA30 for grades I, II and III, respectively. Comparable OS and RFS were observed; OS (log-rank test p = 0.742), the OS at 2 years were 66% and 68% in the FMA 60 and FMA 30, respectively. The hazard ratio for death in FMA 60 versus FMA 30 was 1.121 (95% CI 0.518-2.426) p = 0.772. RFS (log-rank test p = 0.698), the hazard ratio for death in FMA 60 against FMA 30 was 1.208 (95% CI 0.465-3.140) p = 0.698. Conclusions: This retrospective analysis reports the in vivo level of Alemtuzumab on day 0, +7 and +14 in recipients of 60mg (delivered on days -4 and -2) and 30mg (day -1) in MUD transplant. The results confirm that 30mg of Alemtuzumab on day -1 is effective in preventing GVHD in MUD transplant. Disclosure: No conflicts of interest to declare.
Background: Total body irradiation (TBI) is a part of the standard myeloablative conditioning regimen before allogeneic stem cell transplantation (allo-HSCT) for patients with acute lymphoblastic leukemia (ALL) but not available in all center. This study aimed to compare TBI-based conditioning to chemotherapy in term of graft-versus host disease (GVHD), overall survival (OS), event-free survival (EFS), non-relapse mortality (NRM) and cumulative incidence (CI) of relapse. Methods: Retrospective study was conducted in adult patients who underwent allo-HSCT from HLA-identical sibling donors between January 2012 and July 2021. Conditioning regimen consisted TBI plus etoposide or cyclophosphamide (Cy). Non TBI-regimen consisted of busulfan (iv) plus Cy (Bu-Cy) or fludarabine plus busulfan plus Cy (FBC) or thiotepa plus busulfan plus fludarabine (TBF). GVHD prophylaxis consisted of cyclosporine and short course of methotrexate +/− antithymocyte globulin (ATG). Results: Sixty patients were included. Patient characteristics were similar between the two groups (table1). Patients conditioned with TBI were more likely to have delayed platelet engraftment and mucositis grade II-IV (p = 0.03, p < 10-3, respectively). Cumulative incidences of acute GVHD grade II-IV and chronic GVHD were not significantly different between TBI and non-TBI groups (53.6% vs 34.4 %, p = 0.11 and 49.7% vs 33.1%, p = 0.42, respectively). CMV infection(s) were not significantly different between TBI and non-TBI groups (35.7% vs 31.3 %, respectively, p = 0.11). The median follow-up was 36 months (range, 2 - 116 months).There were no statistically significant differences between groups in terms of OS and EFS (65.7% for TBI group vs 50.6% for non-TBI group, p = 0.22 and 58.2% vs 49%, p = 0.27, respectively). Patients from both groups had a comparable CI of NRM (25.5% vs 19.1%, p = 0.73, respectively). TBI group had lower CI of relapse compared to non-TBI group with no significant difference (13.3% vs 29.3%, p = 0.79, respectively). Table1. Patient characteristics. Conclusions: Chemotherapy-based conditioning seems to be an alternative to TBI-based conditioning. Prospective studies in adults ALL comparing TBI-based regimen to homogeneous group of chemotherapy-based regimen are warranted to validate this finding. Disclosure: Nothing to declare
Background: For decades, anti-T lymphocyte globulin (ATLG) has been adopted as part of conditioning before hematopoietic stem cell transplants (HSCT). In recent years, the use of post-transplant cyclophosphamide (PT-Cy), initially introduced for haploidentical HSCT, has spread consistently throughout several HSCT settings, showing comparable results. We recently reported increased risk of both acute and chronic graft versus host disease (GvHD) in patients receiving a high CD3+ cell counts within the graft. In this setting, we aimed to investigate the potential benefit of the co-administration with PT-Cy and a low, post-transplant ATLG dose. Methods: Starting from 2019, 21 patients were transplanted using peripheral blood grafts containing more than 300*106/kg and were administered PT-Cy 50 mg/kg on day+3 and day+4 with the addition of ATLG 5 mg/kg on day+5 (Grafalon, Neovii) (study group). Using a 1:2 matched-pair analysis, we compared the outcomes with 42 patients transplanted prior to 2019 with a PBSC graft containing a CD3+ counts above 300*106/kg, who received PT-Cy without additional ATLG (control group). In both groups, GvHD prophylaxis included sirolimus (with mycophenolic acid in HLA-mismatched and unrelated transplants). Results: Patient and transplant characteristics are shown in Table 1. Median follow up was 538 days in the study group and 1450 days in the control group. 30-day cumulative incidence of platelet engraftment was lower in the study group (29% versus 45%, p = 0.03). There was a non-significant trend of higher rate of poor graft function in the study group (29% versus 19%, p = 0.52). In terms of immune-reconstitution, the long-term negative impact of ATLG was evident on the CD4+ subsets. However, we documented no differences in term of CMV, HHV6, EBV, adenovirus and BK virus reactivation incidence, and there was a non-significantly higher incidence of invasive fungal infection (7/21 cases versus 9/42 cases p = 0.36). We observed a non-significant trend toward a lower day-100 CI of grade 3-4 aGvHD in the study group 10% versus 19% (p = 0.48), whereas 1-year CI of cGvHD was significantly lower (15% versus 41%, p = 0.04). Survival outcomes were comparable between the groups: 1-year TRM 19% versus 19% (p = 0.9); 1-year relapse rate 25% versus 24% (p = 0.9); 1-year PFS 56% versus 57% (p = 0.9), 1-year OS was 75% versus 69% (p = 0.49). Table 1 - Patient and transplant characteristics. $ Myeloablative regimen include: Treosulfan-Fludarabine +/−Melphalan/Thiotepa/Total body Irradiation, Reduced toxicity regimen include Treosulfan-Fludarabine Conclusions: Combining PT-Cy with low ATLG dose in high T-cell content PBSC graft translated into a low rate of chronic GvHD incidence, without impacting relapse incidence and survival outcomes. Disclosure: The authors declare no conflict of inter
Background: Allogeneic hematopoietic stem cell transplantation (HSCT) is a potentially curative therapy for several hematological disorders, therefore improving conditioning regimens can have an important impact on outcomes. Hereby, we report a single center experience of HSCT with one or two alkylating agents as conditioning regimen with special focus on chimerism Methods: We collected data from 2010 to 2020, identifying 75 adult patients, diagnosed with either lymphoid or myeloid disease undergoing HSCT from HLA identical siblings or matched unrelated donor (MUD), with reduced-intensity thiotepa-busulfan-fludarabine (TBF) in a dose of 10mg/kg (two days), 3.2mg/kg (two days) and 30mg/kg (3 days) respectively or busulfan-fludarabine (BF) (same doses, but busulfan for 3 days) as conditioning regimen and with available chimerism data. Full donor chimerism was defined as having >95% donor alleles. Graft source was peripheral blood stem cell in all patients. The study aimed to assess the rate of full donor (FD) chimerism at day 30 and 100, in patients receiving TBF or BF as conditioning regimen. Results: Baseline characteristics of the population are described in figure 1. Summarizing, the median age was 58 years, 64% had an intermediate disease risk index and 88% of were in complete remission. Donor type was HLA identical sibling in 65%. Conditioning regimen was BF in 67% patients (n = 50). The median time of neutrophil and platelet engraftment were 16 (range 10–28) and 13 (range 5–45) days, respectively. Thirty-three patients experienced acute GvHD, 12 of them grade 4. The cumulative incidence (CI) of chronic GvHD was 16% (95% CI 9-28) at 1 year, there were no significant differences between BF and TBF conditioning (18% vs 11%, p = 0.90). The CI of non-relapse mortality (NRM) at 100 day and 1 year was 8% (95% CI 4-17) and 21% (95% CI 13-33), respectively. NRM was not significantly different between patients receiving BF vs TBF at 100 day (4% vs 16%) and at 1 year (20% vs 21%) (p = 0.17). The CI of relapse at 100 days and 1 year was 18% (95%CI 11-29) and 35% (95%CI 25-48), respectively. Relapse was not statistically different between the two groups (p = 0.45) at 100 day nor at 1 year. Overall survival and disease free survival were 57% (95% CI 39-72%) and 44% (95% CI 33-56%) at 1 year, respectively. Overall survival was not statistically different for patients receiving BF vs TBF (56% vs 58%; p = 0,61) nor was disease free survival (BF 40% vs TBF 56%; p = 0,51). A FD chimerism at 30 day was achieved for 62% (n = 21) of the patients receiving TBF while 96% of the patients in the BF group had less than FD chimerism (p < 0.001). At 100 days, almost all patients (95%) in the TBF group achieved a FD chimera comparing to BF group (42%) (p < 0.001). Nine patients received donor lymphocyte infusion, 8 of them in the BF group (6 = disease relapse; 1= mixed chimerism; 1= graft failure). Conclusions: The combination of two alkylating agents is associated with a higher chance of achieving a FD chimerism at 30 days and 100 days without significant survival advantages. Disclosure: No discloures
Background: Chemoimmunotherapy in advance stage mantle cell lymphoma is not curative. Autologous hematopoietic stem cell transplantation (ASCT) currently is the standard of treatment for patients in first remission diagnosed with Mantle cell Lymphoma (MCL) OS has been improved by using induction chemoimmunotherapy conventional and higher intensity, and maintenance therapy as by GELA study reports 75%. LyMa Trial has proven improvement in the global survival and progression-free survival. In this study we aimed to describe overall survival (OS) diagnosed Mantle cell lymphoma (MCL) with PEAM as a conditioning regimen. Methods: A retrospective study was conducted on patients diagnosed with MCL and ASCT as consolidation therapy at the Instituto Nacional de Cancerología in Mexico City between 2011 and 2020. PEAM condition regimen: Cisplatin 100mg/m2 (-4), etoposide 750 mg/m2 (-4,-3), cytarabine 800mg/m2 (-4,-3,-2) and melphalan 140mg/m2 (-4). OS results were obtained using the Kaplan-Meier method. Results: We analyzed 15 patients with MCL after ASCT as consolidation therapy, a median follow-up of 4.58 years, the median age at transplantation was 54-years (range 39-68y), with male predominance (86.7%). Nine patients with at least one comorbidity, the most predominant being diabetes mellitus type II (66.6%). All patients with clinical stage III-IV, 20% of them with bone marrow infiltration. All patients received one line of chemoimmunotherapy, most were treated with R-CHOP plus /or R-DHAP(86%). Eleven patients (73.3%) with complete response, three (20%) with partial response before ASCT. Fourteen patients (93.3%) received PEAM plus rituximab as myeloablative conditioning regimen. Neutrophil recovery median was 10 days, nine patients (66.6%) developed febrile neutropenia. Twelve patients (80%) received maintenance with rituximab. Eleven (73.3%) had complete response at last follow up after ASCT, three (20%) with relapsed disease, one stable disease. Three patients relapsed, two (66.6%) died. The OS at 5-years was 84.8%. Graph 1. Overall Survival Conclusions: Conventional chemoimmunotherapy is effective followed by ASCT as consolidation therapy in mantle cell lymphoma with advanced stage disease. We describe higher OS 5-years (84.8%) as compared with other series (57% and 75%) with similar chemoimmunotherapy and conventional conditioning regimen. Disclosure: Nothing to declare
Background: Treosulfan-based conditioning regimens are widely used among children with leukemia. Treosulfan is usually combined with fludarabine and either melphalan or thiotepa. Due to frequent interruptions of market availability of melphalan and thiotepa, there is an unmet need for alternative safe and effective combinations. There are reports of the use of treosulfan/fludarabine/etoposide regimens among adult patients, but this approach is not well-studied in pediatric HSCT. We performed a retrospective analysis of the clinical use of treosulfan/fludarabine/etoposide regimen in a cohort of children with leukemia, treated at two sites in Moscow: Dmitriy Rogachev National Medical Researh Center of Pediatric Hematology, Oncology and Immunology and Morozov Children’s Hospital, Moscow. Methods: Sixty-five pediatric patients (41-male, 24-female) received conditioning with treosulfan (42g/m2), fludarabine (150 mg/m2) and etoposide (60mg/kg) before allogeneic HSCT between 2018 to 2021. HSCT indications included acute lymphoblastic leukemia (ALL), n = 43 (66%) (B-ALL n-31, T-ALL n-12), acute myeloid leukemia, n = 18 (27.7%), bi-lineage leukemia, n = 3 (4.6%) and JMML n = 1 (1.5%). In 8 patients, it was the 2nd HSCT. The source of HSC was PBSC in 60 cases (92%), and BM in 5 cases (8%). Haploidentical family donors were used in 52 cases (80%), matched related and unrelated donors in 10 and 3 cases, respectively. Three GVHD prevention approaches were used: 35 patients received posttransplant cyclophosphamide (PtCy) on days+3, +4 at 50 mg/m2/day, tacrolimus and MMF with unmanipulated HSCs. In 26 cases ex-vivo αβ T cell depletion combined with abatacept and bortezomib was used. In 4 patients with a matched BM transplant calcineurin inhibitor-based regimen was used. Median follow-up was 12.5 months. Results: Neutrophil and platelet engraftment was recorded in 61 of 65 cases. All cases of primary graft failure were seen among ex-vivo depletion cohort, of them 3 were successfully re-transplanted. The median time to neutrophil engraftment was 16 days (8-47), 12 days in ex-vivo depletion and 18 days in PtCy cohorts, respectively. The median time to platelet engraftment was 16 days (11 - 55), 13 in ex-vivo depletion and 25 in PtCy cohorts, respectively. Most common toxicities affected skin, liver and GI. Cutaneous toxicity grade I-IV was seen in 32 patients (49%), of them 5 cases with grade III-IV (15.6%). Hepatic toxicity grade I-II was observed in 19 patients (29.2%), no cases of grade III-IV liver toxicity were recorded. The most frequent type of toxicity was gastrointestinal toxicity, seen in 58 cases (89.2%). GI toxicity was graded as grade I-II in 46 patients (80%), grade III-IV in 12 patients (20%). Renal toxicity grade I-II was observed in 4 cases (6.5%). Acute GVHD II-IV was observed in 30 patients (36.1%), severe forms of acute GVHD, grade III-IV, were recorded in 10 patients (15.4%). Early (day + 100) non-relapse mortality was not recorded. Conclusions: The conditioning regimen based on Treo42/Flu150/VP60 was safe, ensuring engraftment and tolerable short-term toxicity among children with acute leukemia. This regimen can be combined with two key GVHD prevention platforms, ex-vivo αβT cell depletion and PtCy, with minimal non-relapse mortality. Long-term outcomes should be evaluated prospectively. Disclosure: Nothing to declare
Background: Patients with relapsed or refractory (r/r) acute myeloid leukemia (AML) have poor prognosis but cure is possible with allogeneic hematopoietic stem cell transplantation (allo-HSCT). Myeloablative total body irradiation (TBI) based conditioning is often used in AML patients refractory to standard chemotherapy. Feasibility of chemokine receptor 4 (CXCR4) directed endoradiotherapy (ERT) has previously been shown in patients with CXCR4 expression on leukemic blasts. Here, we retrospectively report on six chemo-refractory, relapsed AML patients that received ERT with CXCR4-targeting [177Lu]-Pentixather combined with TBI and chemotherapy prior to allo-HSCT. Methods: In this retrospective, single center analysis data from six consecutive patients with r/r AML treated between February 2019 and September 2021 were included. All patients had active AML and were either refractory to induction and salvage chemotherapy or had refractory relapse. In-vivo CXCR4 expression on leukemic blasts was confirmed in all patients by [68Ga]-Pentixafor PET-imaging. Conditioning consisted of [177Lu]-Pentixather ERT as compassionate use on day (d) -15, TBI (8-10 Gy) on d-9 to d-7 and chemotherapy, based on donor type. Chemotherapy regimens were fludarabine 30mg/m2 d-5 to d-2 or 60mg/m2cyclophosphamide on d-5 and d-4 for matched donors and fludarabine 30mg/m2 d-6 to d-2 plus cyclophosphamide 14,5mg/m2 d-6 to d-5 for haploidentical or mismatch donors. Immunosuppression for matched donors consisted of antithymocyte globulin (5-10mg/kg d-3 to d-1), mycophenolatmotefil and a calcineurin inhibitor. For haploidentical donors post-transplant cyclophosphamide was used according to standard of care. In this retrospective analysis, we assessed response, toxicity, overall survival, engraftment rates and adverse events. Results: Median patient age was 47 (42-57). 5 patients had de novo AML and one secondary (s)AML. In median, patients had previously received 4 (3-7) lines of intensive therapy, including allo-HSCT in n = 3 patients. Median injected activity of [177Lu]-Pentixather was 12.6 GBq (11.5-16.1). All patients received a peripheral blood stem cell graft with a median of 5.9 (4.9-10.3) x 106 CD34 + cells/kg. During hospital stay, n = 4 patients required intensive care treatment and n = 2 mechanical ventilation. Response evaluation by bone marrow biopsy was available for n = 5 patients, n = 4 achieved complete remission with incomplete count recovery (CRi) and n = 1 morphologic leukemia-free state (MLFS). One patient was refractory and regenerated with 11% blasts in the peripheral blood at day 11. Time to leukocyte recovery in the n = 4 responding patients was 25 (16-28) days, one patient had received a stem cell boost after initial graft failure. Two patients are still alive at month 21 and 20 after allo-HSCT, n = 3 died during hospital stay and n = 1 after a relapse. Causes of death were respiratory failure (n = 1), sepsis (n = 1), refractory disease (n = 1) and relapse (n = 1). In the two surviving patients, kidney function remained normal with creatinine levels of 0.8 and 0.9 mg/dl, respectively. Conclusions: Conditioning with CXCR4-directed ERT plus TBI is feasible and response rates in this heavily pre-treated patient cohort were promising. No acute kidney toxicity related to radiation dosage was observed and engraftment was not impaired in this small cohort. The results warrant prospective studies. Disclosure: Nothing to declare
Background: Busulfan (BU) therapeutic drug monitoring (TDM) is performed in order to avoid over exposure and toxicity and/or under exposure and reduce efficacy. In our Center, BU TDM is performed for every patient receiving BU, and the dose is adjusted in order to achieve a target area under the curve (AUC) within the range 3696-5538 ng x h/m. We observed an unexpected increase in BU AUC in children who were treated before HSCT with deferasirox or deferoxamine for iron overload. BU undergoes glutathione conjugation and subsequent oxidative metabolism. Deferasirox inhibits multiple oxidative enzymes of the cytochrome P450 family, and this could explain the decreased clearance of BU. Methods: We retrospectively analyzed 28 pediatric patients (age range, 1-16 years; median 6.7 years) affected by a non-malignant disease (10 thalassemia major, 17 sickle cell disease, 1 thalasso-drepanocitosis), who underwent allogeneic HSCT between 2018 and 2020. They received a Bu-based conditioning regimen (initial BU dose 1 mg/kg every 6 hours), in association with Thiotepa and Fludarabine. 25% of the patients had received pre-transplant iron chelation to treat/prevent iron overload, with deferasirox or deferoxamine, until the beginning of the conditioning regimen. Blood samples were collected at fixed time points after the first dose of BU, in order to calculate the median concentration at steady state (Css) and derivate the value of AUC. Results: In all tested patients, BU exposure after the first dose, based on body weight, had a median level of 6422 ng x h/mL (range, 3604-10758). In the patient who received iron chelation, BU exposure was higher than expected (median 8459; range, 6872-10758), as compared to that of children who did not receive iron chelation (median 6017; range, 3604 - 8250) (Fig.1). A total of 8 patients required a Bu adjustment, 5 of them (62.5%) had received iron chelation before HSCT, whereas the other 3 (37.5%) had not, with a statistically significant difference (Chi-square P = 0.009). After BU dose reduction, AUC was measured again after the fifth/ninth dose and it decreased to 4484-7789 ng x h/mL. Unfortunately, for 2 patients the dose reduction was not sufficient to ensure an adequate BU exposure: in one of the patient BU dose was reduced by 18%, whereas in the other, even with a 40% dose reduction, an adequate plasma level could not be achieved. In the four patients with adequate exposure after dose reduction, Bu dose was reduced by 21% to 50%. Conclusions: Pharmacokinetic studies dealing with drug-drug interactions between BU and iron chelation therapy are extremely limited. The administration of iron chelation immediately before the conditioning regimen may result in a systemic BU over-exposure. An earlier discontinuation of iron chelation treatment may be necessary in these patients, and TDM remains mandatory in order to optimize the dose for each patient. Disclosure: Nothing to declare
Background: The use of post-transplant cyclophosphamide (PTCy) has significantly changed the approach to the graft versus host disease (GvHD) prophylaxis in patients undergoing allogenic stem cell transplantation (allo-HSCT). However, PTCy can increase the rate of infections, in particular of cytomegalovirus (CMV). Methods: We retrospectively analyzed the clinical outcomes of 68 adult patients (AML = 38, MDS = 7, ALL = 9, NHL/CLL = 8, CML = 3, MF = 2, HD = 1) who underwent allo-HSCT (between December 2011 and September 2021) from mismatch unrelated (MMUD, 9/10) (n = 21), and haploidentical (Haplo) (n = 47) donors. All patients received PTCy as GvHD prophylaxis. Seventeen patients with a positive CMV serology, had a CMV prophylaxis with letermovir. Results: With a median follow-up of 1.9 (range. 0.05-9.7) years, the Overall Survival (OS) of MMUD and Haplo was 73% vs 71% . The non-relapse mortality (NRM) was 16% vs. 11% for MMUD and Haplo, respectively. The incidence of relapse was 34% for Haplo and 17% for MMUD. The intensity of the conditioning regimen, reduced (n = 24) or myeloablative (n = 44) had no effect on these outcomes.By uni and multivariate analysis, more aGvHD (grade II-IV) was associated to MMUD than haplo donors (HR 4.77, CI 1.62-14. in univariate analysis; HR 5 .12, CI 1.57-16.7 in multivariate) with no difference in term of cGvHD. The use of PBSC was significantly associated to a better overall survival, better neutrophil engraftment and reduced risk of poor marrow function/ rejection with no impact on GvHD. None of the patients treated with letermovir had CMV reactivation during CMV prophylaxis (Fig. 1). However, those who had less than 50 CD4 + cells at discontinuation (day 100) invariably showed CMV reactivation, subsequently. By multivariate analysis, CMV reactivation was associated with a better DFS (HR 0.34, CI 0.14-0.83) and OS (HR 0.3; CI 0.12-0.96). Conclusions: The main outcomes of allo-HSCT after MMUD and Haplo transplant with PTCy as GvHD prophylaxis, were similarly favorable although transplants with MMUD showed an increased risk of aGvHD. When letermovir was discontinued, patients with less than 50 CD4 + T cells in the peripheral blood were at high risk of CMV reactivation. Disclosure: Nothing to declare
Background: Melphalan toxicity, in particular oral and intestinal mucositis, is increased in patients with low glomerular filtration rate (GFR). Thus, melphalan dose in autologous stem cell transplantation (ASCT) is adjusted from 200 mg/m2 to 140 mg/m2 in patients with adjusted GFR < 30 mL/min/1.73m2. Our aim is to study the incidence of grade 4 mucositis in patients with mild reduction in GFR (GFR < 90 and >30 ml/min/1.73m2) that underwent ASCT conditioned with melphalan 200 mg/m2. Methods: We retrospectively analysed the incidence of oral or intestinal mucositis that required parenteral nutrition (oral mucositis WHO grade 4 or intestinal mucositis CTCAE grade >3) in consecutive patients that underwent ASCT between January 2016 and June 2020, according to the patient GFR on the day of melphalan administration. Secondary variables were time from infusion to discharge, need of intensive care and transplant-related mortality on day +100 (TRM D100). Results: Of the 129 patients included, 90 had a GFR ≥ 90 and 39 had a GFR between 30 and 90 ml/min/1.73m2. 24 (26%) patients with GFR ≥ 90 and 22 (56%) with GFR < 90 presented mucositis that required parenteral nutrition (relative risk 2.11, p = 0.0011). Time from infusion to discharge was also higher in patients with GFR < 90 (median of 21 vs. 18 days, p = 0.035). There was no significant difference in the need of intensive care and there was no TRM D100 in any of the groups. Conclusions: Patients undergoing ASCT conditioned with high dose melphalan are at increased risk of needing parenteral nutrition due to oral or intestinal mucositis if they have reduced GFR (90 to 30 ml/min/1.73m2). This fact, however, does not translate into increased risk of intensive care need or transplant related mortality. Disclosure: Nothing to declare
Background: T-cell lymphomas (PTCLs) constitute a rare and clinically aggressive group of lymphomas, which has more than 20 histologic subtypes according to WHO 2008 classification. PTCL not otherwise specified (NOS), angioimmunoblastic T-cell lymphoma (AITL) and anaplastic large cell lymphoma (ALCL) are called nodal PTCL and account for approximately 60% of cases. The reported median survival ranges from 22 to 49 months and 5-year survival is less than 30%. High-dose chemotherapy followed by autologous stem cell transplantation (ASCT), has been proven to be beneficial both as a consolidation and in the relapse setting. However, the optimal conditioning regimen for ASCT in patients with T-cell lymphoma is not specified. Methods: All adult T-cell lymphoma patients who underwent consolidative or salvage ASCT in our center between 2012 and 2021 have been identified and medical records were retrospectively reviewed. Standard CY (cyclophosphamide) - TBI (total body irradiation) regimen, which consisted of CY 120 mg/kg total dose and 12 Gy TBI in fractionated doses, was utilized for conditioning. Results: A total of 36 patients, of whom 22 were male (61.1%), was identified. The median age at diagnosis was 51 (23-67). The histologic subtypes included: ALCL, ALK(-), n = 9 (25.0%); ALCL, ALK( + ), n = 3 (8.3%); AITCL, n = 9 (25.0%), PTCL, NOS, n = 10 (27.8%), hepatosplenic T-cell lymphoma, n = 2 (5.5%); NK/T cell lymphoma, n = 2 (5.5%), enteropathy-associated T-cell lymphoma, n = 1 (2.8%). All patients with ALCL, ALK( + ) were transplanted at either CR2 or for salvage of refractory disease. Most of the patients had advanced and high risk disease at diagnosis [Ann/Arbor stage 3-4 disease was present in 85.7% and international prognostic index (IPI) was intermediate or high in 80.0%]. Most of the patients underwent consolidative ASCT in CR1 (n = 4, 66.7%). The median follow-up was 10 months (0-106). The median time from diagnosis to ASCT was 7 months (4-45)Overall survival (OS) and progression-free survival (PFS) at 12 months after ASCT were 86.0% [95% confidence interval (CI): 74.2-97.8] and 45.0% (95% CI: 27.4-62.6), respectively. Non-relapse mortality (NRM) at 3, 6, 9 and 12 months after ASCT were 6.0% (95% CI: 0-13.8), 10.0% (95% CI: 0-39.4), 14.0% (95% CI: 2.2-15.8) and 14.0% (95% CI: 2.2-15.8), respectively. OS was better among patients transplanted at CR1, when compared to CR2 and primary refractory patients (p < 0.05). Four patients with aggressive and high risk disease could proceed to allogeneic transplantation, three of whom are still alive with a median follow-up of 14.5 months (7-77). Grade 3-4 adverse events were rare and neutrophil engraftment was achieved in all patients, except one with refractory disease after ASCT. Kaplan-Meier plot comparing the OS for patients at CR1 versus CR2 and refractory disease (p < 0.05, log-rank test). Conclusions: This study cohort included mainly nodal PTCLS (86%) with high risk and advanced disease profile. ASCT utilizing CY-TBI may provide an important benefit when used in consolidation and as a salvage without increasing toxicity and NRM. It may serve as a bridge for allogeneic transplantation in patients with aggressive and high risk disease. Consolidation at CR1 may provide a greater benefit. Clinical Trial Registry: N/A Disclosure: The authors have no conflict of interest to disclose.
Background: High-dose chemotherapy followed by autologous transplantation (ASCT) is currently the standard of treatment for relapsed or refractory non-Hodgkin’s lymphomas (NHL) and Hodgkin’s lymphoma (HL). Despite the absence of direct data from prospective studies, BEAM, consist of carmustine (BCNU) in combination with etoposide, cytarabine and melphalan, is considered the common conditioning. The recent lack of BCNU has led to the search for alternative regimens. One of the most promising seems to be TEAM, in which BCNU is replaced by thiotepa. Thiotepa penetrates the CNS better, but there are concerns about increased, especially gastrointestinal, toxicity. At the same time, there is a lack of strong data demonstrating the comparable effectiveness of the two conditionings. Therefore, we decided to retrospectively compare the GIT toxicity of the TEAM regimen, which we started using in July 2018, with the previously used BEAM regimen. Methods: Retrospective analysis of 142 consecutive patients with lymphomas autologous transplanted after the administration of the BEAM (2014-2018) or TEAM (2018-2021) conditioning at the Department of Hematology and Oncology. Results: In the group of 142 patients with the age median of 58 years (21-74) there were 85 men (60 %). The BEAM regimen was administered to 82 patients (58 %) with the age median of 59 years (22-74), and TEAM to 60 patients (42 %) with the age median of 58 years (21-73) (p = 0.83). Of the diagnoses, the most common was DLBCL - 12 vs 27, HL - 21 vs 8, MCL - 16 vs 4, T-NHL - 12 vs 8, other lymphomas were represented sporadically, in total 16 vs 13 patients. There was no significant difference between the representation of diagnosis. In the first remission of the disease, we transplanted 43 vs 22 patients (p = 0.29), in the second and next remission 26 vs 22 patients (p = 0.74). 13 vs 16 patients (p = 0.22) were transplanted in the primary induction failure, it means progressive and chemorefractory disease. None or only very mild GIT toxicity (grade 0-I) was present in 53 resp. 28 patients (p = 0.32). Grade II GIT toxicity occurred in 11 resp. 12 patients (p = 0.49), grade III in 12 resp. 16 patients (p = 0.21) and finally the most severe grade IV with the need to move to the ICU in 1 resp. 3 patients (p = 0.32). In 5 resp. 1 patient it was not possible to determine GIT toxicity. Parenteral nutrition was used in 13 (16%) resp. 22 patients (37%) (p = 0.04). TRM during hospitalization was 0%, in 3 months 2% for both conditionings (p = 1.0). Conclusions: Our data demonstrate that despite comparable objective GIT toxicity, the TEAM regimen has a significantly higher need for total parenteral nutrition. However, the overall TRM is comparatively low for both regimens. Disclosure: Nothing to declare
Background: Reduced intensity conditioning (RIC) is associated with lower non-relapse mortality (NRM) and significant disease control in alloHCT recipients. The optimal conditioning regimen in alloHCT for lymphoid malignancies remains unclear. To date, no data are available regarding the use of thiotepa, busulfan and fludarabine (TBF) in adult patients allografted for lymphoma. Methods: Between February 2019 and August 2021, 30 patients diagnosed with lymphoid malignancies received a first alloHCT conditioned with a TBF-based RIC regimen at our institution. All consecutive patients were included in the study and data were collected retrospectively. Low-dose TBF RIC was defined as the use of 5 mg/kg of thiotepa (2.5 mg/Kg/day from days -6 and -5) while a high-dose TBF-RIC was considered as the administration of 10 mg/Kg of thiotepa (5 mg/Kg/day from days -6 and -5). Low-dose thiotepa was generally administered to patients with an HCT-specific Comorbidity Index >3 or considered frail. Two doses of 3.2 mg/Kg/day of busulfan and 150 mg/m2 (50 mg/m2 from days -4 to-2) of fludarabine were administered in all patients. Post-transplant cyclophosphamide-based graft-versus-host disease (GVHD) prophylaxis was used for both haploidentical and matched unrelated donors (>9/10 HLA compatibility). Main outcome variables were overall survival (OS), progression-free survival (PFS), NRM, relapse incidence/progression of disease (RI/POD), cumulative incidence of acute (aGVHD) and chronic GVHD (cGVHD), and hematological recovery. Probabilities of OS and PFS were calculated using the Kaplan-Meier estimator method, and NRM, RI/POD, GVHD and hematological recovery as cumulative incidences. Results: Baseline characteristics are summarized in Figure 1. Overall, the median age was 55 (range 33-70) years, and 15 patients (50%) received haploidentical donor grafts. Nineteen (63.3%) patients were in complete remission prior to alloHCT. Median follow-up among survivors was 517 (range 96-993) days. At 18-months, OS, PFS, NRM and RI/POD were 53% (95% confidence interval [CI], 33-70%), 40% (95% CI, 21-58%), 38% (95% CI, 19-57%) and 22% (95% CI, 4-40%), respectively. Of the 13 patients who died during follow up, causes of death were: 2 relapses and 11 transplant toxicities (9 = infections, 1=aGVHD, 1= endothelial complications). Eighty-two % of toxicity-related deaths were observed during the first 3 months from transplant. The cumulative incidence of neutrophil engraftment at day +30 was 87% (95% CI, 75-99%). The cumulative incidence of grade II-IV and grade III-IV aGVHD at day +100 was 20% (95% IC, 6-34%) and 7% (95% CI, 0-16%), respectively. The cumulative incidence of all grade cGVHD at +18 months was 16% (95% CI, 1-31%), with only 3 patients presenting with moderate-severe cGVHD. On univariate analysis, no prognostic factors were observed in terms of OS, PFS and NRM. However, having an interval between diagnosis and alloHCT >2 years was associated to decreased relapse risk (18 months NRM: 7% vs 52%, p = 0.01). Thiotepa dose was not significantly associated to clinical outcomes. Conclusions: In our series, RIC TBF allows efficient disease control at the expense of increased incidence of severe early toxicities. Despite a higher NRM, OS was similar to other RIC regimens used for lymphoid malignancies. This regimen could be considered for fit patients with high-risk lymphoid diseases. Disclosure: The authors declare no conflicts of interest
Background: Bortezomib (Bor) can inhibit the proliferation of dendritic cells (DCs) and block the expression of co-receptors CD80, CD86 and secretion of cytokines IL-12 and TNF-α and hence the ability of DCs to activate T cells. We started a pilot study incorporating the addition of bortezomib to post-transplant cyclophosphamide (PTCY) in the setting of peripheral blood (PB) HLA-haploidentical stem cell transplantation (Haplo-SCT). Methods: This is a single center open label pilot study. Eligible patients received Fludarabine Melphalan TBI 200 cGy as conditioning followed by haplo-SCT and PTCY. Bor was administered at 1.3mg/m2 on day+1, 4 and 7. Tacrolimus and MMF were started at day+5 Results: Seven patients were enrolled so far, five males and 2 females. Median age was 58 years (26-60). Donors were 3 brothers, 3 sons and 1 mother. Disease risk index was high in 3, intermediate in 3 and low in 1. Three patients had AML, two had ALL and MM, one had ALL and one had CML. CMV recipient status was negative in one and positive in 6. Median HCT-CI was 3(1-4). Median CD34 and CD3 infused were 4.13 x10^6 and 1.7x10^8/ kg recipient respectively, all were cryopreserved except 2. Four patients had CRS before Cy infusion with ASTCT grade of 1. Six patients had grade 3 hypokalemia around day+ 4-5. Five patients had grade 3 mucositis and 2 had grade 1. Four patients had neutropenic fever and one patient had engraftment fever. Median neutrophils and platelets engraftment were 16 and 26 days respectively. Chimerism post SCT was > =99% donor at day 30 for all patients. Six patients are off tacrolimus with median time to be off it was 187.5 days. Five pts had aGVHD with maximum grade of I in 3 patients, II in one patient and III in one patient at a median 50days post SCT. None developed early hematuria, four had late hematuria with highest grade of 4. Two patients were positive for BK virus. One patient had reactivation of CMV, 2 had EBV and one had adenovirus, all resolved. Three pts had HHV6 that resolved. Of the 5 patients who were evaluable, one developed moderate chronic GVHD. So far the median time to follow up is 455 days (70-1239) with relapse and subsequently death in one patient who had high risk AML with 3 different inductions prior to SCT. . At 1 year for 4 evaluable patients IgG were >400 mg/dl and CD4 > 350 cells/ul. Survival Probability Conclusions: Cy2Bor3 post PB Haplo-SCT was well tolerated. Although small number of patients and limited but encouraging results so far. The trial is ongoing. Clinical Trial Registry: ClinicalTrials.gov ID: NCT03850366. Disclosure: Nothing to declare
Background: Although allogeneic hematopoietic cell transplantation (alloHCT) remains the only curative option for Hodgkin lymphoma (HL), efficacy and safety of anti-PD1 (Programmed cell death protein 1) inhibitors have revolutionized HL treatment. Importantly, complications of alloHCT post anti-PD1 inhibitors have raised questions regarding its feasibility. Therefore, we investigated efficacy and safety of alloHCT post anti-PD1 inhibitors compared to a historical group of alloHCT before the introduction of anti-PD1. Methods: We retrospectively studied patients that underwent alloHCT for HL at our JACIE-accredited center over the last decade (2010-2020). We divided them into two groups according to the treatment period: anti-PD1 and historical control group. We analyzed pre-transplant (age, disease status, previous lines), transplant (donor, graft, conditioning), and post-transplant (graft-versus-host-disease/GVHD, treatment-related mortality/TRM, relapse, and overall survival/OS) characteristics. Results: In total, 21 patients received alloHCT for HL. Among them, 3 patients from the historical control group received anti-PD1 due to relapse post alloHCT and were excluded from further analysis. We studied 18 patients (7 in the anti-PD1 period and 11 controls), with median age 32.5 years, suffering from classical HL (12:nodular sclerosis, 3:mixed cellularity, 1:lymphopenic). All patients had undergone autologous HCT at a median of 4 years earlier, 14 with primary refractory chemo-sensitive disease and 4 with chemo-resistant disease. Among them, 8 presented with a positive PET/CT scan after autologous and 10 with relapse at a median of 13.4 (range 8-19) months. Brentuximab vedotin (BV) had been administered post autologous HCT only in patients of the anti-PD1 group; with 2 achieving partial remission, 1 complete metabolic remission (CMR) and 1 progressive disease. Upon progression, patients of the anti-PD1 group received nivolumab for 18 (5-32) cycles. The patient that had achieved CMR with BV, received a combination of nivolumab-BV. Historical controls proceeded to alloHCT following chemotherapy upon progression. Both groups received alloHCT at a similar disease, 11 patients had active disease at alloHCT, while 7 CR (p = 0.205). Donors were mainly unrelated (12/18, p = 0.421). Reduced toxicity conditioning regimen was administered in both groups (Thiotepa-Fludarabine-Cyclophosphamide with Antithymocyte Globulin 5mg/kg in unrelated donors). Post-transplant cyclophosphamide (Baltimore’s protocol) was also given in the 4 more recent patients (p = 0.011). All grafts were PBSC with a median of 5.45x106 cells/kg (p = 0.263). With a median follow-up of 16.3 (1.1-94.7) months, cumulative incidence (CI) of acute and chronic GVHD were similar between groups. Furthermore, 2-year TRM CI did not differ (28.6% in anti-PD1 versus 27.3% in historical controls, p = 0.951). Interestingly, the 4 patients that received post-transplant cyclophosphamide presented no TRM. Only one patient relapsed post alloHCT in the historical control group, while no patient from the anti-PD1. Therefore, 2-year OS was similar between groups (66.7% in the anti-PD1 versus 60.0% in controls, p = 0.982). OS was associated only with type of donor, significantly higher in unrelated donors (p = 0.044). Conclusions: Our single-center experience suggests comparable outcomes of alloHCT post anti-PD1 to historical controls. Adoption of novel modalities, such as post-transplant cyclophosphamide, have led to encouraging results. Further studies are needed to determine the optimal tailored approach for chemo-refractory HL. Clinical Trial Registry: ΝΑ Disclosure: Nothing to declare
Background: The prognosis of patients with refractory, advanced stage primary cutaneous T cell lymphoma remains poor particularly in patients with transformed mycosis fungoides (MF). In these cases, conventional cytotoxic regimens induce only short-lived responses and lymphoma relapses occur even more aggressively thereafter. Allogeneic hematopoietic stem cell transplantation (allo-HSCT) provides a potentially curative treatment option for clinically suitable patients. However, the optimal approach to these patients including bridging treatment to conditioning and transplant remains undefined. Here, we report on three consecutive patients with high-risk MF that received tumor reduction and reduced intensity condition (RIC) in a sequential concept prior to allo-HSCT. Methods: Three patients with advanced stage MF including one large-cell transformation were consecutively treated at our transplant center. All patients received multiple skin directed and systemic treatments including PUVA (n = 2), retinoids (n = 3), IFN-alpha (n = 3) and chemotherapy (n = 2) and brentuximab (n = 3) prior to allo-HSCT. All three patients were in PR prior to transplantation. To reduce tumor burden, all patients received clofarabine shortly before RIC in terms of a sequential therapy concept. Conditioning consisted of fludarabine, cyclophosphamide and melphalan. Bone marrow (n = 2) and peripheral blood stem cells (n = 1) were used as graft source. Donor type was HLA-haploidentical in two and HLA-matched unrelated in one patient. In all cases, cyclosporine or tacrolimus and mycophenolate mofetil as well as post-transplantation cyclophosphamide (PTCY) were used for graft versus-host disease (GVHD) prophylaxis. Results: Clofarabine induced rapid treatment responses in all patients and subsequent RIC could be performed without significantly increased toxicities. At day +30, lymphoma staging demonstrated profound remissions (PR in 2 cases) while complete donor chimerism was detectably in the peripheral blood and bone marrow in all patients. All patients engrafted, no primary graft rejection was seen. Acute GvHD > = Grade 2 was observed in 2 patients responding to the application of steroids in both. However, all three patients relapsed after a median time of 2.6 months but responded to subsequent treatments. Conclusions: Using clofarabine as a salvage treatment prior to RIC in a sequential allo-HSCT concept may be a considerable strategy for patients with advanced cutaneous T cell lymphoma. However, as all patients relapsed within the first year after transplantation, further investigation is needed to determine the optimal post-transplant approach including early immunomodulatory maintenance treatments. Disclosure: Nothing to declare
Background: Autologous stem cell transplantation (auto-SCT) is an important therapeutic approach for malignant lymphoma. Conditioning chemotherapy with alkylating agents such as BEAM and TEAM is the most commonly used in this indication. Our study compares the toxicity profiles of the BEAM and TEAM conditioning. Methods: 48 patients (20 women, 28 men), mean age 53.5 years, who underwent auto-SCT for malignant lymphoma were retrospectively analyzed. BEAM (carmustine, etoposide, cytarabine, melphalan) was used in 24 patients between November 2015 and April 2019, TEAM (thiotepa, etoposide, cytarabine, melphalan) was used in 24 patients between June 2018 and September 2021. Results: The indications for auto-SCT included: 16 patients with diffuse large B-cell lymphomas, 9 with mantle cell lymphomas, 10 with T-cell lymphomas, 4 with follicular lymphomas, and 9 with Hodgkin lymphoma. Grade 4 neutropenia was present in all patients in both groups. The median time to neutrophil engraftment for the TEAM and BEAM arm was 10.5 (9-15) days and 10 (5-15) days, respectively (p = 0.322). Grade 4 thrombocytopenia was present in all patients in both groups. The median time to platelet engraftment for TEAM and BEAM was 13 days (9-22) and 11 days (9-16), respectively (p = 0.341). Mucositis grade 4 was observed in 22/24 (91.7%) of patients receiving TEAM compared to 20/24 (83.3%) of patients receiving BEAM (p = 0.383). Mucositis grade 2-3 was observed in the 2/24 (8.3%) in TEAM cohort compared to 4/24 (12.5%) of patients in the BEAM cohort (p = 0.383). Febrile neutropenia developed in 15/24 (62.5%) of patients receiving TEAM compared to 12/24 (50%) of patients receiving BEAM (p = 0.383). Sepsis developed in 7/24 (29.1%) of patients after TEAM compared to 5/24 (20.8%) of patients after BEAM (p = 0.505). The infectious complications were as follows: 1/24 (4.2%) patient had colitis and 1/24 (4.2%) had pneumonia after TEAM ; 1/24 (4.2%) patient had urinary tract infection and 1/24 (4.2%) had soft tissue infection after BEAM. Transplant-related mortality on day 100 was 2/24 (8.3%) of patients receiving TEAM compared to 3/24 (12.5%) of patients receiving BEAM (p = 0.637). The reason for death was sepsis in all cases. Conclusions: Our retrospective analysis documents a similar result regarding engraftment, toxicity profile, and transplant-related mortality between BEAM and TEAM conditioning prior to auto-SCT. Disclosure: Nothing to declare.
Background: Hematopoietic stem cell transplantation (HSCT) from an haplo-identical donor has been demonstrated to provide the best chances of a cure for many children in need of an allograft but who lack a sibling donor. We propose a retrospective study of 36 pts who benefited the “Beijing protocol” using myeloablative conditioning regimen with G-CSF mobilized/primed grafts Methods: From May 2013 to January 2017, 36 haplo-identical HSCT were used in 36 pts with hematological malignancies (7 AML, 20 ALL, 5 CML, 2 AL biphenotypic, 2 MDS). Median age was 24 years (5-55) and sex-ratio (M/F):3. The diagnosis transplant delay was 29 months (6-138). At the time of the transplant, 7 pts were in first complete remission (CR), 20 pts in second CR and 4 pts in active disease. The donors used were parents (21), siblings (14) or offspring (1); with median age: 41 years (13-65). The degree of compatibility (HLA A, B and DR) was 3/6 (24 cases), 4/6 (10 cases) and 5/6 (2 cases). CMV status between donor/recipient was high risk in 36 cases. The ABO incompatibility is major in 8 cases, minor in 10 cases. The conditioning regimen used associated Busilvex 9.6 mg/kg, Aracytine 8 g/m2, Cyclophosphamide 3.6 g/m2 for 36 pts. The GVHD prophylaxis included the combination Ciclosporin Methotrexate, Mycophenolate mofetil and Thymoglobulin (10 mg/kg) and received an G-CSF mobilized/primed grafts: bone marrow and Peripheral blood stem cells. Median dose infused CD34 + cells: 4,42 106/kg (1,35-19,9), nuclear cells: 6,45 x 107/kg (0,59-21,53). At November 2021, the minimal follow-up delay was 58 months and maximal 102 months. Results: Aplasia was observed in all pts with median duration of 15 days (13-34). The median day of neutrophils engraftment was 17 days (11-27). One pt presented transplant associated micro-angiopathy (MAT). Two pts presented an early rejection (5%). Acute GVHD grade II-IV occurred in 14 pts (43%) on average at day 42 (16-100). Chronic GVHD was seen in 9 pts (36%) with extensive form in 4 pts on average at day 210 (150-240). Sixteen pts (50%) showed CMV reactivation on average at day 45 (28-149). Seven cases of haemorrhagic cystitis (21%) (one grade 4) are observed . Ten pts (27%) relapsed, of which 4 pts were blast crisis at the time of the transplant. After follow-up of 42 months (58-102), 17 pts (47%) are alive and 19 pts (53%) died within 9 pts (25%) from TRM (acute GVHD:02, severe infection:4, haemorrhagic cystitis:1, TRALI syndrome:1, early rejection :1) And 10 from relapse (27%) and the overall survival (OS) and disease free survival (DFS) are 47% and 44% respectively. Conclusions: Our study shows that haplo-identical HSCT using the Beijing is a well-validated approach and feasible in patients with advanced malignancies, associated with prompt engraftment, acceptable rates of GVHD, TRM and survivals. Disclosure: Nothing to declare
Background: Autologous stem cell transplantation (ASCT) is a key component of treatment for multiple myeloma (MM) following induction therapy. The efficacy and safety of high-dose melphalan 200 mg/m2 conditioning is well-established in fit, non-elderly (<65-70 years) patients. Although historical data has demonstrated increased toxicity with melphalan 200 mg/m2 in patients with renal failure, there is limited up-to-date literature demonstrating the safety and efficacy of low dose conditioning (140 mg/m2 or 110 mg/m2) in renal failure and other subgroups with high transplant comorbidity index. Methods: A retrospective analysis was performed on 81 MM patients who underwent first ASCT with low-dose conditioning following induction therapy at a single centre between 2006-2020. The primary outcome was ASCT-related toxicity, and secondary outcomes were overall survival (OS); progression free survival (PFS), depth of response and renal function. Univariate and multivariate analysis was performed for age, renal function, co-morbidities, performance status, ISS/R-ISS, high-risk cytogenetics and depth of response prior to ASCT. Results: Of 81 patients, 31 (38%) were female, 34 (42%) were >65 years (mean age 61.5, IQR 56-68). 76 (94%) patients had MM (34 (45%) light chain and 42 (55%) had IgG or IgA MM); the remainder (6%) had other conditions (AL amyloidosis, monoclonal gammopathy of renal significance, multifocal plasmacytoma). ISS was ≥2 in 46 (57%) at diagnosis; 18 (22%) had high-risk cytogenetics. Prior to ASCT, mean GFR was 45 mL/min, IQR 61-27; mean left ventricular ejection fraction was 61%, IQR 66-55; 5 (6%) had poor performance status (Karnofsky < 80). Induction therapy included a PI in 66 (82%) and IMID in 45 (56%). Mean duration of admission was 24.7 days, IQR 18-25; mean time to neutrophil engraftment was 11.4 days, IQR 11-12. 4 (5%) patients had worsening of renal function during their ASCT admission meeting definition of acute kidney injury by RIFLE criteria; mean creatinine clearance at 100 days was 69 mL/min, IQR 89-57. Mortality at 100 days was 1/81 (1%). Disease status as per IMWG criteria prior to ASCT was Very Good Partial Response (VGPR) or better in 57 (70%), Partial Response (PR) in 20 (25%) patients. Staging data at 3 month post-ASCT was available for 71 patients, of which 52 (73%) were in VGPR or CR, and 15 (21%) were in PR. Mean follow-up period was 30 months, IQR 12-42. Median OS was 6.2 years. Median PFS was 2.3 years, with a significantly better median PFS (2.8 years vs 1.6 years) for patients <70 years old vs patients aged >70 (p-value 0.045); at latest review mean GFR was 67, IQR 89-50. Early relapse/progression within <18 months was observed in 17/81 (21%) patients. Conclusions: Our data suggest that autograft with low-dose melphalan conditioning is a feasible, safe and effective therapeutic option in patients deemed unfit for high-dose melphalan. This analysis provides real world data to support clinical decision making in MM that will be benefited from high-dose alkylating agent. The renal status did not significantly impact on toxicity or efficacy outcomes. In this cohort older patients had significantly inferior PFS. Disclosure: Nothing to declare.
Background: Peripheral T-cell lymphomas (PTCLs) are relatively rare and heterogeneous group of lymphoproliferative disorders accounting for 10%–15% of all non-Hodgkin lymphomas. High-dose consolidation therapy with autologous stem cell support (auto-HCT) is considered beneficial for transplant-eligible patients with newly diagnosed and relapsed disease. The evidence is based on retrospective analyses which included heterogeneity of mainly chemotherapy-based conditioning regimens. Achieving complete remission (CR) before auto-HCT is one of the strongest predictors of the outcome. We report on a retrospective, single institution analysis of auto-HCT with total body irradiation (TBI)-based conditioning regimen in patients with PTCLs in CR1. Methods: Patient selection included all 23 consecutive adult patients with PTCLs in CR1 undergoing auto-HCT between 2010-2019 in the Department of Bone Marrow Transplantation and Onco-Hematology, National Research Institute of Oncology, Gliwice Branch, Poland. Eighteen of them were conditioned with 12Gy TBI (given in 3 fractions of 4Gy on 3 consecutive days) combined with 120mg/kg cyclophosphamide. Five patients (aged above 60 y.o. at transplantation) were conditioned with 8Gy TBI given in 2 fractions of 4Gy combined with bendamustine 160-200mg/m2 given on 2 consecutive days. The group characteristics were as follows: median age at transplantation 52 years (range, 22-68); PTCL subtype (PTCL-not otherwise specified-8; angioimmunoblastic T-cell lymphoma-4; anaplastic large cell lymphoma ALK(-)-4, ALK( + )-6, enteropathy associated T-cell lymphoma -1); Ann Arbor stage at diagnosis (II-5; III-12; IV-6); median number of pre-transplant chemotherapy lines: 1 (1-2). Results: All transplants were performed with use of peripheral blood as a source of stem cells. The median number of transplanted CD34 + cells was 8.6 (2.0-24.8) x 10^6 /kg b.w. of the recipient. All patients engrafted and the median time to neutrophil recovery was 10 days (9-13). With the median follow-up of 92 months (26-134) only 1 patient experienced relapse. Six patients died of other causes: one patient died 5 months after transplantation whereas the remaining 5 patients died at a time longer than 1 year after auto-HCT (median 64 months (17-91). The probability of overall survival at 5 and 8 years after auto-HCT was 87% (+/− 7%) and 68% (+/− 11%), respectively. The probability of progression-free survival at 5 and 8 years after auto-HCT was 82% (+/− 8%) and 63% (+/− 12%), respectively. Conclusions: Our long-term follow-up analysis shows that TBI seems to be an effective part of conditioning regimen before auto-HCT for PTCLs allowing potentially to achieve good disease control. Disclosure: Nothing to declare
Background: Second allogeneic stem cell transplantation (SCT2) is associated with a high risk of peri-transplant and post-transplant mortality. Reduced intensity conditioning regimens (RIC), usually based on busulfan and fludarabine (Bu-Flu), are usually chosen in these cases. The aim of our retrospective study was to evaluate the effect and toxicity of a conditioning regimen containing treosulfan (Treo) instead of busulfan. Methods: In our retrospective analysis, 15 patients transplanted at our center between June 2012 and February 2019 were included (9 women, 6 men, mean age 50 years). There were 12 patients with acute myeloid leukemia (AML) (including 7 cases of secondary AML), 2 patients with myelodysplastic syndrome (MDS), and 1 patient with chronic myelomonocytic leukemia (CMML). Complete remission was achieved in 3/15 (20%) of the patients before aloSCT; In 9/15 (60%) of patients, sequential chemotherapy was used before SCT2 due to disease activity. All patients received peripheral stem cells from HLA-matched unrelated donors; RIC Thymoglobulin-Treo-Flu was used as a conditioning regimen. Graft versus host disease prophylaxis consisted of tacrolimus combined with mycophenolate mofetil. The HLA match was 10/10 in 13/15 (86%) of the patients and 9/10 in 2/15 (14%) of the patients. Fourteen patients had a comorbidity index (HCT-CI) 0; one patient had a HCT-CI 3. Results: Neutrophil engraftment was achieved in 13/15 (86%) of patients, in platelets in all patients. The median time to neutrophil and platelet engraftment was 14 (11-21) days and 11 (10-20), respectively. Donor chimerism 99-100% on day +30 was achieved in all patients; on day +100 donor chimerism 100% was achieved in 13/15 (86%) of patients (2 patients had early relapse). Acute GvHD developed in 6/15 (40%) of patients (grade ≥ 3-4 in 13%) and extensive chronic GvHD (grade 4) in 1/15 (6%) of patients. At the median follow-up of 63 months, 10/15 (66,6%) of the patients have died: in 7 cases due to relapse, in 2 cases due to severe posttransplant lymphoproliferative disease, 1 patient died from acute GvHD. GRFS (GvHD-free, relapse-free survival) at 1 year was 33%. The median overall survival was 13.6 months. Conclusions: Our data indicate a favorable safety profile and high efficacy of Treo-containing RIC in a selected group of patients undergoing SCT2. The advantages of this regimen are low peri-transplant and early post-transplant mortality and the ability to induce 100% chimerism early after transplantation. Disclosure: Nothing to declare.
Background: Haploidentical stem cell transplantation (haploSCT) rate is increasing worldwide, mostly due to its clinical efficiency and accessibility. Bone marrow used to be the preferred stem cell source in the haploSCT because it is less immunogenic and causes less graft versus host disease (GvHD) than peripheral blood stem cells (PBSC). To prevent relapse in high risk hematologic malignancies, at UMC Ljubljana, Slovenia we decided to use PBSC with low dose anti-thymocyte globulin (ATG) in combination with posttransplant cyclophosphamide (PTCy). Methods: We analyzed six high risk leukemia patients who underwent haploSCT with low dose ATG (Grafalon) 5 mg/kg on day -3, -2, -1 (15 mg/kg total dose) and thiotepa -6, -5 (total dose 10 mg/kg), busulfan -4, -3, -2 (total dose 9,6 mg/kg), fludarabine -4, -3, -2 (total dose 150 mg/m2), PTCy 50 mg/kg on day +3 and +5 conditioning regimen with cyclosporine and mycophenolate mofetil for GvHD prophylaxis since May 2020. At the time of haploSCT the median patient age was 64 years (26-67). 4/6 (67%) had secondary AML with MDS related changes, two of them resistant to primary induction but in CR after second induction, one (17%) had primary resistant AML, one (17%) patient had early T-ALL. 5/6 (83%) patients were in CR1 before the haploSCT. Results: All patients received a myeloablative TBF protocol with ATG and PTCy and all received PBSC at median dose of 4 x106/kg CD34 + cells. Median time to neutrophil engraftment was 15 days. One patient died due to intracranial bleeding before engraftment. We did not observe acute or chronic GvHD. Two patients had CMV reactivation after letermovir prophylaxis discontinuation. All patients remain in remission. The mean survival time is 15 months (95% CI 9,6-20,3) with OS 83%. Conclusions: For patients with high risk leukemia and no matched sibling or unrelated donor haploSCT with peripheral blood stem cells and low dose ATG combined with PTCy is an encouraging treatment option resulting in low GvHD and disease relapse rate. Disclosure: Nothing to declare
Background: The pre-transplantation conditioning regimen in allogeneic hematopoietic stem cell transplantation (Allo-SCT) is important in early morbidity, non-relapse mortality, and long-term disease control. It is difficult to standardize the conditioning regimens in Allo-SCT. In this sense, the concept of ‘transplant conditioning intensity (TCI) score’, which has been developed recently, has become important [1]. Methods: Eighty-three patients with various diagnoses who underwent allo-SCT at the Hematology Department of Aydın Adnan Menderes University between the years of 2014-2020 were included in the study, which was designed to be single-center, retrospective. Regarding donor compatibility, we have included all allogeneic transplantation procedures, performed as related or unrelated 9-10/10, as well as haploidentical transplantations. Myeloablative, reduced intensity regimen (RIC), and non-myeloablative conditioning regimens were used in accordance with the diagnoses. Of the patients; the rates of myeloid malignancy, lymphoid malignancy, and aplastic anemia were 65%, 30%, and 5%, respectively. Results: The distribution of conditioning regimens according to the TCI score group was as follows: 13 patients had a low, 44 patients an intermediate, and 30 patients a high TCI score. The mean age was 42 ± 3 in the low group, 48 ± 2 in the intermediate group, and 36 ± 2 in the high group, respectively. No statistical difference was found with Kaplan Meier between the three groups (Fig. 1). Low TCI had a mean survival of 42.1 ± 10.5 months, intermediate 61.3 ± 13.7, high 43.2 ± 8.9 months. Conclusions: Although it is not statistically significant, the longer life expectancy may increase the interest in the RIC, although the mean age is higher in those with the intermediate TCI score. It is important to increase the use of TCI in practice. Disclosure: Nothing to declare
Background: Wolman disease (WD) is a lysosomal storage disorder (LSD) that is caused by complete deficiency of lysosomal acid lipase (LAL). There is a pathogenic variant in the LIPA gene which maps to chromosome 10q23. It is an infantile onset lethal disease characterised by lysosomal accumulation of cholesteryl esters (CE) and triglycerides (TG) predominantly in hepatocytes and macrophages. Symptoms and signs develop due to large accumulation of CE and TG in the lysosomes of Kupffer cells and hepatocytes as well as in macrophages throughout the viscera. Untreated, infants do not survive beyond the first year of life. Treatment includes enzyme replacement therapy (ERT) and haematopoietic stem cell transplant (HCT). The long-term use of ERT is limited by ongoing gastrointestinal symptoms, neutralising anti-drug antibodies (ADA), the need for life long central venous access and the financial implications of treatment. HCT has a high transplant related morbidity and GvHD mortality. An engraftment defect has been reported with the majority of patients having a mixed chimerism. Autologous ex vivo haematopoietic stem cell gene therapy (HSC-GT) is an emerging treatment in LSD. HSC-GT eliminates the risk of GvHD and reduces the need for immunosuppression. By delivering a transgene that includes a promotor, there is the capacity to drive overexpression and achieve supra-physiological levels of enzyme. Methods: Wolman CD34 + cells were transduced with a CD11bLIPA lentivirus at a multiplicity of infection (MOI) of 25,50 and 100 with and without transduction enhancers (TE). Transduction efficiency, vector copy number and enzyme levels were evaluated. Additionally Wolman CD34 + and unaffected CD34 + were compared, investigating colony forming unit (CFU) total, distribution and LAL enzyme level activity. Results: As expected Wolman CD34 + cells had minimal LAL enzyme activity. After transduction of Wolman CD34 + cells with CD11bLIPA LV, the deficient CD34 + cells were able to achieve significantly higher levels of enzyme activity to that of unaffected controls (Figure 1). Transduction without TEs achieved a vector copy number (VCN) between 1.5 and 3. With higher VCN (achieved with TEs) the enzyme activity is significantly higher than unaffected CD34 + ; ‘supraphysiological’, however with MOI 100 there was reduction in overall colonies. There was no significant difference in the CFU distribution between Wolman and Unaffected. The transduction efficiency >90% in all CFU’s. Figure 1: Conclusions: We have demonstrated ex vivo transduction of human Wolman CD34 + stem cells drives LAL activity and that the optimal MOI was reduced by transduction enhancers without associated toxicity. To further demonstrate efficacy and to assess toxicity and safety, we aim to show direct macrophage correction and reduction of oxysterols in normal and Wolman macrophages derived from peripheral blood and transduce normal or Wolman CD34 + cells into NSG mice for biodistribution and toxicology outcomes. Disclosure: Nothing to declare
Background: Allogeneic HSCT currently constitutes the only available therapy which can revert the bone marrow failure (BMF) characteristic of Fanconi Anemia (FA). In the absence of a compatible donor, androgens may be used in FA patients, although other agents are being tested in clinical trials. In this respect, there are two ongoing clinical studies evaluating the safety and efficacy of eltrombopag in FA patients. From preclinical models, it has been postulated that this drug promotes DNA repair in FA HSCs through the non-homologous end joining repair mechanism, improving genome integrity, cell survival, and HSC functionality. In addition to these approaches, gene therapy has conferred preliminary evidence of safety and efficacy in phase I and II studies, in patients receiving autologous gene corrected CD34 + cells exceeding threshold levels and during early stages of BMF. The aim of this study is to analyze the safety and efficacy of eltrombopag in FA patients who had been infused in the phase I clinical trial with very low numbers of corrected CD34 + cells. Methods: We report the hematologic and molecular course of four patients initially included in the gene therapy clinical trial FANCOLEN-1 (NCT03157804) who had an evolution of BMF. Two of them were treated with eltrombopag under a “compassionate use program” and the other two were included in the clinical trial FANCREV (EUDRACT 2020-002703-18). During follow-up, peripheral blood counts (PBC), BM studies including cytogenetics, and vector copy number (VCN) analyses were carried out to assess the efficacy and safety of eltrombopag in these patients. Results: Patients received treatment with eltrombopag for a duration between 3 and 12 months. No medication-related adverse events, including clonal evolution, were observed during their follow-up. One patient showed a modest increase in PBC counts after 3 months of treatment and remained free of transfusion requirements. In the remaining cases no sustained hematologic response was observed. In BM aspirations performed during the course of therapy, a decreasing trend in CD34 + cell numbers was observed. Strikingly, the analysis of VCNs in BM showed more marked increases in the proportion of gene corrected cells as compared to analyses performed prior to eltrompopag treatment. Conclusions: No relevant adverse effects, including clonal events, were observed during eltrombopag treatment in FA patients previously infused with very low numbers of gene corrected CD34 + cells. The evolution of PBC counts during the treatment period does not allow us to conclude that eltrombopag mediated clinically relevant hematologic responses. The proportion of gene-corrected cells in BM increased in all patients. Although current data does not enable detection of a sustained eltrombopag-mediated benefit in the hematopoiesis of FA patients previously infused with very low numbers of gene corrected CD34 + cells, this drug promoted a more marked proliferative advantage of gene corrected cells compared to analyses performed prior to eltrombopag treatment. In order to evaluate the potential impact of eltrompopag in FA gene therapy, studies with a longer follow-up will be conducted in patients infused with autologous gene corrected cells, potentially at earlier stages of BMF. Clinical Trial Registry: EudraCT 2020-002703-18 Disclosure: Julián Sevilla: Consultant/Advisor/Honorarium (Amgen, Novartis, Miltenyi, Sobi, Rocket Pharmaceuticals) and has licensed medicinal products from Rocket Pharmaceuticals. Juan Bueren: Consultant/Advisor/Honorarium and has licensed medicinal products from Rocket Pharmaceuticals. Jonathan D. Schwartz: employee and equity shareholder for Rocket Pharmaceuticals.
Background: HLA haploidentical stem cell transplantation using αβ + T Cell /CD19 + B-cell depletion (αβHaplo-SCT) has been utilized in the last decade as an alternative approach to matched donor allogeneic transplantation in children with hematological malignancies1-4. We report a single center experience using αβHaplo-SCT in children with acute leukemias between 2013-2020. Methods: Data regarding donor/recipient, conditioning regimen, and graft characteristics were noted. Study endpoints were overall survival (OS), event free survival (EFS, relapse, death, or rejection), leukemia-free survival (LFS), non-relapse mortality (NRM), acute graft-versus-host disease (aGVHD), chronic graft-versus-host disease (cGVHD) and graft-versus-host disease, relapse-free survival (GRFS)5. Chi-square test or Fisher’s exact test were used to compare categorical variables, and continuous variables were analyzed using Mann-Whitney non-parametric testing. Cox proportional hazards regression models were constructed and analyzed using SPSS 25. Results: Thirty-eight children with acute leukemia underwent αβHaplo-SCT, Most for advanced disease (≥CR2, or refractory) and third had prior HSCT. Patient’s median age was 8.5 years. Conditioning regimen was total body irradiation (TBI) based in 55% of children and 45% were conditioned with combination of Fludarabine, Treosulfan or Melphalan and Thiotepa. Patients received serotherapy as part of the conditioning, using anti-thymocyte globulin. Eighteen patients had more than 3X10^4 /kg of αβ T cells in the graft and received GVHD prophylaxis with mycophenolate mofetil 2. No patient had donor’s HLA antibodies. Thirty-four patients had primary engraftment and four had primary rejection. Median time for neutrophils engraftment was ten days and for platelets thirteen. Six patients had secondary rejection (median time 27 days). Using TBI was associated with higher engraftment and less rejection compared to chemotherapy only but did not reach a statistical significance (94% vs 82% p = 0.3 and 9% vs 23% p = 0.37, respectively). Thirteen patients developed grade 1-2 aGVHD, age was a significant risk factor (11 Vs 6.5 P = 0.023). No patient developed grade 3-4 aGVHD and one developed extensive cGVHD. Twenty-two patients were alive at last follow up with 5 years OS of 51%, and EFS of 42%. EFS was longer for patients with higher graft composition of γ/δ T cells (54% Vs 26%, P = 0.04). Nine patients had relapse, all were males (34% vs 0%, P = 0.026). LFS probability was 72%. GRFS probability was 47.5%. Eleven patients had NRM, mostly due to sepsis. CMV reactivation was common (50%) at a median time of 27 days, treated preemptively according to established guidelines.6 An expansion of NK cells was noted at day 30 of transplantation than reconstituted at three months. CD4/8 T cells and B cells recovered at six months. Immunoglobulins were supplemented as part of infection prophylaxis during immune recovery. Conclusions: αβHaplo-SCT offers a cure for children with multiple relapses, or those who failed prior SCT with OS of 51%. Higher composition of γ/δ T cells had less rejections. Older age was a significant risk factor for grade 1-2 aGVHD. Chronic GVHD was rare. The role of prophylactic immunosuppression needs to be validated through a prospective trial. Immune reconstitution is slow, necessitating close follow up for viral reactivation, and preemptive prophylaxis is crucial for survival. Disclosure: No conflict of interest
Background: Relapse is the most common cause of treatment failure after allogeneic hematopoietic stem cell transplantation (aHSCT) in patients with high-risk myeloid malignancies. Increasing conditioning intensity before aHSCT has not been able to improve transplant results because of increased treatment related mortality (TRM) especially in elderly, comorbid patients. Venetoclax has been shown to greatly enhance efficacy of conventional chemotherapy as well as hypomethylating agents while increasing mainly hematologic toxicity. Experience in combining Venetoclax with intensive conditioning therapy, where hematotoxicity can be suspended by aHSCT so far is limited. Methods: Starting in 2018, we added Venetoclax to Fludarabine/Amsacrine/Ara-C (FLAMSA) + alkylator based sequential conditioning regimen (FLAMSAClax) in individual patients with poor prognosis myeloid malignancies. All patients gave written informed consent for individualized treatment. We now retrospectively collected data on 11 patients (9 AML, 1 MDS, 1 CMML, 6 female, median age 57, range 20-66) who had a minimum follow up of 300 days after transplant. All patients had active disease at transplant (8 refractory, 3 untreated) and 10 had high-risk genetics. Various doses of Venetoclax (100-400mg/d) were given in addition to FLAMSA and stopped one day before alkylator treatment (8 Melphalan, 3 Treosulfan). Results: No additional extrahematologic toxicity was observed. There was 1 laboratory TLS, but no TRM. WBC and PLT reconstitution occurred on day + 12 (median, range 8-19 for WBC > 1000/ul) and on day + 14 (median, range 10-31 for PLT > 20000/ul). aGvHD grade III/IV occurred in 2 and severe cGvHD in 1 patient. At day +30, ten patients were in CR or CRi, 1 with molecular disease persistence. One patient had blast persistence but achieved molecular CR after salvage with HMA + Venetoclax. After a median follow up of 600 days (range 304-1001) for surviving patients, 10 patients (91%) are alive, 8 in molecular CR and 2 currently receive salvage therapy for relapse. So far 3 patients relapsed (day + 175, +267 and +855) and 1 finally died after salvage and 2nd transplant from disease progression. Conclusions: Venetoclax added to sequential FLAMSA + alkylator based conditioning seems to be feasible and highly effective in patients with poor risk myeloid malignancies. The FLAMSAClax approach should therefore be studied in a controlled prospective trial. Clinical Trial Registry: NA Disclosure: GK has received honoraria for an advisory role from Abbvie and Eurocept as well as travel support from Medac. All other authors have nothing to declare.
Background: Adult onset leukoencephalopathy with axonal spheroids (ALSP) is a very rare disease and belongs to the heterogenouos groups of leukodystrophies. ALSP is a hereditary disease caused by a mutation in the CSF1R gene, presenting with progressive neurological symptoms, usually in the 4th or 5th decade and death within 5-6 years after onset of symptoms. The CSF1R gene is associated with phosphatase and kinase proteins regulating the function of macrophages, microglia and neuronal pathways. Dysbalances and disturbances caused by the CSF1R mutation lead to progressive inflammation and white matter lesions. Patients (pts) with leukodystrophies might benefit from allogeneic hematopoietic stem cell transplantation (allo-HSCT) by exchanging part of the host’s microglia through donor cells. Methods: Two male patients with ALSP and CSF1R gene mutations and white matter lesions in the MRI underwent allo-HSCT at our center. Pt 1, a 49 year old male, was diagnosed in 2017, when he developed gait disturbances and a speech disorder, mainly due to a severe dyspraxia. Pt 2, a 47 year old male, underwent extensive neurological assessment in 2016 for left-sided sensomotor dysfunstions, visual disturbances and progressive cognitive deficits. He also had a family history with his mother, maternal grandmother, two maternal aunts and one older brother dying from early onset dementia and one younger, still asymptomatic, brother. The CSF1R mutation was also found in his two brothers. Due to the lack of material, no analyses are available from his mother, grandmother and aunts. Results: Pt 1 was transplanted in 2019 from an HLA-identical sibling not carrying the mutation, while Pt 2 received a transplant from an unrelated HLA-identical male donor in 2020. Both pts were conditioned with busulfan (9.6 mg/kg BW) and fludarabine (180 mg/m²). Immunosuppression (IS) consisted of anti-thymoglobulin, cyclosporine A (CsA) and short course methotrexate. Both pts promptly engrafted and IS could be tapered without signs of acute or chronic GvHD. Transient deterioration of the neurological functions was seen in both pts in the first 6 months after HSCT, followed by improvement to pre-HSCT status. 2.5 years after allo-HSCT, the symptoms of dyspraxia and dysarthria of pt 1 have improved, he is walking without limitations and has stable cognition . Pt 2 presented with progressive MRI changes 6 months after transplantation and deterioration of gait and speech. In the last evaluation 1.25 years after allo-HSCT, he clearly improved in gait and speech, his MRI was stable. Conclusions: ALSP is a usually fatal neurological disease caused by CSF1R gene mutations. Allo-HSCT has the potential to prevent further destruction of brain by replacing the recipient’s microglia through donor cells and thus stabilization of the neurological functions after 6-12 months. Disclosure: Nothing to declare
Background: Haploidentical peripheral blood hematopoietic cell transplantation(HaploSCT) has become the preferred alternative donor transplant program, owing to its logistic and cost advantages. However, the data from developing countries is not sufficient enough to hold this statement true for them. Methods: It is a retrospective observational study hospital record based study where the outcome of Haplo-SCT was analysed using standard statistics. Results: Between March 2015 and April 2021, 74 patients underwent 75 haploidentical transplantations at our institution. Median age was 31(9-60) years and indications included malignant disorders in majority (96%). Conditioning regimens included myeloablative (38.7%), nonmyeloablative regimens (44%) and reduced intensity (17.3%). GVHD prophylaxis was post-transplant cyclophosphamide on day+3,4 with Mycophenolate and Cyclosporine from day+5 onwards. Peripheral blood stem cells were the predominant graft source. Majority (92%) had CMV reactive donor and recipient combination. Median cell dose (CD34) was 5.26 (1.8-8.09) x106/kg. Median engraftment for neutrophils and platelets was 14 (11-32) and 15 (10-43) days respectively. Nine (12.1%) patients had rejection (primary = 8, secondary=1). CMV reactivation was observed in 52 (69.3%) patients. The cumulative incidence of acute GVHD was 37.3% with 32.1% incidence of grade III-IV acute GVHD and 14.2% patients were steroid refractory. Chronic GVHD was seen in 16% and one fourth patients had extensive chronic GVHD. Sixty-four(85.3%) culture positive bacterial infections (Gram positive= 14, Gram negative=50) were observed in 44 patients whereas 32.4% patients had fungal infection and 17.5% had viral infections. TRM within 30 days, 30-100days and >100days was 8(10.6%), 14(18.6%),16(21.3%) patients respectively. Common causes of death were sepsis and relapse. Conclusions: We emphasize haploidentical SCT offers a reasonable hope of cure for patients with hematological malignancies, though infections, GVHD, relapse are still deal-breakers in our experience which we are striving to overcome in our prospective studies. Clinical Trial Registry: Not Applicable Disclosure: No conflict of interest.
Background: Acute graft-versus-host disease affecting the lower gastrointestinal tract (GI-GVHD) is a major life-threatening complication of allogeneic hematopoietic cell transplantation and is frequently resistant to glucocorticosteroid-therapy (SR). Higher overall response rates and failure-free survival of ruxolitinib compared to best available second-line therapy (SLT) was recently reported in a phase-III trial. However, real-world data and external validation of these promising results are lacking. Methods: To determine the outcome of patients with GI-GVHD in the era of ruxolitinib, we retrospectively analyzed patients who developed GI-GVHD over a 6-year period to determine therapy-responsiveness and survival. Results: A total of 144 patients developed GI-GVHD and 83 (58%) were SR. Ruxolitinib was most commonly used (74.3%) as SLT. Overall and complete response (CR) to ruxolitinib on day 28 were similar to what reported in REACH2 trial (60.0% and 27.3% respectively, Figure 1A). A durable CR was observed in ruxolitinib-treated patients at day 56 in 31.6% and in other single-agent SLT in 16.7% (Figure 1B). Around one fourth of patients could achieve a CR even after third-line (25.8%) and fourth-line therapy (25.0%). Moreover, SR-GVHD patients experienced a lower 5-year overall survival (OS) (34.8% vs 53.3%, p = 0.0014) and higher cumulative incidence of 12-months non-relapse-mortality (NRM) (39.2% vs 14.3%, p = 0.016) compared to glucocorticosteroid-sensitive patients. Interestingly, SR-GVHD patients who achieved CR on day 28 experienced higher 5-year OS (56.3% vs 14.9%, p < 0.0001) and lower 12-months NRM (13.8% vs 77.4%, p < 0.0001) compared to non-responders, having an outcome comparable to glucocorticoid-sensitive patients (Figure 1C). Conclusions: These real-world unselected data confirm the response rate to ruxolitinib as SLT in SR-GI-GVHD obtained in REACH2 trial and the poor outcome of SR-GI-GVHD. Additionally they show improved OS of ruxolitinib-CR-responders and indicate that first-line therapy failure still allows for a considerable CR rate upon SLT. Disclosure: Rober Zeiser received honoraria from Incyte, Novartis and Mallinckrodt, was supported by the Deutsche Forschungsgemeinschaft (DFG), Germany for the SFB 1479 OncoEscape P01, Project ID: 441891347 to RZ, SFB1160 to RZ, ZE 872/4-2, TRR167 to RZ, Deutsche Krebshilfe (grant number 70113473), the Jose-Carreras Leukemia foundation grant number DJCLS 01R/2019.
Background: Allogeneic (allo) hematopoietic stem cell transplant (HCT) has curative potential in multiple myeloma (MM) but remains hampered by high rates of relapse and chronic (c) GVHD. We recently completed a prospective phase II study (LeBlanc R, BMT 2021) in newly diagnosed MM using bortezomib (BTZ) maintenance after tandem autologous (auto) + alloHCT aimed at decreasing relapse. Based on previous clinical observations, we hypothesized that BTZ could also decrease incidence and severity of cGVHD. Methods: Using the 2015 NIH criteria, we retrospectively reviewed the incidence and organ distribution of cGVHD, as well as duration of immunosuppression in 2 contemporaneous cohorts: patients receiving BTZ maintenance q2 weeks for 1 year after alloHCT, and tandem transplants without BTZ maintenance. After autologous HCT, patients from both cohorts received an outpatient nonmyeloablative conditioning followed by G-CSF mobilized donor stem cells. GVHD prophylaxis consisted of mycophenolate mofetil and tacrolimus tapered by D + 100 (sibling donors) or D + 180 (unrelated donors) in both groups. Cumulative incidences of cGVHD were estimated using competing-risk methods including relapse, second transplantation and death. Results: Between 2014 and 2018, 41 patients received BTZ maintenance, whereas 57 patients did not. Myeloma subtypes were similar in both groups. Baseline characteristics showed no difference except that patients in the BTZ group had younger donors (40 years vs. 52 years) and more unrelated donors (59% vs. 12%). Incidences of grade II-IV acute GVHD at day+180 were similar in both cohorts (17.1% vs. 26.6%, p = 0.518). At 2 years, incidences of overall (61.0% vs. 84.2%, p = 0.001) and moderate/severe cGVHD (44.7% vs. 66.7%, p = 0.003, Fig. 1) were significantly lower in BTZ than in non-BTZ recipients. After univariate analysis, overall mouth (56% vs. 79%, p = 0.025), skin (34% vs. 56%, p = 0.041) and liver (32% vs. 54%, p = 0.039) involvement were less frequent in BTZ patients. We elected to choose donor age, sex, CD34 + cell dose, major ABO mismatch, recipients’ CMV status and grade II-IV acute GVHD as variables to perform multivariable analysis. Following multivariate Fine-Gray regression, not receiving BTZ was associated with higher overall incidence of cGVHD (HR 2.38, p = 0.002) and moderate/severe cGVHD (HR 2.39, p = 0.004). The cumulative incidence of prednisone initiation at 5 years was 42.2% in BTZ and 78.3% in non-BTZ recipients (p < 0.001). The cumulative incidence of tacrolimus resumption at 5 years was also lower in BTZ than in non-BTZ recipients (30.1% vs. 73.6%, p < 0.001). Probability of being alive and off immunosuppressants at 3 years were 86% for BTZ patients vs. 47% for non-BTZ patients (p < 0.001). NRM at 2 and 5 years were 4.9% and 8.8% in BTZ recipients vs. 1.8% and 5.4% in non-BTZ recipients (p = 0.575). We observed no impact of BTZ on 5-year OS (82.9% vs. 83.4%, p = 0.938) and PFS (49.5% vs. 58.5%, p = 0.277) respectively in patients receiving or not BTZ. Conclusions: Although it had no impact on survival, BTZ maintenance led to a significant reduction in incidence and severity of cGVHD with shorter duration of immunosuppressants. BTZ maintenance should be considered as a valid option in MM receiving upfront auto-alloHCT. Clinical Trial Registry: N/A Disclosure: The authors declare no conflicts of interest.
Background: Belumosudil is a novel oral selective rho-associated coiled-coil–containing protein kinase-2 inhibitor designed for the treatment of cGVHD following an allogeneic hematopoietic cell transplant. We report the 2-year, long-term, follow-up safety results from the ROCKstar study. Methods: This open-label, randomized, multicenter study evaluated belumosudil 200 mg QD (n = 66) and BID (n = 66) in patients with cGVHD (aged 21-77 years) who received 2 to 5 prior LOTs; cutoff date for the long-term follow-up was August 19, 2021. Results: Median age was 56 years, median time from diagnosis to enrollment was 28 months, median prednisone dose was 0.19 mg/kg/d, 67% of patients had severe cGVHD, 52% had ≥4 organs involved, 73% had received ≥3 prior LOTs (including ibrutinib [34%] or ruxolitinib [29%]) and 73% were refractory to their last LOT. Median treatment duration was 10 months, with 29% of patients receiving belumosudil for ≥24 months. The ORR (95% CI) with belumosudil 200 mg QD and BID was 74% (62%-84%) and 77% (65%-87%), respectively. Belumosudil continued to be well tolerated (Table). The primary reasons for drug discontinuation were adverse events [AEs] (n = 17 [13%]; 7 experienced AEs that were not drug related), progression of underlying malignancy (n = 5 [4%]) and progression of cGVHD (n = 16 [20%]). Twenty percent and 10% of patients experienced ≥1 drug-related AE that led to dose modification and interruption, respectively. Forty-eight percent and 32% of patients experienced ≥1 AE not drug related that led to dose modification and interruption, respectively. AEs observed at all grades in ≥30% of patients included fatigue (39%), diarrhea (35%), nausea (31%) and cough (30%). Grade ≥3 AEs observed in ≥5% of patients included hypertension (8%), pneumonia (8%) and hyperglycemia (5%). Grade ≥3 cytopenias included anemia (4%), neutropenia (2%) and leukopenia (1%). At least 1 serious AE occurred in 44% of patients. There was 1 case of cytomegalovirus reactivation. Of all patients, 5% had ≥1 grade ≥3 drug-related hepatic disorder, 14% had increased gamma-glutamyltransferase, 12% had increased aspartate aminotransferase, 10% had increased alanine aminotransferase and 1% had increased bilirubin. The median corticosteroid dose reduction was 50%. Corticosteroid discontinuation was observed in 27% of patients. Calcineurin inhibitor dose reduction and discontinuation was observed in 54% and 27% of patients, respectively. Table Conclusions: Belumosudil remained well tolerated, with low rates of cytopenias. AE and drug discontinuation rates were comparable to those reported in Cutler et al, 2021. Patients have continued to sustain belumosudil therapy and achieve clinically meaningful responses. Clinical Trial Registry: NCT03640481 Disclosure: Corey Cutler is a consultant and advisor for Janssen, Mesoblast, Syndax Pharmaceuticals Inc, Omeros, Incyte Corporation, Jazz Pharmaceuticals, Mallinckrodt, CareDx and Pfizer; has been a pro bono consultant for Kadmon Corporation; and has not received any payment for consulting in the past year. Stephanie J. Lee is on a steering committee for Incyte Corporation and has received research funding from Amgen, AstraZeneca, Incyte Corporation, Kadmon Corporation, Novartis Pharmaceuticals Corporation, Pfizer, Syndax Pharmaceuticals Inc and Takeda. Steven Pavletic received research support from the Center for Cancer Research at the National Cancer Institute through the National Institutes of Health Intramural Research Program, which includes Clinical Research Development Agreements with Celgene, Actelion, Eli Lilly, Pharmacyclics and Kadmon Corporation. Bruce R. Blazar is a cofounder of Tmunity Therapeutics, is a consultant and advisor for Magenta Therapeutics and Blue Rock Therapeutics and has received research funding from Blue Rock Therapeutics, Children’s Cancer Research Fund and Kids First Fund. Laurie Green, Zhongming Yang, David Eiznhamer and Jonathan Ieyoub have stock options in and are employees of Kadmon Corporation.
Background: Loss of intestinal bacterial diversity, a relative shift toward bacterial monocolonization (i.e. with Enterococci) and colonization with antibiotic-resistant bacteria (ARB) before allogeneic hematopoietic stem cell transplantation (alloHCT) are observed in gastrointestinal (GI) acute graft-versus-host disease (aGvHD) patients. Allogeneic gut microbiota may modulate systemic immune responses, which has been proven in many microbiota dependent diseases. Fecal microbiota transplantation (FMT) is able to restore gut microbiota diversity and has been successfully used for recurrent Clostridioides difficile infection as well as for the treatment of autoimmune diseases (i.e. ulcerative colitis). Recently, studies of the use of FMT to treat (steroid-refractory (SR) intestinal) aGvHD have shown promising results. Our aim was to identify EBMT centers who perform FMT in this indication and summarize procedure modalities, application regimens, indications and outcome. Methods: The study was performed by Transplant Complications Working Party (TCWP) between May 2018 and May 2020 and used a 2-step approach. In the first step we conducted a survey among EBMT centers with >100 alloHCTs since 2010 to identify cases, while in second, we retrospectively collected details of performed FMTs. Results: A total of 10 centers reported 32 patients. Among these, 27 patients (25 adults and 2 children) met inclusion criteria and had undergone 52 FMTs. Most of the reported patients were already published elsewhere separately. 24/2/1 patients had steroid refractory/dependent/de novo GI aGvHD, respectively and concomitant indications to perform FMT were C. difficile infection in 3 and ARB colonization in 14 of subjects. 22 patients (81%) had grade III-IV gut aGvHD and 24 (89%) overall III-IV aGvHD. Almost all patients (25, 93%) had an unrelated stool microbiota donor and obtained FMT as an infusion (25, 93%) or capsules (1, 4%). Most of the patients received FMT through gastrointestinal/duodenal tube (23, 85%) and the procedure was performed as multiple administration regimen (FMT sessions; in 20 patients, 74%). Overall aGvHD response rate (ORR) at day 28 post-FMT reached 63% (17/27); with 12 patients achieving complete response (CR; 44%) and additional 5 patients achieving partial response (PR; 19%). Additionally, successful ARB decolonization was achieved in 8 out of 14 previously colonized patients (57%). It is also important to highlight that 20 patients (74%) were treated with antibiotics during the first two weeks after FMT, which could have negatively impacted the ORR. In total, 2 cases of sepsis as severe adverse events were reported. Conclusions: In this one of the largest cohort of patients published to date, undergoing FMT after alloHCT, we found high response rates of aGVHD as well as decolonization of ARB. To enable a broader clinical use of this promising approach, more evidence from prospective clinical trials as well as a standardization of FMT modalities is needed. Disclosure: OP has no COIs directly related to this manuscript. OP has received honoraria or travel support from Astellas, Gilead, Jazz, MSD, Neovii Biotech, Novartis, Pfizer and Therakos. He has received research support from Gilead, Incyte, Jazz, Neovii Biotech and Takeda. He is member of advisory boards to Jazz, Gilead, MSD, Omeros, Priothera, Shionogi and SOBI.
Background: Long-term follow-up from the prospective randomized phase III multicenter[PDJF1] trial (RCT) comparing a standard GvHD prophylaxis with cyclosporine A and methotrexate with or without additional pretransplant anti-human T-lymphocyte immunoglobulin (ATLG, Grafalon) (20mg/kg/day, days -3 to -1) in unrelated donor hematopoietic cell transplantation after myeloablative conditioning resulted in a significant reduction of acute and chronic GvHD without compromising relapse and survival (Finke et al., Lancet Hematol 2017). Methods: ATOS is a subsequent prospective non-interventional observational study evaluating the outcome of patients receiving ATLG in unrelated donor transplantation in routine clinical practice without the selective measures of a clinical trial. No control group was included. Patients’ characteristics and outcomes were compared to 103 patients in the ATLG treatment arm of our RCT. Primary endpoint was severe GvHD and relapse-free survival (SGRFS). Results: Between May 2013 and March 2015, 13 transplant centers included 165 patients with haematological malignancies (age median 54, range 18-77 years) in early (45%), intermediate (18%) or advanced (37%) disease receiving marrow (N = 6) or PBSC (N = 159) from 10/10 matched (78%) or mismatched (22%) donors after myeloablative (51%) or reduced intensity conditioning (RIC) (49%). GvHD prophylaxis consisted of calcineurin inhibitors, mainly CSA (93%) with MTX or MMF and ATLG. Different dosing regimens were allowed according to current center practice. Median total ATLG dose was 46 (IQR 32-60, range 15-91) mg/kg. Median follow-up was 70 months (range 11-91 months). ATLG dose differed strongly between centers, so dose effects cannot be separated from center effects. As compared to our RCT, patients in ATOS were older, had a more advanced disease status, RIC, HLA 10/10 match and PBSC transplantation were more frequent, and given median ATLG dose was lower. Incidences of aGvHD (0.56), aGvHD III-IV (0.13), relapse (0.33), relapse mortality (0.24), non-relapse mortality (0.24), and disease-free (0.43) and overall survival rates (0.52), (all 5-years), were similar to the results in the ATLG arm of our RCT. Five-year incidences of cGvHD (0.42), and severe cGvHD (0.27) were higher as compared to results in the ATLG arm of our RCT (any 0.31, severe 0.14), which may be due to different reporting procedures. As a result of these differences, also the 5-year rate of SGRFS was lower in ATOS (0.27) as compared to the ATLG arm in our RCT (0.34). In general, the comparison of outcomes in ATOS and the RCT has to take into account the differences in patient characteristics and treatment procedures. In multiple regression models adjusting for these differences, the largest difference in outcome was seen with respect to severe cGvHD (ATOS vs ATLG arm RCT: hazard ratio 2.79, 95%-confidence interval [1.20,6.51], p = 0.017). All other adjusted comparisons resulted in 95%-confidence intervals of the hazard ratio overlapping the value of one. Adverse drug reactions occurred at a rate and severity that are consistent with the known safety profile, and are clinically manageable. Conclusions: The long-term experience in routine clinical practice confirms the results shown in our RCT, namely the GvHD protective effect of ATLG without compromising relapse and non-relapse mortality rates. Clinical Trial Registry: German clinical trials register DRKS00004581 Disclosure: JF: Neovii, Medac Riemser
Background: Chronic Graft Versus Host Disease (cGVHD) simulating eosinophilic fasciitis (EF) is a rare complication after allogeneic transplantation of hematopoietic progenitors (allo-TPH). EF related cGVHD is an underdiagnosed and challenging complication due to the lack of knowledge about its pathogenesis, refractoriness to traditional immunosuppressive agents and their negative impact on physical function and quality of life. The aim of this study is to describe the clinical-biological characteristics and response to treatment of a case series of EF related cGVHD. Methods: Prospective observational study to describe the clinical and diagnostic evaluation characteristics of patients with EF-like follow-up in our multidisciplinary cGVHD consultations since March 2014 to present. 118 patients were evaluated, 39 of whom (33%) developed fasciitis. Joint and fascial cGVHD was diagnosed if the patient had NIH joint and fascia score ≥1. Clinical variables analyzed in the entire cohort were the baseline and transplant-related characteristics and clinical assessment of cGVHD including time from all-HCT to enrollment, cGVHD type, organs affected and NIH global score. In addition, in the fasciitis group, complementary laboratory and imaging tests as well as the therapeutic approach and response were detailed. Diagnosis and classification were performed according to 2015 NIH and treatment response in EF-like according to the response criteria redefined by Inamoto 2020. Regarding statistical analysis, a descriptive analysis of frequencies was performed and nonparametric tests were used for comparisons (X² or Fisher’s exact test for categorical variables or the Mann-Whitney test for continuous variables). The analyses were performed using the SPSS 25.0 statistical package (SPSS, Chicago, IL, USA). Results: From March 2014 to present, 118 patients were evaluated in the multidisciplinary cGVHD consultation and 39 patients (33%) developed fasciitis. No differences regarding baseline and transplant-related characteristics neither clinical assessment of cGVHD was found between patients with or without fascial involvement. The clinical characteristic of EF related cGHVD are described in Table 1. After a 3 median lines of treatment, the vast majority of patients achieved some degree of response, with a complete response rate of 41%. Table 1.- Clinical-biological characteristics and therapies administered in patients with fasciitis (n = 39). Conclusions: Fascial/articular involvement needs to be recognized and evaluated early with validated scales. In our knowledge, our cohort is the second largest series reported. Literature addressing fascial/joints complications related to cGVHD are scarce. The search for new biomarkers, the use of advanced imaging techniques and multidisciplinary approach may help to improve the prognosis of patients with cGVHD. Disclosure: This work was partially supported by the Education Council and Health Council of the Junta de Castilla y León (GRS 2183/A/20), Spain.
Background: Graft-versus-host disease (GVHD) prophylaxis in matched unrelated donor (MUD) allogeneic hematopoietic stem cell transplantation (allo-HSCT) is mainly based on the use of a calcineurin inhibitor with either short course methotrexate (MTX) or mycophenolate mofetil (MMF). When using peripheral blood as stem cell source (PBSC), addition of antithymocyte globulin (ATG) significantly reduces the incidence of chronic GVHD (cGVHD). Reduced-intensity conditioning regimen (RIC) with fludarabine and busulfan (BuFlu) is largely used in this setting. Methods: Included were adults ≥18 years undergoing first allo-HSCT for acute myeloid leukemia (AML) in complete remission (CR) from a MUD and receiving PBSC and a BuFlu-RIC with ATG, transplanted between 2010-2019. Patients receiving cyclosporine (CsA) with either MTX or MMF were included and transplant outcomes with these GVHD prophylaxis were compared. Results: We identified a total of 1001 patients, including 517 receiving CsA+MMF (MMF group) and 484 receiving CsA+MTX (MTX group). Patients in the MMF group were younger (61 versus 63 years, p < 0.01) and less frequently seropositive for CMV (60% versus 72%). No imbalances were observed for other characteristics. Most patients were transplanted in first CR (85% versus 81% in MMF and MTX groups 1 and 2, respectively, p = 0.12). With a median follow-up of 3 years for both groups, 2-years (2y) relapse incidence (RI) was 26% versus 32% in MMF and MTX groups (p = 0.20) while non-relapse mortality (NRM) was lower in the MTX group (9% versus 15%, p = 0.02). No differences were observed in 2y-overall survival (OS, 64% versus 69% in MMF and MTX groups, respectively, p = 0.10) and 2y-leukemia-free survival (LFS, 59% in both groups, p = 0.70) while a higher 2y-GVHD/relapse-free survival (GRFS) was found in the MTX group (52% versus 46%, p < 0.01). Of note, both grade II-IV and III-IV acute GVHD (aGVHD) were lower in the MTX-group (18% and 4% compared to 37% and 11% in the MMF group, p < 0.01). Similarly, cGVHD of all grades and extensive cGVHD were lower in the MTX group (29% and 8% compared to 36% and 17% in the MMF group, p < 0.05 for all grades and p < 0.01 for extensive cGVHD). These results were confirmed in multivariate analysis with lower NRM (HR 0.63, 95% CI 0.44-0.92, p = 0.01) and higher GRFS (HR 0.80, 95% CI 0.65-0.99, p = 0.04) in the MTX group. Use of CsA+MTX was also associated to lower grade II-IV (HR 0.45, 95% CI 0.32-0.62, p < 0.01) and grade III-IV (HR 0.39, 95% CI 0.23-0.67, p < 0.01) aGVHD and lower cGVHD (HR 0.71, 95% CI 0.56-0.90, p < 0.01) and of extensive cGVHD (HR 0.39, 95% CI 0.22-0.68, p < 0.01). Neither RI (HR = 1.20, 95% CI 0.94-1.52), p = 0.14) or LFS (HR 0.99; 95% CI 0.81-1.21, p = 0.93) or OS (HR 0.85, 95% CI 0.69-1.05, p = 0.13) differed significantly between the two groups. Conclusions: In MUD allo-HSCT GVHD prophylaxis containing CsA+MTX and CsA+MMF were not significantly different in term of LFS and OS. However, CsA+MTX better prevented both acute and chronic GVHD, subsequently reducing NRM and providing higher GRFS compared to CsA+MMF when a BuFlu RIC with ATG is used in MUD-PBSC allo-HSCT. Disclosure: No COI to disclose
Background: HLA matched donor has represented the most used stem cell source in last decades. The main graft-verus-host disease (GvHD) prophylaxis platform includes calcineurin inhibitors with methotrexate (MTX) or mycophenolate mofetil (MFA), but recently cyclophosphamide post-transplant (CY) emerged as reliable alternative, not only for haploidentical donors. Methods: We retrospectively analyzed post-transplant outcomes in HLA matched allogeneic stem cell transplantation (HSCT) between March 2015 and May 2021, comparing two GvHD prophylaxis platforms. The first included cyclosporine A 3 mg/Kg from day 0 (CSA), MFA 30 mg/Kg from day +1 to day +36 and CY 50 mg/kg on day +3 and +5 (n = 91). The second one included CSA 3 mg/kg from day 0 to day 60 and then tapering until day +180, MTX 15 mg/m2 on day +1, 10 mg/m2 on day +3, +6 and +11, and rabbit thymoglobulines 2.5 mg/kg on day -1 (ATG) (n = 111). Results: One year (1-yr) cumulative incidence of moderate/severe chronic GvHD (cGvHD) was of 14.7% (95% CI 8.7-24.9) in the CSA/MFA/CY group and 26% (95% CI 18.5-36.5) in the CSA/MTX/ATG group (p = 0.04). In multivariate analysis, CY-based prophylaxis (HR 0.45, p = 0.02), complete remission status of the underlying disease at transplant (HR 0.40, p = 0.007) and a previous acute GvHD (aGvHD) (HR 2.14, p = 0.003) resulted as independent variables for moderate/severe cGvHD occurrence. Moreover, 1-yr graft-relapse free survival (GRFS) was of 58.2% (95% CI 47.4-67.6) in the CSA/MFA/CY group and 43.2% (95% CI 33.9-52.2) in the CSA/MTX/ATG group (p = 0.01). In multivariate analysis, CY-based prophylaxis (HR 0.59, p = 0.01) complete remission status of the underlying disease at transplant (HR 0.65, p = 0.03) and the use of a female donor (HR 1.51, p = 0.05) emerged as independent variables for GRFS. Considering the occurrence of viral infections after transplant, 1-yr EBV viremia occurred in 22.6% (95% CI 15.2-33.7) in the CSA/MFA/CY group and in 64.3% (95% CI 55.5-74.7) of the CSA/MTX/ATG group (p < 0.0001). Multivariate analysis identified CY-based prophylaxis (HR 0.30, p < 0.0001), a previous aGvHD (HR 1.74, p = 0.01) and a previous CMV infection (HR 2.88, p < 0.0001) as independent variables for EBV infection. Finally, 1-yr cumulative incidence of CMV viremia was of 23.6% (95% CI 14.3-39) in the CSA/MFA/CY group and 57% (95% CI 48.4-67.2) in the CSA/MTX/ATG group (p < 0.0001). Multivariate analysis revealed CY-based prophylaxis (HR 0.41, p = 0.007), a diagnosis of acute leukemia or myelodysplasia (HR 2.10, p = 0.02), a familial donor (HR 0.22, p = 0.0001) and CMV seropositive recipient (HR 5.73, p = 0.0008) as independent variables for CMV infection.No differences among the two groups were identified for aGvHD occurrence, disease free survival, transplant-related mortality or overall survival. Conclusions: Patients receiving HLA matched transplant experienced a low incidence of moderate/severe cGvHD, EBV viremia and CMV viremia and a better GRFS when GvHD prophylaxis is realized using CY with CSA and MFA. Disclosure: Nothing to declare
Background: Steroid refractory acute Graft-versus-Host Disease (SR-aGvHD) in children after allogeneic hematopoietic stem cell transplantation (alloHSCT) is associated with high morbidity and mortality. We aimed to assess clinical course and outcomes of pediatric SR-aGvHD. Methods: We performed a retrospective nationwide multicenter cohort study in the Netherlands. All patients aged 0-18 transplanted between 2010 and 2020 with SR-aGvHD were included. For each patient, weekly clinical aGvHD grade and stage, immunosuppressive treatment and clinical outcomes were collected. The primary study endpoint was clinical course of SR-aGvHD over time, which was graphically analyzed. As a secondary outcome, factors influencing overall survival and remission were identified using a multistate Cox model. Results: Between 2010 and 2020, 786 children received an alloHSCT. 158 patients (20%) suffered from grade II-IV aGvHD, which occurred after a median of 34.5 days. 81 patients (51%) required second line therapy after first line treatment with steroids (Table 1). Second line therapy was started after a median of 8 days after aGvHD diagnosis. 42 patients (52%) required three or more lines of therapy. One year after start of second line therapy, 34 patients (42%) were alive and in remission of their SR-aGvHD and 33 patients (41%) had died. 14 patients (17%) had persistent GvHD. Figure 1 displays clinical course since start of second line therapy. Cord blood (CB) grafts were associated with a significantly lower chance of achieving remission of SR-aGvHD than bone marrow (BM) or peripheral blood stem cell (PBSC) grafts (HR 0.51, 95% CI 0.28-0.94, p = 0.032). Older age was associated with higher mortality: children aged 13.9-17.9 (fourth quartile) had a significantly higher hazard of death compared to children aged 0.175-3.01 (first quartile) (HR 2.64, 95% CI 1.05-6.63, p = 0.04). When modelling the interaction between age and graft source, we found that in BM/PBSC grafts older age was also significantly associated with lower remission rates (HR 0.89, 95% IC 0.83-0.96, p = 0.003). Underlying diagnosis, donor matching or choice of second line therapy were not associated with outcome. Pulmonary manifestation of GvHD leading to respiratory insufficiency was an important cause of death in our cohort, accounting for 10/38 deaths (26%). Table 1. Highlight of patient and treatment characteristics. Figure 1. Clinical course after start of second line therapy Conclusions: Our study demonstrates that SR-GvHD confers a high mortality risk in pediatric HSCT. Older age and use of CB grafts are associated with an unfavorable outcome. Novel treatment strategies to prevent SR-GvHD and timely initiation of second line interventions are pivotal to further reduce GvHD-related mortality. Disclosure: Nothing to declare
Background: Bronchiolitis obliterans syndrome (BOS) is a life-threatening pulmonary complication of chronic graft versus host disease (cGVHD) after allogeneic hematopoietic stem cell transplantation (HSCT). The classic first-line therapy of BOS is systemic steroids to prevent progression. However, patients with steroid-refractory BOS did not get a significant improvement in pulmonary function. Furthermore, long-term systemic steroids usage may cause serious complications such as infection. In this study, we retrospectively investigated the outcome of ruxolitinib as first-line therapy in treating newly-diagnosed BOS patients. Methods: All patients who underwent an allogeneic HSCT for a hematological malignancy between January 2019 and June 2021 at the Institute of Hematology and Blood Diseases Hospital, CAMS and PUMC were retrospectively screened. BOS diagnosis uses the criteria of the National Institute of Health (NIH) consensus. Ruxolitinib therapy was begun with an initial dosage of 5 mg twice daily (BID), then a maintenance dosage of 10 mg. The dose of ruxolitinib could be reduced if severe adverse events occurred. Steroids and other immune-suppression agents were added according to the clinical situation. All patients received anti-fungus prophylaxis and FAM therapy in addition to the ruxolitinib therapy. Treatment response included both symptoms response (SR) and disease responses (DR). Symptom response (SR), which was evaluated in the first two weeks after ruxolitinib administration, was defined as relieving respiratory symptoms, elevated peripheral blood oxygen saturation (SpO2 ≥ 96%), and significant improvements in CT scans. We evaluated ruxolitinib disease response (DR) in the 3rd month. The ruxolitinib administration to DR was defined as CR (Complete Response) when clinical symptoms significantly alleviated and FEV1% pred (FEV 1% prediction) increased by more than 75%; partial response (PR) was defined by FEV1% pred levels increased or symptoms improved with stabilization of FEV1% pred. Nonresponse (NR) was defined by worsened clinical status and PFTs, or FEV1% pred decreased to less than 5% with stable symptoms. Results: We identified seven BOS patients. A median time of 300 days (ranged from 103 to 489 days) elapsed between the time of HSCT and diagnosis of BOS. Five patients were treated with steroids at the same time. The average initial daily dose of methylprednisolone was 48.4 milligrams per day (ranged from 6–80 milligrams). It is inspiring that all patients achieved SR within only two weeks after ruxolitinib therapy. Concerning disease response, six patients (85.7%) achieved CR, and one (14.3%) achieved PR (Figure 1). The mean FEV1% pred at diagnosis of BOS was 58.05%, and increased to 79.47% three months after ruxolitinib therapy, suggesting the therapy was effective. At the same time, the steroid dose was reduced to 50% of the initial dose in about two weeks (ranged from 7 to 16 days) and ended within two months of ruxolitinib treatment (ranging from 23 to 58 days). Conclusions: All patients taking ruxolitinib as first-line therapy achieved remarkable responses with a CR rate of 85.7%. It is also noteworthy that ruxolitinib as first-line therapy in BOS could significantly shorten steroid therapy duration and reduce total steroid dose. Additionally, ruxolitinib was well tolerated, no severe infection or relapse was reported. Disclosure: The authors declare no conflicts of interest.
Background: In a haploidentical setting, graft-versus-host disease (GVHD) prophylaxis is now widely based on the combination of post-transplantation cyclophosphamide (PTCY), cyclosporine-A (CsA), and mycophenolate mofetil (MMF). It has been shown that high concentration of CsA in the first week after haploidentical hematopoietic cell transplantation (HCT) is associated with a reduced incidence of acute GVHD. However, the optimal timing of CsA initiation remains controversial. We performed a single-center retrospective study comparing the incidence of GVHD and patient outcomes after haploidentical HCT according to the timing of CsA initiation. Methods: All consecutive adult patients undergoing haploidentical HCT from November 2013 to December 2020 were included according to the following criteria: (1) peripheral blood stem cell graft, (2) hematological malignancy, and (3) thiotepa-based conditioning regimen with a total thiotepa dose of 5 mg/kg. GVHD prophylaxis consisted of a combination of CsA, MMF, anti-thymocyte globulin (ATG, 2.5 to 5 mg/kg) and PTCY in all patients. CsA was either initiated at day – 3 before HCT (group 1, from November 2013 until July 2017) or the day after last administration of PTCY (group 2, from August 2017 until December 2020). Results: The study included 131 patients (57 in group 1, 74 in group 2). The median age was 58 years (range, 15-74) and 78 (60%) patients were male. Patients in group 1 were younger (median age 53 versus 60 years, p = 0.007) and received a graft with a lower number of CD34 + cells (5.5x106/kg versus 7.2x106/kg, p = 0.015). The sequential conditioning regimen was the most used in group 1 (52.6%), whereas a reduced intensity conditioning was the most used in group 2 (47.3%) (p = 0.042). One hundred and twenty-six patients (96%) engrafted, with a median time for neutrophil recovery of 18 days (range, 9-30) in group 1 versus 17 days (range, 13-55) in group 2 (p = 0.016). At day + 180 after HCT, there was no difference in terms of incidence of grade II-IV acute GVHD (21% in group 1, 22% in group 2, p = 0.83) or grade III-IV acute GVHD (11% in group 1, 7% in group 2, p = 0.29) between the two groups. At 2 years, the incidence of chronic GVHD was also similar in both groups (32% in group 1, 21% in group 2, p = 0.21). With a median follow-up of 49 months (95% CI 48-59) for group 1 and 19 months (95% CI 15-27) for group 2, non-relapse mortality was 23% and 17%, relapse incidence 23% and 15 %, disease-free survival 56 % and 68%, overall survival 67% and 76%, GVHD-free, relapse-free survival 32% and 47% at 2 years in group 1 and group 2, respectively (p values are non-significant). In multivariable analysis, the timing of CsA initiation had no significant impact on survival outcomes and on the risk of acute or chronic GVHD. Conclusions: These results suggest that in haploidentical HCT with peripheral blood stem cells, and GVHD prophylaxis combining CsA, MMF, ATG and PTCY, CsA can be initiated either at day-3 before HCT or the day after the last administration of PTCY, without impacting the risk of GVHD or survival outcomes. Disclosure: nothing to declare
Background: Chronic graft-versus-host-disease (cGVHD) is one of the major complications after allogeneic hematopoietic stem cell transplantation (allo-HSCT) with an incidence of 40% to 70%. cGVHD is also the common cause of late non-relapse-related death in patients undergoing allo-HSCT, and seriously affects their quality of life. SHR0302 is a Janus kinase (JAK) 1 selective inhibitor that has demonstrated efficacy in preclinical models of GVHD. Herein, we reported the safety and efficacy of SHR0302 in combination with prednisone as first-line therapy for newly diagnosed cGVHD after allo-HSCT. Methods: This was a single-center, open-label, and phase I study. The study enrolled patients who had a confirmed diagnosis of first-episode moderate/severe cGVHD requiring systemic immunosuppressive therapy after allo-HSCT with an age limitation of 18-70. cGVHD was defined according to national institutes of health (NIH) criteria. Patients were treated with SHR0302 plus prednisone daily. For every patient, prednisone was administered at an initial dose of 1 mg/kg/d, and was tapered according to patient’s response after two weeks of treatment. Dose-escalation of SHR0302 was performed in a 3 + 3 design at doses of 1 mg/d, 2 mg/d, 4 mg/d, 6 mg/d, and 8 mg/d. Primary endpoints are safety and tolerability of SHR0302 and prednisone. Secondary endpoints include the overall response rate (ORR) at week 4 of treatment and the recommended Phase II dose (RP2D). Dose-limiting toxicities (DLTs) were defined as grade 4 hematologic toxicity or grade 3 non-hematologic toxicity related to SHR0302 that occurred in the first 28 days of study treatment. Results: As of December 1st, 2021, 15 patients were enrolled in 5 dose levels with 3 patients in every dose level. The median age was 48 (31-64) years, and the median follow-up was 17 (4 -59) weeks. 5 patients (33%) had moderate cGVHD, 10 patients (67%) had severe cGVHD, and the median cGVHD NIH score was 4 (3-7). As of data cutoff, only 1 patient (7%) had discontinued therapy due to lack of efficacy. Only one DLT, grade 4 hypercholesterolemia, was observed among 3 patients who received 8 mg/d of SHR0302. The patient who experienced DLT had preexisting hypercholesterolemia. 3 additional patients would be enrolled to receive 8 mg/d of SHR0302 plus prednisone to determine the maximum tolerated dose. 14 patients experienced adverse events (AEs) related to SHR0302 (93%), and 2 patients experienced grade ≥3 AEs related to SHR0302 (13%). The most common SHR0302-related AEs included hypercholesterolemia (67%), hypertriglyceridemia (33%) and platelet count decreased (27%). No SHR0302-related serious adverse event occurred. At 4 weeks after being treated with SHR0302 and prednisone, 3 patients (20%) achieved complete response, and 11 patients (73%) achieved partial response among 15 evaluable patients. The ORR at week 4 was 93%, just 1 patient showed no response to treatment. Conclusions: In summary, SHR0302 plus prednisone was well tolerated and demonstrated encouraging efficacy in patients with steroid-naive cGVHD, warranting continued clinical investigations. Clinical Trial Registry: NCT04146207 Disclosure: Nothing to declare.
Background: There is a need for new therapies to prevent and treat GvHD following allo-HSCT; however, contemporary evidence on the burden of the disease in France is not available. This study aimed to investigate the clinical outcomes, healthcare resource utilization (HCRU), and costs associated with GvHD in France, by type – acute (aGvHD), chronic (cGvHD), or both (a + cGvHD). Methods: A nationwide cohort study using administrative claims from the French Health Insurance database, SNDS, identified 6385 adult patients who received allo-HSCT for hematologic malignancies between January 2012 and December 2018. Relapse was explored using re-admission diagnoses and treatments. Propensity score matching was undertaken to compare patients who developed GvHD (by type) vs patients who did not develop GvHD (‘noGvHD’) for occurrence of severe infection during follow-up (defined using hospital discharge diagnosis codes), all-cause death, HCRU, and costs. Results: The mean age of the cohort was 51.1 years; 58% were male; 2668 (42%) patients had no recorded diagnosis code for GvHD, 2002 (31%) experienced an aGvHD episode, 411 (7%) had cGvHD, and 1304 (20%) had a+cGvHD. Patients with GvHD had slightly lower rates of relapse, with 276 (14%), 61 (15%), and 220 (17%) patients with evidence of relapse in the aGvHD, cGvHD and a+cGvHD subgroups, respectively, vs 486 (18%) patients in the noGvHD subgroup. For comparisons, 1934, 408, and 1268 matched pairs were retained for the aGvHD, cGvHD and a+cGvHD subgroups, respectively. Patients with aGvHD and a+cGvHD had an increased rate of hospitalization for severe infection, with a rate ratio (RR) (95% CI) of 1.32 (1.23-1.41) and 1.14 (1.05-1.24), respectively vs noGvHD; rate of severe infection was similar for patients with cGvHD vs without GvHD. Patients with aGvHD had an increased mortality rate, (RR [95% CI], 1.55 [1.41-1.70] vs noGvHD); mortality rate was slightly higher (although not statistically significant) for a+cGvHD vs noGvHD and similar between patients with cGvHD and those without GvHD. Patients with aGvHD and a+cGvHD had significantly more overnight hospitalizations per patient-year (mean rates: 4.3 vs 3.3 and 4.2 vs 3.2 admissions, respectively; p < 0.001) than those without GvHD. Total direct costs (including hospitalizations, outpatient visits, drugs dispensed) were 1.18, 1.53, and 1.89 times higher (p < 0.001) for patients with aGvHD, cGvHD, and a+cGvHD, respectively, vs noGvHD. Inpatient care (including drugs dispensed during hospitalization) cost was the primary driver of increased HCRU and costs. Conclusions: GvHD was associated with significant clinical and economic burden post-allo-HSCT. Patients with GvHD, and in particular, patients with aGvHD and a+cGvHD, had a higher rate of infection and higher mortality. This clinical burden translated into increased HCRU and costs, with patients with aGvHD, cGvHD, and a+cGvHD having a statistically significant higher total direct cost vs noGvHD patients. There is a continued need for effective prophylaxis and treatment options for GvHD, which could prevent clinical burden for patients, as well as the increased cost of allo-HSCT due to GvHD. Disclosure: David Michonneau reports consultancy for Novartis and Incyte, and honoraria from Jazz Pharmaceuticals. Nadia Quignot reports consulting fees paid to Certara contracted with CSL for implementing the analyses. Heng Jiang reports consulting fees paid to Certara contracted with CSL for implementing the analyses, and stock ownership from Certara. Dawn Reichenbach reports employment and stock options from CSL Behring. Maebh Kelly reports employment from CSL Behring. Anita Burrell reports consultancy for CSL Behring and Neumentum Xiang Zhang reports employment from CSL Behring. Kris Thiruvillakkat reports employment, stock options and salary from CSL Behring. Mohamad Mohty reports honoraria from Adaptive Biotechnologies, Amgen, Astellas, BMS, Celgene, Gilead, GSK, Janssen, Novartis, Oncopeptides, Pfizer, Sanofi, and Takeda; and research funding from Celgene, Janssen, and Sanofi.
Background: The use of post-transplant cyclophosphamide (PTCY) is becoming prevalent in alloHCT due to it efficacy for GVHD prevention. Intravenous cyclophosphamide-associated hyponatremia is an uncommon adverse effect attributed to an indirect inappropriate antidiuretic hormone release followed with a reduction in the ability of the kidney to excrete water. This study investigates the incidence and risk factors for hyponatremia in adults undergoing PTCY-based alloHCT. Methods: Between January 2018 and December 2020, 90 adults with hematological disorders underwent first alloHCT combined with PTCY-based GVHD prophylaxis at our Institution. Intravenous PTCY was administered at a dose of 50 mg/kg/day IV on day +3 and +4 at 9 am and followed by tacrolimus from day +5, alone; for matched related and unrelated donor alloHCT (n = 79, 87.8%), and combined with tacrolimus and mycophenolate when haploidentical donors were selected (n = 11, 12.2%). Patients received intravenous fluid therapy with glucosaline 5% 1L/8h and bicarbonate 1/6M 500ml/12h from day +2 to +4. Following our standard Institutional protocols, sodium level was routinely monitored two hours before and 12 hours after every dose of cyclophosphamide. Hyponatremia was defined as plasma sodium levels < 135 mEq/L), and severe hyponatremia was defined as plasma sodium levels < 125 mEq/L). Data was collected retrospectively and updated in November 2021. Risk factors for hyponatremia were explored using Regression Logistics Models. Results: Overall, the median age was 51 years, 40 (44.4%) patients were females, and 49 (54.4%) underwent MAC alloHCT. Hyponatremia was diagnosed in 80% of patients, and in 5.6% of the cases was severe. The majority of episodes were diagnosed 12h after the first dose of cyclophosphamide (73.3%). Four patients (4.4%) had symptomatic hyponatremia, and in all these cases the sodium levels were inferior to 125 mEq/L. Forty-one (45.6%) patients required specific treatment: all were started on oral sodium supplementation or fluid therapy with glucosaline 5% was replaced by normal saline. Hypertonic serum supplementation was given for symptomatic severe hyponatremia. The second dose of PTCY had to be reduced to 40 mg/kg in 1 patient and delayed in an additional one. Out of the overall 72 patients presenting with hyponatremia following PTCY, 57 (79.2%) successfully recovered in a median of 2 days from diagnosis. Hyponatremia did not result in non-reversible sequelae in any patient. Higher doses of cyclophosphamide (>5000mg and >7000 mg) were not associated with higher rates of hyponatremia (p = 0.32 and p = 0.346, respectively). Patient’s age (>60 years) (p = 0.205), sex (p = 0.0.601) and the conditioning regimen intensity (p = 0.249) were not found to be predictors for hyponatremia. Conclusions: This study reports for the first time the incidence of hyponatremia after PTCY administered for GVHD prevention. Hyponatremia was found to be a prevalent adverse effect after PTCY infusion, although the incidence of symptomatic hyponatremia was low. Based on these results, sodium levels after PTCY infusion should be carefully monitored, and hyperhydration using intravenous normal saline may be prioritized to decrease the rates of this complication. Disclosure: No conflict of interest to disclose.
Background: HLA matching is crucial to donor selection in allo-HSCT. HLA-DPB1 mismatches between donor and recipient are known to increase GVHD and can be unidirectional in the Graft-versus-Host or Host-versus-Graft direction. Recent data highlight the potential of host resident T-cells to cause cutaneous inflammation in xenograft models. In this study we sought to determine whether unidirectional HVG mismatches could cause clinical manifestations of GVHD. Methods: 183 patients transplanted at a single UK centre between 2013 and 2018 with 10/10 matched unrelated donor (MUD) PBSC transplants for malignant and non-malignant conditions were retrospectively scored for DP mismatch permissivity and directionality using the T-cell epitope (TCE) algorithm (https://www.ebi.ac.uk/ipd/imgt/hla/dpb.html), and compared with 71 12/12 sibling donor transplants (Sib) for onset of any acute-type GVHD within 6 months of transplant including classic acute, late onset and acute/chronic overlap. Conditioning regimens included MAC (n = 4), RIC (n = 175), Kroger (n = 15), FLAMSA (n = 38) and Seattle (n = 22) protocols. T cell depletion strategies included alemtuzumab (n = 180), ATG (n = 48) and T-replete (n = 26). Cumulative incidence of GVHD by DP mismatch status was adjusted for death from any cause as a competing risk. A multivariate regression was performed controlling for age, diagnosis, T-cell depletion, conditioning protocol and CMV status. Results: With death from any cause as a competing risk, there was a significant difference in GVHD incidence according to DP mismatch group (p < 0.001) (Figure). Multivariable hazard ratios were calculated demonstrating 12/12 MUD and 12/12 sibling transplants had the lowest GVHD incidence, then permissive DP mismatch, then non-permissive HVG with the highest incidence of GVHD in the non-permissive GVH group (Table). 12/12 MUD transplants demonstrated no significant increase in GVHD incidence compared to 12/12 sibling transplants. 6 month overall survival was adverse for HVG HLA-DPB1 mismatches compared to the other groups (p < 0.05). The only co-variate predicting GVHD by multivariate competing risks regression was the permissivity and directionality of DP mismatch (p = 3.2x10-9), with age, diagnosis, T-cell depletion, conditioning protocol and CMV risk group failing to reach significance. The maximal grade of GVHD reached in the HVG group was grade I in 40% and grade II in 60%, with no grade III or above GVHD in the HVG group. Conclusions: These data show that DP mismatching is the major cause of excess GVHD in patients receiving unrelated donor transplants in this cohort with mostly T-depleted transplant regimens. The surprising observation that HVG DPB1 mismatches are associated with a higher risk of GVHD supports recent findings that host resident T-cells may play a role in GVHD pathogenesis. The role of HVG reactions in the pathogenesis of GVHD or as a separate entity which is indistinguishable from clinical GVHD, requires further elucidation. Disclosure: Nothing to declare
Background: The gastrointestinal (GI) graft-versus host disease (GVHD) remains to be challenging and life-threatening complication of hematopoietic stem cell transplantation (HSCT) in children. Steroid based treatment of this condition remains a universal standard first-line therapy. Despite authorization of new agents for second-line therapy, steroid refractory GI GVHD awaits safer and more effective therapeutic approach. Methods: Clinical and laboratory data from 9 children (5 boys and 4 girls) aged from 1 to 17 years treated with anti-α4β7 integrin monoclonal antibody (vedolizumab) for severe (grade 3-4) steroid-refractory GI GVHD were analyzed. The diagnosis of GVHD was proven by biopsy in all cases. The patients and their parents gave informed consent for the off-label use of the medication and publication of the results. Results: The indications for HSCT were acute lymphoblastic leukemia (ALL) in 3 cases, acute myeloid leukemia in 4 cases, Diamond–Blackfan anemia (DBA) in 1 case and metachromatic leukodystrophy (MLD) in 1 case. HSCT from haploidentical family donors (haplo) were performed in all 7 cases of leukemia and HSCT from matched unrelated donor (MUD) were performed in DBA and MLD cases. Source of HSC were PBSC in cases of haplo and BM in cases of MUD. In all cases posttransplant cyclophosphamide (PtCy) on days+3, +4 at 50 mg/m2/day, CNI and MMF with unmanipulated HSCs was used as GVHD prevention approach. Grade III-IV GI GVHD occurred between day +25 to day +320 (median time day +100). In 5 cases we used vedolizumab as a third line therapy after unsuccessful treatment with methylprednisone and ruxolitinib, and in 4 cases we started vedolizumab as a fourth line therapy (after methylprednisone, ruxolitinib and tumor necrosis factor inhibitors). Median time of start of vedolizumab therapy was 14 days after manifestation of GI GVHD. In all cases we continued ruxolitinib course and performed withdrawal of steroids with slow gradual dose reduction during vedolizumab treatment. In 2 cases we combined the course of anti-α4β7 integrin antibodies and extracorporeal photopheresis. We use vedolizumab as intravenous infusion 6 mg/kg and repeated this dose weekly until resolution of GI GVHD symptoms. The number of injections varied from 1 to 4. Four patients received 2 injections, 3 patients -3, 1 patient – 1 and 1 – 4 injections. The median time of response was 33 days after start of vedolizumab treatment. Eight patients survived with complete resolution of GI GVHD symptoms. Six of them during vedolizumab treatment and two demanded additional lines of immunosuppressive drugs after discontinuation of vedolizumab. One patient died from GI GVHD associated complications. We did not see any significant side effects of vedolizumab in any case, but it was quite difficult to assess in this heavily pretreated group of patients. Conclusions: Targeted approach to the treatment of steroid-refractory grade 3-4 GI GVHD with the use of anti-α4β7 integrin monoclonal antibody (vedolizumab) can be promising curative option in pediatric HSCT patients. Further prospective evaluation of this approach is clearly warranted. Disclosure: Nothing to declare
Background: Recent studies describe protein biomarkers in peripheral blood associated to allogeneic-stem-cell transplantation (allo-SCT) outcomes. To date, two studies have focused on haploidentical allo-SCT with post-transplant cyclophosphamide (haplo-SCT). These works evaluate two and seven biomarkers - including ST2 and REG3α- in limited time-points (days + 15 and +30) but do not include the analysis of the MAGIC algorithm, validated with the combination of these 2 biomarkers as a predictor of higher Non Relapse Mortality (NRM). We are analysing ST2, REG3a and MAGIC algorithm early weekly after haplo-SCT and its association with Graft-versus-host disease (GVHD) and transplant outcomes. Methods: Prospective study with 151 consecutive patients who underwent haplo-SCT at University Hospital of Salamanca (2012-2020). The panel was analysed in serial serum samples collected on days 0, +3, +7, +14 and +21 in 81 patients. ST2 and REG3α serum concentration was established by Lumynex X-MAP, comparing the median luminescence levels between groups using Wilcoxon-Mann-Whitney test. Cut-off points for each cytokine were estimated using Cutoff-Finder application in R. MAGIC algorithm was determined from the ST2 and REG3α values considering a threshold of =0.16. Log-rank was used to compare survival curves. Multivariable analyses were carried out with Cox regression, including the most significant clinical variables. Results: Patients’ and transplant characteristics are shown in table 1: Table 1. Patients’ and transplant characteristics Higher levels of ST2 were associated with aGVHD (II-IV day +21, III-IV day +14), higher NRM 1-year (+0, +7, +14), lower OS 2-years (+7, +14) and GRFS 2-years (+14, +21). Higher levels of REG3α were associated with aGVHD III-IV ( + 14), higher NRM 1-year (0, 7, 14, 21), lower OS 2-years (0, +7, +14, +21) and lower GRFS-2y (+14). Similarly, the inclusion of MAGIC algorithm in multivariate analysis, distinguished two statistically significant risk groups for GRFS ( + 14), OS (0, +14) and NRM 1-year (0, +14) (Image 1). OS was independently associated with HCT-CI ≥ 3, ST2 (0, +7, 14, +21) and REG3α (0, +14, +21) levels, NRM with ST2 (0, +14), REG3α (14) levels and HCT-CI ≥ 3. ST2 ( + 7, +14, +21) REG3α ( +14) levels and HCT-CI ≥ 3 were the only variables independently associated to GRFS. Multivariate analysis with the MAGIC algorithm revealed and independent association to GRFS ( +14) and OS (0, +14) together with HCT-CI ≥ 3. Conclusions: These results confirm the prognostic role of ST2, REG3α and MAGIC algorithm in haplo-SCT outcomes in the largest single-centre cohort to date of a homogeneous series of haplo-SCT. We also demonstrate, for the first time, the MAGIC algorithm prognostic impact in haplo-SCT on day +14. Standarization in prospective and larger series is required before its incorporation into the clinical practice. Disclosure: Nothing to declare
Background: Pancreatic atrophy after allogeneic hematopoietic cell transplantation (HCT) is one of symptoms associated with chronic graft-versus-host disease (GVHD). Although pancreatic atrophy has been considered to cause exocrine insufficiency and weight loss, it remains to be elucidated what kind of recipients could recover their body weight (BW) or pancreatic thickness. In addition, the prognostic effect of pancreatic atrophy has not been clarified. Methods: We retrospectively analyzed 171 recipients who received allogeneic bone marrow transplantation or peripheral blood stem cell transplantation at Jichi Medical University Saitama Medical Center between January 2008 and December 2018. The measurement of pancreas was performed every year after their transplantation if the recipients received CT scan. Pancreatic thickness was defined as a sum of the widths which were perpendicular lines to the long axis of pancreas in the following three regions of pancreas: the head, body, and tail of pancreas. We evaluated them using the closest CT scan images to the time-points of 1, 2, 3, and 4 years after HCT. Pancreatic atrophy was defined as 20% or more loss of thickness. Results: Fifty-five recipients demonstrated pancreatic atrophy after HCT. While the BW of the recipients without pancreatic atrophy recovered gradually (P < 0.001), those with atrophy did not show that trend (P = 0.12) by linear mixed models. The 3-year simple cumulative incidence of pancreatic atrophy was 31.3%, while the 3-year current cumulative incidence of pancreatic atrophy, treating pancreatic atrophy as a reversible event, was 15.9%. Moderate and severe chronic GVHD tended to be slightly higher in the atrophy group (47.3% vs 37.9%), whereas these recipients tended to show the recovery of pancreatic thickness (30.8% vs 10.3%). HCT from female donor to male recipient showed superior pancreatic recovery than other donor and recipient sex combination. Although their pancreatic thickness seemed comparable between the recipients who continued and stopped immunosuppressant (IST) at one year (P = 0.87) and 2 years (P = 0.11) after HCT, that tended to decrease at 3 years (P = 0.064), and finally, the difference became significant at 4 years (P = 0.027).Pancreatic atrophy treated as a time-dependent covariate was significantly associated with inferior overall survival (OS) (HR 4.85, P < 0.001) and an increased risk of non-relapse mortality (NRM) (HR 5.38, P < 0.001), while it was not associated with disease relapse (HR 1.45, P = 0.47). Conclusions: The recipients with pancreatic atrophy did not tend to recover their BW after HCT, and those who could stop IST demonstrated the recovery of pancreatic thickness. Moderate and severe chronic GVHD was associated with both pancreatic atrophy and recovery, which indicated that pancreatic atrophy might be mainly developed as a manifestation of chronic GVHD and could be reversible if the allogeneic response was controlled. Moreover, pancreatic atrophy was significantly associated with an increased risk of NRM, leading to inferior OS. These results suggest the importance of monitoring pancreatic thickness after HCT. Further prospective investigations are warranted to clarify the significance of pancreatic atrophy on clinical outcomes. Disclosure: Nothing to declare.
Background: Home care has been associated with fewer infections, decreased acute GVHD, TRM, and improved survival in patients receiving allogeneic SCT (allo-SCT) (Gutiérrez-García et al., 2020; Svahn et al., 2008). The effect of previous autologous SCT (ASCT) on those advantages is unknown. We analyzed outcomes of home-cared allo-SCT patients with prior ASCT. Methods: Since 2015, consecutive adult patients with hematological malignancies undergoing at-home allo-SCT with prior ASCT were included and classified considering GVHD prophylaxis: post-transplant cyclophosphamide (PTCy) (group 1) or tacrolimus plus mofetil mycophenolate (TK/MMF) (group 2). Groups 3 and 4 included matched hospitalized patients to groups 1 and 2, respectively. Results: Since 2015, 56 patients pursued home-cared allo-SCT in our center and, of those, 15 had previously received an ASCT. Baseline characteristics were comparable between home-cared and the respective matched hospitalized series (table 1). Median follow-up was 1.4 years (0.6-3.3). Similar mucositis, renal failure, and haemorrhagic cystitis incidence and grade distribution were observed across the 4 groups. Home care did not provide benefit in neutropenic fever, pathogen detection, multidrug-resistant microorganisms, aspergillosis, or viral reactivation. Similar time to engraftment and length of stay were observed between matched at-home and inpatient groups. Graft rejection was not observed. Acute and chronic GVHD incidence and grade distribution were homogeneous except for comparisons between groups 2 and 4 (grade 3-4 acute GVHD cumulative incidence of 0% vs 40%, q = 0.003) (figure 1). No differences in relapse, TRM, PFS, or OS were observed. Table 1. Patient characteristics. 1Differences only observed between unmatched groups. oConsidering sample size, medians are given with their respective minimum and maximum values. Figure 1. Cumulative probability of grade 3-4 acute GVHD for home-cared patients vs. controls. Conclusions: Patients with previous ASCT can safely receive an allo-SCT in home care units regardless GVHD prophylaxis and without negative effect on toxicity or survival. Benefit in grade 3-4 acute GVHD incidence was observed in home-cared patients receiving TK/MMF compared with matched hospitalized patients. Other characteristic benefits associated with home care were not detected. Disclosure: M.G.R. received honoraria from Janssen and Takeda. L.G.R.L. received honoraria from Janssen and travel grants from Janssen and Amgen. A.C.P., C.J.V, A.M.R., C.G., P.A., T.S., M.S.L., C.M., L.R., M.Q.S., M. R., G.G.G. and F.F.A. have nothing to declare.
Background: A baseline total lymphocyte count (TLC) has been reported to correlate with the development of graft-versus-host disease (GvHD) in patients undergoing T-cell depleted (TCD) allogeneic hematopoietic cell transplant (allo-HCT) using antithymocyte globulin (ATG). We aimed to study the role of TLC and ATG on the development of GvHD and viral reactivations (VR) in patients undergoing ex vivo CD34 + selected allo-HCT. Methods: We retrospectively analyzed data of patients undergoing a CD34 + selected allo-HCT between 2016 and 2021 at our institution. All patients signed a written inform consent. The CD34 + selection allo-HCT platform consisted on ex vivo CD34 + selection plus in vivo TCD with ATG 2.5 mg/Kg/day (2 days in HLA matched donors and 3 days in HLA mismatch donors). Primary endpoint was to study the association of the ATG dose and TLC with the development of acute GvHD (aGvHD) and VR. Secondary endpoints were NRM, RI, PFS and OS. Prognostic variables were: age, conditioning regimen, ATG, TLC, type of donor, disease diagnosis, disease status, HCT-CI, VR (CMV, Adenovirus and EBV), CD3 + , CD19 + and CD16 + CD56 + (NK) cells and CMV status. Associations between ATG, TLC with the number of VR and GvHD were calculated using the logistic model. Survival outcomes were calculated using Kaplan Meier method. The Proportional Hazards model was used to calculate HR and risk associations. We used R-software 4.1.1 for statistical analysis. Results: A total of 51 patients were included, with complete data in 39 patients. The median follow up was 32.9 months (range, 20.2-42.3). The median dose of ATG per patient was 154.5 mg (range 121.5 – 189) and the mean of the TLC at allo-HCT was 161.5x10^6/L (SD ± 420.16).The cumulative incidence of aGvHD at 100 days was 15.8% (CI95%, 5.1%-25.2%). We did not observe any associations between the ATG dose and the TLC with GvHD. Interestingly, we observed a trend towards an increased risk of adenoviremia in those patients with higher ATG exposure (p = 0.07). Nevertheless, we did not observe any further association between ATG, TLC with the number of viral reactivations. The 2-year NRM was 26% (CI95%, 12%-37.7%), the 2-year RI was 22.5% (CI95%, 6.1%-36.1%), the 2-year PFS was 59.1% (CI95% 46.7%-74.8%) and the 2-year OS was 60.5% (CI95%, 48% -76.3%). RIC (p = 0.008) and older patient’s age (p = 0.031) were associated with higher NRM. In contrast, a lower NK cell count at 3 months was associated with lower NRM (p = 0.004). RIC allo-HCT was associated with lower OS (p = 0.005). In MVA, a lower NK cell count at 3 months was associated with lower NRM (HR = 0, CI95% 0-0,21, p = 0.030) and RIC was associated with lower OS (HR = 8.57, CI95% 1.6 - 45.78, p = 0.012). RI and PFS MVA did not identify any prognostic variables. Conclusions: In this homogeneous cohort of CD34 + selected allo-HCT, ATG and TLC were not associated with GvHD or VR. However, the use of a RIC was associated with a lower OS. Further studies with a larger number of patients are warranted. Disclosure: Guillermo Ortí Pascual: BMS, Incyte, Novartis, Pfizer.
Background: Higher HLA mismatch has been associated with worse clinical outcomes in the context of allogeneic stem cell transplantation (alloSCT). Traditionally, HLA disparity has been evaluated at the allele level. Recent in silico tools (PIRCHE and HLA-EMMA) are capable of assessing molecular mismatch (MM) differences. PIRCHE can predict the number of peptides presented by HLA molecules. On the other hand, HLA-EMMA compares HLA polymorphic amino acids between mismatches exposed on the surface of an HLA molecule. However, in the alloSCT the association of MM according to these tools and clinical outcomes has not been extensively investigated so far. The aim of this study is to analyze the relation between MM graft-versus-host disease (GvHD), relapse and early immune reconstitution. Methods: We conducted a retrospective analysis of patients who underwent alloSCT in our center between 2018 and 2020 comparing those who received HLA-identical with HLA mismatched transplants from both related and unrelated donors . High resolution HLA typing was used to analyzed HLA genes and MM was evaluated with both PIRCHE and HLA-EMMA softwares in GvH and host-versus-graft (HvG) directions. PIRCHE and HLA-EMMA scores were divided in low or high categories according to the analysis of receiver operating characteristic (ROC) curves. The data of early immune reconstitution included the levels of lymphocytes and monocytes on day 15, 30, 45, 60 and 90 after transplant. Results: 103 patients (10/10 HLA identical=67, HLA non-identical=36) had undergone alloSCT with a median age of 51 years (20-71). Patient characteristics are detailed in table 1. Median follow-up was 1.6 years. There were no differences in Overall Survival (OS) and Relapse-free survival at 3 years. Patients who developed acute GvHD obtained a significantly lower score in PIRCHE in class I in the HvG direction (p = 0.033). Non-relapsed patients presented significantly higher PIRCHE score (>5 MM) in HLA class I in HvG direction (p = 0.047). However, no differences were found in HLA-EMMA nor in GvH direction. Early immune reconstitution in patients with greater PIRCHE score (>5 MM) displayed higher levels of lymphocytes on day 60 after transplant in HvG direction (p = 0.024). Cumulative incidence of relapse was higher in >5 MM PIRCHE in this direction (p = 0,0558). Conclusions: HLA-MM in PIRCHE in the HvG direction is associated with a higher risk of acute GVHD and relapse and an improvement in early immune reconstitution. HLA molecular analysis could be useful for donor selection and management of patients post-transplant . The implications of HLA-MM according to these in silico tools in alloSCT outcomes deserve further investigation. Disclosure: No conflicts of interest to disclose
Background: Haploidentical (HI) hematopoietic stem cell transplant (HSCT) substantially expands the pool of available donors. However, a recent large registry study showed inferior outcomes of HI HSCT compared to MUD HSCT when both groups received identical post-transplant cyclophosphamide-based graft-versus-host-disease (GvHD). This was mainly due to higher rates of GvHD, underscoring the need for improved GvHD prophylaxis following HI HSCT. Abatacept (A) prevents T-cell co-stimulation by blocking the CD80I86-CD26 axis via its extracellular CTLA4 domain. Shortening the duration of tacrolimus may alleviate the burden of its unwanted effects. Methods: We therefore initiated a clinical trial for patients with hematological malignancies undergoing HI HSCT. Patients received mobilized peripheral blood grafts from first-degree HI related donors. GvHD prevention consisted of PTCy (50mg/kg IV on day +3 and +4), A (10mg/kg IV on day +5, +14 and +28) and tacrolimus (T) (starting on day +5 at 0.02mg/kg/day, by continuous IV). The dose of T was adjusted to maintain a trough level of 5-12 ng/mL. Tacrolimus taper was started on day +60 over a period of 4 weeks. Results: Since September 2020, 28 patients have been enrolled. Median age was 60 (18-73) years. There was 17 males and 11 females. Treated conditions included: AML (9), MDS (5), ALL (9), T-cell NHL (3), others (2). Disease risk index was intermediate in 18 and high in 9 patients. Nine patients had active disease at enrollment. Seventeen patients received myeloablative conditioning. CMV serology for recipients and donors were −/− (9), +/+ (14), −/+ (1,) and +/− (4). For the 27 patients already evaluable for engraftment, median time to ANC and platelet engraftment are 18 (14-30) and 27 (16-67) days. All 27 patients achieved full whole blood donor chimerism by day +30. Median follow-up was 6.6 months. Five patients developed grade II-IV acute GVHD and 2 patient developed grade III acute GvHD. There was no case of grade IV acute GvHD. Two patients developed chronic, 1 mild (skin only) and 1 severe (skin, eyes and oral cavity). There was no case of steroid-refractory GvHD. Only 3 patients did not complete the planned T taper by day +90. Six patients required systemic steroids. CMV reactivation rate was 33%. One patient had EBV reactivation and required preemptive therapy with 2 weekly rituximab doses. Seven patients developed BK reactivation. There were no cases of adenovirus or HHV-6 virus reactivation. Five patients developed transient renal insufficiency (4 in the setting of acute sepsis and 1 with thrombotic microangiopathy that resolved after tapering off T). One patient developed sinusoidal occlusive disease that resolved with therapy. Other toxicities included self-limiting rise in bilirubin. One patient with adult T-cell leukemia/lymphoma and 1 patient with ALL relapsed. All other patients remain disease-free. Conclusions: Our ongoing study suggests that CAST with abbreviated course of T is safe and yields low rates of acute GvHD. Based on a planned interim analysis, the study continues to enroll patients. The results of a larger cohort with longer follow-up will be presented at the meeting. Clinical Trial Registry: NCT04503616 Disclosure: A S Al-Homsi Advisory Board: BMS and Celyad Consultancy: Daiichi Sankyo
Background: Post transplant cyclophosphamide (ptCy) has been shown to improve outcomes of hematopoietic cell transplantation (HCT) from HLA mismatched related and unrelated donors. We analyzed retrospectively outomes of HCT from matched unrelated donors (MUD), mismached unrelated donors (MMUD) and haploidentical (HAPLO) donors using uniform GvHD prophylaxis with ptCy and compared these to a contemporary cohort of HCT from matched sibling donors (MSD) using standard prophylaxis. Methods: Data on transplants performed at the Institute of Hematology and Blood Transfusion in Prague were retrieved from local transplant databases. All patients signed informed consent with data collection and anonymous analysis. Conditioning was either myeloablative (Bu/Flu or Bu/Cy) or reduced intensity (Flu/Mel). GvHD prophylaxis consisted of PtCy, cyclosporine and mycophenolate in MUD, MMUD and HAPLO groups. Standard prophylaxis in MSD group was tacrolimus and mycophenolate. Kaplan-Meier survival estimates, Cox proportional hazard models and competing risk cumulative incidence estimates were calculated. Results: 218 patients were included in the analysis. Patient characteristics are summarised in Table. OS at 2 years was 82% (95% CI 73-92%) in MSD group and 83% (95% CI 74-93%), 82% (95% CI 72-94%) and 73% (95% CI 62-86%) in HAPLO, MMUD and MUD groups (P = 0.4). In multivariate analysis, only increasing age (HR 1.05, 95% CI 1.01-1.08 for each year) and commorbidities (HCT-CI > 1, HR 1.91 – 95% CI 1.09-3.33) were are associated with decreased overall survival (Figure). Non relapse mortality and incidence of relapse did not differ significantly between groups (P = 0.34) and were 10% and 27% for all patients, respectively. Acute GVHD grade III-IV was seen in 8% of patients in MSD group and 10%, 10% and 3.4% after HAPLO, MMUD and MUD HCT (P = 0.5). Chronic GVHD was seen in 69%, 62%, 64% and 31% of MSD, HAPLO, MMUD and MSD groups (P = 0.04). Conclusions: GvHD prophylaxis with ptCy led to equivalent outcomes of HCT from MUD, MMUD and haploidentical donors when compared to a cohort of transplants from matched sibling donors without ptCy in a retrospective, single center analysis. Table. Figure Disclosure: Nothing to declare
Background: Steroid-refractory graft versus host disease (SR-GvHD) remains a major complication leading to a high morbidity and mortality post allogeneic hematopoietic stem cell transplantation (allo-HSCT), despite significant advances in the last few years. Therefore, an effective way to identify SR-GvHD as early as possible could provide an opportunity to start second-line therapy at an early stage. In our study, a comprehensive immune profiling was performed to analyze the phenotype of cell subsets in different GvHD and healthy donor models. Machine learning was used to find critical factors from the huge multidimensional immune profiling dataset and to construct an accurate prediction model for SR-GvHD. Methods: Samples of ten patients without GvHD post allo-HSCT, eleven patients with steroid-sensitive aGvHD, 19 patients with SR-aGvHD ≥ II° and twelve with moderate to severe SR-cGvHD were included in this study. Glucksberg and NIH criteria were used for clinical staging of aGvHD and cGvHD. A comprehensive phenotypical analysis of monocytes, T cells, B cells, and NK cells was evaluated by multicolor flow cytometry. Unsupervised dimensional reduction and clustering algorithms were used to unearth the immunological and clinical data. Several different machine learning algorithms were used for the construction of prediction models. Results: A pipeline for the discovery of biomarkers using machine learning was established for the FACS data analysis. Several distinct disease-specific subsets of monocytes, T cells and NK cells were discovered by unsupervised analysis strategies, and were further validated by manual analysis strategy. Clinical parameters had no influence on these disease-specific subsets. Moreover, the SR-GvHD groups, showed significant higher expression of CD62L on T, NK cells compared to the other two groups, CD3+CD62L+ T cells (no aGvHD vs. SR-aGvHD: 14.75% vs. 54.78%, p < 0.001; steroid-sensitive aGvHD vs. SR-aGvHD: 18.03% vs. 54.78%, p < 0.001), CD56+CD62L+ NK cells (no aGvHD vs. SR-aGvHD: 15.96% vs. 60.46%, p < 0.001; steroid-sensitive aGvHD vs. SR-aGvHD: 18.52% vs. 60.46%, p < 0.001),suggesting its important role in SR-GvHD. The groups of no aGvHD post allo-HSCT and steroid-sensitive aGvHD were similar in their immune profiling. Furthermore, lasso regression and random forest algorithms were used to screen potential predictors for SR-aGvHD. A prediction model was constructed based on the CD62L expression of CD3 + T, CD4 + T, NK and B cells. The specificity and sensitivity of the model was examined by ROC analysis showing a good prediction with an AUC of 0.867. Conclusions: An effective algorithm for the definition of biomarkers was established using machine learning in our current study. The upregulation of CD62L on T and NK cells was defined as a robust predictor for SR-GvHD. Disclosure: The authors declare no competing financial interests, except the following: Funding was provided by Mallinckrodt to AS and MS for the documentation of the clinical course and for the analysis of patient immune cells; MS received funding for collaborative research from Apogenix, Hexal and Novartis and travel grants from Hexal and Kite; he received financial support for educational activities and conferences from bluebird bio, Kite and Novartis; he is a board member for MSD and (co-)PI of clinical trials of MSD, GSK, Kite and BMS. AS received travel grants from Hexal and Jazz Pharmaceuticals. MS and AS are co-founders and shareholders of TolerogenixX Ltd. AS and LW are part-time and full-time employees, respectively, of TolerogenixX Ltd.
Background: Acute graft-versus-host disease (GvHD) remains a major cause of morbidity and mortality after allogeneic hematopoietic cell transplantation (HCT). We aimed to investigate whether cytokine trajectories early post-HCT were associated with developing acute GvHD. Methods: We measured 25 cytokines by ELISA or Luminex in 254 stored plasma samples obtained around day +7 (N = 92), +14 (N = 77) and +28 (N = 85) in 116 patients who underwent HCT with myeloablative conditioning between 2015 and 2018 (median age: 47 [min–max: 18–71] years, 52% transplanted for acute leukemia, 71% with a matched unrelated donor, 25% received anti-thymocyte globulin [ATG] and 41% received 12 Gy total-body irradiation [TBI]). All 254 plasma samples were obtained before an eventual diagnosis of grade II–IV acute GvHD. Longitudinal cytokine levels were smoothed using polynomial loess regression with 95% confidence interval (CI) bands. For each cytokine, a joint survival model was fitted, in which longitudinal mixed-effects model estimates of the cytokine level were used in a proportional hazards spline model to estimate hazard ratios (HR, all per log increase) of grade II–IV acute GvHD (adjusted for donor type, receipt of ATG and receipt of 12 Gy TBI). Results: 35 (30%) patients developed grade II–IV acute GvHD at a median of 32 (Q1–Q3: 27–42) days after HCT. Stratified crude cytokine trajectories early post-HCT (Figure) revealed that patients who later developed acute GvHD had higher interferon-γ (IFNγ) levels from around day +20 to +35, higher interleukin-2-receptor-α (IL2Rα) levels from around day +10 to +35, a smaller decline in interleukin-6 (IL6) levels from around day +10 to +35, higher interleukin-17A (IL17) levels from around day +10 to +30, and more increasing tumor necrosis factor-α (TNFα) and suppression of tumorigenicity-2 (ST2) levels from around day +20 to +35. In the converging 22 joint survival models (the models for interleukin-1, interleukin-22, and E-selectin did not converge), we found that—after adjusting for donor type and receipt of ATG and TBI—higher levels of IL17 (HR: 1.22, CI: 1.00–1.49, p = 0.05) and lower levels of glycoprotein 130 (gp130, an interleukin-6 signal transducer, HR: 0.22, CI: 0.05–0.95, p = 0.04) and transforming growth factor-β1 (TGFβ1, HR: 0.37; CI: 0.15–0.94, p = 0.04) were associated with subsequent acute GvHD. Other cytokines associated with acute GvHD, albeit not with statistical significance at the 0.05 level, were TNFα (HR: 2.29, CI: 0.84–6.21, p = 0.11), IL2Rα (HR: 2.16, CI: 0.71–6.56, p = 0.17) and IL6 (HR: 1.40, CI: 0.95–2.07, p = 0.09). Conclusions: Crude early post-HCT trajectories of IFNγ, IL2Rα, IL6, IL17, TNFα and ST2 differed according to later development of grade II–IV acute GvHD in adults undergoing myeloablative HCT. After adjustment for donor type and receipt of ATG and TBI, high levels of IL17 and low levels of gp130 and TGFβ1 were associated with increased risk of acute GvHD. Our findings warrant further prognostic and mechanistic research of the candidate cytokine trajectories to determine their role in predicting and causing or preventing acute GvHD. Disclosure: Nothing to declare.
Background: Post transplant cyclophosphamide (ptCy) has been shown to improve outcomes of HLA mismatched hematopoietic stem cell transplantation (HCT). Role of ptCy in HLA matched unrelated donor HCT is less well established. We analyzed retrospectively outomes of transplantation from matched unrelated donors using ptCy based GVHD prophylaxis and compared these outcomes to standard ATG based prophylaxis. Methods: Data on transplants performed at the Institute of Hematology and Blood Transfusion in Prague and in Pilsen Teaching Hospital were retrieved from local transplant databases. All patients signed informed consent with data collection and anonymous analysis. Conditioning was either myeloablative (Bu/Flu or Bu/Cy) or reduced intensity (Flu/Mel). GvHD prophylaxis consisted of PtCy, cyclosporine and mycophenolate (ptCy group) or ATG, cyclosporine and either mycophenolate or methothrexate (ATG group). Kaplan-Meier survival estimates and competing event cumulative incidence curves were calculated and compared using log-rank and Gray’s tests. Results: 383 patients were included, 315 in the standard ATG group and 68 in the ptCy group. Patient characteristics are summarized in Table 1. OS at 2 years was 75,6% (95% CI 63-90%) and 72,6% (95% CI 65,8-80%) after ptCy and ATG, respectively (Figure 1A). Cumulative incidence of NRM within 2 years was 9% after ptCy and 19% after ATG (P = 0,023); incidence of relapse was 28% after ptCy and 38% after ATG (P = 0,089). Incidence of acute GVHD grade 1-2 was 35% and 28% (P = 0,56); incidence of grade 3-4 aGVHD was 3,5% and 7,5% (P = 0,28) in ptCy and ATG groups, respectively. Incidence of moderate and severe chronic GvHD was 4,2% after ptCy and 18% after ATG (P = 0,03 for moderate and 0,3 for severe cGVHD); mild cGVHD was seen in 17% (ptCy) and 25% (ATG) of cases. Table 1 Conclusions: GvHD prophylaxis with ptCy and ATG led to similar outcomes after allogeneic hematopoietic cell transplantation from matched unrelated donors in this retrospective analysis of data from two centers. Disclosure: Nothing to declare
Background: GVHD is the main cause of morbidity and mortality associated with allogeneic hematopoietic stem cell transplantation (HSCT) with an incidence rate of 40-60%. Addition of ATG has shown to reduce its incidence. We report the results of the prophylactic use of ATG in a group of patients (pts) undergoing HSCT. Methods: From March 2018 to June 2021 we conducted a prospective multicenter study including 95 consecutive pts.: 33 transplanted with an identical sibling donor(ISD), 33 with an HLA-matched unrelated donor(MUD) and 18 with an HLA 1 miss-matched donor (MMUD). Conditioning regimen was according to institutional protocol. GVHD prophylaxis included tacrolimus and methotrexate plus ATG (Timoglobulina®Sanofi) 2.25 mg/kg days -3 and -2. GVHD incidence, relapse and death were recorded. Acute GVHD (aGvHD) and chronicGVHD (cGVHD) were defined using Glucksberg criteria and NIH clinical grading respectively. Median and interquartile range (IQR) were used to describe non-parametric data, group comparison with X2, Kaplan Meyer curve and multi-sample log Rank test for survival analysis, considering a statistically significant p value of less than 0.05. Results: 42 pts were male and donors were female in 56% (n:54). Conditioning regimen was myeloablative in 81% (n:77). Median time to neutrophil and platelet engraftment were 17 days (range 15-21) and 17 days (range 12-20) respectively. 6/95 pts presented graft failure. aGVHD incidence was 36% (n:34), grades I-II 19% (n:19) and III-IV 16% (n:15). Incidence of grades III-IV did not varied according to donor: 15% (n:7) for ISD, 9% (n:3) for MUD and 27% (n:5) for MMUD (X2 3.06, p0.21). Overall incidence of cGVHD was 23% (n:21): Grades mild 6% (n:6), moderate 11% (n:11) and severe 4% (n:4). According to donor type, severe cGVHD was 2% (n:1), 3% (n:1) and 11% (n:2) using ISD, MUD and MMUD respectively (X2 2.65, p0.26). At one year, 15/79 pts (19%) were under immunosuppressive treatment. Transplant related mortality at day 100 was 9% (n:9). Most frequent cause was sepsis (n:4). In 31 pts CMV reactivation was detected (more than one reactivation in 6 pts of which 5 had GVHD) and 7 reactivated EBV infection. None evolved to PTLD. Median follow-up time was 524 days (IQR 168-833). cGVHD–free and relapse-free survival (combined endpoint) at 1 year was 57% (n:54) of 79 evaluables patients. cGVHD and relapse free survival at 1 year was 48% for IDS (33-63%CI), 66% for MUD (47-81%CI) and 38% for MMUD (21-60%CI). No significant difference was found according to donor type (X2 4.98; p0.083) Fig1 Conclusions: No difference was observed in the incidence of aGVHD, cGvHD and combined endpoint at 12 months between the different types of donor. This could suggest that ATG could equalize transplants results using different types of donor. To confirm these findings, studies with a higher level of evidence are required. Disclosure: Nothing to declare
Background: Since early studies of graft-versus-host disease (GVHD) (Sullivan et al., 1981 and Kennedy MS et al., 1985) no agent was randomized against corticosteroids (CS), but rather additional immunosuppressive agents in combination with CS were tested against CS alone. Nonetheless these early studies identified comparable efficacy of calcineurin inhibitor (CNI) cyclosporine A (CsA) to CS, which was subsequently abrogated by introduction of CNIs in GVHD prophylaxis. Recently several CNI-free protocols were introduced based on posttransplantation cyclophosphamide (PTCY) (NCT02294552, NCT02806375) and alpha/beta ex vivo depletion (NCT02337595). Under these protocols steroid-sparing regimens were evaluated. Here we report the outcome of first line GVHD treatment without CS in patients from these protocols. Methods: The study comprised 74 patients, 44% were allografted from matched related donor (MRD), 42% from matched unrelated donor (MUD) and 14% from haploidentical donor. Underlying disease was acute myeloid leukemia in 21%, acute lymphoblastic leukemia in 27%, primary myelofibrosis in 15%, chronic myeloid leukemia in 11% and other malignant diseases in 26%. Twenty patients were treated for acute GVHD (aGVHD) after single-agent PTCY, 23 were treated for chronic GVHD (cGVHD) after single-agent PTCY, 11 patients were treated for de novo cGVHD after PTCY, tacrolimus and MMF combination, 13 – after PTCY and ruxolitinib combination and 7 after ex vivo depletion. Overall 34 patients were treated for aGVHD (grade II – 44%, grade III- 47% and grade IV – 9%) and 40 for cGVHD (60% moderate and 40% severe by NIH criteria). CNIs were used as the first line treatment in 80% of patients, sirolimus in 10%, and TKIs, rituximab and ruxolitinib in another 10%. Results: Overall response rate (ORR) was significantly higher in chronic GVHD than in acute (80% vs 47%, p = 0.031). However the incidence of complete response (CR) was not different (43% vs 35%, p = 0.53). Median time to partial response (PR) was 35 days in aGVHD and 6 months in cGVHD. Median time to CR was 67 days in aGVHD and 16 months in cGVHD. 2-year failure-free survival was 21% in aGVHD and 81% in cGVHD (p < 0.001, figure A), while overall survival was 76% and 95% in these groups, respectively (p = 0.017). In refractory aGVHD the response to CS administration was 39%, and ORR to all lines in aGVHD was 94% (figure B). In refractory cGVHD 71% achieved a response after CS administration and ORR to all lines was 98%. Among aGVHD and cGVHD patients 47% and 63% did not receive systemic antibiotics, 79% and 90% systemic antimycotics, 35% and 73% systemic antivirals, 62% and 85% did not require hospital re-admission for complications or progressive disease during the course of treatment. GVHD severity in acute (HR 4.69, 95%CI 2.08-10.13, p < 0.001), in chronic disease (HR 5.0, 95%CI 0.96-26.22, p = 0.057) was predictive for response. Conclusions: An attempt to achieve a response without CS in the first line is justified for patients with cGVHD and grade II aGVHD in CNI-free patients after novel GVHD prophylaxis regimens. This approach does not compromise subsequent response to steroids or second-line agents and is associated with low incidence of complications and re-admissions. Clinical Trial Registry: NCT02294552, NCT02806375, NCT02337595 Disclosure: Nothing to disclose
Background: Pembrolizumab is safe and effective in persons with rrHL and has been used as a bridge to allo-SCT. There is concern, prior to allo-SCT pembrolizumab use might increase the risk of graft-versus-host disease (GvHD) and other IRAE [2-3]. Methods: Retrospective comparative cohort study,22 subjects included(Jan,2017 to July, 2019). 2 cohorts(prior to SCT pembrolizumab use,n-9), and 13 with no prior pembrolizumab. All received RIC SCT and PBSC grafts. aGvHD, cGvHD, CRS, reported, compared, and survival outcomes calculated. All had similar baseline characteristics. Subject-, disease- and transplant-related variables displayed in Table 1. Results: Median follow up was 9mo (range:7.7-44.5mo). The median time from the last dose of pembrolizumab to SCT 67 d (range, 42- 135d). All subjects (n-9) in pembrolizumab cohort achieved CR after SCT, compared to 84.6%(n-11) in the no pembrolizumab cohort; 100-day grade 2-4 aGvHD was 63.6% (n-14), more in pembrolizumab (89%,46%; P = 0.04); and 75%(9) vs 37.5%(3),P = 0.062) had grade 3-4.1-year cumulative incidence of moderate-severe cGvHD was 41%(n-9), more in pembrolizumab cohort (55.5% vs 30.7%; P = 0.378). GvHD was not influenced by any variable, but with a trend toward higher GvHD with prior pembrolizumab use (P = 0.074; P = 0.08). No VOD in either cohort, however, there was 1 subject with TAM, 2 CRS reported in pembrolizumab cohort, within 14 days post-SCT. All treated with steroids (1mg/kg) within 14 days with a quick benefit. 2- year OS, PFS for the entire cohort were 76.7%(95% CI:56.9-91.8) and 51.3%(95%CI: 29.8-72.6) respectively. No difference in survival outcomes in univariate analysis. However, when we analyzed subjects, who attained CR, or PR before SCT, 2-year OS 92.3 vs 33.3%; P = 0.05) and 2-year PFS 57.0% vs 33.3%; P = 0.03) respectively. 2-year CIR was 27.3%(n-6/22).When we analyzed subjects according to prior pembrolizumab use, no significant difference was evident between 2 cohorts (33.3%, 23.1%; P = 0.807). Taking into account subjects in CR at SCT, 2-year CIR was 14.9%, with no significant difference between 2 cohorts (46.15%, 36.51%; P = 0.93). 2-year NRM 18%(n-4/22) for the entire cohort; more in no pembrolizumab (11% vs 23%; P = 0.0093).In the pembrolizumab cohort, deaths were attributed to GvHD of the gut and lung(n-2), recurrent pneumonia(n-2), compared with lung cGvHD and DP (n-1), CMV colitis and DP (n-2) in the no pembrolizumab cohort. Conclusions: Pembrolizumab PRIOR to all-SCT results in favorable outcomes, enhanced PFS, in subjects who received SCT in CR, and decrease the risk of relapse, but at the cost of increased grade 2-4 aGvHD without increased NRM. Clinical Trial Registry: NA Disclosure: All authors has nothing to disclose
Background: Air-leak syndrome (ALS) occurs when there is leakage of gas from the alveoli resulting in clinical symptoms that include cough, dyspnea and hypoxemia. ALS is an independent poor prognosis factor among adult patients who have received hematopoietic stem cell transplantation (HSCT), which the 5-year overall survival (OS) is less than 30%. However, the clinical features of ALS among post-transplant pediatric patients have rarely been explored. Methods: We retrospectively reviewed 2,206 pediatric patients who received an allo-HSCT between January 2013 and December 2019 at the Hebei Yanda Lu Daopei Hospital and analyzed the role of ALS in their prognosis following HSCT. Results: Twenty-eight pediatric patients (16 male and 12 female) were diagnosed with ALS: 16 patients with acute myeloid leukemia, 9 patients with acute lymphoblastic leukemia, and 3 patients with aplastic anemia. Twenty-one of the patients received a complete remission status prior to HSCT and 7 patients were in non-remission. The median patient age was 12 years (1-16) and the follow-up time was 871 days (55-2,973). The median OS time was 429 days (55-1614). The most frequent adverse event accompanying the ALS was hypoxemia which occurred in 92.8% of the patients, followed by wheezing and coughing, which occurred in 67.9% and 53.5% of the patients, respectively. Chest pain related to breathing occurred in 21.4% of the patients. ALS types included mediastinal emphysema, subcutaneous emphysema, pneumothorax, scrotal emphysema, peritoneal meteorism, pneumopericardium, intestinal wall and intraspinal emphysema. Following treatment of the ALS, 18 patients survived (18/28, 64.3%) and 10 patients died of respiratory failure or infection (10/28, 35.7%). We divided ALS into two categories: 15 cases of bronchiolitis obliterans syndrome (BOS) and 13 cases of idiopathic pneumonia syndrome (IPS). Patients with BOS-related ALS had a better prognosis compared to those with IPS, who were more likely to die in the early stage of ALS (80% versus 46% in the IPS and BOS groups, respective; P = 0.037). Logical regression analysis showed that, greater than 7 years of age (P = 0.028), non-remission prior to HSCT (P = 0.022), use of tacrolimus (P = 0.005), pulmonary graft-versus-host disease (GVHD) before +163 days (P = 0.004), Grade III-IV acute GVHD (P = 0.001), extensive chronic GVHD (P <0.001), were independent risk factors for ALS. Primary GVHD before +23.5 days (P = 0.021) and IPS type (P = 0.037) were independent risk factors of poor prognosis following an ALS diagnosis. Disease state prior to HSCT, pulmonary infection, TBI pre-treatment, donor type did not have a statistically significant (P > 0.05) effect on patient survival. Additionally, we found that fluticasone, azithromycin, and montelukast (FAM) could significantly improve the prognosis following ALS in our patients (P = 0.005). Compared with IPS, patients with BOS appeared to benefit from imatinib (P = 0.055), ruxolitinib (P = 0.009), and pirfenidone (P = 0.044). Conclusions: ALS is a rare and poor prognosis complication of HSCT, particularly IPS-related ALS. The OS of ALS in our hospital is significantly higher than that cited in previous reports which may be related to early diagnosis and timely FAM treatment. Disclosure: Nothing to declare
Background: Antithymocytic globulin (ATG) and post-transplant cyclophosphamide (PTCy) are frequently used regimens for graft-versus-host disease (GVHD) prophylaxis. However, there is lack of data about the difference of regulatory T cell (Treg) subpopulation between these two regimens. Methods: We collected peripheral blood sample at day+21 after allogeneic hematopoietic stem cell transplantation (Allo-HSCT). We analyzed Treg subpopulation by flow-cytometer and classified Treg into 3 subgroups: naïve, effector and non-suppressive Treg. And, we compared overall survival (OS), the cumulative incidence of acute and chronic GVHD, relapse rate between ATG and PTCy group. Results: We enrolled 39 patients (25 in ATG, 14 in PTCy) in total. In ATG group, 9 and 16 patients underwent human leukocyte antigen (HLA) matched-sibling donor and unrelated donor HSCT, respectively. In PTCy group, 9 patients underwent haplo-identical HSCT and 5 patients underwent HLA-matched unrelated donor HSCT. The conventional CD25 + FOXP3 + Treg count of CD4 + T cell was 6.49% in ATG and 13.34% in PTCy (p = 0.0086). The naïve Treg count of CD4 + T cell was 4.87% in ATG and 5.95% in PTCy (p = 0.32). The effector Treg count of CD4 + T cell was 3.89% in ATG and 6.31% in PTCy (p = 0.16). The non-suppressive Treg count of CD4 + T cell was 23.67% in ATG and 19.00% in PTCy (p = 0.25). The cumulative incidence of Grade 2 to 4 acute GVHD was 16.2% in ATG and 36.5% in PTCy (p = 0.15) and extensive chronic GVHD was 19.1% in ATG and 16.7% in PTCy (p = 0.955) And, OS and relapse rate were not statistically different between two group. Conclusions: There were more conventional CD25 + FOXP3 + Tregs in PTCy group than in ATG group, which was a result of the fact that there were more naïve and effector Treg in PTCy group. PTCy showed similar clinical outcomes with ATG group, although there were more haplo-identical HSCT in PTCy group. This may be because there were more Tregs in the PTCy group, which effectively prevents GVHD. Disclosure: Nothing to declare
Background: Extracorporeal photopheresis (ECP) is frequently used in clinical practise to treat moderate to severe chronic Graft versus Host Disease (cGVHD), however there is limited data available to describe treatment patterns and outcomes associated with this treatment outside clinical trials. Methods: Patients ³18 years with a record of an allogeneic hematopoietic stem cell transplantation (HSCT) in the Swedish Patient Register between January 2006 and July 2020 were identified (n = 2708). Among these, patients with ECP treatment from 3 months post HSCT (index) and onwards were included (n = 183). The data was linked to other nationwide longitudinal and population-based registers; the Prescribed Drug Register, the Cause of Death Register, the Cancer Register and the Longitudinal Integrated Database for Health Insurance and Labor Market Studies (LISA) for follow-up until December 2020. The median follow-up time was 2.7 years. Results: The median age at index was 58 years (interquartile range [iqr] 1-3; 38-61), and 65.6% (n = 120) were male. The median time from index date to the first ECP exposure was 7.8 months (iqr 1-3; 3.0-19.0) and the median number of ECP treatments given was 18 (iqr 1-3; 4-39). The average time spent in healthcare decreased over follow-up time; 68.9%, and 22.1% the first and fifth follow-up year, respectively. During the 3-month period before the first ECP treatment, 90.0% (n = 165) received cyclosporine, 9.8% (n = 18) received tacrolimus and 3.8% (n = 7) received ruxolitinib, respectively. During the same period 52.5% (n = 96) received prednisolone and/or prednisone. When comparing the 3-month period prior to the first ECP treatment [reference] with 3-month periods during the first year after ECP initiation, there was a decrease in the cumulative dose dispensed by pharmacies per patient-time for prednisone/prednisolone (1,381 mg [reference] versus 658 mg [9-12 months post ECP initiation], p = <0.001) and cyclosporin (12,242 mg [reference] versus 3,501 mg ([9-12 months post ECP initiation] p < 0.001). The incidence rate of infections during the 3-month period prior to ECP initiation was 79.2% and decreased over time to 59.1% ([9-12 months post ECP initiation] p < 0.001). The median overall survival time from index was 6.0 years. The direct medical cost per patient-year decreased from 27,719 euro to 1,981 euro when comparing the first versus fifth follow-up year from index. Similarly, among patients < 66 years at index (n = 130), illness-related time absent from work decreased from 73.2% to 31.9%, with a corresponding decrease in productivity loss from 20,358 euro to 7,211 euro per patient-year. Conclusions: ECP treatment was associated with reduced use of corticosteroids, immunosuppressive agents, and fewer infections. Furthermore, cost and healthcare utilization decreased over time. Disclosure: Frida Schain is an employee and own stocks in Schain Research AB. Christina Jones in an employee of Schain Research AB. Constance Boissin, Tamas Laczik and Stefano Fedeli were interns at Schain Research AB and have received payments for analytical work. Mallinckrodt has been the sponsor of the study and Schain Research AB has received payments from Mallinckrodt.
Background: Steroid–resistant graft-versus-host disease (GvHD) is a major challenge after allogeneic stem cell transplantation and associated with significant morbidity and mortality There is no therapeutic standard defined beyond calcineurin inhibitors (CNI) and steroids. Pre-clinical evidence indicates the potent anti-inflammatory properties of the ruxolitinib and efficacy of ruxolitinib against GVHD have been described recently. Methods: In this retrospective study, 16 patients had received ruxolitinib as salvage therapy for corticosteroid–refractory chronic GVHD. All patients had moderate (9/16, 56.2%) to severe (7/16, 43.8%) cGVHD. The most frequent underlying diseases were: acute myeloid leukemia (8, 50%), acute lymphoblastic leukemia (5, 31.3%), Hodgkin’s disease (1, 6.3%), myelodysplastic syndrome (1, 6.3%), or T-cell lymphoma (1, 6.3%). All of the patients received myeloablative conditioning regimens. Grading of cGVHD and response (complete and partial organ based on clinician assessments) was performed Results: The median age was 48 years (range, 30-67). Median cGVHD time was 12 months (range: 2-44). Types of GVHD involvements: liver 7/16 (43.8%), lung 2/16 (12.5%), oral mucosa 1/11 (6.3%), oral mucosa and liver 1/11 (6.3%), skin 2/16 (12.5%), oral mucosa and skin 2/11 (12.6%), skin and gastrointestinal system 1/11 (6.3%). The median number of previous GVHD-therapies was 2.5 (range 1-5). The median dose administered was 20 mg (7 patients) and 10mg (9 patients) daily divided in two doses. Overall response rate was 75.1% (12/16) which was obtained after a median of 40 days of treatment, 43.8% (7/16) reached complete response, and 31.3% (5/16) partial response. cGVHD exacerbation developed in 4 patients during ruxolitinib therapy. Median follow-up and ruxolitinib-treatment was 15 months (range: 2-54). While 68.8% (11/16) of patients were alive, 30.2% (5/16) of patients died (2 of the 5 patients developed disease recurrence). Cytopenia, CMV-reactivation and infections were observed during ruxolitinib-treatment for cGVHD (respectively, 7/16, 43.9%; 6/16, 68.9%; and 11/16, 37.6%) Conclusions: Ruxolitinib in the real-life setting is an effective and safe treatment option for cGVHD, with an overall response rate of 75.1% for chronic graft-versus-host disease, in heavily pretreated patients. Disclosure: Nothing to declare
Background: Tacrolimus (TK) is a pivotal immunosuppressant agent used for GVHD prevention after allogeneic hematopoietic stem cell transplantation (allo-HSCT). TK is usually given intravenously (IV) in the early phase after allo-HSCT and then switched to twice-daily (BID: bis in die) oral administration. In addition to IV and BID TK formulations, once-daily modified-release (QD: quaque die) formulation is available. In solid organ transplantation, BID and QD formulations have been shown to have the same efficacy and safety profile. However, in alloHSCT there is little experience with the use of QD. We analyzed pharmacokinetics of the classic IV TK vs. the oral (BID and QD) formulations used from the beginning of transplantation. Methods: Two-hundred and thirty-eight patients were enrolled: 74 started immunosuppression with IV TK (Prograf®, 0.03mg/kg by continuous perfusion), 73 with BID (Tacni®, 0.6mg/kg/12h), and 91 with QD (Advagraf®, 0.12-0.18mg/kg/24h). The dose was subsequently modified in order to maintain a TK through level (C0) of 5-15ng/mL. Those patients receiving IV formulation were switched to either of the two oral formulations as soon as they could tolerate oral administration. For the purpose of this analysis, C0 at 48 hours, 7, 14, 21, and 28 days after d0 were recorded as well as acute GVHD and adverse effects of TK (acute kidney injury [AKI], thrombotic microangiopathy [TMA], and neurotoxicity within the first 3 months). Results: With the exception of the first 48h, only a small percentage of patients had sub-therapeutic TK levels (23% at 48h, 3% at 7d, 6% at 14d, 3% at 21d, and 3% at 28d). Additionally, despite close C0 monitoring and dose adjustments, a higher proportion of patients presented TK overdosing (>15ng/mL) at different timepoints after alloHCT (23% at 48h, 28% at 7d, 22% at 14d, 20% at 21d, and 26% at 28d) (Figure 1).At 48 hours, C0 levels were significantly higher with IV TK than with oral formulations, resulting in lower proportion of patients with C0 < 5ng/mL (IV TK 14% vs. BID 27%, p = 0.037; and vs. QD 28%, p = 0.027) and higher proportion with C0 > 15ng/mL (IV TK 23% vs. BID 11%, p = 0.027; and QD 11%, p = 0.042). Sub-therapeutic TK levels at 48 hours and at 7d were associated to a higher incidence of grade II-IV GVHD (31% vs. 21% at 48h, p = 0.088; and 8% vs. 0.6% at 7d, p = 0.001). AKI was observed in 180 (76%) patients (grade 1 n = 77, grade 2 n = 79, and grade 3 n = 24) with no significant differences between IV TK, BID, and QD. Ten (4%) and 14 (6%) patients developed TMA and neurotoxicity, respectively, with no differences between groups. Figure 1. TK C0 levels according to different TK formulations (IV TK, intravenous; BID, twice-daily oral; QD, once-daily modified-release). Conclusions: IV TK allows reaching therapeutic levels more quickly than oral formulations. Our results show that a quarter of the patients have supra-therapeutic TK levels regardless of the formulation used, and despite close monitoring. Considering that sub-therapeutic levels are associated with increased risk of GVHD, the use of TK IK subsequently switching to VO appears to be the best option. Disclosure: All authors declare no conflicts of interest.
Background: Defibrotide is an antithrombotic, anti-inflammatory, profibrinolytic and antiischemic drug composed predominantly of single-stranded polydeoxy ribonucleotides. It is mainly used to prevent and treat sinusoidal endothelial cell damage in sinusoidal obstruction syndrome (SOS) after allogeneic stem cell transplantation (Allo-SCT), with acceptable toxicity. As the data showing that defibrotide may have a protective effect on activated endothelial cells become widespread, and it has been shown in preclinical studies that defibrotide suppresses heparanase gene expression, which is thought to have a great effect on acute graft versus host disease (aGvHD) the role of this drug in aGvHD has also become evident, the hypothesis that defibrotide may have a role in aGVHD has been developed. In this study, we investigated the relationship between defibrotid and aGvHD based on this hypothesis. Methods: Between January 2014 and May 2021, 345 patients who underwent allogeneic stem cell transplantation (Allo-SCT) were included for this analysis in our center. In 116 (33.6%) of these patients, the risk of SOS was found to be high (busulfan will be used in the preparation regimen, gemtuzumab ozogamicin was used in the 3 months before the transplant, the abdominal region will be irradiated, in these patients with active hepatitis B or C infection, the preparation regimen will be started. Similarly, 25 mg/kg/dose prophylactic defibrotide was used in 4 equal doses for 21 days. Patients who used defibrotide for therapeutic purposes were not included in the study. The patients who underwent ASCT from a fully matched sibling donor, fully matched unrelated, or a 9/10 HLA-matched unrelated donor were included whereas cord blood transplants were not. Results: The female to male ratio was 143/202 and the median age was 43 (18-72). The distribution of ASCT indications according to defibrotide absent and present groups were as follows: acute myeloid leukemia(AML,n = 142) 88(62%)/54(38%), acute lymphoblastic leukemia(ALL,n = 64) 46(71,9 %)/18(28.1 %), myelodysplastic syndrome(MDS,n = 26) 17(65.4%)/9(34.6%), aplastic anemia(AA, n =18) 12(66.7%)/6(33.3%), mycosis fungoides(MF,n = 13) 9(69.2%)/4(30.8 %), chronic myeloid leukemia(CML,n = 13) 10(76.9%)/ 3(23.1%), primary myelofibrosis(PMF,n = 11) 6(54.5%)/5(45.5%), Hodgkin’s lymphoma(HL,n = 7) 6(85.7 %)/1(14.3), peripheral T cell lymphoma(PTCL,n = 7) 7(100%)/0 and other(n = 44) 28(63.6%)/16(36.4 %), respectively. The distribution did not differ statistically between the groups (p = 0,537). We performed a multivariate analysis to examine the effect of defibrotide on the risk of developing aGvHD and the degree of aGvHD, and observed that prophylactic defibrotide significantly reduced the risk of developing aGvHD (p = 0.001). Similarly, in patients who have developed aGvHD, total Glucksberg degree of aGvHD is attenuated with prophylactic defibrotide, too (p = 0,049). Conclusions: aGvHD is one of the life-threatening early complications of AKHN and is still the most important cause of non-recurrent mortality. Therefore, any approach that can reduce the risk and degree of aGvHH may be clinically important.To our knowledge, this is the largest study that evaluate the effectiveness of defirotide on GvHH, but prospective randomized controlled studies are warranted to verify our data. Disclosure: Nothing to declare
Background: Chronic Graft-versus-Host Disease (cGvHD) is the most serious late complication of allogeneic hematopoietic stem cell transplantation (allo-HSCT). Our aim was to capture the current incidence of cGvHD and late acute GvHD (laGvHD) based on the 2014 NIH consensus criteria and its impact on transplant related mortality (TRM), relapse (R), and overall survival (OS) within a multi-center analysis including transplant centers from Regensburg, Mannheim, Dresden, Vienna, Zagreb and Gdańsk. Methods: The analysis was performed on 317 consecutively transplanted patients, 296 adults and 21 pediatrics, who underwent first allo-HSCT in 2017. Endpoints were OS, TRM and R at last follow-up and second transplantation with the latter being censored. Patients with TRM or R before day 100 after allo-HSCT were excluded from cumulative incidence (CI) analysis of cGvHD. Results: CI of laGvHD were 9.5% (adults) and 4.8% (pediatric) (median d onset 137, range 100-415), while CI of cGvHD of the patient cohort at risk was 43.6% (adults) (median d onset 198, range 68-1051) in a median observation time of 397 days. The type of onset of cGvHD was de novo in 45 (41.3%), quiescent in 54 (49.5%) and progressive in 10 (9.2%) patients, respectively. In adults, the use of ATG (n = 137) or post-transplant cyclophosphamide (n = 62) as prophylactic agents led to a significantly (p < 0.01) lower incidence of cGvHD compared to standard prophylaxis (n = 116) (33.3% and 31.9% vs. 61.5%). By exclusion of the early mortality (d 100: TRM 7.6%, R 6.3%, OS 91.2%) and start of CI on day 100 of the remaining 269 patients at risk for cGvHD or laGvHD TRM was significantly higher in patients with aGvHD and cGvHD compared to no cGvHD (19.3% and 9.0% vs. 5.4%; p = 0.0036). OS was significantly higher in patients with cGvHD compared to patients without cGvHD (77.7% vs. 61.1%; logrank test p = 0.0006; HR 0.3396, 95%CI 0.1939-0.5945) since diagnosis of cGvHD resulted in a significant lower relapse rate compared to patients without cGvHD (17.9% vs. 32.6%; logrank test p < 0.0001, HR 0.216, 95%CI 0.115-0.405). We didn’t find a significant influence of onset type or maximum severity grade of cGvHD on TRM and R. We also found a significant better OS in patients with mild and moderate cGvHD compared to patients without cGvHD, but not for severe cGvHD forms (mild: 94.3%; moderate: 70.7%; severe: 55.6% vs. no cGvHD: 61.1%; p = 0.001; p = 0.0426 and p = 0.9645). OS for adults after diagnosis of cGvHD Conclusions: The analysis revealed an improved survival with mild and moderate GvHD compared to patients without cGvHD due to a reduced relapse rate. In contrast, severe cGvHD negatively affected OS and future aims should focus on prevention of severe forms to improve OS and quality of life. Interestingly, the prognosis of patients with cGvHD appeared to be better compared to past cohorts. Disclosure: Prof. Dr. med. Daniel Wolff received research funds from Novartis and honoraria from Amgen, Neovii, Novartis, Mallinckrodt, Incyte and Takeda. Radovan Vrhovac received honoraria from Novartis, Pfizer, Abbvie and MSD. Dr. Anita Lawitschka, MD received honoraria from Novartis. Dr. med. Jan Moritz Middeke received honoraria from advisory board Novartis. Univ.-Prof. Dr. med. univ. Greinix, Hildegard received honoraria for participation in advisory boards and speakers bureau from gilead, BMS/celgene, sanofi, takeda, Therakos. Prof. Dr. med. Ernst Holler received honoraria from Advisory board MEDAC, Maatpharma, pharmabiome and Novartis, speakers bureau Neovii.
Background: Acute graft versus host disease (aGVHD) remains one of the principal causes of nonrelapse mortality (NRM) following hematopoietic stem cell transplantation (HSCT). Systemic corticosteroids are often used as first-line therapy for aGVHD, but nearly 50% of the aGVHD patients are resistant to steroids. It has been reported that ruxolitinib combined with steroids was tolerable in patients with newly diagnosed aGVHD with improved overall response rate (ORR). However, the optimal predictor for predicting response to this novel first-line treatment has not been established clearly. Myeloid-derived suppressor cells (MDSC) are a group of myeloid cells with immunosuppressive activity, including granulocytic MDSCs (G-MDSC), monocytic MDSC (M-MDSC) and early-stage MDSC (e-MDSC). This study focused on the kinetics of MDSC during the treatment of ruxolitinib combined with steroids for aGVHD, and explored the relationship between MDSC and response to treatment. Methods: Peripheral blood (PB) samples from patients who underwent HSCT were prospectively obtained for regular evaluation of MDSC reconstitution after transplantation. Flow cytometry was utilized to monitor MDSC recovery at different time points. And in cases with aGVHD, MDSC frequency before and after aGVHD treatment (+3 days, +7 days, +14 days and +28 days) were also monitored. MDSC suppression assay was performed to verify the inhibition of MDSC on T cell proliferation through the co-culture of purified CD8 + T cell and MDSC isolated from the PB of patients. Results: Changes of G-MDSC kinetics observed during ruxolitinib-corticosteroids treatment are significant and showed an upward trend after initiation of therapy (P = 0.012). The ratio of G-MDSC to PB CD45 + cells on day 3 after aGVHD therapy was significantly higher than that of day 3 prior to aGVHD. In contrast to G-MDSC, M-MDSC showed a decreasing trend after aGVHD treatment (P = 0.004). Ratios of E-MDSC at different time points were also obviously different during aGVHD treatment (P = 0.010). The G-MDSC of the ruxolitinib-corticosteroids sensitive group increased significantly from baseline after treatment, while the G-MDSC of the combined treatment resistant group showed no noticeable change (Figure 1) . In the MDSC suppression assay, MDSC markedly inhibited T cell proliferation (proliferation index: no MDSC, 9.24 ± 8.10 vs. with MDSC, 5.45 ± 4.05; P = 0.037). Conclusions: MDSC recovery is closely related to the response to ruxolitinib-corticosteroids as first-Line therapy for newly diagnosed aGVHD. Patients with lower G-MDSC levels at baseline and 7-21 days after HSCT were more likely to develop aGVHD resistance to ruxolitinib combined with corticosteroids than controls. The kinetics of MDSC subpopulations may be used to predict the duration of response to this novel first-line treatment. Clinical Trial Registry: None Disclosure: Nothing to declare
Background: Severe digestive graft versus host disease (GVHD) is a topic of great importance after allogeneic Hematopoietic Stem Cell tTransplantation (allo-HSCT), since it is difficult to treat and the involvement of the digestive system is described in almost all cases of fatal acute GVHD. It is also known that the recruitment of inflammatory cells is dependent on bacterial translocations in the intestinal wall, leading to an increasing interest in factors that could take part in this process. Methods: We performed an observational and retrospective analysis in a single-center study, of all patients that underwent to allo-HSCT from January/2019 to March/2021 (N = 71) and analyzed all the possible pre-transplant conditions and during it, which could determine the development of digestive GVHD, such as, antibiotics guided by multi-resistant bacteria isolated in rectal exudates, clostridium difficile infection and mucositis. The primary outcome was the risk of developing upper and lower acute GVHD. We adjusted logistic regression model included transplantation characteristics, GVHD risk factors and adjunctive antibiotic exposures as covariates. Results: A total of 71 patients were included in the full cohort. There were no differences regarding the distribution by sex. The average age was 47. The main transplanted pathology was acute leukemia. The types of HSCT were: 56.3% (40/71) haploidentical, 24% (17/71) non related, 17% (12/71) HLA identical and 2 mismatch. The standard prophylaxis of GVHD was High Dose Cyclophosphamide on days + 3/+ 4 followed by anticalcineurinic and Mycophenolate mofetil from day +5. We used standard antibiotic prophylaxis with quinolones. Multi-resistant (MR) bacterias were discovered in 20% (13/64) of patients at admission date, and from the rest, 15% acquired resistance during hospital stay; no relationship was observed between colonization with MR bacteria and the development of digestive GVHD. Empirical antibiotic therapy in our center is with third generation cephalosporins (cefepime). 93% (66/71) suffered from febrile neutropenia and of these, 82% (54/66), were treated with cefepime combined or in monotherapy. 33% (21/66) used combined therapy, being the main group of antibiotics: glucopeptides (28), piperacilina tazobactam (15), carbapenems (20) and Ceftazidime-avibactam (4). None of them was associated, in adjusted models, with an increased risk of GVHD. Mucositis grades II-IV was present in 41% (29/71) of patients, having received 76% (22/29) of those, a myeloablative conditioning regimen. Clostridium difficile infection was discovered in 18% (13/71) of patients. 20 patients developed digestive GVHD: 30% (6/20) grades III-IV and 75% (15/20) grades II-IV, and we observed an association between the infection with clostridium difficile and an increased risk of GVHD (adjusted odds ratio (aOR) 6.33; 95% confidence interval (CI) 1.7 – 23.5), as well as with an increased risk of grade II-IV GVHD (not statistically significant). Conclusions: Clostridium difficile must be carefully studied in allo-HSCT patients and prevention strategies should be made, in order to control this entity; as it has been related to digestive GVHD according to the bibliography and as it has been shown in our study. No recommendations could be made about the antibiotic strategy and more studies should be performed in this way. Disclosure: Nothing to declare.
Background: Maternal and collateral donors were associated with higher incidence of acute graft-versus-host disease (aGvHD) after haploidentical hematopoietic stem cell transplantation (haplo-HSCT). Methods: To improve the efficiency of GvHD prophylaxis in haplo-HSCT with maternal and collateral donors, a novel regimen which was composed of low-dose ATG and low-dose PTCy combined with CsA and MMF had been developed in our center. We performed a retrospective study on 50 patients diagnosed with hematological malignancies after maternal/collateral haplo-HSCT (20 with ATG-based regimen for GvHD prophylaxis and 30 with the low-dose ATG/PTCy-based regimen). Results: The 180-day cumulative incidences (CIs) of grades II-IV and III-IV acute GvHD were 24.1% and 7.6% in the low-dose ATG/PTCy-based group, which were significantly lower than that in the ATG-based group (51.6% and 27.7%). In low-dose ATG/PTCy-based group, the 2-year probability of overall survival (OS) and relapse-free survival (RFS) were 70.9% and 71.1%, which were higher than that in ATG-based group with OS of 43.2% and RFS of 44.1%. Furthermore, according to multivariate analysis, the low-dose ATG/PTCy-based regimen significantly reduced the risk of grade II-IV (HR = 0.237, 95% CI 0.067-0.829; P = 0.024) and grade III-IV aGvHD (HR = 0.015, 95% CI 0.000-0.899; P = 0.044) as a positive risk factor. Conclusions: The results suggested that low-dose ATG with low-dose PTCy could be a novel regimen to effectively prevent acute GvHD after maternal/collateral donor transplantation. Disclosure: Nothing to declare.
Background: Graft-versus-host disease (GVHD) remains a major complication after allogeneic hematopoietic stem cell transplantation (HSCT). The diagnosis of chronic GVHD is complex, as it can potentially affect almost any organ/tissue1. Rare forms of GVHD have been described in the literature including neurological, myofascial and cardiac involvement among others2,3; to date, few reports have evaluated pancreas involvement in cGVHD. The major difficulty is to be suspicious in the face of the huge number of different clinical presentations of this postHSCT complication, as the one we show in our case report. Methods: We report a case of pancreatic atrophy as a manifestation of cGVHD. Results: A 42-year-old woman was diagnosed with AML in 2018 with NPM1 and FLT3/ITD + . Abnormal karyotype 47XX + mar, MLL negative. The patient had a morphologic complete remission after induction chemotherapy cytarabine and anthracycline, with positive minimal residual disease (MRD). It was followed by early consolidation with 2 cycles with high dose cytarabine and Midostaurin, achieving MRD negative before transplantation. She received and allogeneic peripheral blood HSCT from a matched unrelated female donor, with Cyclophosphamide and Busulfan (CyBu) conditioning regimen. Prophylaxis for GVHD with standard course of methotrexate and cyclosporine. She experienced acute grade II skin GVHD and diarrhoea that cleared after 2 weeks of prednisolone therapy. Six months after transplantation cyclosporine was discontinued. For the next three months she gradually presented with weight loss (4 kg), steatorrhea and abnormal liver function tests. Due to GVHD suspicion 0.5 mg/kg/day course of Prednisone was started. The stool examination was negative for infectious causes of diarrhoea. Faecal elastase level was 5 mcg/g (normal > 200mcg/g). Breath test with 13C-mixed triglycerides was 20.75% (normal > 29% exhaled). Magnetic resonance imaging (MRI) showed remarkable diffuse pancreatic atrophy. The clinical diagnosis was exocrine pancreatic insufficiency due to pancreatic atrophy, most likely as a form of cGVHD after allogenic stem cell transplantation. The patient received pancreatic enzyme supplement (Kreon 10.000 U, containing lipase 10.000U, protease 600U and amylase 8.000U) as required, starting on day +258, and nutritional support. After three months, steatorrhea was fully resolved, being able to taper prednisone without worsening, with progressive weight gain, stabilizing at her initial weight 6 months later. Despite the clinical improvement, the pancreas atrophy persisted in the following MRIs. She didn’t suffer GVHD relapse nor needed to restart immunosuppressive treatment at any point of the follow-up. Conclusions: There are few cases of pancreatic insufficiency after HSCT reported in the literature, being the most common form of presentation weight loss and steatorrhea. The etiology of pancreatic atrophy after HSCT is not fully known, although it shows a significant association with cGVHD, with greater loss of pancreatic glandular tissue than patients without cGVHD. In this population, the presence of gastrointestinal (GI) cGVHD is significant, suggesting that pancreatic atrophy might be a part of GI cGVHD. HSCT receptors that develop persistent fat malabsorption symptoms should be tested for exocrine pancreatic insufficiency, liable to be treated with oral enzyme supplements. All cases should be reported in order to better define and diagnose this rare entity. Disclosure: Nothing to declare.
Background: Allogeneic hematopoietic stem cell transplantation (HSCT) is associated with several complications, including chronic graft-versus-host disease (cGVHD) in 30-70% of HSCTs. Chronic GVHD has impact on quality of life and mortality, often requiring multiple therapeutic lines. Among available therapeutic options, methotrexate is an attractive one as it is a potentially low-cost, effective alternative associated with reduced toxicity. However, previous studies were relatively small or included heterogeneous groups of patients. The aim of this analysis was to assess the impact of methotrexate on the treatment of cGVHD in adult patients. Methods: Retrospective evaluation of electronic medical charts of patients. Eligible patients were those with diagnosis of cGVHD according to the 2014 NIH consensus criteria treated with methotrexate in second line or beyond between January 2014 and November 2020. Additionally, patients were required to have received at least 4 doses of methotrexate and have a minimum follow-up time of 3 months after starting this therapy. Best overall response (BOR) at 6 months was the primary endpoint, whereas secondary endpoints included failure-free survival, cumulative incidence of steroid withdrawal, overall survival, and toxicity. Results: Twenty-one patients receiving methotrexate for treatment of cGVHD were identified; two of whom were excluded for having less than 3 months of follow-up period. The analysis included 19 patients with a median follow-up of 18 months (range, 3 to71). The cumulative incidence of BOR at 6 months was 63% (11/19 patients), 16% of which (3/19 patients) corresponding to complete response, and 43% (8/19 patients) to partial response. Among patients with severe and moderate cGVHD, BOR were 37% (3/8 patients) and 45% (5/11 patients), respectively. The cumulative incidence of steroid discontinuation at 6 months was 60% (95% confidence interval [CI] 29-81%). Skin, mouth and liver were the most affected organs in evaluable patients; only two patients presented fascia/joint involvement. Failure-free survival at 6 months was 90% (95% CI 64-97%), and the overall survival at 1 year was 88% (95% CI 59-97%). Little methotrexate-attributable toxicity was observed, with only one case of renal toxicity and one case of liver toxicity, both at grade. One patient had disseminated adenoviral infection on methotrexate and other concomitant immunosuppressive drugs, which was not fatal. The median time between the start of methotrexate and BOR was 92 days (17-180, n = 11), and the maximum dose was 7.3 mg/m² (min-max3.2-13.4). Conclusions: Patients receiving methotrexate for the treatment of cGVHD had an overall response rate comparable to previous studies in the literature and showed favorable toxicity and tolerance profiles. These advantages combined with its low cost make this medication an interesting option in resource-constrained countries. Apart from its retrospective nature, this analysis has relevant limitations: lack of a comparative group, organ-specific responses, and a formal evaluation of cost-effectiveness. Studies defining the ideal dose of methotrexate in cGVHD and encompassing a larger number of participants are necessary for a more accurate assessment of the role of this medication in the treatment of cGVHD. Disclosure: Nothing to declare.
Background: Extracorporeal photopheresis (ECP) is an immunomodulatory therapy mainly indicated in cutaneous T lymphoma treatment, acute graft-versus-host disease (aGVHD) and chronic graft-versus-host disease (cGVHD) after allogeneic hematopoietic stem cell transplant (AHSCT), solid organ transplant rejection and conventional treatment resistant autoimmune disorders. Among its advantages, it stands out that it does not produce immunosuppression or drug interactions with complementary immunosuppressive treatment. Although it has been shown to be effective, safe and well tolerated, reported responses are highly variable. The objective of this study is to describe the experience in our center in the treatment of graft-versus-host disease (GVHD) with ECP. Methods: Medical histories of those patients who received treatment with ECP for both aGVHD and cGVHD in our center were retrospectively reviewed. The analysis period was from 2014 to 2021. Results: 28 patients were included in the study. The average age was 41,21 years (5-68). For all of them, 20/28 (71,42%) were men. Related to type of AHSCT, patients who received an unrelated donor transplant, HLA-matched sibling donor transplant and haploidentical donor transplant were 17/28 (60,71%), 8/28 (28,57%) and 3/28 (10,71%) respectively. The diagnosis that motivated the transplant was Acute Lymphoblastic Leukemias in 8/28 (28,57%), Acute Myeloblastic Leukemias in 8/28 (28,57%), Chronic Myeloproliferative Neoplasms in 4/28 (14,28%), Non-Hodgkin Lymphomas in 4/28 (14,28%), and other diagnoses in 4/28 (14,28%). All patients (28/28; 100%) received at least one adjuvant immunosuppressant treatment and 17/28 (60,71%) received three. Many patients (24/28; 85,71%) had cGVHD. Clinical involvement related to GVHD that patients presented most frequently was cutaneous (25/28; 89,28%) followed by digestive (12/28; 42,85%), joint (5/28; 17,85%), hepatic (4/28; 14,28%) and respiratory (2/28; 7,14%). Most patients (25/28, 89,28%) required the placement of a central venous catheter. Mean number of procedures performed was 26,53 (2-51). 3/28 (10,71%) patients performed more than one cycle of procedures. The response to ECP was complete, partial and non-response in 8/28 (28,57%), 12/28 (42,85%) and 8/28 (28,57%) respectively. Of the 5/28 (17,85%) patients who died, the cause was aGVHD in 2/5 (40%) and intercurrent infection was the cause in 3/5 (60%). No complications were detected with ECP except catheter-related infection. It was detected in 8 episodes (28,57%) that involve 7/28 (25%) patients. A patient died because of catheter-related infection. Conclusions: ECP is an immunomodulatory procedure, safe and excellently tolerated in patients with GVHD that offers the advantage of being able to be administered together with other immunosuppressive treatments without interactions. Two thirds of patients with GVHD experience a total or partial response so the treatment is effective and should be considered associated with immunosuppression. Disclosure: Nothing to declare
Background: ECP is an immunomodulatory therapy for T-cell mediated diseases. Although the immunological mechanisms are not fully understood, it is known that the induction of apoptosis is a central mechanism of ECP. Following ECP, the cytokine profile is modified toward upregulation of immunosuppressive factors and downregulation of co-stimulatory molecules. These immune responses can help attenuate inflammatory conditions such as GvHD. We performed a prospective analysis of apoptosis and cytokine secretion by flow cytometry in ECP treated cells of steroid refractory cGvHD patients. ECP was performed with the Amicus Blue™ online system (Fresenius Kabi, Germany). Methods: Seventy-five ECP procedures (n = 75) were performed in 3 female patients from May 2021 to December 2021. Samples (2mL) of the collected cells were taken before the addition of 8-methoxypsoralen (8-MOP) and after UVA photoactivation. The samples were re-suspended in 3mL of RPMI 1640 culture medium with 10% AB serum and incubated at 37°C in 5% CO2 for 24, 48, and 72 hours. Apoptosis measurement was performed by flow cytometry using the DxFlex flow cytometer (Beckman Coulter, USA) and the Beckman Coulter Annexin A5 FITC/7AAD kit (IM3614) according to the instructions, and with the additional use of reagents Beckman Coulter CD45-APC AF 750 (A79392) and CD3-APC (IM2467). The expression of two cytokines, TNFα and IFNy, was assessed by flow cytometry using the DxFlex flow cytometer on CD4 + T and CD8 + T cells before and after 24hr in the culture at 37°C in 5% CO2. The samples were either stimulated with CytoStim™ (Miltenyi Biotec), or with PBS (negative control), and incubated at 37°C and 5% CO2 for 2hr. Brefeldin A was added and incubated for 6hr, and fixation and permeabilization were performed. Staining was performed with monoclonal antibodies (Miltenyi Biotec): CD3-APC, CD4-Vio® Bright B515, CD8-VioGreen™, IFNγ-PE, TNFα-PE-Vio® 770 Fixed Dye and Viobility 405/452. Data were processed using Kaluza C Software v1.1 with a minimum of 150,000 acquired events. The one-way ANOVA test for paired samples was performed with GraphPad Prism v.8 (La Jolla, USA) and considered statistically significant for p < 0.05. Results: We used simultaneous staining with annexin V-FITC and 7ADD to measure apoptosis. After in vitro culture of ECP treated cells, 13% (5-20%) of CD3 + T cells were apoptotic at 48hr compared to 31.3% (23-46%) at 72hr, with only 30.3% (24-40.1%) viable cells (p = 0.03). Untreated cells maintained up to 80% viability after 72hr. The number of TNFα-secreting TCD8 cells was significantly higher in pre-ECP stimulated samples (p = 0.001). A decrease in T cells secreting IFNy was also observed in the post-ECP stimulated samples without statistical significance. TCD3 apoptosis induced by ECP and (B) Intracellular flow cytometric enumeration of IFNу/TNFα secreting CD4, CD8-T cells Conclusions: Our results for ECP-induced apoptosis at 72hr with the Amicus Blue ECP system were comparable to published literature. Reduction of TNFα after ECP treatment may have a direct role in reducing the progress of cGvHD. ECP performed with the Amicus Blue system resulted in a reduction of TNFα and IFNу levels in the treated cells, as expected. Disclosure: Nothing to declare
Background: Anti-thymocyte globulin (ATG, Grafalon) is a mix of polyclonal rabbit anti T-cell antibodies used to reduce the risk of graft rejection and acute and chronic graft-versus-host disease (GVHD) in the setting of an allogeneic hematopoietic stem cell transplantation (alloHSCT). The aim of this study was to find out the cumulative incidence (CI) of GVHD while using ATG at our institution, and to compare this incidence among various patient subgroups. Methods: From all the patients who underwent their first alloHSCT at our department between 2006 and 2020 we excluded those who did not receive ATG. We studied the cumulative incidence and severity of acute (100-day CI) and chronic GVHD (24-month CI), and differences depending on HLA matching, conditioning regimen intensity, and the underlying disease. Results: We identified 481 patients in the defined time period. Eighty-three of them were excluded for not having received ATG due to heterogeneous reasons (e.g. obsolete conditioning regimens, non-malignant diseases, bone marrow stem cell source, high risk of relapse), making this subgroup unevaluable. A total number of 398 patients were included into this analysis, 203 with acute myeloid leukemia (AML) or myelodysplastic syndrome (MDS), 69 with acute lymphoblastic leukemia (ALL), 44 with lymphoma, 40 with myeloproliferative neoplasms, 30 with chronic lymphocytic leukemia (CLL), and 12 others. Acute GVHD was observed in 44.7% patients (grade III and IV in 11.6%), chronic GVHD in 37.6% (extensive in 7.1%). Incidence of GVHD using an unrelated donor was higher than in related donor HSCT (acute GVHD 49.0% vs. 34.8%, respectively, p = 0.01; chronic GVHD 40.5% vs. 30.6%, respectively, p = 0.02). Higher incidence of both acute and chronic GVHD was observed in the case of HLA mismatch (53.0%, 44.6%, respectively) compared with the group of fully matched (10 out of 10) unrelated donor HSCT (45.1%, 34.9%, respectively). Myeloablative conditioning was administered in 27.6%, and reduced intensity in 72.4% patients. Incidence of acute GVHD was higher among the patients who received reduced intensity conditioning than in the subgroup with myeloablative conditioning regimen (48.9% vs. 34.0%; p = 0.02). The same was observed for chronic GVHD (42.0% vs. 26.0%; p˂0.01). Incidence of GVHD was also higher in myeloid malignancies (statistically insignificant), but this can be explained by more frequent use of reduced intensity conditioning in this subgroup. Conclusions: With the routine use of ATG the incidence of severe grades (III to IV) acute and chronic GVHD was as low as 11.6% and 7.1%, respectively. There were significant differences in the incidence according to conditioning regimen intensity and HLA matching. Disclosure: Nothing to declare.
Background: Graft-versus-host disease (GVHD) is the major cause of non-relapse mortality (NRM) after allogeneic hematopoietic stem-cell transplantation (HCT). The response rate to first-line corticosteroid therapy for aGVHD patients is approximately 50%. The overall survival of steroid-refractory aGVHD patients is only approximately 30%. A new first-line therapy to improve the efficacy of treatment for aGVHD is of great significance. Methods: We performed a prospective, open-label trail (NCT04061876) in my transplantation center and prospectively observed the kinetic characteristics of lymphocyte subsets and MDSC were monitored, and then we compared them in steroids-ruxolitinib group (n = 23), free-aGVHD group (n = 20) and steroids group (n = 23). Healthy human PBMC was treated with different concentrations of JAK 1/2 inhibitor (Ruxolitinib) to detect the effects on differentiation and function of MDSC. Results: In all aGVHD patients treated with Steroids-Ruxolitinib therapy, the Day 28 CR rate was 78.26% (18/23). On day 28 after treatment, patients had lower level of CD4+CD29+ T cells (P = 0.08) than that of pre-treatment, whereas levels of other lymphocyte subsets in this study were higher than that of pre-treatment; CD8+CD28- T cells (P = 0.03) significantly increased in patients with aGVHD than that in patients without aGVHD, so did CD8+CD28- T/CD8+CD28+ T cell ratio (P = 0.03). Compared with patients without aGVHD, patients with aGVHD had lower level of G-MDSC, especially on day 14 after allo-HSCT (P = 0.04). Compared with pre-treatment, M-MDSC was higher in CR patients on day 3 and 7 post-treatment(P3 = 0.01, P7 = 0.03), e-MDSC was higher on day 28 post treatment (P = 0.01). Moreover, compared with CR patients, M-MDSC was lower in refractory aGVHD patients on day 3 post-treatment (P = 0.01) and e-MDSC was lower on day 28 post-treatment (P = 0.01). Compared with steroids group, MDSC in steroids-ruxolitinib group was higher, with the most significant difference in M-MDSC (P3 = 0.03; P7 = 0.01; P14 = 0.04). In the vitro differentiation model of peripheral blood mononuclear cell (PBMC), JAK 1/ 2 inhibitors can promote MDSC and Treg most (Pe-MDSC = 0.01; PG-MDSC = 0.05; PM-MDSC = 0.02; PTreg = 0.04), at 1.0 μM JAK 1/2 inhibitor (Ruxolitinib) treated for 72 h; We also found JAK 1/ 2 inhibitor can increase the expression of S100A8(P = 0.004), S100A9 (P = 0.04) and NO(P = 0.03), at a concentration of 1.0 μM. Conclusions: The response rate of the novel first-line therapy, Steroids-Ruxolitinib, for aGVHD was promising in patients with moderate and high "Minnesota and MAGIC" risk. Moreover, the novel first-line therapy has a small impact on the immune reconstitution of patients after allo-HSCT. Patients with a better response with novel aGVHD first line therapy have higher level of MDSCs, compared to refractory ones. JAK 1/2 inhibitor can promote the differentiation and function of MDSCs. Disclosure: This work was partially supported by grants from the National Natural Science Foundation of China (Nos. 82070178, 81770203,81700122, 81270610), Military Translational Medicine Fund of Chinese PLA General Hospital (ZH19003). Medical big data and artificial intelligence development fund of Chinese PLA General Hospital (2019MBD-016, 2019MBD-008). Military medical support innovation and generate special program(21WQ034). Special Research Found for health protection(21BJZ30).
Background: Pathogenesis of chronic graft-versus-host disease (cGVHD) is incompletely defined. Standard treatment is lacking for steroid-dependent/refractory cases; Recent insights suggest that several players and different pathways are involved, including imbalance of T and B cells and exaggerated collagen production. therefore, the potential usefulness of tyrosine kinase inhibitors (TKIs) has been suggested, based on their potent antifibrotic effect. Methods: We performed in our clinic including 11 patients with refractory cGVHD, given imatinib at a starting dose of 100 mg per day. All patients had active cGVHD with measurable involvement of skin or other districts and had previously failed at least 2 treatment lines. The most frequent underlying diseases were: acute myeloid leukemia (7/11, 63.6%), acute lymphoblastic leukemia (3/11, 27.3%), and chronic myeloid leukemia (1/11, 9.1%). All of the patients received myeloablative conditioning regimens. Grading of cGVHD and response (complete and partial organ based on clinician assessments) was performed Results: Patient median age was 42 years (range, 30-57 years), and median duration of cGVHD was 24 months (range, 2-300 months). All patients had moderate (37/11, (63.6%) to severe (4/11, 36.4%) cGVHD. Patients developed sclerotic skin lesions (8/11, 72.1%), lichen planus-like lesions (1/11, 9.1%), gastrointestinal and sclerotic skin lesions (1/11, 9.1%), liver and skin lesions (1/11, 9.1%). The median number of previous GVHD-therapies was 2 (range 2-5). Median imatinib treatment was 54 months (range: 3-108). Median follow-up was 77 months (range: 3-107). Overall 10 patients achieved a partial response and 1 patient complete response (CR) at 6 months of treatment. 3 of the patients responding as a partial response in the first year turned into a complete response during their follow-up. 2 patients discontinued imatinib therapy at year 4 of CR. They have been followed for a year without cGVHD treatment. While 1 patient responding as a partial response discontinued imatinib treatment after two years, another 1 patient discontinued imatinib treatment after three years. However, these two patients stopped treatment by themselves and one has been followed for 2 years and the other for 6 years in partial remission. 1 patient responding as a partial response in the first year turned into a flare after two years of imatinib treatment. All of the patients included in the study are still alive and 6 of the patients are still in a partial response. Of those patients responding to imatinib, the rate of GVHD-relapse was 9% (1/11) for fibrotic cGVHD. Imatinib-related, grade 3 to 4 toxicity included fluid retention, infections, and anemia were observed during the cGVHD treatment (respectively 2/11, 18.2%; 1/11, 9.1%; and 1/11, 9.1%). Conclusions: Our findings suggest that imatinib is a promising treatment for patients with refractory fibrotic cGVHD Disclosure: Nothing to declare
Background: Acute graft-versus-host disease (aGVHD) is a major cause of morbidity and mortality after hematopoietic stem cell transplant (HSCT). High dose steroids remain the standard of care as first-line treatment for severe aGVHD, however less than 40% of patients show a long durable remission following such approach. Methods: We retrospectively analyzed the outcome of 23 consecutive patients who developed grade 3-4 aGVHD and received an escalating combinatory first-line treatment at our institution. Results: Donor was matched sibling (n = 1), matched unrelated (n = 12) or haploidentical (n = 10). Stem cell source was PBSC in 90% of the patients. Conditioning was myeloablative (MAC) or reduced-intensity (RIC) in 35% and 65% of the patients, respectively. aGVHD involved skin, GI, and liver in 87%, 83% and 17% of the patients, respectively. All patients received prednisone or methylprednisolone at a dose > = 1 mg/kg as first treatment. Response was evaluated at day 3 and 5 since start of steroids. In patients with symptoms progression at day 3 or stable disease at day 5 extracorporeal photopheresis (ECP) was added (n = 12 patients). In all patients with stage > =2 lower GI involvement, Ruxolitinib 10 mg twice daily was added within 5 days since start of steroids (n = 7 patients). ECP schedule included two treatments per week for 8 weeks with a subsequent individual reduction of treatment frequency. Response to first line treatment was evaluated at day 28 and day 56. In patients treated with steroids + ECP the overall response rate (ORR) at day 28 was 75% (n = 9), with 58% (n = 7) achieving CR. In patients treated with steroids + ECP + Ruxolitinib the ORR was 86% (n = 6), with 67% (n = 4) achieving CR (Figure 1). The durable ORR at day 56 was 58% (n = 7/12) in patients treated with steroids + ECP and 71% (n = 5/7) in patients treated with steroids + ECP + ruxolitinib. Median time on steroids was 12 days. ECP was well tolerated; the only adverse event recorded was mild thrombocitopenia in 4 patients. 5 patients receiving ruxolitinib experienced grade 2 anemia or thrombocytopenia, the latter leading to drug discontinuation in 2 patients. 13 patients received ECP and 3 patients received Ruxolitinib as second line treatment for steroid refractory or steroid dependent GVHD. 2 patients with lower GI involvement received vedolizumab as second line therapy. Median time to second line treatment was 24 days. 5 patients died; 4 due to infectious complications secondary to refractory GVHD, 1 of pulmonary embolism with GVHD in complete response. Median overall survival of the global population was 20 months. Conclusions: In conclusion, an escalating combinatory first-line treatment with a backbone of steroids and ECP, and the addition of Ruxolitinb in patients with lower GI involvement seems feasible and associated with a promising response rate, allowing a rapid steroid taper. Disclosure: Nothing to declare
Background: Steroid-refractory acute Graft-versus-Host-Disease (SR-aGvHD) after allogenic hematopoietic stem cell transplantation (allo-HSCT) is challenging and associated with high morbidity and mortality. So far, no standard second-line therapy for SR-aGvHD has been established. Response rates of second-line immunosuppressive drugs are often disappointing. Also toxicity and especially infective complications are common issues. Alpha 1 antitrypsin (AAT1) is a serine protease inhibitor with antiapoptotic, anti-inflammatory and immunomodulatory effects, which showed efficacy in animal models on inflammatory disorders and autoimmune diseases, as well as promising results in a prospective clinical trial as second-line therapy for SR-GvHD. Safety of long-term AAT1 substitution in patients with inherited AAT deficiency without increased rates of infection was previously shown. Methods: In a single center retrospective analysis we investigated the use of AAT1 in patients with grade III-IV SR-aGvHD of the gut. Steroid-refractory was defined by missing clinical benefit after the administration of 2 mg /kg prednisolone over at least 5 days. All patients had previously received at least one off-label second-line treatment for SR-aGvHD. AAT1 was administered intravenously twice a week for 4 weeks with 60 mg/kg. We analysed overall response rate (ORR), drug related toxicity and overall survival (OS). The response was assessed clinically using MAGIC criteria for aGvHD. Results: We retrospectively analysed 14 patients with a median age of 53 (36-69) years treated for SR-aGvHD after allo-HSCT at our center. 4 patients had grade III aGvHD and 10 patients had grade IV GvHD with at least grade III gut involvement in all 14 patients. Biopsies were performed to confirm a histological GvHD. One patient received a bone marrow graft while the other 13 patients received peripheral blood stem cell grafts. 12 patients had matched unrelated donors (MUD) and 2 patients had haploidentical sibling donors. GvHD prophylaxis after allo-HSCT consisted of anti-thymocyte globulin (ATG), ciclosporin A and mycophenolatmofetil (MMF) for MUD and post-transplant cyclophosphamide, tacrolimus and MMF for haploidentical donors. In median, patients previously received 3 (1-6) lines of GvHD treatment before AAT1 administration, including ruxolitinib, methotrexate, calcineurininhibitors, etanercept, extracorporal photopheresis and mesenchymal stromal cells. 12 patients received all 8 doses of AAT1. One patient died before treatment completion due to infective complications. One other patient even received 2 cycles of AAT1 (16 doses) after responding to the first cycle and relapsing 5 weeks after administering the 8th dose. GvHD manifestations improved in 8 of 14 (57%) patients by day 28 after first dose of AAT1. 3 patients achieved complete remission (CR) and 5 achieved partial remission (PR). AAT1 was well-tolerated and no toxicities attributable to AAT1 were observed. At the last follow-up 5 of the 14 patients were alive with a follow-up of up to 77 weeks after allo-HSCT. Causes of death were infections in 5 patients and progression of GvHD in 4 patients. Conclusions: In this retrospective analysis, AAT1 showed good efficacy in heavily pre-treated patients with SR-GvHD and gut involvement with an ORR of 57% and a CR rate of 21% while having a well-tolerable safety profile. Clinical Trial Registry: Disclosure: Nothing to declare
Background: Bone marrow-derived mesenchymal stromal cells (MSCs) are one of the salvage therapy options for steroid-refractory acute graft versus host disease (SR-aGVHD). Herein we present 8 years of experience in Vilnius University Hospital Santaros Klinikos. Methods: All patients developed biopsy-proven grade III-IV SR-aGVHD after allogeneic hematopoietic stem cell transplantation (HSCT) or donor lymphocyte infusion (DLI). SR-aGVHD was managed with bone marrow-derived MSCs manufactured in a hospital setting. MSCs target dose was 1x10e6 cells/kg body weight administered intravenously once weekly. Treatment response was evaluated on day 7, day 14, and day 28. The patients were evaluated for overall survival. The data were collected prospectively. Results: 45 adult SR-aGVHD (Grade III-41 and Grade IV-4) patients, with a median of 54 (19-66) years, received MSCs as salvage treatment. 34 patients (76%) were transplanted due to acute leukemia. Involvement of the gastrointestinal tract, skin, and liver were 93%, 44%, and 29 %, respectively. One organ was affected in 18 (40%) (gastrointestinal involvement in 94 % of the cases), two organs in 24 (53%), and three organs in 3 (7%) patients. The median time from HSCT/DLI to MSCs treatment was 11 (2-217) days and the median dose was 1x10e6/kg (0,68-1,33). 19 patients received ≤ 3 MSC doses and 26 patients > 3 doses. The median observation time was 2 months (range – 0-97) and the median observation time of surviving patients was 57 months (range – 7-97). Response rates and overall survival are represented in Table 1 and Figure 1. Table 1. Response rate and overall survival. OS – overall survival, CR-complete response, PR-partial response, SD-stable disease, PD-progressive disease, LFU-lost to follow up The next line treatment was initiated in 27 (60%) patients who failed to respond to MSCs: R-ECP 15, Tocilizumab 7, Vedolizumab 3, Ruxolitinib 1, Methylprednisolone re-initiation 1. Regardless of additional therapy, 21 (78%) patients died, all due to infectious complications alone or together with concomitant GVHD. At the last follow-up, 12 of 45 patients were alive. Causes of death were infectious complications alone or with concomitant GVHD – 25 (76%), primary disease relapse – 7 (21%), asystole – 1 (3%). No adverse events that would be clearly attributed to MSCs infusion were observed. Conclusions: The day 14 and day 28 III/IV grade SR-aGVHD responders have a significantly better prognosis, therefore we suggest considering salvage treatment in non-responders on day 14. Novel therapies for refractory SR-aGVHD patients are in urgent need. Clinical Trial Registry: ISRCTN18091201. https://www.isrctn.com. Disclosure: Nothing to declare.
Background: Chronic graft versus host disease (GvHD) is common side effect of allogeneic stem cell transplants and can cause a significant reduction in patients quality of life and a leading cause of transplant related mortality. Severe GvHD can be very difficult to manage with second line treatment options being associated with significant risk of infectious complications. There is emerging evidence that Ruxolitinib, an oral JAK-1 and JAK-2 inhibitor, can improve response rates in terms of improvement in symptoms and severity of GVHD by NIH score and reduction of steroid doses and has impact on GvHD severity in both the acute and chronic settings. Here we share the experience of 15 patients who have been prescribed Ruxolitinib for chronic GvHD at The Royal Marsden hospital NHS Trust, London. Methods: We retrospectively collected data on 15 patients (aged over 18 years) who were commenced on Ruxolitinib for chronic GvHD over the preceding 4 years. Haemoglobin, lymphocyte, neutrophil and platelet counts were assessed at day 1, day 30 and day 90 from starting treatment. In addition, the ALT level and CMV reactivation were also measured. The degree of GvHD at day 1 and day 90 was assessed and graded in terms of mild, moderate or severe disease by NIH criteria. Results: From our cohort of patients, 2 (13.3%) patients required the Ruxolitinib to be withdrawn within 30 days of starting due to toxicity. One patient experienced pulmonary oedema and shortness of breath. The second patient developed acute reversible kidney injury. Of the 13 remaining patients, all tolerated Ruxolitinib with no reported significant side effects. The patients all had steroid refractory chronic GvHD which affected a combination of organs including skin, liver, eyes, mouth and joints. 12/13 (92.3%) patients had an improvement in their GvHD symptoms by at least 1 grade, 3 (23.1%) patients had complete resolution of their symptoms. 4 (26.6%) patients have been able to stop all other immune suppression including steroids following commencement of Ruxolitinib. There was no significant change in the patients full blood count or liver function tests within the 3 months of starting Ruxolitinib. CMV reactivation was noted in 2 patients (13.3%) within 3 months of starting. Both were recipient/donor CMV IgG positive. Conclusions: From this small study, it can be seen that Ruxolitinib is a safe and effective treatment for chronic GvHD which is well tolerated and does not appear to have significant side effects. Only 2 patients (13.3%) required cessation of the treatment due to toxicity. The remainder of the patients either experienced significant improvement in their symptoms or had stabilisation of disease. There was no evidence of significant bone marrow suppression, liver dysfunction or CMV reactivation in patients assessed. This study demonstrates both clinical efficacy and tolerability of Ruxolitinib for the management of chronic GvHD. Disclosure: Nothing to declare
Background: The pathophysiology of GvHD describes the activation of antigen-presenting cells (APC) and the activation, differentiation, and migration of T cells. Dendritic cells (DC) are professional APCs promoting antigen-specific T cell responses. Tolerogenic DCs can play a fundamental role in GvHD by exerting an immunomodulatory or even immunosuppressive effect on T cells. We performed a prospective analysis of the immune profile of peripheral blood (PB) and ECP treated cells (ETC) of steroid refractory cGvHD patients undergoing treatment exclusively with the Amicus Blue™ online ECP system (Fresenius Kabi, Germany). Methods: ECP was given for a minimum of 24 of 28 planned procedures between May and December 2021, with an accelerated treatment plan consisting of three 8-week blocks: 2 sessions/week, 1 session/week, and finally tapered to 1 session every 2 weeks. Immune profiles were measured for PB and the ETC for procedures 1, 6 (21 days) and 16 (2 months). Samples were analyzed within 24 hours of collection, by flow cytometry using the Navios EX flow cytometer (Beckman Coulter, USA) and the Beckman Coulter Duraclone, IM Phenotyping Basic (B53309), IM Treg (B53346), IM T cells subsets (B53328), and IM TCR (B53340) kits. For mass cytometry immunophenotyping, Helios™ (Fluidigm) was used with the Maxpar® Direct™ Immune Profiling Assay™ kit. Statistical analyses were performed with GraphPad Prism v.8 (La Jolla, US). The one-way ANOVA test for paired samples was used to compare immune profiles in PB and ETC over time and considered statistically significant p < 0.05. Results: Three female patients completing at least 24 ECP sessions were included in the analysis. Patients received outpatient treatment and reported only mild adverse events (AEs) typical of apheresis procedures. All patients had received HLA–identical sibling grafts and were classified as severe cGvHD. Comparison of the PB immunophenotypes did not show statistically significant differences, however, the relative count of FoxP3 + /Helios+ Treg cells and TCD8 naive increased over time when compared with baseline. A decrease of TCD8 terminal effector cells was observed, and there was no change in the dendritic cell subsets. In the ETC, a statistically significant increase (p = 0.012) of relative frequency of FoxP3 + /Helios+ Treg cells was observed compared to the baseline. We also saw the same tendency of TCD8 terminal effector decrease. The ratio of myeloid dendritic cells (mDC)/plasmacytoid dendritic cells (pDC) increased. The reduction of effector cells is beneficial because CD8 + T cells are a mechanism by which cGvHD is sustained and persists. Still, there has been little information about the action of ECP on these subsets. Shiue, et al. have reported that the ratio mDC/pDC in GvHD patients is favorably altered after ECP (increase). Conclusions: Our findings align with the previously reported increase in the relative frequency of FoxP3 + /Helios+ Treg cells, a non-significant decrease in the TCD8 end effector, and the increased mDC/pDC ratios in the ECP treated cells. Further research is needed in larger cohorts of patients. Disclosure: Nothing to declare
Background: Vitamin D (VD) may influence outcomes following allogeneic HSCT due to its impact on immunity, including GvHD. In some inflammatory diseases, VD levels correlate with steroid response. Elafin, ST2 and REG3α are GvHD biomarkers that predict therapeutic response and disease prognosis. The association between VD, GvHD biomarkers and response to immunosuppression (steroids) in the acute GvHD (aGvHD) setting has never been explored. Methods: This exploratory, observational study obtained research ethics approval for sampling and analysis. Serum from 16 patients with clinical diagnosis of aGvHD following allogeneic HSCT/DLI was taken at diagnosis and 1 month later. GvHD biomarkers and 25(OH)D3 were measured by ELISA. Vitamin D deficiency (VDD) was defined as 25(OH)D3 serum levels <50 nmol/L . Modified Seattle Glucksberg criteria were used to grade aGvHD. Complete remission (CR) was defined as complete resolution of aGvHD-derived signs/symptoms. Statistical tests performed were Chi-square and Mann-Whitney U tests (SPSS), and Kaplan-Meier method (R). Results: Baseline Twelve patients (75%) had VDD at diagnosis of aGvHD (14 skin, 7 gut and 5 liver, individually or in combination). Ten patients (63%) had grade I-II and 6 (37%) III-IV aGvHD. At diagnosis, there was a trend in ST2 levels, higher in patients with grade III-IV aGvHD compared to those with grade I-II (180.4 vs 47.3 ng/ml; p = 0.051). There was an association between skin involvement and elafin, higher in stage II-IV compared to stage 0-I (32.2 vs 19.9 ng/ml; p = 0.029). No other significant differences were identified. At this time point, none of the variables could predict GvHD grade at 1-month. 1 month response Response 1-month after starting on steroids was assessed in 12 patients (75%). Six patients (50%) achieved CR (responders) whereas 6 did not (non-responders). Two responders (33%) were on systemic and 4 (67%) on topical steroids. Three non-responders (50%) were on systemic and 3 (50%) on topical steroids. There was no significant difference in the median baseline levels of elafin, ST2 and REG3α between the 1-month responders vs non-responders. However, baseline 25(OH)D3 was significantly higher in responders vs non-responders (53.1 vs 32.7 nmol/L; p = 0.037) (Graph 1). Patients with grade 0-II had a higher concentration of 25(OH)D3 compared to those with grade III-IV (41.6 vs 23.3 nmol/L; p = 0.032). Twelve-month survival from diagnosis was 52%. In the deceased cohort, REG3α levels at diagnosis were significantly higher (287.6 vs 23.4 ng/ml; p = 0.007), with significantly higher ST2 levels at 1-month (82.9 vs 24.3 ng/ml; p = 0.019) and REG3α levels also at 1-month (117.9 vs 61 ng/ml; p = 0.008) compared to those alive at 12 months. Conclusions: VDD is common following allogeneic HSCT in reflection of patient-related factors (sunlight exposure, malnourishment). Our small exploratory study provides some support for the association of VD and steroids response in aGvHD. We also found that ST2 correlates with GvHD severity and elafin with organ involvement, and REG3α and ST2 may have prognostic value at early stages of aGvHD. Routinely monitoring and early management of VDD after HSCT is inexpensive and may benefit GVHD outcomes. However, our small exploratory study cannot provide definitive conclusions thus larger observational studies are warranted. Disclosure: Nothing to declare
Background: Allogeneic hematopoietic cell transplantation (allo-HCT) with a myeloablative conditioning (MAC) is a curative option in acute leukemia (AL) patients, However GVHD (graft versus host disease), and recurrence of diseases remains the main causes of morbidity and mortality. Objective of this study was to evaluate the impact of addition of anti-thymocyte globulin (ATG) in the prevention of graft versus host disease (GVHD) after HLA matched related allo-HCT. Methods: We retrospectively evaluated all consecutive acute leukemia patients undergoing allo-HCT from matched related donors who were received ATG (5 mg/kg) as part as conditioning regimen. Primary endpoints were grade III/IV acute GVHD rate, moderate/severe chronic GVHD rate and cumulative incidence of relapse Results: Between February 2013 and November 2020, 62 patients (AML = 54, ALL = 8) were included. Median age was 37 years (18- 62). At time of transplant, all patients were in complete remission. Conditioning regimen was FB4 in 57 patients including; Fludarabine 40 mg/ m3/day for 4 days, iv Busulfan (Bu)130 mg/m3/day for 4 days, and busulfan /melphalan 140 for 5 patients. GVHD prophylaxis consisted of rabbit ATG (Thymoglobulin 1, Genzyme) iv.5 mg/kg/day (days -2, -1), cyclosporine (CsA) and methotrexate 15 mg /m3 day +1, and 10 mg/m3 (days + 3, +6,+ 11). In the absence of GVHD, CsA was tapered from day 100–180. All patients achieved hematopoietic reconstitution except one patient who died before engraftment. The median time to neutrophil engraftment was 12 days (range 5–25 days), and the median time to platelet engraftment was 14 days (range 10–43 days). CMV reactivation occurred in 15 patients (24 %). One patient died from CMV infection. At the end of follow up, incidence of overall acute GVHD was 44 % including 17 patients (27 %) grade II-IV and 5 patients (8 %) grade III-IV. Chronic GVHD occurred in 34 % of patients; including 11 patients (18%) moderate grade and only 4 patients (6 %) presented a severe grade. Relapse occurred in 16 patients (26 %). With a median follow up of 63 months (15-104 months) overall survival rate was 53% (33 patients). Conclusions: Incorporation of an intermediate dose of ATG in the context of HLA matched related allo-HCT reduces severe acute and chronic GVHD without impact graft versus leukemia effects in acute leukemia patients. Disclosure: no conflict of interest
Background: Although the Amicus Blue™ ECP system (Fresenius Kabi, Germany) has received marketing authorization in Europe for treatment of CTCL, there is evidence supporting its benefits for treatment of cGVHD. This system includes the Amicus Separator®, the Phelix photoactivation device, a functionally closed disposable kit, and 8-MOP (20 µg/mL) to perform ECP therapy. We evaluated the characteristics and outcomes of steroid-refractory cGvHD (SR-cGVHD) patients, aiming to characterize the safety and effectiveness profile of the Amicus Blue™ ECP system with the implemented regimen. Methods: All patients were evaluated before starting ECP therapy against inclusion/exclusion criteria. The U.S. NIH severity scoring was performed at baseline and after cycle 2, and classified as mild, moderate, or severe global GvHD disease. ECP standard intensity was performed within 6 months between May and December 2021, with an accelerated treatment plan consisting of 2 sessions/week for 8 weeks, followed by 1 session/week for 8 weeks, and tapered to 1 session every 2 weeks for the last 8 weeks. Amicus v6.0, Phelix v2.0 and double-needle disposable kits were used with central access for all patients. A 12:1 whole blood (WB) to ACD-A anticoagulant ratio was used, 1.24 mg/kg/min citrate infusion rate. We intended to process 1.0 total blood volume (TBV), with minimum of 0.5 TBV and maximum of 1.2 TBV. Haematology counts were performed on patient WB and the treated MNCs, and lymphocyte apoptosis was measured at 72hr. Results: Three female patients age 24-35 years with severe cGvHD (2 de novo, 1 progressive) received ECP treatment. The patients had received HLA–identical sibling peripheral blood transplants for sickle cell anemia/β-thalassemia, blastic plasmacytoid dendritric cell neoplasm, and hypogammaglobulinemia approximately 3-4 years prior to treatment. Cutaneous cGvHD with significant sclerodermal changes was the leading indication for ECP. Two patients received concomitant corticosteroids, and 1 patient received ruxolitinib. No procedure-related severe adverse events (AEs) were reported, only mild AEs typical of apheresis procedures. Median (range) total procedure time including collection, photoactivation and reinfusion was 103 (76-240) minutes. WB processed was 3001 (1830-4044)mL. The yield Hct was 2.84 (2.3-4.1)%. Median treated cell doses (x109) were: WBCs 19.94 (8.7-31.3), lymphocytes 15.7 (3.3-28.9), monocytes 3.5 (0.5-7.2) neutrophils 0.6 (0.1-0.63). MNC collection efficiency was 51.2 (23.4-83.6)%. Lymphocyte apoptosis was 31.3% (23-46%) at 72hr. All 3 patients showed partial response after 2 cycles (24-28 procedures), detailed outcome data is presented in table 1. Table 1. Clinical outcome of SR-cGVHD Patients Undergoing ECP. Conclusions: Our pilot experience confirms that the use of the Amicus Blue™ ECP system was safe and efficacious for treating SR-cGVHD patients with the reported regimen. Disclosure: Nothing to declare
Background: Acute graft-versus-host disease (aGVHD) that is refractory to glucocorticoids (SR) is a major cause of death after allogeneic hematopoietic cell transplantation (allo-HCT). Human bone marrow -derived mesenchymal stromal cell (MSC) have shown activity in patients with SR-aGVHD in retrospective patient series indicating safety of this approach. Prospective data on the efficacy of the MSC product MC0518 for SR-aGVHD are lacking. Methods: The IDUNN-study is a randomized, open label, multicentre, phase 3 trial comparing MSCs MC0518 with Best available therapy (BAT) in patients with SR-aGVHD. Adult and adolescent patients with acute skin, intestinal or liver GvHD > grade 1 and failure of previous steroid treatment are eligible. The trial aims to include 210 patients who will be randomized in a 1:1 ratio and stratified by GvHD grade (grade 2 versus grades 3/4), underlying disease (malignant versus non-malignant), and age group (<18 years of age versus ≥ 18 years of age). Results: The primary endpoint is the overall response rate (ORR) at day 28, defined as: Partial Response (improvement of at least one stage in the severity of aGvHD in one organ without deterioration in any other organ), or Complete Response (disappearance of any GvHD signs from all organs without requirement for new systemic immunosuppressive treatment). Secondary objectives include freedom from treatment failure until 6 months, overall survival until visit month 24, aGVHD response at visit days 60, 100 and 180, change of aGvHD grade at visit days 8, 15, 22, 28, 60, 100 and 180, time to response, duration of response, best OR until and at day 28, cumulative dose of steroids from baseline until visit days 28, 60, and month 24, incidence of and time to cGVHD and graft failure, relapse or progression in subjects with underlying malignant disease, event-free survival, non-relapse mortality, incidence and severity of adverse events and adverse reactions, performance score, and quality of life. In addition, changes in serum levels of pro-inflammatory cytokines and aGvHD-related biomarkers soluble suppression of tumorigenicity 2 (sST2) and regenerating islet-derived 3α (Reg3α) will be assessed. The trial will be conducted across approximately 40 trial sites in approximately 7 European countries. Conclusions: This randomized prospective trial will provide evidence if the retrospectively collected data demonstrating activity of MSC for SR-aGvHD can be reproduced in a prospective trial setting and if MSC show higher efficacy compared to BAT. A major advantage of MSC could be the limited toxicity profile observed in previous applications of MSC in SR-aGVHD patients. This trial will investigate candidate biomarkers to predict and monitor responses to MSC. Clinical Trial Registry: ClinicalTrials.gov Identifier: NCT04629833. Disclosure: R.Z. received honoraria from Novartis, Incyte and Mallinckrodt.
Background: Extracorporeal photopheresis (ECP) is an apheresis based immunomodulatory therapeutic procedure. It utilizes separated white blood cells from the patient, threated with a photoactivated agent – methoxypsoralen, exposed to UVA and then reinfused back, to make an immunosuppressive effect as a addition to the therapeutic activities against chronic GvHD.In our bone marrow transplant unit, we perform hematopoietic stem cell (HSC) transplants from year 2000, and so far have made over 600 interventions. We recognized the therapeutic potential of ECP and have implemented it in 2021. So far we have made 38 procedures. We used peripheral venous access in 22(58%) and had to use central venous access in 16 (42%). So far no serious adverse events were noted. We have treated 3 patients with chronic GvHD and had an attempt in treating 1 patient with Non Hodgkin lymphoma – PTCL. Methods: Unfortunately, in only 1 patient with chronic GvHD we performed the whole treatment protocol. It`s a male, 24 years old, diagnosed with Severe Aplastic Anaemia in 2017. Allogeneic unrelated matched HSC transplant was done in 2018 with successful engraftment. But in may 2019, he complained of stomach ache and yellow skin. The CBC Hgb 119 g/L WBC 11.2 x 109/L PLT 247 x 109/L AST 238 U/L ALT 178 U/L AP 800 U/L GGT 1996 U/L LDH 691 U/L Total Bilirubin 209 µmol/L Direct 126 µmol/L Indirect 83 µmol/L. There was a complete chimerism of the graft. HBV and HCV excluded. The liver biopsy showed chronic GvHD of the liver. We started as first line treatment Methylprednisolone + Cyclosporin A. Only partial response was met, AST 137 U/L ALT 280 U/L AP 588 U/L GGT 1756 U/L Total bilirubin 53 µmol/L Direct 25 µmol/L Indirect 28 µmol/L. As second line we used Rituximab for four doses, and as third line mycophenolate mophetil, but still the results were similar (AST 146 U/L ALT 238 U/L AP 880 U/L GGT 1200 U/L LDH 390 U/L T.Bil 92 µmol/L Direct 87 µmol/L Indirect 5µmol/L). Results: As fourth line we started ECP alone. Treatment plan was 2 procedures on 2 consecutive days equals 1 cycle, and 1 cycle every 2 weeks for 4 months, then 1 cycle a month. All procedures were done successfully, with no adverse events. We used only peripheral venous access. The patient now has normal CBC, AST 51 U/L ALT 55 U/L AP 400 U/L GGT 250 U/L Total Bilirubin 22 µmol/L Direct 12 µmol/L Indirect 10 µmol/L and stabilization and a slight regression of the skin changes that accompanied the chronic GvHD spectrum. Conclusions: This successful treatment story encourages us to maintain on this course and utilize the therapeutic potentials of ECP and implementing it in a number of our patients, even earlier in the treatment plan, mainly for chronic GVHD Disclosure: No disclosures
Background: SER-155, an oral investigational microbiome therapeutic composed of cultivated human-commensal bacterial strains, is being developed to reduce the risk of bloodstream infection and graft-versus-host disease (GvHD) in allogeneic hematopoietic stem cell transplant (HSCT) recipients. SER-155 was rationally designed to restore gastrointestinal homeostasis by restoring colonization resistance, reducing gastrointestinal inflammation and improving epithelial barrier integrity. Methods: SER-155 was evaluated in vitro and in vivo for specific pharmacological properties to mitigate drivers of morbidity and mortality following HSCT. In vitro, functional properties of SER-155 were assessed utilizing culture supernatants to test for a) production of anti-inflammatory metabolites, b) anti-inflammatory activity measured by suppression of IL-8 secretion by TNF-ɑ treated HT29 cells and modulation of inflammatory pathways in IFN-ɣ treated primary colonic epithelial organoids, and c) protection of epithelial barrier integrity in a trans-well culture system of differentiated primary human colonic epithelial cells treated with IFN-ɣ. In vivo, germ-free mice were used to assess the ability of SER-155 colonization to modulate gut immune cell populations towards a noninflammatory phenotype; specifically the ratios of regulatory T cells (Tregs) to Th1 and Th17 effector T cells in the colonic lamina propria. Results: Development of SER-155 included preclinical screening in vitro and in vivo of over 50 designed candidate consortia containing combinations of >150 species. Analysis of culture supernatants showed that SER-155 produces diverse anti-inflammatory metabolites including short- and medium-chain fatty acids and a variety of tryptophan and bile acid metabolites. In vitro, SER-155 supernatants inhibited IL-8 secretion in TNF-α stimulated HT29 cells and induced transcriptional changes in colonic organoids that reduced IFN-ɣ driven inflammatory gene and pathway expression. In an in vitro intestinal epithelial barrier model, SER-155 supernatants protected barrier integrity as shown by a significant reduction in IFN-ɣ mediated barrier permeability. In vivo, SER-155 colonization led to a significant expansion of regulatory T cells (Tregs) and an increased ratio of Tregs to Th1 and Th17 effector T cells. Conclusions: Preclinical assessments in vitro and in vivo support the ability of SER-155, an investigational cultivated microbiome therapeutic, to promote epithelial barrier integrity and reduce local inflammation to restore immune homeostasis in the gut. These data support the ability of microbiome therapeutics to affect diverse pathways important to disease pathogenesis. A phase 1b study evaluating SER-155 in allogeneic HSCT patients is currently enrolling (NCT04995653). Disclosure: This research was sponsored by Seres Therapeutics. Elizabeth Halvorsen, Asuncion Martinez, Marin Vulic, Swarna Pandian, Kathleen Cieciuch, Jennifer Black, Keith Halley, Mary-Jane Lombardo, Christopher Ford, and Matthew Henn are current employees and shareholders of Seres Therapeutics. Divya Balasubramanian, Ambar Pina, and Tim Nelson were past employees and shareholders of Seres Therapeutics.
Background: Ruxolitinib has been demonstrated to be effective in the treatment of steroid-resistant acute graft-versus-host disease (aGVHD) as a JAK1/JAK2 inhibitor. MDSCs represent a heterogenic population of immature myeloid cells to have a protective effect on aGVHD via suppressing T cell functions. Ruxolitinib’s effect on inflammatory cells such as regulatory T cells, dendritic cells and natural killer cells is known. However, its effect on myeloid derived suppressor cells (MDSCs) competency plausibly involved in pathogenesis of GVHD has not been explored. Methods: We aimed to define the effect of ruxolitinib on the immunobiology of MDSCs in the pathogenesis of aGVHD. The ratios and functions of MDSCs were analyzed after ruxolitinib administration compared with controls in vivo and in vitro. Meanwhile, its downstream effector molecules were tested by flow cytometry, western blot and phosflow techniques. Results: In this study, we demonstrate that in vivo administration of ruxolitinib results in the expansion and functional enhancement of polymorphonuclear MDSCs (PMN-MDSCs) in a murine model of aGVHD and the effects could be partially reversed by anti-Gr1 antibody. Ruxolitinib treatment can enhance the suppressive function of PMN-MDSCs via up-regulation of reactive oxygen species (ROS) and activated MAPK/NF-κB signaling pathway. Ex vivo experiments demonstrated that ruoxlitinib can prevent differentiation of mature myeloid cells and promote accumulation of MDSCs via inhibiting STAT5. Moreover, ruxolitinib can also induce a strong immunosuppressive function in PMN-MDSCs utilizing the NF-κB transcription factors as well as NOX2 upregulation. Conclusions: In summary, impaired MDSCs are involved in the pathogenesis of aGVHD, and ruxolitinib corrected PMN-MDSC functions via a mechanism underlying JAK/STAT and MAPK/NF-κB signaling pathways. Our findings may also explain the outstanding immunomodulating activity of ruxolitinib currently used in the treatment of aGVHD and other autoimmune diseases. Disclosure: Nothing to declare
Background: Graft versus host disease (GVHD) remains a major obstacle to successful haematopoietic stem cell transplant. Although donor T cells are necessary in animal models, recent data indicate that the full expression of tissue inflammation in transplanted humans may depend upon myeloid cells, and even recipient T cells. Furthermore, the signals that drive recruitment from blood and the circuits that exist between immune cells, targets and supporting stroma, are not well defined. In this study we sought to understand these mechanisms in detail by defining the landscape of GVHD in human skin and blood at single cell transcriptomic resolution. Methods: We collected paired skin biopsy and blood samples from five patients at the onset of acute GVHD, two transplant recipients without acute GVHD at day 100 post-transplant and samples from healthy untransplanted donors as controls. Skin biopsies were split into epidermis and dermis by enzymatic digestion. Separated tissues were then digested into single cell suspensions. Peripheral blood mononuclear cells were isolated from whole blood by density gradient separation. Samples were then loaded onto 10x genomics single cell 5’ platform to generate gene expression and T-cell receptor libraries. Data was processed using Seurat R package (v4.0.3). Poor quality cells and multiplets were first removed. Samples were then normalized, dimensionally reduced and clustered using the SCTransform approach. To identify the donor or recipient origin of cells, germline single nucleotide polymorphisms were defined by exome sequencing of DNA samples from each donor and recipient. The output was then passed into Demuxlet, a computational tool that calls the origin of each cell base on its genetic identity. Results: It was possible to identify various cell types in each anatomical location. In the epidermis without GVHD, we identified Langerhans cells, keratinocytes and melanocytes. In dermis, there were cells from the non-immune compartment (fibroblasts, Schwann cells, pericytes and endothelial cells) and the immune compartment (T cells, NK cells, innate lymphoid cells, macrophages, dendritic cells, etc). Additionally, we identified the leukocyte subsets in blood. Skin with acute GVHD contained a vastly altered cellular composition, compared to skin unaffected by GVHD. Most notably there was an influx of donor myeloid cells and lymphocyte. Myeloid cells were mainly of donor origin, while a mixed population of donor and recipient T cells were found in both epidermal and dermal compartments. There was an elevated proportion of monocytes in the blood of GVHD patients compared to controls. Populations of monocytes expressing activation markers were also identified. T cell receptor analysis revealed that T cell clones were less diverse and highly shared between skin and blood of each patient with GVHD, consistent with a systemic distribution of T cells mediating GVHD. Conclusions: Single cell RNA sequencing allowed detailed information to be obtained from small clinical biopsies of GVHD-affected tissue. There was a vast difference in cellular composition and gene expression between GVHD-affected tissues compared to controls. By simultaneously profiling the landscape of GVHD in human skin and blood at single cell resolution, we aim to obtain new insights of classical and novel mechanisms of GVHD. Disclosure: Nothing to declare
Background: The GI tract is a primary tissue system damaged by GvHD, leading to a compromised mucosal barrier, mucosal protein loss, and nutrient/fluid absorption failure. Glucagon-like peptide-2 (GLP-2) has demonstrated intestinotrophic effects, enhanced barrier function, and decreased intestinal permeability. Apraglutide, a novel, long-acting synthetic GLP-2 analog, represents a potential regenerative approach to GI-GvHD prevention and treatment. Using two mice models of GvHD, we assessed the effects of apraglutide on engraftment and GI protection following irradiation and allogeneic transplantation. Methods: In Study 1, total-body-irradiated (TBI) immunodeficient (NOG) mice (Day 0) were injected with human peripheral blood mononuclear cell (hPBMC; 3x107; Day 2) and treated with apraglutide 3.3 mg/kg or vehicle (Days -6 to 18). Engraftment rate was determined through CD45 expression (human vs. mouse) in blood, bone marrow, and spleen. In Study 2, TBI-induced intestinal damaged BALB/cJ mice received allogeneic transplantation from C57BL/6 strain and were treated with apraglutide (3.3 mg/kg) or vehicle (Days -9, -7, -5, -3 -1, +1, +3, +5, +7). Intestinal damage indicative of GvHD (histological changes, length, hemorrhage, inflammation), body weight, and survival were assessed. Results: In study 1, hPBMC were successfully engrafted. The engraftment rate in blood, spleen, and bone marrow was not affected by apraglutide (range 22.2-47.6% at D20 in blood). hCD45+ cell infiltration was observed in the intestinal wall with no difference between apraglutide vs. vehicle. In study 2, lymphocyte engraftment was successfully achieved in both apraglutide- and vehicle-treated mice. Weight loss and median survival were similar in both groups, but apraglutide-treated mice had significantly higher overall survival vs. vehicle on Day +9 (40% vs. 0%, respectively; p = 0.0134). Post-mortem histological examination revealed less mucosal degenerative/inflammatory changes (villous atrophy, mononuclear/neutrophilic cell infiltrate in the lamina propria/intra-cryptal epithelium, crypt necrosis) in apraglutide-treated mice vs. vehicle. Mean colon length in the apraglutide group (8.6 ± 0.35 cm) was comparable to mice that did not undergo irradiation or transplantation (9.6 ± 0.33 cm), whereas a significant reduction was apparent in the vehicle group (7.19 ± 0.10 cm; p < 0.05). Conclusions: These results suggest that apraglutide treatment before allogeneic transplantation in immunodeficient mice does not affect engraftment rate. Furthermore, apraglutide showed a significant protective effect in TBI- and allogeneic-transplant-induced GvHD with reduced villi atrophy, less colon shortening, less severe intestinal damage, and showed a survival advantage. These findings support the beneficial role of apraglutide in reducing GI damage and limiting mortality from GvHD. Disclosure: Violetta Dimitriadou is an employee of VectivBio, AG.; Geneviève Chabot-Roy, Cindy Audiger, Ianula Banu, Jean-Sébastien De, and Sylvie Lesage have no relevant disclosures.
Background: Chemotherapy-induced mucositis is a common condition caused by the breakdown of the mucosal barrier. Administration of exogenous glucagon-like peptide 2 (GLP-2) has been associated with reduced epithelial damage, decreased bacterial infection, and decreased mortality or gut injury in rodents with chemically induced enteritis. GLP-2 decreases chemotherapy-induced mucositis via inhibition of drug-induced apoptosis in the small and large bowel. Apraglutide is a novel, long-acting synthetic GLP-2 agonist that has been shown to promote intestinal growth and repair. Two preclinical studies aim to evaluate the efficacy of apraglutide (3.3 mg/kg) as pre-treatment or concomitant treatment in models of chemotherapy-induced intestinal damage with cytarabine or melphalan; both extensively used in hemato-oncology. Methods: Study 1 included four groups of Balb/c mice: (A) vehicle only; (B) cytarabine on Days 5-9, no apraglutide given; (C) cytarabine on Days 5-9; concomitant apraglutide on Days 5-18; (D) cytarabine on Days 5-9; pre-treatment apraglutide on Days 1, 3, and continued as a concomitant treatment on Days 5, 8, 11, 14, and 17. Study 2 included three treatment groups of Balb/c mice: (A) vehicle only; (B) melphalan on Day 9, no apraglutide; (C) melphalan on Day 9; pre-treatment apraglutide on Days 1, 3, 5, 7 and continued as a concomitant treatment on Days 9, 11, and 13. In both models, mice that received the vehicle without any treatment served as controls. Intestinal tissue histology, body weight, survival, and plasma citrulline, a marker of total mucosal mass and intestinal growth, were assessed in both models. Results: Histological examination showed that the degenerative intestinal changes (villi and crypt atrophy) caused by cytarabine or melphalan were reduced by apraglutide co-administration, as demonstrated by similarities in tissue morphology between vehicle-treated and apraglutide-treated mice. In addition, the duodenum, ileum, and jejunum increased in weight with apraglutide. The intestinal protective effects of apraglutide were further supported by preserving plasma citrulline levels (a biomarker of intestinal mass): apraglutide-treated mice had similar levels to animals that did not receive chemotherapy. Apraglutide attenuated chemotherapy-induced weight loss and improved overall survival vs. vehicle-only or chemotherapy-only groups. The effects of apraglutide were optimal when it was administered as pre-treatment before chemotherapy. Conclusions: Microscopic examination showed apraglutide protected GI epithelium structure from chemotherapy-induced injury, improved survival, and prevented severe body weight loss in mice undergoing chemotherapy. Apraglutide also maintained plasma citrulline levels, a marker of intestinal mass, comparable to that in mice who did not undergo chemotherapy. Disclosure: Violetta Dimitriadou is an employee of VectivBio, AG.; Mark Minden has no relevant disclosure.
Background: Hematopoietic stem cell transplantation patients experience profoundly altered gut microbiota composition due to dysregulation of intestinal homeostasis by conditioning regimens, broad-spectrum antibiotics, immunosuppressants, and the introduction of foreign lymphocytes from the donor. A growing body of evidence shows reduced microbiome diversity increases the incidence and seriousness of graft versus host disease (GvHD) and bacteremia. Apraglutide, a novel long-acting synthetic glucagon-like peptide 2 (GLP-2), has been shown to protect gastrointestinal (GI) epithelium structure from chemotherapy-induced injury, improve survival, and allowed better body weight maintenance in mice undergoing chemotherapy. The study aimed to evaluate the protective effect of apraglutide on gut microbiota during chemotherapy with cytarabine. Methods: Balb/c mice received 30 mg/kg of cytarabine on Days 5-9 and apraglutide 3.3 mg/kg on Days 1-18. Control mice received the vehicle on Days 1-18. Fecal samples were collected over 24 hours for bacterial phenotyping at pre-treatment and the day before scheduled termination and for found dead or pre-terminally euthanized animals. Microbiota composition was determined by 16S taxonomical meta-sequencing. Results: Bacteroidetes and Firmicutes were the two leading bacterial phyla identified. Chemotherapy with cytarabine caused significant changes in the composition of bacterial species, increasing the Bacteroidetes population and decreasing the proportion of Firmicutes bacteria. The change in Bacteroidetes and Firmicutes bacteria levels from Days 0 to 18 was significantly greater in the cytarabine-only and cytarabine + apraglutide mice vs. vehicle. However, this effect was reduced by apraglutide co-administration. The difference in the change between cytarabine-only and cytarabine + apraglutide groups reached statistical significance for both Bacteroidetes (0.2486; p < 0.0001) and Firmicutes (0.2037; p < 0.0001). In addition, the ratio of Bacteroidetes to Firmicutes bacteria present remained more constant in cytarabine + apraglutide than in the cytarabine-only group. Conclusions: Chemotherapy profoundly impacted bacterial homeostasis in the mouse intestine, with a notable increase in opportunistic pathogenic bacteria populations. The proportions of different bacterial phyla in feces remained closer to normal when apraglutide was co-administered with chemotherapy. Treatment with apraglutide resulted in the preservation of the global homeostatic environment of the intestinal microbiota. Prevention of intestinal dysbiosis may contribute to the improved outcomes (reduced body weight loss, increased survival) observed in mice when apraglutide is administered concomitantly with chemotherapy agents. Disclosure: Violetta Dimitriadou is an employee of VectivBio, AG.
Background: Conditioning chemotherapy reduces tumor burden and provides immunoablation to prevent graft rejection with hematopoietic cell transplantation, but often induces mucosal barrier breakdown and mucositis. Apraglutide is a novel, long-acting synthetic glucagon-like peptide-2 (GLP-2) analog that protects the GI epithelium from chemotherapy-induced injury, improves survival, and allows better weight maintenance in mice undergoing chemotherapy. Preclinical studies aim to evaluate the impact of apraglutide on chemotherapy’s efficacy in reducing tumor load and inducing immunosuppression. Methods: Study 1 assessed cytarabine’s antitumor effects in leukemic NOD/SCID mice. Apraglutide or vehicle was administered on Days -4 to 4. Cytarabine or vehicle was administered on Days 0-4. Bone marrow and spleen samples were collected on Day 7, and the percentage of hCD45+ cells was determined. Study 2 assessed the effect of apraglutide on cytarabine-induced immunosuppression and included three groups of Balb/c mice: (A) vehicle; (B) cytarabine on Days 5-9; (C) cytarabine on Days 5-9, concomitant apraglutide on Days 5-18. RBC, platelets, WBC, NEU, and LYMPH, were assessed. A cohort was allowed to survive for four weeks to evaluate the effect of apraglutide on immunosuppression recovery. Study 3 assessed the effect of apraglutide on melphalan-induced immunosuppression. Three groups of Balb/c mice were included: (A) vehicle; (B) melphalan on Day 9; (C) melphalan on Day 9, apraglutide pre-treatment on Days 1, 3, 5, 7 and continued as co-administration on Days 9, 11, and 13. WBC, NEU, and LYMPH were assessed. Results: Study 1 showed that human leukemia cells reduction did not differ significantly between cytarabine-only and cytarabine + apraglutide and were significantly greater than in the vehicle-only group. The percentage of hCD45 in bone marrow after chemotherapy was 35.5 ± 4 with cytarabine-only and 33.9 ± 4.2 with cytarabine + apraglutide. A dramatic decrease in leukocytes at the end of the treatment period in Study 2 indicated that cytarabine-induced immunosuppression was not impaired by apraglutide co-administration (91% reduction in lymphocytes with both cytarabine + apraglutide and cytarabine-only). Apraglutide did not impact the recovery of hematological parameters four weeks after the end of treatment. Study 3 showed that melphalan elicited immunosuppression as evidenced by leukocyte decrease. Mice treated with melphalan, with or without apraglutide, had severe reductions in WBC and LYMPH vs. vehicle. Conclusions: Pre- and concomitant apraglutide did not impair the efficacy of cytarabine in destroying human leukemia cells in vivo. Moreover, combination with apraglutide had no negative impact on cytarabine- or melphalan-induced immunosuppression. Apraglutide did not negatively impact the antitumor or immunosuppressive effects of cytarabine or melphalan. Disclosure: Violetta Dimitriadou is an employee of VectivBio, AG.; Mark Minden has no relevant disclosures.
Background: Vaccines against the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) have been approved rapidly. Long-term data in recipients of allogeneic hematopoietic cell transplantations (allo-HCT) after SARS-CoV-2 vaccination are lacking. Methods: We examined longitudinal antibody responses to SARS-CoV-2 vaccination with BNT162b2 (BioNTech/Pfizer) or mRNA-1273 (Moderna) in allo-HCT recipients and healthy controls. Seroprofiling recording IgG, IgA, and IgM reactivities against SARS-CoV-2 antigens (receptor-binding domain (RBD), spike glycoprotein subunits S1 and S2, and nucleocapsid protein (N)) was performed prior to vaccination (T0), prior to the 2nd dose (T1), and 1 (T2), 3 (T3), and 6 (T4) months (m) after the 2nd dose by using the immunoassay ABCORA, an in-house developed assay that allows differentiating immunity after vaccination versus immunity after infection. Based on computational methods high neutralization potency was predicted above a sum of S1 threshold of 17. Results: We enrolled 110 allo-HCT patients (median age 57y (interquartile range (IQR: 46-65 y)) between March 2021 and May 2021 at the University Hospital Zurich. AB responses are available for 101 (T1), 101 (T2), 96 (T3) and 78 (T4) patients, respectively. Patients were stratified to three groups (A) 3-6m post-HCT, (B) 6-12m post-HCT and (C) > 12m post-HCT. In addition, AB responses are available for n = 72 healthy controls (median age 35.5y (IQR 23-64 y)). The study cohort includes 10 patients and 5 healthy controls with a reported preinfection. Individuals early post allo-HCT (3-6m and 6-12m post HCT) developed statistically significant lower antibody titers after vaccination compared to patients >12m post allo-HCT and healthy controls (p < 0.001, Fig 1A-D). Antibody titers achieved the highest levels 1m after the 2nd dose but declined substantially in all transplanted and the healthy group over time: At 6m after the 2nd dose only in 3/15 (20%) of group A, 3/9 (33%) of group B, 29/54 (54%) of group C and 42/66 (64%) of the healthy controls protective neutralization titers were measurable. In a multivariate linear regression analysis on factors associated with antibody response in allo-HCT patients at 1m after the 2nd dose, we found consistently lower immune response in the group 3-12m post-HCT (coefficient = -0.66, 95% CI [-1.06, -0.25], p = 0.002); age >65 years (coefficient = -0.59, 95% CI [-1.11, -0.07], p = 0.030); patients under immunosuppressive treatment (IST) (coefficient = -0.44, 95% CI [-0.84, -0.04], p = 0.033); and whether patients suffered from relapse of the underlying disease (coefficient = -0.55, 95% CI [-0.98, -0.12], p = 0.014), (Fig 1E). Statistically significant higher antibody levels were seen in preinfected patients (coefficient = 0.78, 95% CI [0.21, 1.35], p = 0.009). In contrast, presence of moderate or severe chronic GVHD was not found to directly influence AB levels. Conclusions: Allo-HCT patients early post-HCT displayed impaired antibody formation to vaccination against SARS-CoV-2. A remarkable decline of protective antibody levels has been observed in all groups of patients as well as healthy control in the follow-up until 6m. This analysis of long-term vaccine antibody response is of critical importance to allo-HCT patients and transplant physicians to guide treatment decisions regarding re-vaccination and social behavior during this pandemic. Clinical Trial Registry: The study was conducted according to the Declaration of Helsinki and was approved by the Cantonal Ethics Committee Zurich, Switzerland (BASEC No 2021-00261). Disclosure: Nothing to declare.
Background: Pediatric core binding factor acute myeloid leukemia (CBF-AML) positive for the RUNX1-RUNX1T1 fusion gene is generally associated with favorable outcomes. However, outcomes for patients who also harbor an additional c-KIT gene mutation remain unclear. This study explored the impact of an additional c-KIT gene mutation on the prognosis of CBF-AML pediatric patients with a positive RUNX1-RUNX1T1 fusion gene and who underwent an allogeneic hematopoietic stem cell transplantation (allo-HSCT). Methods: We retrospectively analyzed 119 patients with RUNX1-RUNX1T1 fusion gene positive CBF-AML who received an allo-HSCT and achieved first complete remission (CR1) between February 2012 and August 2021. The median age of patients was 8 years old (range: 2-14 years old). The male to female ratio was 74 to 45. Prior to transplant, 54 of the 119 patients harbored a c-KIT gene mutation and 21 of 54 c-KIT-positive patients harbored a D816 mutation in the c-KIT gene. Ten of the transplant donors were matched siblings, 13 were matched-unrelated, and the remaining 96 were haploidentical. 117 patients received conditioning regimens with busulphan (Bu), cyclophosphamide (CTX) and antilymphocyte globulin (ATG). The other two patients underwent total body irradiation (TBI) and received cyclophosphamide (CTX) and antilymphocyte globulin (ATG) regimens. Cyclosporine or tacrolimus, mycophenolate mofeil and short-course methorexate were the most frequently administrated graft versus-host disease (GVHD) prophylaxis regimens. Results: After a median follow-up of 32 months (range: 1-93 months) after transplant, all patients were successfully engrafted and had 100% donor chimerism. Following the allo-HSCT, 45 of 119 patients (37.8%) developed Grade II-IV acute GVHD and 20 of 119 patients (16.8%) had Grade III-IV acute GVHD. The 3-year overall survival (OS) after allo-HSCT was 81.5%. Eighteen patients died (15.1%) after a median of 10 months (range 2-36 months) post-transplant. Compared with patients without c-KIT mutations (N = 65), patients who harbored c-KIT mutations (N = 54) exhibited a lower OS, although the difference was not statistically significant (72.4% 3-year OS and 89.5% 3-year OS in the c-KIT positive and negative, respectively; p = 0.061). Of the 54 patients with c-KIT gene mutations, 21 had a D816 mutation and the remaining 33 patients had other c-KIT mutations. We observed a worse outcome among those c-KIT mutation-positive patients who harbored the D816 mutation compared to the other c-KIT mutations (3-year OS of 54.7% compared to 83.9%, respectively; p = 0.024). Patients without c-KIT mutations had a prognosis comparable to those patients who harbored a non-D816 c-KIT mutation (3-year OS of 83.9% compared to 89.5% respectively; p = 0.701). Conclusions: Allo-HSCT is an effective therapy among high-risk CBF-AML pediatric patients positive for the RUNX1-RUNX1T1 fusion gene. However, we found that an additional D816 c-KIT mutation is strongly associated with a poor prognosis among these pediatric AML patients. Disclosure: Nothing to declare
Background: Despite trends showing improved outcomes after allogenic hematopoietic cell transplantation (allo-HCT), non-relapse mortality remains a major concern. The prediction of survival is therefore crucial. Large cohorts of patients are needed to develop and validate prognostic scores and the same must be periodically revised in response to changes in clinical practice. The aim of this study was to evaluate the ability of the revised pre-transplant assessment of mortality (PAM) score in predicting survival in an independent large cohort of patients receiving allo-HCT using post-transplant Cyclophosphamide (PTCy) as graft-versus host disease prophylaxis. Methods: All consecutive adult patients with hematological malignancies who underwent a first allo-HCT with PTCy regardless the type of donors at San Raffaele Hospital in Milan between January 2013 and June 2020 were included. Patients with missing PAM score were excluded. Results: A total of 373 patients met the inclusion criteria and were retrospectively analyzed. Median patient age was 54.97 (IQR 41.32-64.64) years. Median follow-up among survivals of 30.49 (range, 2.98-80.13) months. The 2-year overall survival (OS) was 66 +/−4%. Median PAM score was 18.60 (IQR 15.80-24.00). Patients were analyzed according to the PAM score into 4 groups, using the cut-points of the original study. The 2-year OS was 78 +/−4% for group 0 (PAM < 17), 71 +/−5% for group 1 (PAM 17-23), 52 +/−6% for group 2 (PAM 24-30) and 30 +/−8% for patients in group 3 (PAM > 30) (p < 0.0001) As OS for group 0 and 1 were superimposable, we identified a novel cut-point of 25 that allowed us to better stratify overall survival of our patients (p < 0.0001). Matched donors (related or unrelated) were similarly represented in group 0 and 1 (group 0: 65.6% vs group 1: 52.4%; p = 1) but they were less represented in group 2 and 3 (group 0-1: 60.1% vs group 2-3: 16%, p < 0.0001). Haploidentical donors was more frequent in group 2 and 3 (group 0-1: 29.2% vs group 2-3: 69.7%, p < 0.0001). Analyzing OS by donor type (matched vs non-matched) we were able to confirm the ability of PAM score to predict OS for patients receiving a mismatched donor (p < 0.0001) but not for patients receiving a matched one (p = 0.125). In multivariate analysis risk factors for lower OS were PAM (as continuous variable for each point increase: HR: 1.068, CI: 1.041-1.096; p < 0.0001) and HCT-CI ≥ 4 (HR 2.055, CI: 1.380-3.059; p = 0.001). Conclusions: Based on our result, PAM score confirmed to be a useful tool for predicting survival especially in high risk patients (PAM group 2 and 3) and in particular in patients receiving a mismatched donor. Recipients of matched donor showed increased OS but PAM score could not identify patients with different outcome. These results could be likely addressed to the beneficial effects of PTCy use in the matched donor setting. Further validation of our results in different transplant centers and cohorts of patients are needed. Integration of PAM with other pre-transplant scores, in particular with HCT-CI, remains crucial to better define the main outcomes of each individual patient. Disclosure: Nothing to disclose
Background: The introduction of tyrosine kinase inhibitors (TKIs) has significantly improved the prognosis of Ph-positive (Ph + ) B-cell acute lymphoblastic leukemia (B-ALL). However, there are still a significant proportion of patients will relapse and allogeneic hematopoietic stem cell transplantation (allo-HSCT) are the most effective treatment for them. In current study, the efficacy and safety of allo-HSCT for the patients with relapsed/refractory (r/r) Ph+ B-ALL were evaluated. Methods: Between November 2018 and November 2021, 29 consecutive patients with r/r Ph+ B-ALL who received allo-HSCT in our hospital were included. The median age was 28 (3-59) years old. The median disease course was 21 (5-59) months. All patients were in complete remission and eight patients (27.6%) were MRD positive (2 detected by flow cytometry, and 6 detected by RT-PCR) before HSCT. 21 (72.4%) get remission by chimeric antigen receptor T-cell (CART) therapy. Eleven patients (37.9%) had T315I mutation. Five patents (17.2%) underwent the second transplantation. Two patients (6.9%) received allo-HSCT from sibling matched donrs, nineteen patients (65.5%) from haploidentical donors and eight patients (27.6%) from unrelated donors (HLA 10/10 or 9/10 matched). Myeloablative conditioning regimens with total body irradiation (fractionated, total 10 Gy) /etoposide (200mg/m2 x 3) /fludarabine (30mg/m2 x 5) or cyclophosphamide (1.8g/m2 x 2) /rabbit anti-T-cell globulin were used. Cyclosporine, mycophenolate mofetil and short-term methotrexate were employed for graft-versus-host disease (GVHD) prophylaxis. All patients received maintenance regimens with sensitive TKIs based on their ABL1 gene mutations up to 2 years post-transplantation. Results: The two-year OS and LFS of all included patients. All patients achieved durable engraftment. the median follow-up time was 8 (1-36) months and two-year OS and LFS were both 79%. Only two patients relapsed, one patient died of relapse and the other one is receiving post-transplantation CART therapy. Three patients developed grade III~IV aGVHD and two patients had extensive cGVHD. All of them were resolved with immune-suppressants except two patients died from grade IV aGVHD. One patient died of severe viral pneumonia seven months after HSCT. Seven patients had CMV reactivation and no patient had EBV reactivation. Five patients had mild hemorrhagic cystitis. One patient occurred severe bacterial pneumonia and was cured with antibiotics. The non-relapse mortality (NRM) was 10.3% (3/29). For the eleven patients with T315I mutation, only one patient relapsed and died of relapse. The two-year OS and LFS were both 87.5%. There was no patient with T315I mutation died of transplant-related adverse events. For the 21 patients who got remission by CART therapy, the two-year OS, LFS and NRM were 85.3%, 85.3% and 9.5% (2/21), respectively. For the 8 patients with MRD positive before HSCT only one patient relapsed 28 months after HSCT and there was no transplant-related death. Conclusions: Our results indicate that allo-HSCT is an excellent therapeutic method for r/r Ph+ B-ALL. The two-year OS and LFS can reach to 79%, in addition, NRM is quite low 10.3% (3/29). Even for patients with T315I mutation or MRD positive before HSCT, survival was also remarkedly good with the two-year OS and LFS both more than 70%. Disclosure: There is on conflicts of interesting to disclosure.
Background: The use of post-transplant cyclophosphamide (PTCY) as a graft-versus-host disease (GvHD) prophylaxis has led to important improvements in the haploidentical setting. Following this, PTCY was introduced as a safe and feasible option in the mismatched and matched unrelated donor settings. Nevertheless, the main GVHD strategy to date remains T-cell depletion with ATG in one antigen HLA-MMUD (9/10 MMUD). Data comparing the two GVHD strategies in patients undergoing MMUD 9/10 for lymphoproliferative disease are limited. Methods: We compared PTCY versus ATG as GvHD prophylaxis in patients with lymphoproliferative diseases undergoing a first 9/10 MMUD HSCT with a reduced intensity conditioning regimen from 2009 to 2019. Adult patients in all disease status for which high-resolution HLA-allele typing was available in the LWP/EBMT data registry were included. Patient receiving PTCY were matched to patients receiving ATG for age, disease status at transplant, source of stem cells, CMV serology and gender. Results: A total of 322 patients were identified (n = 287 for ATG group and n = 35 for PTCY group). According to the above mentioned variables, 56 patients receiving ATG were identified and matched with 31 patients receiving PTCY. Among the group of 87 patients paired up, diagnosis was Hodgkin Lymphoma in most of the cases (27% and 40% in the ATG and PTCY group, respectively). The majority of patients had received a previous autologous HSCT (62% in the ATG and 68% in the PTCY group, respectively). Graft stem cell source was peripheral blood in all patients of both matched groups. The majority of patients were in complete remission at HSCT (67% and 66% in the ATG and PTCY group, respectively). The most frequent GvHD prophylaxis in the ATG group was cyclosporine A (CsA) and methotrexate (MTX) (n = 24; 43%), followed by CsA and mycophenolate mofetil (MMF) in 29% of the cases. In the PTCY group, the majority of patients (n = 18; 58%) received CsA and MMF, while tacrolimus and MMF (19%) were the second most used associated immunosuppressive agents. The median follow-up was 5 (3.6-6.8 IQR) years for the ATG group and 2.6 (2-4.1 IQR) years in the PTCY group. No significant differences were detected across the two groups. Karnofsky performance status was > = 80% in 100% and 90% of the ATG and PTCY groups, respectively (p = 0.04). A similar engraftment rate was observed for both groups (98% and 100% in ATG and PTCY groups, respectively). The two year- progression free survival was 48% in patients receiving ATG and 41% in those receiving PTCY (NS). Grade III-IV acute GVHD and GRFS were not significantly different in patients receiving ATG or PTCY. No differences were observed in relapse incidence (35% versus 26%, p = 0.14), non-relapse mortality (17% versus 33%, p = 0.39) and overall survival (64% versus 51%, p = 0.25). Conclusions: In patients with lymphoproliferative diseases undergoing 9/10 MMUD HSCT, PTCY as GVHD prophylaxis could be a safe and feasible option. Prospective randomized trial will help to better understand the effectiveness of adding PTCY into different platforms, comprising MMUD 9/10 HSCT. Clinical Trial Registry: not applicable Disclosure: Nothing to declare
Background: Transplant-associated thrombotic microangiopathy (TA-TMA) is an endothelial injury syndrome with a wide range of presentations that could lead to multiorgan injury after allogeneic hematopoietic stem cell transplantation (HSCT). Morbidity and mortality from TA-TMA remain high. Therefore, identifying patients at the highest risk for severe disease and proper management is critical. Recently, a prospective study with children and young adults (Jodele et al, Blood 2014) has proposed new diagnostic criteria, including preceding proteinuria (>30 mg/dL) and elevated sC5b-9 levels, and risk/prognostic factors for TA-TMA, which needs to be validated in adult population. Thus, we investigated risk and prognostic factors for TA-TMA in recent large adult cohorts with acute myeloid leukemia (AML). Methods: We used Cho’s criteria for TA-TMA diagnosis previously published by our group (Cho et al., Transplantation 2010) and analyzed the incidence, risk, and prognostic factors in two independent cohorts of training (n = 382, 2012 to 2015, retrospective) and validation (n = 231, 2016 to 2017, prospective). A total of 613 patients with AML who received allogeneic HSCT from matched siblings (n = 260), unrelated donors (n = 167), or haploidentical family donors (n = 186) was analyzed. Results: TA-TMA developed in 72 patients, and the cumulative incidence was 12.6% (95% confidence interval (CI) 10.0 – 15.5). The validation cohort presented with higher TA-TMA incidence than the training cohort (18.8% vs. 8.9%, p < 0.001). Both cohorts had no significant difference in pre-transplant characteristics with an exception of the higher number of haploidentical transplantation in the validation cohort. In terms of risk factors for TA-TMA, multivariate analysis revealed that preceding proteinuria (≥30 mg/dL) was a significant factor for TA-TMA in both the training cohort (HR 5.94, 95% CI 2.77 – 12.77, p < 0.001) and validation cohort (hazard risk (HR) 5.29, 95% CI 2.51 – 11.16, p < 0.001). Preceding hemorrhagic cystitis was a significant risk factor only in the validation cohort (HR 1.99, 95% CI, 1.07 – 3.71, p = 0.003). In entire cohort, elevated LDH x1.5 above normal (HR 1.66, 95% CI, 1.04 – 2.64, p = 0.034), proteinuria (≥30 mg/dL, HR 5.69, 95% CI 3.30 – 9.82, p < 0.001), and CMV disease (HR 1.99, 95% CI, 1.18 – 3.37, p = 0.010) were significant risk factors. In terms of prognostic factors, preceding proteinuria (≥300 mg/dL), concurrent hemorrhagic cystitis in any grades, acute GVHD grade II to IV, and bacterial and/or fungal infections were associated with poor overall survival (OS) and non-relapse mortality (NRM), but only preceding proteinuria (≥300 mg/dL) remained significant in the multivariate model (OS; HR 2.54, 95% CI 1.45 – 4.43, p = 0.001 and NRM; HR 2.98, 95% CI 1.57 – 5.66, p < 0.001). Conclusions: This study found out potential risk and prognostic factors for TM-TMA in a recent adult retrospective cohort and validated them in an independent prospective cohort. Our data highlight that the preceding proteinuria as a marker for renal involvement of TA-TMA would be an important risk and prognostic factor for TA-TMA in adults. Prospective multicenter studies are warranted to confirm our findings in an adult population. Clinical Trial Registry: CRIS KCT0002261 https://cris.nih.go.kr/cris/search/listDetail.do Disclosure: Nothing to declare
Background: Patients status post stem cell transplant (SCT) are at high risk for severe COVID-19 infections (1). During this pandemic, SARS-CoV2 vaccines have been considered as the priorities for this vulnerable population. Previous reports shed light on the poor immune response after SARS-CoV2 vaccines in recipients of SCT especially in those patients on immunosuppressive medications(2). In this study, we report SARS-CoV2 IgG after COVID19 vaccinations in patients post autologous and allogeneic SCT. Methods: This is a cross-sectional study enrolling 43 patients post SCT (autologous 20, allogeneic 23). who received COVID-19 vaccination (Pfizer-BioNTech or Oxford/AstraZeneca). The participants were recruited between 1st of May to the end of November 2021. The blood samples were collected and tested for the presence of IgG antibodies against SARS-CoV2 spike protein using enzyme-linked immunosorbent assay (ELISA). Results: Around 60% of post SCT patients have COVID19 protective humoral immunity after 1st dose of COVID19 vaccination(1A). This immune response has increased dramatically after the second dose regardless of the type of the vaccine (1A,B). No observed significant differences in immune responses in patients post autologous or allogeneic SCT (1C). A similar response was observed in patients with Non-malignant and malignant conditions (1D). Most of post SCT patients have good immune response after the 2nd dose of COVID19 vaccinations apart from one patient who had poor T cell engraftment despite holding immunosuppressive medications and another patient who is currently on B cell ablative therapy (1F). No major concerns regarding immune protection was observed in patients either on GVHD prophylactic medications (Cyclosporine, Tacrolimus, Sirolimus and Ruxilitinib) or biologics (Nivolumab, Bortezomib, Ixazomib) apart from B cell ablative medications. Conclusions: Post SCT patients have good response to SARS-CoV2 vaccination. This response was observed to be increasing after subsequent dosing regardless of the original diagnosis. Our results provide evidence that the SARS-CoV2 vaccination generates protective immunity in this high risk population even if they are on immunosuppressive medications. References: 1- Q. Wang, N.A. Berger, R. Xu, Analyses of risk, racial disparity, and outcomes among US patients with cancer and COVID-19 infection, JAMA Oncol. 7 (2) (2021) 220–227. 2- Oluwafeyi Adedoyin, Sharmela Brijmohan, Ross Lavine, Fausto Gabriel Lisung. Undetectable SARS-CoV-2 active adaptive immunity-post-vaccination or post-COVID-19 severe disease-after immunosuppressants use. BMJ Case Rep. 2021 Nov 29;14(11). Disclosure: No conflict of interest
Background: Multiple myeloma (MM) is the most common indication for autologous stem cell transplantation (ASCT), and outpatient models have been widely developed in this setting. In addition, the use of oral and subcutaneous treatments both in induction and maintenance, as well as the development of new technologies, has allowed these patients to continue their treatment without going to the hospital, thus improving their quality of life. Methods: Since March 2021, we have put in place a comprehensive home-care program for patients with multiple myeloma in a tertiary hospital. It is a multidisciplinary project in which nutrition, rehabilitation, psychology, pharmacy, hematology and nursing services collaborate to offer the patient individualized and quality care. Results: A total of 59 patients with MM have benefited from our program (Figure 1). Two patients have received an ASCT, going from a hospital stay of about 3 weeks to just 4 days (none of the patients had to be readmitted, completing the treatment at home).A total of 276 analytical extractions have been carried out at home, 427 calls by nurses, and an approximate saving of 30 face-to-face medical consultations per month, doing them virtually (videoconference or phone call).The entire program is coordinated by two hematologists specializing in multiple myeloma and transplantation. 2 nurses are in charge of the analytical extraction and assessment of the patients at home. There is 1 pharmacist who sends the medication to the home. It is a program where the optimization of resources and the use of new technological tools have made it possible to offer the patient with MM a personalized and quality treatment. Furthermore, external companies have collaborated in this innovation project (the car company “KIA” donated the car with which we make home visits) and the Spanish Association Against Cancer (AECC) has given the use of two homes close to the hospital for patients who live outside the hospital area can undergo ASCT and have all the benefits adhered to the program.The implementation of this program has allowed, on the one hand, to carry out multidisciplinary work (hematologists, specialist nurses, nutritionists, pharmacists and psychologists) focused on the patient with MM. On the other hand, the MM patient continues their treatment outside the hospital, thus improving their quality of life and having more self-control of the treatment they receive. Conclusions: The implementation of a home program for patients with MM (ASCT and oral treatment) is possible in a tertiary hospital with availability of digital technology. The multidisciplinary work and the personalization of the treatments in our patients increases their quality of life and even reduces the associated complications. Disclosure: No conflict of interest
Background: The use of post-transplantation cyclophosphamide (PT/Cy) is a well-established strategy that has been proved to achieve excellent results in haploidentical transplants with donor bone marrow cells. On the other hand, the use of PBSC in this setting is limited by the risk of an increased incidence and severity of GVHD compared to the use of bone marrow cells. Recently, new data have been reported describing the use of PT/Cy both in combination and without traditional immunosuppression, also in MFD and MUD donor transplants with promising data, such as reduced risk of GVHD and fewer viral reactivations. At our center, the use of PBSC has been consolidated for several years by performing a procedure that provides the selection of CD34 + and later addback of a controlled number of T lymphocytes (30x106/kg/recipient). This technique leads to rapid engraftment, rapid immunological reconstitution and low incidence of acute and chronic GVHD. Methods: In recent years, 4 transplant procedures have been carried out at our center using peripheral cells as a source of haploidentical donor stem cells and performing product manipulation with CD34 + selection and CD3 + addback with controlled number and subsequent post-transplant Cyclophosphamide. Results: Average neutrophil engraftment was reported on day +18, while that of platelets on day +30. There was no documented presence of significant acute GVHD or chronic GVHD. Only one case of invasive infection (Adenovirus pneumonia), resolved after targeted therapy. Conclusions: In our opinion this technique represents a promising alternative in the setting of haploidentical donor transplantation as it guarantees rapid engraftment, accordingly to the greater number of CD34 + cells (>10 x 106/kg), while not causing an increase in the incidence and severity of GVHD. Indeed, it combines the advantages of administering a high number of CD34 + cells, obtainable through PBSC, and those of the reduced number of lymphocytes T of the donor with the lowest risk of GVHD. Certainly, further data and research will be needed to consolidate this procedure. Disclosure: Nothing to declare
Background: Hematopoietic cell transplantation (HCT) remains the only curative option for aplastic anemia or MDS/AML in Fanconi anemia (FA) patients. We were interested in performing a predictor analysis for outcomes of HCT for FA patients across four large FA referral institutions using different transplant platforms. Methods: We conducted a retrospective analysis of prospectively collected data of FA patients undergoing their first HCT at Memorial Sloan Kettering Cancer Center (New York, USA), Princess Maxima Center, University Medical Center Utrecht, and Leiden University Medical Center (the Netherlands) between 2007 and 2019. No restrictions applied in terms of age, gender, indication (SAA, MDS/AML), HLA matching, conditioning regimen, and graft source or manipulation. These variables were also considered in analyses. Main outcomes of interest were event free survival (EFS) (events: relapse, graft failure (GF), treatment related mortality) and overall survival (OS). Other outcomes of interest were treatment related mortality (TRM), acute graft vs. host disease (aGvHD) grade II-IV, extensive chronic GvHD, and posttransplant malignancies. COX proportional hazard models and Fine and Gray models for competing risk were used for analyses. Results: 89 patients were included: 64 SAA +/− cytogenetic abnormalities and 25 MDS/AML. Median age at transplant was 9.2 years (1.7 - 44.0 years). 52 (58.4%) received a T-replete HCT: 40 (77%) from bone marrow and 12 (23%) from cord blood (10 HLA-mismatched; 19.2%). 37 (41.6%) underwent a TCD-HCT of whom 20 (54.0%) were HLA-mismatched. Conditioning regimens included Cyclophosphamide (Cy) and Fludarabine (Flu) (n = 52), Total Body Irradiation (TBI) /Cy/Flu (n = 11) and Busulfan/Cy/Flu (n = 26). The 5-year OS and EFS were 83.2% (75.3-91.9%) and 74% (65-84.2%), respectively. Age >18 was found to be the only multivariate (MV) predictor for OS (HR 9.1, 95%-CI 1.3– 61.7, p = 0.024), while for EFS, in addition to age >18 (HR 8.9, CI 2.2 – 36.7, p = 0.002), HLA-matching was a MV predictor (HR 4.7, 95%-CI 1.7–12.6, p = 0.002). In the pediatric group (age < 18, n = 73), TCD was a borderline MV predictor (HR 8.4, CI 0.9–76.6 p = 0.059) with 5-year OS of 73.0% (54.7 – 97.4%) in TCD vs 100% for T-replete HCT. For TRM the only MV predictor was age >18 (HR 20.1, CI 1.7 – 236.5, p = 0.017). Age above or below the median within the pediatric cohort was not a predictor for OS, EFS and TRM. The cumulative incidence of day100 grade II-IV aGvHD and 5-year cGvHD was 6.7 % and 4.5%, respectively. Relapse in the MDS/AML subgroup was only seen in 4 (16%). GF was seen in 9 patients (TCD 6/37; 16%; T-replete 3/52; 5.7%). Six patients developed malignancy after HCT; of these, two had preceding GvHD. Conclusions: Survival chances after HCT for FA are excellent and associated with low toxicity. Age above 18 is the main predictor for inferior survival (driven by TRM). For pediatric patients undergoing a T-replete transplant with an HLA-matched donor (or HLA-mismatched cord blood) survival was excellent (100% survival at 5yrs). For patients without a good matching donor, TCD offers a very good alternative. Clinical Trial Registry: None Disclosure: Nothing to declare
Background: Allogeneic hematopoietic cell transplant (HCT) recipients are highly susceptible to COVID-19 and related complications. This vulnerable population has been prioritized for vaccination despite limited safety data. Preliminary studies including heterogeneous series of HCT recipients reported variable frequency of adverse events (AE) including GVHD flares and vaccine-related cytopenias. Methods: This single center prospective study describes frequency and severity of mRNA-1273 SARS-COV2 vaccine AE in 54 alloHCT recipients less than two years away from transplantation date. AE were assessed through calls during the seven days after each dose and graded from 0 to 4 according to the original phase III study (Baden. N Engl J Med 2021). Results: Patients’ characteristics are exposed in Table 1. Median age was 53.5 years (25-73) and 48.1% were females. Median time from HCT to first vaccine dose was 13 months (3-26). AE incidence of any kind (94.4% at dose 1 and 85.2% at dose 2) was similar to that reported in the pilot study. Local AE were the most common, pain at site of injection being the most frequent one (92.6% and 85.2%, dose 1 and 2, respectively). Systemic AEs were rarer, affecting 48.1% and 57.4% (1st and 2nd dose respectively) of patients. Figure 1 shows incidence and grade for every AE. Systemic AEs were more common and severe after the second dose (p = 0.007). Only 5.6% of patients presented grade ³3 AE, none requiring hospitalization. Neither vaccine-related deaths nor cytopenia or GVHD flares were observed. Female sex was significantly associated with a higher degree of AEs at the first dose (OR 3.94, 95% CI 1.14-13.58, p = 0.03). Time from HCT was associated with a higher degree of systemic AEs at the second dose (OR 1.09, 95% CI 1.01-1.18, p = 0.04), specifically with higher degree of chills (OR 1.15, 95% CI 1.01-1.31, p = 0.04). Prior COVID-19 infection correlated with fever at the second dose (OR 10.22, 95% CI 1.21-86.59, p = 0.03), though only five patients had had COVID-19. Age, graft source, donor type, donor age, number of CD34 + cells infused, conditioning type, GVHD prophylaxis, disease type and status, GVHD (either past or active), immunosuppression (neither active/inactive nor its type), living with vaccinated people, T-cell chimerism and some other lab values (lymphocyte, IgG, CD4+, CD8+, and CD19+ counts) had no relation to any type of AE severity. Conclusions: mRNA-1273-related AE in early alloHCT recipients were comparable to the general population. None presented new-onset cytopenias or GvHD flares. Female sex, prior COVID-19 infection and longer time from HCT were associated with a higher rate of AE. Disclosure: Nothing to declare.
Background: Despite offering an opportunity of cure to patients with hematologic diseases, allogeneic stem cell transplantation (aSCT) and their related complications significantly affect quality of life (QoL). The purpose of this study was to evaluate QoL of aSCT long-term survivors (five-years from the aSCT) and determine those factors that could have an impact in it. Methods: This is an observation study of sixty-seven patients who had undergone a single aSCT in our institution during January 2011 to December 2015. Participants answered in a telephone interview two validated aSCT-QoL questionnaires (FACT-BMT (score from 0 to 148) and EQ-5D-5L (score from 0 to 100)), and questions about employment, medications and comorbidities after transplantation. Results: Long-term survivors’ characteristics are shown in Table 1. Median time from aSCT to interview was 7 years (IQR 6-9). FACT-BMT and EQ-5D-5L mean scores were 115 to 148 and 73 to 100 respectively. FACT-BMT score seemed higher in allogenic bone marrow stem cell transplantation (SCT) compared to allogenic peripherical blood SCT – 118 versus 107 points respectively (p-value 0.022). An association between QoL-working status and QoL-drugs intake was also observed. Statistically significant higher QoL FACT-BMT scores were present in patients who were working (123 vs. 111, p-value 0.002) and who were not taking drugs (125 vs. 113, p-value 0.026) as is summarized in Image 1. Patients with active graft-versus-host disease (GVHD) had worse EQ-5D-5L score (78 vs. 65, p-value 0.002). We did not find differences in QoL regarding age and gender of patients and donors, previous autologous SCT, disease status at aSCT or previous acute/chronic GVHD. Patients with current pulmonary complications (mainly obliterative bronchiolitis and recurrent infections) have worse FACT-BMT score (102 vs. 117, p-value 0.010). By contrast, cardiovascular, hormonal, and neoplastic comorbidities do not impact FACT-BMT score. Factors associated with a worse QoL score in the multivariate analysis were drug-intake, active GVHD and SCT source. Having an active GVHD at the time of the interview increased the risk of a worse QoL (defined as having under a 75th percentile score on one or both scores) by 1,7 (95%CI 1,1-9,4 p = 0,029) and using PB as stem cell source by 2,4 (95%CI 1,1-5,8 p = 0,042). Table 1. Image 1. Conclusions: QoL for adult long-term aSCT survivors is close to the 75th percentile in our study. Drug-intake, previous chronic or active GVHD and not having reincorporated to active work are associated with a worse QoL. Among factors related with the procedure, just the use of peripheral blood seems to influence the aSCT QoL scores. Disclosure: Nothing to declare.
Background: The spectrum of indications to HSCT in the pediatric age group is wide, starting from hemoglobinopathies, which are the most frequent life-threatening non-communicable disease of children. Transplantation can cure over 85% of low risk children with severe thalassemia directly in middle income countries and is highly cost-effective. Methods: In 2016 the first HSCT Unit both for adult and children was developed in Iraqi Kurdistan at Hiwa Cancer Hospital of Sulaymaniyah, thanks to a capacity building project funded by the Italian Agency for Development Cooperation. Here we present a 3 years follow up on 47 thalassemia pediatric patients transplanted from matched sibling donors from Oct 2016 to July 2021 with granulocyte colony stimulating factor priming. Follow-up was updated in Nov 2021. Results: Mean age was 6.6 years; mean total nucleated cells (TNC) received was 17.2 x 108/Kg; mean time to engraftment of neutrophil > 500 x 109/L was 17 days and of platelets >50 x 109/L was 19 days. With a median follow-up of 2.6 years, the 3-years event free survival (EFS) and survival (OS) were 82.8% (SE 5.5) and 87.1% (4.9); the 3-yrs cumulative incidence risk of rejection was 4.3% (2.9). Treatment related mortality (TRM) was 12.9% (4.9); 3/6 patients died due to SarsCov2 infection. 10 patients (21%) presented acute graft-versus-host-disease (GVHD) grade III-IV and 14 (30%) moderate/severe chronic one. No statistically significant correlation was found between incidence of aGVHD grade III-IV and median count of white blood cells of the marrow (p = 0.84), white blood cells of the peripheral blood of the donor at the time of the donation (p = 0.98) and CD3 + cells in the marrow (p = 0.81). The same analysis was done for moderate/severe cGVHD (p = 0.55, 0.82 and 0.57 respectively). Even looking to mean values of plasma cyclosporine before the onset of the GVHD (week 1-2-3-4-8-12-16-20) there was no statistical difference between patients with/without aGVHD grade III-IV (p = 0.44) and moderate/severe cGVHD (p = 0.97). Conclusions: Based on our experience HSCT is a feasible and safe procedure in MICs.The incidence of both acute and chronic GVHD was not correlated to donor’s marrow WBC and CD3 + counts or peripheral WBC count at the moment of donation or recipient’s mean values of cyclosporine levels. However GvHD resolved in the vast majority of patients who are now free of any immunosuppression. Compared with our previous analysis on 35 patients, TRM was higher than expected due to SarsCov2 infection; for this reason the BMT program has been temporarily discontinued. Disclosure: No disclosure
Background: Hematopoietic stem cell transplantation (HSCT) is the only curative treatment for a number of malignant and non-malignant disorders. However, only in the 25% of cases is possible to identify a matched family donor. In lack of this, the probability to find a fully matched unrelated donor (MUD) is about 30-70% of cases. Patients given an allograft from a donor with a single antigenic or allelic disparity (mismatch, MM) had an increased risk of both acute GVHD and TRM as compared to patients receiving the transplant from fully matched donors; moreover, disparities at two or more loci further increase this risk. A possible strategy to minimize risks associated with HLA-mismatch is to add Abatacept to standard GVHD prophylaxis [Watkins, 2021]. Abatacept is a fusion protein that selectively inhibits T‐cell co-stimulation by binding to CD80/CD86 on antigen‐presenting cells and blocking CD28-mediated signaling. Methods: From February 2021 to October 2021, Abatacept was administered to 12 pediatric patients (age 2-20 years) who received a transplant from a mismatched unrelated donor (mMUD) or PBSCs from a 10/10 MUD. The drug was administered intravenously at the dose of 10 mg/kg on days -1,+5,+14 and +28; moreover, one patient received an additional infusion on day +42 and two children two administrations on days +42 and +60 (see below for details). Results: Seven patients were affected by malignant disorders and five by non-malignant diseases. Ten patients out of 12 were transplanted from mMUD, while 2 received the transplant from 10/10 MUD. The source of stem cells was peripheral blood in 4 cases(2 patients received PBSCs from fully matched donor). With a median follow up of 6 months, 3 of 12 patients developed grade III-IV aGVHD. In details, one patient presented acute grade IV GVHD on day +70 (gut stage 4); the second one developed acute grade IV GVHD on day +85 (gut stage 3 ed hepatic stage 4) and the last late acute grade IV GVHD on day +200 (gastrointestinal stage 4). The first two patients received PBSCs from 9/10 mMUD with a MM in locus A; the last one underwent HSCT from mMUD with MM in locus B and stem cell source was represented by bone marrow. After the occurrence of these cases of GVHD, we decided to administer additional doses of abatacept to the following patients; none of these additional 3 patients developed acute GVHD. No adverse events was related to the infusion of Abatacept. The comparison with an historical cohort of 29 patients treated with standard prophylaxis showed comparable cumulative incidence of aGVHD; however, GVHD occurred later in patients receiving Abatacept (Figure 1). Conclusions: Our data confirm the efficacy and safety of Abatacept in the prevention of aGVHD in the context of HSCT from mMUD and/or using PBSCs as source of stem cells. Notably, the administration of additional doses (on days +42 and + 60), could further extend the protective effect of this treatment strategy. Disclosure: No disclosure
Background: Clinical efficacy of autologous stem cell transplantation (ASCT) in treating malignancies has been attributed to high-dose chemotherapy. However, ASCT is not curative for a number of patients with malignant diseases. Many hypothesis for the high relapse rate after ASCT have been proposed. In brief, an early posttrasplant lymphocyte recovery could play a critical role for general outcomes. Some previous reports demonstrated that early lymphocyte recovery at day 15 after ASCT (ALC-15) had an impact on overall survival (OS) and disease-free survival (DFS). Methods: Between January 2015 and December 2020, all consecutive patients diagnosed with multiple myeloma (MM) and non-Hodgkin lymphoma (NHL) who underwent an ASCT in the Hospital Universitari Mutua Terrassa were retrospetively enrolled in our study. All the stem cell products collected were processed in the same cell therapy laboratory at Banc de Sang i Teixits. Median (range) follow-up for the entire cohort was 35 (0-81) months. The schema for stem cells mobilization was granulocyte-colony stimulating factor (G-CSF) alone for patients diagnosed with MM or G-CSF plus chemotherapy for patients diagnosed with NHL. Additionally, plerixafor was administered in poor mobilizers with the aim to collect a sufficient cell dose required for an ASCT. We considered an ALC-15 as predict factor for OS and DFS as primary endpoint. We registered demographic, clinical and blood test data to perform the analysis. Results: A total of 142 patients underwent an ASCT during the period of study. Median (range) age was 60 years (23-71) and 86 (61%) patients were male. Underlying diseases were MM (n = 81, 57%), diffuse-large B-cell lymphoma (n = 32, 23%), primary central nervous system lymphoma (n = 7, 5%), follicular lymphoma (n = 9, 6%) and mantle cell lymphoma (n = 13, 9%). A total of 75 patients (53%) achieved a complete response (CR) and 67 patients (43%) achieved a partial response (PR) at time of transplantation. Median (range) for neutrophil and platelet engraftment was 13 days (range 8-27) and 18 days (range 8-57), respectively. Median (range) cell count for ALC-15 recovery was .55/µL (range 0-2.7). Univariate analysis for survival at 80 months after ASCT demonstrated that disease status (CR vs. PR) at time of transplantation [(OS 72% vs. 60% (P = .04) and DFS 73% vs. 48% (P = .05)], infused CD34 + cell dose (⋝ 3 x 106/kg vs. < 3 x 106/kg) [OS 79% vs. 62%, (P = .03) and DFS 69% vs. 58%, (P = .02)], absolute neutrophil engraftment (⋝ 500 cells/μL vs. < 500 cells/μL), [OS 79% vs. 67%, (P = .02) and DFS 72% vs. 61%, (P = .015)] and ALC-15 (⋝ 500 cells/μL vs. < 500 cells/μL) [OS 73% vs. 56%, (P = .001) and DFS 65% vs. 36%, (P = .001), were predictors of higher OS and DFS (Figure 1). Conclusions: We concluded that patients who achieve an ALC-15 ⋝ 500/μL after ASCT have a superior OS and DFS than patients who have < 500 lymphocyte/μL. ALC-15 is correlated with clinical outcomes and requires further study to demonstrate its impact as an independent prognostic factor in a multivariate analysis. Disclosure: Nothing to declare
Background: To investigate the efficacy and safety of preemptive/salvage therapy with venetoclax (Ven) in patients with recurrence after allogeneic hematopoietic stem cell transplantation (allo-HSCT). Methods: Retrospective analysis the clinical data of 25 patients with molecular biology or morphological recurrence after allo-HSCT treated with Ven in our center from 2021.2 to 2021.11, 15 patients were treated preemptive (P-group) and 10 patients were salvage (S-group). In the P-group, the median time from recurrence to the application of Ven was 2.5 (0-12.5) months. The median course of treatment was 2 (1-4). On the 7th day of the first course of treatment, the median concentration of Ven was 1945 (688-5383) ng/ml. In the S-group,the median time from recurrence to the application of Ven was 0 (0-1) months. The median course of treatment was 1 (1-2). On the 7th day of the first course of treatment, the median concentration of Ven was 2419 (1200-6155) ng/ml. Results: In the P-group, after one course of Ven treatment, 8 cases of minimal residual disease (MRD) turned negative, 4 cases of MRD decreased by 50% compared with that before treatment, 3 cases were ineffective, and the overall response rate (ORR) was 80%. The concentration of Ven < 1000 ng/ml or > 3000 ng/ml was 33.3%, and the concentration > 1000 ng/ml and < 3000 ng/ml was 83.3%. Two patients with TP53 mutation before transplantation turned negative after treatment. Grade 3/4 neutropenia occurred in 5 patients (33%) and grade 3/4 thrombocytopenia occurred in 5 patients (33%). No fatal cases of severe infection occurred. In the S-group, after one course of Ven treatment, 1 case was in complete remission (CR) and MRD turned negative, 2 cases were CR but MRD was still positive, 3 cases were in partial remission (PR), 4 cases were ineffective, and the oRR was 60%. The concentration of Ven >3000 ng/ml was 25%, and the concentration > 1000 ng/ml and <3000 ng/ml was 33.3%. One patient with TP53 mutation before transplantation reached CR after ven salvage treatment. Grade 3/4 neutropenia and grade 3/4 thrombocytopenia occurred in 10 patients (100%). One patient died of severe pulmonary infection. The median follow-up was 4.5 (1-8.5) months. No acute graft-versus-host disease (aGVHD) occurred after Ven treatment; There was no obvious abnormality in liver and kidney function. The overall survival rate (OS) of the P-group was (70.2 ± 12.7)%, and that of the S-group was (50.0 ± 15.8)%, P = 0.171. Conclusions: The preemptive therapy with Ven after all-HSCT in patients with hematological malignancies is a promising treatment method. The early application and monitoring of drug concentration is expected to improve the curative effect, and the toxic and side effects can be tolerated. Ven can achieve short-term curative effect in the salvage therapy of allo-HSCT. Monitoring the drug concentration is expected to improve the curative effect, but other treatments still need to be bridged in the follow-up to maintain the curative effect. Disclosure: Nothing to declare
Background: PGF is a severe complication of HSCT occurring in 5 to 27% of patients. We aimed to determine administration modalities of hematopoietic stem cell boost for patients with PGF, hematological response and tolerance. Methods: We realized a retrospective, multicenter, observational study from 2013 to 2019. The data were collected from the registry of the French Society SFGM-TC to evaluate the outcome of patients with PGF who received a stem cell boost. Results: Fifty-five patients were included, with a median follow-up of 346 days. Data from 12 HSCT centers in France were analyzed. Median age was 14 years (0 – 66), with 28 children, 5 adolescents and young adults and 22 adults. Thirty-three patients were transplanted for non-malignant diseases. Thirty-two patients had received a myeloablative conditioning. Grafts were from 16 matched related, 14 matched unrelated, 10 mismatched unrelated and 15 haploidentical donors. Eleven patients had a major ABO mismatch. Twenty five patients experienced acute Graft Versus Host Disease (GvHD) and 15 experienced chronic GvHD after HSCT. Forty-eight patients had a complete donor chimerism post HSCT, and 7 patients a mixed chimerism. There were 45 thrombocytopenia (<30G/L), 28 neutropenia (<0.5G/L) and 19 anemia (Hb < 8g/dl). Sixteen patients had a monocytopenia, 23 a bicytopenia and 10 a pancytopenia. The median delay between allo-HSCT and boost was 119 days (9-1949). Among 55 patients, 43 received a stem cell boost with CD34 positive selection. For the CD34 positive selected boosts, median cell number reinjected was of 5.91.106 CD34/kg (IQR 3.22 – 9.15) and of 3.14.103 CD3/kg (IQR 0 - 62.5). Complete response (CR) at 1 month, defined as platelets counts above 50 G/L, hemoglobin above 10 g/dL and neutrophils above 1.5 G/L, without death, was of 39.58%. Hematological response was stable during 12 months following boost, regardless of the hematopoietic lineage initially involved, as shown by Figure 1. Very few patients with no response or partial response at 1 month experienced a later response. A longer delay between HSCT and HSC boost was associated with a lower rate of complete response at 1 month (p = 0.004). Only 2 patients experimented GvHD de novo after CD34 + selected stem cells boosts. Overall mortality was of 47.2%. Sixteen patients (29%) died from infections. All patients who died from infections were non responders at 1 month. Conclusions: Hematopoietic stem cells boosts (mainly CD34 + selected) were effective for nearly 40% of patients with PGF after HSCT, and safe. However, mortality rate was very high in this cohort of patients and mostly caused by infections, occurring in patients with no CR at one month post boost. Therefore, another treatment should be considered for patients without CR at 1 month. Disclosure: Nothing to declare
Background: Incidence of adverse reactions (ARs) during the infusion of peripheral blood stem cells (PBSC) and bone marrow (BM) products depends, not only on patient’s characteristics, but also on cellular and non-cellular elements of the product. Aim is to evaluate ARs reported in recipients of allogeneic haematopoietic stem cells (HPC) grafts. Methods: A retrospective analysis of infusion-related toxicity of allogeneic PBSCs and BMs was conducted on paediatric and adult patients. The study included a data analysis of infusion reports for patients transplanted from July 2013 to July 2021. Allogeneic PBSC and BM grafts were usually infused fresh, but during COVID-19 pandemic due to logistic reasons PBSC were cryopreserved using dimethyl sulfoxide (DMSO) as a cryoprotective agent. According to institutional standard operating procedure, all patients received intravenous premedication before graft infusion consisting of chloropyramine-chloride, and in the case of ABO incompatible graft also methylprednisolone. Patients were monitored for vital signs and symptoms of the toxicity during and after infusion. ARs were classified according to Common Terminology Criteria for Adverse Events (CTCAE) version 5.0. Results: Total number of patients included in the study was 507: 265 males (52,3%) and 242 females (47,7%), median of age 47 years (range 0-70). They were divided into three groups according to HPC source: 110 (21,7%) patients received BM, 298 (58,8%) fresh PBSCs and 99 (19,5%) cryopreserved PBSCs. The incidence of ARs was 14,5% in BM group, 13,5% in fresh PBSC group, and 23,2% in the group that received cryopreserved PBSC. Patients’ characteristics including gender, ABO incompatibility and diagnosis were not significant predictors for ARs occurrence in either group. However, younger age was significant predictor for occurrence of ARs during infusion in the patients that received cryopreserved PBSC (p = 0,03). According to CTCAE classification, grade 1 ARs were the most common in all three groups (59%). In BM group, febrile reactions (25%) and blood pressure rise (35,7%) were the most common. One case of Takotsubo cardiomyopathy after infusion of BM was reported and confirmed by echocardiography with asymmetry of regional function and positive enzymes. According to our knowledge it is the first case of Takotsubo cardiomyopathy reported after HSCT so far. Febrile reactions were the most common (59%) ARs reported in fresh PBSC group. Cytokine release syndrome was reported in two patients during infusion of fresh PBSC, and both patients required monitoring in the intensive care unit. One patient recovered successfully, but the other one deceased due to infectious complications. In cryopreserved PBSC group, ARs were mostly related to DMSO toxicity, such as nausea/vomiting (34,8%), rash (13%), hot flushes (13%), and cough (13%). Grade 5 ARs were not reported in any group of patients. Conclusions: The highest incidence of ARs was observed in patients that received cryopreserved PBSCs. ARs were mostly mild, classified as grade 1, and they resolved spontaneously or after symptomatic treatment. However, considering that severe, life–threating ARs were reported, we strongly recommend careful patient monitoring during the graft infusion to recognise potential ARs and prevent possible further complications. Disclosure: Nothing to declare.
Background: Allogeneic hematopoietic stem cell transplantation (allo-HSCT) is one of the consolidation modalities for patients with T-cell lymphoblastic lymphoma (T-LBL), however, the best conditioning regimen needs to be explored to decrease the relapse risk and improve the survival of patients with T-LBL Methods: 40 patients with T-LBL undergoing allo-HSCT in our center were retrospectively analyzed. Results: 40 patients with T-LBL undergoing allo-HSCT in our center were retrospectively analyzed. 23/40(57.5%) received total body irradiation (TBI)-based conditioning regimen, while 17/40(42.5) received busulfan(BU)-based conditioning regimen. All patients achieved donor engraftment and neutrophil and platelet engraftment time were similar between TBI- and BU-based groups (P = 0.283andP = 0.368, respectively).TBI-based conditioning regimen significantly increased the cumulative incidence (CI) of grade II-IV aGvHD as compared with BU-based regimen (13.04%vs 0%, P = 0.000). TBI-based regimen significantly decreased the CI of relapse (CIR) after transplantation, the 1-year and 2-year CIRs were 9.11% and 9.11% in TBI-based group respectively, which were significantly lower than that of 41.18% and 49.58% in BU-based group (P = 0.006). The 2-year probabilities of over survival (OS) and relapse-free survival (RFS) were 83.0% (95%CI, 63.4--100%) and 74% (95% CI, 54.4-93.6%) in TBI-based group respectively, which were both higher than that of 35.0%(95%CI,0.0%-72.2%) and 50.0% (95%CI,24.5-75.4%) (P = 0.020; P = 0.081, respectively)in BU-based group.In multivariate analysis, The presence of B symptoms at diagnosis and BU-based conditioning regimen significantly increased the risk of relapse (HR7.662, 95% CI, 1.056-55.593, P = 0.040; HR 33.32, 95% CI, 6.662-166.67, P = 0.000). Conclusions: These results suggested that TBI-based regimen was an optimal conditioning regimen for patients with T-LBL receiving allo-HSCT with a decreased risk of relapse and an improved OS. Disclosure: The authors declare no conflicts of interest
Background: To investigate the survival efficacy of allo-geneic hematopoietic stem cells in patients with MDS/AML with TP53 state changes (deletion, mono-mutation, multi-hit). Analyzing the clinical characteristics in patients with different TP53 gene alterations, and further exploring the influencing factors of clinical prognosis of TP53-change MDS/AML after allo-HSCT. Methods: A retrospective analysis was conducted on 42 patients with TP53-MDS/AML who underwent allo-HSCT from 2006.1 to 2021.7.These patients may have deletion of the TP53 gene and/or mutations in the TP53 gene.The 42 patients were divided into 3 groups,TP53 deletion MDS/AML group (group A), TP53 mono-mutation MDS/AML group (group B), and TP53 multiple-hit group (group C), and the differences in clinical features and the effect on the prognosis of inhibition were analyzed.The changes in the characteristics of second-generation sequencing of 137 genes in bone marrow specimens of 29 patients with mutations were also discussed. Results: There were 42 MDS/AML patients, 30 males (71.4%), 12 females (28.6%), 21 MDS patients, 1 MDS-AML patient, and 20 AML patients. The average age of patients at the time of transplantation was 42 years, of which 11 patients with only TP53 deficiency (26.1%), 25 patients with TP53 single mutation (59.5%), and 6 patients with TP53 multiple hits (14.2%). Among the 42 patients, 14 (33.3%) died, 10 cases relapsed (23.8%), and the overall patients who received hematopoietic stem cell transplantation had 3 years OSå 40%, and there was no statistical difference in 3 years OS in group A, B and C. Univariate analysis shows,hemoglobin、age of HSCT、complex karyotype、pre-HSCT MRD were risk factors for survival (p = 0.003,p = 0.042,p = 0.009, p = 0.004), and TP53 stage、co-mutation、treatment before HSCT、type of Diseases not. Complex karyotype、hemoglobin、pre-HSCT MRD were also risk factors for relapse (p = 0.038, p = 0.02, p = 0.023),besides,pre-HSCT BM blast% also have an impact on relapse, p = 0.027.Among the 42 patients, 12 patients (28.5%) with complex karyotype, including 2 patients with TP53 deletion alone (16.6%), 5 patients with TP53 single mutation (41.6%), and 5 patients with TP53 multiple hit (41.6%). Of the 12 patients with complex karyotypes, 7 died, 5 relapsed, and 4 of the 7 deaths were patients with TP53 multi-hits. Survival and relapse of TP53 mutations may be associated with complex karyotype (p = 0.009,p = 0.038). Conclusions: Allo-hematopoietic stem cell transplantation can overcome the poor changes of MDS/AML patients with TP53 mutation/deletion, but complex karyotype is still a poor prognostic factor for TP53-MDS/AML patients receiving allo-HSCT. Disclosure: Nothing to declare
Background: Combination of carmustine, etoposide, cytarabine, melphalan (BEAM) is a commonly used regimen for autologous stem cell transplantation (auto-SCT) in patients with Hodgkin (HL) or non-Hodgkin lymphoma. Due to both a shortage of carmustine in the past decade and to reduce its pulmonary toxicity, this agent has been replaced in several centers by thiotepa (TEAM). In our center, to further reduce gut toxicity, namely mucositis, since 2015 we use a reduced TEAM (dose reduction of etoposide and cytarabine). Methods: We retrospectively analyzed transplant outcomes of adult patients undergoing auto-SCT conditioned with reduced TEAM (thiotepa 5mg/kg/12h on day −7; etoposide 100 mg/m2/12h and cytarabine 100 mg/m2/12h on days −6 to −3 (instead of the commonly used 200 mg/m2/12h); melphalan 140 mg/m2 on day -1). Results: Included were thirty-nine patients (males, n = 24; females, n = 15) transplanted during the period 2015-2021. Median age was 43 (range 22-65) years. Most patients were transplanted for refractory (n = 10) or relapsed (n = 15) disease, while 12 patients were transplanted after first line treatment. The most frequent histology was diffuse large B-cell lymphoma (n = 15) followed by HL (n = 10), follicular lymphoma (n = 5), mantle cell lymphoma (n = 5) and primary mediastinal B-cell lymphoma (n = 4). The median number of chemotherapy lines prior to transplant was 2 (range 1-5), with a median interval from diagnosis to transplant of 16 months (range 5-218). Disease status at auto-SCT was complete response (CR) in 37 (CR1 = 22, CR2 = 14, >CR2 = 1) and partial response (PR) in 2 patients. Eight patients needed plerixafor for CD34 mobilization, with a median dose of CD34 + collected of 3.36 (range 0.70-17.00) x 106 /kg. All but 1 patient engrafted, with a median time to neutrophil and platelet engraftment of 11 (range 8-16) and 19 (range 9-40) days, respectively. The median duration of hospitalization was 19 days (range 15-27). Regimen related toxicities included mucositis in 32 patients (82%) (grade 1-2, n = 29; grade 3, n = 3; no grade 4 mucositis) and febrile neutropenia in 26 patients (67%). Blood cultures were positive for Gram-positive or Gram-negative bacteria in 3 and 8 patients, respectively. One patient developed resolutive pneumonia. Five patients (13%) underwent maintenance therapy after transplantation (brentuximab, n = 3; rituximab, n = 1; ibrutinib, n = 1) while twelve patients relapsed with a median time after auto-SCT of 5.5 (range 3-19) months, with a 1-year cumulative incidence of relapse of 38% [95% CI 20-56]. Two patients underwent allogeneic SCT with a median time after auto-SCT of 11 and 23 months, respectively. The 100-day and one-year non-relapse-mortality (NRM) was 2.5% [95% CI 0.2-11]. Eight patients died: one 5 days after auto-SCT due to septic shock; 5 due to disease progression and 2, after disease relapse, due to suicide and cerebral hemorrhage, respectively. At last follow-up, 29 patients (81%) were in CR. With a median follow-up of 18 (range 3-77) months, 2-year progression-free and overall survival were 59%[95% CI 38-75] and 81% [95% CI 74-88]. Conclusions: Reduced TEAM is a feasible and valid regimen prior to auto-SCT, with low toxicity and NRM and acceptable survival rates. Disclosure: Nothing to declare
Background: Dimethylsulfoxide (DMSO) is toxic to both cryopreserved cells and patients. We are using a method for freezing at - 80ºC with 5% final DMSO concentration. In an attempt to further decrease the amount of infused DMSO we tested viability of cells and hematologic recovery of patients following cryopreservation with different degree of cell packing. Methods: We compared two groups of myeloma patients with low and high cryopreserved cells concentration. Cell suspension in the first group of 88 patients contained average 175 x 106 cells/ml (maximum 250 x 106 cells/ml) and in the second group of 175 patients the cells were completely packed by total plasma removal (average 391 x 106 cells/ml and maximum 597 x 106 cells/ml) before adding the cryoprotectants resulting in a 60% decrease of the volume to be frozen. Cells were non-programmed frozen and stored at −80 °C in a solution with final concentrations of 5% DMSO, 3.6% hydroxyethyl starch (HES 450 000 mw) and 3% of human serum albumin. Stem cell viability was evaluated by trypan blue exclusion test. Cell dose in the first patient group was 2,62 x 106/kg (0,8–6,6), while in the second group it was 2,67 x 106/kg (1,5-6,8). Results: There was no statistically significant difference between the two myeloma patient groups. Viability was 97,0% (85-99) and 96,5 % (70-99) respectively. The neutrophil recovery was on day 11 (9-19) for the first and on day 11 (9-18) for the second group. Platelets recovered after 12 days for the first and the second group. Conclusions: Dimethylsulfoxide is toxic and induces many side effects following transplant (cardiac, neurologic, respiratory, etc.), which are dose dependent. Reducing them could be achieved by lowering the final DMSO concentration (5%) and by decreasing the volume of the frozen suspension. Our approach does not affect the cellular viability or the hematologic recovery of the patients following transplantation. Disclosure: Nothing to declare
Background: Early lymphocyte recovery (ELR), defined as an absolute lymphocyte count (ALC) ≥ 500 cells/µL at day 15 after autologous stem cell transplantation (ASCT), impacts on overall survival and progression-free survival in patients with hematological and non-hematological diseases. However, limited data are available to predict immune recovery after ASCT. Methods: To investigate which factors determine ELR following ASCT, we conducted a multicenter retrospective study in adult and pediatric patients who underwent ASCT with grafts provided by a single-cell processing laboratory during the period 2014 to 2016. Patients were divided in two groups according to whether ELR was achieved (ELR + ) or not (ELR-). Patient demographic and transplant characteristics, and cell collection and quality control parameters of the stored cell product (purity, viability and potency tests) were compared between the two groups. A receiver operating characteristic (ROC) curve was constructed to predict ELR with statistical variables. Results: We included 379 patients, of whom 278 (73%) achieved ELR + . Male sex (63% in the ELR + group versus 48% in the ELR- group) showed a statistical difference in the bivariate analysis that remained in the multivariate analysis (odds ratio [OR], 1.92; 95% confidence interval [95% CI], 1.19-3.10; P = .008). Regarding the cell graft, the median total lymphocyte dose (2.7 versus 1.6 × 108/kg in the ELR + and ELR- groups, respectively) was associated with ELR + in both the bivariate and multivariate analyses (OR, 1.34; 95% CI, 1.15-1.57; P < .001). Higher median clonogenic efficiency (CLONE), measured as the number of colony-forming units (CFU) scored per CD34 seeded, was also associated with a higher likelihood of ELR + in the bivariate and multivariate analyses (42% versus 38%) (OR, 1.02; 95% CI, 1.00-1.03; P = .040). ROC curves showed that infusion of more than 2.2 total lymphocytes × 108/kg (OR, 2.77; 95% CI, 1.72-4.48; P < .001) and CLONE values higher than 42% (OR, 1.75; 95% CI, 1.07-2.87; P = .026) were the best cut-off values for predicting patients who will achieve ELR + (area under the curve [AUC], 0.68; 95% CI, 0.63-0.74). No statistically significant differences in ELR were found for the transplant conditioning regimen, number of chemotherapy treatments prior to ASCT, medical device used during collection, or CD34+ cells/kg in graft. Conclusions: In summary, our study shows that the total lymphocyte content of the graft, CLONE and male sex may be used as predictors for ELR after ASCT. These data suggest that not only the CD34+ cell dose per kg should be considered the target of peripheral blood cell collection, but the lymphocyte dose should be taken into account as well. Routine potency tests might help to predict not only myeloid engraftment, but also lymphocyte reconstitution. Further studies are needed to corroborate these findings not only in the autologous, but also in the allogeneic setting. Disclosure: The authors state that they have no conflict of interest.
Background: Central nervous system (CNS) complications during hematopoietic stem cell transplantation (HSCT) in sickle cell disease (SCD) patients are a major cause of morbidity, as: posterior reversible encephalopathy syndrome (PRES), seizures, strokes or subarachnoid haemorrhage (HSA). These complications can appear for several causes, but mostly due to variations on hemodynamic status of the patient. Conditioning regimen-related toxicity, graft-versus-host disease (GvHD) and the use of calcineurin inhibitors also play an important role. Methods: A retrospective single center study was conducted in children with SCD, who underwent allogeneic HSCT from an HLA-identical sibling donor since January 2010 to December 2021. Implementation of arterial blood pressure (ABP) Holter, few weeks prior to HSCT, was established in December 2017 providing a better understanding of patient’s hemodynamic status and easing early treatment of arterial hypertension prior to HSCT. We analyze CNS transplant complications between two different periods: May 2010 to November 2017, and December 2017 to June 2021. Besides that, a change on conditioning regimen was established. Until June 2015 we used busulfan, cyclophosphamide and alemtuzumab. Afterwards, we changed to myeloablative but reduced toxicity conditioning: thiotepa, treosulfan, fludarabine, antithymocyte globulin. Also, GvHD prophylaxis used until 2019 was CsA and MTX, changing later to tacrolimus and mycophenolate mofetile (MMF). Seizure prophylaxis was provided during calcineurin inhibitors treatment, as well as maintenance of platelet threshold above 50.000/mcL, hemoglobin 11 g/dL and avoid hypomagnesemia. Epidemiological and clinical parameters were collected. Data are presented as percentages and quartiles. For the comparison of the variables under study, a bivariate analysis with non-parametric Fisher test was used. R Statistical Software was used for the numerical analysis and Survminer to represent Kaplan-Meier curves. Results: 48 allo-HCST were performed in 47 patients, median age 6.0 years (p25 2;p75 9). 22 patients in first period, 10 males (55%); and 26 patients in second, 13 males (50%). Prior to HSCT, 14/48 of HSCT had cerebrovascular disease (Moya-moya vasculopathy, silenct infarction, stroke or leukoencephalopathy); 64% (9/14) on first period and 36% (5/14) on second. During transplant, 11/48 had acute CNS complications: 91% (10/11) on first period (9/10 seizures, 5/10 HSA, 5/10 PRES and 1/10 other complications), and 9% (1/11) on second (mild toxicity to tacrolimus). HTA is present in 89% of total HSCT and in 100% with post-HSCT neurological complications, suggesting his major role in these type of situations. Global event-free survival of post-HSCT CNS complications at the end of follow-up period (10.69 years) was 77% (0.65-0.89). Statistically significant decrease on post-HSCT neurological complications on second period was observed compared to the first (seizures p < 0.001; HSA p0.02; PRES p0.049), after the implementation of ABP Holter. Also, we couldn’t observe a statistically significant predisposition to have post-HSCT CNS complications according to their pre-neurological history. Conclusions: Even with reduced-toxicity conditioning and the switch to tacrolimus, neurological events still happen. Recent modifications in our center, mainly since the implementation of ABPH, have decreased acute CSN complications and improved SCD event-free survival rates during transplant, with less toxicity, morbidity and mortality. Disclosure: Nothing to declare.
Background: AlloHSCT remains the only potentially curative treatment for myelofibrosis (MF) and myelodysplastic syndromes (MDS), but there is no consensus on the best conditioning treatment. The use of treosulfan as alkylating agent in a RIC (fludarabine-treosulfan) regimen demonstrated low toxic profile associated with excellent disease eradication in patients with acute myeloid leukaemia (AML) and MDS. On the other hand, the combination of two alkylating agents with the addiction of thiotepa seems to increase the chance of achieving engraftment with full donor chimerism in MF patients. Therefore, the replacement of busulfan by treosulfan in a dual alkylator regimen with thiotepa and fludarabine (TTF) could be a promising toxicity-reduced but myeloablative conditioning regimen for a setting of patients characterized by an advanced median age and usually with high comorbidity index (HCTI), therefore at high risk for relapse and transplant-related mortality.To date no data about the use of a dual-alkylator treosulfan-based regimen are reported in this setting. Methods: We analyzed retrospectively 10 patients (median age: 61, range 44-69, 60% male) affected by MF (50%) or MDS (50%) who underwent alloHSCT (between December 2020 and November 2021) with TTF (9 pts) or TF (1 pts) as conditioning regimen. Seven patients had primary disease, 2 patients had MF secondary to essential trombocythemia (ET) and one patient had a MDS after autologous HSCT for mantle-cell lymphoma. Patients median EBMT-risk-score (for MDS) and MTSS (for MF) were 4 and 6, respectively. Results: The median time from diagnosis to alloHSCT was 11.5 months (range, 6-158). Graft source was peripheral blood stem cells in all patients. Donor type was HLA-matched related (n = 3), matched unrelated (n = 5) and mismatched unrelated (n = 2). Treosulfan cumulative dose was 30g/m2 in 60% and 36g/m2 in 40% of patients. Graft-versus-host disease (GVHD) prophylaxis consisted of a calcineurin inhibitor (8 pts cyclosporine, 1 pt tacrolimus) plus methotrexate and ATG for 9 patients, while combination of cyclosporine with micophenolate mofetil and post-transplant cyclophosphamide was used in a patient who underwent HSCT from a mismatched unrelated donor. Full donor early engraftment was achieved in all patients except 2, who died during aplasia. The median time to neutrophil recovery was 16 days (range, 16-21). The median time to achieve platelet engraftment >20 G/L was 22 (range, 15-40) days. The median follow-up was 5.6 (range, 1-12) months. Complications after HSCT included mucositis grade 3-4 in 3 patients, one diarrhea grade 3, one grade 3 systolic disfunction, neutropenic fever. Two patients died: 1 for cerebral hemorrhage (at day 12) and 1 due to acute kidney disease and subsequent multi-organ failure (at day 32). One patient experienced grade II acute GVHD at day 35 while mild chronic GVHD occurred in another patient. No relapse was seen. A trend towards better survival was observed for patients who underwent alloHSCT before the median time period of 24 months (OS 100% versus 33.3%, p = 0.06). Conclusions: These data suggest feasibility, safeness and efficacy of TTF myeloablative regimen for MF/MDS patients, with excellent early full donor engraftment and manageable transplant related toxicity. Larger cohort and longer FU are needed for survival analysis. Disclosure: Nothing to declare
Background: Hematopoietic stem cell transplantation is a curative treatment option for various hematologic malignancies. The conventional cryopreserved stem cell grafts are time-consuming, expensive, and may be associated with dimethyl sulfoxide toxicity. Several studies demonstrated the successfulness of non-cryopreserved hematopoietic stem cell transplantation in the resource limited centers. The objective of this study is to evaluate the feasibility, efficacy, and safety of non-cryopreserved autologous stem cell transplantation (ASCT) in patients with multiple myeloma and lymphoma. Methods: A retrospective study conducted in Songklanagarind Hospital, a tertiary university hospital in southern Thailand. The medical data of consecutive patients aged 18 years old or older diagnosed as multiple myeloma or lymphoma who underwent ASCT using non-cryopreserved stem cell graft during January 1996 to March 2021 were enrolled. Mobilization was performed mainly with cyclophosphamide and granulocyte colony stimulating factor (G-CSF) and the collected stem cells were stored for 1–7 days in a blood bank refrigerator at a temperature 4°C. Stem cells were reinfused into patients after complete conditioning regimen infusion for 24 hours. All aphesis products were assessed for stem cell viability at the time of infusion. Results: A total of 62 non-cryopreserved ASCT was performed (40 myeloma and 18 lymphoma cases). The median total CD34+ cell count was 7.59 million/kg in myeloma and 6.9 million/kg in lymphoma with the stem cell viability before infusion was greater than 99% and 97% in myeloma and lymphoma, respectively. All myeloma patients were engrafted, whereas, 2 cases of lymphoma were failure to engraft. The median time of neutrophil engraftment was 9 (range; 7–19) and 13 (range; 8–53) days and platelet engraftment was 11 (range; 7–18) and 14 (range; 8–61) days in myeloma and lymphoma, sequentially. Regarding extra-hematopoietic toxicities, 65% of myeloma and 83% of lymphoma patients had mucositis. All patients experienced any grade of nausea or vomiting, while the diarrhea occurred in two-third of myeloma and half of lymphoma patients. More than 95% of patients developed febrile neutropenia, mostly were grade 1–2. Severe complication such as septic shock and respiratory failure was observed in only one myeloma and four lymphoma patients. With the median follow up time of 60 months, the median progression free survival (PFS) was not reached and overall survival (OS) was 130 months in lymphoma patients. For myeloma, the median PFS was 99.5 months and OS was 157 months, with the median follow up time of 38.6 months. The 100-day transplant-related mortality was 2.5% and 11.1% in myeloma and lymphoma, consequently. Cancer-related was the cause of death in half of myeloma and 14% of lymphoma patients, whereas, 28% of lymphoma patients were died from therapy-related complication. None of any factors were significant predicted PFS and OS in this study. Conclusions: Non-cryopreserved ASCT was effectively and safely performed in multiple myeloma and lymphoma patients which resulted in a short duration of neutrophil and platelet engraftments, acceptable complications, and long-term survival outcomes. Disclosure: Nothing to declare.
Background: Patients with acute myeloid leukemia (AML) relapsed after allogenic hematopoietic stem cell transplantation(allo-HSCT) have poor prognosis although some therapeutic options including hypomethylating agents, chemotherapy, donor lymphocyte infusion (DLI), or a second allo-HSCT could be adopted. Daratumumab, which is a therapeutic targeted drug to CD38 antigen for multiple myeloma, might be a new way for relapsed AML with CD38-positive after allo-HSCT. Methods: Between January 2021 and October 2021, 4 adult patients (2 patients were hematologic relapsed, 2 patients were bone marrow minimal residual disease(MRD)-positive) with CD38 + AML relapsed after allo-HSCT were enrolled, including 2 sibling-matched stem cell transplantation and 2 haploidentical transplantation. Two out of the patients had received chemotherapy followed by DLI before the treatment of Daratumumab (Table). The dosage of Daratumumab was 8mg/kg/d, on day1, day2, day15 and day16; 4-weeks was one cycle. Clinical manifest, routine blood tests, Liver and kidney functions were recorded and evaluated. The bone marrow examinations were performed to assess the response 2 or 4 weeks after the Daratumumab treatment including bone marrow smear, MRD detection by flow cytometry, donor chimera rate by short tandem repeat (STR). Results: The 4 patients did not show serious adverse reactions such as shiver or dyspnea during Daratumumab infusion. After Daratumumab infusion, Patient 2 occurred fever due to agranulocytosis which was cured by antibiotics and the number of neutrophils recovered 2 weeks later. No serious abnormal liver and kidney function were recorded for the 4 patients. Patient 1, who was hematological relapse before Daratumumab treatment, achieved complete remission and complete donor chimerism, but MRD was positive with CD38 negative after one cycle of Daratumumab. Then she received Desitabine maintenance treatment. Patient 2 and Patient 3 achieved MRD negative remission and complete donor chimerism after one cycle. Subsequently, Patient 2 received two cycles of venetoclax maintenance treatment and MRD was persistent negative. Patient 3 did not achieved maintenance treatment after Daratumumab due to cytomegaloviremia and acute graft versus host disease. Patient 4 showed no response to Daratumumab and refused further treatment because of economic reasons. She passed away at the 12th-month after relapsed. Conclusions: Daratumumab treatment seems to be an effective and safe treatment for AML with CD38-positive relapsed after allo-HSCT, especially for the MRD( + )-relapse, which may be confirmed with more clinical studies. Disclosure: Nothing to declare.
Background: Umbilical cord blood (UCB) has been used as an alternative to bone marrow for transplantation of hematopoietic stem cell purposes. Cord blood transplantation (CBT) can be used successfully in patients with marrow failure, immunologic deficiencies, malignancies, inborn errors of metabolism, and other genetic diseases. Cord blood transplantation in children show similar outcome data compared to transplantation of stem cells from other sources. The primary confinement factor for the wide utilization of UCB for hematopoietic progenitors for transplantation is cell dose. Though it is assumed that delayed cord clamping would reduce the volume of placental blood that could be gathered for banking, the extent to which delayed cord clamping decreases total nucleated cells (TNCs) counts and as such impacts cord banking activities remains unknown. The current study aimed to investigate the duration of delayed umbilical cord clamping that impacts cord blood units (CBUs) volume, TNCs and CD34 + cells counts that can affect the quality of CBUs and banking efficiency. Methods: Cord blood units collected at Mansoura University Hospitals from January 2020 to May 2021 were analyzed. The delay in cord clamping after birth was timed and classified as group 1 (30 to 60 seconds), and group 2 (61 to 90 seconds). The collected CBUs were evaluated as regard volume, TNCs and CD34 + cells count. Results: Out of 252 attended deliveries, only 98 cord blood units were collected excluding 61% of donors that didn’t achieve requirements of inclusion in the study. Group 1 was conducted on 52 donors and group 2 was 46 donors. The mean volume, TNCs and CD34 + cells counts in units among group1 were significantly greater than these among group 2 (p < 0.05). Conclusions: Delayed cord clamping greatly diminishes the volume and TNCs and CD34 + cells counts of CBUs collected for a public cord blood bank. Aiming for good quality of collected cord blood units, our results suggest that better stem cell yield is obtained with a practice of delay in cord clamping after 30-60 seconds. Disclosure: Nothing to declare
Background: Anxiety and depression represent a relevant problem for allogeneic hematopoietic stem cell recipients (HCT). Recent studies found that the COVID-19 risk perception is associated with emotional distress. Identification of predictors associated with higher COVID-19 risk perception among HCT recipients would enable provision of tailored psychological support following HCT. Methods: The main aim of the study was to assess changes in COVID19’s risk perception after vaccination in recent HCT recipients. We measured risk perception using the shortened Brief Illness Perception Questionnaire (BIP-Q5) in 54 HCT recipients after dose 1 and 2 of the mRNA-1273 SARS-CoV-2 vaccine through phone calls. This test consists of five items (consequences, timeline, identity, concern and emotional response; see Table 1) with a score from 0 (none) to 10 (a lot). Results: Participants’ median age was 53.5 (25-73) and 48.1% were females. Five out of 54 (9.3%) had suffered from COVID-19 prior to vaccination. Median time from infusion date at first vaccine dose was 13 months (range 3-26). COVID-19 risk perception tended to decrease over time (p = 0.077) to an extent that it was clinically meaningful (p < 0.05), see Table 1. *Statistically significant (p < 0.05) A post hoc exploratory analysis of the BIP-Q5 items (consequences, timeline, identity, concern and emotional response) revealed that, at time of the second dose, participants expected COVID-19 would last less over time and reported being less concerned about COVID-19 relative to prior to vaccination, with these differences being both statistically and clinically significant (p < 0.05). Females (r = 0.42, p < .001), patients receiving myeloablative conditioning -MAC- (r = −0.300, p = 0.027), post-transplant cyclophosphamide-based GVHD prophylaxis -PTCy- (r = −0.371, p = 0.006), having lower absolute neutrophil count (r = −0.288, p = 0.034), development of acute GVHD (r = −0.355, p = 0.008) and having a relative who suffered from COVID-19 (r = 0.285, p = 0.036) were significantly associated with greater disease risk perception in the univariable analysis. Conclusions: Vaccination led to decreased COVID19-risk perception over time in HSCT recipients. Despite several characteristics seem associated with increased risk perception (i.e., females, myeloablative conditioning, PTCy, lower absolute neutrophil count, aGVHD, knowing a relative diagnosed with COVID-19), additional factors, such as patients’ psychological symptoms, should be considered to further understand COVID-19 long-term effects on HCT recipients’ mental health. Disclosure: Nothing to declare.
Background: Engraftment syndrome (ES) is an inflammatory response that occurs during neutrophil recovery after hematopoietic stem cell transplantation (HSCT). The reported incidence of ES has been variable due to the lack of universal diagnostic criteria and its similar presentation to acute graft versus host disease (aGvHD). Several studies have shown an increased risk of aGvHD in patients who were treated for ES. Our primary objective is to determine the incidence of ES in pediatric patients who underwent allogeneic HSCT at a large pediatric institution. Secondary outcomes include determining the characteristics of ES, duration and dose of steroid treatment, and relationship between ES and aGvHD. Methods: This is a retrospective chart review of pediatric patients who underwent allogeneic HSCT at Nationwide Children’s Hospital from 01/2009-09/2020 and were diagnosed and treated for ES. Patients who received HSCT or post-transplantation care at other institutions were excluded. Data collection included patient demographics, details of HSCT, symptoms of ES, staging of GvHD, and treatment characteristics. Univariate analyses were performed using nonparametric methods. Results: There were a total of 261 allogeneic transplants from 01/2009-09/2020; 29/261 (11.1%) developed ES, aGvHD observed in 105 (40.2%), and 17/261 (6.5%) had both ES and aGvHD. Among the 29 patients with ES, 17 (58.6%) developed aGvHD, whereas 88 (37.9%) patients without ES developed aGvHD (p = 0.052). The median (interquartile range; IQR) time to development of ES was 15 days (IQR: 12-19), median duration of treatment with steroids for ES was 3 days (IQR: 3-5), and median steroid dose was 2 mg/kg/day of methylprednisolone or equivalent (IQR: 1-2). Patients presented with a median number of 3 clinical features at time of diagnosis (IQR: 2-3); rash: 75.9%, fever: 62.1%, respiratory symptoms: 62.1%, gastrointestinal symptoms: 37.9%, capillary leak: 41.4%, and liver dysfunction: 10.3%. Patients presenting with rash more often developed aGvHD compared to those who did not present with a rash (82.4% vs 33.3%; p = 0.018). A steroid wean was done in 9/29 (31%) of the patients with ES and did not differ among those who did and did not develop aGvHD (35.3% vs 25%; p = 0.7). Systemic steroids were required in 15/17 (88.2%) of the patients who developed aGvHD after ES. Of the 17 patients with ES and aGvHD, 6 (35.3%) developed steroid refractory (SR) aGvHD and 8 (28.6%) developed chronic GvHD. Conclusions: In conclusion, 11.1% of patients who underwent allogeneic HSCT at our institution developed ES. Most patients with ES developed aGvHD and required systemic steroids. Approximately one third of patients with ES, developed steroid refractory or chronic GvHD. A steroid wean for ES treatment was not found to be associated with the development of aGvHD. Fever, rash and respiratory dysfunction were the most common presenting symptoms of ES. Future studies should examine the associations between ES and other endotheliopathies after HSCT in pediatric patients and further characterize the relationship between steroid duration and dosage used in treatment of ES and aGvHD. Disclosure: Nothing to declare.
Background: The South African National Blood Service (SANBS) provides haematopoietic stem cell transplant (HSCT) collection and processing facilities to 18 public and private clinical institutions across South Africa. The SANBS JACIE Quality Department (JQD) is responsible for overall quality of these two facilities and has implemented a quality management system in line with JACIE standards. In addition to annual mandatory audits, additional scheduled audits and staff competency audits, the JQD reviews and reports on occurrences on a 6 monthly basis. The findings of this report allow for continuous quality improvement. Methods: During the period of 1st March 2021 to the 30th September 2021 all occurrences that were logged by the collections and processing facilities onto a Systems Applications and Products (SAP) platform, an enterprise resource planning (ERP) centralised software system, were reviewed. The number, type, risk rating, problem description and cause of each occurrence per facility were documented. The number of occurrences in this reporting period were compared to the previous 6 month reporting period. Results: Collections facility: During this 6 month period 97 occurrences were logged: the top 3 occurrences were as follows: 63.94% (n = 62) due to delay in starting the procedure (documentation incomplete, access flow problems, blood results not available), 15.5% (n = 15) due to incorrect or incomplete records and 15.5% (n = 15) due to an adverse event. There were no serious adverse events, 66.7% (n = 60) were due to citrate reactions and 25.6% (n = 23) due to hypotension. Processing facility: During the 6 month period 63 HSCT reinfusions were performed with no reinfusion related adverse events. A total of 28 occurrences were logged. Significant occurrences included: bacterial contamination due to patient source (n = 2), not following procedure (n = 7) and incomplete/unavailable/inadequate records (n = 7). 42.9% (n = 12) of the notifications were risk rated as major, followed by 21.4% (n = 6) that were risk rated as moderate. In comparison to the previous 6 months, bacterial contamination occurrences increased due to the patients’ medical condition and recording keeping occurrences decreased. Conclusions: Evaluation of all occurrences on a six monthly basis is a critical part of assessing the implementation and effectiveness of a quality management system and a useful tool to identify trends in a HSCT collections and processing facility. Clinical Trial Registry: N/A Disclosure: Nothing to declare
Background: FLT3 mutations concern 30% of patients with newly diagnosed AML. They are associated with lower rates of remission, high recurrence rates and unfavorable prognosis. The recent use of tyrosine kinase inhibitors against FLT3 mutations has altered outcome. The present study analyzed the outcome of alloHCT in patients with FLT3-AML and the factors affecting it. Methods: 41 patients (18 men, 23 women) underwent alloHCT from 01/01/2010 to 05/15/2021 and were retrospectively studied. Median age was 52 years (range 33-65) and median follow-up duration 32.8 months (range 5-122). 36/41 (83%) patients were FLT3-ITD + while 5/41 (12%) TKD + . Six patients had high allelic load (>0.5) and 15 low. Data was not available for the remaining 20. 27/41 (66%) patients had normal karyotype while 13/41 (32%) had various karyotypic abnormalities. Median time from diagnosis to alloHCT was 8.7 months (range 2.6-25.5). 25/41 (61%) were transplanted in CR1, 11/41 (27%) in CR2 and 5/41 (12%) had refractory disease. 90% received myeloablative conditioning regimen while 10% reduced intensity. In 33/41 patients (80%) the graft source was peripheral blood, in 4/41 bone marrow and in 4/41 double umbilical cord blood. 21/41 (51%), 14/41 (34%) and 2/41 patients were transplanted from matched unrelated donor, histocompatible sibling and haploidentical donor respectively. 12/41 (29%) with FLT3-ITD relapsed, 17/41 (41%) died, 7 from disease and 10 (24%) from TRM. 7/41 (17%) received sorafenib, 6 as prophylaxis and 1 for relapse after alloHCT. The following factors were studied: coexistence of NPM1 mutation, type of FLT3 mutation, allele load, age, karyotype, donor type, presence of MRD at transplantation, number of white blood cells at diagnosis, ELN risk classification, conditioning regimen and pre-treatment with midostaurin. LFS and OS were determined by Kaplan - Meyer and multifactorial analysis was performed by CoxRegression analysis. RStatistics (Rcran) software was used. Results: 2-year CIR was 22.5% (95% CI12.4-40.1%), 2-year NRM 13.6% (95% CI5.9-31.3%), the estimated 2-year OS 66% (95% CI52.4-3.1%) and the estimated 2-year LFS 64.2% (95% CI35.8-72.6%). In unifactorial analysis, statistically important factors for LFS were age >60 years (p = 0.006), disease phase in alloHCT (p = 0.017) and previous administration of midostaurin (p = 0.01). Multifactorial analysis for LFS revealed age> 60 (p = 0.017) and disease phase in alloHCT (p = 0.03) as significant factors. Only age >60 years (p = 0.005) was statistically significant for OS. Midostaurin administration tends to favorably affect LFS (p = 0.07) and allthough statistically insignificant, it decreased CIR to 7.7% (95% CI 1.1 -54.6) versus 29.6% (95% CI 16.3 -53.8, p = ns) and to NRM 0% versus 15.6% (95% CI6.1-39.4). Conclusions: AlloHCT is the treatment of choice for patients with AML-FLT3 + in CR1 regardless of other factors. The best outcome seems to be achieved in younger patients, possibly due to the ability of receiving myeloablative conditioning regimens. The addition of midostaurin, despite the small number of patients, tends to improve the outcome of alloHCT. Larger number of patients and longer follow-up time are required to evaluate the effectiveness of maintenance with sorafenib. Disclosure: There are no disclosures
Background: The Baltimore group has recently reported a transplant platform in patients with aplastic anemia (AA) undergoing a haploidentical transplantation (HAPLO) (De Zern et al. 2020); the conditioning regimen is based on fludarabine cyclophosphamide (FLU-CY) TBI 2 Gy GvHD prophylaxis is based on 4 drugs : ATG on day -9-8-7, post transplant CY (PTCY) 50 mg/kg on days +3 + 4 and tacrolimus mycophenolate. The Baltimore group reports 37 patients, with 35 surviving without GvHD. The Brasilian group has confirmed these results in a multicenter study on 87 AA patients, with survival in excess of 90%, especially for those receiving an intensified TBI dose of 4Gy (instead of 2 Gy) and an increased dose of CY pre transplant (50 instead of 30 mg/kg) (BBMT, 2020; e222-e226). Methods: If this platform allows the engraftment of an HLA HAPLO mismatched marrow, with little or no GvHD, then the same platform should be successful in patients with AA undergoing an unrelated donor (UD) transplant, or in elderly patients grafted from matched siblings.We have grafted 11 patients with AA with this platform.The conditioning was as follows: CY 14.5 mg/kg days -6-5, Fludarabine 30 mg/m^2 days -6-5-4-3-2, TBI 2 Gy day -1 and unmanipulated bone marrow on day 0 (1 patient was grafted with PB cells from a HAPLO donor). GvHD prophylaxis was ATG 0.5, 2 and 2 mg/kg days -9-8-7, PTCY 50 mg/kg day +3 + 4, cyclosporine (CSA) on day +5 onward and mycophenolated (MMF) day 5-day 35. The median age was 30 years (range 20-60). The donor was unrelated, matched (8/8, n = 5), or mismatched (7/8, n = 4), HAPLO (n = 1), or a matched sibling (n = 1). In the latter this transplant platform was chosen because of the patients age (60 years). Results: All patients had 100% donor chimerism within 30 days from transplant. One patients died of a septic shock on day+34. One patient (UD 7/8 matched donor) (9%) experienced a rejection on day +88; underwent a second transplant from the same UD with peripheral blood as a stem cell source, on day +152 from the first transplant; the patient is currently alive and well with trilineage recovery on day +423. The median time to a neutrophil count of 0.5 x 10^9/L was 20 days (15-26) and for 20 x 10^9/L platelets, it was 24 days (13-30). Acute GvHD grade I was recorded in 1 patient (9%), minimal chronic GvHD was reported in 2 patients (18%); moderate severe chronic GvHD was not reported. EBV reactivation was seen in 5 patients (45%) and was treated in 2 patients with rituximab. CMV reactivation was seen in 3 patients (27%). With a median follow up of 323 days, the one year actuarial survival is 95%. Conclusions: This small experience with the Baltimore quadruple GvHD prophylaxis, in patients undergoing unrelated donor transplants, confirms a very high degree of engraftment, low early mortality, low rate of rejection, and little or no GvHD. Whether the dose of CY in the conditioning should be increased from 29 mg/kg to 50 mg/kg, as suggested by the Brazilian group remains to be determined. Disclosure: No disclosure to declare
Background: Autologous hematopoietic cell transplant (auto-HCT) is standard of care in multiple myeloma (MM) and should be performed within 4-6 months after the start of induction therapy in eligible patients. Unfortunately, in resource-constrained countries, the waiting time for auto-HCT is longer due to the small number of HCT beds and difficulties in completing the pre-HCT process. Our objective was to evaluate the incidence, risk factors and impact of progression/relapse (P/R) in transplant waiting list among patients with MM. Methods: Patients with MM referred to auto-HCT at our public institution between 2010 to 2018 were included. The primary outcome was cumulative incidence (CI) of P/R in waiting list, defined as P/R requiring or not treatment after 4 months from the start of the most recent line of systemic treatment. Secondary outcomes were time in waiting list, disease-free survival (DFS) after auto-HCT, % of referred patients actually proceeding to auto-HCT, and overall survival (OS). Results: 304 patients deemed eligible for auto-HCT were included in the analysis. Of these, 70 patients progressed or relapsed while waiting for transplant. Baseline characteristics were similar between patients with P/R or not in waiting list, except that the former were more likely to have non-IgG MM. The CIs of P/R in transplant waiting list at 3, 6 and 12 months were 5, 11 and 17%, respectively. In a univariate analysis, ECOG 2-4, non-IgG MM, serum monoclonal protein ≥1.7g/dL, having high school (vs. elementary school) and no prior use of immunomodulator were significantly associated with higher risk of P/R. In multivariate analysis, non-IgG MM was the only risk factor for P/R in waiting list (HR 1.74 [1.08-2.82], p = .02). Median waiting time was 231 days (IQR 131-429) for patients with P/R in waiting list compared to 149 days (95-213) for their counterparts (p < 0.001). Only 10% of patients were autografted within 6 months after the start of systemic therapy. Of the 70 patients with P/R in waiting list, 20 (29%) subsequently proceeded to auto-HCT compared to 214/234 (92%) of patients without P/R (p < .001). Post-transplant DFS of the 20 patients receiving auto-HCT after P/R in waiting list did not significantly differ from those proceeding directly to auto-HCT (p = .10). OS was significantly inferior for those not receiving auto-HCT (Fig. 1). Conclusions: Patients with P/R in waiting list waited longer for auto-HCT than their counterparts and were less likely to be autografted after P/R. This finding suggests that waiting longer may increase the risk of P/R, making these patients lose performance status to undergo auto-HCT subsequently. Prioritizing patients with non-IgG MM and expanding outpatient transplantation may be strategies to avoid P/R in waiting list. These data may also help stakeholders address hindrances in the pre-HCT process and increase the number of HCT beds in the public setting. Disclosure: none to declare
Background: Plasma cell myeloma (PCM) is the second commonest haematological malignancy. Poorer countries have several folds higher mortality in some cancers compared to those in affluent countries. However, data related to geographical differences, clinicopathological and global survival variability are not well documented and there is a correlation between poverty and lack of accessibility to health care and vice versa in low-income countries. Methods: Sri Lanka is a developing country with a diverse healthcare structure with no dedicated Haemato-Oncology/Clinical Haematology centres, transplant facilities or access to novel anti-cancer agents at the time the first ‘blood cancer centre’ was established in the Island nation in 2013. We have previously published data related to survival of patients treated in the centre. The aim of the study was to analyse patient and disease characteristics and evaluate survival parameters of patients who had peripheral blood stem cell transplant (PBSCT) in the first Haemato-Oncology centre in Sri Lanka. Results: A total of 20 patients received PBSCT during study period. However, one each had the diagnosis of plasmablastic lymphoma and plasma cell leukaemia and one with creatinine clearance of less than 30ml/min at presentation were not included in the final analysis. The median age of the study population was 57 years (range 41,66). There were 9 (53%) males and 8 (47%) females. Melphalan 100mg/m2 was used for conditioning. The median infused haematopoietic stem cells (HSC) per Kg was 4.25 x 106. Median duration for neutrophil and platelet engraftment was 11 days and the median in-patient hospital stay was 18 days. The average cost of PBSCT for PCM was US$ 7500. After a median follow up of 38.33 months, the median overall survival (OS) was not reached (restricted mean was 77.42 with s.e. 8.12) and the estimated 3-year OS rate was 0.9. There was no difference in the survival according to remission status, dose of HSC, age or gender. In comparison, three year over survival (OS) rate of the entire cohort of PCM patients (n = 79) treated in LHBCC was 0.760 (95% CI (0.662,0.873). Table 1. Conclusions: Our study has shown comparable survival parameters as reported in high-income countries using 100 mg/m2 dose of melphalan and non-cryopreserved HSC. This is the only documented study related to PBSCT in any type of blood cancer in Sri Lanka. Clinical Trial Registry: Not applicable Disclosure: Nothing to declare
Background: Bone marrow deficiencies ıs a group of dıseases wıth dıfferent etıologıes. Supportive therapy, immunosuppressive therapy (IST), and allogeneic hematopoietic stem cell transplantation (SCT) are among the treatment options. This study aimed to retrospectively evaluate the demographic characteristics, treatment, and transplantation results of patients who underwent allogeneic SCT due to bone marrow failure in the Uludağ University Hematology Department and review HSCT in bone marrow failures. Methods: Of the seven patients who underwent SCT, 6 had SAA, and 1 had unclassifiable MDS. Transplantation was performed from a fully HLA-matched sibling donor in 5 cases, syngeneic in one and 9/10 compatible in one. Peripheral blood was used as a stem cell source in only 2 of the patients, while bone marrow was used in the other four patients. Fludarabine/cyclophosphamide/anti-thymocyte globulin (Flu/Cy/ATG) was used as the conditioning regimen. Results: 513 SCT were performed in our center between 2009 and 2021. Of these, 140 (27.2%) were allogeneic SCT, and the remaining 373 were autologous SCT. The 140 patients who underwent allogeneic SCT 7 (5%) were diagnosed with bone marrow failure. The median age was 37 (21-47). Four patients were <40 years old at the time of transplantation. Six of the patients received cyclosporine as IST, and one patient received ATG. The median time to transplantation was five months (3-34). Flu/Cy/ATG conditioning regimen was used in all patients. The median time for neutrophil engraftment was 25 days (14-31) and platelet engraftment>20,000 was 18 days (11-21). No correlation was found between the number of cells administered and the ferritin level, and the engraftment time. The patients were discharged on the mean of 29 days (23-39). The chimerism rates at the 1st month of transplantation were 100% in 2 patients, 98% in 1, and 86% in 1 (increased to 99.7% at three months) donor profile. Graft failure, acute or chronic GVHD, was not observed in any of the patients. Cytomegalovirus (CMV) reactivation was observed in two of our patients. One of them is the 4.5 of the transplant. The other was on the 50th day and 5th month. In both patients, CMV-DNA negativity was obtained after four weeks of oral valganciclovir treatment. During the five-year follow-up, the survival rate of the patients was 100%. The median overall survival was 39.5 months (25.5-75). The median survival time after transplantation was 35 months (10-71). All of the patients are still under follow-up, and their complete hematological responses continue. Conclusions: All seven patients who underwent allogeneic SCT are alive and have complete hematological responses. There was no increase in mortality and morbidity in those over 40 years of age. Although the number of our patients is small, it was concluded that allogeneic SCT should be considered as the first-line treatment in the patient group with a fully compatible sibling donor with severe aplastic anemia and between the ages of 40-50 and without comorbidity. Disclosure: Authors declare no conflict of interest.
Background: Umbilical cord blood (UCB) has been used as an alternative source of hematopoietic stem cells for bone marrow transplantation (BMT). The advantages of cord blood include ready availability, no risk to the donor, low rate of viral contamination and low risk of GVHD. Egypt suffers from a large unmet need for BMT, especially for thalassemia being the most common chronic hemolytic anemia in Egypt. This need for BMT could be lessened by establishing a public cord blood bank (CBB). Public banks are about 53 banks in 37 countries around the world not including Egypt. However, many public cord blood banks have stated that they are struggling to maintain their financial sustainability. Methods: The aim of this study is to prove the possibility of providing a cord blood unit (CBU) with the least cost that can ensure the sustainability of a public CBB in a developing country. The study adopts an Activity Based Costing (ABC) system using a model of six steps in the first public cord blood bank in Egypt which is located in Mansoura University (the largest university in the Nile Delta) and comparing it with the traditional method. This is done through the measurement of the cost of each step in the banking process separately (collection, processing and cell enumeration, cryopreservation). Where there are common activities between those stages and therefore indirect costs. Results: The cost of producing a unit of umbilical cord blood using the traditional method was about 17,704 pounds. While its cost was about 16,505 pounds using the ABC method, a difference of 1199 pounds per unit. Thus, the ABC method has contributed to reducing the cost of producing a stem cell unit derived from cord blood by up to 7%. In addition, on separating stem cells from UCB, the processing step produces by-products such as platelet-rich plasma and red blood cells, which are considered an indirect return. Conclusions: This study concludes that using an activity-based costing method can reduce the cost of production of a CBU without sacrificing the value. The pooling of costs by activities or activity areas provides information that may help managers to better plan and control costs. Disclosure: There are no conflicts of interest
Background: Trypan blue exclusion and flow cytometry were compared for testing of cell viability of hemopoietic stem cells (HSCs) cryopreserved at -80ºC. Stem cells were frozen and stored with 5% final DMSO concentration with different degree of cell packing. Methods: We analyzed 47 samples stored at -80ºС in a cryoprotectant solution with final concentrations of 5% DMSO, 3.6% hydroxyethyl starch (HES 450 000 mw) and 3% of human serum albumin. The cell concentration in the frozen suspension ranged between 69х106/ml and 665х106/ml (median 310,25х106/ml). The cell viability was tested simultaneously by trypan blue exclusion test and by flow cytometry with 7 -aminoactinomycin D (7-AAD) without lysing solution. Results: Both viability tests gave similar results. When tested by trypan blue, the viability was 95,5% (80-99%), and by flow cytometry was 95,92% (89,1-98,4%). The additional statistical Spearman’s Rho analysis does not show statistically significant correlation between viability results, performed with either test, and the degree of cell packing. Conclusions: Flow cytometry and trypan blue tests show similar viability results. Since trypan blue testing is well established, has lower cost, and can be performed faster, we accepted it as a routine for our further analyses of cell viability of packed prior to freezing stem cell concentrates. HSC viability after storage at -80ºС with 5% final DMSO concentration is very good and is not affected by higher cell packing thus allowing the reduction of infused DMSO and the side effects (cardiac, neurologic, respiratory, etc.) during transplantation. Disclosure: Nothing to declare
Background: Congenital sideroblastic anemia (CSA) is characterized by anemia and intramitochondrial iron accumulation in erythroid precursors (Ring sideroblasts). The most common recessive form is caused by mutations in SLC25A38. Methods: We described the clinical course and outcomes of CSA patients receiving allogeneic hematopoietic stem cell transplantation (allo-HSCT) from 2016 to 2021 in Mofid Children’s Hospital, Tehran, Iran. Results: Case 1 was a 3-year-old female, the second child of close-relative parents. She presented at 2 months with severe anemia and later developed splenomegaly, large head, and frontal bossing. The blood tests and bone marrow aspiration (BMA) indicated CSA. Due to the transfusion-dependency and lack of response to pyridoxin, at 3 years of age, she received allo-HSCT from HLA-identical matched sibling donor (MSD). The conditioning regimen (CR) included busulfan (Bu), cyclophosphamide (CY), and anti-thymocyte globulin (ATG) with graft-versus-host-disease (GvHD) prophylaxis by cyclosporine (CS) and methotrexate (MTX). She was engrafted on day +21 but later developed GvHD in gastro-intestine (GI) and skin, cytomegalovirus (CMV) reactivation, and posterior reversible encephalopathy syndrome (PRES). Now five years post-transplant, she is drug-free with > 95% donor chimerism. Case 2 was a 2.5-year-old female with first-degree consanguineous parents. She was found to suffer from severe anemia and head deformity at 1.5 months. Her blood tests and BMA indicated CSA, confirmed by a homozygous variant in SLC25A38. She underwent allo-HSCT at the age of 2.5years, from her HLA-identical matched related donor (MRD) with a CR consisting of BU, CY, and ATG and GvHD prophylaxis by CS and mycophenolate mofetil. She engrafted on day +18, however, was later complicated by skin GvHD. She is now, 3 years post-HSCT, stable, without blood transfusion, with 100% donor chimerism. Case 3 was a 3.5-year-old male born to close-relative parents. He manifested with severe pallor at 2 months old and then splenomegaly. His blood tests and BMA suggested CSA. He showed transfusion-related complications (Anti IgG C3d) requiring IVIG, corticosteroids, and four courses of rituximab. At the age of 3.9 years, he received allo-HSCT from his HLA-identical MSD. The CR included Bu, CY, and ATG, and CS, MTX, and then methylprednisolone as GvHD prophylaxis. He engrafted on day +16. Although later developed CMV reactivation (day + 26), PRES, and high ferritin level, he is now 5 years post-HSCT stable with 75% donor chimerism. Case 4 was a 2.6-year-old male born to first-degree relative parents. He was initially presented with severe anemia at 2.5 months old. Following blood and bone marrow analysis, he was diagnosed with CSA, later confirmed by homozygote mutation in SLC25A38. He received allo-HSCT from HLA-identical MRD with a CR consisting of BU, CY, and ATG and GvHD prophylaxis by CS and MTX. He engrafted on day +12 with a 96% chimerism. He later developed bacteremia, GI complications, and thrombotic microangiopathy. He has now mild GI GvHD on day +45 and is on GvHD treatment with corticosteroids and CS. Conclusions: In patients with transfusion-dependent and pyridoxine-resistant severe CSA, HSCT is the only curative option and can result in favorable outcomes particularly when used in a timely manner. Disclosure: Nothing to declare.
Background: Pulmonary complications following allogeneic hematopoietic stem cell transplantation (HSCT) have been reported to occur in 12% of stem cell recipients. The aim of this study was to investigate whether pre-HSCT pulmonary comorbidity and pulmonary function test (PFT) abnormalities in children with hemoglobinopathies are associated with pulmonary complications after HSCT. Methods: We performed a retrospective chart review in eighty-five children (35 SCD and 50 THAL) who were transplanted between 01-01-2010 and 31-12-2020. The primary determinants were pre-transplantation PFT results, radiographic imaging of the lungs, sonographic imaging of the heart and a history of pulmonary disease. PFTs were performed in children from 4 years and up and included spirometry, carbon monoxide (CO) diffusion capacity and lung voluminal tests. PFTs 120 days before up to 2 years after HSCT were modeled using a mixed effects model with splines. Z-scores for PFT measurements were calculated based on reference values corrected for age, height, gender and ethnicity. A multistate cox model was applied to determine the association between pre-HSCT covariates and pulmonary complications post-HSCT. Results: Pre-existing pulmonary abnormalities during pulmonary screening tests were more prevalent in children with SCD than with THAL (74% vs 20%, p-value: < 0.001). Forty-eight percent of SCD patients had restrictive lung disease, 40% had a history of pulmonary disease (acute chest syndrome, pneumonia or asthma) and 17% had abnormal findings during radiographic imaging pre-HSCT. Pulmonary complications occurred in 21 (25%) patients in the first two years after HSCT. This rate was not significantly different between SCD and THAL patients (23% vs 26%). Moreover, 14 out of these 21 patients had an infectious pulmonary complication and 13 patients developed a non-infectious complications (8 bronchiolitis obliterans, 2 peri-engraftment respiratory distress syndrome, 2 idiopathic pneumonia syndrome and one case of cryptogenic organizing pneumonia). Pre-transplantation pulmonary screening abnormalities, including PFT results, radiographic imaging results and history for pulmonary disease, were not significantly associated with the occurrence of pulmonary complications after HSCT. However, the occurrence of acute graft versus host disease (GVHD) as a time-dependent covariate was associated with subsequent pulmonary complications (HR 2.85 (1.18 – 6.88), p-value = 0.02). Furthermore, the usage of Busulfan based conditioning and a matched unrelated donor showed an increased risk of pulmonary complications up to 2 years post-HSCT. Lastly, lung function tests before and up to 2 years after HSCT showed significantly lower levels of alveolar volume (VA) (p-value < 0.001) in patients with SCD as compared to THAL patients. Conclusions: SCD patients had more frequent pulmonary abnormalities pre-HSCT and worse pulmonary function tests post-HSCT than THAL patients. The development of acute GVHD is associated with subsequent pulmonary complications post-HSCT in hemoglobinopathy patients. Disclosure: Nothing to declare
Background: Haploidentical HCT allows universal availability of HCT to achieve long-term cure of sickle cell disease. The main barrier to this approach is graft failure. To minimise this risk, we introduced pre-HCT suppression of haemopoiesis with hydroxycarbamide 30 mg/kg (HU) and hypertransfusions. We present a case series of 10 patients who underwent haploidentical HCT with reduced intensity conditioning and this pre-transplant approach. Methods: Between August 2017 and May 2021, ten consecutive haploidentical related (4 maternal, 3 paternal, 3 sibling) HCT were performed at St. Mary’s Hospital, London. Patients were transplanted for stroke or recurrent vaso-occlusive crises and/or acute chest syndrome not responding to hydroxycarbamide. After 8 weeks suppression of haemopoiesis with hydroxycarbamide and hypertransfusions, patients were conditioned with fludarabine 150 mg/m2, thiotepa 10 mg/kg, cyclophosphamide 29 mg/kg, TBI 2Gy and ATG (Thymoglobulin) 4.5 mg/kg. GvHD prophylaxis was provided with post-transplant cyclophosphamide (PTCy) 50 mg/kg on day +3 and day +4, sirolimus and MMF. MMF was weaned over two weeks after day +35 once evidence of molecular haemopoiesis was available. Stem cells were harvested from the bone marrow for all transplants. Results: The median age was 12 years (3 – 19). The median cell dose was 4.04 x 108 TNC/kg (range 1.85 – 10.26), 4.78 x 106 CD34/kg (range 2.1 – 13.71) and 39.7 x 106 CD3 + /kg (range 15.3 – 81). The median survival was 11.5 months (5.7 – 45.1). No patient suffered transplant related mortality although one patient died on day +361 from Pneumococcal sepsis at a time where there was good donor haemopoiesis, no GvHD or immunosuppression. Primary or secondary graft failure was not seen. Median neutrophil engraftment was 19 days (range 15 to 22). The median platelet engraftment >50 x 109/L was 35 days (range 30 to 52). One patient required a top-up CD34 + selected PBSC on day +725. All patients achieved donor chimerism in T cells >50% by day +90 and were 100% donor in whole blood at day +180. There was no incidence of VOD or idiopathic pneumonia syndrome. One patient suffered transplant associated microangiopathy and two macrophage activation syndrome. Five patients had ≥grade 2 aGvHD (50%), two patients had limited cGvHD (20%) and four patients extensive cGvHD (40%), although no patient had GvHD at 18 months post HCT. The median duration of immunosuppression was 191 days (range 160-525 days). The overall survival and disease free survival in this cohort of children is 90%. Conclusions: In conclusion, haploidentical RIC HCT with thiotepa and PTCy in conjunction with pre-HCT suppression of haemopoiesis with HU and hypertransfusions abrogates the risk of graft failure in paediatric patients and leads to long-term donor haemopoiesis with low risk of mixed chimerism. The incidence of alloreactive complications was low. Although the incidence of GvHD during the treatment period was not insignificant, it responded to standard treatment in all patients with no patients suffering cGvHD long-term or requiring prolonged courses of immunosuppression. Haploidentical RIC HCT with PTCy using thiotepa and pre-HCT suppression with HU and hypertransfusions is effective and represents a feasible curative therapy for children with severe SCD. Clinical Trial Registry: Not applicable Disclosure: Nothing to declare
Background: Despite the progress in medical management, sickle cell disease (SCD) is still associated with severe morbidity and early mortality in adults. Waiting for further development in gene therapy, allogeneic haematopoietic stem cell transplantation (HSCT) is currently the only curative therapy. Methods: A single center descriptive study was conducted on patients with SCD who underwent HSCT from an HLA-identical sibling donor between May 2010 and December 2021. Stem cells source: bone marrow in all patients. Transfusion therapy was started 3 months before HSCT and since 2015 hydroxyurea was increased or started one month before HSCT in order to maintain reticulocyte count under 100 000/mm3. Conditioning regimen: Until January 2015: Busulfan, cyclophosphamide, alemtuzumab. Graft versus host disease (GVHD) prophylaxis with cyclosporine and methotrexate. After January 2015: Thiotepa, Treosulfan, Fludarabine, antithymocyte globulin (myeloablative conditioning but reduced toxicity). GvHD prophylaxis with cyclosporine and mycophenolate mofetil (MMF) (January 2015 – February 2019) or tacrolimus and MMF (February 2019 – December 2021). Seizure prophylaxis during immunosuppression: phenytoin until January 2015, afterwards levetiracetam. To decrease neurological complications: platelet threshold 50 000/mm3, hemoglobin level 11 g/dL, avoid hypertension and hypomagnesemia. Ovarian tissue cryopreserved since 2015. Epidemiological, clinical, and analytical parameters were collected. Data are presented as percentages and medians (p25-p75). Results: 48 HSCT was performed in 47 patients (24 females), median age of 6.3 years (2.9-9.8); 11 of them with the first conditioning, 37 with the second one (GVHD prophylaxis with cyclosporine/MMF in 10/37 and tacrolimus/MMF in 17/37). Donor median age: 6.5 years (4.5-9.2), 48.9% sickle cell trait, 19% with major group and 19% with minor group incompatibility. All patients grafted but 3 patients experienced autologous recovery: 2 patients without symptoms (87% and 79% of receptor cells respectively); 1 patient suffered a vaso-occlusive crisis on day +180 but achieved complete donor chimerism after a second HSCT from the same sibling donor with the second conditioning regiment. Complete chimerism in 40/48 HSCT and stable mixed chimerism in 5/48. The overall survival (OS) and event-free survival (EFS) considering dead, or graft failure were 97.2% and 90.5%, respectively, with a median follow-up of 3.4 years (2.0-5.7). First conditioning: OS 90.9% and EFS 81.8%. Second conditioning: OS 100% and EFS 93.3%. Median time to neutrophil and platelets recovery were 18.5 days (17-22) and 23 (19.7-22.2) respectively. Complications of HSCT were: arterial hypertension 42/48 (87.5%), CMV reactivation 34/48 (71%), acute renal failure 11/48 (23%), neurological complications 11/48 (23%) (subarachnoid haemorrhage, seizure, posterior reversible encephalopathy syndrome or toxicity related with drugs), and acute GVHD 20/48 (41.7%): 55% with stage I (10 patients), 35% with stage II (7 patients), one patients with stage III but good response to treatment and only one patient developed refractory grade IV causing his death on day 51. None of the patients developed chronic GVHD. Conclusions: The outcome of our center, the larger HSCT series in Spain is similar to the international cohort and confirms the role of HSCT for children with SCD. Since 2015 we have improved our results, with less toxicity and without mortality. Disclosure: "Nothing to declare".
Background: Paroxysmal nocturnal hemoglobinuria (PNH) is a rare, potentially life-threatening hematologic disease characterized by complement-mediated hemolysis and thrombosis. Pegcetacoplan, an FDA-approved C3-targeted therapy for PNH, controls both intravascular and extravascular hemolysis. This analysis reports hemoglobin, lactate dehydrogenase (LDH), and Functional Assessment of Chronic Illness Therapy (FACIT)-Fatigue normalization rates in patients from the PEGASUS (NCT03500549) and PRINCE (NCT04085601) phase 3 trials. Methods: PEGASUS enrolled patients with hemoglobin levels <10.5 g/dL at screening, despite stable eculizumab treatment ≥3 months. Patients were randomized 1:1 to eculizumab or pegcetacoplan (1080 mg subcutaneously twice weekly) during the randomized controlled period (RCP) through Week 16. Patients who received pegcetacoplan (PEG) during the RCP continued with pegcetacoplan monotherapy (PEG-to-PEG) and eculizumab (ECU) patients switched to pegcetacoplan monotherapy (ECU-to-PEG) during the open-label period (OLP) through Week 48. PRINCE compared pegcetacoplan treatment (1080 mg subcutaneously twice weekly) in complement-inhibitor naïve patients (i.e., no eculizumab/ravulizumab within 3 months prior to screening) to standard of care (SoC; excluding complement-inhibitors). SoC patients could escape to the pegcetacoplan group if hemoglobin levels decreased ≥2 g/dL from baseline. Hemoglobin normalization (≥the lower limit of the gender-specific normal range in the absence of transfusions), LDH normalization (≤the upper limit of normal in the absence of transfusions), and FACIT-Fatigue normalization (≥population norm [43.6]) were determined at Week 16 and 48 (PEGASUS) and Week 26 (PRINCE). Patients who withdrew, were lost to follow-up without providing efficacy data at the specified timepoints, or escaped from SoC to the pegcetacoplan group in PRINCE were classified as non-responders. Safety endpoints included incidences of adverse events (AEs). Results: At Week 16 (PEGASUS) and Week 26 (PRINCE), pegcetacoplan groups achieved significantly higher rates of hemoglobin, LDH, and FACIT-Fatigue normalization compared to eculizumab and SoC groups (Table). At Week 48, PEGASUS patients who received eculizumab during the RCP achieved similar results to the pegcetacoplan group at Week 16 after switching to pegcetacoplan monotherapy during the OLP (Table). The most common AEs for pegcetacoplan in PRINCE were hypokalemia (11.4%) and dizziness (11.4%). In ≥12.0% of PEGASUS patients, diarrhea (RCP, 22.0%; OLP, 13.2%), abdominal pain (RCP, 12.2%), nasopharyngitis (OLP, 15.8%), upper respiratory tract infection (OLP, 13.2%), hemolysis (OLP, 18.4%), cough (OLP, 13.2%), and headache (OLP, 13.2%) were commonly reported. Injection site reactions were experienced during both trials (PEGASUS: RCP, 36.6%; PEGASUS: OLP, 18.4%; PRINCE, 31.4%). Conclusions: Overall, pegcetacoplan treatment leads to higher rates of normalization in hemoglobin and LDH, as well as improvements in FACIT-Fatigue in patients with PNH compared to SoC or eculizumab. This further supports the efficacy of pegcetacoplan in improving clinical parameters and quality of life while also demonstrating a favorable safety profile. aPatients with missing data at the specified timepoint were classified as non-responders Clinical Trial Registry: PEGASUS (NCT03500549): https://clinicaltrials.gov/ct2/show/NCT03500549 PRINCE (NCT04085601): https://clinicaltrials.gov/ct2/show/NCT04085601 Disclosure: Brian Mulherin: "nothing to declare". Michael Yeh reports current employment and current equity holder in publicly-traded company for Apellis Pharmaceuticals. Mohammed Al-Adhami and Jessica Savage report current employment with Apellis Pharmaceuticals. David Dingli reports consultancy and advisory board with Alexion, Apellis, Janssen, Milleneum/Takeda, Novartis, R-Pharm, and Rigel and Sanofi; and research grant with Juno and Karyopharm.
Background: Paroxysmal nocturnal hemoglobinuria (PNH) is a rare and potentially life-threatening disease characterized by chronic complement-mediated hemolysis, thrombosis, and some degree of bone marrow dysfunction. In May 2021, pegcetacoplan, a C3 complement-inhibitor, was approved by the FDA for the treatment of adults with PNH. This post hoc analysis evaluated the efficacy and safety of pegcetacoplan in a subgroup of patients with PNH with baseline hemoglobin levels ≥10.0 g/dL at 16 and 48 weeks from the PADDOCK (NCT02588833) Phase 1b and PEGASUS (NCT03500549) Phase 3 studies. Methods: PADDOCK evaluated pegcetacoplan therapy (270-360 mg/day subcutaneously) in complement-inhibitor naïve patients. PEGASUS enrolled patients that remained anemic despite stable eculizumab treatment (≥3 months) with hemoglobin levels <10.5 g/dL at the screening visit. Patients were randomized 1:1 to eculizumab or pegcetacoplan (1080 mg subcutaneously twice weekly) during the randomized controlled period (RCP) through Week 16. Patients who received pegcetacoplan during the RCP continued with pegcetacoplan monotherapy through Week 48 of the open-label period. The post hoc analysis included adult patients with PNH with baseline hemoglobin levels ≥10.0 g/dL and no transfusions within 14 days of the baseline measurement. For PEGASUS, only patients treated with pegcetacoplan in the RCP were included. Mean hemoglobin levels, absolute reticulocyte count (ARC), lactate dehydrogenase (LDH) levels, Functional Assessment of Chronic Illness Therapy (FACIT)-Fatigue scores, and percentage of patients with hemoglobin response (≥1 g/dL hemoglobin increase without transfusion) were evaluated at Week 16 and Week 48. Results: Overall, 12 patients were included in the post hoc analysis: six PADDOCK and six PEGASUS patients (baseline hemoglobin range: PADDOCK, 10.0-11.0 g/dL; PEGASUS, 10.0-10.8 g/dL). In this subgroup of patients treated with pegcetacoplan, improvements from baseline to Week 16 were seen in mean hemoglobin levels, ARC, LDH levels, and FACIT-Fatigue scores (Table). Similar results were also seen at Week 48 in both trials (Table), demonstrating the sustained effect of pegcetacoplan. A majority of patients in this subgroup also achieved a hemoglobin response at Week 16 and Week 48 (Table). Clinically significant increases (≥ 3-points) in mean FACIT-Fatigue scores were observed at both Week 16 and Week 48 (Table) and no thrombotic incidents occurred in this post hoc patient population. Conclusions: Overall, these results suggest pegcetacoplan can be efficacious long-term in patients with PNH with less severe anemia regardless of prior complement-inhibitor treatment, which results in further clinical improvements in markers of hemolysis and quality of life. The safety profile of pegcetacoplan was similar to results from previous studies. *One PADDOCK patient stopped dosing at Day 29 and left the study due to physician decision; therefore Week 16 and Week 48 data are out of a total N = 5. Clinical Trial Registry: PADDOCK (NCT02588833): https://clinicaltrials.gov/ct2/show/NCT02588833. PEGASUS (NCT03500549): https://clinicaltrials.gov/ct2/show/NCT03500549. Disclosure: Jens Panse reports consultancy, honoraria, membership on an entity’s Board of Directors or advisory committees with Blueprint Medicines, MSD, Grunenthal, Bristol Myers Squibb, Apellis Pharmaceuticals, and F. Hoffmann-La Roche Ltd; consultancy, membership on an entity’s Board of Directors or advisory committees with Amgen; speakers bureau with Chugai and Pfizer; and membership on an entity’s Board of Directors or advisory committees, speakers bureau with Novartis, Alexion, and Boehringer Ingelheim. Nicolas Daguindau and Sonia Okuyama: “nothing to declare”. Régis Peffault de Latour reports consultancy, honoraria, research funding with Novartis, Pfizer, and Alexion Pharmaceuticals Inc.; research funding with Amgen; and consultancy and honoraria with Apellis Pharmaceuticals Inc. and Swedish Oprhan Biovitrum AB. Philippe Schafhausen reports membership on an entity’s Board of Directors or advisory committees with Blueprint Medicines and Swedish Orphan Biovitrum AB; Honoraria, Membership on an entity’s Board of Directors or advisory committees and Speakers Bureau with Alexion and Bristol Myers Squibb; Honoraria and Membership on an entity’s Board of Directors or advisory committees with MSD and Novartis. Nicole Straetmans reports membership of advisory committee with Alexion. Mohammed Al-Adhami reports current employment with Apellis Pharmaceuticals. Temitayo Ajayi and Michael Yeh report current employment and equity holder in publicly-traded company with Apellis Pharmaceuticals.
Background: Both from an ethical and sustainability perspective, centers initiating allogeneic transplantation should start with cases least likely to develop severe transplant-related complications. In countries with a population in the 2-5 million range, e.g. Armenia, having an allogeneic transplantation program is justified but the availability of appropriate start-up candidates at low transplant risk may be limited. On the other hand, children with severe sickle cell disease (SCD) have one of the highest success rates after BMT (Iqbal et al. TCT 2020) and may have a very poor quality of life and life expectancy (Nnodu et al. Lancet Haematol 2021). Methods: Suitable initial candidates for allo HCT were defined as children < 10 years at low risk of pre-BMT active infections, poor prognosis with standard care, established indication for BMT, and full understanding and cooperation from their families. Free buccal swab DNA-based HLA typing was offered to families of children with SCD living in Sub-Saharan Africa and the implications of BMT were thoroughly explained. The start-up phase was carried out in collaboration with the Cure2Childen Foundation (C2C) which has been involved in the start-up of 10 HCT units across the Indian subcontinent and the Middle East, were more than 800 allogeneic transplants have been performed over the past 12 years. Intensive and structured collaborations with condition-specific experts combining both online and on-site training was implemented (Faulkner et al. BMT. 2021). A total of 4 children aged 1.9 to 10.6 years, three from Nigeria and one from Cameroon, were offered free HCT at the Haematology center after prof. R.H.Yeolyan, Yerevan – Armenia equipped with a state-of-the-art HCT unit used for autologous HCT in adults, and an active pediatric hematology-oncology unit treating high-risk patients with intensive chemotherapy. Families were fully informed that their child would be the initial allo HCT patient of that center but also that he/she would be followed by highly experienced HCT specialists. Results: All 4 patients are doing well at 164, 158, 39 and 12 days post BMT. Two patients had steroid-responsive acute GVHD grade II and III, one patient had a CVL-associated infection with staphylococcus aureus. The initial 2 patients have over 95% donor chimerism and hemoglobin electrophoresis consistent with a sickle cell carrier as their donors, they returned to their home country on days +100 and +106. Conclusions: This limited experience suggests that within structured cooperation with experienced professionals and organizations, offering BMT to children with SCD may facilitate BMT start-up and create a win-win situation for families whom otherwise would have not had the opportunity to cure their children from a life-threatening disease. Disclosure: Nothing to declare
Background: Sickle cell disease (SCD) is the most common, inherited red blood cell disorder (RBC) worldwide with significant debilitating and life-threatening complications such as stroke, acute chest syndrome and multiorgan failure. Conventional care has not demonstrated to match long-term survival of healthy peers. Hematopoietic stem cell transplantation (HSCT) is currently the only curative option for SCD and is the standard of care for the 20% of patients with a matched sibling (MSD) or matched unrelated donor (MUD). For the remaining 80% of patients, alternative curative options are a significant unmet need. T cell depleted haploidentical HSCT (T-haplo-SCT) is an established transplant modality with major advantages for SCD patients. T-haplo-SCT at least doubles the donor pool, provides an accelerated engraftment, and is associated with a low incidence of acute and chronic graft-versus-host disease (GvHD). In a pilot series feasibility, efficacy, and low toxicity of ab T cell depleted haplo-SCT for SCD has been shown. In order to provide prospective evidence for non-inferiority of T-haplo-SCT compared to MSD HSCT, a large international, controlled trial has been set up, the T-Haplo for SCD (T-haplo-SCT, EudraCT number: 2018–002652-33). Methods: The control arm of this stratified trial consists of eligible SCD patients with a MSD. For patients with no MSD, haploidentical relatives will be identified as potential donors according to established criteria and will be enrolled in the T-haplo-SCT experimental arm. The myeloablative conditioning for both arms consists of Fludarabin-Treosulfan-Thiotepa (FTT) with an ATG-Grafalon based in-vivo immunotherapy upfront in T-haplo-SCT versus prior to day 0 in MSD. Post-transplant immunosuppression consists of MMF and Tacrolimus. The primary endpoint is disease-free (DFS)/GvHD free survival (acute/chronic GvHD 12 months after omission of immunosuppression). Key secondary endpoint(s) are graft failure, hematological and immune-reconstitution, quality of life (QOL) and fertility. Results: The trial intends to enroll 212 patients in both arms and started recruitment in June 2021 in Germany. So far nine patients have been enrolled in three active centres. In total 27 centres in Germany, Austria, Italy, UK, Finland, Sweden and other European countries will be activated. Conclusions: Alternative curative approaches are needed in SCD. This trial intends to analyze in a large group of patients the efficacy and safety of T-Haplo-SCT with standard MSD-HSCT in a direct comparative design. A successful trial will offer a curative option for the majority of patients with no MSD, available at young age prior to development of SCD related irreversible damage and low transplant-related morbidity. Clinical Trial Registry: ClinicalTrials.gov: NCT04201210, https://clinicaltrials.gov/ct2/show/NCT04201210 EudraCT number: 2018–002652-33, Disclosure: Nothing to declare
Background: Hematopoietic cell transplant (HCT), the only standard curative option for patients with hemoglobinopathies, is limited by lack of unaffected HLA-matched donors, graft failure (GF), and graft-versus-host-disease (GVHD). Pre-transplant immunosuppression (PTIS), reduced toxicity conditioning (RTC) with related haploidentical (r-haplo) HCT has been associated with favorable outcomes among beta thalassemia (B thal) patients; Reports in sickle cell anemia (SCA) are evolving. Methods: Retrospective study of pediatric patients with severe SCA and B thal who underwent HCT from 2019-2021 at our institution. Results: Five patients (SCA n = 3, B thal n = 2; Table 1) with median age of 12 (2-16) years received 2 cycles of PTIS with fludarabine/dexamethasone followed by myeloablative r-haplo HCT with busulfan [AUC 18,000 µM.min with median clearance 108 (88-122) ml/min/m2, and median marrow total nucleated cell dose was 4.65 (1.72-5) x108/kg](Figure 1). Median time to neutrophil and platelet engraftment of 50,000/uL was 16 (15-17) and 32 (25-54) days respectively. DFS within 100 days was 100%. No GF, seizures, PRES, >grade II acute or any chronic GVHD were observed. PTIS cycles were well tolerated with asymptomatic viral detection noted upon routine surveillance. Patient 1 developed post-HCT hemolysis and decreasing chimerism on day 234, received rituximab and bortezomib (1125 mg/m2 and 5.2 mg/m2), required no further PRBC transfusions and was 100% donor on follow-up. Patient 3 had CMV viremia pre-PTIS, developed early CMV reactivation, severe sinusoidal obstruction syndrome, pulmonary fibrosis and subsequently died. No other patients required ICU admission and median hospitalization was 46 (42-113) days. Median absolute CD19, CD3/4, CD3/8, CD3/56 around 100 days post-HCT were 252 (64-415), 199 (96-282), 158 (50-465), and 5 (1-90) cells/uL, respectively. Neurocognitive testing and neuroimaging in two patients > 1-year post-HCT were unchanged from prior with no evidence of stroke. At median follow-up of 174 (95-898) days, all patients were 100% donor on most recent chimerism. CMV = cytomegalovirus, Rh = Rhesus antigen, R = recipient, D = donor, TBI = total body iron, LIC = liver iron concentration Figure 1. Treatment Schema for pre-transplant immunosuppression, reduced toxicity conditioning, and GVHD prophylaxis received by all 5 patients in this report. Conclusions: We report 100% DFS within 100 days of r-haplo HCT among patients with SCA and B thal. Significantly, there was no GF, severe acute or any chronic GVHD associated with this regimen. Vigilant viral monitoring and mitigation strategies may further improve outcomes. Disclosure: Nothing to declare.
Background: Inborn errors of immunity (IEIs) are a heterogeneous group of monogenic defects that manifest as increased susceptibility to infectious diseases, immune dysregulation (autoinflammatory or autoimmune conditions, atopic manifestations), and hematopoietic or solid tissue malignancies. Although conservative treatment may be effective for some IEIs, hematopoietic stem cell transplantation (HSCT) still remains the only curative approach for the vast majority Methods: Monocentric retrospective analysis of 220 patients treated with HSCT for IEIs at the Pediatric Bone Marrow Transplant Unit of Brescia between year 2000 and 2020. We report post-HSCT complications, evaluating their incidences according to conditioning strategies, use of graft source and donor. The incidence of viral reactivations and their correlation with long-term immunological outcome were also assessed. HSCT immunological outcome has been evaluated as donor engraftment and lymphocytes’ proliferative responses. Results: On a 20-year experience on 220 IEIs patients, overall survival was 73,2%, with lower survival rate in patients treated with non-myeloablative regimens (p < 0,001) or receiving HSCT without preconditioning regimen (p = 0,047) rather than those treated with myeloablative conditioning with Busulfan (p < 0,001) and Treosulfan (p = 0,003) or Reduced Intensity Conditioning (RIC) (p = 0,004). Graft failure occurred mostly in severe combined immune deficiency (SCID) patients, both T-B- and T-B + (31,8% and 22,8% respectively), followed by combined immune deficiency (CID) patients (18,2%) and congenital defects of phagocyte and osteopetrosis (13,6%). Median time of graft failure after first HSCT was 1,8 months. Overall, immunological reconstitution showed T-cell restoration in 93,7% of patients and B-cell restoration in 87,17% of patients, with median time of replacement therapy of 20,32 months. Use of anti-thymocyte globulin for graft versus host disease (GvHD) prophylaxis resulted also in an higher incidence of immune reconstitution on CD4 + (p < 0,05) and CD19 + (p < 0,05) cells and a lower median time of immunoglobulin replacement treatment (p < 0,005). Patients with mixed chimerism on long-term follow-up were mainly treated for SCID and CID. Patients with previous EBV infection showed a reduced number of CD3 + (p < 0,001), CD8 + (p = 0,005) and CD19 + (p = 0,014) when compared to all the patients who suffered from post-HSCT infectious episodes. GvHD occurred in 53,3% of all cases, with major prevalence in Wiskott-Aldrich syndrome patients; hepatic veno-occlusive disease occurred in 3,4% of the patients; transplant-associated microangiopathy was witnessed in 2,7% of all patients. Malignancies occurred in 4,7% of patients, lymphoproliferative disorders in 1,35%. Mean age from HSCT in patients presenting malignancy was 5,7 years. Cases of infection at HSCT or viral reactivation as post-HSCT complication mainly affected patients transplanted from cord blood units and treated with RIC conditioning (p = 0,002). Using human leukocyte antigens-mismatched donor was associated with a reduction of the average time of reactivation compared to other donors (p = 0,006) related to an increased use of immunosuppressive therapies in this subgroup. Conclusions: Our results confirm the effectiveness of HSCT as a curative treatment for IEIs, with excellent long-term survival rate and effective immunological reconstitution. Disclosure: No conflict of interest to declare
Background: Allogeneic hematopoietic stem cell transplantation (HSCT) provides an option as life-saving and curative treatment for a subset of primary immunodeficiency disorders (PIDs). However, allogeneic HSCT is accompanied by high rate of morbidity and mortality.To establish and evaluate a model that can predict prognosis of patients with PIDs who received allogeneic HSCT by machine learning. Methods: Clinical and lab data from 194 pediatric patients with PIDs who received allogeneic HSCT between February 2014 and June 2020 at the Children’s Hospital of Fudan University were retrospectively analyzed. Random forest was employed for constructing prediction models with Leave-one-person-out cross-validation. Model performance was evaluated by area under the receiver operating characteristic curve (AUC). Results: With a median follow-up of 32 months (range 0 to 92 months), estimated overall survival (OS) and disease-free survival (DFS) for the whole cohort at 3 years were 76.3% and 71.6%, respectively. We developed 3 models to predict patients’ prognosis after HSCT. The first model was to predict the mortality within about 2 months (early mortality) by 4 variables which achieved an AUC of 0.63 (95% confidence interval [CI], 0.53-0.74). The second model was built to explore the relationship between factors and DFS by cox proportional hazards regression with all variables, significant factors including patients’ height and transplantation age. The third model was built for risk event prediction model, consisting of all variables, demonstrated an AUC of 0.68 (95% CI, 0.58-0.78). Conclusions: The machine learning approach provided clinically reasonable and robust model to predict prognosis of PIDs patients who received HSCT. Our findings may be helpful for transplantation window selection, stem cells selection and supportive care adjustment. Clinical Trial Registry: No Disclosure: Nothing to declare
Background: Receptor Interacting Serine/Threonine Kinase 1 (RIPK1) is a widely expressed protein kinase, crucial in inflammatory and cell death signalling. RIPK1-knockout mice die perinatally secondary to tissue-wide apoptosis. Recently described in humans, RIPK1-deficiency results in impaired MAPK activation and reduced NF-κB activity causing uncontrolled necroptosis, and cytokine dysregulation, principally raised IL-1β and reduced IL-10. The phenotype includes lymphopenia, severe very-early-onset inflammatory bowel disease (VEOIBD) and arthritis. Haematopoietic cell transplantation (HCT) has been suggested as a potential curative therapy. We previously reported four children that were transplanted in the Great North Children’s Hospital (GNCH). To expand upon the role of HCT as a curative option we performed a multi-centre international review of the presenting characteristics and clinical outcomes of transplanted RIPK1-deficient patients. Methods: A retrospective case review of HCT for 7 children RIPK1-deficiency between 2011-2021 in: GNCH, UK; The King Faisal Specialist hospital & Research Centre (KFSH&RC), Saudi Arabia; Dmitry Rogachev Medical Research Centre of Paediatric Haematology, Oncology and Immunology (DRMRCPHOI), Russia. Results: Table 1 summaries the transplant characteristics and outcomes. P1-P5 presented with lymphopenia, VEOIBD, and arthritis, P6-P7 had VEOIBD alone. All except one received treosulfan-based conditioning and one had busulfan-fludarabine. Median CD34 + cell dose 17.09 x 106/kg (range 6.73-38.2). One event of Grade-I Acute Graft versus Host Disease. Viraemia post-HCT in P3: adenovirus, and P6: adenovirus, rhinovirus, and cytomegalovirus. Overall survival was 71%. One had secondary autologous reconstitution and underwent a successful second HCT. Most have led relatively disease-free lives. All are free of arthritis and only one has residual effects from IBD; at the most recent follow up P4 continues regular immunoglobulin replacement. Conclusions: Within the limitations of a retrospective small case series, the findings from this international, multi-centre review support the safety and efficacy of HSCT for RIPK1-deficiency, even with significant pre-transplant comorbidities. Disclosure: The authors certify they have no relevant financial or non-financial interests to disclose.
Background: Targeted busulfan (45-65 mg/L.h)-based reduced intensity conditioning (RIC) HSCT has been used successfully in chronic granulomatous disease (CGD) (Gungor et al. Lancet 2014). The use of this approach in MHC-class II deficiency (MHC-II-D) has not been reported. We describe our experience using this regimen in MHC-II-D and CGD. Methods: All patients underwent HLA matched HSCT using the following conditioning regimen: busulfan to target a cumulative dose of 50-70 mg/L.h, fludarabine (180mg/m2) and serotherapy (ATG for MRD and Alemtuzumab for MUD). GVHD prophylaxis included calcineurin inhibitor till at least day +180 and mycophenolate mofetil till day +100 if no GVHD. Bone marrow was the source of stem cells. Supportive care was consistent among all patients. Results: A total of 16 patients (8 MHC-II-D and 8 CGD) underwent HSCT at our institution. Patient and transplant characteristics are shown in the Table. All patients engrafted successfully. All CGD patients had normal oxidative burst oxidative test post-transplant without developing infections. All MHC-II-D patients expressed HLA-DR on lymphocytes with no history of post HSCT infections except for one patient who underwent MRD and had secondary graft failure associated with multiple viral infections and the cumulative busulfan AUC was 52 mg/L.h. Acute GVHD was reported in 2 patients, both of grade II and none had chronic GVHD. There was no mortality reported among our patients. Median whole donor chimerism at day 30-60, 100, 365 post HSCT were 88 (57-100), 78(0-100), 80 (0-100) respectively in MHC-II-D patients and were 94 (53-100), 97 (48-100), 88 (57-100) in patients with CGD. Median lymphocyte donor chimerism at day 30-60, 100, 365 post HSCT were 96(47-99), 84(0-100), 96 (0-100) respectively in MHC-II-D patients and 76 (53-100), 99(53-100), 82(60-100) in CGD patients. Median follow up duration was 1112 days (49-1839) in MHC-II-D patients and 409 days (111-1730) in patients with CGD. Conclusions: HSCT using targeted busulfan conditioning regimen was safe and effective in MHC-II-D and CGD patients. Larger sample size and longer follow up is warranted. Clinical Trial Registry: Not applicable Disclosure: Nothing to Disclose in relation to this abstract
Background: Allogeneic haematopoietic stem cell transplant is currently the only curative treatment for the recently described Autoinflammatory Periodic Fever, Immunodeficiency and Thrombocytopenia disease (PFTI) caused by mutations in WD repeat domain-1 (WDR1). This mutation inherited in an autosomal recessive manner causes abnormalities in actin-interacting protein-1 affecting neutrophil morphology, motility and function. Methods: We describe the case of a Pakistani 10 years old female from consanguineous parents diagnosed of PFTI from a Spanish tertiary University Hospital. She presented periodic fevers, thrombocytopenia (≤16 x 109/mm3), pyoderma gangrenosum and chronic multifocal osteomyelitis requiring steroids and Adalimumab; accompanied of multiple recurrent infections despite antibiotic prophylactic treatment. A mutation in WDR1 was identified through whole exome sequencing. The family study revealed both parents carried the mutation. Due to the lack of response to standard treatment she got an allogeneic stem cell transplant from an unrelated identical donor. She received a myeloablative conditioning regimen based on busulfan, fludarabine and rabbit antithymocyte globulin (ATG); and GVHD prophylaxis with cyclosporine and mycophenolate. Four months later she lost the engraftment requiring a second transplant this time from a related identical donor (mother). She received a reduced intensity myeloablative conditioning based on tiotepa, fludarabine, treosulfan and ATG. Cyclosporine was used as GVHD prophylaxis. Results: After the first transplant, the patient had mixed chimerism (figure 1) progressively decreasing until final rejection of the graft, not improving despite immunosuppression suspension and two donor lymphocytes infusions. After the second transplant she achieved total chimerism and maintains it three years later. Clinically the thrombocytopenia resolved and has not presented new infectious complications or any other symptom of her autoinflammatory disease. Conclusions: PFTI is a rare recently described disease and there is still not enough data to decide the best treatment for these patients. HSCT may be a valid therapeutic option, allowing restoring the immune system and decreasing their risk of fatal infection episodes. In cases where a related non carrier donor is not available, a heterozygous donor could be suitable as seen in our patient. Disclosure: Nothing to declare
Background: Results of a randomized trial suggest that autologous HSCT is effective in adult patients with severe, therapy-refractory Crohn’s disease (CD). However, relapse of the disease is frequent. In contrast, allogeneic (allo) HSCT has resulted in long-term cure of CD in few affected patients who underwent HSCT to treat life-threatening haematological malignancy. AlloHSCT is curative for many patients with inflammatory bowel disease (IBD)-like conditions due to monogenic inborn errors of immunity (IEI). Here, we describe alloHSCT in 3 children with severe treatment-refractory IBD in whom no genetic etiology could be found. Methods: Retrospective analysis of 3 pediatric IBD patients receiving alloHSCT at our institution. Results: All patients shared the following clinical features: disease manifestation at young age (1-7 years), severe systemic disease including failure to thrive, as well as refractoriness to various anti-inflammatory and immunosuppressive agents and/or steroid-dependency, requirement of surgical intervention, and failure to identify a genetic cause of IEI despite extensive immunophenotyping and whole exome sequencing including parents (trio-approach). Patient 1 presented at the age of 7 with severe CD-like disease, refractory to anti TNF, 5-aminosalicylic acid (ASA), azathioprine (AZA) and methotrexate (MTX). Rectal stenosis required partial colectomy and permanent ileostomy. At age 18 she underwent alloHSCT (Table). 6 months post HSCT she developed a toxic megacolon of the stenotic and unused colon requiring colostomy. After 6 years of follow-up, she remains in excellent clinical condition, has regained normal weight and is off all medication, but required colectomy 2 years post HSCT. Patient 2 presented at the age of 15 months with unclassified IBD, refractory to AZA, anti-TNF, MTX, 5-ASA, ustekinumab and ruxolitinib. Ileostomy was performed at the age of 8, followed by alloHSCT at the age of 9. At 1.5 years post HSCT she is in clinical remission, shows good catch-up growth and is off all medication. Endoscopy 6 months post HSCT showed colonic fibrosis with residual stenosis and mild inflammation. Patient 3 presented at the age of 2 with CD-like disease, requiring ileostomy until the age of 7, refractory to anti-TNF, AZA, MTX, vedolizumab, ustekinumab and cyclosporine A. At age 12, steroid dependency required re-ileostomy, followed by alloHSCT. 1 year post HSCT he is well without clinical signs of intestinal inflammation, shows good catch-up growth and is off all medication. Conclusions: While it is desirable to define the genetic etiology and immune-mediated pathomechanisms in greater detail, our experience shows that selected patients may benefit even in the absence of a molecular diagnosis. Early alloHSCT may avoid excessive morbidity caused by refractory disease and ineffective treatment. Table: Disclosure: Nothing to declare.
Background: WASp interacting protein(WIP) mutation is an autosomal recessive disorder, molecularly characterised by premature degradation of Wiskott Aldrich syndrome protein (WASp), since WIP is a stabilising chaperone protein to WASp. It is a rare mutation which has been reported only in three kindreds and 6 patients. WIP deficiency, though expected to classically recapitulate classic Wiskott Aldrich Syndrome(WAS), confers a phenotype with slight differences. We report the clinical features of a seventh case of WIP deficiency. We extend the phenotype of WIP to include transient, Juvenile myelomonocytic leukemia (JMML), which has also previously been reported rarely in WAS. Methods: Retrospective review of the patient records and laboratory investigations were undertaken. Results: A 4 weeks old male infant born to consanguineous parents of Libyan ethnicity, was referred with bloody vomiting and was found to have isolated thrombocytopenia. The blood smear confirmed thrombocytopenia, and the platelets were small. Since there was also history of an earlier male sibling death with infection and bleeding tendency, a diagnosis of classical WAS was considered. Flow cytometric studies showed absent WASp but no genetic mutation within the WAS gene was identified, and immunoblot revealed the presence of severely reduced amount of WASp in the cytoplasm. Panel sequencing showed a novel mutation in WIP, which is a chaperone protein for the WASP. On follow up, at 60 days of life, he developed signs of myeloproliferative disorder with gross hepatosplenomegaly, leucocytosis with monocytosis and a leukoerythoblastic blood picture with circulating myelocytes and erythroid precursors, qualifying for a diagnosis of Juvenile myelomonocytic leukemia according to 2016 WHO criteria. The HbF wasraised for age. The bone marrowshowed expanded left shifted myelopoiesis. The karyotype was normal. The molecular mutations for JMML including NRAS, KRAS, CBL, PTPN11, and NF1 were negative. In view of the underlying life limiting condition, he was worked up for a stem cell transplant. He had a matched sibling donor transplant with Fludarabine, Treosulfan, Alemtuzumab and Thiotepa conditioning, and with Ciclosporin alone as GVHD prophylaxis at 7 months of age and engrafted after 11 days and became transfusion independent from D25 of transplant. He had a mild steroid responsive gut GVHD which settled down quickly. He remains GVHD free and donor cell engrafted, now several years after HCT, and is fully donor immune-reconstituted and all infection and GVHD prophylaxis has been stopped. Conclusions: The dysregulation in RAS signalling caused by mutations of NRAS, KRAS, PTPN11 and CBL and NF1 can be found in 90% of the cases of JMML.However, in the remaining 10% of patients without a molecular mutation, the diagnosis is largely reliant on a combination of clinical and laboratory observations, as stringently laid by the 2016 WHO criteria.It has been well recommended to consider Wiskott-Aldrich syndrome in male infants with JMML where none of the 5 canonical molecular mutations of JMML can be identified. Certainly, a strong degree of suspicion should be exercised and investigations for WAS-WIP complex mutations should be actively looked for, particularly in children in JMML where the classic driver mutation cannot be identified. Disclosure: Nothing to declare.
Background: Autosomal recessive osteopetrosis is a rare disease caused by osteoclast malfunction. This results in a lack of bone resorption, thus leading to bone marrow suppression, extramedullary haematopoiesis, and compression of the cranial nerves, foremost the optic and vestibulocochlear nerve. As the disease proceeds, this causes visual and hearing impairment, as well as psychomotor developmental delay. Untreated it leads to death within the first decade of life. For most forms of autosomal recessive osteopetrosis haematopoietic stem cell transplantation is an option for curative treatment. Methods: We present data from 62 patients with malignant infantile osteopetrosis, transplanted at Ulm University paediatric clinic between 03/1984 and 02/2019. We analyse the relationship between neurological level prior to transplantation and short as well as long term neurological outcomes. We created a questionnaire and a corresponding ranking score scheme to collect longitudinal neurologic data on the following six topics: visual function, optic atrophy, auditory function, cognition, autism spectrum disorder, motor function. Data were gathered during pre- and follow-up appointments at Ulm University paediatric clinic. Results: Under 62 patients the share of females is slightly elevated. We find no evidence that the age of patients at the beginning of transplantation correlates with the occurrence of an autism spectrum disorder, diminished cognitive abilities, or motor function. There is a strong correlation between age and the occurrence of visual impairment, in particular the atrophy of the optic nerve. By exceeding the age of 12 months, we observe that optic atrophy occurs among all patients in our study. Furthermore, we find that patient’s age correlates negatively with auditory function. Conclusions: We conclude that the earlier beginning of the transplantation affects the neurological outcome of patients positively, especially concerning visual and auditory function. Hence, a precocious diagnosis and early treatment are of high prognostic relevance. Disclosure: Nothing to declare
Background: Mitochondrial neurogastrointestinal encephalomyopathy (MNGIE) is a rare autosomal recessive disease due to TYMP mutations that result in thymidine phosphorylase (TP) deficiency, leading to accumulation of nucleosides thymidine and deoxyuridine and subsequent mitochondrial dysfunction. Patients develop clinical manifestations of peripheral and autonomous neuropathy and myopathy. MNGIE is uniformly progressive and fatal, leading to premature death due to cachexia and infections. Normal leukocytes and thrombocytes have abundant TP, and it can therefore be permanently replaced through allogeneic HSCT. Methods: We present the case of a 23 years old man who presented with peripheral neuropathy. Diagnostic work-up revealed ptosis, ophtalmoplegy, leukencephalopathy and exercise-induced rhabdomyolysis. He was functioning normally, but was severely underweight (BMI 14.2 kg/m2) with evidence of malabsorption, but no gastrointestinal autonomous dysfunction, and intestinal biopsies were normal, consistent with an early stage of disease. He was homozygous for TYMP-mutation (c.866A > C) with complete TP deficiency and increased urine excretion of thymidine. He was transplanted with a BMSC graft from an unrelated male 10/10 HLA-matched donor after RIC (fludarabine 160 mg/m2 and busulfan 6,4 mg/kg intravenously). GvHD prophylaxis was antithymocyte globuline 4 mg/kg, cyclosporine and methotrexate. Medications with potential toxic effects on mitochondria were avoided if possible. Results: Post-transplantation follow-up was uneventful. Urine excretion of thymidine immediately fell to physiological levels, where it has remained. Neutrophil engraftment occurred on day +10, and by 3 months he achieved full unfractioned donor chimerism (>95%), but persistent mixed T-cell chimerism, which has been steadily improving after tapering of cyclosporine and subsequent removal at 10 months. At 17 months after transplantation he is well, has resumed full-time studies and working part-time. His body weight has increased slightly. Peripheral polyneuropathy and leukencephalopathy are unaltered and does not interfere with his everyday life. He has not experienced acute or chronic GvHD or other significant transplant related complications. Conclusions: Allogeneic HSCT was successfully performed in an adult MNGIE patient with malabsorption but no evidence of severe gastrointestinal involvement. Restoring TP through HSCT may repair mitochondrial function in MNGIE and improve clinical symptoms over time. However, there is conflicting data to support whether symptoms of progressive disease can be improved or not, and irreversible gastrointestinal changes, such as loss of interstitial Cajal cells, cannot be amended. Mortality after HSCT is reported in the literature as high as 62,5%, either due to TRM or progression of disease. Therefore, HSCT should be performed early, before irreversible gastrointestinal manifestations occur, to minimize risk and maximize recovery potential. Disclosure: Anders Myhre: Advisory board Takeda. Tobias Gedde-Dahl: Advisory board Takeda, Novartis, Incyte.
Background: In the last decade, many studies have tried to introduce innovations in the diagnosis and treatment of cytomegalovirus (CMV) infection and disease after allogeneic stem cell transplantation (allo-SCT). The IDWP undertook in 2020 a survey to describe the approach to the management of CMV infection among EBMT centers. Methods: A questionnaire was mailed to 579 EBMT centers performing allogeneic stem cell transplantation (SCT) in January 2020 with the deadline for the response by April 2020. A reply was returned by 180 centres (31%). The data relate to CMV infections occurred in 2019. Results: Among responding centers, 58%, 24% and 18% were adult, pediatrics, and mixed adult/pediatric transplant centers, respectively. CMV surveillance: CMV-DNAemia by PCR in blood plasma or serum was used in 175 (97%) of centers to diagnose CMV infection (missing data 1%); moreover, in 80 centers (46%) CMV-DNAemia was also tested at pre-transplant work-up. The patient CMV monitoring was performed in all types of allo-SCT in 97% of centers, mostly once (61%) or twice (30%) a week. The duration of CMV surveillance was limited to day + 100 in 27% of centers while in the remaining centers it continued for at least 6 months post-SCT or was modulated on duration of GVHD, immunosuppressive therapy and/or immune recovery. CMV prophylaxis: in high-risk patients (R-or-D-CMV + ), drug prophylaxis was used in 56% of centers with higher prevalence in adult/mixed than pediatric centers (62% and 61% vs. 37%), with the following drugs: letermovir, 61.4% (almost exclusively in adult patients), acyclovir/valacyclovir, 18.8%, gancyclovir/valgancyclovir, 7.9%, foscarnet 1%, CMV-CTLs, 1%, other (several combinations), 9.9%. CMV infection and pre-emptive treatment: the median annual number of pre-emptive treatments for CMV infection was 12 per center, range 1-600. The drugs used as first-line treatment were: gancyclovir/valgancyclovir in 79%, foscarnet in 4 %, gancyclovir + foscarnet 13%, other or missing 4%; CMV-immunoglobulin were used together with drug prophylaxis in 3.3% of centers. The most frequent threshold to start pre-emptive therapy was: CMV-DNAemia of any positivity, >102, >103 and >104 copies/ml in 12.8%, 16.1% 56.7%, 8.4%, other 6% for unmanipulated allo-SCT, Pre-emptive treatment lasted until 2 consecutive CMV-DNAemia negative tests (76.7%), or CMV-DNAemia inferior to the threshold used to start treatment (14.4%), or other (9.9%). CMV disease and treatment: 64.4% (116) of centers diagnosed > 1 episode of CMV disease (median 2, range 1-50) for a total of 605 episodes, classified as proven, 217 (35.8%), probable, 183 (30.2%) and possible (33.8%). The first-line treatment used was gancyclovir/valgancyclovir, gancyclovir+foscarnet, foscarnet in 71.6%, 15.5%, 3.3% of centers, respectively (missing 9.6%). CMV-immunoglobulin were used in 7.2%. Fifty centers (28%) declared to use CMV-CTL in case of rescue treatment or in combination with antiviral drugs. CMV drug resistant infections were reported in 70 of 180 centers (median 2 episodes/center). Conclusions: PCR-CMV-DNAemia monitoring and pre-emptive therapy remain the key interventions-More than half of centers treating adult patients have adopted the policy of letermovir prophylaxis. Further studies are needed to assess how prophylaxis impacts the burden of CMV infection in allo-SCT. Disclosure: No conflict of interests to declare
Background: Meningococcal ACWY and B vaccinations are recommended after HCT. However, there is so far no data on MenB vaccination after HCT. We evaluated the safety and immunogenicity of the 4CMenB vaccine (Bexsero®) and the persistence of response 12 months later in allogeneic adult, HCT recipients (NCT03509051). Methods: Patients were eligible from 6 months post-HCT, if they had no relapse of their underlying disease, and had not received antiCD20 antibodies since >6 months. They received 2 doses of 4CMenB at 2 months interval. Adverse events (AE) were prospectively collected. Blood samples were collected before the first vaccine dose (V1), 1 month after the second dose (V3), and 12 months after V1 (V4)(Fig. 1). Sera were immediately frozen at -80°C. The serum bactericidal activity (SBA) using human complement (hSBA) was assessed against fHbp, NadA, PorAP1.4 and NHBA antigens (Caron, LID 2011). The response was defined by > one of these two criteria for > one of the 4 vaccine antigens: (1) In patients with a hSBA titer <4 on V1: a titer >4 after vaccination; (2) In patients with a hSBA titer > 4 on V1: at least a x4 increase from baseline. hSBA titers were described as % of responders. Geometric mean titers (GMT) [95%CI] were also calculated. Quantitative and qualitative variables described with median (ranges) and proportions respectively were compared with Kruskal Wallis test and chi-2 or Fisher test as appropriate. Univariate and multivariate analysis were performed using logistic regression. Results: 40 patients were included a median of 2.14 years post-transplant. The median age at transplant was 52 y. Most patients had acute leukemia and were transplanted with an unrelated donor. Four patients had GvHD at inclusion, 8 were receiving immunosuppressive drugs. At V1, most patients had hSBA titers < 4 (93%, 100%, 95% and 90% for fHbp, NHBA, NadA and PorAP1.4, respectively) and 8/40 (20%) had titers > 4 on > 1 antigen. The proportion of patients with a titer > 4 was significantly increased between V1 and V3 (primary objective) for fHbp, NadA, and PorA but not for NHBA for which only 6/40 (15%) patients were responders. At V3, 36/40 (90%) patients were responders to > 1 antigen, with 27 patients (67.5%) responding to 2 (25%), 3 (30%) and 4 (12.5%) antigens. At V4, 23/37 (62.2%) patients were still seroprotected although at lower titers and mainly against NadA (51.4%). Moreover, GMT did not differ significantly between V1 and V4 for fHbp, NHBA and PorA antigens suggesting rapid decline of hSBA titers. Age and CD4 counts were associated with a response at V3 in univariate analysis. AE were all minor, without any severe AE. Conclusions: Considering a response rate of 90% for > one vaccine antigen and the favorable safety data, our findings fully support the 4CMenB vaccination of HCT recipients from 6 months after transplant with 2 doses. However, a booster dose may be required. Meningococci B vaccination should be combined with ACWY vaccination for which data are already available. Clinical Trial Registry: NCT03509051 Disclosure: none
Background: The role of antibacterial prophylaxis in the pre-engraftment phase of allogeneic haemopoietic stem cell transplantation (HSCT) is still a matter of debate: prophylaxis may reduce bloodstream infection (BSI) incidence, but may also increase the rate of multidrug resistant (MDR) bacteria. Based on these observations, fluoroquinolone (FQ) prophylaxis has been withheld in our Center from April 2016 onward. The aim of this retrospective single center study was to evaluate the impact of the omission of FQ prophylaxis on the incidence of BSI in the first 30 days after HSCT. Methods: Overall, 592 consecutive patients who received an allogeneic HSCT between January 2011 and August 2021 were included in the study. Patients who received an antibacterial prophylaxis other than FQ or were given broad spectrum antibiotic treatment at the time of transplant were excluded from the analysis. Among the 444 evaluable patients, 195 received FQ prophylaxis (group A) while 249 did not (group B). Baseline characteristics were superimposable in the two groups, except for bone marrow as stem cell source (23.1% in group A and 9.2% in group B, p < 0.001) and reduced intensity conditioning regimen (14.8% in group A and 21.7% in group B, p = 0.043). Median duration of neutropenia was 16 days in both group A (range 9-49) and group B (range 4-35). Results: Overall, BSI was detected in 100 patients (22.5%), 32 (16.4%) in group A and 68 (27.3%) in group B (p = 0.008). Cumulative incidence of BSI at day 30 post transplant was 16.4% in group A and 27.5% in group B. In multivariate analysis, FQ prophylaxis was the only factor associated with the risk of BSI (SHR 0.59; 95% IC 0.39-0.91; p = 0.016). Nine patients in group A (4.6%) and 41 patients in group B (16.5%) developed a gram-negative BSI (p < 0.001); 20 patients in group A (10.3%) vs 17 patients (6.83%) developed a gram-positive BSI (p = 0.13); a polymicrobic BSI occurred in 13 patients, 3 in group A (1.5%) and 10 in group B (4%) (p = 0.1). Gram-negative bacteria accounted for 28.1% (n = 9) of BSI in group A and 60.3% (n = 41) in group B (p = 0.005). Overall, 8 patients in group A (25%) and 6 patients in group B (8.8%) developed MDR-gram negative BSI, unveiling a marginally significant trend towards the reduction of MDR gram-negative BSI after withholding FQ prophylaxis (p = 0.06). Death attributable to BSI occurred in 4 of 100 patients (4%); 2 in group A and 2 in group B. Neither antibacterial prophylaxis (p = 0.56) nor the occurrence of BSI (p = 0.9) had a significative impact on overall survival (OS). Conclusions: The results of our study show an increased rate of BSI, mostly caused by Gram-negative bacteria, in patients who did not receive FQ prophylaxis, with no impact on OS. By contrast, a lower incidence of MDR gram-negative BSI has been observed in patients not receiving FQ prophylaxis. Additional prospective studies are needed to confirm our data. Disclosure: Nothing to declare
Background: We recently reported favorable short-term outcome in patients with leukemia after HSCT and a history of COVID-191. Considering the key role of endothelial-damage in HSCT causing transplant associated thrombocytopenic-microangiopathy (TA-TMA), veno-occlusive disease (VOD)/SOS and even more in COVID-192, we analyzed incidence and outcome of endothelial complications after HSCT following COVID-19 in 14 patients. Methods: Patients with advanced leukemia and a history of COVID-19 were transplanted at the University-Medical-Center-Hamburg-Eppendorf UKE (n = 9) and University-Hospital-Frankfurt (n = 5). Patients experienced COVID before, during or after induction chemotherapy with persistent lung infiltrates in nine and required ICU admission in six patients. The time interval from diagnosis of COVID-19 to HSCT was median 143 (46-212) days and resolution of COVID-19 to HSCT 94 (35-136) days. Patients (median age 52.5 (33-69) years) had high risk AML (n = 8), in PR or higher CR (n = 2), high risk ALL (n = 3) or blast crisis of CML (n = 1). Donors were matched related (n = 3), haploidentical related (n = 4), matched unrelated (n = 4) or mismatched unrelated (n = 3). All patients received fludarabine in combination with total body irradiation (8 or 12 Gy, n = 7), thiotepa/busulfan (n = 3), melphalan (n = 3) or treosulfan (n = 1) + ATG (n = 10). TA-TMA was defined as previously described3. VOD was diagnosed according to Seattle-criteria. Results: After a median follow-up of 221 (range 69-492) days, 11 (79%) of the 14 patients are alive. One patient died on day +146 from AML relapse, one from cardiac (d + 208) and one from liver complications (d + 179). Three female patients (out of 8 female and six male patients) had VOD, TA-TMA or both, all of them associated with polyserositis, a median of +67.5 (9-242) days after HSCT. All three are alive a median of +451 (range 221-492) days. One patient(H1) with a history of severe pulmonary COVID-19 developed histologically confirmed TA-TMA eight months post-haplo HSCT. Cytomegalovirus (CMV) reactivation, bacterial urogenital and clostridium difficile infection may have triggered TA-TMA. After successful treatment of infections, the patient was readmitted for CMV reactivation and polyserositis.The patient is alive 492 days after HSCT. One patient(H4) recovered from COVID-19 after treatment with reconvalescent serum (7 d BID) and received a haplo-identical HSCT. On day +59 post-HSCT, CMV reactivation, BKV cystitis, polyserositis and ascites were detected. Pathological liver enzymes and liver histology confirmed VOD, which was treated successfully with defibrotide. The patient is alive 451 days post-HSCT. One patient(F4) with AML had a HSCT from a mismatched (9/10) unrelated donor after COVID-19. The patient was diagnosed with VOD/SOS (day + 9) and treated successfully with defibrotide. On day +76, EBV reactivation, BKV cystitis and TA-TMA was diagnosed. The patient was treated with eculizumab, but remained on dialysis (+221 days). Conclusions: After an uneventful early post-transplant period, three patients out of 14 were diagnosed with severe endothelial-damage and polyserositis. Interestingly, all patients were female, transplanted from non-matched donors and had viral/bacterial infections. The incidence of TA-TMA observed in this two-center study is 21% and in the range of complications observed after COVID and HSCT alone2,4. This observation should be confirmed in a larger cohort of patient receiving allogeneic HSCT after resolution of COVID-19. Clinical Trial Registry: References 1. Christopeit M, Reichard M, Niederwieser C, et al. Allogeneic stem cell transplantation in acute leukemia patients after COVID-19 infection. Bone Marrow Transplant. 2021;56:1478–81. (https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7871512/pdf/41409_2021_Article_1225.pdf). 2. Tiwari NR, Phatak S, Sharma VR, Agarwal SK. COVID-19 and thrombotic microangiopathies. Thromb Res 2021;202:191–8. 3. Dandoy CE, Rotz S, Alonso PB, et al. A pragmatic multi-institutional approach to understanding transplant-associated thrombotic microangiopathy after stem cell transplant. Blood Adv 2021;5:1–11. 4. Pagliuca S, Michonneau D, Sicre de Fontbrune F, et al. Allogeneic reactivity-mediated endothelial cell complications after HSCT: a plea for consensual definitions. Blood Adv 2019;3:2424–35. Disclosure: No conflict of interest, G. Bug: Honoraria from Jazz Pharmaceuticals
Background: High-level BKPyV replication in patients after allogeneic hematopoietic cell transplantation (HCT) indicates failing immune control and increased risk of hemorrhagic cystitis. We investigated BKPyV-DNA genome loads in plasma and urine of HCT-patients, and sequenced large tumor-antigen (LTag) or capsid Vp1 epitopes predicted to mediate CD8 T-cell killing and antibody neutralization. Methods: BKPyV-loads and human genome-loads were assessed with and without DNAse-I digestion prior to nucleic acid extraction in longitudinal urine (N = 61) and plasma samples (N = 64) from 17 HCT-patients with detectable BKPyV and hematuria (grade-1 N = 8; grade-2 N = 3; grade-3 N = 4; grade-4 N = 2). For BKPyV, three quantitative nucleic acid tests (QNAT) with different amplicon lengths (88bp, 133bp and 239bp) were used (Leuzinger et al., 2019 J Clin Virol 121:104210 PMID:31759262). Variation in BKPyV genome sequences was determined by next-generation-sequencing with different amplicon lengths (250bp, 1000bp and 5000bp). Results: DNAse-digestion resulted in significant reductions of >90% of urine and plasma BKPyV-loads in HCT-patients with all three amplicon sizes (p < 0.001; Figure 1). Significantly higher BKPyV-loads were obtained with the 88bp compared to the 133bp and 239bp BKPyV QNAT in both urine and plasma samples (p < 0.001; Figure 1). Little sequence variation was determined by NGS in LTag and Vp1 when using large amplicons of 1000bp and 5000bp. In contrast, NGS of 250bp amplicons identified LTag and Vp1 minority variants with frequencies in up to 15%. This included non-synonymous aa-exchanges in immunodominant LTag-9mer T-cell epitopes reflecting different BKPyV-genotypes (I and IV) as well as genotype-independent variants. For example, genotype-IV dependent variation in the LTRDPYHTI LTag 9mer T-cell epitope altered HLA-A/HLA-B-binding scores, predicting reduced 9mer epitope presentation and escape from T-cell activation (Table 1). Genotype-IV dependent H244Y/T245I exchange has been previously reported in 22.3% of LTag sequences in the NCBI protein database (Leuzinger et al., 2020 Viruses 12:1476 PMID:33371492). Moreover, we detected mutations at highly conserved positions in the BC-loop of Vp1 (A72, E73, E82), previously associated with escape from neutralizing antibodies. Table 1. BKPyV genotype-I and -IV in 9mer T-cell epitopes and predicted HLA-A/HLA-B-binding scores. Amino acid, aa; human leukocyte antigen, HLA Amino acid starting position 238 (BKPyV-WW numbering [acc. no. AB211371.1]) HLA binding was predicted with the Immune Epitope Database and Analysis Resource tool (http://tools.iedb.org/main/). Conclusions: Urine and plasma BKPyV-loads in HCT-patients are mostly derived from DNAse-I sensitive, non-encapsidated BKPyV-DNA fragments of <100bp. This reduced diagnostic sensitivity by QNAT with larger amplicon-targets leading to under-quantification of BKPyV-loads. Moreover, BKPyV-diversity may be underestimated including immune escape in immunodominant LTag and Vp1 epitopes from CD8 T-cell killing (Wilhelm et al., 2020 J Infect Dis 223:1410 PMID:32857163) and antibody mediated naturalization. Importantly, the results change current models by indicating that BKPyV-loads in plasma are a direct marker of viral replication and cell/tissue damage releasing genomic-fragments and not virions, and hence being not susceptible to neutralizing antibodies. We discuss the implications in an updated model on BKPyV replication and disease for treatment approaches. Clinical Trial Registry: Not applicable Disclosure: Nothing to declare
Background: Endemic human coronaviruses (HCoV) are frequent causes of respiratory tract infections after allogeneic stem cell transplantation, and preexisting HCoV immunity possibly protects against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infections because of cross-reactive antibodies binding to the spike protein. We therefore investigated whether pre- and postvaccination levels of antibodies directed against various community-acquired respiratory viruses correlates with antibody responses following SARS-CoV-2 vaccination. Methods: Serum samples were collected from allotransplant recipient at Oslo University Hospital and analyzed for SARS-CoV-2 specific antibodies directed against the spike protein receptor binding domain (RBD), full-length spike protein and nucleocapsid. Antibody levels were also determined for four endemic human corona viruses (HCoV-229E, HCoV-OC43, HCoV-NL63, HCoV-HKU1) as well as four influenza A subtypes (H1N1, H3N2, H5N1, H9N2), Rhinovirus and Epstein-Barr-virus. Results: The study included 16 females and 15 males (median age 58 years, range 21-73years) who had received at least two doses of mRNA-1273 (Moderna) or BNT162b2 (BioNTech/Pfizer). The median time from transplant to the second vaccine was 371 days (range 149-2443 days, IQR 715); 10 patients received systemic immunosuppression at the time of the first vaccination. One patient had pre-existing borderline protective levels of antibodies against RBD and spike protein without any history of coronavirus infection or increased levels of nucleocapsid antibodies. Twenty-five patients showed a detectable RBD response; which was classified as a strong response for 18 individuals. Preexisting levels of antibodies against endemic corona viruses varied considerably and were highest for HCoV-229E and HCoV-OC43. Prevaccination antibody levels against HCoV-229E showed a significant difference only with the full length spike protein vaccine response. The post-vaccination HCoV-229E antibody levels showed both a weak correlation with the post-vaccination RBD response (Kendall’s τ 0.33/p = 0.016) and a moderate correlation with the full spike protein response (Kendall’s τ 0.43/p = 0.003). No correlations with antibody levels for influenza A subtypes, EBV or Rhinovirus were observed. Both previous ATG exposure and short time from transplant to first vaccination were associated with weaker vaccine responses and lower levels of full-length antibody levels. Ongoing systemic immunosuppression at time of first vaccination and chronic GVHD were not associated with lower vaccine response. Conclusions: 18 of 31 allotransplant recipients showed a strong antibody response to SARS-CoV-2 vaccination. The antibody response was associated with pre-vaccination antibody levels to HCoV-229E, previous ATG therapy and time from transplant to vaccination. Our study suggest that this vaccination regimen for allotransplant recipients should be individualized and possibly be guided by the RBD antibody responses. Clinical Trial Registry: N/A Disclosure: THAT: Advisory Board/ Honorary: Sanofi, Novartis, Sobi, Janssen AEM: Advisory Board: Takeda
Background: Fecal microbiota transplantation (FMT) is now considered a potentially efficient option for microbiota reconstitution and recovery from different intestinal syndromes including intestinal GVHD. Hence, the aim of present study was a search for informative bacterial markers associated with clinical response to FMT in severe gut GVHD. Methods: The prospective, non-randomized study enrolled 27 children and adults (1 to 52 years) with intestinal form of GVHD (acute, chronic, or overlap syndrome) following allogeneic HSCT. FMT was used in the cases of severe refractory GVHD. In most cases, FMT was performed using ingestible gelatin capsules containing either third-party fecal biomaterial, or placebo in controls. On D + 3, D + 16, D + 30, D + 60, and D + 120 after FMT, 16 major bacterial groups were assayed in fecal DNA samples using real-time multiplex PCR (Colonoflor test system). Biodiversity of the bacterial microbiota was also studied by means of 16S rRNA sequencing (NGS platform). Clinical response was assessed by common scales for intestinal syndrome and GVHD evaluation. Results: Complete response, according to Bristol intestinal scale, was registered in approx. 50% of cases following FMT, being less common in the placebo group (13%) . Multiplex PCR of fecal DNA has shown significant recolonization of fecal microbiota after FMT. Such positive shifts were demonstrable since D + 30 for total microbial mass (p = 0.002); Escherichia coli (p = 0.001); Bacteroides fragilis group (p = 0.05); Faecalibacterium prausnitzii (p = 0.005). In particular, the median copy numbers of B.fragilis group and F.prausnitzii showed sufficient increase along with evidence of clinical response after FMT. Meanwhile, a significantly lesser recolonization with these bacterial species was revealed in the patients with poor clinical response to FMT. Over 120 days of observation, the subgroups with complete response versus partial/no response showed significant increase for B. fragilis group (p < 0.0001), F.prausnitzii (р < 0.0062). To confirm this finding, we compared the results of multiplex PCR with the data on general Bacteroides class by means of 16SrRNA sequencing using NGS. The difference between FMT and placebo-treated patients was revealed on the days 16 and +30. By day +30, an increase over pre-FMT values was found for the Bacteroidetes phylum; Bacteroidia (Class); Bacteroidales (Order) which include B.fragilis group. Both B.fragilis and F.prausnitzii represent sufficient part of microbiota, promote immune tolerance. Therefore, they are considered potential probiotic microorganisms. Other groups of intestinal bacteria, e.g., Lactobacillus spp., and Bacteroides thetaiotaomicron, generally, were not changed over this time period. In the placebo group we did not find significant changes against initial levels over 120 d post-FMT. Conclusions: 1. Quantitative PCR of major bacterial groups of, especially, B. fragilis group, F. prausnitzii could be used as useful tool for evaluation of gut microbiota shifts after HSCT followed by FMT. 2. Multiplex PCR of the common bacterial species allows routine semi-quantitative monitoring of intestinal dysbiosis and its recovery. 3. The genocopy counts of B. fragilis group correlate with clinical response in the patients with intestinal GVHD after HSCT, either with, or without FMT procedure. 4. Our results argue for potential usage of novel probiotics based on the non-toxic strains of B. fragilis and/or F. prausnitzii. Disclosure: No conflicts of interest declared
Background: Patients with hematologic malignancies are at high risk of severe illness and mortality from SARS-CoV-2 infection. Evolving data illustrates that patients with hematologic malignancies may have diminished responses to SARS-Cov-2 vaccinations further increasing their risk of severe infection. Several monoclonal antibody (MOAB) combinations have been approved under an emergency use authorization (EUA) as treatment of mild to moderate COVID-19 in non-hospitalized high-risk patients. The safety and efficacy of MOABs in recipients of hematopoietic cell transplantation (HCT) and cellular immunotherapy (CI) is undetermined. The aim of this study is to describe the safety and effectiveness of anti-SARS-CoV-2 MOAB in HCT/CI recipients at our institution. Methods: We retrospectively reviewed the charts of HCT/CI recipients who were treated with MOAB for a diagnosis of SARS-CoV-2 between December 1, 2020-Sepetember 30, 2021. The primary end point was hospitalization and/or death related to severe SARS-CoV-2 infection. Results: Fifty patients were identified and included in this analysis with a median of 360 days (12-4790) from time of HCT/CI to diagnosis of COVID19. The median age was 60 years (25-78 years) with 54% of patients being males. Twenty-eight patients (56%) were recipients of allogeneic HCT, 30% (15/50) autologous HCT and 14% (7/15) CAR-T cell therapy. Most patients in the allogeneic HCT group (89%, 25/28) were on immunosuppressive medications at time of diagnosis. Three of the 15 autologous HCT recipients had received rituximab within 90 days of diagnosis. Twenty-four patients (48%) had previously completed 2 doses of SARS-CoV-2 vaccine. All treated patients met EUA criteria for the administration of MOAB for mild to moderate COVID19 infection. Casirivimab/imdevimab was the MOAB used in 76% (38/50) of patients, bamlanivimab in 18% (9/50) of patients and 6% (3/50) received bamlanivimab/etesevimab. Most patients (68%) received the MOAB infusion on the same day of diagnosis. There were no reported infusion reactions. With a median follow up of 81.5 days, the COVID-19 related hospitalization rate was 6% (3/50). Two of these 3 patients had received two COVID-19 vaccines. None of the hospitalized patients required intubation despite 67% (2/3) experiencing acute hypoxia requiring ≥2L of supplemental oxygen. The median duration of hospitalization was 5 days (4-23). There were no deaths related to SARS-CoV-2 infections. Table 1: Baseline Demographics and Clinical Characteristics of Patients N = 50. * Myeloproliferative disorders and aplastic anemia ‡ After two doses of mRNA SARS-CoV-2 vaccine Conclusions: Treatment with anti-SARS-CoV-2 MOAB in fifty HCT-CI patients at our institution demonstrates encouraging efficacy in a high-risk patient population. We show low rates of severe illness (6%) requiring hospitalization and no SARS-CoV-2 related deaths. Disclosure: Nothing to declare.
Background: Patients with hematologic malignancies (HMs) are at higher risk for severe Covid-19 disease (CVD), reflected on twofold increased mortality than the affected general population. Vaccination against SARS-CoV-2 remains the most important measure for CVD prevention. However, HMs patients are characterized by lower antibody responses (AR), while they were excluded from the initial studies for BNT162b2 mRNA efficacy. Moreover, the immunological response and actual benefit of vaccination in hematopoietic stem-cell transplantation (HSCT) recipients has not yet been clarified. The aim of this study was to analyze the AR after vaccination against SARS-CoV-2 in HMs patients treated with HSCT. Methods: HMs patients treated with HSCT beyond 6 months and within the last 5 years in our Department were included. Antibody-titers (AT) against SARS-CoV-2 prior to the first dose as well as 1 and 3 months after the second dose of BNT162b2 mRNA vaccine were assessed using Elecsys®Anti-SARS-CoV-2 S immunoassay against spike protein RBD [positivity limit ≥0.8IU/mL]. Median AT in healthy individuals 1 month after vaccination are reported ≥ 1000U/mL, measured by the same method. Results: Patients’ characteristics are depicted in Table 1. No patient had detectable antibodies prior to vaccination, while one month and three months after vaccination, 84.6% and 87.8% of them had detectable antibodies, respectively. A decline in AT between 1- and 3 months was observed: median AT at 1- and 3-months post-vaccination were 480.5(0.4-25000) and 293(0.4-7869)U/mL, respectively. Specifically, AT decreased in 75% of the patients, increased in 17%, while antibodies remained undetectable in 11%. The distribution of AT is shown in Table 1: AT ≥ 1000U/mL were detected in 38.5% and 30.6% of the patients at 1-month and at 3 months post-vaccination, respectively. Concerning potential influencing factors of AR, age, sex, underlying hematologic disease, type of HSCT and absolute lymphocyte/mononocyte counts did not prove significant. On the contrary, hypogammaglobulinemia [IgG < 500mg/dL], time from HSCT ≤ 18 months and disease-related treatment at the time of vaccination proved significant unfavorable factors for AR (p = 0.01, p = 0.03, p = 0.001, respectively). Median AT for those with no treatment was 1488U/mL, while those treated with rituximab, lenalidomide or other regimens were 0.4, 210 and 18.5U/mL, respectively. Table 1. Basic features of HM/HSCT patients included in this cohort. Conclusions: Over 80% of HM/HSCT patients developed positive AR after BNT162b2 mRNA vaccine. Consequently, the vaccination of these patients is highly recommended. However, the presence of a decreasing trend in AT 3 months post-vaccination suggests the need for a third dose in this group. Disclosure: Nothing to declare
Background: In-vivo lymphocyte depletion methods such as anti-thymocyte globulin (ATG) and post-transplantation cyclophosphamide (PTCy) are used in unrelated donor (MUD) hematopoietic stem cell transplantations (HSCT). In this retrospective study, we compared the incidence of clinically relevant infections (necessitating intervention) and infections/reactivations related readmissions within one year following HSCTs with ATG or PTCy-based conditioning. Methods: Patients undergoing MUD HSCT in two centers each using ATG-based (Center A) or PTCy-based (Center B) (nonmyeloablative) conditionings were compared with each other for incidence of infections/reactivations and readmissions. Incidence of bacterial/fungal infections was evaluated for the periods 0-30 vs. >30 days, as PTCy is associated with a significantly longer neutropenic phase in the first weeks post-HSCT. Viral infections/reactivations were evaluated for 0-100 vs. >100 days. Infection density per infection category, accounting for number of infections in a patient per 1000 person days, were compared over three periods (0-100 days, 101-365 days and overall, 0-365 days). Results: We analyzed 118 patients with ATG and 79 with PTCy who were transplanted between 2009 and 2020. Cumulative incidence of bacterial infections was significantly higher in the PTCy group in the first 30 days (22% vs. 7%), but higher in the ATG group after >30 days (35% vs. 14%)(Table 1). The incidence of fungal infections was similar in both groups/phases. Patients receiving ATG had a higher incidence of CMV reactivations and disease as compared to PTCy (37% vs. 16% and 10% vs. 0%, respectively), which mostly occurred within 100 days post-HSCT. Other viral infections were comparable in the first 100 days but significantly higher in the ATG group after 100 days post-HSCT (22% vs. 6%, p = 0.003). Infection densities of bacterial, fungal and viral infections and reactivations are shown in Figure 1. Infection density analyses showed higher bacterial infection density (per 1000 person days) by day 100 for PTCy (4.83 vs. 2.25, p < 0.01) and it was higher in the ATG group after 100 days (2.03 vs. 0.17, p < 0.01). The overall density for bacterial infections was similar in the first year post-HSCT. The density of fungal infections was not significantly different for both treatment groups. Viral infections were significantly higher in ATG compared to PTCy patients from day 100 to 365 (1.54 vs. 0.29, p < 0.01) and in the overall density analysis in the first year post-HSCT (3.09 vs. 1.41, p < 0.01). Viral reactivations remained higher in the ATG group in all periods within 1 year. The incidence of readmission due to infections/reactivations after 30 days post-HSCT was 24% vs. 16%, for ATG and PTCy respectively. Table 1. Cumulative incidence of infections. Conclusions: As both viral reactivations/infections and readmissions are more common in patients receiving ATG than in those receiving PTCy, replacing ATG with PTCy as GvHD prophylaxis might result in decreased disease burden and hospitalizations. Disclosure: David de Leeuw: Takeda: Membership on an entity’s Board of Directors or advisory committees. Bart Biemond: Celgene: Honoraria; Global Blood Therapeutics: Honoraria, Research Funding, Speakers Bureau; Novartis: Honoraria, Research Funding, Speakers Bureau; Novo Nordisk: Honoraria; CSL Behring: Honoraria; Sanquin: Research Funding. Jeroen Janssen: Bristol-Myers Squibb: Consultancy, Research Funding; Novartis:Consultancy, Research Funding; Incyte Biosciences Benelux BV: Research Funding, Speakers Bureau; Pfizer:Consultancy; Uppsala County Council: Research Funding; Glycomimetics: Research Funding; Avillion: Research Funding; Ellipses Pharma: Research Funding; Roche: Speakers Bureau; Celgene: Membership on an entity’s Board of Directors or advisory committees, Speakers Bureau. Erfan Nur: Novartis: Research Funding, Speakers Bureau; Roche:Speakers Bureau; Celgene: Speakers Bureau.
Background: The incidence of coronavirus disease 2019 (COVID-19) is less in the pediatric than in the adult population. Although children with cancer, bone marrow transplantation recipients are considered a high-risk population for COVID-19 infection, published data specifically addressing the pediatric oncology are still limited. Methods: We analyzed the clinical course and outcomes of COVID-19 in a pediatric cohort of RM Gorbacheva Research Institute. The PCR test was performed at the admission to the hospital for all patients, weekly during hospitalization and in cases of developing symptoms of infection. Results: A total of 54 (29 male and 25 female) pediatric patients (pts) had a laboratory-confirmed SARS-CoV-2 infection between April 2020 and August 2021. Median age was 7 years (4 months – 18 years). There were 3 diagnosis groups: hematological malignancies (33, 61%), solid tumors (13, 24%), non-malignant diseases (8, 15%). Twenty-seven (50%) pts received hematopoietic stem cell transplantation (auto HSCT – 2, 8%, MRD-HSCT – 2, 7%, MUD-HSCT – 2, 7%, haplo-HSCT – 21, 78%; median interval to COVID-19 infection: 94 days, range -2-2711). Chemo or IST were used in 24 (44%) pts with median interval to COVID-19 infection of 19 days (range, 4-39); 3 (6%) pts were without specific therapy (COVID-19 and cancer were diagnosed at the same time). The majority of pts (33, 61%) had asymptomatic forms, while 13 (24%) pts had mild (leukemia – 5 pts, SAA – 2, PNH – 1, solid tumors – 5); 3 (6%) - moderate (all pts with leukemia) and 5 (9%) – severe disease (leukemia – 3, SAA – 1, MPS 1 type - 1). Severity and clinical manifestation were the same in transplant and chemo/IST groups. The typical clinical manifestations were fever (90%), fatigue (50%), tachypnoea (30%) and cough (20%). Seventeen pts (31%) were hospitalized in infection units. Twelve (22%) pts received COVID-19-directed treatment (convalescent plasma – 8, ruxolitinib – 3, steroids – 2, tocilizumab – 1). Fifty (93%) pts recovered. Median time between symptom resolution and negative PCR was 25 days in symptomatic group, 14 days – in asymptomatic. Persistent SARS-CoV-2 PCR positivity (more than 21 day) observed in 16 pts (30%). Overall survival was 92%. Overall mortality was 8% and did not differ in HSCT and chemo/IST groups[MP1]. The only death was attributed to COVID-19, while ALL progression and complications after HSCT were causes of death in 1 and 2 patients, respectively. Conclusions: In our cohort immunocompromised pediatric patients the incidence of severe COVID-19 does not exceed 10%, overall mortality was 8% and the only one death was attributed to COVID-19. There was no difference in the course of COVID-19 in subgroups analysis. Although this patient population is managed as high risk, according to our initial experience the clinical features of COVID-19 are milder and prognosis is relatively good. Nevertheless, further research should detail the therapeutic tactics for the underlying disease and HSCT complications in establishing the diagnosis of COVID-19. Disclosure: Nothing to declare
Background: HCST recipients present high risk of severe COVID-19 infection and have been considered for priority vaccination. However, as they usually remain immunosuppressed for months after HSCT, vaccine efficacy might be compromised. Reports on serological response after vaccination in hematological patients confirm the lower antibody response rate, compared to the general population (>90%). The characterization of the response of transplanted patients could help to design more efficacious vaccination programs. Methods: SARS-CoV-2 vaccination with mRNA1273 vaccine (Moderna) was prospectively evaluated in patients vaccinated from 6 months to 5 years after HSCT. Underlying disease, HSCT and vaccination characteristics were collected. The SARS-CoV-2 serological status and immunological conditions were determined before vaccination. Vaccines were administered as two doses 4 weeks apart and serological response was assessed 2-4 months after complete vaccination. In cases without serological response, we reassessed humoral and cellular response after a third dose vaccine. Results: Eighty-seven patients (56 auto-HSCT and 31 allo-HSCT) received two doses between 23 March - 5 May 2021. No severe adverse effects were reported. Patients’ characteristics are summarized in Table 1. At 2-4 months after vaccination, 75/87 (86%) patients presented seropositivity without differences between auto-HSCT and allo-HSCT. Considering patients with previous negative serological status, 65/77 (84%) patients presented seroconversion, without differences between both groups. The antibody response rate was higher in the allo-HSCT group, with a median of 2578 (40.8-42793.41) UI/mL, compared to 1675.5 (67.2-10686.09) UI/mL for auto-HSCT patients. Twelve patients showed a persistent negative serological status after vaccination. 6/12 patients had a NHL as HSCT indication and 8/12 were auto-HCST recipients. We found an association between negative serological response and B-lymphocyte count <113.5/mm3 (29% vs 3% in the group with B-lymphocyte count ≥113.5/ mm3, p = 0.002), IgG <700 mg/dL (31% vs 4% in the group with IgG ≥700 mg/dL, p = 0.002) and receiving anti-CD20 therapy in the last year before vaccination (100% vs 12% in patients without anti-CD20 therapy, p = 0.003). Eleven of twelve patients without seroconversion received a third dose of the vaccine. At 2-6 weeks, 5/11 patients achieved seroconversion and 6/11 presented cellular response (2 of them without seroconversion). At 6 months follow-up, no cases of COVID-19 infection have been reported. Table 1. Conclusions: SARS-CoV-2 vaccination seems to have clinical benefit in preventing COVID-19 infection in HSCT recipients, although seroconversion rate after mRNA SARS-CoV-2 vaccination is lower than in the general population. Receiving anti-CD20 therapy in the last year before vaccination, a low B-lymphocyte count and hypogammaglobulinemia are associated with no serological response after vaccination. Cellular and humoral response monitoring after SARS-CoV-2 vaccination could help to identify booster dose candidates. Disclosure: Nothing to declare.
Background: Several studies have compared the outcome of patients undergoing a haploidentical transplant (HAPLO) or an HLA-matched transplant (MATCHED). In most cases, HAPLO grafts received triple post-transplant cyclophosphamide (PTCY)-based GvHD prophylaxis, whereas MATCHED grafts received a conventional cyclosporine, methotrexate prophylaxis with or without ATG. This has also been the case for studies comparing the risk of infections in these two different cohorts of patients. Methods: The aim of the study was to assess the risk of infections in the first 100 days, in patients grafted from HAPLO or MATCHED donors, all of them receiving a homogenous GvHD prophylaxis: PTCY, mycophenolate, and cyclosporine. Eligible for this study were patients with hematological malignancies, with triple PTCy-based GvHD prophylaxis: This included 117 HAPLO and 68 MATCHED patients (29 from HLA identical siblings and 39 from 8/8 matched unrelated donors). HAPLO grafts were unmanipulated bone marrow transplants. The two groups were comparable for donor age (p = 0.2), intensity of the conditioning regimen (p = 0.5) and disease phase (Early, advanced) (p = 0.4). Data on post-transplant infections, including bloodstream infections (BSI), invasive fungal infections (IFI), and viral infections (CMV and EBV), were obtained retrospectively. For each infection type, a competing risk analysis was performed, with mortality from any cause as the competing risk. A confirmatory analysis with propensity matching was performed and included 68 HAPLO transplants and 68 MATCHED transplants. Criteria for propensity matching included donor and recipient age, disease phase, and intensity of the conditioning regimen (Myeloablative, reduced intensity). Kaplan Meier curves were used to compare actuarial survival. Results: Patients who received a HAPLO transplant had an increased incidence of BSI (HR 2.9; 95% CI 1.7–5.1; p < 0.001); sub-analysis revealed an increased incidence of gram-positive BSI (HR 2.8; 95% CI 1.4–5.6; p = 0.003) and a trend for increased incidence of gram-negative BSI (HR 1.9; 95% CI 0.9–3.8; p = 0.08). Among patients with BSI, the most frequently isolated gram-positive bacteria in the early post-transplant period (Days 0 to +20) were coagulase-negative Staphylococcus, and the most frequently isolated gram-negative bacteria were E. coli, both in HAPLO and MATCHED transplants; Klebsiella spp. were also frequently isolated in HAPLO patients. There was also a trend for increased incidence of IFI in HAPLO grafts (HR 1.9; 95% CI 0.9–3.9; p = 0.08), of CMV infections (HR 2.0; 95% CI 0.9–4.6; p = 0.08), and of EBV infections (HR 4.2; 95% CI 0.5–34; p = 0.2). In the propensity matched analysis, these results were confirmed. The actuarial 1-year survival was comparable: 74% for HAPLO and 78% for MATCHED grafts (p = 0.2). Conclusions: In this single center study, patients with a HAPLO donor have an increased risk of early bloodstream infections and a trend for increased risk of invasive fungal and viral infections. Disclosure: Nothing to declare
Background: Fluoroquinolone prophylaxis (FQ-P) has been largely adopted worldwide in hematological patients undergoing allogeneic hematopoietic stem cells transplantation (allo-HSCT) with expected protracted neutropenia. However, sepsis sustained by Gram-negative bacteria (GNB), particularly if multidrug resistant (MDR), still affects mortality in neutropenic hematological patients. In recent decades, Italian epidemiological data has shown worrisome rates of fluoroquinolone (FQ) resistance. Moreover, alterations of the intestinal microbiome have been reported in patients receiving FQ-P, potentially affecting the occurrence of bloodstream infections (BSI) after allo-HSCT. In such a context, the benefit of FQ prophylaxis is controversial. Methods: We prospectively analyzed a cohort of consecutive 223 adult allo-HSCT performed at our Bone Marrow Transplant Unit from January 2018 to December 2020. Since February 2019 FQ-P was withdrawal. During study period an active microbiological surveillance was performed weekly according to local practice and piperacillin/tazobactam was the first-line therapy of febrile neutropenia (FN). We collected data of FN according to FQ-P: 71 allo-HSCT receiving FQ-P (levofloxacin-group, January 2018-January 2019) and 152 allo-HSCT without FQ-P (withdrawal-group, February 2019-December 2020). Study’s outcomes were cumulative incidence function (CIF) of GNB pre-engraftment BSI (PE-BSI) and any changes in antimicrobial resistance, CIF of FN and infection-related mortality (IRM). Results: Overall, 221 patients underwent 223 allo-HSCT. The levofloxacin-group and the withdrawal-group were superimposable for characteristics (age, sex, disease, disease status at allo-HSCT, comorbidity-index score). The majority (73.5%) of patients was affected by myeloid disorders. The graft source was mainly (91%) unmanipulated peripheral blood, using a post-transplant cyclophosphamide strategy. Stem cell donors were matched unrelated volunteer (n = 105, 47.1%), family haploidentical (n = 61, 27.4%), HLA-identical sibling (n = 44, 19.7%), or cord blood (n = 13, 5.9%). One FN episode occurred in 96.4% of transplants, with no differences among the two groups according to the usage of FQ-P [95.8% vs 96.7%; p = 0.72]. At least one PE-BSI occurred in 45.3% of allo-HSCT and a significant difference was observed in the 30-day CIF according to FQ-P [36.4% levofloxacin-group versus 51.9% withdrawal-group; p = 0.019]. At least one GNB PE-BSI occurred in 26.5% of allo-HSCT and a significant difference was observed in the 30-day CIF according to FQ-P [14.7% levofloxacin-group versus 34.4% withdrawal-group; p = 0.003]. Regarding GNB-PE-BSI etiology, among the levofloxacin-group 10 single-species GNB PE-BSI occurred in 10 patients; the most represented pathogens were Escherichia coli (n = 4), Klebsiella pneumoniae (n = 4) and Pseudomonas aeruginosa (n = 2).In the withdrawal-group 55 GNB PE-BSI occurred in 49 patients; the most represented GNB were Escherichia coli (n = 27), Klebsiella pneumoniae (n = 13) and Pseudomonas aeruginosa (n = 8). Comparing antimicrobial resistance among GNB, in the withdrawal-group a significantly higher proportion of pathogens was susceptible to piperacillin/tazobactam (71% versus 30%, p = 0.026) and FQ (49% versus 10%, p = 0.03), and a lower proportion was resistant to carbapenems (5% versus 50%, p = 0.001). At 30-days CIF of IRM was 5%, superimposable in both groups [p = 0.62]. Conclusions: In allo-HSCT setting, the FQ-P reduced GNB PE-BSI, with no impact on IRM; its withdrawal concurred to decrease significantly antimicrobial resistance in GNB. These data confirm the safety of an approach based on FQ withdrawal in the in-patient setting where active surveillance is applied. Disclosure: Nothing to declare
Background: COVID-19 has resulted in high morbidity and mortality among hematopoietic stem cell transplant (HCT) recipients and CAR T cell treated patients. These population have therefore been regarded as high priority for vaccination. Little is known of the severity and outcome of COVID-19 contracted after vaccination. The IDWP has collected data on this topic through the continuing prospective data collection on patients with COVID-19. This abstract summarizes current results. Methods: The EBMT registry has collected information on COVID-19 infection since end of February 2020. In February 2021 questions regarding COVID-19 vaccinations were added. To date, the registry has received reports on 28 patients contracting COVID-19 after the date of the first vaccine dose. 20 patients were after allo HCT, 6 after auto HCT, and 2 had received CAR T cells. The median age was 54.5 years (20-74). 11/20 allo HCT patients had active GVHD and 10 received immunosuppression at the time of COVID-19. Results: The median time from the first vaccine dose to diagnosis of COVID-19 was 24 days (2 – 242 days). 10 patients required hospitalization while 16 were cared for as out-patients (data missing = 2). Four patients required ICU (data missing for 2 patients) and five patients died. For all 5 patients, the primary cause of death was COVID-19. Four patients were treated with monoclonal antibodies and one with hyperimmune plasma. Out of patients receiving one dose of vaccine, 7/14 (50%) were hospitalized, 4/14 (28.6%) required ICU, and 5/16 (31.2%) died including the patient, who had received one dose just before the HCT. Two patients having received one dose had not resolved COVID-19 at the time of reporting. Of patients receiving two doses of vaccine, 3/12 (25%) were hospitalized, none required ICU or died, and all COVID-19 infections had resolved. The overall survival probability was 79.1% at 6-weeks from the diagnosis of COVID-19. Excluding the patients vaccinated before HCT the survival probability at 6-weeks was 81.6%. The survival was superior in patients having received two doses (Fig. 1). Conclusions: Two doses of any vaccine against COVID-19 resulted in lower risks for complications requiring ICU and death. Therefore, it is of uttermost importance to pursue vaccinations of HCT and CAR T cell treated patients. Disclosure: Per Ljungman: Speaker for Pfizer
Background: Bloodstream infections (BSIs) after alloHCT are prevalent secondary to the profound immunosuppression needed to preserve graft function and to prevent GVHD. The use of PTCY for GVHD prophylaxis is becoming more widespread in the transplant community, and further studies are needed to determine the incidence of BSIs among transplanted patients. This study investigates the incidence and risk factors for BSIs in adults undergoing alloHCT, and explores the effect of PTCY on the probability of presenting this complication. Methods: Between January 2014 and March 2021, 334 adults with hematological malignancies underwent first alloHCT at our Institution, and 204 (61.1%) received PTCY-based GVHD prophylaxis. Antibiotic prophylaxis with levofloxacin was given to all patients during the aplastic phase. The diagnosis of at least 1 episode of BSI was considered the main dependent variable. And, among those patients that had more than 1 BSI, only the first episode was accounted. Data was updated in November 2021. The cumulative incidence of BSI was calculated considering death a competing event. Results: Baseline characteristics between patients receiving PTCY vs those that did not were balanced, except for the proportion of patients transplanted during 2018-2020 where the use of PTCY was more prevalent. Overall, 165 (49.4%) patients had at least one episode of BSI. Of the 165 patients with BSI, the majority of them were diagnosed during the first 30 days after alloHCT (70%); and with an estimated cumulative incidence at day +30 of 34.7%. The median of days to the first BSI was 14 days (range: -5 – 1083). BSIs caused by Gram-positive bacteria were more prevalent that those cased by Gram-negative bacteria (50.9% vs 43.6%). With a median follow-up of 2 years, the estimated one-year OS and NRM of the entire cohort were 72.7% and 13.9%, respectively; and the 1-year incidence of first BSI mortality was 3%. The cumulative incidence of BSI was higher for patients receiving PTCY compared with those receiving others GVHD prophylaxis (Day + 30 and +100 incidences of 46.6% vs 16.2% and 52.0% vs 20.8%, respectively; P < 0.001), and the median of days to the first BSI was shorter for patients treated with PTCY (13 vs 36 days, P < 0.001). The multivariate analysis confirmed that the use of PTCY-based GVHD prophylaxis can be considered a risk factor for being diagnosed with BSIs (HR 2.37, P = 0.001). Other risk factors were age at transplant (HR 1.02, P = 0.01), KPS ≤ 80% (HR 1.58, P = 0.008), and the use of MAC regimens (HR 1.47, P = 0.05). Conclusions: The incidence of BSI at our institution was 49.4%, but the overall mortality rate attributed to this complication was 2%. The inclusion of PTCY for GVHD prevention was found to be an independent risk factor for being diagnosed with BSI; in fact, those patients were 2.37 times more likely to be diagnosed with BSI compared with patients that did not received PTCY. The use of PTCY for GVHD prevention is becoming prevalent, so further study will be required to refine antimicrobial prophylaxis and improve supportive care. Clinical Trial Registry: No applicable Disclosure: Conflicts of interest: PP-A has received honoraria for talks on behalf of Merck Sharp and Dohme, Gilead, Lilly, ViiV Healthcare and Gilead Science. CG-V has received honoraria for talks on behalf of Gilead Science, MSD, Novartis, Pfizer, Janssen, Lilly as well as a grant from Gilead Science and MSD. The rest of the authors have nothing to declare.
Background: Clinically significant CMV infection (csCMVi) frequently complicates post-alloHCT care. We hypothesise that anti-CMV drugs may be better tolerated with improved neutrophil and immune recovery occurring after several weeks. The impact of csCMVi commencement for first CMV infections in the earliest period post-alloHCT compared to a later period post-alloHCT has not been thoroughly evaluated. Methods: We conducted a national multi-centre retrospective cohort study across 6 adult HCT centres across Australia. Patients receiving an allo-HCT were included between the study period of 2015 to 2020. Patient and transplant demographics were collected as well as details of acute graft versus host disease, relapse and overall survival. csCMVi was defined as CMV DNAemia which was treated with specific anti-CMV treatment. HCT centres varied in the viral threshold with which to commence anti-CMV treatment depending on the type of transplant received. Early csCMVi was defined as <50 days from stem cell infusion based on the median time to csCMVi from previous studies. Results: A total of 830 allo-HCT recipients were included with a median age of 53 years (IQR 42-60). The most common indications for transplantation were AML (38%), MDS (12%) and ALL (12%). Donor relationship included unrelated (54%), related matched sibling (38%) and haploidentical donors (8%). Reduced intensity conditioning was used in 42% and T-cell depletion used in 44% of the cohort. Peripheral blood stem cells were the most common source of cells (93%). Baseline CMV serostatus included R + /D + (42%), R + /D- (25%), R-/D + (11%) and R-/D- (22%). Pre-emptive CMV monitoring was the preventative strategy used in 99% of patients. Detectable CMV DNAemia occurred in 54% of patients at a median time of 20 days post-HCT (IQR 8-33). Two-hundred and ninety patients (35%) had csCMVi where the median time to anti-CMV treatment was 49 days (IQR 40-63). The first prescribed anti-CMV treatment was valganciclovir (57%), ganciclovir (29%), foscarnet (0.7%), others such as trial products and CMV-specific T cells (10%). The median neutrophil count at the start of CMV treatment was 2.8x109/L (IQR 1.6-5). Grade 3-4 neutropenia occurred in 50% of patients during the course of treatment. AGVHD developed in 38% of the cohort of which 65% were Grade 2-4. Patients who received CMV treatment in the period 50+ days compared to the early period post-HCT had a significantly increased risk of all-cause mortality (HR 1.52 95% CI 1.04-2.22, p = 0.03) (Figure 1). In the R + /D- cohort, the risk of mortality was also increased (HR 2.2 95% CI 1.17-4.18, p = 0.01). In an adjusted model for AGHVD, late versus early CMV treatment remained an independent risk factor for all-cause mortality (adjusted HR 1.48 95% CI 1.01-2.17, p = 0.04). Conclusions: There is a significant all-cause mortality burden on allogeneic HCT recipients who develop csCMVi in the period >50 days following transplantation. Severe neutropenia is common in patients using valganciclovir/ganciclovir as first line anti-CMV treatment. Disclosure: MY, JL, AG, SvH, PB, JS, DR and MS have received consulting fees from MSD. Financial declaration: This was an investigator initiated study sponsored by MSD
Background: The efficacy of Covid-19 vaccine, and the optimal vaccination time in recipients of allogeneic hematopoietic stem cell transplants (allo-HSCT) are still unknown. Methods: Our analysis involved adult outpatients after allo-HSCT, who have been receiving the mRNA-based SARS-CoV-2 vaccine. Vaccine-induced antibody responses against the SARS-CoV-2 were assessed in serum using the chemiluminescent immunoassay (CLIA) test, validated according to the WHO standard (the quantification range was 4.81- 2080 BAU/mL, and a cut-off for a positive result was 33.8 BAU/mL). Peripheral blood lymphocyte subpopulations CD3 + CD4 + , CD3 + CD8 + , CD19 + , and NK cells were analyzed using flow cytometry. Statistical analysis was performed using Spearman’s Rho correlation coefficient to measure the strength and direction of correlation between antibody response and the lymphocyte subpopulations. Results: Retrospective analysis involved a group of 57 patients (median age - 48 years), transplanted from HLA-identical siblings (35%), matched unrelated donors (57%) or haploidentical donors (8%) in the years 2006-2021 (31% of patients in the years 2020-2021). There were 53 patients (93%) transplanted for hematological malignancies of whom 30 (56%) received myeloablative conditioning, and 4 (7%) patients with aplastic anemia. Patients have been vaccinated against SARS-CoV-2 with two doses of vaccine. Ninety-five percent of patients were vaccinated with Comirnaty® (BNT162b2, Biontech/ Pfizer), and five percent of patients with mRNA-1273 (Moderna) vaccines according to the EBMT guidelines 2021 (v.4). Five patients (8%) had previously mild or moderate COVID-19 according to the WHO guidelines. Immune responses were analyzed between 1-4 months after the second dose. Fifty one patients (89%) were receiving immunosuppressive treatment at the time of vaccination. Eleven patients (19%) were supplemented with immunoglobulins at least 4 weeks before the first dose of vaccine. Altogether, vaccine-induced antibody responses were achieved in 42 patients (74%). Nineteen patients (33%) achieved high level of anti-SARS-CoV -2 titer above 2080 BAU/ml, 8 patients (14%) - antibody titer between 1000-2000, 12 patients (22%) between 100-1000, and the remaining 3 (5%) between 33-100 BAU/ml. We did not detect anti-SARS-CoV-2 antibodies in 2/3 (66%) of patients vaccinated within 6 months after transplantation, in 6/13 (46%)- within 6-12 months, 7/41 (17%) – above one year after allo- HSCT. Most of them (14/15, 93%) suffered from Graft versus Host Disease. No detectable SARS-CoV-2 antibodies were observed in 7/11 (64%) of patients with CD19 + deficiency, 11/33 (30%) patients with CD3 + CD4 + deficiency, and 5/11 (45%) – with NK cells deficiency. In the multivariate analysis, a correlation was found between the number of CD3 + CD4 + lymphocytes, and the obtained anti- SARS-CoV-2 titer (p = 0.012). Conclusions: These results indicate that the ability for humoral response after 2 doses of anti-SARS-CoV-2 vaccination develops during the 1st year after allo-HSCT provided the low intensity of graft versus host disease. CD3 + 4 + T-cells deficiency may play a role in the antibody response to SARS-COV2 vaccine. However, none of our patients succumbed to COVID19. Apparently, both isolation procedures, and vaccinations provided sufficient protection even in this group composed of patients at particular risk of a severe course of COVID 19. The advisability of administering additional doses of the vaccine to non-responding patients requires further research. Disclosure: Nothing to declare
Background: Invasive pulmonary aspergillosis (IPA) is the most common invasive fungal disease in patients with AML and allogeneic stem cell transplant recipients. Without antimould prophylaxis, the incidence of IPA in these populations is 10-20%, with a fatality rate between 20-38% 6 to 12 weeks after diagnosis. Optimizing the management of IPA is key to reduce mortality and morbidity. At Ghent University Hospital, a diagnostic driven approach is followed for IPA. With this retrospective cohort study, we wished to evaluate the added value of baseline chest CT before start of classical induction 3 + 7 chemotherapy, a common practice in newly diagnosed AML patients in our centre since 2015. Methods: All adult patients with newly diagnosed AML, MDS or myelofibrosis without intensive pretreatment, who were eligible for intensive chemotherapy and who had a baseline chest CT around start of chemotherapy (+/−7 days) were included. Data were collected retrospectively from the electronic health record (EHR) from patients admitted between January 2015 and October 2019. Diagnosis of IPA was classified using the EORTC/MSG-criteria. Statistical analysis was performed using SPSS statistic version 25. Results: Ninety-nine patients were included (table 1). Baseline chest CT was abnormal in 61/99 patients (62%): 14/61 patients (23%) had radiological signs that met EORTC/MSG-criteria. Only 6/99 patients received mould-active antifungal prophylaxis. No patient was diagnosed with IPA at baseline. During treatment, 29/99 patients (30%) developed IPA (no proven, 29 probable). Of the patients with normal baseline CT, 8/38 (21%) developed IPA, versus 21/61 (34%) patients in the group with abnormal baseline CT. Mortality 12 weeks after start induction chemotherapy was 50% (7/14) in patients with IPA and 19% (5/27) without IPA. Seven patients with abnormal lesions at baseline received BAL, but no IPA was found (BAL GM and culture were negative, no PCR performed). However, 4 of these patients developed IPA during their first two cycles of chemotherapy (figure 1). Table 1: baseline characteristics. Figure 1: incidence of IPA vs baseline CT Conclusions: Patients with abnormal baseline CT had a higher incidence of IPA during treatment. Baseline chest CT did not lead to any probable/proven IPA at admission however only few BAL were performed following abnormal baseline CT. Disclosure: Nothing to declare
Background: Children undergoing allogeneic hemaptopietic stem cell transplantation (HSCT) face a higher risk of severe and lethal blood stream infections (BSI). Rapid and sensitive diagnosis of acute infectious complication represents an unmet clinical need; the T2MR-technology is a direct, non-culture assay for rapid identification of 6 common BSI-pathogens (P. aeruginosa, K.pneumoniae, E. coli, A. baumanii, S. aureus, E. faecium). Methods: We retrospectively analyzed 58 consecutive T2Bacteria performed in febrile patients undergoing HSCT from May 2018 to September 2020, with concurrent blood-culture (BC) requested as standard-of-care for suspected BSI. We wanted to test diagnostic real-life performance of T2Bacteria panels in the identification of proven, probable and possible BSI, and evaluation of possible benefits provided by T2MR-technology in children. Results: Clinical characteristics are listed in fig.1. T2Bacteria provided definitive microorganism identification in a mean time of 4.4 (SD: 0.7) hours, compared to 65.7 (SD: 24.5) hours for BC (p < 0.001). BC and T2Bacteria resulted positive in 7 and 11 cases, respectively, with 53/58 concordant results (91%). T2Bacteria identified E.coli in 3 cases, P. aeruginosa in 4 cases, E. faecium in 2, K. pneumoniae in 2. Four out of 7 proven BSI were concordant; one positive BC with T2MR negativity was considered blood contaminant, while probable- and possible-BSIs, defined by compatible cultural and/or laboratory characteristics, represented 2 and 5 additional T2MR-positive cases (7/11, 64%), respectively. T2Bacteria had a sensitivity of 67% (95%CI: 1-0.28) and 86% (95%CI: 0.95-0.75) specificity in the identification of 4 BC-positive proven-BSI; relatively low sensitivity is probably due to the low number of BC-positive cases. Thus, when probable-, or probable/possible-BSI are classified as true-positives, sensitivity and specificity of T2Bacteria rose to 85% and 95%, respectively Conclusions: T2Bacteria panel rapidly and accurately diagnosed pediatric BSIs caused by 6 common bacteria, with small blood volumes. These findings support its clinical use in the setting of pediatric HSCT, which is characterized by high risk of occurrence of serious and life-threatening infections. Disclosure: Nothing to declare
Background: Patients with hematological diseases are at higher risk of developing carbapenem-resistant Enterobacteriaceae (CRE) bloodstream infection (BSI) and associated with high mortality. Prediction model for a subsequent CRE BSI in hematological patients with CRE isolated from previous perianal swab samples can provide timely and useful target treatment strategies. Methods: The data were extracted from patients with CRE isolated from perianal swab samples at the Hematopoietic Stem Cell Transplantation Center of Blood Diseases Hospital, Chinese Academy of Medical Sciences between July 2017 to October 2020 January. Patients who developed subsequent CRE BSI were compared with those who did not develop BSI. Univariate logistic analysis, multivariate logistic analysis and stepwise regression analysis were carried on a variety of clinical factors. Results: A total of 215 cases were included and 29 (13.4%) patients of them with CRE isolated from perianal swab samples developed CRE BSI subsequently. Of the 29 patients with CRE BSI, 9 (31%) died of CRE BSI within 30 days. The CRE strains isolated from perianal swab samples and blood cultures of these 29 patients were consistent, and the resistance to commonly used antibiotics was highly similar. Multivariate analysis showed that C-reactive protein(CRP)>30mg/l(OR 10.613, 95%CI 2.965~37.985, P = 0.000), perianal infection(OR 6.450, 95%CI 2.223~18.714, P = 0.001), concomitant gastrointestinal symptoms (OR 4.175, 95%CI 1.476~11.813, P = 0.007), age>4 years (OR 3.415, 95%CI 1.222~9.541, P = 0.007)and neutrophil count <0.025×109/L (OR 4.583, 95%CI 0.939~22.369, P = 0.060) were risk factors for CRE BSI in patients with CRE isolated from perianal swab samples (P < 0.01). They were included in the Logistic regression model to predict BSI. According to receiver operating characteristic (ROC) curve analysis, the cut-off value of the model was 0.921 (0.851-0.968, P < 0.001). Conclusions: Hematological patients with CRE isolated from perianal swab samples have a relatively low incidence of subsequent BSI but a relatively high risk of death. The risk prediction model based on CRP, perianal infection, concomitant gastrointestinal symptoms, age and neutrophil count can effectively predict subsequent CRE BSI in patients with isolated from perianal swab samples and provide timely and effective treatment reference for this kind of patients. Disclosure: Nothing to declare
Background: Cytomegalovirus (CMV) infection is a serious concern for transplant recipients; a Phase 3 trial (SOLSTICE) in transplant recipients with refractory CMV infection with/without resistance (R/R) demonstrated that maribavir treatment was superior to investigator-assigned therapy (IAT; ganciclovir, valganciclovir, foscarnet, cidofovir) in clearance of CMV viremia at Study Week 8, and in clearance at Week 8 with maintenance through Week 16. We report subgroup efficacy and safety data by baseline renal impairment. Methods: Hematopoietic cell/solid organ transplant recipients (aged ≥ 12 years) with R/R CMV infection were randomized 2:1 to either maribavir (400 mg BID) or IAT for an 8-week treatment phase with 12 weeks of follow-up. The primary endpoint was confirmed CMV viremia clearance (2 consecutive post-baseline confirmed plasma viral loads of <137 IU/mL, ≥5 days apart) at Study Week 8. Baseline renal impairment was defined by creatinine clearance per Cockcroft–Gault equation: none (>80 mL/minute), mild (50 to 80 mL/minute), moderate (30 to <50 mL/minute), and severe (<30 mL/minute). Central clinical laboratory creatinine concentrations were assessed every two weeks. Results: Overall, 352 patients were randomized: 235 to maribavir and 117 to IAT. Baseline renal impairment status was balanced between the maribavir and IAT arms (% patients maribavir and IAT, respectively): none (34.5% and 33.3%), mild (30.2% and 35.9%), moderate (25.5% and 18.8%), severe (3.4% and 2.6%), and missing data (6.4% and 9.4%). In all renal impairment categories, a greater proportion of patients in the maribavir arm than in the IAT arm achieved CMV viremia clearance at Study Week 8 (Table). There was no change in median serum creatinine level from baseline to last on-treatment visit in patients receiving maribavir in all renal impairment categories. In the IAT arm, an increase in serum creatinine (µmol/L) from baseline to last on-treatment visit was observed across all renal impairment categories (median change: none, 8.80; mild, 9.00; moderate/severe, 9.00); for foscarnet-treated patients, a greater increase in serum creatinine than in the overall IAT arm was observed across all baseline renal impairment categories (median change: none, 9.00; mild, 18.00; moderate/severe, 35.50). Conclusions: In these post-hoc analyses, a greater proportion of patients on maribavir achieved CMV viremia clearance at Week 8 than IAT in all renal impairment categories consistent with the primary endpoint analysis observed for the overall population. No changes in creatinine levels from baseline to last on-treatment visit were observed in the maribavir arm while an increase was seen in the IAT arm. Funding: Takeda Development Center Americas, Inc. Clinical Trial Registry: NCT02931539 Disclosure: Sanjeet Dadwal: consultant: Allovir, Cidara, Merck; research support (to institution): Allovir, Amplyx, Ansun Biopharma, Gilead, Karius, Merck; speakers bureau: Astellas, Merck; advisory board: Aseptiscope, Merck. Rafael Duarte: research support (to institution): Janssen, Merck, Novartis, Omeros, Roche Diagnostics; speakers bureau: Bristol Myers Squibb, Gilead, Incyte, Jazz Pharmaceuticals, Merck, Omeros, Pfizer, Sanofi, Shire, a Takeda Company, Sobi; advisory board: Bristol Myers Squibb, Gilead, Incyte, Jazz Pharmaceuticals, Merck, Omeros, Pfizer, Sanofi, Shire, a Takeda Company, Sobi. Alosiyus Ho: nothing to declare. Nassim Kamar: consultant: AstraZeneca, Biotest, ExViR, Hansa, Merck Sharp & Dohme, GlaxoSmithKline, Novartis, Sandoz, Takeda; honoraria/travel expenses: Astellas, Biotest, Chiesi, Novartis, Sandoz, Takeda. Joan Gu and Aimee Sundberg: employee and stockholder: Takeda.
Background: Loss of vaccine immunity after hematopoietic stem cell transplantation (HSCT) is well established for some vaccines such as polio, varicella, rubella or tetanus. However, to date, there is no evidence assessing the loss or persistence of vaccine immunity against SARS-CoV2 after transplantation. The aim of the study is to evaluate SARS-CoV2 vaccine-induced immunity after HSCT. Methods: Fourteen patients who received allogenic (n = 6) or autologous (n = 8) HSCT due to an oncohematological disease (3 acute leukemias, 3 myelodysplastic/myeloproliferative syndromes, 4 lymphomas, 4 multiple myelomas) after having received one or two-dose of the COVID-19 vaccine schedule (13/14; 92.8% mRNA-1273-Moderna) were recruited. In the case of allogeneic transplants, all donors were fully vaccinated. Sixteen healthy donors who had received the complete vaccination regimen were used as controls (87,5% BNT162b2 mRNA-Pfizer; 12,5% mRNA-1273). The immune response was analyzed pre-transplant and 2.5 months post-transplant. The mean time from vaccination to analysis was 64 days. In healthy subjects, the immune response was analyzed 1 month and 3 months after receiving the full vaccine regimen. IgG titers against SARS-CoV-2 were quantified by Euroimmun-Anti-SARS-CoV-2 ELISA. Direct cellular cytotoxicity (DCC) was determined against Vero E6 cells infected with pseudotyped SARS-CoV-2, measuring caspase-3 activation after co-culture with peripheral blood mononuclear cell (PBMCs). Antibody-dependent cellular cytotoxicity (ADCC) analyses were performed using Annexin V on Raji cells as a target. Results: Patients in the allogeneic HSCT group showed reduced levels of specific IgGs compared to healthy donors in the pre-transplant baseline sample (-2.6-fold; p = 0.0011) (Fig.1a). In the post-transplant sample, results were variable, with some patients showing a decrease in antibody levels after transplantation while others show an increase in comparison with the baseline sample. IgGs levels were also decreased (2.3 fold; p = 0.0476) in patients with autologous HSCT and did not change significantly after HSCT. Unspecific ADCC response was decreased (-1.5-fold; p = 0.0072) before HSCT compared to healthy donors. However, previous response was maintained after HSCT (Fig.1b). DCC response against SARS-CoV-2-infected cells increased 2.1-fold in healthy donors 3 months after two-dose vaccine regimen (statistical significance was not reached); similar response was observed in individuals with autologous HSCT, but this response was reduced 2.7-fold after allogenic HSCT (Fig.1c). Consequently, PBMCs from individuals with allogenic HSCT showed a reduced capacity to eliminate SARS-CoV-2 infected cells (Fig.1d). DCC was reduced 7.9- and 6.2-fold (p = 0.0043) before and after HSCT, respectively, in comparison with healthy donors. In individuals who underwent autologous HSCT, this capacity was reduced 4.0- and 5.1-fold (p = 0.0403) before and after SCT, respectively. Fig1. Analysis of immunity against SARS-CoV2. a)Humoral response. b)ADCC. c)DCC. d)DCC- viral replication. Conclusions: IgGs levels developed after receiving one- or two-dose vaccination schedule were maintained after autologous HSCT and increased in some patients with allogeneic HSCT, possibly due to donor vaccination. ADCC in PBMCs from oncohematological patients was slightly decreased in comparison with healthy donors before HSCT and it was recovered after HSCT in both groups with allogenic or autologous HSCT. DCC was mostly impaired in patients who received allogenic HSCT, but both groups showed a reduced capacity to eliminate SARS-CoV-2 infected cells before and after HSCT. Disclosure: Nothing to declare
Background: Human Adeno Virus (HAdV) infections are associated with significant morbidity and mortality in patients undergoing allogeneic hematopoietic stem cell transplantation (HSCT)(1)(2), Whereas the gastrointestinal (GI) tract is recognized as a major site of persistence and reactivation in pediatric HSCT recipients, the prevalence and consequences of HAdV GI infection in the adult setting are not well defined(3). Here we studied the prevalence, risk factors, and consequences of HAdV GI-tissue infection in HSCT recipients with GI symptoms undergoing GI biopsy. Methods: Eighty-eight HSCT recipients (73 adults and 15 pediatric) who presented with GI symptoms leading to GI biopsy between 2012 and 2017, were retrospectively studied. The presence of HAdV DNA in the tissues was analyzed by Real-Time PCR. Patients’ clinical data were retrieved from the patients’ electronic files. Results: HAdV GI tissue infection was detected in 23.9% of patients examined (33.3% and 23.9% of the pediatric and adult population respectively). No significant differences were found in patients’ characteristics between the adults and pediatric population, except for a higher percentage of ATG used in the pediatric population. A higher prevalence of cGVHD was detected in the population presenting with GI positive HAdV-PCR before day +100. Twenty-one patients were found to have HAdV-PCR positivity in their GI biopsy, while twenty-seven patients were diagnosed with HAdV infection in other sites (14 of them were positive also in their GI biopsy). Arab ethnicity (p = 0.001), cGVHD (p = 0.023), the presence of bloody diarrhea (p = 0.033), and positive cytomegalovirus (CMV) PCR in the GI tissue (p = 0.033) were significantly correlated with HAdV detection in GI biopsies. Over a median follow-up of 9.3 (5.0-13.6) months. Detection of HAdV at any site was not associated with a lower survival rate. Conclusions: To our knowledge, this is the first study reporting on the prevalence of PCR positive HAdV infection in GI symptomatic HSCT recipients. Our data shows a trend towards a higher sensitivity of HAdV PCR in GI biopsies in detecting HAdV infections, compared to HAdV PCR in feces samples. These results might suggest that there is an under-diagnosis of GI HAdV infections in symptomatic patients following allogeneic stem cell transplantation. Real-time diagnosis might alter the treatment approach to GI symptomatic patients thus preventing further deterioration. Our results should be confirmed in larger prospective studies. Disclosure: Nothing to declare
Background: Multidrug-resistant gram-negative bacteria (MDRGNB) are still one of the actual problems in oncology and hematology. The question remains whether colonization by MDRGNB leads to an increased risk of subsequent bloodstream infections (BSI) especially in patients after allogeneic hematopoietic stem cell transplantation (allo-HSCT) Methods: The retrospective study included 288 patients received the first allo-HSCT between 2018 and 2019. The median age was 32 (18-66) years, male – 152 (53%). Majority of patients had acute leukemia (n = 178, 62%) and received transplant from matched unrelated (n = 120, 42%) or haploidentical (n = 75, 26%) donor. We used a screening program with detecting bacteria by microbiological culture from non-sterile sites (stool, throat, urinary) twice a week during the HSCT. Results: Colonization of non-sterile sites before allo-HSCT by at least one MDR bacteria was detected in 64 (28%). In most cases resistance is due to extended spectrum beta-lactamases (ESBL) – 55 (86%), while carbapenemases in combination with ESBL were detected in 9 (14%) of patients. Etiology of colonization was presented by K. pneumoniae -35 (55%), E. coli – 14 (21%), Pseudomonas spp. - 7 (11%), Citrobacter spp. – 2 (2.5%), Enterobacter spp. – 1 (1.5%), Acinetobacter spp. – 1 (1.5%) and 4 (7.5%) of patients were colonized with more than one bacteria including K. pneumonia. After allo-HSCT the colonization was significantly higher than before transplantation (n = 161, 56%, p = 0.001), mainly due to carbapenem - and ESBL - producing bacteria – 118 (73%) (p = 0.001). The etiology of colonization after allo-HSCT was presented by K. pneumoniae – 99 (61.5%), E. coli - 12 (7.4%), Pseudomonas spp. - 23 (14%), Enterobacter spp. - 1 (0.6%), Chryseobacterium indologenes – 1 (0.6%), Proteus spp. - 3 (1.9%) Acinetobacter spp. – 6 (3.7%) and 16 (10.3%) of patients were colonized with more than one bacteria including K. pneumonia. BSI in the early period after transplantation developed in 76 (26%), and in 43 (56%) was caused by MDRGNB. The etiology of BSI included K. pneumoniae - 51%, E. coli - 26.5%, Pseudomonas spp. - 11.6%, Acinetobacter spp. - 4.6%, as well as K. pneumonia in combination with other bacteria - 6.3%. The etiology of BSI was the same bacteria that colonized non-sterile sites 2 weeks before the detection bacteria in bloodstream in 30 (69%) patients. Colonization by MDRGNB was associated with the development of BSI (p < 0.0001). The 100-day overall survival was significantly lower in patients with colonization of non-sterile sites compared with patients without colonization: 60.6% vs 88.2% (p = 0.001). Conclusions: Colonization of MDRGNB after allo-HSCT reached 56%. K. pneumonia was predominant etiology in both colonization and bloodstream infections. Colonization by MDRGNB was significantly associated with the development of BSI in the early period after HSCT. Disclosure: Nothing to declare
Background: Invasive fungal infections (IFI) pose one of the most important infectious complications in hematopoietic stem cell transplantation (HSCT). Isavuconazole (ISV) is a new generation azole with a more favourable adverse and interaction profile approved for the treatment of invasive aspergillosis and mucormycosis. Methods: To analyse the indications, effectiveness, adverse event profile and drug interaction management of ISV in the real-world setting in adults who received allogeneic-SCT within the Spanish Group of HSCT and Cell Therapy (GETH-TC). Results: 166 adult patients treated from 2017 to 2021 were retrospectively analysed (Table 1). Median age was 48 years, 57% were male. The most common stem cell source was peripheral blood in 91%, the most frequent donor were haploidentical (39%) and matched siblings (35%). 57% used reduced intensity conditioning. GvHD prophylaxis consisted in post-transplant cyclophosphamide in 64%. In 81 patients ISV was used for treatment of IFI, and in 85 patients ISV was used as off-label prophylaxis (secondary in 25%). The median duration of ISV for the treatment of IFI was 86 days, 57 days as prophylaxis.Among patients who received ISV as treatment, it was empirically initiated in 5%. The most frequent indication was invasive aspergillosis (78%), followed by mucormycosis (6%), and in 5% a Geotrichum spp IFI or invasive Candida infection. In 52% ISV was used as second line (or more), usually as savage therapy (32%), due to adverse events of prior antifungals (26%) or as a directed de-escalation therapy (26%). 74% of the patients had received prior antifungal prophylaxis. The most frequent reason for ISV discontinuation was therapeutic success (45%). 58% of the patients showed at least one pharmacological interaction, mainly associated to immunosuppressive drugs (49% of the interactions). Only 9% of the patients reported ISV-related adverse events (AE), mainly related to liver enzyme alterations (71% of the AE). ISV was not discontinued due to these AE. In the prophylaxis group, the most frequent reason for ISV withdrawal was the resolution of IFI risk in 62%. 6 breakthrough IFI were reported among the 81 patients (aspergillosis in 5 cases). 16% of the patients reported ISV-related AE: cholestasis (11%), hypertransaminasemia (9%) and hyperbilirubinemia (5%). 3% of the patients developed cytopenia (1 case of neutropenia and 1 case of thrombopenia). ISV was discontinued in 8% of the patients due to AE. 84% of the patients showed at least 1 interaction (multiple in 41%), mainly related to immunosuppressive drugs (53%), and in 10% of the cases, due to small molecules (mainly ruxolitinib and gilteritinib). With a median follow-up of 321 days since ISV initiation, the 1-year OS was 50% for the treatment group and 64% for the prophylaxis group (Figures 1 and 2). Conclusions: ISV is an effective option for IFI treatment and prophylaxis after allogeneic HSCT. The favourable interaction profile allowed a safe use of concomitant drugs, especially immunosuppressants. Of note, a significant proportion of its use in the real- world setting is in prophylaxis, and the incidence of breakthrough IFI is low. Adverse events were not common, and most of them resolved with ISV withdrawal. Disclosure: Research funding: Pfizer.
Background: Seasonal and non-seasonal respiratory viral pathogens have potential to cause serious morbidity and mortality following HSCT, as well as health resource demands including in-patient hospital admissions and anti-viral treatment. In March 2020, outbreak of the COVID-19 pandemic in the UK led to several government-enforced measures aimed at reducing the spread of SARS-2-CoV, including shielding of the clinically vulnerable, social distancing, and face-masking in public, along with specific measures in healthcare and HSCT units (NICE Guidance NG164 and EBMT/BSBMTCT Guidelines). We considered that these measures may have impacted upon the incidence of non-SARS-2-CoV respiratory viral infections in HSCT patients, and associated morbidity, mortality and healthcare resource utilization. Methods: We retrospectively evaluated in-patient admissions for positive non-SARS-2-CoV respiratory viral PCR tests in adult HSCT patients (16 and over) in Sheffield Teaching Hospitals in two 6-month autumn/winter cohorts 1) the 6-month period prior to COVID-19 measures (01/09/2019-29/02/2020) and 2) the same 6-month period one year later, when measures had been enforced (01/09/2020-28/02/2021). PCR tests were performed on respiratory samples, including for Respiratory Syncytial Virus (RSV), Parainfluenza Virus (PIV) 1-4, Human Metapneumovirus (HMPV), Seasonal Coronavirus, Rhinovirus, Adenovirus, and Influenza A&B. In those testing positive, admission days, days on HDU/ITU, mortality, and costs of admission were compared between the two periods. Results: During period 1 (01/09/2019-29/02/2020), there were 90 episodes of non-SARS-2-CoV respiratory viral infections in 263 admissions (34.2 per 100 admissions), due to RSV (n = 26), Rhinovirus (n = 22), PIV 1-4 (n = 14), Influenza A (n = 12), Seasonal Coronavirus (n = 9), Adenovirus (n = 4), and HMPV (n = 3). In period 2 (01/09/2020-28/02/2021), there were 13 episodes in 159 admissions (8.2 per 100 admissions) with Rhinovirus (n = 12) and Adenovirus (n = 1). Total number of in-patient bed days with non-SARS-2-CoV respiratory viral infections in period 1 was 555, including 19 HDU/ITU bed days. In period 2, there were 196 in-patient days, with HDU/ITU 5 bed days. There were 3 deaths within 28 days of non-SARS-2-CoV respiratory viral infection in period 1, compared to 1 death in period 2. Based on NHS costings, there was a major reduction in total cost of admissions relating to non-SARS-CoV respiratory viral infections (including HDU/ITU) in period 1 versus period 2. Conclusions: Our data support a significant reduction in incidence of non-SARS-2-CoV respiratory viral infections consequent upon introduction of nationwide (societal and healthcare) infection control measures, with a reduction in ward admission days, HDU/ITU bed days, mortality, and associated costs. Whilst the COVID-19 pandemic also impacted on our HSCT activity (with 151 procedures in 2018, 140 in 2019, 97 in 2020, and 117 in 2021, with reduction in autografts, but allograft activity stable) and may explain some difference, it is likely that the impact of wide-ranging infection control measures resulted in significantly reduced infections, admission rates and healthcare resource utilization in HSCT centres. Analysis of a third cohort is ongoing, covering the current equivalent 6-month period (01/09/2021 – 28/02/2022), during which different UK COVID-19 measures are in place, with some national relaxations following SARS-2-CoV vaccination of majority of UK adults, whilst previous levels of HSCT activity within our programme is resumed. Disclosure: Nothing to declare
Background: Infective complications represent a relevant cause of morbidity and mortality in patients undergoing allogeneic haematopoietic stem cell transplantation (Allo-SCT). The BATMO (Best-Antimicrobial-Therapy-TMO) is an innovative program for infections prevention and management, adopted in our Center since 2019. Table 1. Main characteristics of the BATMO program. Methods: Data on infective complications of 116 transplanted before BATMO protocol (cohort A; 2016 - 2018) were compared with those of 84 patients transplanted following BATMO protocol (cohort B; 2019 - 2021). The clinical and transplant characteristics of the 2 cohorts are well comparable, with the exception of a significantly higher proportion of myeloablative regimens and haploidentical donors in cohort B. Results: No change in the incidence of infections with organ localization was observed between the two cohorts. A significant reduction in Clostridioides difficile infections by day +100 was observed in cohort B (47% vs 15%; p = 0.04). Neither the incidence of BSI and of the various etiological agents, nor the mortality from Gram negative bacteremia (Figure 1) and the incidence of invasive fungal infections was different in the two cohorts. The incidence of CMV reactivation by day +100 dropped drastically in patients of cohort B, following letermovir registration (51% vs 15%; p = 0.00001). Figure 1. OS of the 84 patients undergoing allo-SCT from 2019 and 2021 (BATMO program), according to the development of Gram negative bacteremia by day + 100 (OS at 1 and 2 years – event vs no event: 65.1% vs 74.5% and 54% vs 53.4%, respectively; p = 0.54) Conclusions: The results of this study suggest that the BATMO program is safe. In particular, the choice to avoid prophylaxis with fluoroquinolone was not associated with an increased Gram negative mortality and was associated with a significantly reduction of Clostridiodes difficile infections. Anti-CMV prophylaxis with letermovir confirmed its efficacy in significantly reducing CMV reactivation. Even though patients of cohort B were at higher risk of developing fungal infections (more haploidentical transplants with more myeloablative regimens), the extensive use of posaconazole for prophylaxis balanced this risk and no increase in incidence and mortality from fungi was observed. Clinical Trial Registry: NA Disclosure: All the Authors declare no conflict of interests
Background: Introduction: Respiratory viral infections in children undergoing haematopoietic stem cell transplantation (HSCT) can result in mortality rates of 33%. We report a retrospective outcome analysis following an outbreak of respiratory viral infection in our transplant unit during a heavy monsoon season and its impact beyond the respiratory tract. Methods: We collected retrospective data on the respiratory viral panel screening for influenza, parainfluenza, RSV, adenovirus, rhinovirus, coronavirus and enterovirus and its impact on respiratory, haematological, hepatic systems and mortality. Results: A total of 42 HSCT patients developed respiratory viral infections between August 2021 and November 2021. All patients had upper respiratory tract symptoms. Amongst this cohort, we documented Parainfluenza-3 (PIV-3) in 23/42 (54.7%), Influenza in 2/42 (4.7%), RSV in 7/42 (16.6%), rhinovirus in 8/42 (19%) and enterovirus in 2/42 (4.7%). Adenovirus coinfection was seen 3 children with PIV-3. Sepsis with multi-organ failure and the presence of pulmonary co-pathogens caused mortality in 4 children and one child had refractory autoimmune haemolytic anaemia. All the 5 children who died had PIV-3 and although the overall mortality was 5/42 (11.9%), the PIV-3 associated mortality was higher at 5/23 (21.7%). In the second week of the infection all children had profound pancytopenia and altered liver enzymes was seen in 7/42 (16.6%) and significant autoimmune haemolytic anaemia in 6/42 (14.3%). Our series confirms that high mortality is associated with PIV-3 infection especially those with coinfection with other viruses. In HSCT recipients, significant hepatitis and autoimmune haemolytic anaemia were seen during this outbreak. Conclusions: Despite strict enforcement of infection control policies, respiratory viral outbreaks do occur in HSCT units and children are the most vulnerable. We need to manage the respiratory effects with careful risk assessment, and effective broad-spectrum anti-microbials in those who are at risk of secondary infection. However, we also need to watch for hepatic dysfunction during the viraemia and provide supportive care with ursodeoxycholic acid, N acetyl cysteine and adequate nutrition. Life threatening autoimmune haemolytic anaemia needs urgent introduction of steroids and rituximab if refractory. The non respiratory complications result in morbidity and mortality in PIV-3 infection. There are no effective antivirals and aggressive healthcare interventions to contain and outbreak and isolation of actively infected patients to prevent spread is the only way forward. Disclosure: NIL
Background: To determine factors influencing the vaccination response against SARS-CoV2 is of great importance in recipients of allogeneic hematopoietic cell transplantation (alloHCT) as they were reported to experience (1) an increased mortality after SARS-CoV2 infection, (2) an increased risk of extended viral persistence and (3) reduced vaccination response. Methods: We retrospectively collected real-life data on humoral response after vaccination within Germany licensed vaccines (BNT™, Moderna™, AstraZeneca™, Johnson/Johnson™, heterologous scheme vector/mRNA) in recipients of alloHCT at our center. Anti-SARS-CoV-S1-IgG was determined by ELISA (SARS-CoV-2 IgG, Siemens™). Anti-S1-IgG titres of >100 BAU/ml were classified as sufficient response, values of 21.8-100 BAU/ml as low response and values <21.8 BAU/ml as no response. Lymphocyte subsets were quantified 0-3 months prior to the last documented vaccination with flow cytometry. Results: Anti-SARS-CoV-S1-IgG titres were available in 192 adults vaccinated between January-September 2021. Median age at alloHCT was 54 years (range 19-79 years). More than 90% of patients were transplanted for malignant diseases. Half of patients received a graft from a MUD, one third from a MRD. Graft source was peripheral blood and conditioning intensity was MAC/ reduced toxicity MAC in the majority of patients. An mRNA based vaccination scheme was used in 58%, a vector based in 10% and 5% of patients were vaccinated with onetime vector and mRNA-based combination. Details on the vaccine were not available in ¼ of patients and 1% received a non-conventional combination. Median time between alloHCT and best vaccination response was 3.3 years (range 0.35-26.03 year). A sufficient humoral response was achieved in 131 (68%), a low response in 19 (10%) and no response was observed in 42 (21%) patients. All patients (n = 10, 100%) with onetime vector and mRNA combination responded sufficiently. Immunosuppressive treatment was used in 74 patients. Of those, 56% responded sufficiently compared to 75% in the non-immunosuppression group (p < 0.05). Patients under ruxolitinib treatment (n = 23) had a sufficient humoral response rate in 61%, patients under glucocorticosteroids (n = 14) in 50%. Individuals receiving an ATG based GvHD prophylaxis showed a sufficient response in 63% compared to 78% without ATG (p < 0.05). Overall sufficient responders showed higher B-cell (median 183 vs 64 cells/ul, p < 0.05) and CD4 + T-cell counts (median 339 vs 240 cells/ul, p < 0.05) than non-suffcient responders 0-3 months before vaccination. Concurrent with these findings is that only 52% of individuals vaccinated in the first year after alloHCT responded sufficiently while 76% of patients vaccinated more than one year after alloHCT had a sufficient response (n = 136, p < 0.05). Conclusions: Overall the sufficient humoral response rate of 68% in our single center retrospective cohort is comparable to published data of prospective studies after vaccination with mRNA vaccines. Humoral response was dependent on B- and CD4 + T-cell counts at vaccination, time after transplant, active immunosuppression and the use of ATG. Interestingly, the heterologous vaccination scheme (vector, mRNA) showed a 100% effectiveness in our cohort pointing towards possible strategies for vaccination in non-responders. Disclosure: Robert Zeiser received honoraria from Novartis, Incyte and Mallinckrodt, all outside of the present work.
Background: Prophylaxis with letermovir has become part of mainstream practice to prevent reactivation of human cytomegalovirus (CMV) in patients undergoing allogeneic haematopoietic stem cell transplantation (allo-HSCT). We reviewed the impact of letermovir prophylaxis in our institution by comparing two highly-matched cohorts of patients over a 100-day and 12-month period before and after its introduction. Methods: We compared the incidence of CMV disease, DNA titres meeting the threshold for additional antiviral treatment and any detectable CMV DNA titres in a cohort who had received letermovir prophylaxis once daily for 100 days (n = 29) with a control cohort of patients treated before its introduction (n = 29). The cohorts were matched for conditioning regimen (stratified for T cell-deplete and T cell-replete regimens: n = 22 and 7, respectively), donor CMV IgG status (72% positive in letermovir cohort, 69% control), diagnosis and age. All patients were positive for CMV IgG prior to transplant and had DNA monitoring following transplant according to local protocols. We assume no other differences in clinical care between the cohorts. Results: In the 100 days post-transplant, letermovir reduced the number of patients who met the threshold of > 3000 IU/mL to start antiviral treatment compared to the controls (6.9% and 82.8%, p = <0.00001). This was also seen over 12 months (27.6% and 82.8%, p = 0.0051). Furthermore, letermovir reduced the number of patients who developed any detectable CMV DNA in the first 100 days (31.0% and 82.8%, p = 0.00015), but results over 12 months were not significant (p = 0.14). In the cohort who received T cell-depletion conditioning, letermovir reduced the number of patients meeting the threshold over 100 days and 12 months (4.5% and 81.8%, p = <0.00001; 31.8% and 81.8%, p = 0.0019, respectively). Overall, peak viral titres recorded over both 100 days and 12 months was reduced in patients taking letermovir compared to the controls (p = 0.00008 and p = <0.00001, respectively). This was also seen in the T cell-deplete cohort (p = 0.00058 and p = <0.00001, respectively). The reduction in DNA titres did not translate into any significant difference in CMV disease, as only 1 patient (in the letermovir cohort) developed this. Results in the small (n = 7) T cell-replete regimen cohorts were also not significantly different. 17 patients died during 12 months (10 letermovir, 7 controls) - 7 during 100 days post-transplant (4 letermovir, 3 controls). Conclusions: Letermovir significantly reduces peak and above-threshold CMV titres during the 100 days of treatment and 12 months following transplant in our cohort. This is also seen in the T cell-depletion subset, who are at greater risk of reactivation. Letermovir is well-tolerated and has low renal and myelotoxicity, in contrast to agents traditionally used for pre-emptive treatment. Our data shows that the benefits of letermovir persist beyond the 100-day window, reducing the risk of CMV reactivation and from antiviral treatment for 12 months. Disclosure: Nothing to declare.
Background: Hematopoietic stem cell transplantation is a curative approach for a constantly increasing number of non-malignant disorders. However, serious post-transplant complications remain a relevant obstacle for the broader and more common use of HSCT in the treatment of benign diseases. CMV reactivation/CMV disease is one of the most common infectious complications following HSCT, correlating with high morbidity and mortality. This issue is well investigated in transplant recipients with underlying malignancy; however, the data regarding the outcome of CMV viremia in pediatric non-malignant transplant recipients are sparse. This single-center retrospective study aimed to evaluate the impact of CMV reactivation on the length of hospital stay and patients’ general outcome. Methods: The study incorporated all children and adolescents with a non-malignant disorder who underwent allogeneic HSCT in the Department of Pediatric Hematology, Oncology and Bone Marrow Transplantation during years 2015-2020. CMV reactivation was defined as CMV viremia at the level >1000 copies/ml (measured by qPCR) whereas patients with more than 10000 copies/ml were classified as individuals with high level CMV reactivation. Ganciclovir resistance was defined as an increase in CMV viremia followed by the need for second-line antiviral therapy. Results: Among 94 patients (60 males/34 females; median age 3.8 years), 27 (29%) presented CMV reactivation, including 13 (14%) with high CMV viremia. Seventy HSCT recipients were CMV seropositive prior to transplant. Fifty-six received stem cells from a seropositive donor. Fourteen (15%) patients developed resistance for Ganciclovir. Nine (9.6%) presented with multiple CMV reactivations. Patients with CMV reactivation required longer hospitalization in comparison with those without CMV reactivation (48 days vs 33 days, p = 0.007). We did not observe any correlation between CMV reactivation and the incidence of GvHD or invasive fungal infections. The survival analysis revealed significantly decreased overall survival (OS) and event-free survival (EFS) in those with high CMV viral load compared to patients with either low levels or no CMV reactivation (OS 0.7 vs 0.93, p = 0.016; EFS 0.6 vs 0.85, p = 0.007). Similarly, resistance for Gancyclovir correlated with worse survival (EFS 0.4 vs 0.87, p = 0.0004; OS 0.72 vs 0.93, p = 0.023) as well as multiple CMV reactivation (EFS 0.25 vs 0.84, p < 0.0001; OS 0.44 vs 0.94, p < 0.0001). Conclusions: CMV reactivation is a common complication in non-malignant HSCT recipients. It correlates with a significantly longer hospital stay, affecting the patients’ quality of life in early stage post-HSCT. Patients presenting with high CMV viremia, developing Ganciclovir resistance, or experiencing multiple reactivations had significantly lower chances for survival. Targeted anti-CMV prophylaxis should be strongly considered in non-malignant pediatric transplant settings, particularly for patients at risk of high CMV viral load reactivation. Disclosure: Nothing to declare
Background: Diarrhea is a common and often debilitating complication of HSCT. Data regarding the longitudinal assessment of the infectious etiology of episodes of enterocolitis are limited. Methods: Prospective, observational, and multi-center study. Between April 2017 and November 2018, all consecutive adult patients who underwent a reduced-intensity HSCT in 10 Spanish tertiary University Hospitals were included. Our objective was to determine the frequency and etiology of enterocolitis. Acute diarrhea episodes (grade ≥2 CTCAE) and the diagnostic yield of routinely performed microbiologic stool studies occurring in the first 6 months post-HSCT were collected. Results: One-hundred-forty-two patients were included, of whom 54 (38%) developed a total of 75 diarrhea episodes, (Table 1). Thirty-seven out of 54 (69%) had a single episode, while 19 (35%) had two, and 2 had ≥3 (4%). The median time from HSCT to the first episode was 21 days (4-88). Sixty-seven % of the episodes occurred in hospitalized patients, while an additional 16 (43%) with an outpatient onset required admission for a median of 11 days (3-60). Causative enteropathogens were identified in only 13/75 (17%), including: C. difficile (n = 10), Rotavirus (n = 2) and Campylobacter jejuni (n = 1). CMV-colitis was confirmed by biopsy in 1 (6%). Two patients had colitis during a severe influenza A and systemic HHV-6 infection. Recurrent C. difficile-related infection occurred in 2/10 (20%), without occurrence of gastrointestinal (GI) GvHD. The most common cause of enterocolitis was acute GI-GVHD, in 28 (38%), followed by infections in 16 (21%), drug-related toxicity in 6 (8%) and others in 3 (4%). In 22 (29%) the cause of the diarrhea was not found (Figure-1). Of note, 80% of the patients had recent or active non-enteric infections. At data cutoff—October 2020, the median follow-up for survivors was 32 months (5-41). The incidence of non-relapse mortality (NRM) at 100 days and 1 year was 11% (95% CI: 6-17%) and 20% (95% CI: 14-28%). Causes of NRM included non-enteric infections (n = 14), aGVHD (n = 5), cGVHD (n = 1), bleeding (n = 3), cardiac toxicity (n = 2), and other organ toxicity (n = 5). Grade 2-4 diarrhea was not associated with higher NRM (p = 0.37). The one-year overall survival and relapse free survival was 69% (95% CI: 61-77%) and 78% (95% CI: 70-86%). Conclusions: This real-life multicenter study confirms that the diagnosis and management of acute diarrhea early after allo-HSCT is challenging. Previously reported infections, such as C. difficile, may be less common due to a more rational use of antibiotics and viral enteropathogens may be under recognized because of the lack of sensitivity of classical diagnostic methods. Disclosure: There are no conflicts of interest to report.
Background: Retrospective studies of allogeneic hematopoietic cell transplantation (alloHCT) recipients have suggested rifaximin as an antibiotic allowing higher microbiota diversity even in the presence of systemic broad-spectrum antibiotics. Although loss of diversity has been clearly associated with acute graft-versus-host-disease (GVHD) and poor outcomes in alloHCT, it remains unclear whether the use of rifaximin would improve outcomes. Therefore, we designed a prospective randomized study to compare our standard-of-care for neutropenia prophylaxis (ciprofloxacin) with rifaximin. Methods: We prospectively enrolled consecutive adult patients that underwent alloHCT according to EBMT indications, at our JACIE-accredited Unit and provided written informed consent to participate in this study (2019-2020). Patients that received secondary prophylaxis due to pre-transplant infections with resistant bacteria were excluded. Patients were randomly assigned to receive standard doses of ciprofloxacin or rifaximin at day -1. Treatment of neutropenic fever was administered according to our Unit’s protocol in both groups and included cessation of ciprofloxacin or rifaximin. The following variables were analyzed: pre-transplant (age, gender, disease risk index/DRI), transplant (donor, graft, conditioning) and post-transplant (infections, GVHD, treatment-related mortality/TRM, disease-free survival/DFS, overall survival/OS) characteristics. Results: We studied 38 patients, the majority of whom were transplanted from matched unrelated (17/38) or alternative (5 haploidentical and 3 mis-matched unrelated) donors. After randomization, 20 received ciprofloxacin and 18 rifaximin. As expected, pre-transplant and transplant characteristics did not differ between groups. Cumulative incidence (CI) of acute graft-versus-host disease (GVHD) grade II-IV and moderate/severe chronic GVHD was similar in both groups (60% in ciprofloxacin versus 44.4% in rifaximin, p = 0.516; 58.3% versus 59.9%, p = 0.84, respectively). Bacterial, viral and fungal infections were similar between groups. With a median follow-up of 13.2 months (range 6.8-30.2) in surviving patients, 1-year CI of relapse was 20.8% in ciprofloxacin versus 17.8% in rifaximin (p = 0.616). Importantly, 1-year CI of TRM was significantly reduced in ciprofloxacin group (10.2% versus 27.8%, p = 0.032). This led to higher 1-year overall survival (OS 88.9% versus 74.6%, p = 0.038). In Cox-regression multivariate analysis, antibiotic prophylaxis remained the only predictor of OS (HR = 5.847, 95% Confidence Intervals 1.053-32.478, p = 0.044), independently of donor type, DRI and chronic GVHD. Conclusions: We present the first open-label randomized controlled study to evaluate rifaximin as neutropenia prophylaxis. Our results suggest that standard-of-care with ciprofloxacin was superior leading to survival advantage. Further studies are needed to assess effects on microbiota diversity and confirm these outcomes. Clinical Trial Registry: ΝΑ Disclosure: Nothing relevant to disclose
Background: Respiratory viral infections represent an important cause of morbidity and mortality in immunocompromised patients, especially those recently submitted to allogeneic stem cell transplantation (HSCT) and receiving immunosuppressive drugs. The main common seasonal respiratory viruses (CSRV) who affected immunocompromised host are influenza, respiratory syncytial virus, parainfluenzae virus and, recently, severe acute respiratory syndrome coronavirus (SARS-CoV-2). Methods: During flu season, hospitalized patients and outpatients coming at the clinic with respiratory symptoms or fever are routinely tested with nasal swab for respiratory viruses. Multiplex polymerase chain reaction (PCR) is usually performed on nasal swabs to detect the presence of respiratory viruses. The used panel includes: influenza A, influenza B, parainfluenzae 1-4, respiratory syncytial virus, coronaviruses NL63, OC43, 229E, HKU1, enterovirus/rhinovirus, adenovirus. Here we reported positive cases detected with nasal swab in the last three seasons among patients who had received a stem cell transplant in our unit. Results: Between November 2018 and March 2021 a total number of 210 HSCTs were performed in our unit. Other 240 patients submitted to HSCT before January 2018 were followed during that period as outpatients in our clinic. A total of 90 positive swabs were detected. We identified three seasons, according to the local epidemiology: the first from November 2018 to April 2019, the second one from September 2019 to March 2020 and the third from October 2020 to march 2021. Positive swabs were distributed as follows: 44 cases in the first season, 31 cases in the second season and 15 cases in the third season. In the first season the identified viruses were Influenza A and B (n = 11), Parainfluenzae virus 1-3 (n = 4), RSV (n = 25), Coronavirus NL63 (n = 1), Coronavirus OC43 (n = 1), Rhinovirus/Enterovirus (n = 2). In the second season the viruses were Enterovirus/Rhinovirus (n = 6), Parainfluenzae virus 1-2 (n = 5), RSV (n = 6), Influenza A and B (n = 10), Coronavirus NL63 (n = 1), Coronavirus 229E (n = 1), Adenovirus (n = 1), Bocavirus (n = 1). In the third season, only SARS-CoV2 was identified. Figure 1: Respiratory virus episodes of RSV, Parainfluenzae, Influenza A and SARS-CoV2. Prevalence of each common seasonal respiratory virus (CSRV) type by months. Conclusions: Starting from the SARS-CoV2 pandemia onset, there has been a dramatically disappearance of the others common seasonal respiratory virus infections. In particularly, during the first lockdown period we have not even registered SARS-CoV2 cases, that come to the light starting from October 2020. This indicates that prevention measures adopted to prevent SARS-CoV2 spread, such as wearing masks, frequently washing of the hand, reducing inter-personal contact and maintaining social distances, resulted as weel effective in reducing others CSRV infections. However, at the beginning of the current season, we are observing a new rise of the CSRV infections, particularly due to Parainfluenzae virus, Rhinovirus and RSV. Disclosure: Nothing to declare
Background: Nowadays antimicrobial resistance is one of the most important medical and epidemiological problems. Patients after hematopoietic stem-cell transplantation (HSCT) are considered a high-risk group for infectious complications, caused by multidrug-resistant bacteria (MDR). The enteric microbial flora is considered a major source for these complications. The routine fecal screening for colonization by MDR bacteria helps the adequate choice of empirical antibacterial therapy, especially in the era of the COVID-19 pandemic and the increased consumption of antimicrobial agents. The aim of this study is to investigate the spectrum of the MDR gut colonizers and to detect the genes associated with resistance to beta-lactam and glycopeptide agents. Methods: During a two-year period (November 2019 – November 2021), 74 patients were studied. A total of 44 non-duplicate MDR bacterial isolates were obtained from fecal samples of 28 patients after HSCT. All studied samples were inoculated on media containing cefotaxime (1 mg/L), CHROMagarTM CPE (BD BBLTM, USА) and blood agar (BD BBLTM, USА) and were incubated at 37˚C for 24 hours. Species identification and antimicrobial susceptibility were determined by the Phoenix Automated System (BD, USA). PCR was used to identify the genes, encoding resistance to 3rd generation cephalosporins, carbapenems (blaSHV, blaCTX-M, blaTEM, blaKPC, blaNDM, blaVIM, blaIMP, blaOXA-48) and glycopeptides (vanA, vanB, vanC, vanD). DNA was obtained by SaMag Bacterial DNA Extraction Kit (Sacace, Italy). Epidemiological typing by ERIC and RAPD PCR was performed to determine the genetic relationship between the isolates. The PCR products were resolved by gel electrophoresis. Results: A total of 44 MDR fecal isolates were collected: Enterococcus faecium, n = 14; E. coli, n = 10; Pseudomonas spp., n = 8; Enterobacter cloacae complex, n = 7; Klebsiella pneumoniae, n = 4 and Serratia marcescens, n = 1. All E. faecium were vancomycin and teicoplanin - resistant and vanA positive. In the group of Gram negative bacteria, resistant to 3rd generation cephalosporins and/or carbapenems, the following genes were identified: blaSHV (n = 4, K. pneumoniae), blaCTX-M (E. coli, n = 9; K. pneumoniae, n = 3; E. cloacae complex, n = 7; S. marcescens, n = 1), blaTEM (E. coli, n = 5; K. pneumoniae, n = 2; E. cloacae complex, n = 6; S. marcescens, n = 1), blaVIM (Pseudomonas spp., n = 6; E. cloacae complex, n = 1). In 13 isolates (46.7%) more than one resistance gene were found: blaCTX-M + blaTEM (n = 13), blaCTX-M + blaTEM + blaSHV (n = 2) and blaCTX-M + blaTEM + blaVIM (n = 1). The epidemiological typing of E. faecium, E. coli and Enterobacter cloacae complex revealed both unique profiles and clusters of closely related strains, demonstrating identical profiles. All K. pneumoniae isolates exhibited unique ERIC profiles. Conclusions: А high rate of fecal colonization by MDR bacteria was detected (38.3%). Resistance to 3rd generation cephalosporins was associated with blaSHV, blaCTX-M, blaTEM genes and carbapenem resistance - with blaVIM. Glycopeptide resistance was mediated by vanA gene. The identification of epidemiologically related isolates is an indication for possible intra-hospital dissemination of these MDR pathogens and a risk factor for nosocomial infections associated with these problematic bacteria. Disclosure: Nothing to declare.
Background: Bloodstream infections (BSI) are common and serious complications after allogeneic hematopoietic cell transplantation (allo-HCT). This study aimed to analyze the incidence, etiology, risk factors, and outcomes of pre-engraftment BSI after the first and the second allo-HCT. Methods: The retrospective study included 284 patients who underwent first allo-HCT and 37 patients who underwent second allo-HCT at the National Research Center for Hematology in Moscow, Russia, from January 2018 till September 2021. Patients in the second allo-HCT cohort had higher age adjusted hematopoietic cell transplantation-specific comorbidity index (p < 0,0001), were more likely to receive an allograft from haploidentical donor (p = 0,006) and reduced-intensity conditioning regimen (p = 0,042) as compared to the first allo-HCT cohort. Median graft cellularity (p = 0,005) and the rate of primary graft failure (p = 0,002) were also higher in the second allo-HCT group. Results: Cumulative incidence of pre-engraftment BSI was 29.9% after the first allo-HCT and 35.1% after the second (p = 0,805). Median time to the first BSI was 9 days (range 0-61 days) after the first and 16 days (range 1-28 days) after the second allo-HCT (p = 0.014). A total of 111 pathogens were isolated during 94 BSI episodes after the first allo-HCT (gram-negative bacteria 51.3%; gram-positive bacteria 48.7%). Fourteen pathogens were isolated during 14 BSI episodes after the second allo-HCT (gram-negative bacteria 50.0%; gram-positive bacteria 50.0%). The rate of BSI caused by carbapenem-resistant gram-negative bacteria was higher after the second allo-HCT compared to the first (57.1% vs. 14.0%; p = 0.048). Mismatched unrelated donor transplantation was the only independent risk factor associated with a higher risk of pre-engraftment BSI after the first allo-HCT (HR = 2.93; 95% CI:1.55-5.51; p = 0,008). No risk factors associated with a higher risk of pre-engraftment BSI after the second allo-HCT could be identified. Overall 30-day survival after all BSI episodes was 94.4%. Survival was lower after the second allo-HCT compared to the first (71.4% vs. 97.9%; p < 0,0001), especially after BSI caused by carbapenem-resistant gram-negative bacteria (25.0% vs. 100.0%; p < 0.0001). Non-relapse mortality (NRM) rate at day +60 was 4.0%. The NRM risk was highly associated with primary graft failure (HR = 9.62; 95% CI: 1.33-71.43), second allo-HCT (HR = 6.80; 95% CI: 1.36-34.48), and pre-engraftment BSI caused by carbapenem-resistant gram-negative bacteria (HR = 32.11; 95% CI: 4.91-210.15). Figure 1. A – 30-day survival after all BSI episodes; B - 30-day survival after all BSI episodes according to allo-HCT number; C – 30-day survival after BSI caused by carbapenem-sensitive and non-ESBL producing bacteria according to transplant number; D – 30-day overall survival after BSI caused by carbapenem-resistant gram-negative bacteria according to transplant number. ESBL - extended spectrum beta-lactamase producing bacteria; CR – carbapenem-resistant gram-negative bacteria Conclusions: Pre-engraftment BSI is still common after allo-HCT, particularly after mismatched unrelated donor transplantations. BSI incidence was higher after the second allo-HCT with significantly higher rate of carbapenem-resistant BSI. Although pre-engraftment BSI would generally follow benign clinical course, survival was dramatically lower during the second allo-HCT especially in case of carbapenem-resistant BSI. Disclosure: none
Background: With the use of PTCy, the high risk of CMVi in haploidentical HSCT has been widely described. Our aim was to analyse whether under the same GvHD conditioning and prophylaxis (PTCy) there are differences in CMVi, and its consequences between haplo and non-haplo-HSCT groups. Methods: We conducted a multicentre, observational and retrospective study of three hundred patients (January 2013 – December 2018) from three Spanish hospitals. Results: All patients received TBF ablative or reduced conditioning regimen and PTCy plus calcineurin inhibitors +/− Mycophenolate as GvHD prophylaxis. There were no statistically significant differences in age (53 vs 56 years), stem cell source (71% peripheral blood), ablative vs reduced TBF, diagnosis, HCT-CI, DR index or pre-transplant status between haplo (n = 191) and non-haplo-HSCT (n = 109) groups. One hundred and eighty-seven patients developed CMVi (113 – 59.2% – haplo-HSCT vs 74 – 67.9% – non-haplo-HSCT). There was no difference in day of onset or duration of CMVi episode between the two groups (+46/31 days haplo vs +50/32 days non-haplo-HSCT respectively). CMVi CI (cumulative incidence) requiring PET at day +365 was 50.3% (haplo-HSCT) and 61.5% (non-haplo-HSCT) with similar incidence also in recurrent CMVi for both groups (17.3% vs 20.2%). The CMVi CI at 365 days was higher for CMV-seropositive recipients, a total of 74% (95% CI: 67%-78%) vs 3.9% (95% CI: 0%-9%) in CMV-seronegative recipients (p < 0.0001), regardless of donor CMV serostatus or HSCT type. The NRM CI at +365 day was 24.7%, with no differences in both groups. In the first year after HSCT, overall mortality (OM) was 32.7% (33.5% haplo-HSCT vs 32.2% non-haplo-HSCT). CMVi had no differential impact on OS, OM, NRM or relapse by study group. CI for II-IV aGvHD and severe moderate cGvHD was 30.3% vs 17.4% (p 0.05) and 16.8% vs 23.9% (p 0.007) at +100 and +365 days for haplo and non-haplo-HSCT respectively. However, the existence of aGVHD or cGVHD did not differentially impact CMVi. We also did not find differences in hospitalisation days due to direct or indirect effects of CMVi. Among the infections most frequently associated with CMVi, the frequencies by haplo- and non-haplo-HSCT groups were: bacterial 16.2% vs 15.1%, viral 15.8% vs 10.4% and fungal infections 7.1% vs 2.8%. Eighteen patients developed CMV disease (14 haplo and 4 non-haplo-HSCT), 68% of whom developed gastrointestinal disease. Nine patients (3% of the total group of patients) died from direct or indirect effects of CMVi (5 in the haplo-HSCT group). Conclusions: In our study, apart from the recipient’s CMV seropositivity, no other risk factors were identified for the development of CMVi. The homogeneity of conditioning and GVHD prophylaxis could have contributed to this result. In the PTCy as GVHD prophylaxis era, this finding may help to better define the target population at higher risk of CMVi in order to optimise the indication of CMVi prophylaxis with letermovir and thus reduce the morbimortality due to CMVi. Larger series are needed to confirm these results. Clinical Trial Registry: FIM-CIC-2020-01 Disclosure: B. Herruzo: Nothing to declare A. Esquirol: Nothing to declare C. Martín: Nothing to declare B. Gago: Nothing to declare A. Gallardo: Nothing to declare M Cuesta: Nothing to declare A. García: Nothing to declare I Sanchez: Nothing to declare S Martín: Nothing to declare A Doblas: Nothing to declare D Muñoz: Nothing to declare M Pérez: Nothing to declare A Mena: Nothing to declare MJ Pascual: Merck Sharp & Dohme SA
Background: Allogeneic hematopoietic stem cell transplant (allo-HCT) recipients are particularly at risk of severe COVID-19 when infected by SARS-CoV-2. We previously reported that while >80% of allo-HCT recipients developed binding antibodies (Ab) to SARS-CoV-2 receptor binding domain (RBD) after 2 doses of BNT162b2 mRNA vaccine, only half of them developed neutralizing Ab against wild type SARS-CoV-2 (Canti et al., J Hematol Oncol 2021, 24: 174). Importantly, response rates correlated with counts of memory B cells and of naive CD4 + T cells. Methods: Here we assessed the impact of a booster dose of the BNT162b2 vaccine on SARS-CoV-2 humoral immunity in a cohort of 41 allo-HCT included in a prospective vaccination study (EudractCT # 2021-000673-83). Results: Data from 41 patients given the booster vaccine were analyzed. The booster dose was the third vaccine dose in 40 patients and the second vaccine dose in 1 patient (who was diagnosed with Covid-19 six days after the first dose). Median time from transplantation to first vaccine dose was 1037 days (range, 209-1903 days). Median time from first dose to booster dose was 153 days (range, 104-226 days). RBD binding Ab increased from a median of 141 (range: <5 – 2914) IU/mL the day of booster vaccination to a median of 3082 (range: <5 – 47131) IU/mL three weeks later (P < 0.0001). Five patients had undetectable RBD Ab after two vaccine doses and 4 of them seroconverted after the third dose. The non-seroconverter had received rituximab 298 days before third vaccine dose. Conclusions: The administration of a third dose of the BNT162b2 vaccine was highly efficient at increasing anti-RBD IgG titers in allo-HCT recipients. The impact of the booster dose on neutralizing Ab response as well as correlations between baseline immune parameters and Ab response will be presented. Clinical Trial Registry: EudractCT # 2021-000673-83 Disclosure: The authors have no COI to disclose in regards to this abstract
Background: The use of immunosuppressive conditioning regimens in allogeneic hematopoietic cell transplantation (allo-HCT) leaves recipients with prolonged T cell-specific immunodeficiency in the post-transplant period, rendering them susceptible to severe viral infections and death. The most common viral infections following allo-HCT include adenovirus (AdV), BK virus (BKV), cytomegalovirus (CMV), Epstein-Barr virus (EBV), human herpesvirus 6 (HHV 6), and JC virus (JCV). Up to 70% of patients experience a clinically significant infection post allo-HCT, highlighting the serious unmet need for improved preventive antiviral therapies in high-risk allo-HCT recipients. Methods: We are conducting a two-part, phase 2/3, multicenter, randomized, double-blind, placebo-controlled trial to assess the safety and efficacy of posoleucel compared to placebo for the prevention of clinically significant AdV, BKV, CMV, EBV, HHV-6, and JCV infection in high-risk pediatric and adult allo-HCT recipients. Posoleucel is an ex-vivo expanded, partially HLA-matched, off-the-shelf, multivirus-specific T cell investigational product targeting CMV, EBV, HHV-6, AdV, BKV, and JCV. The open-label cohort of the trial is fully enrolled for DSMB review of the Phase 2 data to commence the randomized Phase 3 cohort. To date the seven doses of posoleucel administered in the Phase 2 study have generally been well tolerated with a low incidence of breakthrough infections through 14 and 26 weeks. In the Phase 3 cohort approximately 300 patients will be randomized 1:1 to receive posoleucel or placebo to achieve statistical power of 90%. Patients must be >1 year of age and have undergone high risk allo-HCT from 15-42 days before randomization and have demonstrated clinical engraftment. High risk allo-HCT is defined as receiving a transplant from a mismatched related donor, haploidentical donor, unrelated donor, cord blood donor, or undergoing T cell depletion from ATG, alemtuzumab, or ex-vivo graft manipulation. Patients with Grade ≥3 acute GVHD and those receiving ongoing high-dose corticosteroids, or ongoing clinically significant AdV, BKV, CMV, EBV, HHV-6, or JCV infections are excluded. The Primary Study Period is 14 weeks for evaluation of efficacy, the primary endpoint is a composite of the number of clinically significant infections or episodes of end-organ disease per patient due to AdV, BKV, CMV, EBV, HHV-6, or JCV through Week 14, with the key secondary endpoint consisting of the same criteria through week 26. An independent, blinded CAC will adjudicate all potential cases of clinically significant infection or end-organ disease for the primary and key secondary endpoints. Figure. A) Study schematic. B) Definitions of clinically significant infection and end-organ disease for primary endpoint Results: Planned enrollment is approximately 300 patients at approximately 70 international sites. The Phase 3 cohort of the study is due to commence enrollment first half of 2022 and to be completed in 2024. Conclusions: This phase 3 trial cohort will provide pivotal data on the efficacy and safety of posoleucel compared with placebo for the prevention of clinically significant infection and disease from CMV, EBV, HHV-6, AdV, BKV, and JCV in high risk allo-HCT recipients. Clinical Trial Registry: Clinicaltrials.gov: NCT04693637 Disclosure: Simona Sica has received honoraria from Jazz, Alexion, Novartis, Gilead, and Incyte, and has served on advisory boards for Jazz, Novartis, Astellas, and Alexion. Caroline Besley has received honoraria from Novartis and Kite (Gilead), and has served on advisory boards for Novartis and Kite. Gerard Socie has received honoraria from Novartis, Elsalys, and Incyte, and has served on advisory boards for Novartis, Elsalys, and Incyte, and has received research support from Alexion. Keith Boundy, Michelle Matzko, and Nikola Tripkovic are employees of and hold stock in AlloVir.
Background: Antibiotic resistant bacteria have spread worldwide and, therefore, increased rates of mortality and treatments cost are seen. Patients who undergo Hematopoietic Stem Cell Transplantation (HSCT) are specially exposed due to immunosuppression and long hospitalizations. However, data of the incidence of multidrug-resistant (MDR) colonization and related bloodstream infections (BSIs) are scarce and there is a wide distribution in resistance rates between countries. Methods: This prospective observational study aimed to describe risk factors and incidence of colonization, relationship with GNR bacteriemia and impact on survival in 113 patients who underwent HSCT (88 Auto-HSCT and 25 Allo-HSCT) at Hospital Universitario Central de Asturias from April 2019 to September 2020. Median lenght of hospitalization was 24 days and all of them had routine weekly surveillance rectal cultures beginning at admission. Results: 44 patients (38,9%) presented MDR rectal colonization, particularly Gram-negative strains and median lenght of colonization was 28,9 days. Thirty patients were colonized by extended spectrum beta-lactamase (ESBL) and 13 by carbapenem resistant (CR) enterobacteriae. The most common isolated bacteria were: Klebsiella (24), Escherichia (25), Enterobacter (19) y Citrobacter (7). Colonization was observed at admission in 25% with significant higher incidence in patients with previous betalactamic antibiotic exposure and mucositis. There was no seen a significant higher incidence of fever between colonized patients. Table 1 shows risk factors of colonization. Overall, 100 patients developed fever during admission and 38 patients developed at least one episode of gram-negative BSI. Ten of them developed BSI due to MDR bacteria (35,7% of all isolated pathogens); 6 due to the MDR-colonizing pathogen. In colonized patients, length of antibiotic therapy was higher (11 vs 8,5 days), need to change antibiotic therapy was more frequent and as well as use of meropenem, vancomycin and amikacin. Only one patient died before day 100 due to MDR BSI (CR Klebsiella), that there was not been detected in surveillance cultures previously. We did not find significant differences in overall survival between colonized vs not nor between CR vs ESBL. Table 1: Risk factor for colonization Conclusions: We found higher resistance rates in our media compared with Northern countries and similar to reported in other South Europe countries. We did not demonstrate higher mortality rates caused by MDR bacteria. Knowledge of colonization and pathogen resistant patterns can help to direct appropriate antibiotic therapy. In high risk hospitalized patients (previously treated with betalactamic antibiotic or mucositis) is mandatory the development of a screening programme for MDR strains and guidelines of contact isolation and antibacterial use. Disclosure: Nothing to declare
Background: Allogeneic stem cell transplant recipients have been considered as high-risk patients for Covid-19 due to their immunosuppression (IS) status. However, most of published studies are referred to the first wave of the pandemic when diagnosis capability was limited, so a better understanding of Covid-19 in AlloSCT is needed now that we have a higher diagnosis power and almost 100% of AlloSCT recipients have received complete vaccination. Methods: All AlloSCT recipients from our center who tested positive for Covid-19 by PCR or serological test between March 1st2020 and December 15th 2021 were included. We included patients diagnosed after presenting symptoms, history of close contact or routine screening before any procedure or visit to the hospital. We collected data related to AlloSCT, immune status and Covid-19 severity (mild: symptoms without pneumonia; moderate: pneumonia without or with low oxygen requirements; severe: pneumonia with high oxygen requirements or mechanic ventilation). Results: Thirty-six AlloSCT patients had a confirmed diagnosis of Covid-19, 32 by PCR and 4 by serological antibodies test. Nine cases were diagnosed during the 1st wave (March-June 2020), 5 during 2nd (August-Nov 2020), 12 during 3rd (Dec-Feb 2021), 2 during 4th (Apr-June 2021), 3 during 5th (July-Sept 2021) and 4 during the 6th wave (From Nov2021). Five (14%) had received SARS-CoV-2 vaccine with 1 (n = 1), 2 (n = 1) or 3 (n = 3) doses. Diagnosis were MDS/AML (n = 18), NHL (n = 8), MPD (n = 4), Hodgkin lymphoma (n = 3), MM (n = 1) and ALL (n = 2). Median time from AlloSCT to Covid-19 was 43 months (2-204), and only 6 patients were diagnosed in the first 12 months after AlloSCT, suggesting higher standards in prophylactic measures taken by patients at early post-AlloSCT period. Thirty-two patients (89%) were in complete remission and 17 of them (47%) had finished IS more than 6 months before contagious. Five (14%) and 13 (36%) patients respectively had active acute or chronic GVHD under treatment at the moment of Covid-19 diagnosis. Median CD4/uL was 321 (50-762), median IgG was 1170 (300-1470) and median lymphocytes count was 3100 (400-4100), and there were not significant differences in Covid-19 severity based on these parameters. Seven patients (19%) were asymptomatic and PCR was performed as screening before some medical procedure (n = 4) or after a close contact (n = 3). Covid-19 was mild in 20 (56%), moderate in 4 (11%) or severe in 5 (14%). Thirteen patients were admitted to the hospital, 10 due to Covid-19 and 4 because causes different from Covid-19 (GVDH, colon surgery, bacterial coinfection and hemorrhagic cystitis). None of these patients required mechanical ventilation Only 3 patients who were not candidates for mechanical ventilation due to severe comorbidities (2 post-AlloSCT relapses and 1 secondary lung neoplasm), died from Covid-19. Fatality rate was 8% among diagnosed patients. Conclusions: Our data suggests that AlloSCT patients in complete remision and without significant comorbidities have a similar outcome than general population, independently of their immunosuppression status. Considering percentage of asymptomatic cases, probably Covid-19 incidence in underestimated in AlloSCT patients. Contagious is possible, even after a SARS-CoV-2 booster, but most of cases are asymptomatic or mild. Disclosure: Nothing to declare
Background: Cytomegalovirus (CMV) infection is the most important complication after allogeneic hematopoietic stem cell transplantation (Allo-HSCT) and threatens the prognosis of patients seriously. There are limited data on CMV drug-resistant gene mutations in CMV infection after transplantation in China currently. In order to further understand the expression of CMV drug-resistant gene mutations in patients with poor efficacy of CMV infection, this study was conducted. Methods: Clinical data of 41 patients with CMV infection after allo-HSCT were retrospectively analyzed from January 2019 to July 2021 in transplantation Center of Hematology Hospital, Chinese Academy of Medical Sciences, due to poor clinical efficacy or the possibility of clinical drug resistance, who were tested for CMV resistance gene mutation. Results: ①Of 41 patients included, 1 patient (2.4%) had UL97 mutation, 19 patients (46.3%) had UL54 mutation, no patients occur simultaneously with UL54 and UL97 mutation. The overall mutation frequency was 48.8% (20/41), and all mutation types were missense mutation.② UL54 gene were detected 8 cases of T691S mutation (8/41, 19.5%), 3 cases of M827I mutation (3/41, 7.3%) and 1 case of A786V mutation (1/41, 2.4%). All above these 3 mutation sites had been reported, but the drug resistance significance was unknown. There were 1 site (A543S) of UL97 gene and 19 sites of UL54 gene that had not been reported. In all the 41 patients, only one patient exhibited a mutation with definite anti-CMVdrug resistance significance: Q578H mutation of UL54 gene. The patient was diagnosed CMV DNAemia on Day 31 after transplantation, T691S mutation was found in the first sequencing on +43d. The patient still in CMV DNAemia on +98d and Q578H drug-resistant mutation was found in the second sequencing with T691S mutation,who was successfully treated with cytomegalovirus specific T cells.③Compared with patients without UL54 mutation, patientswith UL54 mutation strains had a higher proportion of CMV-DNA repositive rate in 10 days after remission of CMV DNAemia (42.1% vs.9.5%, χ2 = 5.647, P = 0.028),and had a significantly longer complete clearance time of CMV DNAemia (40 days vs. 24 days, Z = 2.198, P = 0.027).④ Compared with those patients without CMV gene mutation, the patients with T691S mutation had a higher incidence in terms of CMV-DNA turned repositive in 10 days and 14 days after remission of CMV DNAemia (71.4% vs. 19.0%, P = 0.008; 71.4% vs. 9.5%, P = 0.001; 71.4% vs. 9.5%, P = 0.001, respectively). The median time of CMV-DNA reaching the peak was significantly later (14 days vs. 10 days, P = 0.016). The median clearance time of CMV DNAemia was significantly longer (56 days vs. 24 days, P = 0.002). Compared with the non-T691s UL54 mutation, the patients with T691S mutation also had similar results. Conclusions: Most of CMV gene missense mutations occurred in UL54 gene and T691S mutation is a common site. The incidence of missense mutations with clear drug resistance was low and might be induced by antiviral therapy. The median clearance time of CMV DNAemia in UL54 mutated patients was significantly longer, and the proportion of CMV-DNA turned repositive was increased which might be caused by T691S mutation in UL54 gene. Disclosure: Nothing to declare.
Background: There are limited data on outcomes of allogeneic hematopoietic stem cell transplantation (allo-HSCT) recipients with prior COVID-19. Methods: This single-center retrospective study included 54 adult patients who received allo-HSCT from July 2020 to September 2021 after the previous COVID-19. The median age was 33 years (18–76), there were 30 (56%) females and 24 (44%) males. The underlying disease were AML (n = 22, 40.8%), ALL (n = 17, 31.5%), MDS (n = 6, 11.1%), AA (n = 4, 7.4%), CML (n = 3, 5.5%) and HL (n = 2, 3.7%). The median follow-up was 138 days (11 - 391). The control group included 122 patients without a history of COVID-19 who underwent allo-HSCT during the same period. The median age was 35 years (18 – 69) and the median follow-up time - 223 days (13 – 502). We assessed the transplant-related mortality (TRM), overall survival (OS) and GVHD, and relapse-free survival (GRFS). Results: The median time from COVID-19 onset to the diagnosis of underlying disease was 129.5 days (-183 - 5135). To the date of COVID-19 manifestation 23 (42.6%) patients were in remission of the underlying disease, 18 (33.3%) in relapse/progression and remaining 13 (24.1%) had a simultaneous manifestation. The median time of COVID-19 onset since the last chemotherapy was 11 days (0 - 616). The severity of COVID-19 is known in 36 patients: mild in 11 (30.5%), moderate in 24 (66.7%) and severe in 1 (2.8%). The median time from COVID-19 to allo-HSCT was 211 days (31 - 447). The comorbidity index of HSCT was 0 points in 55.5% (n = 31), 1 point - 33.3% (n = 18), 2 points - 5.5% (n = 3), 3 points - 5.5% (n = 3). Donors were haploidentical – 16 (29.6%), MUD – 16 (29.6%), MMUD – 11 (20.4%) and MRD – 11 (20.4%). The source of HSC: PBSC – 41 (75.9%) and BM – 13 (24.1%). RIC and MAC were used in 41 (75.9%) and 13 (24.1%) recipients, respectively. Majority of patients (n = 48, 88.9%) received posttransplantation cyclophosphamide-based GVHD prophylaxis. The median time to the engraftment was 20 days (13 - 28). Acute GVHD developed in 16 (29.6%) patients, including grade 3-4 in 10 cases, chronic GVHD - in 4 (7.4%) patients. The main complications of post-transplant period included venous thrombosis (n = 4, 7.4%), TMA (n = 1, 1.85%), VOD (n = 1, 1.85%), bloodstream infections (n = 28, 51.8%), pneumonia (n = 8, 14.8%), soft tissue infections (n = 8, 14.8%), viral infections (n = 26, 48.1%), invasive mycoses (n = 5, 9.2%). The 100-day OS, GRFS and TRM were 88.9%, 74.1% and 9.3% respectively. The control group demonstrated the same short-term outcomes of allo-HSCT: 100-day OS – 89,3%, GRFS – 80,3% and TRM – 8,2%. Conclusions: Allo-HSCT is feasible in patients with a history of COVID-19 and characterized by common post-transplant complications. The history of COVID-19 did not affect the short-term results of allo-HSCT. Nevertheless, further studies are required in subgroups of patients with different consequences of COVID-19. Disclosure: Nothing to declare.
Background: In the context of hematopoetic stem cell transplantation (HSCT) the CD20-specific monoclonal antibody Rituximab is used as a highly effective treatment for EBV infection and EBV-associated post-transplant lymphoproliferative disease. To a lesser extent, it is also used in other situations (I.e. autoimmune hematologic diseases or included in the treatment regimen for CD20 + lymphoma). However, knowledge of immunological consequences and impact on immune reconstitution after pediatric HSCT are insufficient. Methods: To produce further insight we performed a retrospective analysis of a cohort of pediatric patients treated with rituximab within 365 days after HSCT. We included 44 (17,8%) out of the 248 patients that received HSCT in our clinic in the period from 01/01/2015 to 12/31/2019. Patients with a drop out event (I.e. relapse or death) before d30 were not included. We compared this group to a second cohort of 44 matched control cases from the same population. To eliminate immortal time bias, potential control patients were only considered if no drop out event occurred before the first dose of rituximab was administered to the corresponding rituximab patient. Data was collected and analyzed for an observation period of up to 3 years after HSCT. Results: Evaluating general outcomes, we did not find differences regarding overall survival. We could confirm, however, that rituximab therapy led to an increased time to recover normal CD19 + B-lymphocyte levels in the blood (Median B cell recovery: d282 vs d120) and impaired lymphocyte function making prolonged intravenous immunoglobulin (IVIG) substitution necessary (Median last day of IVIG: d254 vs d109). Analyzing the occurrence of other complications, we observed higher rates of other viral infections (75% vs 43%) and signs of increased rates of bacterial infections, longer re-hospitalization durations and higher necessity for transfusions. We did not find differences regarding the development of GvHD. Furthermore, we were able to identify a subgroup of 9 patients (21%) in the rituximab group compared to 0 in the control group, that received IVIG substitution during the entire observation period. Patients in this subgroup showed especially high rates of occurrence of the aforementioned complications. Conclusions: To our knowledge, this is the first systematic analysis of implications of rituximab therapy after pediatric HSCT in a larger cohort. In line with reports from rituximab applications in non-HSCT related settings, our findings provide strong evidence that rituximab harbors a risk for prolonged B cell damage when administered shortly after HSCT. Our study moreover shows that regular IVIG substitutions cannot prevent completely the occurrence of secondary complications. The similar overall survival of the compared groups, however, suggests that the occurring complications can be managed via specific treatments. As the observed complications or subgroup allocation did neither correlate with maximal EBV copy numbers in the serum nor with the number of rituximab doses that a patient received, we propose that the initial indication for rituximab treatment should be considered carefully. A repeated dosing over up to 4 weeks on the other hand, does not seem to increase the risk of rituximab-related impairment of immune reconstitution compared to a single dose. Disclosure: Nothing to declare
Background: Post transplant cyclophosphamide (PTCy) is increasingly used as Graft-versus-Host Disease (GVHD) prophylaxis. Hemorrhagic cystitis (HC) is a common complication following PTCy usually linked to BK virus reactivation. Although intravenous cidofovir has been reported to improve and/or reduce BK viraemia or viruria, current data do not support its regular use. Methods: We retrospectively analyzed HC management and outcome data from a consecutive cohort of adult recipients who underwent an allogenic stem cell transplant (allo-SCT) at our institution between October 2012 and October 2021. The inclusion criterion was the use of PTCy-based GVHD prophylaxis. Cidofovir therapy was considered in case of grade III-IV HC with BK viremia without any symptomatic improvement after at least 7 days after adequate supportive measures. The primary endpoint was to assess cidofovir efficacy and safety. Secondary endpoints included comparison of grade III-IV HC duration between the cidofovir cohort and a non-treated cohort. Results: We included 158 allo-SCT recipients. The main clinical and transplant characteristics are shown in table 1. Most recipients received an allo-SCT from alternative donors (78%). Most of recipients (76%) received mycophenolate mophetil (MMF) plus sirolimus whereas the remaining (24%) received MMF plus cyclosporine A as GvHD prophylaxis. The median follow-up was 13 months (IQR, 5 to 35 months). HC grade II to IV was diagnosed in 62 patients (39%) at a median of 26 days (IQR, 13,5 to 48,2) after stem cell infusion. Of them, 30 patients (19%) developed grade III-IV HC. The cumulative incidence of grade II-IV HC was 37% (29-44%). BK viremia was detected in 47 (76%) recipients at the HC onset. The median BK viremia before cidofovir onset was 2749 copies/mL (IQR, 1142-8421), whereas it was 3095 copies/mL (IQR, 820-4931) at the time of HC diagnosis in the non-treated. Intravenously cidofovir was given in 12 (40%) out of 30 recipients with grade III-IV HC, at a median of 13 days (IQR 11-26) after HC onset. Median cidofovir dose was 3mg/kg/week and the median number of doses was 2 (range 1-4). One treated recipient died from sinusoidal obstruction syndrome 10 days after first cidofovir dose. Then, only 11 recipients were evaluable for response and 10 (91%) achieved a complete remission. To evaluate the effect of cidofovir therapy, we compared the symptoms duration from the day of cidofovir in the treated cohort and from HC onset in the non-treated cohort with grade III-IV HC having BK viremia (n = 13). Median time of resolution in the treated cohort was 11 days (range 8-24) and 31 days (range 24-37) in the non-treated (p = 0.09). Two (15%) patients develop cidofovir related nephrotoxicity grade 2 and two (15%) patients develop myelotoxicity (neutropenia grade 2 and 3). Conclusions: Despite the inherent limitations of this study and the reduced number of patients, cidofovir was able to resolve severe forms of HC in most of recipients with uncontrolled HC symptoms despite intensive supportive measures. The safety profile was favourable. Prospective randomized trials are required in this setting. Disclosure: Nothing to declare
Background: The emergence and global spread of Carbapenem-resistant Enterobacteriaceae (CRE) has represented a major threat to public health. Patients with hematologic malignancies, who frequently experience prolonged neutropenia, immunosuppression, chemotherapy-induced mucositis and invasive procedures, are more vulnerable to CRE infection. Although many studies on CRE infection have been reported in the literature, there are only a few on patients with hematologic malignancies. Therefore, we conducted a retrospective study to describe the clinical outcomes in hematological patients with bloodstream infection (BSI) due to CRE. Methods: Our retrospective study included patients with monomicrobial CRE BSI between January 2012 and April 2021. The primary outcome was all-cause mortality 30 days after BSI onset. Risk factors were evaluated by comparing the variables of survivors with those of non-survivors. Results: A total of 94 patients with CRE BSI were documented in the study period. Escherichia coli was the most common Enterobacteriaceae, followed by K. pneumoniae. 66 CRE strains were tested for carbapenemase genes, and 81.8% (54/66) were positive, including NDM (68.5%, 37/54), KPC (29.6%, 16/54), IMP (1.9%, 1/54). Besides, one Escherichia coli isolate was found to express both NDM and OXA-48-like genes. Overall, 28 patients received an antimicrobial treatment containing CAZ-AVI, the remaining 66 patients were treated with other active antibiotics (OAAs). The 30-day mortality rate was 28.7% (27/94). Univariate analysis revealed that patients with high-risk hematological diseases, prolonged neutropenia (≥14 days), pulmonary infection, septic shock and the Pitt bacteremia score≥2 were risk factors for increased mortality. The administration of appropriate empirical therapy within 24 hours of BSI onset was protective factors. For patients treated with CAZ-AVI, the 30-day mortality rate was only 7.1% (2/28), which was significantly lower than those treated with OAAs (37.9%, 25/66, p = 0.003). In multivariate analysis, the presence of septic shock at BSI onset (OR, 10.53, 95% CI, 1.38 to 76.92) and pulmonary infection (OR, 6.29, 95% CI, 1.35 to 29.41) were independently risk factors for 30-day mortality. Comparing different antimicrobial regimens, CAZ-AVI showed a significant survive benefit than OAAs (OR, 14.64, 95% CI, 1.54 to 139.55). Conclusions: Timely appropriate empirical therapy is essential and CAZ-AVI-containing regimen is superior to OAAs for CRE BSI. Disclosure: This work was supported by the Natural Science Foundation of Tianjin City (18JCZDJC34400); the nonprofit Central Research Institute Fund of Chinese Academy of Medical Sciences (2018PT32034); CAMS Innovation Fund for Medical Sciences (CIFMS)
Background: Vaccines or SARS-CoV-2 infection are highly effective in inducing immunological responses in healthy individuals, however, they are able to induce protective immunity in hematological malignances and allogeneic HSCT recipients is still unknown. Methods: For this aim, 83 patients of the Department of the Hematology and Transplant Center of the Lower Silesian Oncology Center in Wroclaw (M/F:35/48, age: range: 20-86, mean: 52 years old, diagnosis: ALL = 13, AML = 21, MDS = 6, NHL = 20, CLL/SLL = 12, HL = 1, MM = 7, SAA = 3, including 18 SIB-, 21 MUD-, 1 haploidentical-, and 14 auto-transplantations) were evaluated for the presence of anti-SARS-COV-2 antibodies (anti S1 antibodies in IgG class) in serum between December 2020 and September 2021. Results: In this group: 21 (25%) patients had documented SARS-CoV-2 infection by PCR and/or antigenic test 59 (70%) patients were vaccinated with one of the available SARS-CoV2 vaccines 39 patients had SARS-CoV-2 antibodies after infection or/and vaccination (from 9 to 161 post-vaccination or infection) 17 (20%) patients had had SARS-CoV-2 antibodies without previous SARS-CoV-2 history or vaccination 11 patients did not have anti-SARS-CoV-2 antibodies despite vaccination and/or a history of confirmed infection (from days 29 to 201 post-vaccination or/and infection) which constitutes 28% of vaccinated and/or previous COVID-19 history patients (4 patients were excluded from antibody analysis due to short time post-vaccination (6-10 days)). It was found: Patients diagnosed with NHL (excluding alloHSCT) lacked more frequently anti-SARS-CoV-2 IgG antibodies after vaccination and/or SARS-CoV-2 infection compared to patients with other diagnoses who were vaccinated and or had SARS-CoV-2 infection (6/14 vs 3/39 p < 0.001). Six of 8 NHL patients who were negative for antibodies to SARs-CoV-2 had reduced IgG antibody levels (225-639 mg/dL, median: 507 mg/dL with a reference value of 700-1600 mg/dL). The absence of anti-IgG SARS-CoV-2 antibodies after vaccination or infection was associated with lower IgG antibody levels. Five out of 11 patients with lower IgG levels had no anti-SARS-CoV-2 antibodies after vaccination or infection (5/11 vs 14/68, p = 0.001). The majority of patients in the entire group had reduced IgM levels at the time of the study - 52/79 patients had levels of IgM antibodies below reference and no association was observed between low IgM levels and lack of anti-IgG SARS-CoV-2 antibodies after vaccination or infection. Group of patients who had SARS-CoV-2 antibodies despite no evidence of a past history of SARS-CoV-2 and were not vaccinated was most commonly comprised of post-HSCT patients (13/40 vs 4/44, p = 0.013). Patients who lacked anti-SARS-CoV-2 antibodies after vaccination and/or infection were not statistically different in WBC and lymphocyte levels compared to patients who had anti-SARS-CoV-2 antibodies after vaccination or infection. 2 patients in this group died due to COVID-19, both patients have lowered IgG levels. Conclusions: Conclusions: NHL patients should be given special SARS-CoV-2 surveillance, in those who do not have anti-SARS-CoV-2 antibodies, the cellular response after vaccination or infection should be examined, and advised to take 3 doses of vaccine. HSCT patients more frequently undergo asymptomatic SARS-CoV-2 infection. Including asymptomatic patients, 45% of subjects underwent SARS-CoV-2 infection. Disclosure: Nothing to declare
Background: Aspergillus fumigatus is the most common etiologic agent of IA reported in severely immunocompromised patients. However, IA caused by A. non-fumigatus species are becoming increasingly important and poorly studied. Methods: We designed the retrospective study in order to investigate the epidemiology of A. non-fumigatus IA vs A. fumigatus in adult patients (≥18 years) underwent allogeneic hematopoietic stem cell transplantation (allo-HSCT), who subsequently developed culture-positive IA from 2016 to 2021. During the observation period 33 patients with IA caused by Aspergillus non-fumigatus (n = 20) and A. fumigatus (n = 13) were identified. Results: The most common underlying diseases in patients with A. non-fumigatus were acute leukemia – n = 6 (30%) and non-Hodgkin’s lymphoma – n = 5 (25%), in A. fumigatus group – acute leukemia – n = 7 (54%). Transplantation from matched related donor was performed in n = 7 (35%) and n = 3 (23%), from matched unrelated – m = 3 (15%) and n = 3 (23%), from haploidentical – n = 8 (40%) and n = 4 (31%), from mismatched unrelated – n = 2 (10%) and n = 3 (23%) in patients with A. non-fumigatus and A. fumigatus, respectively. Myeloablative conditioning regimen was used in n = 4 (31%) patients from A. fumigatus group, in n = 7 (35%) - A. non-fumigatus group. Both groups received primary antifungal prophylaxis in post-transplant period (tabl.1). Etiology agents in A. non-fumigatus group were A. niger – n = 11 (55%), A. flavus – n = 2 (10%), A. terreus – n = 2 (10%), A. versicolor – n = 1 (5%), A. spp. – n = 1 (5%), combination A. fumigatus, A. flavus and A. niger – n = 1 (5%), combination A. fumigatus, A. niger and A. nidulans – n = 1 (5%). The median day of diagnosis IA caused by A. non-fumigatus was day +110 (17 – 2093), for A. fumigatus – day +46 (2 – 866). The main site of infection were lungs – n = 13 (100%) in patients with A. fumigatus, n = 18 (90%) in patients with A. non-fumigatus. Overall survival at 12 weeks was 55% and 59,2% in A. non-fumigatus and A. fumigatus groups, respectively (p = 0.617). Frontline therapy in A. non-fumigatus group was voriconazole in n = 16 (80%) patients, voriconazole in combination with echinocandin – n = 2 (10%), liposomal amphotericin B in monotherapy – n = 1 (5%) or in combination with posaconazole -n = 1 (5%); in A. fumigatus group voriconazole was used in n = 10 (77%) patients, voriconazole in combination with echinocandin – n = 1 (8%) or liposomal amphotericin B – n = 1 (8)%, isavuconazole – n = 1 (8%). Second line therapy received two patients with IA caused by A. non-fumigatus: liposomal amphotericin B in combination with echinocandin or with echinocandin and posaconazole, and two patients with IA caused by A. fumigatus: liposomal amphotericin B and voriconazole in combination with echinocandin. Comparative analysis showed that none of the assessed characteristics (sex, age, diagnosis, disease status, source of HSC, conditioning regimen, donor type, allo-HSCT number, type antifungal prophylaxis, CMV-reactivation, severe acute and chronic graft versus host disease) in patients from the two groups were not significantly differed. Conclusions: A. niger was dominant agent among A. non-fumigatus. IA etiology (A. non-fumigatus vs A. fumigatus) was not associated with specific patient characteristics and did not impact on treatment and outcomes in adults after allo-HSCT. Disclosure: Nothing to declare
Background: Carbapenemase-producing Enterobacteriales (CPE) are a significant risk to stem cell transplant recipients, with mortality rates from CPE blood stream infection (BSI) during transplant of up to 70%. This study assesses the impact of a series of measures introduced by Royal Manchester Children’s Hospital, with the aim to use screening and surveillance to reduce CPE BSI and death, in a paediatric allogenic HSCT population. Methods: This was a retrospective review of the Manchester bone marrow transplant database and microbiology laboratory information management system. Eligibility criteria were age < 18 years and allogenic stem cell transplant performed between July 2009 and October 2021 at Royal Manchester Children’s Hospital. The study endpoints were incidence of CPE BSI within 1 year of transplant and incidence of death from CPE BSI within 1 year of transplant. Results: A total of 585 patients were included. Sixty-two (10.6%) patients were colonised with CPE and 9 (1.5%) patients had CPE BSI within 1 year of transplant; Klebsiella pneumoniae carbapenemase (KPC) was the predominant CPE enzyme, and was detected in 7 patients, a carbapenemase enzyme was not identified in the remaining 2 patients. Five of the patients with CPE BSI died, in 3 patients CPE BSI was thought contributory although all had significant underlying issues. Incidence of CPE BSI within 1 year of transplant for all patients was 0.015. Incidence of death from CPE BSI within 1 year of transplant was 0.005. CPE-infection related mortality rate in those with CPE BSI within 1 year of transplant was 33%. All-cause mortality rate in patients with CPE bacteraemia within 1 year of transplant was 55%. Conclusions: Transplant recipients are at risk of CPE colonisation and infection due to immunosuppression, prolonged hospital stays, central venous access and exposure to broad-spectrum antibiotics. The Royal Manchester Children’s Hospital is within a Trust where transmission of plasmid-mediated blaKPC had become endemic, and a number of measures were introduced to reduce CPE colonisation and subsequent infection. These measures included screening all patients admitted for HSCT on admission and throughout for CPE, isolating patients colonised with CPE and ensuring empiric antibiotic for these patients include appropriate CPE cover. Our study demonstrates the success of this approach as incidence of CPE bacteraemia and death in our cohort was low, despite a relatively high baseline colonisation rate. Disclosure: Nothing to declare.
Background: South Korea is an endemic area of cytomegalovirus (CMV), and the CMV seropositivity of Koreans is estimated to be over 90%. Recently, letermovir has been considered as primary prophylaxis for CMV in CMV-seropositive recipients of allogeneic hematopoietic stem cell transplant (HSCT). Consequently, most HSCT recipients in South Korea are indicated for the use of letermovir. However, its high costs make this strategy non-feasible. In this study, we analyzed the pattern and survival of CMV reactivation in patients undergoing pre-emptive therapy for CMV infection during HSCT, and assessed high-risk patients who can benefit from CMV prophylaxis. Methods: We retrospectively analyzed the patients undergoing pre-emptive therapy for CMV infection during HSCT in the Korea University Transplant Registry from November 2003 to July 2020. CMV monitoring was performed in all enrolled patients using CMV antigen (from 2003 to 2013) or CMV polymerase chain reaction (PCR) (from 2013 to 2020), and pre-emptive therapy for CMV was done using ganciclovir (5 mg/kg, intravenous injection every 12 hours) in the patients who presented CMV antigenemia or PCR titer more than 1,000 copies/ml. Results: A total of 295 patients with HSCT were analyzed. The median age was 47 years (range: 16‒68), and the reasons for HSCT were aplastic anemia (26 patients, 8.8%), acute myeloid leukemia/myelodysplastic syndrome (182 patients, 61.7%), acute lymphoblastic leukemia (64 patients, 21.7%), multiple myeloma (11 patients, 3.7%), and lymphoma (12 patients, 4.1%). CMV-seropositivity was confirmed in 87.2% (donor + or recipient + ). Antithymocyte globulin (ATG) was used in 68.8% of patients. Pre-emptive therapy for CMV was performed in 142 patients (48.1%), and the median time from day 0 of HSCT to the start of therapy was 33 days (range: 3‒192). The overall survival of the patients who were treated with pre-emptive therapy for CMV was not different compared to those who were not treated. Of these 142 patients, 75 patients (52.8%) underwent more than two pre-emptive therapies for CMV during HSCT. The median duration of pre-emptive therapy for CMV was 14 days (range: 1‒74). In the multivariate analysis, the risk of the use of pre-emptive therapy for CMV was high in patients with multiple myeloma (OR: 14.979, 95% CI: 1.436‒156.282, p = 0.024), acute graft-versus-host disease of grade more than 3 (OR: 2.262, 95% CI: 1.175‒4.356, p = 0.015), and ATG. In case of patients with ATG use, the risk escalated with an increase in the total dose of ATG ( < 5 mg/kg, OR: 2.601, 95% CI: 1.227‒5.515, p = 0.013; ≥5 mg/kg and <9 mg/kg, OR: 4.846, 95% CI: 2.235‒10.507, p < 0.001; and ≥9 mg/kg, OR: 17.905, 95% CI: 5.255‒61.012, p < 0.001) compared to that in those with no use. Conclusions: In this study, half of the patients with HSCT were treated with pre-emptive therapy for CMV, without any effect on their survival, reflecting that pre-emptive therapy has enough advantage even in CMV endemic area. However, 50% of the patients experienced CMV reactivation repeatedly with a median duration of 14 days more than twice; therefore, it is necessary to re-evaluate the cost-effectiveness of CMV prophylaxis in high-risk patients. Disclosure: The authors declare that they have no competing interests.
Background: CMV reactivation remains one of the most common and serious infectious complications after allogeneic HCT. Patients transplanted from unrelated or mismatched donors are at high risk of CMV reactivation. We found interesting to compare reactivation rate in this two groups due to different immunosuppressive approach. Methods: All patients were transplanted before letermovir prophylaxis was widely available and none of them received it. 94 patients with hematological malignancies were included in the study – 55 transplanted from haploidentical donor (haplo group) and 39 transplanted from mismatched unrelated donor (mmud group). Conditioning was based on post-transplant cyclophosphamide (with tacrolimus and MMF) in 100% of patients in a haplo group and in 18% (7/39) in a mmud group. 32/39 patients of a mmud group received classical immunosuppression based on ATG/CsA/Mtx. Median age was 38 in a haplo and 41 in a mmud group, F/M ratio – 29/26 and 20/19, respectively. In a mmud group – there were single HLA mismatches in locus A, B, C and DQ in 15, 4, 14 and 6 cases. Results: We did not find any differences between groups in patients’ and donors’ age, conditioning intensity, diagnosis and CMV preemptive treatment. The median number of CD34 cells transplanted was significantly higher in a haplo group (9.84 vs 7.51 x 106/kg b.w., p = 0.007) Overall, CMV reactivation rate was as follows: 23/55 (41,8%) in a haplo group and 23/39 (58,9%) in a mmud group (p = 0.08) and CMV positive donors (D + ) rate was 44/55 and 24/39, respectively (p = 0.04). Acute GvHD was more frequent in a mmud group (20/39 (51,3%) vs 17/55 (31,0%), p = 0.04, including grades 3/4 (6/39 (15,4% vs 1/55 (1,8%), p = 0.04). In opposite, chronic GvHD was more frequent in a haplo group (11/55 (20,0%) vs 2/39 (5,1%), p = 0.03). On the other hand, in a mmud group, HLA-C mismatch was correlated with the highest rate of reactivation (HLA-A - 8/23 (34,8%), HLA-B - 0/23, HLA-C 11/23 (47,8%) and HLA-DQ - 4/23 (17,4%), p = 0.03). Conclusions: We are aware the results are preliminary; the study is still ongoing, we are collecting new data. The results, if confirmed in a larger group, may support a wider use of posttransplant cyclophosphamide instead of ATG in a mmud groups both due to CMV reactivation and acute GvHD rate. On the other hand, the results may be helpful to better define recipient groups for strict CMV prophylaxis. Disclosure: Nothing to declare
Background: Allografted patients are at high-risk for life-threating complications post SARS-CoV-2 infection, as the mortality rates in this group of patients has been reported of approximately 30-35%. The currently available vaccines proved their safety and efficacy by reducing the severity of the COVID-19 infection in the general population however, scant data exist in allografted patients. Methods: We evaluated the safety and efficacy of the commercially available vaccines in 20 allografted patients aged of 29,8 (21-50) years, vaccinated from March to November 2021 according to Saudi Arabia national vaccination program, after a median of 2,7 (0,3-6,7) years post-transplant. At the time of vaccination all patients were in complete remission however, one allografted patient for severe aplastic anemia had delayed engraftment. Fifteen were off immunosuppression without evidence of active GvHD, one was only on Cyclosporine while 5 were on steroids plus Cyclosporine or Mycophenolate MofetiI or Ibrutinib as treatment for chronic GvHD. Eight had additional co-morbidities: hyperglycemia (n = 4), post-transplant metabolic syndrome (n = 2), thyroid dysfunction (n = 2), all on specific treatment. One had received 5 months before vaccination Rituximab because of EBV-reactivation. The side effects post vaccination were estimated according to WHO grading system, while the antibody response was evaluated using automated commercial chemiluminescence immunoassay (CLIA) against spike (S1/S2) protein); antibody detection of >15.0 arbitrary units/ml (AU/ml) was considered as positive, between 12-15.0 as equivocal and less than 12.0 AU/ml as negative. Additionally, blood counts, lymphocytes sub-populations, immunoglobulins and D-Dimers were evaluated before and after each vaccination to evaluate any vaccine-related effect on the above parameters. Despite none patient had prior documented COVID-19 infection, three (15%) found to have detectable anti-SARS-CoV-2 antibodies before vaccination. Within a median of 42 (19-156) days, 2 doses were administered either of Pfizer (n = 17) or combinations of Pfizer/Moderna (n = 2) or AstraZeneca/Pfizer (n = 1) products. Results: After a median follow-up of 5,3 (2,6-7,7) months post 1st vaccination, no side effects grade ≥3 (WHO) reported. No allergy, thrombosis or heart dysfunction were noticed. The commonest complains were generalized fatigue (n = 4, 20%), bony pain (n = 2, 10%) while 2 (10%) patients reported fever <38.5oC (one over 24 hours and one for 5 days). No laboratory abnormalities were found post vaccination on the aforementioned evaluated parameters. Eventually 18/20 patients were available for humoral responses after 1st and 19/20 after the 2nd dose. Twelve (66%) and 18 (95%) achieved antibody responses after the 1st and 2nd dose respectively. One patient (with delayed marrow recovery post-transplant) failed to produce antibodies after completion of 2 vaccinations. Importantly, patients with active cGvHD and intensive immunosuppression, were capable for adequate humoral responses. None of the vaccinated patients developed COVID infection of any severity. Conclusions: Our retrospective study although with small number of patients and with short term follow-up, in agreement with others, confirms that the current commercially available vaccines against SARS-CoV-2 are safe and highly effective in producing detectable humoral responses in allografted patients. Prospective studies with longer follow-up are needed to elucidate the proper timing and the number of necessary doses for a safe and effective approach in preventing severe COVID infection. Disclosure: Nothing to declare
Background: Cytomegalovirus (CMV) reactivation or disease is the most common infectious complication following allogeneic stem cell transplantation (allo-HSCT). All CMV seropositive recipients are in risk for CMV reactivation, especially those who receive a mismatched, T-cell depleted or umbilical cord blood graft, and those who develop GvHD requiring corticosteroid treatment. Prophylactic therapy with letermovir became available quite recently, and is approved for administration in CMV seropositive patients during the first 100 days post HSCT. The aim of our study was to evaluate the efficacy of primary CMV prophylaxis with letermovir and the frequency of late CMV reactivation and disease. Methods: In this retrospective study, we evaluated 29 transplant recipients who received letermovir prophylaxis between August 2018 and February 2021. Letermovir was administered from day +7 through day +100. CMV viral load was monitored by quantitative PCR 1-2 times weekly, for a minimum of 6 months following transplant. We studied the frequency of CMV reactivation during administration of letermovir and after cessation of therapy. Results: Twenty-nine patients with a median age of 55.4 (range, 21-71) years were evaluated. The indication for HSCT was AML/MDS (n = 23), NHL/CLL, (n = 3), MPN (n = 2) or ALL (n = 2). Twenty patients received myeloablative and 9 patients reduced-intensity conditioning. Eighteen of 29 patients were high-risk for CMV reactivation, based on mismatched unrelated (n = 3) or haploidentical (n = 1) transplant, acute GvHD grade ³ΙΙ (n = 15) and/or high-dose corticosteroid treatment (n = 13). During the first 100-day follow-up period, 11 cases of CMV DNA detection were noted in 10 patients (5 of whom were high-risk) at a median time of 25 (8-96) days from transplant. 10/11 events involved low viral load that subsequently resolved without antiviral treatment. Only one case of clinically significant CMV reactivation was observed, which responded to pre-emptive therapy with foscarnet without evidence of CMV disease. Following letermovir discontinuation, 14 patients experienced CMV reactivation at a median of 160.5 (116-195) days post transplant. 8/14 received antiviral therapy with valgancyclovir (n = 7) or foscarnet (n = 1). Two patients required second-line antiviral treatment. No case of CMV disease was documented. The absolute CD3 + cell count was significantly lower in patients with versus without CMV reactivation after completion of letermovir prophylaxis (median, 438/μL vs 701/μL, respectively, p = 0.02). Conclusions: Letermovir is effective as primary prophylaxis, albeit with a considerable risk of CMV reactivation following discontinuation. Patients with better T cell reconstitution had a lower risk of late CMV infection, presumably due to the presence of CMV-specific T cells. T cell immunity could therefore serve as a marker for the optimal duration of letermovir prophylaxis. Disclosure: Nothing to declare
Background: HSCT recipients are profoundly immunosuppressed and health care workers (HCW) of transplant units need to be tested periodically by SARS CoV-2 PCR to avoid patient transmission during hospitalization. Methods: We conducted a prospective cohort study with periodic serology and nasal wash (NW) sampling to estimate the cumulative incidence of COVID-19 in health professionals from HSCT unit before (May 2020 to January 2021) and after COVID-19 vaccination (January to October 2021) and to evaluate the occurrence of hospital acquired COVID-19 in HSCT recipients. In addition to periodic sampling, from inclusion (dzero) onwards, HCWs were daily surveyed for the presence of symptoms. NW was taken if symptoms and/or exposure to a confirmed or suspected case of COVID-19. If tested positive by PCR, they were away for 14 days and returned to work with at least 1 negative PCR test. Detection of SARSCoV-2 was performed by PCR (RealStarÒ SARSCoV-2, Altona Diagnostics/ Germany) and monthly serology by ELISA (Anti-SARSCoV-2 ELISA, Euroimmun/ Brazil). The incidence of SARSCoV-2/COVID-19 was estimated by cumulative incidence. Study participants received the 1st dose of COVID-19 vaccines (Sinovac/Butantan or Oxford/AstraZeneca/Covishield) between January and March 2021, and the 2nd dose between February and June 2021. Vaccine Effectiveness (VE) was identified by the formula VE = (r0-r1)/r0 (r0 = rate in unvaccinated individuals; r1 = rate in vaccinated). Results: Between May 13, 2020 and March 22, 2021, 109 HCWs were included. The median follow-up was 259 (79-309) days. Before vaccination, 29 cases of SARS CoV-2/COVID-19 were diagnosed at a median of 53 days, for a cumulative incidence of 30 %. Thirteen cases (11.9%) were detected at inclusion and 16 during follow-up. Of the 13 cases detected at inclusion, 8 (30.8%) were diagnosed by serology, showing previous infection. During follow-up, 7 individuals dropped out of the study and one was not vaccinated. Thus, 101 HCWs were included in the post-vaccine analysis. Eight PAS (8%) received chAdOx1 (Oxford/Astrazeneca/Covishield) and 93 (92%) Sinovac (Butantan). After vaccination, 76 of the 78 susceptible HCWs tested positive (97.4%), 1(1.3%) had an indeterminate result, and 1(1.3%) had a negative result after the 2nd dose. Within a median post-vaccine follow-up of 153 (91-165) days, 9 HCWs acquired COVID-19 (6 between the first and second dose) for a cumulative incidence of 9.7%. Three (33.35) acquired COVID-19 despite the presence of specific SARS CoV-2 antibodies. Considering only the susceptible subjects at vaccination (n = 78), the rate of COVID-19 in unvaccinated (n = 29) or partially vaccinated (n = 6) was 44.8% (35 of 78) and the rate in those fully vaccinated was 3.8% (3 of 78), for a VE of 91.5%. Due to this intense surveillance of COVID-19 in the HSCT unit, no hospital-acquired COVID-19 was observed in HSCT recipients during the study period. Conclusions: In conclusion, a good serological response was observed after vaccination (97,4%), resulting in a decrease in the incidence of COVID-19 from 30% to 9,7%. The current pandemic scenario continues to represent a great challenge in HSCT units. COVID-19 control policies in HSCT health professionals successfully avoided hospital transmission of SARS CoV-2 to HSCT recipients. Disclosure: C Machado - speaker fee from MSD, Altona and Takeda. Source funding - Virology lab, Institute of Tropical Medicine, University of São Paulo Medical School.
Background: Letermovir (LMV) is the first drug approved for prophylaxis of Cytomegalovirus (CMV) infection in adult CMV-seropositive patients undergoing hematopoietic stem cell transplantation (HSCT). It acts by inhibiting the viral DNA terminase complex, interfering with virion maturation. Clinical efficacy and particularly favorable toxicity profile make the drug attractive in pediatric settings, both for the prophylaxis of CMV infection and for the treatment of resistant cases. Methods: Between July 2020 and November 2021, Letermovir was administered to 20 pediatric patients at the Bambino Gesù Children’s Hospital. The off-label clinical indications for LMV were: primary prophylaxis (n = 9), secondary prophylaxis (n = 2), pre-emptive treatment (n = 7) and CMV-disease treatment (n = 2). Letemovir was administered at median dose of 240 mg/die (range 50-480 mg/die); notably, patients taking cyclosporine (n = 14, 70%) as prophylaxis/therapy of Graft-versus-Host Disease (GVHD), received half of the planned dose. Primary prophylaxis (CMV positive recipient with negative donor) with LMV was administered at least until day 100 post HSCT. Five patients were already taking antiviral therapy (Ganciclovir and/or Foscarnet) for CMV-DNA positivity on blood; two of them had CMV disease (pneumonia and encephalitis). Results: All patients were able to assume Letermovir with only mild nausea and vomiting in 2 patients (14%), and none of them discontinued the treatment. Eight out of 9 patients who assumed LMV as primary prophylaxis did not reactivate CMV viremia up to day 100 after transplant. All 7 patients who started LMV with positive CMV-DNA on blood (median 4400 copies/ml) achieved negative CMV-viremia at a median time of 27 days; 5 of them (71%) received a T-depleted HSCT. Two patients on secondary prophylaxis therapy did not exhibit CMV reactivation until drug discontinuation. One patient with CMV pneumonia treated with Letermovir in combination with Ganciclovir and Foscarnet presented a rapid decrease of CMV-DNAemia, despite subsequent death due to interstitial pneumonia (CMV-), while one patient with CMV encephalitis, already treated with dual antiviral therapy, had CMV-DNA negativity on blood and CSF after initiation of therapy with LMV. Overall, probability of negative CMV-DNAemia at 60 days after start of Letermovir was 90%. Conclusions: With the limit of sample size, Letermovir seems safe and effective in all indications tested in our pediatric cohort. More studies are needed to address precisely the duration of primary and secondary prophylaxis, considering type of transplantation (e.g. T-depleted), immune reconstitution and any concomitant immunosuppression (GVHD prophylaxis and therapy). Preliminary results with Letermovir as third-line therapy for treatment-refractory CMV-reactivation show clinical efficacy which could be assessed in larger trials. Disclosure: Nothing to declare
Background: Follow-up of cytomegalovirus (CMV) reactivation in autologous peripheral stem cell transplantation (ASCT) is not recommended because of the low risk of reactivation. This study created a model for the high risk of CMV reactivation in patients undergoing ASCT, and the results were examined. Methods: The study was conducted with 360 patients who underwent ASCT between August 2009 and August 2021. For the transplant conditioning regimen, melphalan 200 mg/m2 was administered to all multiple myeloma patients and BEAM to lymphoma patients. CMV DNA follow-up after ASCT was checked weekly only in patients with prolonged fever, diarrhea, and pneumonia in the absence of bacterial or fungal infection, unexplained elevations of liver enzymes greater than 1.5-fold, or delay or loss of engraftment neutrophils and platelets. Engraftment delay was defined as an unsupported absolute neutrophil count (ANC) of 500/mm3 and a platelet count of no more than 20000/mm3 at 14 and 21 days after ASCT. In addition, ANC and platelet counts <1000/mm3 and <100000/mm3 after recovery, respectively, were considered as engraftment loss. Results: CMV reactivation was detected in 43 patients, 29 male and 14 female, and the cumulative incidence was 11.9%. The patients’ ages ranged from 25 to 73, with a median of 56. The median time to CMV reactivation was 39 days (8-1825 days). Three patients died within three days of CMV reactivation. All other patients, including six patients with end-organ disease, recovered with anti-CMV therapy. Twenty-one (48.8%) patients with CMV reactivation were treated 12 ganciclovir and 9 valganciclovir. For CMV reactivation, induction and consolidation treatments were each administered for a median of 14 days. Cytopenia in 4 patients and electrolyte disturbances in 6 patients were detected as treatment-related toxicity. In our study cohort with CMV reactivation with a median follow-up of 47.7 (10.2-277) months, the 100-day mortality was 6.9%, and the overall mortality was 46.5%. More detailed information of patients with CMV reactivation after ASCT is shown in table 1. Table 1: Clinical features and distribution of patients with CMV reactivation Conclusions: Our data suggest that high-dose chemotherapy regimens administered with ASCT may also be a reason for the risk of CMV reactivation. Both ganciclovir and valganciclovir are effective in treating CMV reactivation after ASCT. More frequent evaluation of CMV infection may be recommended in high-risk ASCT patients included in the criteria of our study. Thus, with rapid diagnosis, treatment can arise without significant toxicity. Disclosure: Nothing to declare.
Background: COVID-19 infection has been associated with adverse outcomes in hematopoietic stem cell transplantation (HSCT) recipients. In this study, we aimed to evaluate the clinical features and outcomes of COVID-19 in HSCT recipients followed in our center. Methods: Patients who were followed up in our Blood and Marrow Transplantation Center and had post-transplant COVID 19 infection were retrospectively screened. Features such as age, diagnosis, donor type, graft source, conditioning regimens and COVID 19 disease severity were included in the analysis. The main outcome was overall survival 30 days after diagnosis of COVID-19. Survival analysis was calculated using the Kaplan-Meier method. Factors associated with death after diagnosis of COVID-19 were examined using Cox proportional hazards models Results: 76 HSCT recipients diagnosed with COVID-19 were included in the study. The median age of the patients included in the study was 55 (range, 20 - 73) and 41 (54%) were male. The median time from HSCT to diagnosis of COVID-19 was 26 months (range, 3-146) for HSCT recipients. While 43 (57%) of the patients were autologous HSCT receivers, 33 (43%) were allogeneic HSCT receivers. Of the 33 allogeneic HSCT recipients, 5 (15%) were receiving immunosuppression within 6 months of COVID-19 diagnosis. The majority of patients (88%) were in remission for hematological disease. Of 76 patients, 14 (18%) had mild disease severity and 10 (13%) had severe disease requiring mechanical ventilation. While 52 patients used favipravir as treatment, 21 patients used a combination of steroid and favirpravir. Convalescent plasma was given to 3 (4%) patients. Ten (13%) patients died due to covid. Five of these patients were autologous HSCT receivers and 5 were allogeneic HSCT receivers. At 30 days after diagnosis of COVID-19, overall survival for HSCT recipients was 90%. Disease status was associated with a higher risk of death among all HSCT recipients (p = 0.018) When 10 patients who died were evaluated, 4 of them had active hematological cancer when they were diagnosed with COVID-19 Conclusions: COVID-19 infection is a feared situation in hematopoietic stem cell recipients. HSCT recipients should avoid contact with COVID-19 patients as much as possible and carefully maintain routine hygiene practices. Patients with active disease are in higher risk group in terms of mortality. Disclosure: nothing to declare
Background: Midostaurin in combination with chemotherapy become the standard of care of FLT3 positive AML. Although its favorable role is well known in terms of OS and EFS, there is lack of data regarding its impact on infectious complications after HSCT. Methods: We retrospectively evaluated 48 consecutive patients underwent HSCT for FLT3 ITD positive AML over the last 10 years (from 2012 to 2021). All patients from 2018 received Midostaurin as a part of induction and consolidation treatment and we compared them to an historical cohort which did not receive any FLT3 inhibitors. Monitoring of mucositis during aplasia phase, incidence of infectious events (sepsis, septic shock and GI infection), acute or chronic GVHD, relapses and CMV reactivation after HSCT was carried out. Comparison of non-continuous variables was done by Chi-square test. Kaplan-Meyer curves were used to assess time to event analysis. Results: Midostaurin was administered to 29 patients as a part of AML treatment during induction and consolidation before allogeneic transplant. No difference was observed between the two cohorts in age at HSCT, gender, status of disease at time of HSCT, ATG as GVHD prophylaxis, intensity of conditioning regimen, type of donor, graft source, CMV reactivation and incidence of relapse. Midostaurin group reported a higher number of both pre-existent colonization (p = 0,011) and incidence of septic shock (p = 0,019). The incidence of aGHVD was lower in Midostaurin group (p = 0,021). No difference was observed for 1-year OS and 1year PFS between the two groups (49% vs 51,7%, p=ns; 40% vs 44,7%, p=ns). Conclusions: Patients previously exposed to Midostaurin who underwent HSCT reported a higher incidence of pre-existent colonization and thereafter to life threatening infections, such as septic shock. It seems to have a protective role against aGVHD. This result may be explained by the gastrointestinal toxicity related to Midostaurin. However, the small sample size of Midostaurin group might prevent to assess other differences among the two groups. Disclosure: nothing to disclose
Background: Allogeneic hematopoietic cell transplantation (allo-HCT) is the only curative treatment for many malignant and non malignant haematological diseases. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the coronavirus that causes COVID-19 (coronavirus disease 2019), the respiratory illness responsible for the ongoing COVID-19 pandemic. The COVID-19 pandemic led to the fast invention and wide use of mRNA and viral vector based vaccines. While the protection of the general population against SARS-COVID-19 after vaccination is undoubtedly, the question about protection in immunocompromised people remains unknown. The goal of this study is to examine the extent in which a subpopulation of immunocompromised patients, those who have underwent allogenic stem cell transplantation are able to produce immune response against SARS-COVID-19 after vaccination. Methods: Vaccination against SARS-CoV-2 was recommended to all allo-HCT patients after interruption of the immunosuppression. Low dose Campath (10 mg – 20 mg) was used as GvHD prophylaxis along with cyclosporine. Levels of serum IgG antibodies against spike protein S of SARS-CoV-2 were measured at least 2 weeks after the 2nd vaccine dose. The quantitative anti-spike RBD IgG antibody responses were measured using the Abbott SARS-CoV-2 IgGIIQuant assay (cut off ≥50 AU/mL). Results: Totally 35 patients who underwent allo-HCT had serum IgG antibodies levels measured against SARS-CoV-2. Patients characteristics are listed in Table 1. Thirty two (91%) patients were vaccinated with mRNA vaccines (Comirnaty, Pfizer-BioNTech) and 3 (9%) with viral vector based vaccines (Vaxzervia, AstraZeneca). The median day after transplantation that patients were vaccinated was 913.5 (range: 137-4086). Thirty patients (86%) had immune response against SARS-CoV-2 and 5 (14%) hadn’t. The median day of IgG antibodies measurement was 913.5 (range: 137-4086). It is worth of notice that 5 patients had serum IgG against SARS-CoV-2 measured after the 3rd vaccine dose. One didn’t respond to the third dose, one that was negative after the 2nd dose became IgG positive after the 3rd dose and the remaining three continued to be positive. One patient presented a serious adverse event after the first dose of mRNA based vaccine, that was thrombocytopenia that required hospitalization and treatment with immunoglobulins and corticosteroids with complete resolution of the thrombocytopenia. All patients that were vaccinated against SARS-CoV-2 haven’t presented COVID-19 disease and are alive. Table 1. Characteristics of patients who underwent allo-HCT and where vaccinated against SARS-CoV-2. Conclusions: Our experience demonstrates the efficacy of vaccination against SARS-CoV-2. Although further prospective studies with longer follow up are required. Disclosure: Nothing to declare
Background: Cytomegalovirus (CMV) infection is a serious, potentially lethal complication of paediatric hematopoietic stem cell transplant (HSCT). To date, antiviral therapy has been the mainstay of prophylaxis and encouraging results with Letermovir are limited in paediatric transplant by its restricted use to children aged 12 and over. Development of alternative CMV prophylaxis agents such as CMV-specific immunoglobulins could meet a clinical need in younger recipients. So far, however, studies focusing on CMV-specific immunoglobulins (CMV-Ig) prophylaxis have met with conflicting results. Methods: After introducing prophylactic CMV-Ig for HSCT recipients at risk (seropositive recipient and/or donor), we conducted a single center retrospective study comparing incidence and severity (symptomatic infection, peak CMV titres, duration, requirement for treatment, and outcome) of CMV infection between patients who were at risk of CMV reactivation and did or did not receive CMV-Ig (historical controls). Patients in the CMV-Ig group received CMV-Ig at 0.5 g/kg fortnightly for six doses, starting along with the initiation of conditioning. We identified 49 ‘at risk’ recipients from 76 consecutive HSCTs over 3.5 years: 39 did not receive CMV-Ig and 10 did. There was no significant difference in donor type, cell source, intensity of conditioning or CMV status between the groups. Results: We observed a non-significant reduction (p = 0.620) in incidence of CMV reactivation with CMV-Ig (n = 3, 30%) versus without (n = 15, 38.4%). No patient who received CMV-Ig developed symptomatic or lethal infection, all serious infections occurring in the non CMV-Ig group. Formal analyses on infection severity are limited by the small number of CMV infections in the group who received prophylaxis with CMV-Ig, however, duration of infection appeared shorter (21 (+/−7) vs 51.4 (+/− 55) days) and peak titers lower (4578 (+/− 4788) vs 24 131 (+/− 49 257)) with CMV-Ig. CMV infection tended to occur earlier following transplant in patients receiving CMV-Ig (25.6 (+/− 5.5) days following HSCT vs 47 (+/−86.1) days for patients who did not (p = 0.681). Active CMV treatment was started for all patients who developed CMV infection after receiving CMV-Ig, and for 70% of patients in historical control group (p = 0.290), which is more likely to reflect a change in practice rather than severity of infection. No adverse events during CMV-Ig infusions were noted. Conclusions: CMV-Ig administered fortnightly for a total of six doses starting with the initiation of the conditioning regimen appears to be a safe CMV-directed prophylactic measure, and is worth consideration for HSCT recipients at risk of CMV reactivation younger than 12. CMV-Ig potentially reduces CMV incidence, but more convincingly may reduce severity of infection and duration of treatment with myelosuppressive antivirals, sparing transplant-related morbidity, thus potentially improving transplant outcomes. Disclosure: Haydn Munford, PhD is a medical science liaison at Biotest UK, Ldt and has offered support with data analysis suggestions and graphic illustrations.
Background: The SARS-CoV-2 pandemics has been spreading in Czechia since March 2020. The course and mortality of this infection are substantially more serious in immunocompromised patients compared to normal population. The aim of this study was to monitor the course of COVID-19 disease in patients undergoing allogeneic or autologous hematopoietic stem cell transplantation (HSCT) or chimeric antigen receptor T-cell (CAR-T) therapy at our department. Methods: All patients with SARS-CoV-2 positivity detected between October 2020 and April 2021 who previously underwent allogeneic or autologous HSCT or CAR-T therapy were included into this retrospective study. We describe the time interval between infection and transplantation or CAR-T therapy, need and length of in-patient stay, use of mechanical ventilation, antiviral therapy and infection outcomes. Results: A total number of 34 patients (average age 55 years) were included into this analysis, 10 after allogeneic HSCT, 21 after autologous HSCT, and 3 after CAR-T therapy. The average number of days since HSCT or CAR-T therapy to the first positivity was 1324, with a maximum of 7799 and a minimum of -2 days (infection started during the conditioning regimen). Twenty-five (73%) patients were hospitalized, 10 of them were admitted to the intensive care unit (ICU), and three required mechanical ventilation. Average length of in-patient stay was 17 days (range 3 to 44), and 16 days at the ICU (range 7 to 38). Sixty percent of hospitalized patients received convalescent plasma transfusion, 52% antiviral treatment with remdesivir (3 patients were reinfused for prolonged viral shedding), and one patient received monoclonal antibody (bamlanivimab). Twenty-four patients recovered from the infection, and 10 died (29%), all of them during their hospitalization. Conclusions: Although the general COVID-19 mortality (case fatality rate) is about 2%, in patients who underwent hematopoietic stem cell transplantation or cellular therapy it reaches up to 20 to 30%, which corresponds to our cohort. Thorough standard precautions, vaccination of family members, and monoclonal antibodies and antivirals early after contact or at the very beginning of the infection are the inevitable essentials to protect these fragile patients from severe COVID-19 disease associated with high risk of fatal outcome. Disclosure: Nothing to declare.
Background: Adenovirus infections are an important cause of morbidity and mortality after allogeneic hematopoietic cell transplantation (allo-HCT) especially in children. There are no approved antivirals for adenovirus infection. Posoleucel is an allogeneic, off-the-shelf investigational T cell therapy designed to target adenovirus as well as five other common opportunistic viruses: BK virus, cytomegalovirus, Epstein-Barr virus, human herpesvirus 6, and JC virus. In a phase 2 trial of posoleucel in hematopoietic cell transplant recipients, 83% (10/12) of those with adenovirus disease had a clinical response. Methods: We are conducting a phase 3, multicenter, randomized, double-blind, placebo-controlled trial to assess the safety and efficacy of posoleucel for the preemptive treatment of adenovirus infection in pediatric and adult allo-HCT recipients. The table shows eligibility criteria, endpoints, and definitions. Patients will be randomized 1:1 to receive posoleucel or placebo in two infusions separated by 14 ± 3 days stratified by level of adenovirus viremia (≥10,000 or <10,000 copies/mL DNA) and age (≥12 or <12 years) (Figure). Posoleucel will be infused at dosages of 2×107 cells for patients <40 kg or 4×107cells for those ≥40 kg. Patients who progress to active target organ disease or whose existing target organ disease progresses will have the option to cross over to the alternate treatment arm between Day 29 and Week 10, but study treatment will remain blinded. To be eligible for cross-over, patients must not have evidence of GVHD > 2, must not be receiving >0.5 mg/kg/day prednisone or equivalent, and must not have experienced an infusion-related reaction with prior doses of posoleucel or placebo. Results: Planned enrollment is approximately 80 patients at approximately 40 sites in the United States and Europe. The study is currently ready to initiate. Conclusions: This phase 3 trial will provide data on the efficacy and safety of posoleucel compared with placebo for the treatment of adenovirus infection and disease in allogeneic HCT recipients. Clinical Trial Registry: Pending Disclosure: Kanchan Rao is the National Coordinating Investigator for the study in the UK, and is an advisor to AlloVir. G. Doug Myers is an advisor to AlloVir. Iain Fraser is employed by and holds stock in AlloVir. Elizabeth Stoner is a Senior Clinical Advisor to AlloVir. P. Ljungman is the National Coordinating Investigator for the study in Sweden.
Background: Cytomegalovirus (CMV) infection after allogeneic hematopoietic stem cell transplant (allo-HSCT) is a catastrophic complication, as it can result in graft failure and increased mortality. Higher CMV seroprevalence has been reported in low- and middle-income countries (LMIC). Our aim was to describe risk factors, monitoring and treatment strategies, and outcomes of allo-HSCT patients with CMV infection. Methods: Retrospective, single-center, observational study that included patients >18years who underwent allo-HSCT between January 2016 and March 2020. Results: A total of 52 patients were included. Median age was 32.9 years (range 20-60). Most common baseline diagnosis was lymphoblastic leukemia in 57.7% (n = 30), followed by myeloblastic leukemia in 19.2% (n = 10). Disease status was complete remission in 44.2% (n = 23). Regarding transplant procedures a median of 2.3x106CD34+cells/kg (0.9-5.9) were infused, bone marrow source was used in 79.6% (n = 41), donors were matched related in 74.1% (n = 44), and 88.5% received a myeloablative conditioning regimen. Patients developed mucositis in 90.4% (n = 40), acute graft versus host disease (aGVHD) in 30.7% (n = 16), and chronic GVHD (cGVHD) in 28.8% (n = 15). CMV risk was categorized as low in 10.2% (n = 5), intermediate in 18.4% (n = 9), and high in 71.4% (n = 35). A 98% (n = 51) of our cohort proceeded with preemptive strategy. With a median of 4 (1-5) tests per month, a total of 27 patients (51.9%) developed CMV viremia and 21 (77.8%) received antiviral therapy. The median time from HSCT to a positive test was 46 days (20-153) with a median viral load of 1724.22 IU/ml (69-12986). T-cell depletion with post-HSCT cyclophosphamide (p = 0.049) conferred an increased risk for CMV viremia. Median time to resolution of viremia (TV) was 107 days (7-1804), being cGVHD (p = 0.032) related to a longer TV. Median overall survival (OS) was 52 months (8-169). On univariate analysis, CMV viremia was not a factor associated with a decreased OS. Conclusions: There is limited information regarding incidence and outcomes of CMV infection after allo-HSCT in LMIC, in which limited access to diagnostic tests, as well as a high seroprevalence can negatively impact. Interestingly, despite having characterized as high risk >70% of our population, incidence of CMV was similar to the previously reported. Concordant to information of other study groups, increased immunosuppression was the sole factor related to increased risk of CMV incidence and duration. We report good monitoring and no impact of CMV on mortality, with no cases of disease. Further multicentric studies are required to define the most adequate monitoring and treatment strategies in vulnerable populations. Disclosure: The authors declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.
Background: This study investigated the association of CMV viremia and healthcare resource utilization (HCRU) in adult allogenic HSCT patients. Methods: This was a single-center retrospective observational cohort study from the Turku University Hospital Stem Cell Transplantation center. The study included patients with their first allogenic HSCT between January 1, 2013 and December 31, 2018. CMV viremia was defined as detection of CMV DNAemia ≥ 1000 copies/mL or start of preemptive therapy due to CMV DNAemia. Co-variates studied included: age, gender, D/R status, underlying malignancy (grouped as acute leukemias, lymphoproliferative diseases, and myeloproliferative diseases including aplastic anemia), preconditioning, donor type and HLA matching, graft source, and GVHD. Data for CMV viremia, hospital readmissions, hospital days, intensive care admissions, outpatient visits and anti-CMV medication were collected by 1 year. Data analyses included descriptive statistics, Mann-Whitney U-, Pearson’s chi-squared and two-proportions z-test, multiple Cox’s regression model including all co-variates and simple negative binomial regression model. Results: The study included 251 patients. CMV seroprevalence was 69.7%. One hundred thirty-five (77.1%) R + patients and 16 (21.1%) R- patients had CMV viremia, and 76.8% of patients had ≥2 viremias. The median time to CMV viremia was 40 (IQR 33, 53) days. In multiple Cox regression, co-variates associated with higher CMV viremia risk included R + (HR 7.31, 95% CI 3.77-14.19, p < 0.001), lymphoproliferative diseases (HR 1.69, 95% CI 1.09-2.60, 0 = 0.018), and grade 3-4 acute GVHD (HR 1.74, 95% CI 1.11-2.71, p = 0.015). HLA identical donor was associated with lower CMV viremia risk (HR 0.53, 95% CI 0.32-0.89, p = 0.017). Patients with CMV viremia had more hospital readmissions (IRR 1.56, 95% CI 1.24-1.95, p < 0.001) and longer hospital length of stay (IRR 1.77, 95% CI 1.35-2.13, p < 0.001) compared to patients without CMV viremia (Table 1). We did not see differences in ICU admissions or out-patient visits. 149 patients received anti-CMV medication. Valganciclovir (70.5%) and foscarnet (60.9%) were the most used anti-CMV drugs, and valganciclovir was used more often in patients with ≥2 viremias compared to patients with 1 viremia (82.8% vs 36.4%, p < 0.001). Foscarnet was more often used in patients with grade 3-4 acute GVHD compared to patients with grade ≤2 acute GVHD (50.0% vs 33.2%, p = 0.037). Ganciclovir was used in 36.8%, and there was no difference in patients with 1 viremia or ≥2 viremias (30.3% vs 37.9%, p = 0.550). Table 1. Healthcare resource utilization in 1 year post allogenic HSCT. Differences were tested using Mann-Whitney U-test. # Differences was tested with chi-square test. Conclusions: CMV seroprevalence is relatively high among allogenic HSCT patients. CMV viremia occurs commonly and is often repeated. CMV viremia was associated with higher hospital readmission rate and longer additional hospital length of stay. Prevention of CMV viremia may reduce additional HCRU associated with CMV viremia post allogenic HSCT. Disclosure: Funding for this research was provided by Merck Sharp & Dohme Corp., a subsidiary of Merck & Co., Inc., Kenilworth, NJ, USA. JR received Principal Investigator fee, and Turku University Hospital received institutional research funding. RK is an employee of MSD Finland Oy, and owns stocks of Merck & Co, Inc., Kenilworth, NJ, USA.
Background: Human T-lymphotropic virus type I is a retrovirus that infects 10 to 20 million people worldwide. However, is associated with disease only in 5% of infected individuals. Two diseases associated: Adult T cell leukemia-lymphoma and HTLV-I-associated myelopathy. Impact of infection is unclear among patients who undergo allogeneic hematopoietic stem cell transplantation (HSCT) due to limited clinical reports. Methods: We report two HTLV-I leukemia/lymphoma allotransplanted patients. Results: CASE 1: 41-year-old male from Chile, presented with decrease state of consciousness, requiring admission at ICU. Diagnosis of Adult T cell leukemia was done. Laboratory tests showed leukocytosis (11.400/mm3), lymphocytosis with immunohistochemical profile of ATLL. Serological tests showed HTLV-1 antibodies. Bone marrow: 61% blasts; cerebrospinal fluid: normal. Initially treated in ICU with CHOP, subsequently 6 cycles of VCAP-AMP-VECP and zidovudine 200 mg BID. The patient was referred to our center in first CR for HSCT from mismatched unrelated donor, conditioning with TBF and post-cyclophosphamide. On admission, HTLV1 viral load was 32. 000/106 copies. Switch to lamivudine was decided for hepatitis B reactivation prophylaxis and to avoid zidovudine myelotoxicity. HTLV1 viral load monitoring was performed on day +17 with a load of 2.000/106copies. Lamivudine was continued on discharge and zidovudine restarted. Serial viral load monitoring was performed during follow-up at outpatient clinic: negative since day +46. Currently with both antivirals and in CR with full donor chimerism 6 months posttransplant. CASE 2: 54-years-old woman from Dominican Republic, diagnosed with Adult T cell lymphoma/leukemia IV-B in 2018. She presented hypercalcemia, hepatomegaly, lymphadenopathy, and bone marrow infiltration of 25% lymphoid blasts with a ATLL phenotype. No CNS infiltration. Serological tests: HTLV-1-IgG antibodies. She received treatment with Hyper-CVAD scheme. In first CR, she received haploidentical transplantation, TBF conditioning. HTLV-1-load was 47.665/106 copies. Treatment with tenofovir 245 mg/24h and raltegravir 300 mg/12h was started and continued at discharge (day + 23) with undetectable HTLV-1-load. As post-transplant complications: grade II gastrointestinal GVHD treated with steroids, CMV reactivation treated with gancyclovir and grade 2 hemorrhagic cystitis. HTLV-1-load monitoring was performed during follow-up, ending antiviral treatment on day +42. On day +145 the patient required admission for abdominal pain and confirmed relapse, with a HTLV-1-load of 4.935/106 copies. She received GEMOX scheme and restart of raltegravir + tenofovir. Patient returned to her country. No additional follow-up was possible. Conclusions: While most HTLV-I-infected individuals remain asymptomatic, 1–5% will develop ATLL. The experience in european countries is limited and the role of HTLV-1 during treatment and HSCT is not clear. Further studies are needed to assess the role of antiviral treatment, its complications, and their impact on maintaining remission of hematological neoplasia. Disclosure: Nothing to declare.
Background: BK polyomavirus (BKPyV) is a common cause of hemorrhagic cystitis during the early post engraftment phase. It occurs most commonly at 21 to 42 days following HCT. The clinical manifestations of BKPyV-associated hemorrhagic cystitis after HCT may include cystitis, hematuria, renal failure, and rarely, life-threatening bleeding. Methods: We report in this case series the treatment approach in five of our patients who developed symptomatic infection as listed below Our treatment approach was to initiate aggressive hydration, decrease immunosuppression, and start cidofovir, Cipro and IVIG. Cidofovir has a modest in vitro antiviral activity against BKPyV, and has been widely used to treat patients with BKPyV hemorrhagic cystitis. On the other hand, immunoglobulin preparations contain BKPyV neutralizing antibodies against all major genotypes of BKPyV. Our patients had low immunoglobulin level, and for that reason adding IVIG to their treatment regimen was considered. All five patients received the first treatment with cidofovir (5 mg/kg/dose), Our treatment approach was to initiate aggressive hydration, decrease immunosuppressive medications, and start treatment with combination high dose cidofovir, and IVIG. Cidofovir has a modest in vitro antiviral activity against BKPyV, and has been widely used to treat patients with BKPyV hemorrhagic cystitis, even though it is not FDA approved for that indication. On the other hand, immunoglobulin preparations contain BKPyV neutralizing antibodies against all major genotypes of BKPyV. All five patients received the first treatment with cidofovir (5 mg/kg/dose), cipro and IVIG, immediately after their symptoms started, and the diagnosis was confirmed. Results: All five patients noticed significant improvement in their symptoms after the first treatment. Patients 1, 2 and 4, received three treatments with weekly cidofovir, over three weeks, while patient 3 and 5 received 2 treatments only, and the third treatment was held due to complete resolution of the symptoms. As shown in the table above, we can see how the BKPyV level was trending down as the patients were receiving their treatment; this also coincided with the improvement in their macroscopic hematuria, the amount of blood clots in the urine, urinary frequency and bladder spasms that they presented with. Conclusions: BKPyV hemorrhagic cystitis is a significant cause of morbidity, occurring in up to 20 % of stem cell transplant recipients. At the present time treating these patients centers around hydration, reduce immune suppression, and sometimes using cidofovir, which is still not FDA approved for this indication. In this case series we demonstrated that starting our five patients on combination treatment with cidofovir, Cipro, and IVIG, helped improve their symptoms and reduce their BKPyV level, just after the first treatment, with complete resolution of their symptoms in 2-3 weeks. Disclosure: nothing to declare
Background: The different clinical presentations of SARS-CoV-2 infection reflects the diversity of immune responses, especially in immunocompromised patients. Immunosuppression in Multiple Myeloma (MM) raises the question of whether these patients are at a higher risk of developing severe COVID-19, in whom uncontrolled MM was associated with an increased risk of death. We report a case of a MM patient diagnosed with SARS-CoV-2 infection a day before autologous stem cell transplant (ASCT) infusion. Methods: Conditioning regimen used was Melphalan 200mg/m2, in an isolation room with HEPA filters and positive airflow. All patients performed a SARS-CoV-2 PCR screening, with a negative test required for admission, which was repeated at 48h and after, every five days. Results: The patient was a 57-year-old woman with a personal history of obesity and chronic obstructive pulmonary disease. She was diagnosed with light chain MM, International Staging System (ISS) stage I and amplification 1q, with a complete remission after induction with four cycles of Bortezomib, Thalidomide and Dexamethasone. At admission, her SARS-CoV-2 PCR screening test was negative but, on day -1, the screening result was positive. As expected, the patient proceeded with the transplant with a known COVID-19 infection. She did not develop symptoms suggestive of SARS-CoV-2 infection. On day +6 she was started on Meropenem and Vancomycin for a febrile neutropenia with hypotension, of which she completed a course of 7 and 5 days, respectively. At this time, pulmonary tomography scan showed no evidence of respiratory infection. Grafting was recorded at day +15. On day 21 of COVID-19 infection (day + 20), an antigen test for SARSCoV-2 was performed with a negative result and the patient was discharged, maintaining a complete remission at day +100. Conclusions: SARS-CoV-2 infection progressing to acute respiratory stress syndrome in patients with MM has a mortality rate of about 55%. Previous studies have identified age, stage III-ISS, high risk cytogenetic disease, kidney disease, and active or progressive MM as risk factors for higher death rates. Immunosuppressive treatment was not associated with an increased risk of death. Patients with MM undergoing ASCT have severe acute immunosuppression and long-term reconstitution of the immune system. Studies in this matter recognize that in patients who did ASCT and COVID-19 infection within 12 months, the expected and combined adverse outcome of these factors was not significantly high. Although is not consensual, there are no data to support prevention and expectant attitude in any specific treatment for MM, whether steroids or high-dose chemotherapy, as in cases like the reported. In patients with high-risk MM features, even taking account for the immunosuppression, treatment should be continued. This case report exposes the need and difficult management of the MM in the COVID-19 era, as well as reinforces the importance of SARS-CoV-2 monitoring in inpatient care. MM requires careful consideration relatively to the disease and the therapy to be discussed, in order to reduce patient risk of having COVID-19, without compromising disease outcome. Controlled MM is associated with a better outcome, even with SARS-CoV-2 infection. Disclosure: Nothing to declare
Background: Coronavirus disease 2019 (COVID-19) is a serious viral infection associated with high mortality in patients with hematological diseases. Patients after haematopoietic stem cell transplantation are at higher risk of Covid-19 disease due to their immunocompromised status. There is still insufficient information concerning this cohort of patients. Our study presents first single data from Bulgarian BMT unit (859) in patients in whom COVID-19 was confirmed. Methods: This study is the first analysis of patients with haematological malignancies after SCT, with COVID-19 infection in the period of 03.2020 till 11.2021 at BMT Unit 859 in Sofia Bulgaria (SHATHD). Results: The study included 45 patients; aged 23 -69 years (median, 47.6 years); men/women 26 /19 ; who had SCT (autoSCT - n = 28 ; AlloSCT - n = 17), with diagnosis as follows: MM- 21; MX-2; NHL-6; AA – 2; AML-8; ALL- 4; MDS-2. At the time of COVID-19 infection 36 (80%) of patients were in CR/VGPR, 9 (20 %) in progression/relapse, 6 (13.3%) patients were on immusupression therapy. In most patients, the infection was relatively mild, in outpatient settings; 9 (20%) had severe course of infection with bronchopneumonia, and 3 (6.6%) patients required intubation. Over the study period a total of 6 (13.3 %) patients died. The average age of this patients is 56y.old (median 51-67), 3 of them were diagnosed with Covid 19 shortly after the ASCT (2 at +1 months; +3 months respectively). Conclusions: Despite the small group of observed patients, these results confirm the prognostic significance of age for severity and mortality of COVID-19 in hematological patients. The infection is more severe, with higher mortality rates in elderly patients, especially soon after ASCT. Disclosure: Nothing to declare.
Background: Autologous hematopoeitic stem cell transplantation (Auto-HSCT) is part of the first-line treatment of fit patients with Multiple Myeloma (MM). Bloodstream infections (BSI) occur in 5–10% of auto-HSCT. The incidence rates of Non-tuberculous mycobacteria (NTM) as Mycobacterium abscessus among recipients of HSCT ranging between 0.4 and 10%, being reported more frequently in recipients of allografts than in autograft. Methods: We report a case of a 42 years-old male with an Auto- HSCT by Bences-Jones Kappa MM in complete response who developed a catheter-related bloodstream infection (CRBSI) by Mycobacterium abscessus. Results: At day +47 after Auto-HSCT, our patient was accepted at the emergency department whit fever, headache and myalgias. Physical examination was anodyne, the blood test and radiology thorax image showed no findings and he was discharged home with empiric antibiotic treatment with levofloxacin. Five days later, peripheral blood and catheter blood cultures were positive for fast-growing mycobacteria (FGM), so the patient was admitted to the hematology unit for clinical evaluation and directed treatment. Upon reassessment, he had blood pressure 129/79 mmHg, 101 beats per minute, 97% saturation breathing ambient air. The patient reported having been asymptomatic at home, but at the time of the assessment he had a febrile episode with associated shivering, so blood cultures were extracted again. Empiric antibiotic treatment with clarithromycin, ciprofloxacin and amikacin was started. Mycobacterium abscessus was identified, and central venous catheter was removed and cultivated, subsequently confirming colonized by the same microorganism. Clarithromycin was suspended because of a severe hepatitis consisting of cholestasis and hypertransaminasemia. Finally, after 18 days of admission, our patient became afebrile, with an improvement of the liver profile and negative blood cultures, so he was able to continue his triple antibiotic therapy at home until completing a total of 4 weeks. Conclusions: Fast growing NTM species such as M. abscesuss are a rare cause of CRBSI in patients with Auto- HSCT and usually affect lungs and skin. In our patient, despite suffering from MM and being recently transplanted, M. Abscessus infection only represented a febrile syndrome without other complications, which allowed continuing treatment on an outpatient basis with an excellent impact on his quality of life. Disclosure: Nothing to declare
Background: Hemophagocytic Lymphohistiocytosis (HLH) occurring after allogeneic hematopoietic stem cell transplantation (HSCT) is a rare and serious disease. Differential diagnosis may be other complications post-HSCT such as acute graft-vs-host disease (GVHD), thrombotic microangiopathy, engraftment syndrome, graft failure and particularly infection, which induced symptoms and parameters overlapping with the diagnosis criteria for HLH. In the absence of clear guidelines, the management of HLH post-HSCT is challenging (Sandler R. D. et al, Bone Marrow Transplantation 2020; 55: 307-316) We present a case of HLH post-allogeneic HSCT illustrating this critical issue. Methods: Case Report. Results: A 50-year-old female underwent an allogeneic HSCT from her HLA identical brother for a chronic myelomonocytic leukemia. Conditioning consisted in Flu-Bu-ATG and GVHD prophylaxis in cyclosporin. The patient (pt) had persistent pancytopenia after 3 weeks of allogeneic HSCT without symptoms of infection while being on large spectrum antibiotics, anti-fungal and prophylaxis with valacyclovir. On day 23 of allogeneic HSCT, she manifested high grade fever followed by abdominal pain and transfusion resistance for severe anemia and thrombocytopenia. Bone marrow aspirate showed on cytology an increase of activated macrophages with hemophagocytosis. The level of ferritin in serum was high at 2529 ng/mL (reference range: 13-150) as well as LDH at 591U/L (reference range: 135-225). Fibrinogen level was low at 151 mg/dL (normal range: 200-400). CT scanner of the abdomen showed hepatomegaly (23cm) and splenomegaly (20cm). Neither veno-occlusive disease nor thrombotic microangiopathy was documented. The diagnosis of HLH was made and the patient was treated with methylprednisolone pulse followed by one dose of etoposide (100mg/m2) administered on day 25 of allogeneic HSCT. Starting day 28, the pt recieved treatment with ganciclovir after PCR for cytomegalovirus (CMV) came positive (3270 copies/mL). Epstein-Bar virus, human herpes virus-6, parvovirus B19 and adenovirus B11 were not detected. Then, the pt started gradual improvement with progressive normalization of the abnormal parameters simultaneously to hematological recovery and non-detection of CMV in blood by PCR. She was discharged on day 82 of allogeneic HSCT. Conclusions: Our case, with the diagnosis made of HLH post-allogeneic HSCT possibly due to CMV, illustrates the importance of being aware of the clinical manifestations of HLH post-allogeneic HSCT, which is often under-recognized. Our case highlights that the cause of HLH is not always easy to establish. It is also important to treat HLH post-allogeneic HSCT early with an individualized approach for the most optimal outcomes. Disclosure: Nothing to declare
Background: Patients with relapsed/refractory SCNSL have generally poor prognosis with limited treatment options. Autologous stem cell transplant (SCT) is associated with improved survival in patients who achieve remission prior to transplant and/or those who received ≤2 lines of therapy. Refractory and heavily pretreated patients have dismal prognosis. Limited data exist for the role of allogeneic SCT (allo-SCT) in these high-risk patients. We sought from this report to assess the outcomes of SCNSL patients who underwent allo-SCT at our center and identify predictor factors for prognosis. Methods: We included all consecutive adult patients with SCNSL who underwent allo-SCT between July 1993 and November 2018. Excluded patients who received mismatched or cord blood grafts. The primary objectives were to assess progression-free survival (PFS) and overall survival (OS). Secondary objectives included assessment of cumulative incidence of relapse (CIR), non-relapse mortality (NRM), and graft-versus-host disease (GVHD). Results: Sixty-one patients with a median age of 49 (range, 18-69) years, with male predominance (62%), were identified. Overall, patients were heavily pretreated with a median of 3 prior lines of therapy (range, 1-13), 11 (18%) had prior autologous SCT, and 31 (52%) had active disease at time of transplant. Twenty-seven patients had large B-cell lymphoma, 23 had indolent B-cell lymphoma, and 11 had T-cell lymphomas. Thirty (49%) patients had matched related donor (MRD) and 36 (59%) received myeloablative conditioning (MAC). Forty-two percent had HCT-CI > 3. Eighteen (30%) patients transplanted in time period 1993-220, 24 (39%) period 2001-2010, and 19 (31%) in the 2011-2019 period. With a median follow up of 7.09 (range, 0.84-16.72) years, the 2 and 5-year PFS/OS rates were 21%/30% and 19%/23%, respectively. The 2 and 5-year NRM/relapse rates were 31%/48% and 33%/48%, respectively. In MVA for PFS, MAC was the only factor associated with inferior outcome (HR 2.059, 95%CI 1.055-4.016; p = 0.0342). In MVA for OS, age ≥50 years (HR 2.071, 95%CI 1.028-4.176; p = 0.0418), MAC (HR 3.609, 95%CI 1.659-7.852; p = 0.0012), and receiving MUD graft (HR 4.566, 95%CI 1.78-11.712; p = 0.0016) were associated with inferior outcome. OS improved over time (reference, transplant year 1993-2000) with HR of 0.302 (95% CI: 0.128-0.712; p = 0.0062) and 0.413 (95% CI: 0.153-1.112; p = 0.0802) in transplant years 2001-2010 and 2011-2019, respectively. In subgroup analyses to explore predictive factors for prognosis, patients <50 year who received reduced intensity conditioning (RIC) had better OS (67% at 5 years). Likewise, recipients of MRD with RIC conditioning had 2 and 5-year OS of 50% and 38%, respectively, compared to 7% and 0% survival for MUD recipients with MAC (p = 0.0053). The CI of grades 3-4 aGVHD was 14 % at 3 months. CI rates of cGVHD at 1 and 3 years were 15 and 21%, respectively. Conclusions: In this heavily pretreated patient population of SCNSL, allogeneic SCT was associated with durable remissions and potential cure, particularly for younger patients, recipients of matched related donors, and those who received reduced intensity conditioning. Disclosure: Nothing to declare.
Background: Mycosis fungoides (MF) and Sézary syndrome (SS) are the most common subtypes of cutaneous T-cell lymphoma (CTCL) and those with progressive/transformed MF or SS have a median survival of <5 years with conventional treatment. Allogeneic stem cell transplantation (allo-SCT) can prolong overall survival and cure a subset of patients with CTCL but optimal timing, pre-SCT therapy and conditioning therapy remain unclear. Methods: Consecutive patients undergoing allo-SCT for CTCL from January 2010 to December 2020 at a national transplant centre were included in this retrospective analysis. Pre-transplant demographics, pre-SCT therapy, standard SCT data and post-SCT survival, relapse, toxicity and chimerism was collected. Pathology review including CD30 expression and T-cell receptor gene clonality was performed. Statistical analyses were carried out using GraphPad Prism version 9.0.1. Results: Fifteen patients (10 male, 5 female) with a median age at transplant of 49 years were included. Eight patients had SS, while 7 patients had MF, including 4 with transformed MF (tMF) who expressed CD30. The median number of pre-transplant therapies, excluding skin directed treatments and bexarotene, was 2.5. All 8 SS patients achieved a partial response (PR) pre-transplant, while MF patients were in either PR (n = 1), stable disease (n = 3), or progressive disease (n = 3). Median time from CTCL diagnosis to transplant was 12 months. Conditioning used included Cy-TBI (n = 5), Total skin electron beam therapy (TSEBT) based reduced intensity conditioning (RIC) (n = 4) and Flu/ATG/Mel RIC (n = 6). Acute GVHD grade ≥2 (with skin involvement) occurred in 13 patients. Five patients (2 SS, 3 MF) developed chronic skin GVHD (3 extensive, 2 limited stage). There were two early treatment related mortalities (<100 days), and one late (>100 days) treatment related mortality due to infection. Five patients relapsed at a median of 5 months which were managed by combining debulking strategies such as radiotherapy, gemcitabine, brentuximab vedotin with immune modulation. Donor lymphocyte infusion was used in 5 patients (Table 1). One relapsed MF patient died 5 months post allo-SCT from progressive disease, but 4 patients remain alive at a median follow up of 40 months. With a median follow-up of 32 months, overall survival (OS) and progresson-free survival (PFS) at 5 years were 72.7% and 41.0% respectively. Conclusions: We report an improved OS and PFS at 5 years of 72.7% and 41.0% respectively compared to registry data. This may reflect a shorter time from CTCL diagnosis to allo-SCT; use of TSEBT-based conditioning; and higher rates of acute GVHD skin possibly associated with a lower relapse rate. Our data supports early referral to a transplant unit of CTCL patients <70 years old who are failing skin directed therapy. Disclosure: Nothing to declare
Background: The curative treatment for primary central nervous system lymphoma (PCNSL) includes induction consisting of high-dose methotrexate (HD-MTX), rituximab (Rtx), cytarabine (AraC) and thiotepa (TT) followed by consolidation by autologous stem cell transplant (ASCT). Here we report our 12-year experience of patients who have been treated with HD-MTX, AraC, TT, Rtx containing induction protocol and consolidation with ASCT. Methods: We retrospectively reviewed the medical records of all newly diagnosed PCNSL patients who underwent ASCT as consolidation in Vilnius University Hospital Santaros Klinikos over the period of 2009-2021. These patients received a sequential induction therapy consisting of 4 cycles of Rtx (375 mg/m2) and HD-MTX (3,5 - 8 g/m2) every 10 days followed by 2 cycles of high-dose AraC (2-3 g/m2 BID), TT (40 mg/m2) and Rtx (375 mg/m2) every 21 days. MRI was performed after 4 R-HD-MTX cycles and then after 2 R-HD-AraC-TT cycles. Stem cell mobilization and collection were done after first R-HD-AraC-TT cycle. Patients who reached complete remission (CR), unconfirmed complete remission (CRu) or partial response (PR) (based on International PCNSL Collaborative Consensus Guidelines for the Assessment of Response in PCNSL) proceeded to ASCT. The ASCT conditioning regimen consisted of Rtx on day -7, carmustine 400 mg/m2 on day -6 and TT 5 mg/kg on days -5 and -4. Endpoints included progression-free and overall survival (PFS, OS) and grade 3-5 non-hematological toxicity (CTCAE v. 5.0). Results: 57 patients were enrolled. The median age at diagnosis was 55 years (range 36 - 75). 35 % of the patients were older than 60 years. DLBCL was the most common histologic type comprising 96%. 42% of patients had poor performance status (ECOG ≥ 2) and 59% were classified into intermediate or high-risk groups by IELSSG score. The median time to transplantation was 4 months from diagnosis. The median number of hematopoietic stem cells reinfused was 5.5 x 106/kg . All patients achieved prompt hematopoietic recovery with a median duration of hospitalization of 26 days. The most common complications were infectious (26 patients with febrile neutropenia, 9 patients developing bacteremia, 3 – pneumonia, 2 – urinary tract infection). 24 patients had grade 3-4 mucositis and 2 patients had pseudomembranous colitis. There was one treatment-related death caused by sepsis and fungal pneumonia. 65 % (37/57) patients achieved CR or Cru prior to transplant and 35% (20/57) achieved PR. At day +100 post-transplant, the CR rate increased to 93%. After a median follow-up of 64.5 months, 40 of 57 (70%) patients are still alive. The median OS and PFS was not reached at the last follow-up. The 2-year and 5-year PFS were 85% and 71% and the 2- and 5-year OS were 85% and 72%, respectively. Responding patients had similar outcome (Figure 1). Conclusions: Our results with a long follow-up period show that sequential HD-MTX, AraC, TT and Rtx containing induction chemotherapy followed by consolidation with ASCT is safe and leads to high survival rates in transplant eligible patients with newly diagnosed PCNSL. Disclosure: Cernauskiene: Registration fees: Abbvie, Takeda. Ringeleviciute: Registration fees: Abbvie, Roche; Travel expenses: Abbvie. Zucenka: Jannsen: Honoraria, travel-expenses; Takeda: Travel Expenses; Novartis: Honoraria, travel Expenses; Pfizer: Honoraria, Travel Expenses; Astellas: Honoraria; Abbvie: Honoraria, travel Expenses. Peceliunas: nothing to declare. Griskevicius: nothing to declare.
Background: High-dose therapy followed by autologous hematopoietic stem cell transplantation (HSCT) has been implemented as standard first line treatment in younger/fit patients with mantle cell lymphoma (MCL). In recent years R-CHOP has been the preferred regimen for induction therapy and some studies suggest a positive effect of high dose Cytarabine (HDAC) as part of induction therapy as well as Rituximab (R) maintenance. The introduction of Bruton-Tyrosine-Kinase inhibitors currently challenges the high-dose concept in MCL. Methods: We retrospectively analyzed clinical outcome of 47 patients (38 male, 9 female, median age 57 years, range 34-75) with MCL receiving autologous HSCT (autoHSCT) at the UKD, Heinrich Heine University of Düsseldorf between 1993 and 2019. Forty-six of 47 patients presented with advanced stage (III/IV) at diagnosis. Thirty-five patients (74%) received HSCT as first line therapy, 30 (86%) of these following induction with sequential R-CHOP and HDAC (28 Cytarabine alone or with Mitoxantrone but without Platinum, 2 with Platinum). Twelve patients (26%) received HSCT as salvage therapy. In these median time to first relapse/progression from diagnosis was 13 months (5-77). BEAM/TEAM + -R was used for conditioning in 39 patients (83%) and 22 (47%) received R maintenance therapy. Results: Median follow up after HSCT was 4 years for surviving patients. Thirty-one patients (66%) were alive at last follow up. Median event free survival (EFS) was 55 months and overall survival (OS) was 132 months. Twenty-three patients (51%) relapsed at a median of 67 months (2-113). After relapse eight patients received allogeneic transplantation, 10 patients chemoimmunetherapy and four patients were treated with Ibrutinib. Median OS after relapse was 44 months. (0-85). Nine patients died of lymphoma, three died of secondary neoplasms and three died of therapy associated complications after allogeneic transplantation. In univariate analysis EFS and OS were superior when autoHSCT was performed as first line versus salvage therapy (median EFS 88 vs. 7 months, p < 0,01, median OS 132 vs. 29 months, p < 0.01). In the salvage group EFS was significantly longer when pre-transplant relapse occurred later than 24 months after diagnosis (POD24, median 37 vs. 4 months, p < 0.03). Thirteen patients (37%), who received first line autologous HSCT relapsed at a median of 88 months (2-113)) after HSCT and median OS after relapse was 54 months (0-85). Median EFS for Patients receiving R-CHOP and HDAC without use of Platinum was not reached at the end of follow up with 5 year EFS of 59%. EFS after autoHSCT was shorter in patients not receiving Rituximab maintenance (median 18 vs. 89 months, p < 0.04). Conclusions: In younger patients with MCL, response to R-CHOP and R-HDAC without Platinum allows long term EFS and OS after autoHSCT sparing the toxic side effects of Platinum therapy. In addition data suggests a positive effect of Rituximab maintenance therapy on EFS after autoHSCT. Following relapse after first line autoHSCT long term survival may be achieved in individual patients following treatment with Ibrutinib and allogeneic HSCT. Disclosure: Ben-Niklas Baermann: Nothing to declare Paul Jäger: Nothing to declare Ju Hee Chae: Nothing to declare Thomas Ulrych: Nothing to declare Kathrin Nachtkamp: Nothing to declare Roland Fenk: Nothing to declare Ulrich Germing: Institutional Research Support Celgene, Novartis, Speaker honorarium Celgene, Novartis, Jazz, Advice: Celgene Guido Kobbe: received honoraria for an advisory role from Abbvie and Gilead
Background: The B-CD30 + HOdgkin Lymphoma International Multi-Center Retrospective Study of Treatment PractIces and OutComes (B-HOLISTIC) assessed real-world treatment patterns and outcomes in patients with Hodgkin lymphoma (HL) from Latin America, East Asia, Africa and the Middle East, Russia, and Australia. A subgroup analysis of patients with relapsed/refractory HL (RRHL) receiving stem cell transplantation (SCT) is presented here. Methods: The primary B-HOLISTIC study involved a retrospective chart review of newly diagnosed adult patients with previously untreated Stage IIB–IV classical HL (cHL) or RRHL from January 2010–December 2013. This subgroup analysis assessed treatment patterns and clinical outcomes in patients with RRHL who received SCT. Results: Of the 426 patients with RRHL, 302 were SCT-eligible and 222 (73.5%) underwent SCT (52.1% in overall RRHL group). The main reasons for not undergoing SCT were patient refusal (26.3%) and pre-SCT lymphoma progression (16.3%). Autologous SCT (ASCT) was performed in 188 (84.7%) patients, allogeneic SCT (allo-SCT) in 10 (4.5%) patients, and 24 (10.8%) patients received both ASCT and allo-SCT. The median (range) age at RRHL diagnosis was 29.0 (18.0–67.0), 28.5 (19.0–46.0), and 27.5 (19.0–45.0) years in patients receiving ASCT, allo-SCT, and both, respectively. Most patients who underwent SCT had Stage IVB disease (19.0%), and only 49.4% and 39.1% of patients achieved a complete and partial response, respectively, with chemotherapy prior to SCT. Prior to SCT, the regimen consisting of etoposide/methylprednisolone/cytarabine/cisplatin (ESHAP) was the most common salvage chemotherapy (26.3%) in patients with RRHL. The combination of carmustine/etoposide/cytarabine/melphalan (BEAM) was received as pre-SCT conditioning regimen in 61.1% patients undergoing ASCT. Only 4 transplanted patients received consolidation therapy with brentuximab vedotin (BV). A total of 63 (28.4%) patients relapsed post-SCT, and the most frequent salvage regimen in these patients was again ESHAP (45.9%). The most common third-line regimens post-SCT relapse were BV monotherapy (16.4%) and the combination of ifosfamide/gemcitabine/vinorelbine/prednisone (IGEV) (16.4%), and 24 (38.1%) patients received a subsequent SCT. The median PFS was 20.6 (95% CI: 13.2–31.1) months, and the 1-, 3-, and 5-year PFS rates post-SCT from initiation of first treatment for RRHL were 58.4%, 42.4%, and 38.2%, respectively. The 1-, 3-, and 5-year overall survival rates from cHL diagnosis were 100.0%, 88.1%, and 80.4%, respectively. Conclusions: The SCT rate and clinical outcomes with SCT reported in this study were similar to previous reports from Europe and North America. The treatment patterns pre- and post-SCT, and post-SCT relapse, align with standard clinical practice and guideline recommendations at the time of the study. However, the clinical outcomes remained suboptimal. The most important parameter predicting SCT outcomes is the pre-SCT complete metabolic response rate, based on positron emission tomography/computed tomography imaging, which was achieved in only 49% patients with RRHL in this study. Novel agents were mostly used in the post-SCT and third-line settings when patients had poor prognosis or significantly inferior outcomes with SCT or conventional treatments. Introducing novel targeted therapies (alone or in combination) earlier in the treatment continuum, may increase responses pre-SCT, which, in turn, can lead to better long-term outcomes in patients with RRHL. Clinical Trial Registry: NCT03327571 Disclosure: This study was funded by Takeda Pharmaceuticals International AG – Singapore Branch. Mubarak Al-mansour serves as member of a board of directors or advisory committee for Takeda. Burhan Ferhanoglu serves as a member of a board of directors or advisory committee of Takeda, Roche, AbbVie, and Janssen. Marta Zerga has received conference fees from Bristol Myers Squibb, Janssen, Roche, and Takeda, has received support for attending meetings from Sandoz, Amgen, AbbVie, Teva, and Pint Pharma, has received honoraria from Takeda, AbbVie, Astra Zeneca, Teva, and Sandoz, and has participated on the advisory board for Takeda, Janssen, and Merck. David Brittain has received honoraria from Takeda, Roche, AbbVie, and Janssen, and serves as members of a board of directors or advisory committee for Takeda. Su-Peng Yeh has received honoraria from AbbVie, Amgen, Janssen, Astellas, Astra Zeneca, Novartis, Sanofi, Roche, Bristol Myers Squibb, and Takeda, and has served in an advisory role for AbbVie, Amgen, Janssen, Astellas, Astex, Novartis, Sanofi, and Takeda. Gayane Tumyan serves as member of a board of directors or advisory committee for Takeda. Yuqin Song has received conference fees from Takeda and has participated on the advisory boards for Roche, Takeda, and Janssen. Amado Karduss has received honoraria from Takeda, Amgen, and Janssen, and serves as a member of a board of directors or advisory committee of Novartis. Silvia Rivas-Vera has received honoraria from Roche and serves on the advisory board for Takeda. Mark Hertzberg has received honoraria, consulting fees, and serves as a member of a board of directors or advisory committee of Takeda, Roche, BMS, Gilead, and MSD. Lim Soon Thye’s institution has received honoraria from Takeda. Yok Lam Kwong serves as a consultant and has received honoraria or research funding from Amgen, Astellas, Bayer, BeiGene, Bristol Myers Squibb, Celgene, Gilead, Janssen, Merck, Novartis, Roche, and Takeda. Zhongwen Huang and Kwang-Wei Wu are employees of Takeda Pharmaceuticals and hold shares in the company. Tae Min Kim has consulting and advisory roles for AstraZeneca, Boryung, Hanmi, Janssen, Novartis, Takeda, Sanofi, and Roche/Genentech, and receives a grant from AstraZeneca-KHIDI and has received consulting fees from AstraZeneca, Hanmi, Janssen, Novartis, and Takeda outside this work.
Background: Despite the high cure rate with initial therapy, approximately 10% of HL patients are refractory to initial treatment, and up to 30% of patients will relapse after achieving the initial complete remission (CR). Monoclonal antibodies, targeting the programmed cell death 1 (PD-1) receptor showed promising results with high response rates in relapsed/refractory HL (rrHL). Most responders, however, would eventually progress. For those patients, allogeneic hematopoietic stem cell transplantation (alloHSCT) remains so far the last potentially curative option. Its use in rrHL patients has been argued due to the high rates of NRM although the introduction of reduced intensity conditioning regimens has undoubtedly contributed to the better outcomes. Here we report the Swiss experience in alloHSCT for HL from 2001 to 2020. Methods: This retrospective analysis included 62 adult patients with HL, who received the allo-HSCT in one of three University Hospitals of Switzerland (Zurich, Basel and Geneva) between May 2001 and January 2020. The primary endpoint was OS (overall survival). Secondary endpoints were relapse free survival (RFS), non-relapse mortality (NRM), relapse incidence (RI), acute (aGVHD) and chronic (cGVHD) rates, which were assessed in univariate analysis. Results: Median follow-up was 61 months (IQR 59-139). The median age of patients at the time of allo-HSCT was 28 years (24-33), there were more male patients (74%). Prior autologous HSCT was performed in 50 (82%) and 9 (15%) patients have previously undergone an allo-HSCT. Performance status with Karnofsky index ≥80 was reported in 42 (98%) patients. Only 19 (31%) of 62 patients presented CR at the time of allo-HSCT. Regarding conditioning regimen, 48 (77%) patients were treated with a reduced-intensity conditioning regimen (RIC) and 13 (21%) received total body irradiation (TBI). Stem cell source was peripheral blood for 54 (87%) of patients and 8 (13%) patients received bone marrow. Donor was an HLA-identical sibling or an HLA-matched unrelated donor for 29 (47%) and 22 (35%) patients, respectively, 3 (5%) patients had a mismatched unrelated donor and 8 (13%) had a partially matched related donor. Successful neutrophil engraftment occurred in 60 (98%) patients in a median of 15 days (13-17). 2- and 5-year OS was 54% (SE ± 12) and 50.2% (SE ± 13.3), respectively, and 2- and 5-years RFS was 40.7% (±16.3) and 34.4% (SE ± 19.0). NRM was 23.1 % (SE ± 2.2) and 27.4% (SE ± 2.5) at 2 and 5 years respectively. The cumulative incidence of relapse was 36.1% (SE ± 5.6) at 2 years and 38.2 % (SE ± 6.6) at 5 years. Conclusions: Our analysis of allo-HST outcome in the context of rrHL shows encouraging OS and RFS rates with mortality rate reaching plateau at 50% at 2 years after the allo-HSCT. This confirms that allo-HSCT still remains a potentially curative option for half of patients with rrHL. Relatively high NRM rates in this young population could be explained by the accumulated toxicities due to the multiple previous treatments. The better timing with earlier allo-HSCT application in the rrHL setting should perhaps be considered. Disclosure: Nothing to declare.
Background: Adult T-cell Leukemia/Lymphoma (ATLL) is a rare subtype of mature T-cell lymphoma with generally poor outcomes. Hematopoietic stem cell transplant is undertaken to ameliorate disease, but additional data are needed to support its optimal use. Much of the published literature on ATLL comes from Japan where HTLV-1, the retrovirus that causes ATLL, is endemic. Caribbean populations carry a significant portion of the global burden of ATLL and there may also be an underappreciated burden of ATLL in Latin America, Africa, and the Middle East as well. Our center in Bronx, New York provides care to a large population of Caribbean immigrants and we care for a significant proportion of patients with ATLL in the United States. Here, we describe hematopoietic stem cell transplant outcomes in North American ATLL. Methods: Patients were identified from an institutional database of patients with mature T-cell lymphomas. Data were collected by chart review. Subjects were excluded for receipt of investigational cellular therapies or if pathological diagnosis only became available after follow-up ended. Individuals who underwent autoHSCT with subsequent alloHSCT were analyzed with the alloHSCT group. Survival outcomes were assessed using the Kaplan-Meier method and Cox regression. Follow-up began at diagnosis and ended at death or censoring. Subjects were censored at date of last contact or the study end date 6/8/2021. Data were analyzed using Python and lifelines. Results: 117 individuals with ATLL were identified with median follow-up 7.3 months (2 days-23.9 years). Subjects were diagnosed with ATLL 6/30/1995-4/16/2021. Median age at diagnosis is 59.1 years (25.2-87.6). The cohort is 59% female, 66.1% African American, 3.5% Caucasian, and 0.9% Asian. 30.8% are Hispanic. 16/117 underwent alloHSCT, 5/117 underwent autoHSCT, and 96/117 did not have any HSCT (Table 1). Data on alloHSCT suggest superiority over no HSCT, though statistical measures were inconsistent (median OS 3.75 vs. 1.41 years, log rank p 0.0518, 2-year OS 74% [95% CI 45-89] vs. 30% [95% CI 1-72], χ2p = 0.0277, HR 0.50 [95% CI 0.24-1.03, likelihood ratio test p = 0.0430]). Outcomes with autoHSCT were similar to those with no HSCT. AlloHSCT outcomes were not statistically different from those of autoHSCT. Conclusions: Our data lend support to the use of alloHSCT to improve outcomes in ATLL. We hope that as we further characterize the cohort we will be able to optimize selection of ATLL patients for alloHSCT. Disclosure: Gritsman, Kira: iOnctura: Research Funding. •Shastri, Aditi: Onclive: Honoraria; GLC: Consultancy; Kymera Therapeutics: Research Funding; Guidepoint: Consultancy. •Verma, Amit: Acceleron: Consultancy; Novartis: Consultancy; Stelexis: Consultancy, Current equity holder in publicly-traded company; Eli Lilly: Research Funding; Curis: Research Funding; Medpacto: Research Funding; Incyte: Research Funding; GSK: Research Funding; BMS: Research Funding; Celgene: Consultancy; Stelexis: Current equity holder in publicly-traded company; Throws Exception: Current equity holder in publicly-traded company.
Background: Osteoporotic fracture has been shown to be more common in lymphoma patients compared with the general population. Studies have shown that bone mineral density (BMD) decreases after hematopoietic stem cell transplantation, however, few have focused on change in BMD in lymphoma patients after autologous stem cell transplantation (ASCT). We intended to fill this knowledge-gap. Methods: Lymphoma patients in the Västra Götaland region in Sweden aged at least 18 years old, with a planned ASCT in 2015 – 2019 were eligible to participate. Participants did dual-energy X-ray absorptiometry (DXA) one month before, and six, 12 and 24 months after ASCT. DXA-measures were performed in the hip, femoral neck and lumbar spine, and differences in BMD, Z- and T-score were analyzed using paired T-tests. Results: 42 lymphoma patients were included, of whom 24 completed all four DXA-scans. The median age at ASCT was 55.5 years and 42% were women. The greatest change in BMD and Z-score was seen in the first six months and was more pronounced in the total hip and femoral neck compared to the lumbar spine. BMD in the lumbar spine recovered to baseline within the study period, while BMD in the total hip and femoral neck remained decreased. Conclusions: BMD, Z- and T-scores decreased in the first six months, after which they increased, after ASCT in patients with lymphoma. The restoration of BMD was faster in the spine compared with total hip and femoral neck. The spine recovered to baseline levels within the 24-month period while the total hip did not. Further research is needed, particularly to identify which patients might benefit from prophylactic osteoporosis treatment. Disclosure: Nothing to declare.
Background: In the era of novel biologic agents, the role of hematopoietic cell transplantation (HCT) in mantle cell lymphoma (MCL) remains under investigation. Therefore, we aimed to compare strategies and outcomes of MCL patients in the current period of biologic agents versus the early period. Methods: We retrospectively enrolled consecutive adult patients treated with MCL at our center over the last two decades (2000-2020). Patients were divided into two treatment periods: early (2000-2011) and late (2012-2020). According to each period’s department protocol, RCHOP/RCHEPOM was used as first-line treatment in the early period. Although we planned for autologous HCT as consolidation treatment in the early period, this was not feasible due to patient eligibility and poor mobilization. Therefore, intensified treatment with alternating cycles of RCHOP/RDHAP in fit-transplant eligible was given in the late period with autologous HCT as consolidation treatment. Unfit patients received R-Bendamustine, R-VCAP in first line along with ibrutinib in relapse, in the late period. In both periods rituximab consolidation was planned for 2 years. The following variables were analyzed: pre-transplant (age, gender, MIPI, performance status, blastoid variant, cytogenetics, stage, different lines of treatment), transplant (donor, graft, conditioning) and post-transplant (disease-free survival/DFS, overall survival/OS) characteristics. Results: We studied 81 MCL patients, 68 male:13 female, with a median age of 59.4 years (range 18-82). Median MIPI score at diagnosis was 6 (1-12), and performance status 1 (0-4). Patient and disease factors were similar in both treatment periods. Autologous HCT was performed more frequently in the late treatment period compared to the early (44% versus 12%, p = 0.008) due to patient eligibility and stem cell mobilization. Allogeneic HCT was performed at a similar rate in the two periods (7% versus 13%, p = 0.125). With a median follow-up of 40.7 months (4.3-269.3) in surviving patients, disease-free and overall survival (DFS and OS) were significantly higher in the late treatment period (p = 0.002 and p = 0.001, Figure 1). Among studied factors, age and performance status were also univariately associated with DFS and OS. It should be noted that autologous or allogeneic HCT per se was not significantly associated with improved survival. In the multivariate analysis, treatment period remained an independent predictive factor of both DFS and OS (p = 0.034 and p = 0.032), independently of age and performance status. Conclusions: Our study suggests that the integration of autologous HCT as consolidation treatment along with novel agents in the late treatment period was associated with improved survival. Thus, it highlights the need of further refinement of MCL treatment in the era of personalized medicine. Clinical Trial Registry: NA Disclosure: Nothing to declare
Background: Primary CNS lymphoma (PCNSL) induction therapy is based on systemic high dose methotrexate (HD-MTX) followed by consolidative therapy. Consolidation with high-dose chemotherapy and autologous stem cell transplant (HDC-ASCT) is used for younger patients. In this study we evaluate efficacy and tolerability of rituximab, methotrexate, ifosfamide, vincristine (R-MIV) followed by a course of cytarabine with thiotepa and HDC-ASCT. Methods: We evaluated the outcome of 60 immunocompetent adult patients with PCNSL treated at the Maria Sklodowska-Curie National Research Institute of Oncology between February 2015 and March 2021. Six cycles of induction chemotherapy with rituximab, methotrexate (3.5 g/m2), ifosfamide and vincristine (R-MIV/every two weeks) and one additional cycle of cytarabine with thiotepa were given. Patients with a complete or partial response (CR/PR) proceeded to consolidation with thiotepa 5 mg/kg/bid on day -5,-4; carmustine (BCNU) 400 mg/m2 on day -5; and etoposide 150 mg/m2 on day -5,-4,-3, followed by ASCT. Alternatively, whole brain radiotherapy (24-36 Gy) was given to patients not eligible for HDC-ASCT. Results: 39 (65%) patients were eligible for HDC-ASCT of whom 30 patients actually received HDC-ASCT. Two patients with CR are currently awaiting HDC-ASCT. Seven patients did not undergo HDC-ASCT due to progression (n = 2), patient’s refusal (n = 2) and toxicity related to induction treatment (n = 3; one patient died in PR due to SARS-CoV-2 infection). Median age (range) of 30 transplanted patients was 56 years (19-66). 17/7 patients were in CR/CRu following induction therapy and six in PR, based on CT/MRI assessment. For all 21 patients with PET-CT done before ASCT, metabolic-CR was confirmed (initially active changes were seen in 14 of 21 tested patients). Peripheral blood stem cell collection was performed after one of R-MIV cycles. Mean (range) number of 483.4 (187-1040.7) x 106 CD34 + cells were collected corresponding to 5.73 x 106 cells/kg (2.55-11.64x106 cells/kg). Mean hospitalization time from the day of stem cell reinfusion was 14 days. Mean time to hematologic recovery with PLT > 25 G/L and NEU > 0.5 G/L was 9 and 8 days, respectively. Neutropenia and thrombocytopenia grade 3-4 (CTCAE v5.0) were observed in all patients (mean 6 and 7 days, respectively), anemia grade ≥ 3 was observed in 7 patients (mean 1 day), elevated transaminases grade 3 (1 patients for 2 days). No renal toxcicity was observed. The most common grade 3-4 toxicities observed were: diarrhea (10 patients, mean 4 days), mucositis (17 patients, mean 5 days). Febrile neutropenia occured in 17 patients (mean 2.5 days). Blood cultures did not reveal any relevant pathogens except one patient with blood culture with confirmed Enterobacter cloacae-ESBL and Klebsiella oxytoca. Two patients (6.6%) died of transplant-related complications: septic shock and neurotoxicity. At a median (range) follow-up of 22 (2-69 months), 23 transplanted patients are alive in CR, 2-year progression free survival (PFS) and overall survival (OS) post-ASCT was 79% (95% CI: 66-92) and 79% (95%CI: 66-92). Relapse occurred in 4 patients - 2,5,6 and 42 months after ASCT. Conclusions: R-MIV induction therapy followed by HDC-ASCT is a relatively safe and highly effective treatment for PCNSL patients. Disclosure: NO DISCLOSURE
Background: Second-line salvage chemotherapy followed by high-dose chemotherapy (HDT) and autologous stem-cell transplantation (ASCT) is the current standard treatment for eligible patients with relapsed/refractory (R/R) Hodgkin lymphoma (HL). Several multiagent salvage chemotherapy regimens have been used to to provide cytoreduction and stem cell mobilization before HDCT. However the optimal salvage regimen is unclear. We report outcome of patients with R/R HL treated with salvage gemcitabine, cisplatin and dexamethasone (GDP) regimen before ASCT in this study aiming at evaluating efficacy, stem cell mobilization activity and safety of GDP. Methods: Fourty-five patients with R/R HL who were treated with GDP as salvage and mobilization regimen before ASCT in Medicana International Ankara Hospital between february 2013 and february 2021 were analysed retrospectively. GDP regimen was administered in an outpatient setting. Chemotherapy consisted of gemcitabine at a dose of 1000 mg/m2 intravenously (i.v.)on days 1 and 8, cisplatin at a dose of 75 mg/m2 i.v. and dexamethasone at a dose of 40 mg orally on days 1 to 4, of each 3-week course. PBSC were collected after first or second course of GDP. All patients underwent autologous stem cell transplantation after salvage GDP treatment and stem cell mobilization . Results: Median age of patients at study entry was 32 years (15-65 years). Thirty patients (66.7%) had relapse (7 early relapse, 23 late relapse) and 15 (33.3 %) had refractory disease. Fourteen patients (31.1 %) had advanced stage disease (stage III-IV) before salvage therapy. Thirty-six (80%) patients achieved overall response including 24 (53.3 %) CR and 12 (26.7 %) PR. Nine (20%) patients had no response. There was no association between risk factors at study entry and achievement of CR significantly (P:0.353). Peripheral stem cells were collected after the first or second cyle of GDP in 42 of 45 (93.3 %) patients. Peripheral blood stem cell collection were adequate in all patients with a median number of 11.18x106/kg CD34 + cells (range 2.6 to 36.6). No treatment-related deaths have been documented during therapy. The most common grade 3 or 4 hematological adverse event was thrombocytopenia (42.2%), followed by neutropenia (22.2%). Platelet and red blood celltransfusion supports were required in 11% and 11% of patients. There were no febrile neutropenic episodes that required hospitalization or treatment delay. Grade 3 or 4 renal, neurological, hepatic, or cardiac toxicity was not observed. With a median follow up time of 43 months (range 5–94 months), the 3 year PFS and OS for patients receiving 2cyles of GDP followed by ASCT were 72% and 92% respectively Conclusions: Our results suggest that GDP is a viable therapeutic option before ASCT with high response rate, favorable toxicity profile and excellent mobilization potential and may serve as a bridge for ASCT. Applicability of GDP on an outpatient setting also provides advantage over other effective salvage regimen. Disclosure: no disclosure
Background: Autologous peripheral blood stem transplantation (APBSCT) after PD-1 blockade results in favorable outcome among patients (pts) with multiply relapsed/refractory R/R Hodgkin’s lymphoma (HL). Anti-PD-1 monoclonal antibodies (mAbs) can sensitive pts to high dose chemotherapy (HDCT) followed by APBSCT. Response to PD-1 blockade, and not prior chemo-sensitivity, best predicted post – APBSCT outcome (Merryman R.W. et al, Blood adv (2021) 5 (6): 1648-59). Few studies were reported on the impact of anti-PD-1 mAbs immediately followed by APBSCT in multiply R/R HL. Methods: Between October 2017 and June 2021, 8 pts with multiply R/R HL received anti-PD-1 mAbs alone immediately followed by APBSCT. Anti-PD-1 mAbs consisting in Pembrolizumab in 5 pts and Nivolumab in 3 pts. All the pts were in complete remission (CR) before HDCT. HDCT was TEAM regimen (Thiotepa, Etoposide, Aracytine, Melphalan). All the pts were re-evaluated by PET-Scan done every 3 months after APBSCT. Results: The median age was 33 yo (24-38). There were 5 females and 3 males. The pts received a median of 4 systematic therapies (3-5) before APBSCT. Anti-PD-1 mAbs were administered as third line therapy in 2 pts, as fourth line in 4 pts, and as fifth line in 2 pts. The median number of CD34 + cells transfused was 3.95 106/kg (3-4.6). The median time to neutrophil recovery was 14 days (d) (10-24), and for platelets recovery 17d (8-32). All the pts developed febrile neutropenia. On neutrophil recovery, while being apyretic, 5 pts developed maculopapular skin rash of grade 1 in 1 patient (pt), grade 2 in 3 pts and grade 3 in 1 pt. Two pts (with grades 2 and 3 skin rash) developed concomitantly lung infiltrates. The pt with grade 1 skin rash recovered without specific therapy after 5 days; however, the other 4 pts received corticosteroids IV at the dose of 2 mg/kg/d which induced a total resolution of skin lesions and lung infiltrates within 5 to 8 days. Biopsy of skin lesions performed in 2 of these 4 pts showed pathological findings similar to those of acute cutaneous graft - vs - host disease. All these 5 pts are alive in CR at a median of 38 months (m) (18-50) after APBSCT. The 2 remaining pts relapsed at 13 and 17 m after APBSCT; one pt died of HL after 2m of relapse, and the other underwent haploidentical allogeneic bone marrow transplantation (BMT) and is still alive in CR at 5 m after allogeneic BMT, (at 22 m of APBSCT). Conclusions: Anti-PD1- mAbs immediately followed by APBSCT are efficient in multiply R/R HL pts, but further investigations are needed to better elucidate their immunological impact on APBSCT. Disclosure: Nothing to declare
Background: Despite having curative potential in relapsed Follicular Lymphoma (FL), allogenic hematopoietic cell transplantation (AlloHCT) remains underused and the ideal timing for its performance is still to be defined. Although transplant-related mortality (TRM) is not negligible, good overall survival (OS) and progression free survival (PFS) rates have been described. Methods: Characterization of patients (Pts) with FL who underwent HLA-matched AlloHCT at our institution between 2000 and 2020, response assessment and analysis of OS, PFS and TRM. Results: 43 Pts with relapsed FL were included, with male predominance (n = 24; 56%) and median age at diagnosis of 44 years old (yo) (range 28-58). 36 Pts (84%) had advanced stage disease, 20 Pts (47%) with bone marrow involvement and 7 (16,3%) with extra-nodal disease. The median time of follow-up was 134 months (range 2-229). First line treatment with chemotherapy alone or chemotherapy plus rituximab were used in 28 (65,1%) and 15 (34,9%) Pts, respectively. 27 Pts (62.8%) achieved complete responses (CR), 12 (27.9%) partial responses (PR), 1 (2.3%) stable disease (SD) and 3 (7%) progressive disease (PD). 4 Pts performed AutoHCT, with a median event-free survival (EFS) of 10 months (min 3, max 120). A median of 3 lines of treatments (range 1-6) were used before AlloHCT. The median time to AlloHCT was 34 months (range 11-177). At the time of AlloHCT, the median age of Pts was 49 yo (range 30-62), all with ECOG < 2. Forty grafts were obtained from HLA-matched related donor (FluBu conditioning) and three from HLA-matched unrelated donor (FluBuATG conditioning). Graf versus host disease prophylaxis were: Cyclosporine+ mycophenolate mofetil (related donor) and Tacrolimus+ mycophenolate mofetil (unrelated donor). The source of hematopoietic progenitors was peripheral blood in 42 Pts and bone marrow in 1 Pts. No graft failures were reported, with a median recovery of 15 days (range 6-72). Twenty Pts developed acute graft-versus-host disease (GvHDa), with isolated cutaneous involvement being the most frequent. 4 Pts had grade 3 GvHDa. 25 (58%) Pts had chronic GvHD. After AlloHCT, 37 Pts (86%) achieved CR, 2 (4.7%) PR, 1 (2.3%) SD and 1 (2.3%) PD. The response was not evaluated in 2 Pts. During follow-up 3 relapses and 11 deaths were reported. The median of PFS and OS at 5 and 10 years were not reached. The TRM was 4.7%. Estimated OS at 5 and 10 years was 90%. A trend towards reduction of OS was verified on comparative analysis in Pts submitted to more than 2 previous lines of treatment, without statistical significance (p = 0,143). Conclusions: Our study demonstrates the safety and efficacy of AlloHCT in young multi-treated Pts with relapsed FL, as the only curative option. Prospective and comparative studies are still needed to validate this strategy in the era of immunochemotherapy and targeted therapies. Disclosure: No conflicts of interest to declare
Background: Secondary hypogammaglobulinemia (SHG) in CLL is associated with both leukemia-related immunodeficiency and immunosuppression from CLL-specific therapy. Methods: Patients followed with CLL in Erciyes University Faculty of Medicine, Hematology Department between 2010-2021 (73 received treatment and 35 untreated) were divided into two groups. According to BCSH 2012 and ESMO 2015 criteria, patients with IgG<500 g/L and recurrent infections were given IVIG and named SHG. This group was compared with non-SHG. IgG at the time of diagnosis and last follow-up were noted in all patients. The relationship between IgG levels and SHG was examined. In addition, infections that developed in the patients during the follow-up period were reported and its relations with immunoglobulin replacement therapy (IgRT) were examined. Results: The number of infections in those who received IgRT treatment (n = 12) was statistically less than those who did not receive IgRT (n = 25) (p = 0.003) (table 1).In patients with SHG, the IgG level at the time of diagnosis (mean: 909) was lower than the group without SHG (mean: 1125) (p = 0.031). The pre-treatment IgG level was lower in SHG (median 750 versus 1000)(p = 0.001). Post-treatment IgG level (median: 452.5) in patients with SHG was lower than the group without SHG (median: 896) (p < 0.001). While 17 patients (94.4%) of 18 patients who developed SHG were receiving any chemo-chemoimmunotherapy, 56 (62%) of 90 patients who did not develop SHG were receiving treatment. This difference was found to be statistically significant (p = 0.006). SHG development was statistically higher in those treated with Chlorambucil plus Prednisolone, Fludarabine plus Cyclophosphamide plus Rituximab(FCR), Venetoclax and Ibrutinib (p < 0.05). Ibrutinib treatment was observed as a single independent variable in predicting the development of SHG in the multivariate logistic regression analysis (p = 0.002). Table-1. General features of the study population. Conclusions: One of the immunological defects in CLL disease is hypogammaglobulinemia (HG). Studies have reported that 10-44% of CLL develops HG at the time of diagnosis and 25-85% during treatment. Chemotherapy agents such as Anti-CD20 antibodies, BTK inhibitors, Phosphoinositide-3 kinase inhibitors, bcl-2 inhibitors, alkylating agents and purine analogs used in the treatment of CLL have been reported to cause SHG. In the study of Çelik S. et al.in CLL patients using ibrutinib, it was determined that IgG levels decreased during and after ibrutinib treatment. In our study, SHG development was statistically higher in those who received treatment. We also observed that ibrutinib was the only independent risk factor in predicting the development of SHG. Disclosure: Nothing to declare
Background: The Amicus Blue™ ECP System (Fresenius Kabi, Germany) was CE marked and commercially available in Europe since 2019. The system incorporates the Amicus Separator®, the Phelix photoactivation device, a functionally closed disposable kit, and 8-MOP to perform ECP therapy in an online, closed system. A post market clinical follow up study was performed at 3 sites in the EU to obtain data from at least 38 procedures over approximately 24 months. The primary objective of this study was to assess the safety of the Amicus ECP System during routine clinical ECP procedures in CTCL patients by analyzing adverse device effects (ADEs) that are unanticipated. The secondary objective was to assess system performance of the system in CTCL patients. Methods: A minimal sample size of 38 procedures was statistically established to demonstrate a 10% incidence rate of unforeseen ADEs with a confidence level of 90%. Patients were screened for entry into the study according to stated inclusion/exclusion criteria. Subjects received ECP treatment with Amicus Blue as defined by their individual treatment regimen for up to 24 months or according to physician discretion. Each patient could complete more than one procedure. Amicus v6.0, Phelix v2.0 and double-needle disposable kits were used. Procedure parameters were not defined in the study protocol as the intent was to assess system safety in routine use, however that is defined by each site. Most procedures targeted 2000ml WB processed (n = 36), while ca. 4000ml (n = 21) was targeted by 1 site. Sites documented all adverse events, and primary analysis was conducted on ADEs that are unanticipated. Procedure results such as whole blood processed, procedure time, photoactivation time, and percent photoactivation complete were recorded. All data collected were entered directly into the Electronic Data Capture system by authorized and trained personnel from the study sites. Results: Between September 2020 and May 2021, 17 adult patients (12 male, 5 female) enrolled in the study at 3 different sites in 3 different countries. Each site performed a minimum of 12 procedures. No patients were withdrawn from the study. 16 patients were under treatment for Sézary syndrome, and 1 patient for mycosis fungoides. Mean (SD) age of subjects was 71.6 (7.77) years. 57 procedures were completed, and 6 patients received >3 treatments on Amicus Blue. All procedures had photoactivation completed to 100%. One adverse event was recorded (itching) but was determined not to be device related. Zero ADEs were reported. Procedure data is presented in the table, median (range). Conclusions: All 57 ECP procedures were safely completed, and the primary objective of the study was satisfied. Only one non-device related adverse event was recorded. The Amicus Blue ECP System performed as expected in CTCL patients with evaluation of safety and performance. Disclosure: Tarik Kanouni: hospitality fees from Fresenius Kabi
Background: Mature B-cell lymphoma is a group of heterogeneous diseases, and multiparameter flow cytometry (MFC) plays an irreplaceable role in its diagnosis, especially in the identification of chronic lymphocytic leukemia (CLL) and other B cell lymphomas. However, immunophenotypic overlap is still encountered in clinical work. Besides, with the widespread application of chimeric antigen receptor-modified T cells (CAR-T) therapy, more markers with high coverage and expression level need to be found as promising targets for CD19 and CD20 may loss after target therapy. Therefore, we used MFC to simultaneously detect the classic markers CD5, CD23, CD200, CD79b, CD19, and CD20, and the new marker CD268, in an attempt to evaluate the role of these markers in distinguishing CLL from non-CLL small B lymphoma. Methods: From December 2018 to December 2020, 158 mature B-cell lymphoma patients, including 75 CLL patients and 83 non-CLL B lymphoma patients, were admitted to Hebei Yanda Lu Daopei Hospital. The expression of CD5, CD23, CD200, CD268, CD79b, CD19 and CD20 on malignant B cells was detected by 10-color flow cytometry. Results: CD5, CD23, and CD200 were higher in CLL patients (P = 0.000) and CD79b was lower in CLL patients (P = 0.000) compared with non-CLL B-cell lymphomas. CD268 was expressed in both CLL and most non-CLL B-cell lymphomas, but that in CLL was significantly higher (P = 0.001). The expression of CD19 and CD20 showed not significantly differences between the two groups. In addition to the classic differential diagnostic markers CD5, CD23, CD200, and CD79b, CD268 may be one of the new immune markers to differentiate CLL from non-CLL small B lymphoma. CD19 and CD20 lack specificity and cannot be used as immune markers specific to CLL. To further simplify the score, we tried to group patients with CD5 + /CD23 + /CD200 + expression over 80%, and found that the sensitivity, specificity, positive predictive value and negative predictive value of CLL were 80.0%, 98.8%, 98.36% and 84.54% respectively. The sensitivity, specificity, positive predictive value and negative predictive value of CLL were 73.3%, 100%, 100% and 0%, respectively, when CD79b expression was added (≤90%). The sensitivity, specificity, positive predictive value, and negative predictive value of CLL were 70.7%, 100%, 100%, and 0%, respectively, when the expression of CD268 was added (≥90% as the cut-off). Thus, these antibody combinations can be used as one of the combinations to distinguish CLL from non-CLL specific immune markers. Conclusions: The combination of immune markers CD5, CD23, CD200 and CD79b detected by multi-parameter flow cytometry can effectively distinguish CLL lymphoma from non-CLL lymphoma, and the new marker CD268 provides a new diagnostic basis for differential diagnosis. Moreover, it is highly expressed in most mature B-cell lymphomas and is expected to be an effective therapeutic target. Disclosure: Nothing to declare
Background: The primary limitations to successful allogeneic hematopoietic stem cell transplantation (AHSCT) are relapse and non-relapse mortality (NRM). The later is primarily driven by graft versus host disease (GVHD). Despite multiple phase III trials documenting the efficacy of rabbit anti-thymocyte globulin (ATG) in preventing both acute and chronic GVHD, there is no clear survival benefit. It is believed that ATG given over 3-4 days before transplant may result in excessive graft T-cell depletion, delayed immune reconstitution, resulting in a higher risk of infection and relapse. Recent data has shown that residual host T-cells play an important role in acute GVHD pathology. We adopted a new dosing strategy, which focused most of ATG bioactivity towards host immune cell depletion. Methods: This is a retrospective analysis of clinical and immunologic outcomes for 55 consecutive AHSCT patients (Pts) who received Thymoglobulin (ATG) between January 2020 and March 2021. 80% of the ATG dose (total of 5.5 mg/kg) was given early in the preparative regimen on days -6, -5, -4, and 20% on day -1. Pts received tacrolimus (0.03 mg/kg) and mini methotrexate 5mg/m2 on days +1, +3, +6, +11 post-transplant for GVHD prevention. The graft was HLA matched peripheral blood stem cells from related or unrelated donors. Preparative regimen was reduced intensity Fludarabine(Flu)/ Busulfan(Bu)/ TBI (total body irradiation 200 rad) in 70% of Pts, and myeloablative Bu/Flu or Cyclophosphamide/TBI (1200 rad) in 30%. We analyzed day 30 and 100 post-transplant immune reconstitution phenotypes for 49 Pts. Results: Disease diagnoses included AML, MPN, MDS, and ALL.98% of Pts had high or intermediate risk for relapse based on disease risk index, and 64% had a high transplant comorbidity index (≥3). The median age was 61(32-75) years, with 40% of the pts ≥65 years old. The cumulative incidence(CI) of all clinical outcomes is at 1 year post transplant. CI of grades III/IV aGVHD was 0. CI of grade II aGVHD was 94%(95% confidence interval 87-100%), 83% of which was upper gastrointestinal (GI) +/− skin(<50% body surface area). CI of lower GI aGVHD was 18%(10-34%). The CI of cGVHD was 2%(0.3-12%; NIH 2015 consensus criteria). The median follow-up for surviving Pts is 10 months (6-20 months). The CI of NRM was 11%(5-22%) for all the pts, and 2% for pts <70 years of age. CI of CMV viremia was 29%(18-47%), EBV 7%(2-17). The CI of relapse was 20%(12-35%). One year overall survival, relapse free survival, and cGVHD/relapse free survival (GRFS) was 75%(60-85%), 69%(54- 80%) and 67%(52-78%) respectively. We observed a favorable reconstitution of CD4, CD8, total NK cell numbers, and mature NK proportions compared to published literature. In addition, we report for the first time a correlation between increased frequency of day 30 CD8 + CD57 + and CD8 + CD16 + T-cells with no relapse at one-year post-transplant in a univariate and multivariate analysis. Pts with CD8 + CD57 + and CD8 + CD16 + T-cells above 57% & 32% (of total CD8) respectively, did not relapse. Conclusions: This simple and novel GVHD prevention platform shows encouraging clinical outcomes with exciting surrogate immunologic markers supporting of early GVL. Disclosure: No disclosures.
Background: Interferon α-2b is effective for preventing relapse of acute leukemia. We investigated the safety and efficacy of new combination with ruxolitinib and interferon α-2b for relapse or minimal or measurable residual disease (MRD) after allogeneic stem cell transplantation. Methods: Patients with relapse status (NCT02568241) or positive MRD (NCT02185261) are enrolled to receive intervention after day 60 post all-SCT: 1) Interferon α-2b (subcutaneously at dosages of 3 million units 2-3 times per week) for 8 weeks in the absence of disease progression or unacceptable toxicity/GVHD; 2) ruxolitinib (Jakafi) taken orally 5mg Bid. MRD is defined as reemerged fusion gene (≥0.1% for core binding factor) or LAIPs by flow cytometry. Overall response rate (ORR) is defined as patients who get complete remission, or with lower MRD (negative or at least 10-fold-reduction of fusion genes). MAGIC or NIH criteria was applied to evaluate acute or chronic GVHD. Event free survival(EFS) is defined as survival without relapse, mortality, or progression of MRD. Results: In the present phase I/II study between Feb 2020 to Oct 2021, consecutive patients after allo-SCT (n = 26, 5 Rel and 21 MRD + ), including AML (n = 21, Rel 3 and 18 MRD + ), Ph-ALL (MRD + , n = 3) and MDS (Rel, n = 2) were enrolled. 23 and 3 patients received SCT from haplo and MSD, respectively. The median follow-up was 9 months. The median time of intervention was 10 weeks (range: 1-20). The incidence of grade-II aGVHD is 11.5% (n = 3) which resulted in discontinued Interferon, while III-IV aGVHD was absent. NIH mild, moderate, severe cGVHD occurred in 15.3%, 7.7% and 11.5% of enrolled patients. The GVHD related mortality was absent. All the relapsed patients (n = 5, 4 MRD- and 1 CR with MRD + ) and 95.2% MRD + patients (n = 20, 18 MRD- and 2 ten-fold-reduction) responded to the intervention respectively. However, 4 relapsed patients experienced progression at 6, 6, 13 and 39 weeks after response (HLA loss n = 3), while the fusion gene of one MRD + patient increased over ten-fold at 10 weeks after response. The sustained response rate was 20% for relapsed or 90.5% for MRD + patients, respectively. Conclusions: Our results suggested that combination of ruxolitinib and interferon α-2b can be effective for relapse or MRD after allo-SCT without increasing risk of GVHD compared to interferon α-2b alone. Considering HLA loss frequently lead to treatment failure for relapsed leukemia, patients might be bridged to 2nd SCT after intervention. Clinical Trial Registry: NCT02185261; NCT02568241 Disclosure: Nothing to declare
Background: Immunocompromised patients, including patients after allogeneic hematopoietic stem cell transplantation (HSCT), are at excessive risk for severe SARS-CoV-2 infection and were therefore assigned priority for SARS-CoV-2 vaccination. As not all patients developed specific vaccination antibodies, we aimed to assess induction of S1 domain of spike protein-specific (S1-specific) T-cell responses in order to distinguish whether seronegative patients had an isolated B-cell or a more global adaptive immune incompetence. Methods: In our study, we included 17 patients after allogeneic HSCT remaining anti-spike protein antibody-negative after double-vaccination with the BioNTech/Pfizer mRNA vaccine Comirnaty (n = 13) or AstraZeneca vector vaccine Vaxzevria (n = 4). Enumeration of SARS-CoV-2 specific T-cells was performed using SARS-CoV-2 T-cell Analysis Kit (Whole Blood, Miltenyi Biotech). T-cell responses to a TCR-MHC-cross-linking reagent (positive stimulation control) and an S1 peptide library were measured at a median of 55 days (range, 21-127) after the second vaccination and compared to similarly vaccinated patients with malignant B-cell lymphomas and iatrogenic B-cell aplasia (n = 5) or healthy controls (n = 22). Results: At the time of vaccination, 16/17 patients were more than six months out from their HSCT (median 47, range 5-1409 months) from a matched sibling (n = 6) or at least 9/10 HLA matched unrelated donor (n = 11) using T-cell depletion in 11 (65%) patients. B-cells were detected in 14/17 and numerically normal in 10/17 patients. CD8 + T-cell counts were at least normal in 16/17 patients, but absolute CD4 + helper cell lymphopenia was prevalent (14/17) and even in the three patients where CD4 + cell counts were low-normal, CD4:CD8 ratios were skewed in favor of cytotoxic T-cells. Seven/17 seronegative transplanted patients had mounted a CD4 (n = 2), a CD8 (n = 4) or both a CD4 and CD8 response (n = 1) to S1 peptide pool. Of note, all three patients with B-cell aplasia due to previous anti-CD19 CAR T-cell therapy (n = 2) or B-cell depleting post-transplant therapy for EBV reactivation (n = 1) were among the seven T-cell responders. We further distinguished patients receiving systemic immunosuppressive therapy („IS“) or not („no IS“) concurrent to the vaccination. Four/12 IS and 3/5 no IS patients generated spike-protein specific T-cells at least in one of the T-cell subtypes with no difference between groups. For comparison, 5/5 lymphoma patients had T-cell responses, 4/5 in both CD4 and CD8, 1/5 only in CD4 subsets. Similarly, 20/22 healthy volunteers harbored CD4, 16/22 CD8 cells responding to S1 peptide. Conclusions: We conclude that isolated T-cell responses after SARS-CoV-2 mRNA or vector vaccine occur, albeit relatively less frequently after HSCT than in patients receiving specifically B-cell-targeted therapies. Disclosure: Nothing to declare
Background: Immune reconstitution (IR) is important for long term survivors after allogeneic hematopoietic stem cell transplantation (allo-HSCT). However, the characteristics and evolution of IR in steroid-refractory acute graft-versus-host disease (SR-aGVHD) human leukocyte antigen haploidentical donor (HID) patients were unknown. Thus, we aimed to identify the characteristics and evolution of IR in SR-aGVHD patients receiving basiliximab treatment after HID HSCT. Methods: Consecutive HID HSCT recipients achieving overall response (ORR) after basiliximab treatment for SR-aGVHD at the Peking University Institute of Hematology from January 2016 to December 2018 were enrolled in this study. Immune cell subsets were identified and measured by multiparameter flow cytometry at 3, 6, 9, and 12 months following allo-HSCT. This work was supported by the Foundation for Innovative Research Groups of the National Natural Science Foundation of China (grant number 81621001), CAMS Innovation Fund for Medical Sciences (CIFMS) (grant number 2019-I2M-5-034), the Program of the National Natural Science Foundation of China (grant number 82170208), the Key Program of the National Natural Science Foundation of China (grant number 81930004), and the Fundamental Research Funds for the Central Universities. Results: A total of 179, 124, 80 and 92 patients were included in the analysis for IR at 3, 6, 9, 12 months, respectively, after HID HSCT. A total of 399 and 76 patients received < 5 doses and ≥ 5 doses of basiliximab, respectively. We observed that (1) IR was fast for monocytes and CD8 + T cells, intermediate for lymphocytes, CD3 + T cells, and CD19 + B cells, and very slow for CD4 + T cells in the whole cohort; (2) almost all immune cell subsets recovered comparably between patients receiving < 5 doses and ≥ 5 doses of basiliximab; (3) most immune cell subsets recovered comparably between SR-aGVHD patients receiving basiliximab treatment and those without aGVHD; (4) all immune cell subsets except CD4 + T cells achieved comparable level with healthy donor within 1 year after HID HSCT. Kinetics of immune reconstitution in SR-aGVHD HID patients receiving basiliximab treatment. Data are showed as median absolute counts with error bars indicating 25th–75th percentiles. The horizontal dotted lines represent the median value of healthy cohorts, and the gray areas represent the 25th–75th percentiles for the healthy cohorts. *P < 0.05, **P < 0.01, basiliximab low dose (< 5 doses) vs. basiliximab high dose (≥5 doses) Conclusions: In conclusion, most immune cells recovered rapidly in SR-aGVHD HID patients achieving ORR after basiliximab treatment, which was comparable with those without aGVHD after HID HSCT. Thus, basiliximab treatment may not seriously impact the IR of SR-aGVHD patients. Disclosure: Nothing to declare
Background: Relapse remains the primary treatment failure after HCT for acute childhood leukaemia. Analysis of chimerism in blood post‐HCT using STR‐PCR is routinely applied in parallel with quantification of MRD to monitor for relapse. Real time quantitative PCR (RQ-PCR) chimerism is 10‐ to 100‐fold more sensitive than STR-PCR, but clinical studies in children are sparse. We aimed to analyse the clinical applicability of the method by two different cut-offs. Methods: This prospective multicenter study was performed in 64 children consecutively transplanted for leukemia between December 2014 and March 2018 at the paediatric transplant centres in Copenhagen, Denmark, Helsinki, Oslo, Gothenburg, and Lund. RQ-PCR chimerism was performed using a commercial kit (KMRtype/track, GenDx, Utrecht, Netherlands). Increasing mixed chimerism (IMC) was defined based on results in a seperate study as a minimum increase of either 0.1% or 0.05% recipient DNA between two samples or a ≥ 10-fold increase. Samples closer than 30 days to diagnosis of relapse were omitted. Endpoints were overt and molecular relapse. Results: In total, 986 samples of whole blood DNA were analyzed, with a median of 15 (1-23) samples per child. Sixty samples from 27 children had IMC ≥ 0.1%, and 107 samples from 40 children had an IMC ≥ 0.05% recipient DNA. The risk of relapse was higher in children with IMC of both 0.1% and 0.05% compared to children without IMC (27.8 (95% CI 4.4-175.8; P < .001), and 18.4 (95% CI 2.8-120.5; P = 0.002), respectively). From the date of IMC, the 3-year CI of relapse or MRD-positivity was 26.7% (CI 9.4-47.0) and 18.5% (6.4-35.3) for IMC ≥ 0.1% (n = 27) and ≥ 0.05% (n = 40), respectively. In the subset of children without an IMC ≥ 0.1% and ≥ 0.05%, CI of relapse or molecular relapse were 10.8% (3.4 -23.3) and 16.7% (5.0 -34.1), respectively. In all cases with a relapse undetectable by IMC, standard chimerism and MRD both remained negative prior to relapse. In a landmark analysis, neither an IMC ≥ 0.1% nor ≥ 0.05% prior to 90 days post‐HCT was significantly associated with relapse Of six children with detectable MRD during follow-up, one progressed to overt relapse, two received donor lymphocyte infusions and are in complete remission and three had only a transient MRD below 10-3 and are in complete remission. Conclusions: We confirm that by RQ-PCR chimerism analysis, one IMC is associated with a higher risk of relapse and two IMCs imply a poor outcome. These results indicate that serial monitoring of RQ‐PCR chimerism in peripheral blood post-HCT may be a useful analysis rather than STR-PCR chimerism and might allow for fewer bone marrow analyses. Using an IMC ≥ 0.05% provided no benefits compared to ≥ 0.1% but entailed more false positive results. Disclosure: Anna Karen Haugaard: reagents for this study were available at a discounted rate from GenDx, Utrecht, The Netherlands. All other authors: Nothing to declare.
Background: In patients transplanted for acute lymphoblastic leukemia (ALL), decisions about early therapy intensification are based on minimal residual disease (MRD) levels. MRD testing via quantitative PCR (qPCR) using clone-specific immunoglobulin (Ig) and T-cell (TR) receptor gene rearrangements is a standard for MRD detection in ALL and is the most widespread method for post-transplant MRD monitoring. We have previously shown that MRD detection using Ig/TR monitoring via next-generation sequencing (NGS) is more specific than qPCR (Kotrová, BMT 2017). Since then, NGS-MRD detection has been standardized within the EuroClonality-NGS consortium. In the current study, we investigated the clinical outcome of prospectively verifying positive non-quantifiable (PnQ) qPCR results via NGS-MRD. Methods: Sequential post-transplant MRD monitoring in pediatric and young adult ALL patients after hematopoietic stem cell transplantation (HSCT) was performed in our facility for patients from 4 transplant centers in the Czech Republic and Slovakia. For qPCR-MRD monitoring, we used the standardized EuroMRD approach. For NGS-MRD we used the EuroClonality-NGS protocols for library preparation (Brüggemann, Kotrova, Leukemia 2019). The qPCR products with PnQ result were first retested for length using Agilent-on-a-chip analysis (Fronkova, BMT 2008) and if the length did not match the diagnostic sample, the qPCR results were concluded as negative. In cases with irresolvable or unavailable PCR products size comparison, the results were reported as positive according to the EuroMRD criteria and further retesting via NGS was performed. Results: In total, we reevaluated MRD via NGS in 26 patients. In 8 patients (31%), the results were confirmed as positive using NGS and reported to clinicians. Out of these 8 patients, 5 relapsed despite therapeutic efforts to avert relapse (median time to relapse: 2 months). All 3 patients positive by NGS who did not progress to relapse had immunosuppressive treatment (IST) reduced and one received 4 doses of donor lymphocyte infusions (DLI) in reaction to the qPCR-detected positivity. One patient died of GvHD reactivation after IST withdrawal. Among the 17 patients identified as negative by NGS, only one relapse occurred (5 months after testing), despite the fact that only in 5 NGS-negative patients therapy was intensified (IST reduction) on the basis of the qPCR result (including the patient who progressed to relapse, who also received 2 doses of DLI). In one patient (who did not relapse), NGS testing was evaluated as inconclusive due to low sensitivity. Conclusions: Ig/TR monitoring via qPCR still represents the most cost-effective and time-efficient method for post-transplant MRD monitoring. Although the NGS method has comparable cost-efficiency to qPCR, its main challenge remains the longer turnaround time, depending on the laboratory throughput. In our study, we confirmed that the NGS method is more specific for discerning low positive MRD from background in physiological lymphocytes and thus more reliable for clinical decisions. The combination of qPCR measurements and subsequent verification of low positive results via NGS appears to be the safest method for post-transplant MRD-guided clinical decision-making. Supported by NU20-03-00284 and GA UK 318321. Disclosure: Nothing to declare
Background: Chimerism and minimal residual disease (MRD) were suggested to be predictive for relapses after allograft in AML patients (pts). Nevertheless, the predictive values of both approaches remain underinvestigated. Though several studies showed a significant correlation between mixed chimerism and relapses, some pts may require very frequent monitoring that may not be reliable in real-world practice. We suggest the post-transplant MRD may have a higher predictive value for relapses as chimerism and may lead to improved early relapse recognition in intermediate risk AML pts early after allograft. Methods: 79 pts with intermediate risk AML (40 males, median age 57 years (19-77)) transplanted in CR during 2015-2020 at the University Cancer Center Hamburg-Eppendorf with available chimerism measurements during the first 100 days and post-transplant (day + 100) MRD data were included. Majority of the pts received allografts after MAC (75%) from MUD (50%). Post-transplant MRD detection on day +100 was performed in bone marrow according to ELN guidelines (flow, n = 79; qPCR, n = 25). Sensitivities were 10-4/10-5 (flow) and 10-6 (qPCR). Chimerism analysis was performed in peripheral blood samples at least once per week based on deletion/insertion polymorphisms for duplex analysis combined with a reference gene or Y-chromosome specific PCR (sensitivity 10-4). Full donor chimerism (FDC) was defined as persistence of ≥99.9% of donor alleles; mixed chimerism (MC) was defined as persistence of <99.9% of donor alleles during the first 100 days post-transplant. Results: Day +100 flow-MRD status correlated both with qPCR-MRD status (neg/neg: 89% vs pos/pos: 57%, p = 0.032) and with chimerism dynamics (neg/FDC: 70% vs pos/MC: 65%, p = 0.005). The concordances between flow-MRD/qPCR-MRD; qPCR-MRD/chimerism dynamics; and flow-MRD/chimerism dynamics were 80% (20/25), 56% (14/25), and 39% (54/79), respectively. The relapses at 3 years was 21% (13-32%) with a median of 216 days (98-722). MC (n = 32) had unfavourable impact on relapses (HR 3.2, 1.1-9.5, 0.038) and LFS (HR 2.8, 1.1-6.8, p = 0.027) compared to FDC (n = 47). Day +100 flow- and qPCR-MRD negativity had favorable impact on relapses (HR 0.1, 0.02-0.3, p < 0.001 and HR 0.02, 0.01-0.3, p = 0.0023, respectively) and OS (HR 0.1, 0.04-0.4, p = 0.001 and HR 0.1, 0.01-0.4, p = 0.004, respectively). qPCR-MRD showed the highest sensitivity and specificity for relapses (86% and 100%) followed by flow-MRD (70% and 85%), and MC (60% and 34%). Also, positive-predictive and negative-predictive values were highest for qPCR-MRD (100% and 95%), followed by flow-MRD (83% and 79%) and MC (38% and 83%). The area under the ROC curve was highest for qPCR-MRD (0.93, 77-100%, p = 0.001) followed by flow-MRD (0.70, 46-95%, p = 0.13) and MC (0.55, 29-81%, p = 0.72). Day +100 flow-MRD negative pts with MC (n = 17) experienced significantly lower relapse rate at 1 year (6% vs 67%, p = 0.003) and higher 1-y LFS (81% vs 48%, p = 0.017) comparing to positive ones (n = 15). Conclusions: Molecular and flow MRD measurements at day +100 have a stronger predictivity for post-transplant relapses compared to early chimerism dynamics in intermediate risk AML. Intensified MRD monitoring early post-transplant together with chimerism studies may improve the identification of AML pts with increased relapse risk who may be candidates for early post-transplant interventions. Disclosure: Nothing to declare
Background: Genomic loss of mismatched HLA (HLA loss) is a vital immune escape mechanism of leukemic cells after hematopoietic stem cell transplantation (HSCT). However, limited literature has been published, especially in lymphoid malignancies. The methods currently used for HLA loss detection have some limitations. Methods: 162 post-transplant patients from 18 centers in China were selected out for this study. HLA loss analysis was performed with HLA-KMR and next-generation sequencing (NGS)-based methods. Variables of the prognostic risk factors for HLA loss or HLA loss relapse were examined in the proportional hazards model or competing risk regression model. Results: An HLA loss detection system, HLA-CLN (HLA chimerism for loss of heterozygosity [LOH] analysis by NGS), was successfully developed. To our knowledge, the largest scale of 40 (24.7%) patients with HLA loss was reported including 27 with myeloid and 13 with lymphoid malignancies, whereas 6/40 (15.0%) did not relapse. The 2-year cumulative incidences of HLA loss (22.7% vs 22.0%, P = 0.731) and HLA loss relapse (18.4% vs 22.0%, P = 0.571) were similar between patients with myeloid and lymphoid malignancies in partially mismatched related donor (MMRD) transplantation. The genomic loss of both HLA I and II loci occurred in 82.5% of patients (33/40). The number of HLA mismatches (5/10 vs < 5/10) was significantly associated with HLA loss in the whole cohort (HR: 3.15, P = 0.021) and myeloid malignancies (HR: 3.94, P = 0.021). A higher refined-disease risk index (HR: 6.91, P = 0.033) and donor-recipient ABO incompatibility (HR: 4.58, P = 0.057) contributed to HLA loss in lymphoid malignancies. Conclusions: HLA-CLN system for HLA loss detection could cover a higher percentage of patients and have a broader range of clinical applications with a higher accuracy. With the new system, the largest scale of 40 HLA loss patients was reported. The cumulative incidence of HLA loss and other clinical characteristics were similar between patients with lymphoid and myeloid malignancies. The results suggested that a patient with HLA loss was not necessary to relapse. Disclosure: Nothing to declare.
Background: Allogeneic hematopoietic stem cell transplantation (allo-HSCT) offers the best chance for relapse-free long-term survival for most patients with acute myeloid leukemia (AML). Thereby, sequential conditioning regimens are successfully used for high-risk disease. Presence of measurable residual disease (MRD) at time of allo-HSCT has recently been shown to be associated with an increased risk of relapse post-transplant. Yet, the reported effect might vary according to genetic and immunophenotypic diversity of the disease, as well as transplant-setting and MRD method. The negative impact might be overcome with sequential therapy in MRD positive patients. Here, we aim to determine the impact of NPM1-MRD assessed by qPCR on outcome in a homogeneous patient cohort undergoing sequential conditioning. Methods: We retrospectively analyzed sixty adult AML patients harboring NPM1 mutations, who underwent a first allo-HSCT from a matched sibling (n = 17), unrelated donor (n = 26), or HLA-haploidentical family donor (n = 17). All patients were transplanted between January 2015 and November 2021 at our institution. Sequential conditioning using FLAMSA-RIC was the treatment of choice for patients transplanted with either active disease, evidence of molecular marker or high-risk cytogenetics. Nine patients deemed ineligible for sequential treatment due to age or comorbidity. NPM1mutation status was assessed at diagnosis and monitored by qPCR throughout the treatment course. MRD status pre-transplant was correlated with outcome. Results: Sixty patients had an initial diagnosis of NPM1-positive AML (secondary AML n = 15) of whom forty-two were in complete remission (CR)1 or CR2 and eighteen had active disease (primary refractory or relapsed AML) at time of allo-HSCT. Whereas forty-six patients had cytogenetically normal AML, three had a complex karyotype. At diagnosis twenty-one patients were classified as favorable according to ELN risk stratification, thirty-three as intermediate and six as adverse. Median age of the entire cohort was 56 years (21-75). No graft rejection occurred. At a median follow up of 27 months (range 0,6 – 79) the estimated probability of overall survival (OS) and leukemia-free survival (LFS) at 1- and 2-years was 88% and 67% and 70% and 63%, respectively. If stratified in patients transplanted in any remission vs non-remission, there was a trend towards better OS and LFS at one year for patients in CR and molecular CR (molCR) in comparison to non-remission patients (OS 82% and 83% vs 64%; p = 0.07; LFS 74% and 75% vs. 61%; p = 0.21). Yet, if considering only patients in remission (n = 42) no difference could be detected depending on NPM1-MRD status at transplantation (OS p = 0.46 and LFS p = 0.6). Cumulative incidence (CI) of non-relapse mortality (NRM) at 1-year was 11%, 0% and 0% for CR, molCR and nonCR patients, respectively (p = 0.97), whereas CI of relapse was 15%, 25% and 36% within the first year (p = 0.18). Conclusions: Sequential conditioning improved disease control and survival in patients transplanted with NPM1-positive AML, regardless of pretransplant MRD status assessed with qPCR, in this single-center study. Disclosure: Nothing to declare.
Background: The pattern and quality of the immune reconstitution (IR) from the long-lasting immunodeficiency after transplantation may affect the outcome of hematopoietic allogeneic stem cell transplantation (allo-HSCT). However, there are limited data on the association of the quality of the IR on either the development of graft vs host disease (GvHD) or survival. We therefore aimed to explore the factors conditioning the IR and its impact on survival, and on the development of GvHD. Methods: To this end, 163 patients who received a non T-cell depleted allo-HSCT in our center from 2011 to 2019 were prospectively studied. Acute and chronic GVHD were diagnosed according to published criteria. Lymphocyte immunophenotyping was performed on fresh whole blood by flow cytometry to quantify total CD4 + and CD8 + T cells and their subsets: naïve, stem cell-like, memory central, effector memory and CD45RA + effector memory cells. Data were collected at days +30, +60, +90, +180 and +365 after allo-HSCT. The association between pretransplant factors and the IR was studied with ANOVA followed by a Bonferroni test. The association between IR and either the survival or the development of GvHD was studied through generalized estimating equation (GEE) models which included the confounding variables with p < 0.10 in the multivariate GEE models. Results: The absolute counts of CD3 + T lymphocytes reached normal levels within six months. CD8 + T lymphocytes recovered much faster than CD4 + T cells and, after one month, the median CD8 + T cells count was within the normal range. Conversely, it took nearly one year for the CD4 + T cells to reach normal values. Recovery of the different subsets of CD4 + and CD8 + T cells followed the same pattern of their parent populations with the exception of naïve CD8 + T lymphocytes, which hardly recovered normal counts after one year. Naïve and stem cell like CD4 + T cells were identified among the different lymphoid subsets whose reconstitution was strongly affected by the characteristics of the transplant. Their IR was particularly poor in non-identical HLA transplants, with the worst results for haploidentical transplants. Similarly, patients with myelodysplastic syndromes, acute myeloid leukemia and acute lymphoblast leukemia had a worse IR, probably because of a connection with myeloablative conditioning regimens. Regarding the influence of IR in either survival or GvHD, multivariate GEE models showed that the recovery of the absolute number of total lymphocytes and CD3 + T lymphocytes determined the survival at 3 months. In the univariate models we found that higher numbers of CD8 + T cells and naïve CD8 + T cells were associated with survival at 3 months. Similarly, the recovery of CD4 + T cells was associated with a greater survival at 3, 6 and 12 months. Finally, a faster restoration of TEM CD4 + T cells was associated with the development of chronic GVHD in the multivariate analysis. Conclusions: In conclusion, we identified IR variables that could be used as biomarkers for both the survival in the first 3 months and the development of chronic GvHD. Also this study can be the basis for therapeutic strategies to recover immune function. Disclosure: Nothing to declare
Background: The outcomes after allogeneic HSCT has improved significantly but is still challenged by the risk of GvHD and related infections that contributes substantially to morbidity and mortality. aGvHD has been shown to negatively impact B-cell reconstitution by targeting the bone marrow and disrupting the normal B cell ontogeny and/or through the immunosuppressant treatment given to control the disease. A delayed B-cell recovery and impaired B-cell immunity in peripheral blood have previously been described in patients with cGvHD, but the mechanisms are unclear and few studies have looked into the B-cell development in the bone marrow compartment. In this study, we expand current insights by investigating the B-cell reconstitution in the bone marrow after pediatric HSCT and how this relates to alloreactivity. Methods: We included 30 children undergoing HSCT for ALL (n = 19) or AML (n = 11) from 2015-2020 with a median age of 9.3 years (range: 3.1-16.6). All patients received myeloablative conditioning based on TBI (n = 12) or chemotherapy alone (n = 18). Patients received a bone marrow (n = 29) or peripheral blood stem cell (n = 1) graft from MSD (n = 8) or MUD donors (n = 22). GvHD prophylaxis consisted of cyclosporine A alone (n = 8) or in combination with methotrexate and ATG (n = 22). Fresh bone marrow samples were analyzed for specific B-cell subsets using flow cytometry at month +1, +2, +3, +6, +9, +12, +15 and +18 post-HSCT. Results: The B-cell compartment mainly comprised naïve B-cell precursors stage I (BCPI) and BCPII cells during the first months post-transplant and was gradually replaced by more mature subsets, such as BCPIII, mature B-cells, and plasmablasts. The percentage of CD19 B-cells in the bone marrow correlated positively with the same-day CD19 B-cell concentration in peripheral blood from month +2 to +12 (r = 0.5-0.8, all p < 0.05). TBI-based conditioning was associated with undetectable levels of plasmablasts in the bone marrow during the first three months following HSCT (p = 0.03-0.06), while a higher nucleated cell dose correlated positively with the percentage of CD19 B-cells including naïve and mature subsets (all p < 0.05). In patients with aGvHD grade II-IV (n = 15), development of the B-cell lineage appeared generally compromised in bone marrow samples, with reduced CD19 B-cells and BCPII cells at month +3 (both p = 0.03), and fewer plasmablasts from month +6 to +12 (p ≤ 0.05). In children developing extensive cGvHD (n = 3), B-cell reconstitution appeared considerably delayed with less CD19 B-cells and naïve B-cell subsets from month +2 to +6 and fewer plasmablasts from month +9 to +15 (Figure). Patients treated with immunoglobulin-substitution (57.1%) (day 6-786) due to reduced immunoglobulin production post-transplant had significantly less CD19 B-cells at month +2 and +3 (p < 0.05), but not significantly reduced percentages of the other B-cell subsets. Conclusions: Our findings suggest that alloreactivity following HSCT is associated with a delayed B-cell reconstitution in the bone marrow rather than a more targeted loss of specific B-cell precursors. Risk factors for impaired B-cell recovery include TBI-based conditioning and low numbers of transplanted donor cells. Further studies are warranted to further explore deficiencies in B-cell maturation and the potential for prediction of chronic GvHD based on B-cell monitoring. Disclosure: None to declare.
Background: Currently, the relapse after allogeneic stem cell transplantation (SCT) remains the main risk factor in term of transplant outcome of adult patients affected by Acute Myelogenous Leukemia (AML). However, although the detection of minimal residual disease (MRD) in AML patients after allogeneic SCT plays an important role, the cut off of NPM1 positivity related to haematological relapse is still unclear. The aim of this study was to analyze, prospectively, the correlation between NPM1 and recipient chimerism values, after allogeneic SCT in AML-NPM1 + adult patients, in order to identify a predictive cut off for haematological relapse Methods: From June 2019 and December 2021, 57 allogeneic SCT were performed at AORN Cardarelli Single Transplant Program in Naples. Indication to allogeneic SCT was AML in 29 cases, of whom 14 NPM1 + , associated with FLT3-ITD + (N = 10) or TKD + (N = 2) in 12 cases. In this subset of patients, 7/14 were males, the median age was 42,5 (range 22-64), reduced conditioning regimen (RIC) was used in 6/14 allogeneic SCT procedures, the donor was an HLA identical sibling, haplo-identical or unrelated in 7,4 and 3 cases, respectively. Disease status at transplant was: 1st CR, 2nd CR or active disease in 10, 3 and 1 cases, respectively. RIC or myeloablative TBF conditioning regimen was used in all patients whereas GVHD prophylaxis depended on the donor’s type. Overall, 258 bone marrow samples were analysed using quantitative RT-PCR to detect chimerism (N = 128), by insertion/deletion genomic biallelic polymorphisms, and type A NPM1 mutation (N = 130). In this study, the time points for monitoring quantitative chimerism and NPM1 mutation were: before allogenic SCT, as baseline values, every month along 24 months and, then, every 3 months until 5 years after allogeneic SCT Results: Overall, 13 patients are alive and in complete remission after a median follow up of 18 months (range 1-30) while 1 died for relapse. Concerning the relationship between quantitative chimerism and NPM1 mutation, the analysis has shown that day +30 after allogeneic SCT is a too early time point not reliable for prognostic purpose. Conversely, time points included between day +60 and +180 show linear correlation between NPM1 and recipient chimerism with r-value very close to 1. Long term median follow up is necessary to evaluate recipient chimerism and NPM1 mutation relationship after day 180. Moreover, recipient chimerism>1% associated with NPM1 > 100 copies led to haematological relapse whereas the quantitative recipient chimerism value ≥1% with the NPM1 copies value included between 1 and 99 did not Conclusions: The strict and contemporary monitoring and quantification of recipient chimerism and NPM1 mutation positivity, after allogeneic SCT, could led to a better selection of patients who can benefit from immunomodulation and relapse pre-emptive therapy. From these preliminary data, the association between recipient chimerism>1% with NPM1 > 100 copies, may be identified as a risk factor for haematological relapse Disclosure: Nothing to declare
Background: Post-transplant immune reconstitution plays a major role in determining the outcome of allogeneic hematopoietic stem cell transplant (allo-HSCT) recipients. Immune cell counts provide only a general indication of the competence of the patient immune system, whereas the quantitative and functional assessment of virus-specific T-cell responses may be more relevant to patient’s risk stratification and clinical decision-making. However, information is lacking about the current practice across centers. Methods: In September 2021, the CTIWP conducted a survey across EBMT centers to identify current policies to monitor virus-specific immune reconstitution in patients undergoing allo-HSCT. Results: Policies for post-transplant virus-specific immune monitoring have been reported by 152 (30%) EBMT centers, active in 37 countries. Centers perform allo-HSCT in adults only (43%), children only (26%) or both (31%), and use various donor sources (100% HLA-identical related, 94% matched unrelated, 99% mismatched related, 56% cord blood). Although cytomegalovirus (CMV) viremia is monitored after allo-HSCT in all centers, 17 (11%) centers are currently testing CMV-specific T-cell responses, either within clinical (35%) and/or experimental practice. Flow-cytometry based tests (e.g. intracellular cytokines, MHC multimer binding), mainly set up in house, are used in 9/152 centers (6%) to assess CMV-specific T-cells. IFN-γ ELISpot is adopted in 15 (9.9%) centers, either with home-made or commercial assays. CMV Quantiferon is used in only 4 (2.6%) centers and mainly through commercial assays. Additionally, other home-made tests (e.g. proliferation assays or CMV specificities within different T-cell subsets) are rarely applied. Thresholds are not harmonized among centers. Longitudinal monitoring for human herpesvirus 6 (HHV6) viremia in allo-HSCT recipients is performed in 45% of the centers, while 22% search for the virus only in case of clinical suspicion. Tests for HHV6-specific T-cell responses are reported by two centers, adopting home-made IFN-γ ELISpot or limiting dilution analysis for antigen-specific T-cells. In 51% of the centers, adenovirus viremia was monitored in allo-HSCT recipients, whereas 32 (21%) centers only monitored in case of symptoms. Moreover, 8 centers check for adenovirus-specific T-cell responses, using different home-made or commercial assays (e.g. MHC multimer binding, intracellular cytokines, IFN-γ ELISpot, limiting dilution analysis), both as experimental and clinical practice. Frequencies of T cells specific for other viruses, mainly Epstein-Barr virus, are currently tested in 15 (9.9%) centers. Overall, 21 (13.8%) centers are performing at least one type of virus-specific immune monitoring. Furthermore, 47 (31%) centers are planning to start monitoring for virus-specific immune responses in the future. Conclusions: Immune monitoring for virus-specific T-cell responses is currently performed in a limited number of centers and is highly variable in terms of targets, technologies and interpretation. These results underline the need to harmonize and standardize methods, both for routine and investigational purposes, and to stimulate clinical curiosity questing for information. Monitoring of CMV-specific responses should probably be more widely adopted for its clinical value in the new era of letermovir prophylaxis. Overall, improved reporting and communication between centers adopting these technologies is needed to foster collaborative and comparative research studies in the future, which may translate into new immune monitoring guidelines. Clinical Trial Registry: CTIWP survey EBMT study number 8420021 Disclosure: Nothing to declare.
Background: The relapse of malignant disease is one of the main complications of hematopoietic stem cell transplantation (HSCT) in myeloid neoplasms. Mixed chimerism (MC) is considered an important factor to predict relapse. Therefore, reliable detection is crucial for early diagnosis of relapse. The aim of this study is the evaluation of indel-qPCR non-classical chimerism analysis in peripheral blood to predict the outcome of patients with myeloid pathologies post-HSCT by individualized follow-up. Methods: We monitored chimerism of 44 patients diagnosed of AML and MDS considering risk factors and clinical grounds. The analysis was performed by KMR Track kit (GeneDx) and results was represented as host-DNA percentages. We defined complete chimerism (CC) as host-DNA percentage inferior to 0.05% and mixed chimerism as host-DNA percentage above this threshold. Results: Our results show that the kinetic profile of chimerism is heterogeneous and could be influenced by different factors, such as the pre-transplant conditioning regimen. In our series, a statistical difference is only detected between RIC (reduced-intensity conditioning) and cyclophosphamide treatment after transplantation (PTCy) at day 30 post-HSCT. In patients without relapse or progression, CC is detected over a wide range of time, 30-240 days (median 144 days). In a group of 8 patients CC is detected at day +30 and of those, 75% (6/8) had been treated with PTCy. Despite point values of chimerism not correlating with outcome, we observed an association between the evolution of the disease and three different kinetic profiles. The first profile consists of 21 patients with CC (N = 2) or decreasing mixed chimerism (DMC) (N = 19) who achieved CC. Of this group, 71% (15/21) of patients have not relapsed after one year of follow-up and only one patient showed early MRD + despite detecting CC. Late relapse was observed in 23% of patients (5/21) with a median of 846 days post-HSCT. In 4/5 patients, continuous monitoring of chimerism allows to predict relapse by increasing of mixed chimerism (IMC). The second profile consists of 3 patients with stable mixed chimerism (SC-MC) after HSCT. In 2/3 patients a late relapse is observed (400 and 800 days post-HSCT) and 1/3 no relapse is observed. The last profile consists of 20 patients with IMC over time. Early relapse/progression is observed in 19/20 patients (median = 110 days post-HSCT). Moreover, we observed that out of 25 relapsed patients, 52% had continuous IMC and died. Eventually, 24% achieved CC and 24% are still alive with MC. Figure 1. Association of early-relapse, late-relapse or non-relapse with chimerism kinetic profiles. Conclusions: - Post-HSCT follow-up by means of chimerism must be carried out on an individualized basis according to the patient’s evolution, just as it occurs in the transplant procedure. - Our results show that despite the heterogeneity of chimerism, three different kinetic profiles correlate with disease progression. - The short experimental time, relatively cost-effective and minimally invasive nature of the peripheral blood, could make the close monitoring of chimerism by qPCR technique a very useful tool for predicting outcome in AML/MDS allografted patients, especially in the absence of a molecular marker. Disclosure: Nothing to declare.
Background: Chronic granulomatous disease (CGD) is a primary immunodeficiency caused by the inability of phagocytic cells to produce reactive oxygen metabolites required for microbial killing. The dramatic reduction in oxidative burst intensity characteristic of patients with CGD can be demonstrated by flow cytometry (FC) used routinely to analyze expression of various cell surface and intracellular markers. Hematopoietic stem cell transplantation (HSCT) is curative for CGD - the success is based on the engraftment of myeloid lineage and production of functionally competent granulocytes. Chimerism tests allow evaluation of the donor/recipient origin of the post-HSCT hematopoietic system. The purpose of our study was to compare the utility of two methods, quantitative real-time polymerase chain reaction (qPCR) and FC, for chimerism tests. Methods: Clinical samples of peripheral blood (a total of 35) were collected from 15 patients aged 3–13 years (median age 6 years) at different time points after stem cell transplantation, within a period from 2018-09 to 2021-11. The cohort included 14 boys with X-linked CGD (CYBB-mutated) and one girl with autosomal-recessive CGD (CYBA-mutated). All patients received either TCRαβ/CD19-depleted allogeneic HSCT (n = 12) or bone marrow transplantation (n = 3). Chimerism was determined in total leukocytes (35 samples) or CD15 + myeloid cells obtained by immunomagnetic separation (25 samples). Quantitative PCR assays were carried out with test systems for insertion/deletion polymorphisms developed and provided by Research Center for Medical Genetics, Moscow. Functional activity of neutrophils was assessed by FC using FagoFlowEx Kit (EXBIO Praha, a.s.) to measure the oxidative (respiratory) burst after stimulation by detection of fluorescence of rhodamine 123. Pearson correlation coefficients were calculated to estimate relationships. Results: There was a complete concordance between the molecular PCR-based and flow-based assessment of chimerism among samples with complete donor chimerism both in whole blood and selected CD15 + fraction. With the absolute predominance of myeloid donor cells in all tested CD15 + populations (>99% as measured by qPCR), 24 (96%, all but one) of them exhibited unimodal oxidative burst patterns. Among cases with mixed chimerism in the whole blood, as determined by PCR -based method, there was a highly significant positive correlation between the two methods (Pearson r = 0.651, p > 0.0001). The discordance of quantitative assessment by two methods is presumed to be due to different potential sources (CD3 + T cells vs CD15 + granulocytes) of the recipient signal. Conclusions: Direct oxidative burst measurements by FC provide a rapid and sensitive test to assess the proportion of functionally competent granulocytes in peripheral blood. Mixed granulocyte populations produce bimodal negative-positive distributions. Comparison of qPCR results with FC data reveals a strong positive correlation for CD15 + lineage chimerism, albeit a moderate positive correlation for mixed chimerism in total blood leukocytes. Overall, FC provides robust and reliable means for evaluation of HSCT efficacy in patients with CGD. Disclosure: Nothing to declare
Background: Chimaerism monitoring post-HPCT is a powerful tool used to assess engraftment and relapse. An emerging technology for chimaerism analysis is digital PCR (dPCR), reported to have greater sensitivity for target DNA detection. This method has not yet been clinically implemented over the predominant use of STR-PCR based monitoring. Methods: The sensitivity of chimaerism results obtained using dPCR and STR-PCR were compared. As part of the study, an evaluation of the technical applicability of dPCR in a clinical Histocompatibility & Immunogenetics laboratory was carried out. Chimaerism analysis was carried out for a cohort of 50 HPCT recipients aged between 9 months and 65 years and transplanted between 2015 and 2020 as treatment for haematological malignancy (n = 19 multiple myeloma, n = 7 juvenile myelomonocytic leukaemia, n = 24 high relapse risk paediatric AML, HR-AML). A total of 117 samples were selected according to: (1) recipient consistently displaying 100% donor chimaerism (DC) by STR-PCR, two most recent samples, (2) deceased recipient, final two samples available, (3) recipient displaying mixed chimaerism with samples considered ‘borderline’ between 95%-100% DC. Artificial chimaerism mixtures (100, 99.5, 99, 98 %DC) were also generated and evaluated on both technologies. Patient-donor chimaerism was evaluated using the QuantStudio™ 3D Digital PCR System (Applied Biosystems™) with Imegen-Quimera dPCR informative marker kits (Imegen) and compared to STR-PCR results (Geneprint® 24 System, Promega) processed using the ABI 3500XL Genetic analyser (Applied Biosystems™) in accordance with manufacturers’ instructions. Results: dPCR demonstrated higher sensitivity for the detection of very low levels of the artificial ‘HPCT recipient’ DNA when DC is ≥99%. Conversely STR-PCR is more accurate than dPCR when a greater level of artificial recipient DNA is present, ≤98% DC. Above 99.5% DC, the lower values given by dPCR, compared with the values from the STR technique, may identify recipient DNA that would be missed by STR-PCR as the sensitivity limits of STR-PCR are known to be approximately 1-2% of minor component. Direct comparison of technologies showed dPCR quantification situates tightly around 99-100% DC, whereas STR-PCR shows a greater spread of values (92-100% DC; image panel A). In contrast, the results indicate STR-PCR has better performance at increasingly mixed recipient chimaerism, where DC is ≤98% (artificial mixture analysis). The technical evaluation of dPCR and STR-PCR platforms presented several differences which impact performance and the technical considerations for clinical implementation. Aspects include DNA concentration, labour/ technical demands, financial cost, ad hoc versus batch testing suitability and turnaround time. Conclusions: In this single centre study, dPCR was found to supersede STR-PCR for detection and quantification of extremely low-level recipient chimaerism (99-100% DC), whereas STR-PCR was more accurate for increasingly mixed recipient chimaerism (<99% DC). A technical evaluation found dPCR can provide a faster turnaround for small sample numbers. Conversely, STR-PCR is suitable for batch testing. Overall, it is suggested that dPCR is advantageous for rapid chimaerism analysis monitoring in urgent scenarios in early engraftment, early suspected relapse or high-risk leukaemia patients. In contrast, STR-PCR should remain the technique of choice for patients with overtly mixed chimaerism. Disclosure: Nothing to declare
Background: MicroRNAs (miRNAs) has been used to predict outcome after allo-HSCT, most of them related with the development of aGvHD. However, few reports connect miRNA expression to other outcome variables such as relapse. We performed a high throughput profilling in circulating miRNAs. Overexpressed miRNAs were used to identify potential target genes. Methods: We performed in a discovery cohort miRNA profilling using a miRNA array containing 175 miRNAs. We validated individual overexpressed miRNAs using droplet digital PCR (ddPCR) in a validation cohort including patients relapsing after allogeneic hematopoietic transplantation (allo-HSCT). By bioinformatics anaylisis, we identified candidate target genes and analysed the target gene expression in peripheral blood mononuclear cells (PBMCs) in patients at relapse after allo-HSCT. Results: Four miRNAs (hsa mir 363, hsa mir 505, hsa mir 200c and has mir 320c) were identified in the miRNAs profiling of circulating nucleic acids to be overexpressed (>1.5 fold) in patients relapsing after allo-HSCT. In silico analysis, identified DNAJB9 and TUBB as the target genes for the combination of the four overexpressed miRNAs. We further analysed DNAJB9 expression in PBMCs in three groups of patients: complete remission (n = 13), pre-transplant with active disease (n = 5) and relapse after HSCT (n = 11). Mean Log dd Ct DNAJB9 expression was: -0.01, -0.39 and -0.23 for those patients in complete remisssion, pre-transplant with active disease and relapse after allo-HSCT, respectively. A significant DNAJB9 expression difference between patients in complete remission and those patients with either active disease before transplant or relapse after allo-HSCT was observed (p = 0.0056 and p = 0.036). We assessed the assay perfomance of the DNAJB9 expression between the different analyzed groups, using the area under the curve (AUC) of a ROC curve. The AUC was 0.923 and the optimal DNAJB9 expression threshold to discriminate relapse from complete remission was Log dd Ct DNAJB9 -0.17. Within the group of patients relapsing after allo-HSCT, two groups were observed: high (mean Log dd Ct DNAJB9 -0.049, n = 7) and low (mean Log dd Ct DNAJB9 -0.55, n = 4) DNAJB9 expression. The mean time to relapse in patients with a high DNAJB9 expression was 26 months whereas in those with low DNAJB9 expression was 9.6 months. This time difference reached statistical significance (p = 0.011). Conclusions: miRNAs profiling of circulating nucleic acids identified DNAJB9 gene, which is associated with disease progression after allo-HSCT. Disclosure: This work was supported by a grant of the Else Kröner-Fresenius-Stiftung (Nr. 2018-A56).
Background: Late graft failure with mixed chimerism and autologous reconstitution is not uncommon after haematopoietic stem cell transplant (HSCT) for non-malignant disease, and is considered an indication for second transplant. Such second transplant is associated with significant morbidity and mortality and should therefore only be undertaken if necessary. We discuss 3 cases where late recovery of donor chimerism was described, after apparent graft failure with loss of donor chimerism, and without any medical intervention. This observation has implications for the planning of second transplants. Methods: The chimerism data of patients over the last 20 years receiving transplant in Royal Manchester Children’s Hospital was reviewed. The clinical data, including enzyme and substrate levels, were included in the analysis. Results: We examined and highlight the graft experience of 3 patients, who experienced apparent graft loss, with significantly falling donor chimerism, and who experienced later recovery of donor chimerism despite no medical intervention. Patient A HSCT indication ELA2 constitutional neutropenia with GCSF resistance, long –term donor chimerism to less than 10% after BuCyCampath MAC allograft. The donor cells retained sensitivity to post-transplant GCSF and so second transplant not performed, and eventually donor engraftment improved, and GCSF could be discontinued (perhaps as marrow failure associated with ELA2, and donor cells occupying hypoplastic recipient marrow). Patients B + C HSCT indication MPS1H. Patient B initially 95% chimerism, reduced to 15% at 20 months then recovered to 80% donor chimerism at 50 months with graft producing iurondiase enzyme level sufficient to allow substrate reduction and clinical correction. C has experienced a primary graft failure with autologous reconstitution and received a second transplant. He experienced second graft loss, to less than 50% door chimerism but recovered to 90% donor chimerism without intervention. Enzyme and substrate levels mirrored chimerism. Conclusions: Transplant in non-malignant disease is to correct disease, and the level of chimerism required is that to correct the disease. This report emphasises this statement, especially since it demonstrates that donor chimerism can recover without medical intervention. Repeat HCT may be indicated for mixed chimerism after transplant, but not always so, avoiding its associated morbidity and mortality. Disclosure: Nothing to declare.
Background: A retrospective analysis of patients with AML, ALL or MDS, who received allogeneic hematopoietic stem cell transplantation for the period 2017-2019 in TU at SBALHZ was performed. The aim of the study is to identify the cut off value and time point of early lymphocyte recovery, presented as an absolute lymphocyte count (ALC), that would have prognostic significance for the outcome of AloHSCT and whether it can serve as a surrogate marker for NK cell recovery. Methods: The electronic files of 56 patients, followed until 20.01.2021, were analyzed. The demographic, clinical and laboratory data were obtained from HIS. Peritransplantation factors were evaluated. Spearman’s correlation coefficient, chi-square analysis, Mann-Whitney test, Cox regression analysis, log-rank test, ROC-curves were applied in the statistical analysis. Values of p < 0.05 were considered significant. Results: The significance of the factors ALC - D + 21 and D + 30 for overall survival was checked with Cox regression analyze. D21 proved to be significant (p = 0.016, HR = 0.998, 95% CI 0.995-0.999). The limit value of the function was set at 0.5376, which corresponds to ALC 230/μl of D + 21. The risk factor shows that the survival of patients in the group with ALC D + 21 > 230 /μl is 3.78 higher and 2.87-fold higher risk of disease relapse in patients with ALC D + 21 < 230 /μl (fig. 1). We have also investigated the NK cell recovery by FCM at D + 30 ± 7 days, the mediana count is 148/μl. No association of NK D + 30 with OS, PFS, CIR, NRM and ALC D + 21 has been demonstrated. We found a correlation between NK cell recovery and aGVHD. Conclusions: ALC D + 21 ≥ 230/μl in the study group of patients was associated with improved overall survival and reduced risk of recurrence, but a higher incidence of cGVHD. ALC D + 21 ≥ 230/μl may be a surrogate for engraftment. ALC D + 21 is a biologically significant predictor of the outcome of transplantation - universal, affordable, easily measurable and inexpensive. we couldn’t prove that ALC D + 21 can serve as a surrogate marker for NK cell recovery, probably due to small number of patients included in the analysis. Clinical Trial Registry: N/A Disclosure: We have no conflict of interest to declare
Background: Chimerism monitoring is routinely used following haematopoietic stem cell transplant (HSCT) to analyse donor engraftment through detection of donor and recipient cells, and subsequently detect graft failure or relapse of malignant disease. Higher quality monitoring can impact patient care by allowing earlier intervention in the event of disease relapse or graft failure post-HSCT. Short tandem repeat (STR) polymerase chain reaction (PCR) is the current gold standard for chimerism analysis in clinical practice, but its utility is limited by low sensitivity, high variability, and limited throughput. There is a need for chimerism monitoring that incorporates the highest sensitivity testing possible, can be standardised across clinical practice and has the potential to monitor both donor/recipient cells and disease present. Several other innovative methods with higher sensitivity and accuracy are being explored. Next generation sequencing (NGS) has extended applications in genomics, forensic science, and clinical diagnostics and has shown promise as the future of chimerism analysis in both solid and haematological transplantation. Higher sensitivity and precision, combined with the possibility to monitor disease levels in haematological malignancy, present NGS as a viable candidate to explore for routine clinical chimerism analysis within the NHS. This has the potential for better patient outcomes through earlier detection of relapse of disease or loss of donor graft. NGS technology also has the potential to revolutionise monitoring following gene therapy HSCT (HSCGT). Methods: Post-transplant engraftment monitoring by STR-PCR was carried out using GenePrint® (Promega) on an ABI3500xl platform in 4 patients who had previously undergone HSCT for malignant or non-malignant disease. DNA from each peripheral blood sample was also tested using AlloSeqTM HCT (CareDx®) on a MiSeq (Illumina) instrument, a technique which uses NGS to facilitate more accurate quantification of donor and recipient templates present in the sample. Analysis was carried out using AlloSeq HCT Software. Results: The preliminary work undertaken in the Transplantation Laboratory at Manchester University NHS Foundation Trust demonstrated the earlier detection than STR of mixed chimerism in patients. Micro chimerism was detected from week 7 post-transplant in peripheral blood DNA samples using NGS, compared with first detection of mixed chimerism at week 26 post-transplant with STR-PCR. Earlier detection of recipient micro chimerism in CD15+ DNA was observed at week 8 post-transplant, compared with 100% donor DNA detection at 22 weeks using STR-PCR. Conclusions: Further research is required on larger patient sample numbers to further validate this method and demonstrate reproducibility of results on a large scale. This research will support the validation required to meet ISO accreditation standards for clinical laboratory use, and work towards commissioning through the NHS England service development process. Disclosure: Nothing to declare.
Background: Hematopoietic stem cell transplantation (HSCT) is one of the treatment approaches in variety of hematologic malignancies. Its risks include mainly severe infectious episodes. QuantiFERON-monitor (QFM) is a method assessing an unspecific cellular immune function. The method is based on detection of a value of interferon gamma (IFNγ) in plasma after previous stimulation of both innate and adaptive immune system response. Methods: In our work we focused on analysis of the relationship between the dynamics of a QFM value in patients treated with HSCT for a hematooncologic disease and the incidence of severe infectious complications in early post-transplant period. Our hypothesis was increased or stationary QFM in patients without severe infection history. Consecutive patients who underwent HSCT from 2017 to 2020 were included in the study. We performed an analysis of specimens of whole blood from our subjects by QuantiFERON-Monitor testing kit (QIAGEN, Hilden, Germany). Each specimen was processed according to the manufacturer’s manual. The production of IFNγ in plasma was measured after 24 hours of incubation with a specific stimulus by ELISA (normal range 15–1000 IU/ml). Results: Our patients were divided into two groups. In the first group, blood specimens were obtained from 13 subjects for QFM analysis 2 weeks before the conditioning regimen (median value 63.2 IU/ml) and 3 months after the allograft infusion (median value 65 IU/ml). The second group consisted of 26 patients; assessment of a QFM was performed 2 weeks before the conditioning regimen (median value 122.8 IU/ml) and 6 to 9 months after HSCT (median value 57.0 IU/ml). We evaluated the change of IFNγ levels in the samples before and after HSCT. In the patients in the first group with non-decreased QFM, infection was present in 3 cases and absent in only 1 case. Decrease of the QFM value was associated with the infection in 6 cases and was not present in 3 cases (p-value = 0.8238). Corresponding blood samples were compared in the second group. In patients with increased/stationary QFM, the infection was observed in 4 and was absent in 9 out of 26 subjects. Decreased QFM result was not associated with infection in 8, more precisely 5 subjects (p-value = 0.5). Conclusions: We did not confirm an association between the elevation of QFM and the decreased risk of infection in the 3-month follow-up neither at 6 to 9 months. No predictive value of QFM in association with the presence of an infection was seen in our cohort of patients after HSCT. QFM results need to be interpreted with caution mainly because of a wide normal value range. Disclosure: Nothing to declare.
Background: An 18-month-old child affected by KTM2A infant acute lymphoblastic leukemia (ALL) underwent allogenic transplant from Match Unrelated Donor (MUD) and achieved measurable residual disease (MRD)-negative complete remission (CR) with 100% full donor chimerism. Six months post-transplant the patient relapsed, with loss of full donor chimerism (45% donor chimerism), and underwent treatment with azacytidine, again reaching MRD-negative CR2 full donor chimerism. A second allo-SCT from a haploidentical donor (mother) was performed. We correlated CD56-NK chimerism with MRD status after second transplant. Methods: Polymerase chain reaction (PCR)-based short tandem repeat (STR) assays were performed on peripheral blood at +15 days and +19 days after second transplant. For each sample, a MACS (Magnetic-activated cell sorting) for CD56 + isolation (StemCell Technologies™ - EasySep™ Human CD56 Positive Selection Kit) was performed and purity of separated cells was evaluated by flow cytometry (BD FACSCanto II) as recommended by manufacturer. DNA was extracted with Maxwell® 16 instrument (Promega) and Maxwell® RSC Whole Blood DNA Kit (Promega) and a AB 3500 (Applied Biosystem) and ChimerMarker™ (SoftGenetics®) analysis software were used for STR analysis. Results: Chimerism analysis performed on peripheral blood on day+15 day after second transplant revealed 99.5% Total Donor 2 Chimerism (Haplo). Interestingly CD56 + NK cells, isolated from the same sample revealed 98.2% from Donor 2 (Haplo) and 0.24% from Donor 1 (MUD). Same results were also observed at day +19 and day +32, although with a decrease of the CD56 + spike observed on day +15 (110/µl. vs 370/ µl). The patient remains in MRD-negative CR at day +45 Conclusions: NK cells have anti-leukemic activity (GvL) in both adult AML and pediatric ALL. We hypothesize that the persistence of donor NK chimerism can improve transplant outcome through elimination of leukemic cells without GVHD. The rapid reconstitution of NK cells after haplo-HSCT is based on expansion of the cytokine-producing CD56bright NK cell subsets. We hypothesize that this patient with KMT2A + leukemia, which has dismal prognosis, had a quick response to treatment (single-agent hypomethylating treatment), due to the persistence of 1st donor NK cells, which persisted after 2nd transplant. The two populations of cells co-exist with no toxicities for the patient and both participated in achieving the MRD-negative CR response. The two populations of NK cells represent a reciprocal tolerance, and a possible synergistic effect. A clear knowledge and understanding of the number and chimerism of NK cells could drive donor NK cell treatment (NK-add back). The tolerance between the 2 NK populations potentially supports a rationale for third party/off shelf NK cell treatment. Disclosure: Nothing to declare.
Background: POEMS syndrome is a rare paraneoplastic condition associated to an underlying plasma cell dyscrasia. The role of autologous peripheral blood stem cell transplantation (aPBSCT) with the use of alkylating agents as conditioning regimen seems to provide optimal outcomes. At present aPBSCT should be considered the first line therapy in young patients with POEMS, eligible for high-dose Melphalan (HD-Mel), in the absence of organ dysfunction. The best treatment before aPBSCT remains to be defined, because of the disease rarity and the heterogeneity of published case series. We therefore decided to collect the patients from major Italian institutions, to describe and compare results and outcomes of patients with POEMS, eligible for aPBSCT. Methods: We collected clinical and laboratory data of patients with POEMS syndrome from 10 Italian centres. We included all the consecutive patients with diagnosis of POEMS undergoing to aPBSCT from 1998 to 2020. Results: Our data set consisted of 44 patients who underwent aPBSCT with a median follow-up of 77 months (37-169 months). Progression free survival (PFS) and overall survival (OS) rates at 6 years for transplanted patients was 65% (49-85) and 92% (84-100), respectively. The cumulative incidence of transplant related mortality and relapse was respectively 4% and 36%. We then divided patients in three subgroups: front-line patients who did not receive any treatment before transplant (15 patients, Group 0), patients treated pre transplant with cyclophosphamide (14 patients, Group 1) and patients treated with other agents such as lenalidomide, chemioterapics or radiotherapy (15 patients, Group 2). The three groups did not show differences in terms of demographic and clinical characteristics. All patients underwent aPBSCT after Mel conditioning regimen (HD-Mel, 200 mg/m2, in 86% of patients) and achieved a successful engraftment. The response rates after transplantation were complete response (CR) in 46%, very good partial response (VGPR) in 23%, partial response (PR) in 18%, stable disease (SD) in 8% and progressive disease (PD) in 5%. The responses (CR vs PR/VGPR vs SD/PD) showed a significant impact in terms of progression free survival (PFS). When comparing the response rate (CR vs PR/VGPR vs SD/PD) between the 3 groups any differences was found (p 0.25). When analysing PFS and OS, the 3 groups did not show significant differences; there was a tendency to unfavourable PFS for patients of Group 1 but no variable was found to negatively affect PFS, neither the treatment chosen before transplantation. In 10 cases it was necessary a re-admission in hospital: in 5 cases for relapse and in 5 for infectious complications. We then considered VEGF levels after aPBSCT and we found out that patients with VEGF levels higher than 758 pg/mL were at higher risk of relapse (AUC 0.86, sensibility 78% specificity 86%), with very high potency in Group 1. Conclusions: This is a relatively large series of patients with POEMS treated with autologous PBSCT. We show durable and impressive PFS and OS, without significant differences between groups of pre-treated patients and patients who underwent front-line aPBSCT. Disclosure: Nothing to declare.
Background: Despite the considerable improvements in newly diagnosed (ND) Multiple Myeloma (MM) outcomes in recent decades, remission times, in particular after ASCT, remain variable. Better outcome prediction post-ASCT could highlight populations in particular need for individualised post-ASCT management and potentially inform optimal allocation of resources. Genetic biomarkers including chromosomal aberrations t(4;14), t(14;16), and t(14;20) translocations, gain of 1q and deletion of 17p, have been associated with adverse outcome, and co-occurrence of ≥2 such aberrations (a Double-Hit) is predictive of especially aggressive disease. We investigated the prognostic impact of genetics in a real-world setting. Methods: Electronic records of all patients who received an ASCT at the Royal Marsden Hospital between Jan 2014 – Oct 2019, were retrospectively reviewed. Cut-off for record review was May 2021. Only patients with sufficiently long follow-up post stem cell re-infusion were considered (>18 months). Cytogenetics and Fluorescent in situ Hybridisation (FISH) data from diagnostic samples were obtained through our reference laboratory’s database and clinical data were obtained by review of electronic records. Only patients with a full complement of cytogenetic risk reported were included (defined as fully reported probes for lesions t(4;14), t(14;16), t(14;20), gain(1q) and del(17p)). Co-occurrence of ≥2 lesions was classified as Double-Hit, and a single lesion as Single-Hit. Presence of amyloidosis/POEMS, participation in interventional clinical trial and transplant-related death or treatment-associated malignancy were pre-specified as exclusion criteria. Progression-free Survival (PFS) and Overall Survival (OS) were calculated from time of stem cell re-infusion. Groups were compared using the log-rank test. The study was approved by the hospital’s internal review board as a service evaluation. Results: We identified 139 patients eligible for evaluation as per above criteria. Clinical and genetic characteristics were representative of a transplant-eligible cohort with regards to age (median 64 years; range 32-76), sex (62% male), ISS (Stage I 22.3%, Stage II 33.8%, Stage III 17.2%, unknown 26.7%) and number of genetic lesions (No-Hit 51%, Single-Hit 39.6%, Double-Hit 9.4%). Double-Hit patients had significantly shorter median PFS (15.1 months, 95% CI: 2.73-NA) compared with Single-Hit (24.6 months, 95% CI: 20.12-27.6) and No-Hit (35.7 months, 95% CI: 28.8-39.7), (p = 0.00063). Median OS for Double-Hit was 49.2 months (95% CI 40.7-NA), whereas it was not reached for the remaining groups (p = 0.034). In only 1.4% of the No-Hit cohort did the myeloma relapse in the first 6 months post-ASCT, whereas relapses in the same timeframe were observed in 7.3% of Single-Hit and 30.8% of Double-Hit cohorts. On univariate analysis, advanced ISS and high-risk cytogenetic lesions (“hits”), were associated with a shortened progression free survival, and significance was maintained for both on multivariate analysis. Age was not associated with PFS in either univariate or multivariate analysis, although in the UK there is no numerical age-driven cut-off for ASCT consideration. Conclusions: We demonstrate here that detailed genetic profiling, specifically the combined assessment of adverse genetics, can help stratify NDMM patients undergoing ASCT in a standard of care setting. This approach can support identifying patients with particular need for intensified monitoring and post-ASCT therapy in a standard clinical setting already at diagnosis. Disclosure: Aikaterini Panopoulou- nothing to declare. Sandra Easdale- nothing to declare. Mark Ethell- nothing to declare. Emma Nicholson- nothing to declare. Mike Potter- nothing to declare. Asterios Giotas- nothing to declare. Helena Woods- nothing to declare. Tracy Thornton- nothing to declare. Charlotte Pawlyn reports personal fees and non-financial support from Sanofi, personal fees and non-financial support from Janssen, personal fees and non-financial support from Celgene, personal fees and non-financial support from Amgen, non-financial support from Oncopeptides, outside the submitted work. Kevin Boyd reports personal fees from Janssen, personal fees from Celgene, personal fees from Takeda, personal fees from GSK, outside the submitted work. Martin Kaiser reports personal fees from AbbVie, grants and personal fees from BMS/Celgene, personal fees and other from Amgen, personal fees and other from Janssen, personal fees from Karyopharm, personal fees from Seattle Genetics, personal fees and other from Takeda, personal fees from GSK, outside the submitted work.
Background: Risk stratification at the initial approach of newly diagnosed multiple myeloma (NDMM) is essential to predict overall survival and response to therapy, especially in patients in whom front-line treatment with autologous stem cell transplantation (ASCT) is being considered. Fluorescence in situ hybridization (FISH) is the basis for cytogenetic risk assessment in various prognostic scores, and the presence of high-risk abnormalities (HRA), or “hits”, can be used to define a high risk disease. The aim of this study was to evaluate the impact of the cumulative effect of HRA on relapse after front-line ASCT. Methods: We retrospectively studied NDMM patients submitted to ASCT with melphalan conditioning, between 2012 and 2020, after induction with proteasome inhibitor (PI) and/or immunomodulatory drugs (IMiD). Only patients with available data on R-ISS staging and FISH analysis for del(17p), t(4;14), t(14;16) and 1q+ were included. We evaluated the progression free survival (PFS) and calculated hazard ratios (HR) for the groups with one hit NDMM (1 HRA) and double or triple hit NDMM (2 or 3 HRA), including multivariate analysis with other factors with impact on PFS. Results: Within a total of 67 patients, 28 had one hit, 5 double hit and 1 triple hit NDMM. Median age at transplant was 60 years (interval 38-71) and 51% of the patients were male. Most patients (70%) were treated with PI and IMiD during induction while 30% received PI with cyclophosphamide and corticosteroids, achieving complete remission (CR) rate of 48%. Stem cell mobilization was performed with cyclophosphamide and G-CSF in 61%, while the remaining received G-CSF alone. CR after transplant was 66% and maintenance treatment was initiated on 55% of the patients. PFS 3 years after ASCT was not achieved in the group of patients without HRA, while the one hit NDMM and the double/triple hit NDMM groups showed a PFS of 21 ± 2 months and 7 ± 6 months, respectively (p = 0.002). On multivariate analysis, 3-year PFS was negatively impacted by the presence of 1 HRA (HR 5.25, confidence interval [CI] 95% 2.04-13.50), more than 1 HRA (HR 4.52, CI 95% 1.63-12.55) and the absence of maintenance treatment after ASCT (HR 7.15, CI 95% 2,83-18,09). There was no statistically significant impact on PFS for age, R-ISS staging, induction treatment and response, mobilization therapy and response after ASCT. Conclusions: Our study suggests that cytogenetic risk stratification through the cumulative effect of HRA independently influences PFS in NDMM patients that underwent frontline ASCT. This strategy could define biological subtypes of NDMM better than the R-ISS and, thus, individualize therapeutic options for these patients. Disclosure: Nothing to declare.
Background: Deletions involving chromosome 13 identified by chromosome studies were one of the first recognized adverse prognostic factors in patients with newly diagnosed multiple myeloma (NDMM). However, recent evidence has challenged its prognostic value due to cooccurrence with other high-risk abnormalities (HRA). Our aim was to analyse the impact of 13p deletions in early relapsed in NDMM after first line autologous stem cell transplant (ASCT). Methods: Single-centre retrospective study of patients with NDMM submitted to frontline ASCT with melphalan conditioning, between January 2008 and August 2020, with at least one year of follow-up. Genetic abnormalities ((17p and 13p deletion, t(4;14), t(11;14), t(14;16)) were identified by fluorescence in situ hybridization (FISH) at the time of diagnosis. Logistic regression was used to identify the variables of prognostic interest that correlate with early relapse, defined as relapse less than 12 months after ASCT. Results: Out of 298 transplanted patients, 171 had complete FISH data at diagnosis. Median age at ASCT was 60 years (range 37-71) and 55% were male. 21% had hypercalcemia, 21% presented with renal failure, 55% anaemia, 18% extramedullary disease and, considering HRA by WHO, 27% were defined as high risk. Patients were treated with combinations of Immunomodulator and Proteosome Inhibitor (PI) 50% and only with PI 46%. Cyclophosphamide was used for stem cell mobilization in 74%, 13% performed tandem ASCT and 40% did maintenance therapy. Multivariate analysisshowed that patients who had higher odds of early relapse were the ones with paraprotein non-IgG/non-light chain NDMM (odds ratio [OR] 3.23, 95% confidence interval [CI] 1.14-9.15), not achieving complete remission (CR) at day +100 of ASCT (OR 2.79, 95%CI 1.14-6.84), no use of maintenance therapy (OR 3.07, 95%CI 1.10-8.60), presence of HRA (OR 2.41, 95% CI 0.98-5.94) and the 13p delection (OR 2.53, 95% CI 1.05-6.19). Amp(1)q was not included by missing data. Age of diagnosis, presence of anaemia, hypercalcemia, renal failure, bone lesions, extramedullary disease at diagnosis, induction chemotherapy regimen and its response, tandem ASCT and year of transplant were not statistically significant. Conclusions: Our study suggests that 13p deletion is an independent prognostic factor for early relapse, regardless of the presence of other HRA, response at day +100, use of maintenance therapy and NDMM immunological subtype. Our findings support the importance of the loss of 13p as a marker of poor prognostic in patients with NDMM. Whether this alteration is a surrogate of other high-risk genetic alterations or carries true biological poor risk needs to be addressed in future studies. Disclosure: Nothing to declare
Background: Consolidative autologous stem cell transplantation (ASCT), incorporating high dose melphalan (HDM) for conditioning, is the UK standard of care for transplant-eligible multiple myeloma (MM) patients. Melphalan undergoes rapid hydrolysis in plasma with a short half-life (60-90 minutes). The duration between HDM and haematopoietic stem cell return (HSCR) varies between institutions, with limited data on optimal timing for patients with renal impairment (RI). In March 2020 we reduced the time between HDM and HSCR from 48 to 24 hours (h) in patients with a glomerular filtration rate (GFR) of <60 ml/min to improve resource utilisation. We retrospectively compared the impact of 24 versus 48 h HSCR on time to neutrophil engraftment (TTNE), hospital stay (HS), need for renal replacement therapy (RRT), intensive care unit (ICU) admission and death. Methods: We identified MM patients with a GFR < 60 ml/min, assessed by 51Cr-EDTA, who underwent ASCT between June-December 2019 and July 2020-July 2021 in our institution, and compared those with 24 or 48 h HSCR after HDM. HS was calculated from the date of HDM administration to discharge. Neutrophil engraftment date was defined as the first of 2 consecutive days with a neutrophil count > 0.5 x 109/L. High risk cytogenetics were defined as per the International Myeloma Working Group definition. Results: Of the 239 MM patients who underwent ASCT, 37 (15%) had a GFR < 60 ml/min (Table 1). Males represented 53% and 55% of the 24 and 48 h groups, respectively. Age, prior therapy lines and stem cell dose were similar between both groups. Median HS for both groups was 17 days (24 h group range: 15-42; 48 h group range: 14-28). Median TTNE was 12 days in the 24 h group (range: 10-13) and 11 days in the 48 h group (range: 10-13; p = 0.143). No patients required ITU admission or RRT. Three deaths occurred in the 48 h group (2 received HDM at 200 mg/m2; causes included COVID and MM relapse). Table 1: Patients’ characteristics. Conclusions: Our results suggest a 24 h interval between HDM and HSCR in patients with renal impairment is safe. A 24 hour HSCR facilitates better scheduling and resource utilisation. More data is needed particularly in those with a GFR < 40/min. Disclosure: Nothing to declare
Background: Allogeneic hematopoietic stem cell transplantation (HSCT) is considered the only potentially curative treatment for multiple myeloma (MM). Before the approval of anti-BCMA chimeric antigen receptor (CAR) T cells, HSCT was the only available cellular therapy based on the graft-versus-myeloma effect of adoptively transferred allogeneic T cells. However relapses after HSCT are frequent and its role in MM treatment remains unclear. The aim of this work was to evaluate the outcome of patients undergoing allogeneic HSCT for MM at our institution to generate a reference survival curve for new upcoming cellular therapies such as BCMA-CAR T cells. Methods: We retrospectively analyzed the outcome of MM patients receiving allogeneic HSCT at our institution between 1990 and 2021. Results: We included 55 patients in the analysis of which 14% received upfront allogeneic HSCT, while 42% and 38% had a single autologous HSCT and a tandem autologous HSCT prior to allogeneic HSCT respectively. Among patients for which cytogenetics characteristics were available (n = 41), 14% displayed high risk cytogenetics. With a median follow-up for alive patients of 5.6 years(y), the median overall survival (OS) was 3.6y and the median progression-free survival (PFS) was 0.8y. The 5y OS and PFS were 41% (30%-58%) and 17% (9%-31%), respectively. The 5y cumulative incidence of disease progression/relapse was 64%±7% and of non-relapse mortality (NRM) was 22%±6%. Patients’ stratification based on disease status at transplantation, previous autologous HSCT, type of allogeneic HSCT (allo-upfront, tandem auto-allo, later allo), conditioning (MAC, RIC), stem cell source (PBSC, BM), donor type, ATG use, and ex vivo T-cell depletion (TCD) suggested a significant improvement in OS [HR 0.46 (95% CI 0.21-1); p = 0.05] and PFS [0.46 (0.24-0.88); p = 0.019] associated with ex vivo TCD. We observed a trend not reaching statistical significance toward reduced PFS [2.1 (0.92-4.7); 0.078] in patients who received one or more previous autologous HSCT. Use of grafts from HLA-matched unrelated donors (MUD) was associated with a significantly reduced OS [2.6 (1.3-5.2); 0.0095] and PFS [3.1 (1.7-5.9); p = 0.00037] compared with grafts from HLA-identical siblings. The 5y OS for patient receiving grafts from SIB donors was 55% (40%-75%) and the 5y PFS was 26% (15%-47%) compared with a 16% (5%-53%) 5y OS and a 0% PFS for patients receiving grafts from MUD. Multivariable analysis considering previous autologous HSCT and ex vivo TCD confirmed the negative impact of grafts from MUD on both OS [2.13 (0.99, 4.59); 0.05] and PFS [2.65 (1.32, 5.32); 0.006] while the impact of previous autologous HSCT and ex vivo TCD was not confirmed. Use of grafts form MUD was associated with a trend not reaching statistical significance towards an increased cumulative incidence of both relapse and NRM. Conclusions: Our study identifies a subset of MM patients, receiving grafts from SIB donors, displaying an improved long-term outcome after allogeneic HSCT, suggesting the curative potential of this treatment approach for a fraction of patients. Our results may help to evaluate the impact of new cellular therapies, including BCMA-CAR T cells, for relapsing/refractory MM. Disclosure: Nothing to disclose. COI Yves Chalandon : consulting fees from MSD, Novartis, Incyte, BMS, Pfizer, Abbvie, Roche, Jazz, Gilead, Amgen and travel support from MSD, Roche, Gilead. Amgen, Abbvie, Janssen, AstraZeneka, Jazz.
Background: Consolidation therapy with lenalidomide (LEN) after induction therapy and transplantation is one strategy that improves outcomes in patients with multiple myeloma (MM). Toxicity due to LEN could lead to delays or reductions in initial dose treatment plan. The objective was to evaluate modifications in the initial treatment plan during maintenance with LEN after autologous transplant (ASCT) in patients with MM (dose reductions or delays due to its toxicity), and analyze differences on long-term outcome. Methods: Retrospective observational study in an oncohematological hospital between June/2018–October/2020. Patients diagnosed with MM who were treated with LEN after ASCT were included: Treatment data were obtained with prescription software system: starting date, LEN dose in each cycle, preplanned delays in cycles (yes or not), dose reductions (yes or not) and dose intensity. Clinical and demographic data were obtained by means of a electronic medical record software. X2 test was used to analyze categorical variables, T-student test for quantitative variables, progression free survival was calculated by means of the Kaplan Meyer Test. Results: 29 patients were included, length of LEN treatment was 15.47 months (range 0.1–33). Most frequent starting dose was 10 mg, treatment delay was needed in 14 patients and dose reduction in 13 patients. Most patients reduced to 5 mg per day (69.2%). A second reduction was needed in 5 patients. Median dose intensity administered was 78.3% (range 34%–101%); progression was detected in 5 cases (17.2%). Median dose intensity was 80.76% and 64.55% (p = 0.092) in patients without/with progressive disease after transplant. Delayed therapy and dose reductions had also a non-significant impact on disease progression [26.6% vs 7.1%, p = 0.164 and 23% vs 12.5%, p = 0.453, respectively]. Conclusions: In most cases, patients needed a LEN dose reduction of 50% (from 10 to 5 mg po daily). Dose reduction of LEN maintenance therapy seemed to be associated to a non-significant increase in disease progression, probably due to the low sample size. More studies are needed to evaluate if dose intensity could be an independent predictive factor for survival in this setting. Disclosure: Nothing to declare
Background: Multiple myeloma (MM) is a neoplastic entity for which Hematopoietic Stem Cell Transplantation (HSCT) is a therapeutic option. Between 30–60% of patients managed with HSCT have pulmonary complications (PC). The aim of this study was to identify the clinical and sociodemographic characteristics associated with the incidence of PC in patients with MM after HSCT (post-HSCT) at Clinica FOSCAL. Methods: An observational, descriptive, retrospective cohort study was carried out in adults with a diagnosis of MM post-HSCT in a clinic in Colombia, between July 2013 and April 2020. Sociodemographic, clinical and hematological variables related to post-HSCT were established. All patients had a chest image (X-ray or Computed Tomography) prior to HSCT Results: A total of 73 patients were included. 54.8% were women, with a mean age at the time of HSCT of 58 years, and a mean time between diagnosis and HSCT of 2 years. As a history of lung disease, it was found that 15% had presented pneumonia, 6.8% asthma, 5.5% Chronic Obstructive Pulmonary Disease, and 1.4% both bronchitis and pulmonary hypertension. 56.2% had a normal chest image. The overall incidence of PC post-HSCT was 19.2%, with 8.2% occurred before day 100 post-HSCT and were entirely infectious complications, and 10.9% occurred after 100 days post-HSCT, 9.5% being infectious and 1.4% not infectious (asthma). Of the total sample, patients who developed PC post-HSCT corresponded to 18% and 20% of men (RR = 0.98) and women (RR = 1.02) respectively, with a mean age of 62.7 years (RR = 1.02), and 18.2% of patients were from urban areas. In the medical history, these patients with PC post-HSCT corresponded to 20.4% of the patients with normal BMI (RR = 0.94) and 18.2% of those who were overweight (RR = 1.05). Regarding comorbidities, 20.8% had chronic kidney disease (RR = 1.13), 17.8% had hypertension (RR = 1.39) and 7.4% had dyslipidemia (RR = 1.14). Likewise, 25.9% of the smokers/ex-smokers (RR = 1.1) and 20% of those who reported alcohol consumption (RR = 1.36) had PC post-HSCT. Regarding the hematological variables, it was observed that 21% of the patients with MM IgG Lambda (RR = 0,.7), 19.4% with MM IgG Kappa (RR = 1.05), 22.8% in stage 3 according to ISS (RR = 5.7) and 21.3% with Durie Salmon II (RR = 1.75) developed PC post-HSCT, as well as 19.1% of patients with MM who received myeloablative conditioning (RR = 1.8) and 22.2% who received Cybord (Bortezomib + Cyclophosphamide + Dexamethasone) (RR = 0.88). None of these results showed statistically significant differences. Conclusions: This study allowed to identify an incidence of PC of 19.2% in MM patients post-HSCT in a university Clinic in Colombia, without statistically significant relative risk for sociodemographic, clinical, or hematological variables. Clinical Trial Registry: N/A Disclosure: N/A
Background: Serum free light chain (FLC) ratio is a sensitive method to detect light chain hyper-production and is a biomarker of Multiple Myeloma (MM) progression from premalignant conditions. We investigated the relationship between FLC ratio at diagnosis and clinical/MM characteristics and the predicting role of FLC ratio in influencing progression-free (PFS) and overall survival (OS). Methods: A total of 33 MM patients who underwent autologous stem cell transplantation after induction therapy at the Hematology and Transplant Center, University Hospital “San Giovanni di Dio e Ruggi d’Aragona” of Salerno since 2015, was included in this retrospective study (Table 1). Quantification of FLC ratio at diagnosis was performed by nephelometric assays and standardized (sFLC) as follows: involved/uninvolved chain. Correlations between sFLC ratio and clinical/MM parameters were investigated by univariate and multiple linear regression models, while the influence of sFLC ratio on PFS and OS was analyzed by Mantel-Cox proportional hazard regression model. Results: Effects of gender (male, P = 0.28), age (P = 0.75), MM type (micromolecular, P < 0.005), M-protein level (P = 0.05), free light chain type (lambda, P = 0.34), β-2 microglobulin (P < 0.005), LDH (P = 0.66), albumin (P = 0.9), flow cytometry plasma cell count (P = 0.62), and extramedullary disease (yes, P = 0.76) on sFLC ratio were investigated by univariate linear regression analysis. By multiple linear regression, only micromolecular MM (P = 0.05) and β-2 microglobulin (P < 0.005) were significantly associated to sFLC ratio. Furthermore, sFLC ratio inversely correlated with glomerular filtration rate at diagnosis (r = -0.45; P = 0.009) by Pearson analysis. No influences on PFS [median PFS 26 months; HR:0.93 (CI: 0.85-1.03); p = 0.21] and OS [median OS 43 months; HR:0.97 (CI:0.92-1.02); p = 0.27] by sFLC ratio were observed. Conclusions: Diagnostic and predictive roles of serum sFLC ratio in MM has been largely evaluated in recent years. In our study, sFLC ratio was significantly associated with micromolecular MM and β-2 microglobulin likely due to a delay in MM diagnosis ultimately leading to higher disease burden, especially without heavy chain M-protein at serum electrophoresis. Furthermore, the inverse correlation between sFLC ratio and glomerular filtration rate might be related to an early damage of FLC excess on renal tissue. In conclusions, our preliminary results confirmed the importance of early sFLC evaluation for diagnosis of micromolecular and lower disease burden MM, reducing the risk of renal damage. However, further validation on larger and prospective studies are needed. Disclosure: Nothing to declare
Background: Novel drugs induction followed by autologous stem cell transplantation (ASCT) is the standard regimen for transplant-eligible patients with multiple myeloma (MM). However, approximately 20% of patients will have early relapse within 2 years after ASCT, which are defined as high risk myeloma. Tandem ASCT or allogeneic stem cell transplantation (allo-SCT) may overcome the poor prognosis of high risk myeloma. However, prospective controlled studies evaluating the role of tandem ASCT and allo-SCT in high risk myeloma are lacking. Thus we aimed to assess effectiveness of tandem ASCT with allo-SCT. Methods: In our phase 3 biological assignment trial, we enrolled patients with multiple myeloma attending 8 transplant centers in China. Patients (<70 years old) with adequate organ function who had completed four to six cycles of systemic antimyeloma therapy were eligible for inclusion. We assigned patients to receive an myeloablative allo-SCT or tandem ASCT on the basis of the availability of an HLA-matched sibling donor. We used the Kaplan-Meier method to estimate differences in time-to progression survival (TTP; primary endpoint) and overall survival (OS) between the two groups. Results: Between Jan 1, 2018 and Jun 30, 2021, we enrolled 49 patients, of whom 40 were enrolled to tandem ASCT group and 9 were enrolled to allo-SCT group. In intention to treat population, Kaplan-Meier estimates of TTP (not reach and 15.0 months, p = 0.010) and OS (not reach and 17.3 months, p < 0.0001) were better in Tandem ASCT group. TTP of those received double ASCT in Tandem ASCT group was better than those received only one time of ASCT (33.5 months and 25.3 months, p = 0.037). Cumulative nonrelapse mortality after allo-SCT (n = 2, because of GVHD) was higher than tandem ASCT (n = 1, because of infection), p = 0.012. Conclusions: Tandem ASCT is effective that myeloablative allo-SCT for patients with high-risk multiple myeloma. Clinical Trial Registry: ChiCTR2100046510; http://www.chictr.org.cn/index.aspx. Disclosure: Nothing to declare
Background: Standard treatment of MM consists of induction chemotherapy followed by autologous hematopoetic cells transplant (autoSCT), but this scheme doesn’t present a curative potential. The only one modality with curative potential remains allogeneic hematopoetic cells transplant (aloSCT). With the knowledge of high mortality & morbidity related to the tratment (TRM) in comparison to tratment with novel drugs, there is no consensus in indication of aloSCT in MM, also identification of ideal patients profiting from this method is difficult. Therfore a retrospective unicentric study of patients after aloSCT for MM was performed. Methods: 36 consecutive patients with MM transplanted in University hospital in Pilsen between years 2000-2020. Median of age 52 (38-63), 26 men (72%), 19pts transplanted between 2000-2010 including (52,8%). Median of previous regimens 2 (1-5), 92% of pts underwent autoSCT after induction, in 9 patients (25%) elective auto-alo tandem. 12 patients (33,3%) transplanted with resistant disease. Novel drugs before aloSCT in 16pts (44,4%). Conditioning mostly non-myeloablative (Flu-Mel, n = 33, 91,7%), non-related donors (n = 20, 56%), identical related donors (n = 14, 38,9%), haploidentical (n = 2, 5,5%). Results: With median follow up of 85 months (8-178) 27 patients (75%) died: TRM in 11 patients (31%), relaps in 16 pts (44%). 9 patients alive, 6 of them with relaps/profression. OS/PFS medians 30 (10-60), respectively 15 months (11-175). OS in 1 and 5 years 55% and 30,3%. Patients transplanted without resistant disease had statistically significantly prolonged OS (HR 0,43, 95%CI 0,18-1,01, p = 0,05), not proven in PFS (HR 0,75, 95%CI 0,25-2,21, P = 0,57). Neither period of tranplant (2000-2010 vs 2011-2020), nor previous treatment with novel drugs showed the impact on survival. Conclusions: Accordingly to international litarature, our data prove curative potential of aloSCT in some carefully selected high risk patients, it proves the potential to prolong survival in these patients even if with active disease, but not derogating the QoL significantly. GvM efect shows like potential platform for following therapy. Even though we cant precisely define patients profiting from alo SCT, we should still consider aloSCT as an efective treatment in suitable high risc patients. Disclosure: No disclosures
Background: We aimed to evaluate the outcome and cost-effectiveness of salvage ASCT (sASCT) for multiple myeloma (MM) relapsing after prior ASCT in a cohort of patients from China. Methods: Thirtheen patients relapsing after upfront ASCT retreated with sASCT were enrolled. The outcome and cost-effectiveness were compared with those of patients retreated without sASCT. Results: The median age of the patients at the first and second ASCT were 52.0 and 55.0 years. Twelve patients (92.3%) achieved a response of VGPR or above after sASCT. Compared with patients relapsing during the same period who were retreated with novel agents (NAs) (n = 26)or with conventional cytotoxic drugs (CCs) (n = 4), sASCT patients had longer PFS (37.800 months in the sASCT group vs 10.533 months in the NA group vs 1.933 months in the CC group, P <0.001) and longer OS (45.867 months in the sASCT group vs 15.633 months in the NA group vs 1.933 months in the CC group, P <0.001). The sASCT group demonstrated lower cost-effectiveness ratio (¥179126 per life year) than those of patients retreated without sASCT (¥330474 per life year, P = 0.049). Eight (61.5%) patients used cryopreserved stem cells from the first ASCT for sASCT, all of which achieved successful engraftment. Three (60.0%) of the 5 remobilized patients failed to collect sufficient stem cells, 2 of which failed hematopoietic reconstruction. Conclusions: sASCT is a cost-effective treatment option for MM patients relapsing from upfront ASCT. Reserved stem cells from the first ASCT are of significance for hematopoietic reconstruction of sASCT. Disclosure: Nothing to declare
Background: Amyloid light chain amyloidosis (ALA) is characterized by the pathologic production of fibrillar proteins comprised of monoclonal light chains which deposit in tissues and cause organ dysfunction. The therapeutic approach is based on the use of drugs widely used in the treatment of multiple myeloma and performing autologous stem cell transplant (ASCT). Although the median survival of patients undergoing ASCT is quite relevant (10 years), only 20% of patients are eligible for ASCT due to delayed diagnosis and strict eligibility criteria for ASCT. Methods: Retrospective review of ALA patients submitted to ASCT in one institution (2017-2021). The overall survival (OS) and progression-free survival (PFS) was estimated by the Kaplan‐Meier method. ASCT toxicity was evaluated by CTCAEv5.0. Results: A total of 6 patients had undergone ASCT (male = 1, 16.7%), with a median age of 66 years (51-68) and ECOG performance status 0-1. Median visceral organ involvement at diagnosis was 2.5 (1-4). All patients receive induction treatment before ASCT with cyclophosphamide/bortezomib/dexamethasone and peripheral blood progenitor cells (PBPCs) mobilization was accomplished with granulocyte-colony stimulating factor (G-CSF) alone (n = 5, 83.3%) or G-CSF plus cyclophosphamide (n = 1, 16.7%). After induction treatment, haematological complete remission (CR), very good partial response (VGPR) and partial (PR) response were 16.7%, 66.7% and 16.7% respectively, whereas organ response was 66.7%. All patients received melphalan as conditioning [200mg/m2 (n = 3, 50%) and 140mg/m2 (n = 3, 50%)]. Median inpatient stay during ASCT was 24 days (17-52). All patients received supportive care specific for ALA: infusion of PBPC performed through peripheral venous access; graft syndrome prophylaxis with 0.5mg/kg/day prednisolone from day +7 until neutrophils ≥500/mm3 and eviction of G-CSF after PBPC infusion. Toxicity was frequent and severe. All patients developed febrile neutropenia; median time until neutrophils>500/mm3 was 11 days (10-15). Two (33.3%) patients had gastrointestinal mucositis (grade 2-3), and all had grade ≥2 oral mucositis. Three patients developed renal failure requiring renal replacement therapy. The four patients with cardiac involvement at diagnosis, developed cardiac complications (two during hyperhydration of the conditioning regimen). One patient died from sepsis and multiple organ failure. At day +100 post-ASCT, haematological CR and VGPR were 60% and 40% respectively. During monitoring period (9-60 months) median OS and PFS was not achieved. Two patients relapsed after 4 and 9 months after ASCT and one patient died without apparent cause 31 months after ASCT. Conclusions: ASCT in ALA was first performed in our institution in 2017. Therefore, the number of patients included in this analysis is limited, which does not allow us to make comparisons with studies already carried out in this area, nor to establish logistic regression models to assess the clinical factors that may have an impact on transplant success in our population. Although the number of patients, our real-life results demonstrate that ASCT appears to be an effective therapy for patients with ALA, with durable response rates. Additionally, despite the strict eligibility criteria for ASCT, the occurrence of serious complications is still noticed, so these patients must always be guided by a dedicated and trained team. Disclosure: Nothing to declare.
Background: The role of mutations in diagnosis, prognosis, treatment and even follow-up post HSCT in MDS has become increasingly relevant from a diagnostic point of view, especially in cases with cytopenias without blasts. The presence of mutations, epigenetic regulation or splicesseosome regulation can be of great diagnostic value. In treatment, the presence of the SF3B1 mutation indicates the possibility of treatment with luspatercept; in the case of TP53 mutation, the possibility of using APR-246 or even Venetoclax associated with Vidaza can be very efficient, opening up a great discussion of a more rational therapy. Regarding HSCT, despite being a single curative therapy, its precise indication is essential, because even in the best centers, morbidity and mortality is still necessary, and a fundamental aspect is to select the best donor, cell source and type of conditioning, which force us to define post-HSCT goals. In view of these considerations, we decided to study two aspects of this work in patients from the Latin American HSCT registry in MDS: donor type and the correlation of stratification with R-IPSS with overall survival, considering that we do not have molecular analysis as a tool, just like most places in Latin America. Methods: We analyzed data from 331 patients with MDS from the transplant registry of 32 centers in Latin America from 1989 to 2021. Statistics were performed using SPSSv.23.1, considering a significant p < 0.05. Results: There was a predominance of males (59%) and whites (87%). The median age was 46 years. According to the Prognosis Scoring System (IPSS-R), patients were classified as very low (0.6%), low (10.6%), intermediate (24,1 %), high (18.7%) and very high risk (5.1%). About 40% did not have IPSS-R data. In myeloablative conditioning (MAC) (73,7%), the regimens were bulsulfan/fludarabine (39,58 %), bulsulfan/cyclophosphamide (30,51%) and regimes with total body irradiation (7,85%). At reduced intensity/non-myeloablative regimen (RIC) (26,3%) were bulsulfan/fludarabine (40,79%), fludarabine/melphalan (44,71%) and regimens based on total body irradiation (11,48%). The cell source was bone marrow (BM) (54,08%), peripheral blood (PB) (44,11%) and umbilical cord blood (1.8%). In 69,18% of cases, donor was related, 22,96% unrelated and 7,85% haploidentical. The main post-HSCT complications were infections (92.1%) of which 48,8% included CMV infection. Frequency of death was 39,88%(n = 132). When comparing the donor type, haploidentical group had higher Overall Survival (OS) but there was no statistical difference in HSCT for haploidentical (63,30%) related (59,90%) and unrelated (58,70%) p = 0.9614. Regarding R-IPSS stratification, the 5-OS was 57.1%, 50%, 50.5% and 16.4% for Low, Intermediate, High and Very High Risk, respectively. Conclusions: Haplo-hematopoietic stem cell transplantation is a valuable alternative in the absence of an HLA-compatible donor in patients with myelodysplastic disease. R-IPSS stratification is a useful prognostic tool in HSCT, especially in those centers where molecular analysis is not yet a reality. Disclosure: Nothing to declare
Background: Hypocellular variant myelodysplastic syndrome (Hypo MDS) accounts for approximately 10 to 20% of all MDS cases. Diagnosis can be challenging due to clinical and laboratory similarities with aplastic anemia. Some studies indicate a better response to treatment and a better prognosis in this group of individuals. Allogeneic HSCT is indicated for patients with high risk R-IPSS or transfusional dependence. The objective was to evaluate the HSCT results in patients with Hypo MDS from 32 centers in Latin America. Methods: It is multicenter study of Brazil, Uruguay and Argentina. Data of 331 patients (both sexes) from 1989 to 2021 were analysed. Patients were stratified according to the Revised International Prognostic Classification System (R-IPSS). Conditioning regimen and supportive treatment were performed according to the protocol of each institution. Statistical analysis by GraphPad Prism version 5.0 and SPSS software v.23.1 and v.24, considering significance of p < 0.05. Results: The mean age was 46,29 years. There was a predominance of male patients (n = 194; 59%) e Caucasian (n = 288; 87%). Prevalence of Hypo MDS was 11,1% (n = 37). According to R-IPSS patients were Intermediate (24,17%), High risk (18,73%), very hig risk (5,14%) and low risk/very low risk (11,17%). No response was observed in 40,79% cases. The Conditioning regimen used was Myeloablative (244; 73,72%) and Reduced Intensity/Nonmyeloablative (87;26,28%). Most donor type was Related (229; 69,18%). Cell sources were: bone marrow (179; 54,08%), peripheral blood (146; 44,11%) and umbilical cord (6; 1,81%). Treatment Prior to HSCT was performed in 217 (65,56%) cases with predominant use of Chemotherapy (134; 61,75%). Overall survival in 5 years was 28,4%. According to the cell source, OS was higher when used peripheral blood compared to bone marrow and umbilical cord (p < 0,001). There was no difference in overall survival according to treatment prior to HSCT, type of donor and conditioning regimen. Hypo MDS patients had a better 3-years OS with a tendency of statistical significance (p = 0, 07). Specific data analysis is in progress to better understand the factors that may influence these results. Conclusions: Patients with Hypo MDS seemed to present a tendency to better OS. Absence of blasts and the molecular profile as other specific aspects may be the differentials in this response to treatment. Further specific studies need to be carried out to confirm these observations. Disclosure: Nothing to declare
Background: Patients with secondary myelodysplastic syndrome (t-MDS), developing after previous chemoradiation therapy, have a more unfavorable course and prognosis compared to patients with initially diagnosed MDS (de novo MDS). Methods: A single-center retrospective cohort study included 60 patients: 30 with t-MDS (therapy –related myelodysplastic syndrome),30 with de novo MDS. A group of 30 patients (10 m/20 f) with t-MDS who developed after chemotherapy or radiation therapy of a previous malignant neoplasm (MNO) was analyzed. The control group included 30 patients (10 m/20 f) with de novo MDS, matched by age at the time of MDS diagnosis, sex and risk in accordance with IPSS-R and WPSS.Overall survival (OS) was assessed, which was defined as the time from diagnosis of MDS to death from any cause Results: All patients were included in the analysis. Median OS for t-MDS was significantly shorter than for de novo MDS (13 and 48 months, respectively, p = 0.03). The frequency of transformation into acute myeloid leukemia (AML) didn’t differ significantly in the t-MDS and de novo MDS groups (33% and 30%, respectively, p = 0.7). The median time to transformation into AML in the t-MDS group was 2.6 months, and in the de novo MDS group - 8.6 months (p = 0.27). Risk factors have been analyzed in patients with t-MDS. The structure of previous malignant neoplasms included classical Hodgkin’s lymphoma 30% (n = 9), non-Hodgkin’s lymphomas 23% (n = 7), breast cancer 20% (n = 6), acute leukemia 7% (n = 2), chronic lymphocytic leukemia 7 % (n = 2) and other solid tumors 13% (n = 4). Therapy for previous malignant neoplasms included chemotherapy in 37% (n = 11), radiation therapy in 3% (n = 1), and combination therapy in 50% (n = 15). There was no effect of the type, status, and treatment option of prior cancer on OS in patients with t-MDS. At the same time, the predictive value of risk stratification proposed by the MD Anderson Cancer Center group for t-MDS (TPSS) was demonstrated. Allo-HSCT was performed in 37% of patients (n = 11) with t-MDS. The median OS for patients without allo-HSCT was significantly shorter than for patients undergoing allo-HSCT (8 and 38 months, respectively, p < 0.001). There was a tendency to an increase in OS in patients with allo-HSCT in the first year after diagnosis compared with patients who underwent allo-HSCT at a later date, but the statistical significance of the differences was not confirmed, probably due to the small sample size (median OS 38 and 22 months, respectively, p = 0.8). Conclusions: Patients with t-MDS have a significantly poorer prognosis compared to patients with comparable risk with de novo MDS, which makes it inappropriate to use standard prognostic scales for patients with t-MDS.Allo-HSCT increases the survival rate of patients with t-MDS and requires an earlier time frame. Disclosure: Nothing to declare
Background: The study aims to investigate the applicability of a novel method for spleen size reduction and splenic activity down-regulation in patients with myelofibrosis. Partial embolization of splenic artery (PESA), in comparison with conventional methods, such as surgical splenectomy or splenic irradiation, is hypothesized to minimize the risk of known complications - bleeding, thrombosis and infection with encapsulated bacteria - therefore improving outcomes and reducing mortality. Methods: A total of 12 patients were referred for PESA. Two patients with suspected hypersplenism-impaired engraftment had PESA in post-transplantation setting as a salvage procedure with the purpose of maintaining the graft function. Next, seven transplant-eligible patients underwent PESA because of splenomegaly and/or hypersplenism to reduce risk of graft failure. In three transfusion-dependent patients with massive splenomegaly and portal hypertension, PESA was conducted as a preparation for otherwise high-risk surgical splenectomy. The procedure has been evaluated regarding perioperative recommendations, occurrence of infections, bleeding and thrombotic events, management of complications and hospitalization time. Results: PESA causes necrosis of 70-80% of splenic tissue and is associated with severe pain in the following days after the procedure. Successful pain management was achieved by the use of patient controlled analgesia (PCA) with the use of oxycodone and administration of coanalgesics. PESA requires access to splenic artery. Intravascular catheter manipulation within the pancreatic area is suspected to cause symptoms similar to acute pancreatitis, inducing inflammation and thrombosis in splenic and portal vein. All patients received a prophylactic dose of anticoagulant starting 12 hours after the procedure. To maintain anticoagulant treatment in patients with thrombocytopenia, PLT count >30 G/l was sustained through daily platelet transfusion. Two thrombotic events were recorded. One patient, who had PESA with subsequent splenectomy, developed massive splenic vein thrombosis after surgery, ultimately leading to the rupture of the vein, intra-abdominal bleeding and death in hypovolemic shock. We recommend the use of therapeutic dose of anticoagulants in patients that are referred for PESA as a preparation for splenectomy. Additionally, we believe it’s reasonable to keep the patient on the stable dose of JAK-inhibtor throughout the procedure. All patients presented with a rapid increase in inflammatory markers over the first two days after the procedure. One patient has been discharged shortly after the procedure with the use of oral antibiotic prophylaxis only and required rapid rehospitalization due to sepsis. In remaining patients, an i.v. antibiotic (meropenem) was maintained until normalization of inflammatory markers and no detectable source of infection has been identified. We recommend the use of broad-spectrum antibiotics (preferably carbapenem) as a perioperative prophylaxis. The median time of hospitalization for PESA was 22 days. Notably, successful engraftment has been achieved in all patients who had PESA either prior or post transplantation procedure. Conclusions: We revealed several applications of PESA and prepared a management algorithm to ensure patient’s safety. However, the applicability of PESA, including proper patient selection, timing in relation to allo-SCT and long-term outcomes remains to be further investigated. Disclosure: Nothing to declare
Background: Allogeneic HSCT is capable of inducing a significant proportion of patients with MF into a clinical and molecular remission. Less clear is the role of bone marrow morphology beyond 6 months from transplant. We aimed to correlate clinical, hematologic, molecular, chimerism data with marrow morphology from bone marrow biopsies, in MF patients 6-48 months post-allogeneic HSCT Methods: Data were derived from an electronic database into which patient, disease, and transplantation characteristics had been entered prospectively. All patients provided written informed consent for research studies using forms approved by the Institutional Review Board. Eligible for this study were patients with a negative driver mutation and full donor chimerism. Forty two, out of 50 consecutive MF allografts, met the inclusion criteria. They are 21 male and 21 female, median age was 59 years (41-71 range); 7 patients had diagnosis of primary myelofibrosis and 35 secondary MF, 19 had high and 23 intermediate-2 risk disease (DIPSS) 29 have JAK positive disease. Before transplant all the patients showed severe (MF3) in bone marrow biopsy. Median follow up was 642 days (range 210-1907). All patients underwent a routine bone marrow biopsy between 6-24 months post-HSCT. Results: Bone marrow biopsies were scored as follows: moderate severe fibrosis (MF2-MF3), n = 19, 45%; normal or minimal fibrosis (MF0-1), n = 17, 40%; not evaluable n = 4 (10%), non performed n = 2 (5%). When stratified by interval from transplant, the proportion of MF2-MF3 was 53%, 50%, 50% and 29% at <365, 366-730, 731-1095 and >1095 days from transplant (Table 1). Two patient (5%) showed a poor graft function at the time of bone marrow biopsy, all other patients had normal/near to normal blood counts . Nintyfive percent of these patients were off immunosuppressive therapy at the time of bone marrow biopsy. Conclusions: A significant proportion of patients with negatifve driver mutations, complete donor chimerism, and normal blood coutns, still exhibit marrow fibrosis long term post transplant: there seems to be a trend for a reduction of MF2-MF3 patients with time. Additional investigation in these patients may highlight the pathogenesis of fibrosis in patients with MF. Table 1. Driver mutation, Chimerism and Bone marrow fibrosis at differet time after allogeneic transplantation. Disclosure: none
Background: Allogeneic stem cell transplantation (alloHSCT) is currently the only treatment option with curative potential in patients with PMF. Relapse prevention and treatment remains a challenge in these patients. Here were report a case of extramedullary relapse of myelofibrosis and its molecular profile after alloHSCT. Methods: А 39-years old female was diagnosed with CALR type 1-positive PMF, 46XX, IPSS low risk, DIPSSplus intermediate -2 risk, four years before alloHSCT. During 12 months the patient received therapy with pacritinib in clinical trial which was accompanied by reduction of spleen volume and transfusion requirements. Just before alloHSCT splenectomy was performed. After short course of ruxolitinib alloHSCT from 10/10 –HLA matched related donor with peripheral stem cells (10.5 x 109 СD34 + cells/kg) was performed. Conditioning regimen consisted of fludarabine (180 mg/m2), busulfan (10 mg/kg p.o.). Post-transplant cyclophosphamide was administered at 100 mg/kg at day +3, +4, and ruxolitinib 15 mg was used from D + 5 till D + 100 as graft versus host disease (GVHD) prophylaxis. Library for next-generation sequencing was created with QIAact Myeloid DNA UMI Panel, sequencing itself was performed on Illumina MiSeq System. We used our in-house script based on GATK Best Practices for primary data analysis. All variants with population frequency exceeding 1%, confirmed by less than 100 UMI, or with allele frequency less than 2% were filtered out. We used PyClone with standard parameters to infer clonal evolution. Results: Platelet and neutrophil engraftment was documented at D + 35 and D + 38 and full donor chimerism and molecular remission was achieved. Acute GVHD grade 3 with liver (2) and skin (1) evolvement at D + 50 was documented and resolved after cyclosporine A administration. Bone marrow fibrosis regression from grade MF 3 to MF 0-1 was documented at D + 749. At D + 1045 the patient experienced mild pain and additional mass in her right shin. At D + 1344 PET/CT revealed additional metabolic active mass (SUV = 8.66) 38 x 27 x 63 mm with cortical thinning and destruction. Histological examination documented PMF relapse with reactive osteoblasts proliferation. At D + 1401 thrombocytopenia 30-14 x 109/l, anemia – 92 g/l, peripheral blasts – 5%, bone marrow blasts – 5% were documented. Due to hematological and extramedullary relapse fludarabine and cytosine arabinoside – containing chemotherapy was started. However, patient died due to infection complications after chemotherapy completion. Targeted next-generation sequencing was carried out in four samples: (1) blood taken during the onset, (2) spleen after splenectomy, (3) extramedullary hematopoiesis elements in bone in the course of late bone marrow remission, and (4) bone marrow in relapse. Clonal reconstruction revealed three relevant clones: the first, which was present in the onset, contained six mutations: ETV6:p.Asn227fs, ASXL1:p.Glu635fs, TET2:p.His1737fs, EZH2:p.Glu726Lys, EZH2:p.Lys665_Tyr666insValTyrAspLys, and BRAF:p.Asp594Asn. It was discovered in all samples. The second emerged in the third timepoint with SETBP1:p.Ser869Asn mutation, and was also present in the fourth sample. The third clone, characterized by SRSF2:p.Pro95Arg mutation, appeared only in the bone sample (figure 1). Conclusions: Bone marrow and extramedullary PMF relapse after alloHSCT may be characterized by diverse mutational profile. Extended genetic sequencing of PMF relapses may help to identify new therapeutic strategies in case of treatment failure following allo-HSCT. Disclosure: This work was done as a part of Russian Science Foundation grant № 17-75-20145-P.
Background: Primary myelofibrosis (PMF) or post-essential thrombocytosis/polycytemia vera myelofibrosis (SMF) is one of the Philadelphia-negative myeloproliferative neoplasms which has a poor prognosis with a survival of 6 years, approximately. Allogeneic hematopoietic stem cell transplantation (allo-HSCT) can cure a substantial number of patients (pts) but is still not universally applicable due to toxicity which leads to therapy-related morbidity and mortality. Methods: This is a retrospective study to analyze variables associated with pts’ overall survival (OS) after allo-HSCT. The study population included 16 pts who were diagnosed of myelofibrosis from January 2005 to December 2020 and sent to Hospital Universitario Central Asturias to assess allo-HSCT. Nine (56.3%) were male. Median age was 51 years old (range: 32-66). Seven pts (43.75%) received cells from matched siblings, 7 from unrelated donor and the remainder haploidentical-HSCT. Stem cell source were: peripheral blood (n = 14) and bone marrow (n = 2). Results: Only 7 pts (43.75%) had PMF; 6 pts (37.5%) progressed from ET and 3 from PV to SMF with a median time of 12.6 and 13.6 years, respectively. Median time from PMF diagnosis to allo-HSCT was 12.5 months. All pts had one of the driver mutations; 13 pts JAK2 mutation (81.25%) and 3 pts CALR mutations, none MPL or triple-negative. Fourteen pts (87.5%) presented constitutional symptoms and all had splenomegaly with a mean spleen size of 19.3 cm before allo-HSCT. Thirteen (81.25%) pts had anemia (hemoglobin <10g/dL); 3 of them (23%) with transfusion dependency. Ten pts (62.5%) had circulating blasts ≥1%, 2 pts (12.5%) leukocytes higher than 25 × 109/L and another 2 pts (12.5%) platelets lower than 100 × 109/L. Since ruxolitinib is approved for MF, the drug was used in 11 pts (68.75%) before allo-HSCT. Six pts (37.5%) developed poor graft function or graft failure. Median time to achieve 500 and 1.000 neutrophils/µL and 20.000 and 50.000 platelets/µL were 19, 21, 24 and 48 days, respectively. Thirteen pts reached complete chimerism with a median time to achieve it of 7.3 months. Bone marrow biopsy could be performed at day 100 in thirteen pts and 5 (38.46%) had grades 0 or 1 fibrosis. The most frequent complications were GVHD, 62.5% and 18.75% of pts developed aGVHD (≥grade II) and cGVHD, respectively. Six pts (37.5%) died, 3 out of 6 due to aGVHD, remainder due to progression, second neoplasm and sepsis. Median and maximum follow-up were 51 and 131 months, respectively. Overall survival at 5 years was 54%. When using IPSS, one year survival was 100% for intermediate-1, 79% for intermediate-2 and 25% for high risk group, but differences were not significant maybe due to the low number of cases. Conclusions: In our series of pts, only 7 pts had PMF and the remainder were SMF developed more than 10 years after PV/ET. Most patients had constitutional symptoms and splenomegaly that were treated in 68.75% of cases with Ruxolitinib. The most frequent complication of transplantation was GVHD that was involved in 66.7% of deaths. Disclosure: none
Background: Primary myelofibrosis (PM) is a malignant hematologic disease, characterized as chronic myelopriferative disorder. Allogeneic hematopoietic stem cell transplantation (allo–HSCT) is the only therapeutic option with curative potential, but remains reserved for a minority of patients (pts).We report the results of a series of 6 pts who underwent this procedure over a period of 206 months. Methods: between May 2002 and July 2019, allo-HSCT performed in 6 pts with PM. The median age is 37 years (5-48) of which one of them 5 years. The sex-ratio (M/F): 1. The average delay diagnosis-transplant is 29 months (12-89). Four pts (66,6%) had a history of red blood cells (RBC) transfusions, including 1 (16.6%) with more than 20 RBC units. Two pts were splenectomized before the transplant and 2 pts received a previous treatment based on Hydroxyurea. According to the DIPSS score: Intermediate-2 (5 pts), high (1 pt). Conditioning regimens based on Busulfan were used in all pts. Prophylaxis GVHD consisted on Ciclosporin–Methotrexate. The grafts used are peripheral blood stem cells with an average rate of CD 34+ cells: 8,89 x 106/kg (6,11-12,9). At July 2021, the minimal follow-up was 24 months and maximal was 206 months. Results: Aplasia was observed in all pts with median day of neutrophils engraftment was 12 days (8-15). Early rejection was observed in one patient. Grade IV Acute GVHD occurred in one pt (25%). CMV reactivation is noted in 3 pts (60%). After a median follow-up of 30 months (24-36), 2 pts are alive with a strictly normal blood count and 4 pts (66.6%) died within 3 (50%) from TRM (severe infection:2, GVHD:1) and one patient following early rejection. Conclusions: Allo–HSCT remains the only curative treatment in PM, which can restore hematopoiesis on the one hand and prevent progression to acute leukemia on the other. The Fludarabine-Busilvex-antilymphocyte conditioning regimen is well tolerated and seems to give better results (50%). Disclosure: Nothing to declare
Background: Natural Killer (NK) cell-based therapies hold great promise in treating multiple myeloma. One method to enhance NK cell specificity against myeloma is antibody dependent cellular cytotoxicity through CD16 receptor. We targeted B7-H3 (CD276) because its expression in myeloma is associated with decreased progression free survival, it exhibits low expression on healthy tissue, and it is expressed on myeloid derived suppressor cells (MDSC), which promote myeloma growth. Methods: We developed a tri-specific killer engager (TriKE) with camelid single-domain antibody (sdAb) fragments against B7-H3 and CD16, linked by IL-15, to enhance NK cell killing of myeloma. Delivery of IL-15 by TriKE supports NK cell proliferation while avoiding off-target effects on T cells. We found high expression of B7-H3 on the myeloma lines RPMI-8226, U266, and MM1S and relatively low expression on H929 by flow cytometry. We compared the ability of peripheral blood NK cells with or without B7-H3-TriKE to kill myeloma cells in live imaging IncuCyte Zoom assays with escalating doses of TriKE. Maximal killing occurred with 3 nM concentration. We also tested the efficacy of B7-H3-TriKE with the proteasome inhibitor bortezomib (10nM) and the immunomodulatory drug lenalidomide (5mM). Cytotoxicity curves were compared by repeated measures ANOVA and performed in triplicate. We developed MDSC from CD33+ myeloid cells from healthy donors using IL-6 and GM-CSF and these cells suppressed T cell proliferation. MDSC were used at 1:1 ratio with targets in cytotoxicity assays. Results: We found a statistically significant increase in NK cell mediated killing of all four myeloma lines when 3nM B7-H3-TriKE was added. Against U266 and MM1S, B7-H3-TriKE significantly enhanced killing at effector:target (E:T) ratios of 1:1, 2:1 and 4:1. RPMI-8226 showed relatively high resistance to NK cell cytotoxicity but B7-H3-TriKE enhanced killing at E:T of 4:1. H929 cells were more potently killed in the presence of B7-H3-TriKE at E:T of 1:1 and 2:1 but there was no difference in killing at E:T 4:1 likely due to high natural cytotoxicity in both groups. We compared degranulation (CD107a) and cytokine production by flow cytometry after NK cells were incubated with healthy donor NK cells at an E:T of 2:1 with or without 3 nM B7-H3-TriKE. There was an increase in degranulation with TriKE compared to NK cells alone after four hours but no significant change in cytokine production. Combination therapy with B7-H3-TriKE, NK cells, and lenalidomide showed synergistic killing of H929 cells but combination with bortezomib did not further enhance killing compared to NK cells and TriKE alone. Addition of MDSC to cytotoxicity assays enhanced myeloma cell growth but was overcome by B7-H3 TriKE + NK cells. We examined MDSC from a relapsed myeloma patient and found 81% B7-H3 expression on CD14+CD11b+ cells suggesting B7-H3 is highly expressed on myeloma-derived MDSC. Conclusions: In conclusion, B7-H3-TriKE significantly enhances NK cell mediated killing of myeloma cells, even in the relatively low B7-H3-expressing H929 line. Our data also shows it can reverse MDSC-induced myeloma growth. Commercial manufacturing of B7-H3 TriKE (GTB-5550) has begun and a phase I trial will begin enrollment in 2022. Disclosure: Greg Berk: Employee GT Biopharma Martin Felices: Royalties: GT Biopharma Jeffrey Miller: Royalties; Fate Therapeutics, Inc. Fate Therapeutics, Inc. GT Biopharma. Royalties; GT Biopharma. Vycellix. Vycellix. ONK Therapeutics. Honoraria; ONK Therapeutics. Honoraria; Nektar. Nektar. Wugen. Wugen. Magenta. Magenta. Sanofi. Sanofi.
Background: Graft-vs-host-disease (GvHD) is a life-threatening complication of allogeneic hematopoietic cell transplantation (allo-HCT) with limited approved therapies. HLA-G is an immunosuppressive molecule playing a central role in the acceptance of the semi-allogeneic foetus during pregnancy, the expression of which is epigenetically regulated. We have previously reported the small-scale generation of HLAG+CD4+FOXP3- regulatory T-cells through hypomethylation exerting potent suppressive function in vitro (Stamou et al, 2017). Herein, we aimed to produce and characterise a clinical scale HLAG+ product (iG-Treg), assess its safety profile and elucidate the molecular mechanisms of HLAG+CD4+ T cell-mediated suppression. Methods: Peripheral blood mononuclear cells from healthy donors were enriched for T cells through monocyte depletion and were activated with CD3/CD28 beads for 3 days and followed a 3-day decitabine treatment (n = 9). The final product was tested for immunophenotype, expression of exhaustion markers and ability to produce effector cytokines following 4-hour stimulation with PMA/ionomycin via flow cytometry (n = 3) as well as for cytokine secretion in supernatants during production using a multiplex magnetic bead-based immunoassay (n = 9). Sorted HLAG+CD4+ T-cells and HLAG-CD4+ were analysed by RNA-seq and the expression of the key differentially genes were validated via RT-PCR and/or flow cytometry. Results: The iG-Treg product (n = 9) contains 95,8% CD3 + of which 58,6% are CD4 + and 37,8% are CD8 + . Compared to untreated controls (PBS), iG-Tregs are enriched for HLAG+CD4+ T-cells (25.6% vs 0,7%, p < 0,0001), are PD-1 + (61.25% vs 20.46%, p = 0.04), with impaired ability to produce effector cytokines as was evident by diminished intracellular IL-2 (38.1%, vs 63.3% p = 0.0176), IFNγ (45.8% vs 59%, p = 0.008) and IL-17a (2.94% vs 5.87%, p = 0.0368). Assessment of culture supernatants interestingly displayed increased production of IL-13 (653.3pg/ml vs 291pg/ml, p = 0.0496) without concomitant increase of other Th1/Th2 cytokines (p = ns). RNA-seq revealed that HLAG+CD4+ T cells have a distinct and uniform gene expression profile compared to HLAG-CD4+ with highly differentially expressed IDO-1, CCL17, CCL22 and CXCL9 transcripts (log2(fold change)>1.5 & q < 0.05), findings whch were validated via RT-PCR. Notably, the expression of IDO-1 on HLAG+CD4+ cells was further validated with flow cytometry (p = 0.02). Conclusions: Our data indicate that iG-Tregs, which are enriched in HLAG+CD4+ T cells, can be effectively produced through a short and GMP-compatible protocol. iG-Tregs demonstrate a favourable safety profile as depicted in the exhausted phenotype associated with high levels of PD-1, the impaired ability to produce effector cytokines that are typically associated with GvHD exacerbation and the absence of Th1/Th2 polarized cytokine secretion in supernatants despite the increase in IL-13. Moreover, we describe, for the first time, the presence of the predominantly myeloid suppressor gene IDO-1 on regulatory HLAG+CD4+ T cells. The exact effect on immunosuppression mediated through IDO-1 remains to be assessed through functional. In parallel, iG-Tregs are being evaluated for their GVHD and GVL effect in in vivo models. Collectively, iG-Tregs constitute a well-characterized and safe immunosuppressive product able to be administered against GvHD in the initiated phase I clinical trial (EUDRACT number: 2021-006367-26). Clinical Trial Registry: N/A Disclosure: Nothing to declare
Background: Pembrolizumab demonstrated significant efficacy in R/R cHL, resulting in high ORR, prolonged PFS in patients, who relapse after or are ineligible for autotransplant, including patients with chemorefractory disease. Methods: Retrospective cohort study.55 patients included (JAN 2016 to March 2021). Primary objectives were OS, PFS. Secondary objectives ORR, CR, and toxicity (IRAE). patients demographics are detailed in Table-1. Results: 55 included,14/55 (25.5%) received pembrolizumab AFTER Auto-SCT, 41(74.5%) as bridge to auto-SCT. Median pembrolizumab cycles 6 (range: 1-32). Median number to CR 4(range:2- 8). 41(74.5%) had ORR;35 (85.3%) continued response;18(32.7%) went into CR;17 PR-VGPR(30%);4(7.3%) had no response and 15(27.3%) progressed at the end of pembrolizumab. 12/41 (29.2%) received auto-SCT,02/41(4.87%) had no Auto-SCT, 9 (22%) allo-SCT; 2 had no prior auto-SCT(mobilization failure1, short response 1) Six out of 18 patients (11%), who achieved CR remained on pembrolizumab at last encounter with median duration of 7.5mo (range, 2-20.5mo); among patients who discontinued therapy, median treatment duration 3mo (range,1-21mo). DP was the most common reason for pembrolizumab discontination,23/49 (47%), followed by pneumonitis, 5 /49(10%), auto-SCT 9/49 (18.4%), 9/49(18.4%) went to allo-SCT,1 patient based on physician preference. At median FU of 15.3 mo(range: 0.23-48.5mo), MOS and MPFS was NR, 12.5 mo (95% CI, 94.3-35.8mo) respectively. 1-year OS and PFS 92% (95% CI: 76–95%),and 51% (95% CI, 39-67%) respectively, Fig. 1. 1-year PFS for patients who achieved CR or PR or PD was 88%(95% CI:07-75%); PR 60%(95%CI:21-29%) and 5%(95%CI: 5-0%). MPFS for patients in CR and PD was 26.1mo(9.6-NR) and 4.5mo(3.4-7.8mo) respectively. Patients, who received auto-SCT consolidation after Pembrolizumab(n-12), had 1-year OS 100% (95%CI:0-100),PFS of 93%(6-82%), respectively. Only 2 patients had no Auto-SCT, continue to survive and disease free at last follow up. Patients who received allo-SCT(n-9) after failure of auto-SCT in 7 patients had 1-year OS 88% (955CI:10-70),PFS 66% (15-42). 11 (20%) patients deceased at the end of the study. Death attributed to DP in 7,sepsis 1, GvHD 2, and unknown 1 patient Adverse events reported in 26(47%);25(45.5%) IRAEs. Most common were hypothyroidism 10(18%),pneumonitis 9(14.5%). Auto-allo-SCT group had the highest AE(25%).11(20%) required steroids;3(4.5%) discontinued pembrolizumab. All recovered, no deaths attributed to pembrolizumab. 8 out of 9(88.9%) developed aGvHD, 6(75%) cGvHD. 2 patients died due to severe lung and gut GvHD with TRM 22%. Figure: OS and PFS for the whole cohort(N-55) Conclusions: Pembrolizumab demonstrated high remission rates at reasonable follow up time, improved survival outcomes, especially in patients who attained CR and received SCT consolidation with acceptable safety profile. Clinical Trial Registry: NA Disclosure: All authors has no conflict to disclose
Background: HLA-mismatched stem cell microtransplantation (MST), pioneered by Huisheng Ai, is a non-engrafting stem cell immunotherapy for elderly patients with AML/MDS not eligible for allogeneic HSCT, in whom it provides high response rates and survival in de novo AML primed with conventional chemotherapy [Guo M, et al. JAMA Oncology 2018]. Evidence is lacking in more advanced forms of AML/MDS and in elderly patients treated with hypomethylating agents. Methods: Retrospective analysis of all consecutive adult patients with AML/MDS who had HLA-mismatched related PBSC donors available (haplo or lower matching), were not eligible for allogeneic HSCT and underwent MST at our center between 2012 and 2020. Results: Twenty-five patients (19 male; median age 69, 53-77; 10 de novo, 8 relapsed/refractory, 4 secondary AML, and 3 advanced MDS) received 31 MST procedures, including 6 patients who had a second MST upon AML relapse. DRI was high/very high in 18 patients (72%). Ten patients received a first course of chemotherapy without stem cell infusion and initiated them with their second course, when donors were available. Sixty-seven MST infusions were carried out in these series (median 2 per procedure, 1-3, approximately two months between infusions). Most of these infusions were primed with hypomethylating agents either alone (23; 34%) or combined with chemotherapy (13; 19%) or FLT3-inhibitors (2; 3%), and 29 infusions were primed with intensive chemotherapy alone (43%). Overall, patients received 6.16 (2.16-10.7) mononuclear cellsx108/kg, 2.72 (0.64-3.96) CD3 + cellsx108/kg and 6.01 (1.13-15.65) CD34 + cellsx106/kg per MST procedure. Haploimmunostorm symptoms occurred in all patients after the first MST infusion, fever being the commonest (94.03%), followed by skin rash (24%), although only nine (13%) required specific treatment with corticosteroids or anti-IL6. Tolerance overall was good with a low rate of opportunistic infections (18; 27%) and a mortality at 28 days after any infusion of only 7%. Four patients died before the response to MST could be evaluated. Among 21 evaluable patients, 16 achieved complete remission following MST (76%) and five did not respond. Median overall survival (OS) after the first MST was 12 months (range 0-100), with two-year OS of 37% (IC95% 17.7-55.9%; Figure 1A). Five out of six patients who underwent a second MST procedure achieved a complete response, with a median OS of 20 months and 47.12% (IC 95% 25.51-64.49%) at one-year. Median progression-free survival in our population was 10 months. Finally, despite statistical limitations in this small population, it is worth mentioning that the novel use of MST in patients with refractory and secondary AML (Figure 1B), in those primed with hypomethylating agents (Figure 1C), and even as a second MST following prior progression were not associated with worse response rates of survival. Conclusions: MST can be used in combination with hypomethylating agents, in advanced forms of AML/MDS, and can also be offered to patients a second time with good outcomes and no evidence of increasing toxicity, hereby becoming a safe and effective immunotherapy for a broad spectrum of elderly patients with AML/MDS that are not candidates for allogeneic HSCT. Disclosure: Nothing to declare
Background: There is no standardized therapeutic approach for relapsed acute myeloid leukemia (AML) following allo-HSCT. A growing body of evidence exists about the efficacy of venetoclax (VNC) based therapies in AML. VNC is a bcl-2 inhibitor that has shown a composite complete remission rate of 66.4% in treatment-naïve elderly AML patients. There are scarce data about the efficacy of VNC based therapies in patients who relapsed after allo-HSCT. In this study, we retrospectively analyzed the data of relapsed AML after allo-HSCT treated with the combination of VCN and a hypomethylating agent. Methods: In total, 30 patients were included in this study between February 2019 and October 2021. The median age was 43.1 years (range, 20-69). Sixteen (53.3%) patients were female and 14 were male (46.7%). Before transplant 23 (76.7%) patients had de-novo and seven (23.3%) had secondary AML. The distribution according to ELN 2017 risk category was 8 (26.7%) adverse, 20 (66.7%) intermediate, and 2 (6.7%) favorable. At the time of the transplant, 18 patients were in CR1, 2 patients were in ≥CR2, and 10 were with persistent disease. Among 30 patients, 28 received azacytidine (75mg/m2 for 7 days, subcutaneously), and the remaining 2 received decitabine (20mg/m2 for 5 days, intravenously) in combination with VNC . Nine patients (30%) had a history of prior HMA exposure. The median cycle of VNC was 3 (1-6). The targeted VNC dose was 400mg/d reached by rump-up dosing schema. The median maximal VNC dose was 400mg per day (range, 100-400mg). In total, 23 patients achieved at least one cycle of VNC based therapy. Seven patients were excluded from treatment response analysis who failed to complete at least one cycle of VNC. Results: The median time from diagnosis to allo-HSCT was 7.3 (2.5-125) months.The median time from allo-HSCT to relapse was 8.5 (1.7-47.8) months. Fourteen patients (46.7%) relapsed within six months after transplant. The median duration of follow-up was 5.3 (1-22) months. Among 23 patients who were evaluable for assessment, the overall response rate (ORR) was 56% (7/23 CR, 3/23 CRi, 3/23 PR). None of the patients developed clinical tumor lysis syndrome. The median time to best response was 2.1 (0.6-4.4) months.). Median PFS was 3.9 months (95% CI 1.2-6.6) (Figure 1) . Median overall survival (OS) was 5.3 (2.6-8.0) months. Two patients who attained a negative measurable residual disease CR had a survival of more than 20 months despite the interruption of the VNC based therapy after six cycles. At the time of the data cut-off, 6 patients (26%) were alive, and the remaining 17 (74%) had died. None of the patients experienced new-onset acute or chronic GVHD during the VNC based therapy. In total, 5 patients received second allo-HSCT. The vast majority of patients experienced grade2-4 neutropenia (82%) and thrombocytopenia (78%) at any time of the treatment. Conclusions: The combination of VNC and HMA is an effective and safe therapeutic option for adult AML patients who relapsed after allo-HSCT. Disclosure: Nothing to disclose.
Background: The therapeutic apheresis department at CHU Montpellier has performed ECP therapy since 2010 using both offline and online methods. We perform ca. 500 procedures annually to treat GvHD, CTCL, heart and lung transplant rejection. In 2019, the online Amicus Blue™ ECP System (Fresenius Kabi, Germany) was CE marked and introduced in Europe. We have used the Amicus since 2012 for MNC, TPE and RBCx, and so evaluated the new ECP procedure. A retrospective comparison to historical data from our other ECP systems was performed. Methods: We evaluated the system in routine use from November 2020 through October 2021 (n = 131 procedures) for collected cell yield and procedure time in adult patients prescribed ECP. Amicus Blue was incorporated into their existing treatment regimen. Amicus v6.0, Phelix v2.0 and double-needle disposable kits were used. All patients used peripheral access. A 12:1 whole blood (WB) to ACD-A anticoagulant ratio was used, 1.24 mg/kg/min citrate infusion rate, and the default 2000ml WB processed. Hematology counts were performed on patient WB and the treated MNCs. Historical data was summarized for all ECP procedures performed since 2010. For online ECP, we use Cellex (Mallinckrodt, UK) and in years past, its predecessor, UVAR. Offline ECP is performed with UVA-PIT (PIT Medical Systems, Germany) and Macogenic (Macopharma, France) in conjunction with cells collected using standard MNC procedures. The cell separators used include COBE Spectra and Spectra Optia (Terumo BCT, USA), and COM.TEC and Amicus (Fresenius Kabi). Results: For Amicus Blue, no adverse events were reported, and mean total procedure time including collection, photoactivation and reinfusion was 93 minutes. Cellex mean procedure time was 127 minutes, however 50% of these procedures were single-needle. The mean concentration of MNCs collected for each system type is presented in the graph. Amicus Blue had a higher concentration than Cellex, and comparable levels to offline methods even though less WB was processed. Lymphocyte proliferation measured for Amicus Blue system qualification was >90%. We did a subset analysis of ECP methods in aGVHD patients, as they are often lymphopenic. The data indicate that total lymphocytes collected is related to the volume of WB processed. Amicus Blue’s flexible programming allows processing of up to 4000ml WB. Although the optimal cell dose is unknown, this feature is appreciated especially for lymphopenic patients. Conclusions: Results for cell yields are comparable to our historical data for online and offline systems. We observed no differences in clinical response compared to previous ECP treatment. A multi-procedural platform like Amicus Blue provides better return on investment for apheresis departments that require flexibility. Our staff prefer the Amicus Blue ECP for its shorter, predictable procedure time and for its friendly use. We will expand its use when the single-needle option becomes available (in development). Disclosure: tarik kanouni: Hospitalty fees fromFresenius Kabi
Background: Although complete remissions can be achieved in 60-80% of AML patients by conventional chemotherapy, more than half of them experience relapse. Since relapses are thought to evolve from leukemic stem (AML-LSCs) or progenitor cells persisting despite repetitive chemotherapeutic cycles, innovative therapies preferably targeting AML-LSCs leading to deep and long-lasting remissions are highly desirable. SPM-2 is a triplebody carrying single-chain variable fragments, dually targeting CD33 and CD123. The combined surface density of CD33 and CD123 is far greater on AML-LSCs than on non-malignant hematopoietic stem cells. Therefore, SPM-2 seems to be a promising therapeutic agent. Here, we analyze the specific lytic capacity of SPM-2 utilizing different NK cell preparations against MOLM-13 and primary AML blasts. Methods: ADCC mediated by SPM-2 was fluorometrically assessed in calcein release assays. Targets (T): calcein-labeled MOLM-13, blasts from pediatric AML patients. Effectors (E): PBMCs, lymphokine-activated killer (LAK) cells, expanded NK (eNK) cells, cytokine-induced memory-like (CIML) NK cells (CD3-depleted/CD56-enriched). Expression of CD25, CD69, NKG2D (effectors) and of CD33/CD123 (targets) were determined by FACS and/or QIFI Kit. Results: We evaluated the capacity of different effector cell preparations to lyse tumor cells in concert with SPM-2. Using patients’ PBMCs (E:T 30-40:1), specific lysis mediated by SPM-2 (500-1500 pM) against MOLM-13 ranged from 30-40%. LAKs were generated by incubating PBMCs with OKT-3 (10 ng/ml; d1-5) and IL-2 (500 U/ml; d1-21). Lysis capacity of LAKs varied markedly among donors with a maximum of 30% (E:T 20:1; SPM-2 500 pM). eNKs were produced by co-stimulation of PBMCs with IL-2 (50-100 U/ml) and irradiated feeder cells (K562-mbIL15-4-1BBL). Concentrations of SPM-2 paralleled specific lysis of MOLM-13, plateauing at 50% (E:T 10:1). eNKs also lysed (range: 15-50%; 4 donors; E:T 10:1) CD33+CD123+AML blasts in the presence of SPM-2 (500 pM). Cytokine combinations were used to generate CIML-NKs without the need for feeder cells which is of high relevance for potential clinical application: CIMLs stimulated for 16h with IL-12/15/18 (10/50/50 ng/ml) (“triples”); CIMLs stimulated with IL-15 high (10 ng/ml), boosted with IL-21 (25 ng/ml) on days 9-12 (“boosts”); NKs stimulated with IL-15 high and low dose (10 and 1 ng/ml) (“controls”). “Triples” showed a high natural cytotoxicity (55-70%), only slightly increased by SPM-2 (70-75%; 16h). In contrast, “controls” showed low natural cytotoxicity (3-25%), whilst specific lysis was drastically enhanced by SPM-2 (60-80%; 16h) (500 pM; E:T 5-10:1). NKG2D was increased in all three subsets after 16h, whereas relevant expression of CD25 and CD69 was limited to “triples” (100%). “Boosts” displayed robust, but donor-dependent ADCC even at d + 25. Conclusions: SPM-2 mediates efficient lysis of tumor cells in the presence of PBMCs, even without additional NK cell transfer. SPM-2-mediated specific lysis was substantially increased using mono-stimulated (IL-15) NK cells. Thus, SPM-2 appears to be a potent new agent in AML treatment, combining targeted with immunotherapy. Whether highly preactivated CIMLs are also clinically beneficial due to their enhanced natural cytotoxicity should be investigated in vivo. Disclosure: Nothing to declare.
Background: To investigate the safety and efficacy of daratumumab-containing regimen in the management of relapsed hematological malignancies in children after allogeneic hematopoietic stem cell transplantation (allo-HSCT). Methods: From January 2019 to November 2021, thirteen children with refractory/relapsed hematological malignancies who relapsed after myeloablative allo-HSCT in our hospital were enrolled. Diagnosis included acute myeloid leukemia (AML, 5 cases), T-lymphoblastic lymphoma/leukemia (T-LBL/L, 6 cases), and B-acute lymphoblastic leukemia (B-ALL, 2 case). The median age was 10(5-18) years old. The disease status pre-HSCT was either non-remission (7 cases) or minimal residual disease (MRD) positive (6 cases). The fusion genes of recurrence were amL1-ETO positive in 3 cases, MLL gene rearrangement in 2 cases, HOX11 positive in 2 cases, BCR-ABL1 positive in 1 case and no special gene in 5 cases. The expression of CD38 antigen in their tumor cells was all positive by flow cytometry. Five AML patients received at least 1 cycle of chemotherapy or donor lymphocyte infusion (DLI) and failed. Daratumumab-containing regimen was consisted of daratumumab 400mg x 1, cytarabine 100mg/d x 3-5d, etoposide 100mg/d x 3-5d, and venetoclax 10mg bid x 14d. Results: All patients were tolerant this regimen well. The main adverse events during daratumumab infusion were runny nose, cough, chest tightness, transient decrease of blood oxygen saturation. They all experienced pancytopenia for 2-3 weeks. No life-threatening infections and bleeding were noted. One AML patient underwent the second allo-HSCT after complete remission (CR) with daratumumab-containing regimen. With the median follow-up 218 (30-617) days, CR was achieved in 4/6 T-LBL/ALL, 3/5 AML, and 0/2 B-ALL. The disease-free survival rate was 46.2%, and the overall survival rate was 53.8%. Conclusions: The prognosis of refractory/relapsed patients with hematological malignancies who relapsed after allo-HSCT is extremely poor, and effective therapeutic regimens are very limited. Our pilot study has shown that daratumumab-containing regimen is feasible and effective in half of CD38 positive hematological malignancies in children who relapsed after allo-HSCT. It seems that better response is found in the patients with T-LBL/ALL. More patients and longer follow-up are warranted. Disclosure: Nothing to declare
Background: Persistence of Measurable Residual Disease (MRD > 10-4) in Acute Lymphoblastic Leukemia (ALL) is high risk for relapse after allogeneic Hematopoietic Stem Cell Transplantation (allo-HSCT) which has a very dismal prognosis. The role of donor lymphocytes in the treatment of MRD-positive ALL is still debated, while favorable results have been observed with treatment of MRD-positive relapse with the CD3-CD19 bi-specific T cell engager, blinatumomab. Methods: Three pediatric patients with high risk B-ALL, were treated according to AEIOP ALL 2009 and AIEOP ALL 2017 protocols and referred for allo-HSCT. Patients all were MRD-positive after allo-HSCT and received Blinatumomab + DLI. Results: Patient 1, a 17 years old male was primary refractory to 2 lines of chemotherapy and blinatumomab, received 2 courses of inotuzumab and achieved an MRD-positive complete remission (CR). Patient 2, a 16 years old female, had secondary ALL who achieved CR1 after 1 course of chemotherapy and subsequently received 2 courses of blinatumomab, due to treatment chemotherapy-associated toxicity and achieved MRD-negative CR1. Patient 3 developed CNS relapse during maintenance, then received salvage therapy according to the INTREALL HR 2010 and 1 course of blinatumomab, achieving MRD-positive CR1. All patients proceeded to allo-SCT and were MRD-positive at 14, 31 and 8 weeks post-allo-SCT, respectively. All patients discontinued immunosuppressive therapy and received 3, 1 and 4 courses of DLI + blinatumomab respectively . Blinatumomab was started one week after each dose of DLI. Only patient 1 experienced Grade 2 liver GVHD. None of the patients experienced serious adverse events needing Blinatumomab discontinuation . All 3 patients reached an MRD-negative complete remission (CR). One patient is still in CR and 2 patients presented MRD-positive and CNS relapse. All patients still present MRD- Bone Marrow disease. Conclusions: The role of cellular therapy with DLI is still debated ALL. Blinatumomab directs T cells to bind CD19 present on malignant B cells and engages CD3 on T cells causing activation and inducing cytotoxicity against the ALL cells. The use of blinatumomab allowed recruitment of fit donor-derived T lymphocytes (not exposed to immune suppressive agents) against ALL B cells. This hypothesis is supported by the fact that patient 1, who received blinatumomab pre-HSCT and had disease progression, (probably due to lack of T cells showed by flow cytometry), achieved MRD-negative response after receiving DLI + blinatumomab post transplant. All patients reached MRD-negative status and 2 subsequently developed CNS relapse; none received CNS prophylaxis post-HSCT. We hypothesize that blinatumomab + DLI is able to clear the hematological disease, but it is not effective in preventing CNS relapse. Disclosure: Nothing to declare.
Background: Cerebral palsy (CP) is the most common motor disorder in children in which Wharton Jelly Mesenchymal Stem Cell (WJMSC) infusions has become a promising therapeutic strategy for patients with this disease. Our earlier work has shown that application of mesenchymal stem cells derived from Wharton’s Jelly seems to be safe and an effective procedure that improves gross motor functions, muscle tension, communication, attention, and cognitive functions in children with CP. Here we obtain results from a population of patients with cerebral palsy. This population of patients with cerebral palsy was taken from a larger study where patients with neurological/genetic diseases were treated with at least two doses of WJMSC coming from PBKM. Methods: The WJMSC were derived from umbilical cords obtained from unrelated third party donors. The intravenous injections of WJMSC were used as a treatment for 123 patients with CP, who have received at least two infusions of mesenchymal stem cells in dose 20-30 x 106 cells/infusion. The median age of the studied participants at the initiation of the therapy was 6 years of age. Each procedure had an individual bioethical committee approval. The patients were evaluated by the same neurologist both before the first treatment dose and during the subsequent administrations using scales testing for motor and neurological abilities such as GMFM (Gross Motor Function Measure), GMFMS (Gross Motor Function Classification System) scale test, 6MWT (6-minute walk test), Up & Go test, and the CGI (Clinical Global Impression) scales test. The results were analysed using the Wilcoxon Test for pairs of observations. Results: Among 123 patients with CP, there was a statistically significant improvement in motor skills as measured by the GMFM scale, with the median score increasing from 19.5 points before therapy to 24.5 points after therapy (p < 0.05). Also, the 6MWT and Up & Go test results showed statistically significant improvement in 19 and 18 patients, respectively, who were tested before and after umbilical cord mesenchymal stem cell infusion. The median distance walked by pediatric cerebral palsy patients increased from 120 meters (range min 38 m max 220 m) to 180 meters (range min 110 m to max 300 m) p = 0.00025. The median time for children to stand up after hearing a command decreased from 12 seconds (range min 7 sec to max 22 sec) to 10 seconds (range min 8 sec to max 17 sec) p = 0.0005 after cell administration. There were observed no serious adverse events related to WJMSC application Conclusions: The infusion of third party donor WJ-derived MSC is a safe procedure with clinical effects that significant improves CP patients health status in all assessed scales despite the lack of changes in muscle strength. The results from this and our previous studies allow us to predict that the therapeutic effect of WJMSC administration will increase with subsequent infusions of the WJMSC cells. Disclosure: The authors of abstracts are workers of Bank providing cells and Medical Center where cell infusions took place.
Background: In recent years, extensive pre-clinical and clinical experimental data have suggested that mesenchymal stem cells derived from Wharton Jelly (WJMSC) have unique properties that not only invoke minimal immune reactivity, but also possess secretion ability, modulatory and anti-inflammatory effects. As a result, WJMSCs can provide an interesting therapeutic option in the treatment of many neurological/genetic diseases. In this abstract we obtained results from medical experiments where patients with various neurological/genetic diseases were treated with at least two doses of a WJMSC from an unrelated third party from the Polish Stem Cell Bank (PBKM). Methods: The intravenous injections of WJMSC (dosage: 20-30 x 106 cells/infusion) were used as a treatment for 38 patients with spina bifida (SB), 12 with global developmental disorder, and 40 with other neurological disorders (including 8 with neuroinfections, 6 with encephalopathy, 6 with intellectual disability, 5 with malformations, 3 patients with brain injuries, 2 with spinal cord hernia with brain trauma, 2 diagnosed with myasthenia gravis, 2 with epilepsy, and 1 patient each with dystonia, systemic developmental disorders, optic nerve disc disorder, arthrogyposis with myopathy, and 3 diagnosed with other disorders) and 26 patients with genetic defects (including 4 patients with muscular dystrophy, 4 with adrenoleukodystrophy, 4 with leukodystrophy, 2 patients with Down syndrome, 2 with Wolf-Hirschoma syndrome, and 1 patient each with Cornelli de Lange syndrome, Phelan McDermid Pitt syndrome chromosome 3 deletion, propion aciduria, ceroidolipofuscinosis, genetically determined epilepsy, and 3 patients with unspecified defects. Each WJMSC infusion procedure had an individual bioethical committee approval. The patients were evaluated by scale testing motor and neurological abilities such as GMFM (Gross Motor Function Measure), GMFCS (Gross Motor Function Classification System),scale test 6MWT (6-minute walk test), Up & Go test, and the CGI (Clinical Global Impression) scale test. The results were analysed using the Wilcoxon Test for pairs of observations. Results: Among the 33 patients with SB from whom follow ups were obtained, the median GMFM motor ability rating scale showed significant (statistically) improvement upon treatment, where the median value before therapy was 48 points (range 12 -96) and after therapy it increased to 50 points (range 16-99), p = 0.002. Similar results were observed in a group containing 12 patients with global developmental disorder, where, the median GMFM score showed a significant (statistically) increase from 82 points (11-95) to 85 (18-97) p = 00764. There was also a significant increase in the distance that patients were able to walk in 6 minute during the 6MWT test after infusion of WJMSCs compared to their scores before therapy. The median distance increased by almost 60 meters; patients went from 100 meters before therapy to 158 meters after therapy p = 0.025. Among the 20 patients who suffered from other neurological disorders and 16 with genetic disease from whom follow ups was obtained to the present day, there was a statistically significant improvement in GMFM and CGI scales. Conclusions: WJ-MSC administration is promising for the improvement of motor skills, cognitive functions, in patients with various neurological and genetic disease. Disclosure: Authors of abstract are working in stem cell bank and medical center where cells infusion took place
Background: VOD/SOS is a potentially life-threatening complication of haematopoietic cell transplantation (HCT) that can also occur after high-dose chemotherapy. Defibrotide is approved for the treatment of severe hepatic VOD/SOS post-HCT in patients aged >1 month in the EU. The DEFIFrance study collected real-world data on the effectiveness and safety of defibrotide from HCT centres across France. This analysis presents outcomes in patients who received defibrotide treatment for VOD/SOS post-chemotherapy. Methods: This post-marketing registry study collected retrospective and prospective real-world data on patients receiving defibrotide at 53 HCT centres in France. VOD/SOS diagnosis was per the investigator’s typical clinical practice. Disease severity was categorised using adult EBMT severity criteria in adults; paediatric patients (<18 years) were retrospectively/prospectively categorised using paediatric EBMT severity criteria. Survival and complete response (CR; total serum bilirubin <2 mg/dL and multiorgan failure [MOF] resolution per investigator’s assessment) rates by Day 100 post-VOD/SOS diagnosis were calculated. Treatment-emergent serious adverse events (TESAEs) of interest were haemorrhage, coagulopathy, injection-site reactions, infections, and thromboembolic events, irrespective of relationship to treatment. Results: Overall, 46 patients (19 [41%] paediatric and 27 [59%] adults) received defibrotide for VOD/SOS post-chemotherapy. Median age was 5.8 years (range: 2, 17) in paediatric patients and 56.6 years (range: 18, 72) in adults. Paediatric patients were more likely than adults to have a primary diagnosis of ALL (paediatric: 53%; adult: 22%) or neuroblastoma/solid tumour (32%; 0%), while AML (11%; 63%) was more common in adults. Paediatric patients were less likely to have prior exposure to gemtuzumab ozogamicin and inotuzumab ozogamicin (11% and 0%, respectively) than adults (41% and 15%). VOD/SOS was severe/very severe in all paediatric patients and 22 (81%) adults. MOF was present in 5 (26%) paediatric patients and 10 (37%) adults, including renal failure (paediatric: 60%; adult: 70%), respiratory failure (100%; 80%), and cerebral failure (20%; 70%). By Day 100 post-VOD/SOS diagnosis, the Kaplan-Meier (KM)–estimated survival rate was 74% in paediatric patients and 37% in adults (Figure 1A), and the CR rate was 68% in paediatric patients and 37% in adults (Figure 1B). Of 5 paediatric patients who died within 100 days of VOD/SOS diagnosis, all 5 had very severe VOD/SOS. Of 17 adults who had died by Day 100, 10 had very severe VOD/SOS. By Day 100, 2 paediatric patients and 6 adults had died due to relapse/progressive disease and there were no VOD/SOS-related deaths. TESAEs of interest occurred in 15% of patients with VOD/SOS post-chemotherapy (paediatric: 11%; adult: 19%), including infection and haemorrhage (each 9% in the overall group). Conclusions: In this real-world DEFIFrance study, the majority of patients who received defibrotide for VOD/SOS post-chemotherapy had severe/very severe VOD/SOS and a notable proportion had MOF, indicating advanced disease in this patient population. Over half of adult patients had received ozogamicin-containing therapy. These data indicate that VOD/SOS is a concern outside the HCT setting and suggest a need for continuous vigilance for VOD/SOS among those monitoring patients after chemotherapy, especially for signs and symptoms to improve earlier diagnosis. Disclosure: IY-A, DB, J-HD, ML and RPL have received honoraria from Jazz Pharmaceuticals. SC and MM have received honoraria and research funding from Jazz Pharmaceuticals. KA and FD are employees of and hold stock and/or stock options in Jazz Pharmaceuticals. AP has no conflicts to disclose.
Background: The conjucated monoclonal antibodies (c-mABs), inotuzumab ozogamycin (Ino) and gemtuzumab ozogomycin (GO), are novel treatment options for relapsed/refractory acute lymphoblastic and myeloid leukemia, respectively. Nonetheless, they are known to cause sinusoid obstructive syndrome/veno-occlusive disease (SOS/VOD), especially in allogeneic stem cell transplantation recipients (alloHSCT) after bridging with these agents. Currently there is limited data on the incidence and risk factors of SOS/VOD after c-mABs and alloHSCT, as well as no recommended approach to allograft patients after these agents. Methods: A total 82 patients (56 adults and 26 children) with acute leukemia (acute myeloid leukemia (AML) n = 33, acute lymphoblatic leukemia (ALL) n = 49) were included in this study. The treatment consisted of median 2 (range 1-3) cycles Ino for ALL and all AML patients received 1 cycle of GO-FLAG prior to allo-HSCT. Allo-SCT was performed from a matched related (n = 13), unrelated (n = 15) or haploidentical donor (n = 54) between 2017 and 2021. The disease status before allo-SCT were: remission I (n = 8, 9.8%), remission II + (n = 55, 67.1%) and active disease (n = 19, 23.2%). Median time from c-mABs to SOS/VOD development was 75 days (39-287). Results: In 26 patients (32%) we observed SOS/VOD in the posttransplant period: 1 mild (1.2%), 9 moderate (11%), 7 severe (8.5%) and 9 very severe (11%) based on EBMT criteria. Median time to development VOD was 15 days (7-100). Ino therapy in ALL was associated with significantly higher SOS/VOD incidence than GO in AML 41% (n = 19) vs 33% (n = 7) (HR 3.75, 95%CI 1.43-9.8, p = 0.007). Also calcineurin inhibitors (CNIs) in graft-versus-host prophylaxis regimen was strongly associated with increased SOS/VOD incidence (HR 5.58, 95%CI 1.53-20.3, p = 0.009). Other factors as busulfan in conditioning regimen, time interval between c-mABs course and alloHSCT, type of donor, HLA-mismatch between recipient and donor, myeloablative conditioning were not associated with high VOD/SOS incidence. In multivatiative analysis, VOD severity (HR 2.2, 95%CI 1.07-4.67, p = 0.032) and disease status (HR 3.99, 95%CI 1.25-12.76, p = 0.019) were associated with survival differences, while type of disease was not predictive (HR 1.089, 95%CI 0.33-3.56, p = 0.88). In patients developed PT-VOD 1-year OS was 36% (95%CI 17%). Conclusions: The incidence of SOS/VOD is high after c-mABs bridging to alloHSCT. The only potential intervention we identified to reduce the risk of SOS/VOD after c-mABs is to use CNI-free GVHD prophylaxis regimens. Severe SOS/VOD is a negative prognostic factor for survival in this group of patients. Disclosure: M.K. received lecturer fees from Pfizer. I.M. received lecturer and consulting fees from Novartis, J&J, Celgene, BMS, Takeda, Sobi.
Background: The main clinical challenge of hematopoietic stem cell transplantation (HSCT) is treatment-related mortality (TRM). Endothelial dysfunction plays a crucial role in the pathophysiology of major complications contributing to TRM. In the previous adult studies, the Endothelial Activation and Stress Index (EASIX) score was detected as a predictor for survival after HSCT, and also it was shown that EASIX scores were associated with biomarkers of endothelial homeostasis. In this study, we aim to assess if the EASIX might be valuable for the prediction of early HSCT-related complications and survival after pediatric HSCT. Methods: This study is a retrospective analysis of 343 children who underwent HSCT between January 2018–September 2021 in Medicalpark Göztepe Pediatric Stem Cell Transplantation Unit. EASIX was calculated as described before by Luft et al. Veno occlusive disease (VOD) was diagnosed according to EBMT recommendations which were described by Corbacioglu et al. and acute graft versus host disease (aGvHD) was diagnosed according to Glucksberg criteria. Results: The median age of the patients was 81 months (1-248 months), the primary disease was malign in 118 patients and non-malign in 225 patients. According to donor type, 204 transplants were performed from matched unrelated donor (MUD), 73 from match sibling donor (MSD), 23 from match family donor (MFD), and 43 from haploidentical donors. Stem cell source was peripheral blood (PB) for 196 transplants, followed by bone marrow (BM) for 127, combined BM and PB for 12 (for only haplo procedures), and combined BM and cord blood for 8 transplants. Ninety patients (26%) developed aGvHD. Regarding the severity of aGvHD, 47 patients (13%) developed grade 2, 22 patients (6,4%) developed grade 3, and 21 patients (6,1%) developed grade 4 aGvHD. Twenty-one patients (6,1%) developed VOD. VOD is not a cause of death for any of the patients. Analyzing the EASIX scores on different time points; median pre-conditioning EASIX was 0,64 (0,11 – 16,16), median day 0 EASIX was 1,48 (0,17 - 107), median day 15 EASIX was 2,6 (0,12 - 148), and median day 30 EASIX was 1,9 (0,29 -71,3). Pre-conditioning EASIX scores were significantly higher for malignancies (p < 0.001). Regarding the age, patients over 7 years old had significantly higher EASIX scores on pre-conditioning and day 0 (p = 0.001 and p = 0.009 respectively). The pre-conditioning EASIX scores were significantly higher in patients who developed VOD at any time after HSCT (p < 0.001). The EASIX scores in any of the time points were not significantly associated with aGvHD. Mortality was significantly higher for the patients with high EASIX scores in any time point and this association was stronger in early time points (p < 0.001 for pre-conditioning EASIX, p < 0.001 for Day 0 EASIX, p = 0.03 for Day 15 EASIX, and p = 0.05 for Day 30 EASIX) Conclusions: In our study, we showed that higher EASIX scores were associated with higher VOD risk and lower survival rates. We propose that EASIX scores be further investigated as an early biomarker for risk-adapted treatment strategies in larger datasets. Disclosure: Nothing to declare
Background: Poor graft function (PGF) is a life-threatening complication following allo-HSCT characterized by bilineage or trilineage cell deficiency with hypoplastic marrow with full chimerism, in the absence of relapse or GVHD. With the increased use of allo-HSCT, PGF has become a growing obstacle to transplant success. Factors involved in PGF include low dose of infused CD34 + cells, donor specific antibody (DSA), viral infections, iron overload, splenomegaly, GvHD, among others. Emerging evidence demonstrates that bone marrow microenvironment is dysfunctional in PGF patients. The PGF pathogenesis risk factors remain largely unknown and treatments are limited. Here we report PGF incidence in a cohort of patients and analyze risk factors to understand how to prevent or limit PGF. Methods: PGF risk factors investigated were splenomegaly, myelofibrosis, iron overload, DSA, gender mismatch, AB0 mismatch, donor age, viral infections, use of myelotoxic drugs, prophylactic G-CSF and use of cryopreserved graft particularly utilized during COVID-19 pandemic. Other HSCT features were also analyzed (i.e. source, donor, donor age, conditioning, among others). Results: From 2018 to 2020, we performed 231 allo-HSCT in patients with hematological malignancies, receiving myeloablative or reduced-toxicity conditioning, GvHD prophylaxis consisting of ATG or PT/Cy, coupled with Sirolimus and mycophenolic acid. 127 patients received allo-HSCT for AML, 24 for ALL, 32 for lymphoproliferative disorders, 48 for MDS/MPD. Donors were MRD, MMRD, MUD, UCB in 42, 64, 109, 16 patients respectively. According to the Disease Risk Index, patients were stratified in very high (n = 18), high (n = 63), intermediate (n = 130) or low (n = 16) risk. Stem cells were collected from BM (n = 7), PB (n = 208) and CB (n = 16). We reported 1-year probability of OS 74.8% and PFS 70.5%. 1-year CI of TRM was 15.5%, 1-year CI of relapse was 14%, along with 100-day CI of grade II-IV acute and chronic GvHD of 19.5% and 25.3%, respectively. Platelet engraftment was moderately delayed: 30-day CI was 43.7% and 60-day CI was 68.4%. 30-day CI of neutrophil engraftment was 78.8% and at 60-day was 93.5%. In our cohort, 34 patients developed PGF: 21 with primary PGF and failed to achieve sustained graft function and 13 with secondary PGFdeveloped after complete hematologic recovery. The CI of PGF at 60 days was 6.1% and 15.2% at 180 days. Univariate analysis for PGF risk factors showed significance for cryopreserved graft (HR 3.26; 1.65-6.42; p = 0.001) and major ABO mismatch between host and donor (HR 2.43; 1.22-4.86; p = 0.012), whereas donor age (HR 1.02; 0.99-1.04; p = 0.119) and prophylactic G-CSF (HR 1.86; 0.94-3.69; p = 0.076) were borderline significant. Cryopreserved graft (HR 3.59; 1.80-7.2; p < 0.001), major AB0 incompatibility (HR 2.38; 1.18-4.83; p = 0.016) and donor age ≥35 years (patients median age; HR 2.22; 1.03-4.78; p = 0.041) were confirmed using multivariable Cox regression analysis as factors predicting PGF as opposed to the prophylactic G-CSF which didn’t show significance. Conclusions: These findings suggest that donor age ≥35 years, major AB0 mismatch and graft cryopreservation may play a role in PGF onset. This might help to better understand PGF pathogenesis, to establish targeted interventions and to guide risk stratification, excluding donors and transplant with these features when possible. Disclosure: No relevant conflicts of interest
Background: Poor Graft Function (PGF) is a life-threatening complication after allogeneic stem cell transplantation (alloSCT). Few outcomes with a CD34-selected stem cell boost (CD34 + SCB) for PGF in pediatric alloSCT recipients have been reported. Here we report on a single center experience from MSK Kids, Memorial Sloan Kettering Cancer Center. Methods: A retrospective analysis of data was done on all patients who received a CD34 + SCB between 2008-2020 for PGF defined as the need for G-CSF and/or RBC/platelet transfusion support. Peripheral blood stem cells from the original alloSCT donor was the source for CD34 + SCB. Donor chimerism was done by short tandem repeat molecular testing. Patients had bone marrow donor chimerism >85%. Complete Recovery (CR) was defined as achievement of ANC > 500x106/L without G-CSF support, and Hb >7 g/dL and platelets >50 x 109/L without transfusion support. Additional outcomes of interest included development of GvHD and other toxicities. Results: 19 consecutive patients (AL/MDS-9, FA-4, SAA-2, SCID-2, CGD-1, WAS-1) received 22 CD34 + SCBs. Median age was 7.8 years (0.4–24.5 years). Donors were MRD (1), MMRD (3), MUD (6) and MMUD (9). Primary alloSCT: 15 patients received CD34 + enriched PB (12) or BM (3) and 4 patients received unmodified BM following MA (16) or RIC (3) regimens. The median CD34 + /kg was 6.38x106/kg (2.0-8.08). The median time between alloSCT and CD34 + SCB was 152 days (50-461 days). The median T-cell donor chimerism was 89% (0-100%). At the time of PGF, 13 patients had an associated infection: CMV/adenovirus (9), EBV (2), or mycobacterium (2). Three patients received ATG and 1 patient received ATG plus fludarabine prior to CD34 + SCB. The median CD34 + /kg was 5.46 (2.01-19.07). Following CD34 + SCB, only 1 patient developed aGVHD. Four patients with PGF and autoimmune cytopenias were analyzed separately. Among the 15 patients without autoimmune cytopenias, 5 of 5 patients with single (3 patients) or two (2 patients) lineage PGF and 7 of 10 patients with trilineage PGF had durable CR (12 total patients). None of these patients required a second CD34 + SCB. The median time for recovery was 20 days (7-166 days). One patient with late ANC engraftment (166 days) had CGD with 66% donor myeloid engraftment. One patient with CR died of new onset aGVHD. Three patients did not achieve CR: 2 patients transplanted for leukemia died (prior aGVHD 1, EBV 1) and 1 patient transplanted for SAA underwent a successful 2nd alloSCT. The overall survival for these patients is 72.7% at 2 years and 5 years (Figure 1). In patients with autoimmune cytopenias (n = 4), CD34 + SCB aided recovery of other lineages but did not control autoimmune cytopenias despite repeated CD34 + SCBs. Conclusions: For pediatric allogeneic stem cell transplantation recipients with high donor bone marrow chimerism (>85%) and PGF associated with infection or of unknown cause, CD34-selected peripheral blood stem cell boost is safe and can provide for durable trilineage engraftment and long-term survival. CD34-selected peripheral blood stem cell boost for autoimmune cytopenias was not effective in this cohort of 4 patients. Disclosure: Boelens: Consultancy: Race Oncology, Sanofi, Avrobio, BlueRock, Omeros, Advanced Clinical, Equillium, Medexus and Sobi. Not conflicting with content abstract Curran: Consultancy and support from Novartis Fraint, Farooki, Klein, Scaradavou, Prockop, Cancio, Spitzer, Oved, O’Reilly, Harris, Kernan – nothing to declare.
Background: Diarrhea is common in allogeneic (alloHSCT) recipients and may result from multiple causes, including chemo-radiotherapy, drug toxicity, infections, GVHD and others. Gastrointestinal endoscopies and biopsies are part of the broad differential diagnosis in alloHSCT recipients with persistent diarrhea, but their value to help establish the correct diagnosis and treatment of these patients requires thorough evaluation. Methods: In June 2016, we initiated a prospective protocol whereby all alloHSCT recipients with persistent diarrhea (>48h) despite supportive treatment and negative screening tests (stool culture, C. difficile, adenovirus, rotavirus, norovirus and parasites) would undergo esophago-gastro-duodenoscopy and recto-sigmoidoscopy with serial biopsies of the gastric fundus, duodenum, sigmoid and rectum, and urgent processing with a pathology report in 24-48h to guide management. Results: Thirty-nine patients (17 males; median age 55, 19-69; 16 HLA-identical siblings, 10 cords, 9 haploidentical, 4 unrelated; 26% of alloHSCT in this period) were included in this protocol at a median day +90 post-alloHSCT (IQR: 54-121). A total of 59 procedures were carried out, including 8 patients who had 20 additional procedures (median 3.5, range 2-5) for recurrent diarrhea and diagnostic uncertainties. A minority of only 14 procedures (24%) in 8 patients (21%) did not complete the full upper/lower endoscopy/biopsy protocol, primarily a physician’s decision for patient/clinical circumstances. GVHD was the initial cause of persistent diarrhea in 12 patients (31%): 6 out of 15 patients (40%) with GVHD in other organs (skin or liver) and 6 out of 24 (25%) patients without GVHD elsewhere. The vast majority of alloHSCT recipients with persistent diarrhea had causes of diarrhea other than GVHD (69%), including nonspecific colitis with normal histology or isolated nonspecific changes (10; 26%), infectious colitis (7; 18%) including 3 cases of CMV disease (8%), gastritis (6; 15%), pharmacologic diarrhea (3; 8%) and acute pancreatitis (1; 3%). This had major relevance for treatment decision-making to avoid immunosuppressive treatment and its complications in many patients with other causes of diarrhea. Repeated endoscopy/biopsy procedures helped the management and choice of treatment for patients with diagnostic uncertainties or recurrent episodes of persistent diarrhea (Figure 1). Diagnoses may change over time, from nonspecific colitis to CMV disease (#8) or to other infections and GVHD (#16). Some patients may have recurrent episodes of nonspecific, infectious, or pharmacologic colitis over the course of months without GVHD (#9, #23), and others have GVHD alone (#29, #35) or associated with other complications at different time-points (#26, #34). Of clinical relevance, patients with a confirmed diagnosis of GVHD may evolve over time to a pattern of nonspecific regenerative changes without signs of active GVHD (#34, #35), which also helps modulating immunosuppressive intensity and patient management. Conclusions: The prospective, systematic assessment of upper/lower gastrointestinal endoscopies and biopsies in alloHSCT recipients with persistent diarrhea shows that beyond GVHD, most patients have other causes of diarrhea, even among those with GVHD in other organs. These findings have major implications in patient management and treatment decision-making. Repeated endoscopies/biopsies in cases with recurrent episodes of persistent diarrhea are useful to clarify diagnostic uncertainties and improve patient management. Disclosure: None of the authors have conflicts of interest to declare.
Background: Anti-thymocyte globulin (ATG)/Anti-T-lymphocyte globulin (ATLG) enhances graft-versus-host disease (GVHD) prophylaxis in HLA-matched related and unrelated donor hematopoietic stem cell transplantation (HSCT). Its use is frequently accompanied by systemic infusion reactions attributable to cytokine release syndrome (CRS), but current data on this complication are lacking. Methods: Retrospective single-center analysis including consecutive allogeneic HSCT recipients treated with ATG/ATLG to prevent GVHD at the Medical University of Vienna, Austria, between January 1, 2014, and August 15, 2021. Multivariate regression models were constructed to explore risk factors of CRS and its association with clinical outcomes (acute GVHD II-IV, clinically significant cytomegalovirus infection, non-relapse mortality, overall survival) six months after HSCT. Results: A total of 284 patients (median age: 54 [interquartile range: 45-61] years; f:m = 120:164) were included in the study. ATLG was used in 222 (78%) patients, ATG in 62 (22%) patients. 166 (58%) patients developed CRS grade ≥1 according to ASTCT criteria (Lee et al., BBMT 2019) during any ATG/ATLG administration day despite prophylaxis with high-dose systemic steroids (250 mg prednisone) on all infusion days. CRS was mostly mild, with 92% of the cases having experienced grade 1-2 (Figure). Thirteen (5%) patients had CRS grade 3, one patient CRS grade 4, and no CRS-related death (grade 5) was observed. Patients with CRS showed a pronounced systemic inflammatory response as measured by inflammatory markers (i.e., C-reactive protein, interleukin-6, procalcitonin). In multivariate analysis, lymphoma as the underlying disease (subdistribution hazard ratio [sHR]: 4.93 [95% confidence interval: 1.50-22.37]; p = 0.02), high ATG/ATLG dose level (sHR 3.01 [95%CI: 1.42-6.78], p < 0.01) and body weight (sHR 1.02 [95%CI: 1.00-1.04] per kg, p = 0.03) were statistically significantly associated with CRS. Patients with CRS grade ≥1 had a higher 6-month incidence of acute GVHD II-IV than non-CRS patients (24% vs. 14%, p = 0.04) (Figure). This effect remained statistically significant only for CRS grade 3-4 (sHR 3.70 [95%CI: 1.58-8.68]; p < 0.01) after adjusting for relevant confounders (Table). Other clinical outcomes were not affected by the occurrence of CRS. Figure Upper Panels: Distrubution of CRS grades and symptoms. Lower Panels: Cumulative incidence of acute GVHD II-IV and NRM according to CRS grade ≥1 (blue = yes, red=no). Table Conclusions: In our cohort, CRS defined by ASTCT grading was a frequent but mostly mild complication following ATG/ATLG administration for GVHD prophylaxis. Our results suggest a possible interaction of CRS with GVHD risk. Further studies should be conducted to define this relation, as it might be amenable to additional prophylactic interventions (e.g., tocilizumab). Disclosure: Nothing to declare.
Background: There are growing evidence in the literature on the influence of body composition, especially sarcopenia and body fat mass, on overall mortality in various patient groups, including patient with hematological malignancies. The aim of our study was to assess the relationship between complications after allogeneic (allo) or autologous (auto) hematopoietic stem cell transplantation (HCT) and body composition, as well as bone mineral density. Methods: We evaluated total bone mineral density (BMD) and body composition in all consecutive patients who underwent autologous or allogeneic HCT between October 2019 and November 2021 in our center. The data on patients and disease characteristics, as well as the transplant details and outcomes were prospectively gathered. To assess total BMD and body composition, a densitometry using dual-energy x-ray absorptiometry (DXA) method was performed (Horizon A, Hologic, USA, 2017). The BMD was expressed in T score and Z score. Regarding the total body densitometry reports on body composition, the measured and calculated body fat values were: % body fat, fat mass index (FMI), androidal fat deposit (AFD), gynoidal fat deposit (GFD), androidal/gynoidal ratio. The measured and calculated body free fat mass values were free fat mass index (FFMI) and appendicular lean mass index (ALMI). Results: A study group consisted of 209 patients (116 male and 83 female), including 126 patient treated with autoHCT and 83 with alloHCT. The median age of autoHCT and alloHCT patients was 57 years (range, 20-73) and 56 years (range, 18-73), respectively (p ns). The median follow-up time of survivors was 6 months (range, 1-28). No patients died in the autoHCT group during the follow-up time. The non-relapse mortality rate in alloHCT group was 9.6% (8/83 patients), with no association between NRM and BMD or body fat values. With regard to non-infectious complications, no grade 3-4 complications were observed in the autoHCT group. In the alloHCT group grade 3-4 non-infectious complications (including mucositis, toxic diarrhea, cardiac arrhythmia, heart arrest, and orthostatic hypotension) were observed in 22 out of 83 patients (26.5%) with significantly lower BMD Z-score, android/gynoid ratio, FFMI, and ALMI found in this group compared to patients without serious toxicities. Regarding the infectious complications after autoHCT, the significantly lower BMI (27.1 vs 23.1; p = 0.009), as well as lower total fat values and free fat values (i.e. % Fat Trunk/% Fat Legs, FFMI, and ALMI) were observed in the group of patients with febrile neutropenia (FN) in comparison to non-FN patients. In alloHCT group, FN was associated with significantly lower BMI and lower FMI. Conclusions: The patients with post-transplant serious non-infectious complications present with lower values of fat mass and free fat mass, as well as lower BMD compared to patients without these complications. Our preliminary results suggest that fat tissue and lean tissue adjusted to height, as well as android/gynoid ratio might serve as new predictors of early non-infectious complications. Disclosure: Nothing to declare
Background: Hematopoietic stem cell transplantation (HSCT) is curative for many disorders of impaired hematopoiesis, immunity and malignancies. The number of patients undergoing HSCT has grown and incremental improvements have been made in HSCT techniques. However, among HSCT complications, endothelial cell (EC) damage syndromes remain some of the most serious complications causing severe morbidity and mortality, with limited therapies available. They include liver veno-occlusive disease (VOD) and renal thrombotic microangiopathy (TMA). The pathogenesis of the potentially fatal HSCT complication, idiopathic pneumonia syndrome (IPS), is poorly understood, however the role of EC injury may be pivotal. Defibrotide is a drug that has revolutionized the prevention and treatment of VOD, and there is growing evidence of its effectiveness in TMA. This study aimed to elucidate effects of defibrotide on pulmonary EC to evaluate its possible use in treating IPS. Methods: Primary human pulmonary microvascular EC (HPMEC, Promocell) from 3 donors were cultured under recommended conditions and treated as follows: 1) human recombinant tumor necrosis factor alpha (TNFa) (Thermo Fisher Scientific) 20ng/mL for 24hr for HPMECs activation; 2) defibrotide (Jazz Pharmaceuticals) 20-100µg/mL for 24hr; 3) either TNFa or 4) defibrotide at the above concentrations for 24h followed by co-treatment with TNFa and defibrotide for 24h; 5) EC medium alone for 24h as a negative control. Cell viability analysis in HPMEC supernatants was performed using the CyQUANT LDH Cytotoxicity Assay (Thermo Fisher Scientific). Quantitative real-time PCR (TaqMan gene assay, Thermo Fisher Scientific) was used to investigate mRNA gene expression in treated HPMECs for VWF, VCAM1, NOS3, CASP3, BAX and BCL2 compared to controls by DCt method. Expression of endothelial nitric oxide synthase (eNOS; Abcam) and vascular cell adhesion molecule 1 (VCAM1; R&D Systems) proteins were assessed in treated HPMECs using immunofluorescence microscopy and quantified by measuring fluorescence intensity mean of single cell cytoplasm mean in 20 cells per sample, compared between the samples (Nikon NiE microscopy; Fiji J software). Results: HPMEC viability was not significantly affected by TNFa exposure and defibrotide treatment alone or before/after TNFa exposure. TNFa activation of HPMECs caused upregulation of both inflammatory (VWF, VCAM1, NOS3) and apoptotic (CASP3, BAX, BCL2) gene expression. Defibrotide treatment in HPMECs at the above concentrations before and after TNFa application caused downregulation of inflammatory and apoptotic gene expression to levels comparable with controls. Defibrotide treatment before and after TNFa activation reduced expression of VCAM1 and eNOS proteins in HPMEC cultures derived from 2 of the 3 donors investigated. Conclusions: These results demonstrate downregulatory effects of defibrotide on inflammatory gene and protein expression in HPMECs in response to TNFa activation. This suggests a possible mechanism for the effect of defibrotide on IPS in HSCT recipients that is worthy of further investigation. Disclosure: AL received a non-restrictive educational grant from JAZZ Pharmaceuticals
Background: Since graft failure (GF) is associated with dismal outcomes, novel strategies aimed at prevention/pre-emptive treatment of this complication are desirable. Several studies showed that IFNγ might have an important pathogenic role in immune-mediated GF [Merli, 2019]. We hypothesized that inhibition of this cytokine through emapalumab, an anti-IFNγ monoclonal antibody approved in US for refractory/relapsed primary hemophagocytic lymphohistiocytosis (HLH) [Locatelli, 2020], may improve engraftment in patients at risk for GF. Methods: We retrospectively collected HSCT data from different centers who treated patients with emapalumab (on a compassionate use/off-label basis) in the peri-HSCT period to reduce the risk of GF. Patients at risk of GF were defined based on one or more of the following criteria: previous GF, disease know at risk for GF [e.g., primary HLH], use of reduced-intensity or non-myeloablative conditioning, T-cell depletion, and HSCT from a haploidentical donor. All patients who received at least 1 dose of emapalumab between day -1 and +28 were considered eligible. Patients were treated either during a second HSCT after experiencing a first episode of GF or upfront during the first HSCT. Treatment schedule varied in terms of dose (median 3 mg/kg/dose, range 1-10 mg/kg/dose) and frequency/number of administrations. Five patients underwent monitoring of CXCL9 (a chemokine specifically induced by IFNγ and used as marker of IFNγ neutralization) levels during the first 28 days after HSCT. Results: Between 07/2016 and 01/2021, 10 patients were treated at 3 centers (Bambino Gesù Children’s Hospital in Rome, Helsinki University Central Hospital and Karolinska Institutet in Stockholm). Main indication for treatment was primary hemophagocytic lymphohistiocytosis (n = 8); two patients affected by leukemia (T-ALL and JMML) were treated because of previous GFs. Most of patients were transplanted from a haploidentical donor after ex-vivo T cell depletion. Emapalumab was well tolerated with no infusion reactions reported. Adverse events recorded, including infectious complications, did not differ from common toxicities encountered after HSCT in pediatric patients; no treatment-related emergent adverse events were noted. Eight out of ten patients engrafted at a median of 17 days; platelet recovery was fast (median 10 days). Chimerism at day +28 was full donor in all patients who engrafted. Two patients developed chronic GVHD; 4 patients suffered from CMV infection and 1 had adenovirus infection. The patient with T-ALL engrafted, while that with JMML rejected and was rescued with a fourth allograft; none experienced relapse. However, one of the leukemia patients succumbed due to uncontrollable adenovirus infection. With a median follow-up of 40.3 months (range 10.0-65.1), 9/10 patients are alive and well. In patients who engrafted the levels of CXCL9 resembled those of the control group (i.e., that of patients who achieved sustained engraftment) of our previous report (Merli, 2019; Figure 1). Conclusions: Emapalumab seems to have a good safety profile also when used in the setting of HSCT. Preliminary data from this small cohort suggests that emapalumab may be effective in promoting engraftment. A prospective clinical trial is ongoing (#NCT04731298). Disclosure: PM: Advisory board: Sobi, Speaker’s bureau: Bellicum. Honoraria: Jazz. RS: Honoraria from Amgen and Novartis (advisory board) SM: personal fee (consultancy): MSD; Speaker’s bureau: Celgene, Kiadis, Jazz, Miltenyi. PL,:personal fees from AiCuris and grants from Astellas, Oxford Immunotech, Takeda (Shire), and MSD. FL: Research support: Bellicum; Speaker’s bureau: Miltenyi, Bellicum, Amgen, Medac, Neovii, Novartis, Sanofi, Gilead, bluebird bio; Advisory board: Bellicum, Amgen, Neovii, Novartis, Sanofi.
Background: Secondary failure of platelet recovery (SFPR) is a life-threatening complication that may affect up to 20% of patients after allogeneic hematopoietic stem cell transplantation (HSCT). The effect of recombinant human thrombopoietin (rhTPO), a naturally occurring glycosylated peptide growth factor, in treating SFPR after allo-HSCT has not been reported in detail yet. Here, we describe a single center experience with rhTPO for the treatment of SFPR. Methods: In this study, to evaluate the efficacy of rhTPO, we retrospectively analyzed 29 patients who received continuous rhTPO for the treatment of SFPR. SFPR is defined as a decline of platelet counts below 20 ×109/L for 7 consecutive days or requiring transfusion support to maintain a platelet count above 20 ×109/L after achieving sustained platelet counts ≥ 50 × 109/L without transfusions for 7 consecutive days after HSCT. Between January 2017 and April 2020, 741 patients underwent allo-HSCT in our institution. 29 patients who consistently received rhTPO for the treatment of SFPR were analyzed in this study. Patients who did not develop SFPR or developed SFPR but received other therapy or different schedule of rhTPO were excluded. RhTPO was injected at 300 IU/kg/day for 42 consecutive days at most or until platelet counts were ≥50 × 109/L, independent of platelet transfusion. Overall response (OR) and complete response (CR) was defined by platelet ≥ 20 × 109/L and ≥50 × 109/L for 7 consecutive days without platelet transfusion support within 2 months after initiation of rhTPO, respectively. Patients did not meet the criteria of response were defined as "no response". Results: Patients were treated with rhTPO immediately after diagnosis of SFPR. The median duration of rhTPO treatment was 18 days (range, 8-42 days). The platelet count in peripheral blood significantly increased after 7 days post-rhTPO initiation. Of note, platelet counts continued to increase when compared 14-day vs 1-month (21.46 ± 2.32 vs 25.21 ± 2.426 ×109/L, P = 0.03) and 1-month vs 2-month (25.21 ± 2.426 vs 34 ± 3.983 ×109/L, P = 0.01). In addition, the number of total megakaryocytes and thromocytogenic megakaryocytes in bone marrow was significantly higher after rhTPO treatment (7.03 vs 21.58, P = 0.04; 0.31 vs 1.35, P = 0.048). In total, 24 (82.8%) patients responded to rhTPO treatment and 10 (34.5%) patients further achieved CR. The 30-day and 50-day cumulative incidence of OR was 55.2% and 72.4%, respectively, and of CR was 7.0% and 28.5%, respectively, since the start of rhTPO treatment. Multivariate analysis indicated that CR to rhTPO was associated with higher CD34+ cells (≥3 × 106/kg) infused during HSCT (HR:7.22, 95% CI: 1.53-34.04, P = 0.01) and decreased ferritin after rhTPO treatment (HR: 6.16, 95% CI: 1.18-32.15, P = 0.03). Importantly, rhTPO was well tolerated in all patients without side effects urging withdrawal and clinical intervention. Conclusions: Our results emphasize the essential role of rhTPO as a safe and effective treatment option for SFPR after HSCT. Further prospective randomized comparative studies are required to make accurate assessment on the efficacy of rhTPO for treating SFPR. Disclosure: Nothing to declare.
Background: High rate of graft failure and delayed hematologic recovery are the major limitations of cord-blood transplantation (CBT), which lead to high morbidity and mortality. Prolonged thrombocytopenia may cause long-term transfusion dependency and hospitalization. Romiplostim, a thrombopoietin receptor agonist (TPO-RA), has shown to promote not only megakaryopoiesis in chronic ITP but also tri-lineage hematopoiesis in aplastic anemia. After allogeneic stem cell transplantation (AlloSCT), it has been tested for delayed platelet recovery and secondary thrombocytopenia in some previous studies. However, whether romiplostim promotes hematopoiesis if administered early after CBT is poorly understood. Here, we conducted a phase 1 trial in order to investigate the safety of starting romiplostim immediately after CBT with 6 adult patients. Methods: This study was a phase 1, open-label, single center, dose escalation study (PROMPT-1; UMIN000033799). Adult patients with hematologic malignancy in remission undergoing single-unit CBT as the first AlloSCT were eligible for this study. Patients were excluded if they had impaired organ function, donor-specific anti-HLA antibody, prior history of thrombosis, or bone marrow fibrosis. Romiplostim was administered a day after CBT and then once a week for 14 weeks or until platelet recovery. The initial dose of romiplostim was 5 µg/kg (first 3 patients) or 10 µg/kg (subsequent 3 patients), and could be escalated to maximum 20 µg/kg. The primary endpoint was the incidence of any adverse events related to romiplostim. Secondary endpoints included hematologic recovery, incidences of relapse, non-relapse mortality, thrombosis, bone marrow fibrosis, and romiplostim-specific antibody. Results: Seven patients were enrolled between April 2019 and August 2020, and romiplostim was administered except for a patient who met the exclusion criteria. The median age of the evaluable 6 patients was 40 years (range, 19–57). The diagnoses were AML (2), ALL (3), and MDS (1). Four patients received myeloablative conditioning and two received reduced-intensity conditioning. Tacrolimus and mycophenolate mofetil were used as GHVD prophylaxis. The median number of romiplostim administration was 5.5 (range, 3–15), and the maximum dose was 20 µg/kg. The administration was terminated due to platelet recovery in 5 patients, and due to non-relapse death in 1. The events possibly related to romiplostim were bone pain in 3 patients, and injection site reaction in 1. A total of 10 serious adverse events were reported in 5 patients: febrile neutropenia (4), acute GVHD (2), pneumonia (1), HHV6 encephalitis (1), CMV antigenemia (1), and arrhythmia (1). Relapse, thrombotic complications, or bone marrow fibrosis were not observed. All achieved neutrophil recovery, in whom the median day was 14 (range, 12–32). Platelet recovery was recorded in all patients except for one who died of pneumonia on day 48. The median days for platelet ≥ 50×109 /L was 34 (range, 29–98). Anti-romiplostim antibody was detected in 1 of 6 patients, which did not have neutralizing activity. Conclusions: Romiplostim can be safely administered in the early phase of CBT. Further investigation with a larger prospective trial is warranted to evaluate its safety and efficacy. Clinical Trial Registry: UMIN000033799 Disclosure: Romiplostim was provided by Kyowa Kirin. Mamiko Sakata-Yanagimoto has received research funding from Eisai, Bristol Myers Squibb, and Otsuka. Shigeru Chiba has received research funding from Kyowa Kirin, Chugai, Ono, Astellas, Beyer, Eisai, and Thyas.
Background: Pure red cell aplasia (PRCA) is a possible complication after allogeneic stem cell transplantation (HSCT) with major ABO incompatibility. Patients experience a delayed engraftment of the erythroid series, with prolonged transfusion dependent anemia and iron overload. Methods: We conducted a retrospective study, over the last 10-years, which included all consecutive major ABO mismatched HSCT performed in our unit, with the aim to assess PRCA prevalence and response to different treatment. One hundred-four patients received a major ABO mismatched transplant between May 2010 and August 2020. For each patients data about demographic and transplant characteristics, engraftment, blood transfusion and possible treatment received for transfusion dependent anemia were collected. Results: A total number of 17 cases (12%) of PRCA were diagnosed: group A donor for group 0 recipient (n = 12), group A donor for group B recipient (n = 1) and group B donor for group 0 recipient (n = 4). Stem cell source was bone marrow from haploidentical donor in 10 cases, bone marrow from HLA matched donor in one case and peripheral blood from matched donor in 6 cases. IgG antibodies titer was available as dilution ratio before conditioning start for 13 patients: 1/64 (n = 1), 1/128 (n = 4), 1/256 (n = 2), 1/512 (n = 1), 1/1024 (n = 3), 1/2048 (n = 1), 1/4096 (n = 1). Four patients did not received specific treatment but only transfusion and recombinant erythropoietin (rEPO), with a median time to reticulocytes engraftment of 91 days (61 to 92). Median number of red cells packets received during the first three months was of 19.5 (18 to 21). Six patients had received peripheral plasmapheresis before transplantation, with a median number of 2 procedures (1 to 3). Among these, one patients did not receive specific treatment other than rEPO and transfusions after transplant. The other five patients received 4 weekly administration of rituximab without obtaining a response, followed by 6 procedures of plasmapheresis each other days combined with double dose of rEPO in a week. Three patients were treated with 4 weekly administration of rituximab, two of whom were then submitted to 6 procedures of plasmapheresis each other days combined with double dose of rEPO. Three patients received only 6 procedures of plasmapheresis each other days combined with double dose of rEPO in a week and another patient received only steroid therapy and rEPO. Among patients who received specific treatment, median time to reticulocytes engraftment was of 189 days (68 to 308) and median number of infused red cell packets was of 26 (14 to 80) in the first three months after transplant. All but one patients resolved PRCA with sensitive reduction in IgG antibodies dilution ratio (p = 0.07). Conclusions: PRCA occurred in 12% of ABO mismatched patients, with a moderate prevalence among patients who had received bone marrow as stem cell source and haploidentical donor. Even if PRCA does not directly affect survival, it impacted strongly the quality of life of the affected patients. Specific treatments are needed for patients with prolonged PRCA duration and heavy transfusion support. Disclosure: Nothing to declare
Background: Endothelial syndromes (ESy) is emerging as a common but often underdiagnosed complication of hemopoietic stem cell transplantation (HSCT), with a relevant impact on morbidity and mortality; the most frequently reported ESy are: TA-TAM, Diffuse Alveolar Hemorrhage (DAH), Engraftment syndrome (ES) and VOD. Moderate-severe arterial hypertension, renal failure, neurological symptoms and capillary leak syndrome or fluid overload have been commonly recognized as recurrent clinical pictures (here defined as major signs) of endothelial damage (ED), outside of diagnostic criteria for specific ESy; they are often associated with laboratory alterations (here defined as minor signs) such as LDH increase and albuminuria, PLT drop and haptoglobin consumption. We performed a retrospective study to evaluate the incidence of damage and endothelial syndrome in our center. This study has been approved by our IRB. Methods: We retrospectively evaluated 155 consecutive patients (pts) underwent HSCT from January 2015 to December 2020. Characteristic of pts: median age 52 y (20-70 y); M/F recipient sex 57,4%-42,6%, conditioning MAC/RIC 54,5%-42,5%; ATG 56%, PTCy 39%, donor sibling 22,5%, MUD 46,5%, haploidentical 31%; source PBSC 62,5% and BM 37,4%. The median follow up was 31 months (14-54). Results: We observed 45 (29%) ESy (13 TA-TAM, 15 VOD, 1 DAH, 16 ES) with a median onset of 39 days; 121 ED (79,2%) of wich 84 without ESy (median onset: 7,5 days) and 37 associated with ESy (median onset: 31 days). We observed 69,7% aGVHD (17% grade 3-4); the aGVHD prevalence was 77,8% in ESy vs 66,4% in no Esy; 70,5% in ED vs 65,6% in no ED. The OS was 48,3, 24 and 129,5 months in whole cohort, in ESy and in no ESy setting respectively (p-value 0.109). The one year overall NRM was 27,2% with a RI of 21%; in ESy we observed NRM of 44,7% versus 20,5% in no ESy (p-value 0.003); pts developing ED without ESy, did not show increased NRM at 1 y (27,9% versus 22% in no ED. At univariate analysis, the significant risk factors for NRM were: the presence of ESy, PBSC source, aGVHD, PTCy prophylaxis, value of bilirubin; for OS: stem cell source, donor type, PTCy prophylaxis, G-CSF administration, bilirubin value, VOD, DAH and capillary leak syndrome. Conclusions: We have analyzed here both the incidence of ESy and the incidence of ED in a real life contest; ESy and ED are often underdiagnosed complications of HSCT that represent an important risk factors for OS and NRM. Both clinical signs and some simple biomarkers alterations of ED, in some cases may anticipate the ESy; although the identification of ED seems not to have a significant impact on OS, it represents a complication to be taken into account in the usual management of the transplant patient and should be captured in a prospective fashion in order to better correlate its real impact on the HSCT outcome. Disclosure: Nothing to declare
Background: High-dose chemotherapy (HDT) and autologous hematopoietic cell transplant (AHCT) is the standard-of-care (SOC) therapy for patients with aggressive lymphoma. Reporting of severe toxicities is sparse in literature but suggests high rates that increase with age despite improvements in supportive care. Clinically, severe toxicities involving the gastrointestinal (GI) tract lead to breakdown of the mucosa and translocation of the normal GI flora into circulation, increasing the risk of febrile neutropenia (FN) and severe infections. This may limit the use of the life-prolonging HDT-AHCT in older adults. Therefore, this study was conducted to document the extent of the GI toxicities in a rapidly aging population. Methods: We conducted a retrospective analysis of 143 adults from 2 academic transplant centers undergoing HDT carmustine or bendamustine with etoposide, cytarabine, melphalan (BEAM or BeEAM) followed by AHCT for aggressive lymphoma between 2018 and 2020. Severe GI Regimen Related Toxicities (GI SRRT) were defined as grade 3 or higher (≥G3) oral/GI toxicities (NCI CTCAE v5). Results: The median age of all patients was 58 years (yr) (range, 20–77). 69% were male with HL (29%) and NHL (71%). Median prior lines of therapy were 2 (1-4). BEAM was used in 99% of patients. GI SRRT occurred in 45% of patients overall. The most common GI SRRTs were diarrhea (D) (26%), nausea/vomiting (N/V) (20%), and oral mucositis (OM) (10%). GI SRRT rate increased with age: <40 (N = 32), 40-64 (N = 79), ≥65 (N = 32) yr from 31% to 44% and 63%, respectively. Frequency of D (9%, 25%, 44%), N/V (16%, 19%, 28%), and OM (3%, 10%, 16%) increased with age. When compared to the youngest group <40 yr, patients ≥65 yr had significantly higher risk of GI SRRT (OR 3.67, p = 0.0227), D (OR 7.50, p = 0.0046), and a trend towards more N/V and OM. Older patients were more likely to experience 2 or more GI SRRTs (9%, 13%, 25%). FN occurred in 66% of patients overall, increasing with age: 56%, 67%, 75% in respective age groups. Patients with GI SRRT developed FN more frequently than those without SRRT (74% vs 60%, OR = 1.86, p = 0.088). Age group 40-64 and ≥65 yo compared to <40 yo Conclusions: We demonstrate that in a contemporary cohort of patients receiving HDT-AHCT, rates of GI SRRT and FN remain high and increase in frequency with age, despite advances in supportive care. The study provides support that GI SRRT and FN may be linked. The findings underscore the need for a new therapy that could prevent or reduce these clinically meaningful toxicities so that HDT-AHCT, a potentially curative therapy, can be provided to a broader group of older adults where toxicity concerns remain paramount. Disclosure: Geoffrey Shouse has nothing to declare. Annabel Kate Frank has nothing to declare. Edward Kavalerchik is currently employed by and a current holder of stock options in Angiocrine Bioscience. Sanjay K. Aggarwal is a current holder of stock options in Angiocrine Bioscience. John K Fraser is currently employed by and a current holder of stock options in Angiocrine Bioscience. Paul Finnegan is currently employed by and a current holder of stock options in Angiocrine Bioscience. Bita Fakhri has nothing to declare.
Background: Transplant-associated thrombotic microangiopathy (TA-TMA) is a complement activation disease, considered to be caused by “multiple hits” that includes abnormal genetics of alternative pathway, calcineurin inhibitors, conditioning regimens, infections and graft-versus-host disease. It is characterized by thrombocytopenia, microangiopathic anemia with schistocytes on the blood smear, and varying organ impairment such as renal failure and gastrointestinal symptoms. Mannose residues on fungi and viruses activate mannose-binding lectin pathway, and hence activation of lectin pathway could be the prime reason for infection-driven TA-TMA. Narsoplimab, a human monoclonal antibody targeting MASP-2 is a potent inhibitor of lectin pathway. Hence, it could be an effective way to treat infection-driven TA-TMA. Methods: We describe the transplant course of a pediatric patient who developed TA-TMA following Candida-driven Macrophage activation syndrome that was treated with Narsoplimab. The data collection was performed prospectively. Results: Six-year old girl without HLA matched donor underwent haploidentical transplant for severe aplastic anaemia. Transplant conditioning included John Hopkins protocol with post-transplant cyclophosphamide and cyclosporine as graft-versus-host disease prophylaxis. In the second week of transplant, the patient developed macrophage activation syndrome necessitating treatment with steroids and intravenous immunoglobulin (IVIg). USG abdomen and blood fungal PCR revealed the diagnosis of a hepatosplenic candidiasis (Candida tropicalis) at a later date. Although macrophage activation and Candida infection was controlled with steroids, IVIg and caspofungin, the clinical course was further complicated by thrombotic microangiopathy. Patient developed hypertension in the 2nd week, followed by high LDH (1010 U/L), schistocytes (5 per hpf), low haptoglobulin (<5 mg/dl), thrombocytopenia and anaemia in the 3rd week. Ciclosporine was stopped and the patient was treated with 10 days of defibrotide without any response. The course was further complicated by microangiopathy of the gastrointestinal tract and kidneys. She had per rectal bleeding with frequent but low volume stools and severe abdominal pain, and hypoalbuminemia (1.8 g/dl) and proteinuria with high urine protein:creatinine ratio (2.8). Narsoplimab was started in the 5th week. A dramatic fall in LDH was observed after starting Narsoplimab (Figure 1). This was followed by resolution of gastrointestinal symptoms, proteinuria and recovery of cytopenia. Figure 1 LDH levels before and after starting Narsoplimab Conclusions: Lectin pathway inhibition could be useful in treating the fatal complication of TA-TMA. Lectin pathway inhibitor should be considered in Infection-related TA-TMA since lectin pathway is activated by viral and fungal infections. Clinical Trial Registry: Not Applicable Disclosure: No conflicts of the interest to declare.
Background: The mechanism of action of DNA-containing drug defibrotide in venoocclusive disease (VOD) after HSCT is not precisely known despite its proven clinical efficacy. Extracellular DNA (ecDNA) has been implicated to play a role in pathopysiology of several disorders associated with endothelial dysfunction. We aimed to test the hypothesis that defibrotide increases total, but decreases patient-derived ecDNA in VOD. Methods: Plasma samples from 10 pediatric VOD patients were collected before and shortly after initiation of defibrotide therapy. Plasma samples were collected at various timepoints between 3 hours to 48 hours since the first defibrotide infusion. Defibrotide was administered at standard dose of 25mg/kg/day divided in four 2-hours infusion per day. The underlying diagnoses included 7 neuroblastoma, 1 B-ALL, 1 JMML and 1 MDS patient. The VOD diagnosis was established based on updated pediatric EBMT criteria for VOD. EcDNA was isolated from double-centrifuged EDTA plasma and quantified using Qubit fluorometry. Nuclear (ncDNA) and mitochondrial (mtDNA) ecDNA was assessed using real time PCR. Wilcoxon non-parametric related sample test was used to compare pre- and post-defibrotide ecDNA concentrations. Results: The interindividual variability in plasma ecDNA (13-820 ng/ml), ncDNA (38-204724 genome equivalents (GE)/ml) and mtDNA (5493-367409 GE/ml) was very high already before treatment. The median concentrations of ecDNA, ncDNA and mtDNA before and after defibrotide administration were 73 vs. 124 ng/ml (p = 0.14), 4287 vs. 9203 GE/ml (p = 0.07, 53062 vs. 118142 GE/ml (p = 0.96). The pre- and post-defibrotide concentrations of total ecDNA did not follow a uniform pattern (Figure 1), similarly to ncDNA and mtDNA (data not shown). Only one of ten patients showed a steep decrease of ecDNA, ncDNA and mtDNA concentrations in plasma after start of defibrotide therapy. Conclusions: The results of this small but focused study do not suggest that defibrotide in VOD after HSCT does affect plasma ecDNA. Further analyses shall test ecDNA, its fractions and its metabolism as potential prognostic biomarkers but should also prove whether ecDNA has a role in the pathogenesis of VOD, as it does in sepsis and acute liver injury. Disclosure: Nothing to declare
Background: Allogeneic Stem Cell Transplant (allo-SCT) patients often experience weight loss as a result of impaired oral intake from gastrointestinal toxicities, taste dysfunction, and psychological effects. To date, there is currently no study in Canada examining the clinical impact of weight loss on allo-SCT outcomes. Methods: A retrospective review of 246 patients transplanted at Princess Margaret Cancer Centre from 2016-2018 was performed. Patients were categorized into <10% weight loss or > =10% weight loss from SCT to 3 months and SCT to 6 months post-transplant Clinical outcomes included 2-y rate of OS (overall survival), transplant related mortality (TRM), and relapse free survival (RFS). Cox PH and Fine and Gray’s models [CS1] examined the associations between weight loss and survival outcomes. [CS1]Cox PH models and Fine-Gray’s models. Results: The overall incidence of patients who experienced weight loss of ≥10% from SCT to 3 months post-transplant was 45.9% and from SCT to 6 months was 56.6%. In univariate analysis, there was no difference in 2-y OS in patients who had ≥10% weight loss at both 3 and 6 months post transplant compared to those who did not, p = 0.11 and p = 0.27, respectively. Similarly, there was no difference in RFS in patients who had ≥10% weight loss at both 3 and 6 months post transplant compared to those who did not, p = 0.22 and p = 0.31, respectively. However, patients with ≥10% weight loss at 3 months post transplant had significantly higher 2-y TRM, 36% versus 17%, p = 0.0007. This was also found in patients with ≥10% weight loss at 6 months post transplant, with TRM rates of 22% versus 8%, p = 0.0034. In multivariate analysis, weight loss of more ≥10% remained statistically significant for increased TRM at both 3 months (HR = 1.91 (95% CI: 1.13-3.22)), p = 0.015 and at 6 months post-transplant (HR = 3.29 (1.47-7.35)), p = 0.004. Conclusions: Patients who experienced ≥10% weight loss at 3 and 6 months post allo-SCT had significantly higher 2-year TRM. Ongoing monitoring of weight and nutritional status pre and post-transplant is imperative to allow for early intervention. More prospective studies are needed to examine specific strategies to address this potentially modifiable risk factor. Disclosure: Nothing to declare.
Background: Multi-organ failure due to transplant associated thrombotic microangiopathy (TA-TMA) is often fatal after hematopoietic cellular therapy (HCT). Narsoplimab is a human monoclonal antibody which inhibits MASP-2, the effector enzyme of the lectin pathway of the complement system. In a phase 2 trial (NCT NCT02222545), 61% of adults with TA-TMA responded to narsoplimab. While experience in children is limited, we initiated treatment in an infant with refractory TA-TMA complicated by diffuse alveolar hemorrhage (DAH). Methods: While experience in children is limited, we initiated treatment in an infant with refractory TA-TMA complicated by diffuse alveolar hemorrhage (DAH) via a compassionate use eIND. Results: A 9-month-old girl with leukemia (KMT2A-MLL) underwent a 10/10 unrelated bone marrow transplant conditioned with busulfan and cyclophosphamide, complicated by anicteric veno-occlusive disease (day + 16). Despite treatment with defibrotide, she required a peritoneal drain, ventilator support, and renal replacement therapy (CRRT). On day +18 she was diagnosed with high-risk TA-TMA (proteinuria, elevated sC5b-9 multiorgan dysfunction) and started on eculizumab. Her hematologic parameters and organ function improved on eculizumab; she was extubated and recovered renal function. On day +34 she developed acute respiratory failure requiring re-intubation and was diagnosed with diffuse alveolar hemorrhage (DAH). She was started on inhaled tranexamic acid, and CRRT again. While her CH50 remained appropriately suppressed and eculizumab level was appropriate, she had evidence of worsening microangiopathy and organ dysfunction including recurrent DAH. She was treated with meropenem and vancomycin though her infectious work up was unrevealing. She demonstrated elevated C3a and Bb, confirming activation of the alternative pathway of complement. After obtaining an eIND, the family enrolled in a single patient protocol to use narsoplimab 4mg/kg IV starting on day + 57 (eculizumab discontinued to prevent additional risk of infections). Hematologic markers of TMA improved, she was extubated without any recurrent pulmonary bleeds, CRRT was stopped, and her proteinuria is improving. She continues twice weekly dosing (received 6 weeks). Narsoplimab was well tolerated. While there are no standard MASP2 pathway complement markers, after 3 weeks of treatment, she had normalization of Bb, C3a, C5a, and sC5b-9, and decreasing platelet and hemoglobin transfusion needs, suggesting the rapid reversal of organ dysfunction seen in this child was likely from lectin pathway blockade. As of the most recent follow up, day +91, she is on ¼ L nasal cannula for oxygen support and is working toward discharge home. Conclusions: We describe an infant who developed severe TA-TMA that progressed on adequate blockade of terminal pathway using eculizumab, who had an excellent response to narsoplimab, emphasizing the role of lectin pathway activation in the pathophysiology of TA-TMA. Additional clinical trials of narsoplimab to treat pediatric TA-TMA are warranted. Disclosure: Nothing to declare
Background: Hepatic sinusoidal obstruction syndrome/veno-occlusive disease (SOS/VOD) is a life-threatening complication following allogeneic hematopoietic cell transplantation (alloHCT). The reported incidence of SOS/VOD in the literature ranges widely depending on conditioning regimen, type of transplant, patient characteristics and other factors. The aims of the study were to identify retrospectively the incidence of SOS/VOD, estimate its impact on survival and, finally, validate EBMT severity scoring in population of Polish adult transplant patients. Methods: To identify SOS/VOD patients, pilot survey study was undertaken within Polish adult transplant centers aligned within the Polish Acute Leukemia Group (PALG). Based on review of institutional medical records, we have gathered the detailed data on SOS/VOD patients who underwent alloHCT from January 2012 to November 2021 in PALG centers. EASIX pretransplant and day 0 scores were assessed using EASIX calculator. SOS/VOD severity was evaluated using novelized EBMT criteria. Results: As a result of a pilot survey study, the data was obtained from 7 centers. A total of 56 cases of SOS/VOD in 55 patients were reported, resulting in a SOS/VOD incidence rate of 1.8%. Thirty-three patients were conditioned with myeloablative regimens (59%), 21 with reduced intensity (37.4%) and 2 with non-myeloablative conditioning (3.6%). Thirty-five patients were conditioned with chemotherapy-based regimen (62.5%), and 21 patients received radiation-containing conditioning (37.5%). The median EASIX pretransplant, and EASIX 0 score were 2.59 (range 0.67-138.82) and 7.325 (range 0.95-685.15), respectively. According to EBMT grading system, 49 patients (87.5 %) presented with severe/very severe SOS/VOD. Regarding the SOS/VOD treatment, defibrotide was used in 13 out of 56 SOS/VOD cases (24%) – all these patients were classified as severe or very severe. Other therapeutic modalities included fluid and sodium restriction, diuretics, heparin, UDCA, steroids, and tocilizumab. More than two EBMT risk factors were present in almost all cases (54/56). Half of all SOS/VOD patients presented with platelet refractoriness (28 cases). Similarly, multi-organ failure (MOF) was observed in 51.7% of patients (29 cases). After the median follow-up time of 38 months (range 1-102), the outcomes were poor, with the overall survival (OS) rate of 33% (95%CI 21.8-46.5) at 2 years, and corresponding cumulative incidence (CI) of non-relapse mortality (NRM) of 65.1% (95% CI 53.6-79.6). Of note, CI_NRM was significantly lower in patients with mild/moderate SOS/VOD compared to patients with severe/very severe disease according to EBMT score (20.0% vs. 71.1%, p = 0.032). In univariate survival analysis, good performance status, low disease risk index (DRI), non-severe/very severe EBMT severity score and low EASIX scores were associated with longer OS. In a multivariate Cox proportional hazard model, a good performance status before transplantation remained an independent prognostic factor for OS. Conclusions: The incidence of SOS/VOD in adult patients after alloHCT is low, however, prevalence of severe and very severe disease in the analyzed cohort might suggest that SOS/VOD is still underdiagnosed in adults. EBMT severity scoring allows adequate NRM risk assessment. Furthermore, pretransplant performance status, DRI, and EASIX scores might predict survival in SOS/VOD patients, with performance status confirmed in our study as an independent prognostic factor. Disclosure: The study is supported by grant JAZZ Pharmaceuticals IST 10682
Background: Allogeneic stem cell transplantation (alloSCT) is a curative treatment option for patients with many malignant and non-malignant hematological diseases, being the high treatment-associated mortality the main clinical challenge to overcome. Endothelial dysfunction plays a crucial role in major complications of alloSCT, such as the graft-versus-host disease (GvHD), sinusoidal obstruction syndrome (SOS/VOD) and transplant-associated microangiopathy (TA-TAM). The Endothelial Activation and Stress Index (EASIX) is a marker of endothelial damage that has been recently validated to identify a patient population at high risk for SOS/VOD and to predict the risk of death after acute GvHD (aGvHD). The study’s primary end-point was to test the capacity of EASIX and modified EASIX (m-EASIX) to predict these alloSCT complications Methods: We performed a retrospective cohort analysis of 72 adult alloSCT recipients at a single Spanish transplant center (Fundación Jiménez Díaz University Hospital) between January 2017 and December 2020. EASIX was calculated by the formula: LDH (U/l) x Creatinine (mg/dL)/Thrombocytes (nL), and modified EASIX (m-EASIX) was calculated replacing creatinine with CPR. Both scores were assessed prior to conditioning in all patients. To analyze the differences between the values pre-transplant and the onset of complications, we use the U-Mann-Whitney Test and the independent T-test. All the statistical analysis were done with SPSS program 25.0. Results: The baseline patient characteristics by cohort are given in Figure 1A. Median PRE-EASIX values were 1.22 (interquartile range [IQR] 0.74 to 2.10). Median PRE-m-EASIX values were 0.60 (IQR 0.14 to 1.98). SOS/VOD was diagnosed in 7 patients (9.7%, median onset day +14). Median EASIX in patients who develop SOS/VOD was higher than in patients who did not develop VOD/SOD (4.48 vs. 1.15, p < 0,014). There were no differences in the m-EASIX values. Grade II-IV aGvHD was diagnosed in 19 patients (26.4%, median onset day + 71). Median EASIX in patients who develop aGvHD was higher than in patients who did not develop aGvHD (1.58 vs. 1.08, p < 0,034). There were no differences in the m-EASIX values. TA-TAM was diagnosed in 3 patients (4.2%, median onset day +46). There were no differences both in EASIX and m-EASIX values between patients who develop TAM and who did not develop TA-TAM (Figure 1B). Conclusions: In our cohort, higher PRE-EASIX values were associated with the development of SOS/VOD and grade II-IV aGvHD. No association was found between PRE-EASIX values and TA-TAM. We did not find differences between PRE-m-EASIX values. Disclosure: The authors declare no conflicts of interest.
Background: Neuroblastoma is the most common paediatric extra-cranial solid tumour and accounts for 12% of cancer-related deaths in children. Autologous stem cell transplant following Busulfan-Melphalan (Bu-Mel) conditioning has shown superior survival compared to other regimens and is currently the standard of care for high-risk (HR)-neuroblastoma. High incidence of Veno-occlusive disease (VOD) of up to 38% has been reported in patients undergoing this treatment. To identify potential new factors associated with VOD, we undertook a single centre analysis, assessing incidence as well as selected clinical and laboratory parameters. Methods: A retrospective analysis of all patients undergoing Bu-Mel conditioning with autologous stem cell return for HR-Neuroblastoma over a 10 year period (August 2011-2021) was performed. VOD was defined as per modified Seattle criteria. All patients received intravenous Busulfan and prophylactic Ursodeoxycholic acid. The following variables were extracted: age, sex, percentile of weight at start of conditioning, presence of liver impairment (ALT/AST/Bilirubin)/liver metastasis at time of starting conditioning, CD34 cell count, number of stem cell bags transfused (to account for DMSO content), number of days over which stem cells were infused, platelet counts on day 0 and day 8 along with platelet refractoriness (≥1 platelet transfusion/ day for 3 consecutive days to achieve a platelet count > 20x 109/L or control bleeding), parenteral nutrition required in immediate post-transplant period (yes/no); duration of Defibrotide therapy and VOD related mortality within 100 days of transplant. For numerical factors median, range and percentages were tabulated. Appropriate statistical tests were performed and significance calculated. Results: Of a total of 44 patients 19 (43%) fulfilled the diagnostic criteria for VOD and received Defibrotide. Two novel factors were identified in our cohort which showed significant correlation with VOD: 1) platelets <20 x 109/l within 8 days of stem cell return (14/19 in the VOD group versus 10/25 in the No-VOD group, p = 0.031); 2) weight <25th percentile (9/19 in VOD group versus 3/25 in No-VOD group, p = 0.014). No statistical correlation was identified for other factors as outlined above. Interestingly, 63% (12/19) of VOD patients were platelet refractory 7 days prior to starting Defibrotide but reversal of portal flow on doppler was only seen in 52% (10/19) at time of diagnosis. Defribotide treatment was started at a median of 20 days (range: 7-27) after stem cell return and was administered for a median of 14 days (range: 4 -25). VOD related mortality was 6.8% (3/44). Conclusions: Compared to previous studies, we report a higher incidence of VOD in our patient cohort. We report a clinical association of early drop in platelet count within 8 days post autologous transplant and VOD development. If proven significant in larger studies, this might be beneficial for early diagnosis of VOD. Further, we identified for the first time a weight <25th percentile as a potential factor contributing to VOD. Optimizing patient weight prior to transplant might be beneficial. More data including assessment of other measures of nutritional status are needed to better assess the potential clinical association and a larger multi-centre cohort study is required. Disclosure: Nothing to declare
Background: VOD/SOS is a potentially fatal complication of HCT. Its diagnosis is challenging, due to the many patient- and transplant-related VOD/SOS risk factors, and the dynamic presentation of symptoms. This study identifies the clinical manifestations of VOD/SOS post-HCT and determines how these relate to the features that comprise standard VOD/SOS diagnostic criteria using an SLR. Methods: Medline and Embase were searched up to 4 March 2021 for studies of VOD/SOS post-HCT and were supplemented with guidelines on VOD/SOS diagnosis and management. English language reports of observational and database studies were included if they studied adults or children with any HCT-related disease, therapies aimed at preventing/treating VOD/SOS, or HCT-associated VOD/SOS outcomes. Publications were evaluated based on inclusion of patients with features related to VOD/SOS diagnostic criteria and changes in diagnostic features over time; reports were categorised by specific subgroups within inclusion criteria. In cohort studies, an unweighted mean was calculated for the proportion of patients with each VOD/SOS feature; for case studies, the number of case reports where the feature was present was divided by the total number of cases where any features were reported. Results: Overall, 204 publications were included in the SLR. Among cohort studies (n = 22,121 patients receiving HCT), the mean proportion of patients with reported VOD/SOS features was: weight gain (any) 87%, hyperbilirubinaemia 82%, hepatomegaly 76%, thrombocytopaenia 70%, ascites 61%, and right upper quadrant [RUQ] pain 61%. Among case studies (n = 280 patients), the proportion of patients with each reported feature was: hyperbilirubinaemia 79%, ascites 59%, hepatomegaly 47%, RUQ pain 21%, thrombocytopaenia 9%, and weight gain (>5%) 6%. There was no consistent pattern on which VOD/SOS feature appeared first (Table), although there was a trend for hepatomegaly and RUQ pain as early manifestations, with hyperbilirubinaemia, weight gain and ascites observed subsequently, consistent with the pathology of progressive VOD/SOS. VOD/SOS features changed rapidly in some patients after the first symptoms developed. Conclusions: Presentation of VOD/SOS symptoms post-HCT in individual patients is heterogeneous, underscoring the difficulty in VOD/SOS diagnosis. VOD/SOS features are not reported in a distinct sequence, requiring a high index of suspicion for VOD/SOS and continuous vigilance by those involved in patient monitoring post-HCT. Table. Typical VOD/SOS featuresa in case reports by order of occurrence aFor each report, ≥1 feature could count as a first/subsequent manifestation (e.g., if a report listed ascites and hepatomegaly as the first manifestations, both were counted as first). bThe denominator was the total number of occurrences of each feature. cTotal number of cases that reported a first, second, third, or fourth manifestation of VOD/SOS. Disclosure: JA is an employee of and holds stock ownership/stock options in Jazz Pharmaceuticals. LR and KR are employees of Crystallise Ltd., which received funding from Jazz Pharmaceuticals to conduct this literature review. LM was an employee of Crystallise Ltd. at the time this study was conducted. AM is a director and employee of Crystallise Ltd.
Background: The importance of vitamin D deficiency to transplant outcomes is largely unknown, however vitamin D is known to be critical for bone health and has known immunomodulatory roles. Several retrospective analyses have been performed examining the association between vitamin D deficiency and transplant outcomes, with variable findings related to overall survival, rates of graft-versus host disease (GVHD), CMV disease, and relapse risk. In addition to contradictory evidence, there is variability in the definition of deficiency, and the optimal marker of clinically significant deficiency remains unknown. A recent EBMT study of practices relating to peri-transplant vitamin D supplementation showed a lack of consensus regarding optimal management and absence of guidelines in this area. In New South Wales, it is recommended to maintain vitamin D levels above 75nmol/L following allogeneic stem cell transplant, for optimal bone health. Methods: A retrospective audit of peri-transplant vitamin D levels in patients undergoing allogeneic stem cell transplant at St Vincent’s Hospital in Sydney between January 2016 and December 2020 was performed. Rates of testing within 60 days pre-transplant and 100 days post-transplant were assessed. The incidence of vitamin D deficiency (defined as 25-hydroxyvitamin D level <50nmol/L) pre and post transplant were calculated. Testing and deficiency rates at any time post transplant were also assessed and the proportion with vitamin D < 75nmol/L was determined. Results: A total of 209 transplants were performed in the five year study period. Rates of pre transplant vitamin D testing increased each year over the study period, being performed in 26% of patients undergoing transplant in 2020. Post transplant testing rates were higher than pre-transplant rates across all years, varying between 24-66%. Rates of pre-transplant deficiency generally decreased over time, whilst rates of post transplant deficiency remained high across the study period at 50-70%. As pre-transplant testing increased, the rate of vitamin D deficiency decreased, however post-transplant vitamin D deficiency was seen in 50-70% of patients across the study period. Of the 209 patients included in this analysis, 154 (74%) had at least one vitamin D level tested post transplant. Of these, 131 (85%) were deficient relative to local guidelines recommending a vitamin D level of >75nmol/L for maintenance of optimal bone health. Conclusions: The importance of vitamin D deficiency to transplant outcomes remains controversial with little prospective research to date. Most studies focus on pre-transplant deficiency, however the development or worsening of vitamin D deficiency in the acute post transplant period has received little attention, despite this likely being a critical period for development of GVHD and infection. Our findings suggest that even in Australia, a country with long sunlight hours by global standards, pre and post transplant vitamin D deficiency is common. While the importance of this to transplant outcomes remains to be determined with prospective research, there is an existing mandate for supplementation due to the impacts of vitamin D deficiency on bone health. Disclosure: No funding sources or conflicts of interest to declare.
Background: Hematopoietic stem cell transplant-associated thrombotic microangiopathy (HSCT-TMA) is a serious and potentially fatal complication of HSCT with no approved treatment. Frequent co-existing transplant complications can affect outcomes for patients with HSCT-TMA. A systematic literature review was conducted to better understand outcomes in this patient population. This literature review describes the natural history of HSCT-TMA in adults who have not received any specific pharmacological treatment for this condition, and the impact of co-existing complications on that history. Methods: A protocol-driven, retrospective systematic literature review was conducted by searching PubMed for unique, English-language articles published between 2000 and 2020 using specific and relevant search terms. The criteria for including patient data from the articles identified were: age ≥18 years; diagnosis of HSCT-TMA following allogeneic HSCT; and had identifiable patient-level data that included any of the following: 1) any relevant laboratory or clinical variable (i.e., platelet count, lactate dehydrogenase [LDH], organ function, or transfusions) OR 2) report of “response” AND at least one risk factor (i.e., graft versus host disease [GVHD], neurological dysfunction, infection, pulmonary dysfunction, renal dysfunction, gastrointestinal dysfunction, or relapse or persistence of underlying malignancy). To assess the natural history of HSCT-TMA, patients from the published literature were excluded if they received pharmacological treatment for HSCT-TMA. Data from the literature were subjected to meta-analysis statistical methods using random effects logistic regression. Resolution of HSCT-TMA was defined as 1) evidence in the article of reported “response” OR 2) improvement in platelet count, LDH, and either organ function or transfusion burden, if reported in the article. Unreported measures in all articles were imputed as improvement. Rates of HSCT-TMA resolution and 100-day survival from HSCT-TMA diagnosis were estimated with 95% confidence intervals (CI). Results: The literature search identified 459 articles that were subsequently reviewed in duplicate by independent medically qualified reviewers, with adjudication by a physician. Twenty-five articles, which included 149 distinct patients, met the predefined criteria. Patient counts in these 25 studies ranged from 1 to 22 with a median of 4 patients. Median patient age was 41.5 years (range, 18–66 years). Fifty-one percent were male, 39% were female, and sex was not reported for 10% of patients. Median time from HSCT to TMA diagnosis was 48 days (range, 1–2500 days). The overall HSCT-TMA spontaneous resolution rate from the identified articles was 23.3% (95% CI, 15.1–34.2%); in patients with co-existing GVHD or infection, HSCT-TMA resolution rates were 20.8% and 12.7%, respectively. The overall 100-day survival following HSCT-TMA diagnosis was 37.3% (95% CI, 22.8–54.6%). Conclusions: This systematic literature review provides a comprehensive approach for determining the natural history of adult patients with HSCT-TMA in a real-world setting. HSCT-TMA resolution rates and 100-day survival in the absence of pharmacological treatments are generally low, highlighting the unmet medical need for an approved treatment for HSCT-TMA. Disclosure: Steve Whitaker, Narinder Nangia, Kathy Sotolongo, William Pullman: Employment (Omeros Corporation); Edmund Ng: Consultant (Omeros Corporation)
Background: Hematopoietic cell transplantation (HCT) represents the only curative choice for children with certain malignant and non-malignant diseases. Nevertheless, HCT remains a high-risk procedure, and can lead to severe complications, requiring transfer to pediatric intensive care unit (PICU) Methods: We retrospectively reviewed the medical charts of pediatric patients who underwent HCT between January 2014 and August 2020 and required PICU admission in the first 100 days after HCT at King Hussein Cancer Center (KHCC). The aim of our study is to describe incidence, causes, and outcomes related to PICU admission in the first 100 days post-HCT Results: From January 2014 to August 2020, 530 children and young adults (0-19 years) underwent HCT at KHCC. Thirty seven patients (7%) were admitted to PICU in the first 100 days post-HCT for a total of 46 PICU admissions. The main causes of PICU admission were respiratory failure (32%), septic shock (21%), Gastrointestinal bleeding (19%), multiorgan failure (MOF) (16%), and seizure (11%). The overall 100-day cumulative probability of survival after PICU admission was 84% and six patients died. Causes of death were sepsis and MOF in 4 of the 6 patients, 1 with pulmonary hemorrhage, and one with acute respiratory distress syndrome (ARDS). Five of the 6 patients who died within 100 days after PICU admission were admitted within 10 days of stem cell infusion and 3 of the 6 patients had severe aplastic anemia. Seven patients (19%) required invasive ventilation and only 1 patient died. Four patients (11%) required dialysis and 1 died. On univariate analysis, only MOF had a negative impact on survival (p = 0.02) Conclusions: These findings may provide support for the clinical decision making process on the opportunity of PICU admission for severely immunocompromised patients after HCT Clinical Trial Registry: NA Disclosure: Nothing to declare
Background: The presence of donor-specific antibodies (DSA) has a known impact on graft rejection and delayed immune recovery. However, it is unknown if recipient-specific antibodies (RSA) affect transplant-related complications. Here we present the incidence and impact of RSA on transplant outcomes in 17 consecutive pairs of recipients and their mismatched related donors. Methods: At the donor selection, we checked both donors (D) and recipients (R) for the presence of DSA and RSA. Cytometric Luminex screen was performed as a first step. If any anti-HLA antibodies were detected, the next test involved a Luminex single antigen beads array (SAB) in determining the type of DSA/RSA. The results were presented as a mean fluorescence intensity value (MFI). All patients received a myeloablative conditioning regimen, peripheral blood stem cells (except one bone marrow), the same type of immunosuppression with post-Cy and tacrolimus/MMF, except one treated with sirolimus. Results: The majority of patients, 15/17, were transplanted from haploidentical donors, two from mismatched related donors (1 mismatch). DSA were detected in 2 recipients (2/9; 22% patients with positive screen) whereas RSA in six donors (6/9; 66%). There was no difference in sex between DSA and RSA groups – R: 1 man/1woman, D: 3men/3women. We didn`t find differences in MFI values. Among 3 RSA-positive female donors, two were pregnant in the past. None of the donors had a prior blood transfusion history. The median follow-up was 141 days (13-401); 3 patients are still in the early post-transplant period (before engraftment) and were excluded from further analyses. Among the remaining 14 cases, two patients didn`t have engraftment and finally died due to transplant complications, 4 had poor graft function. Ten out of 14 patients (71%) experienced CMV reactivation; in 11/14, transplant-associated thrombotic microangiopathy (TA-TMA) was diagnosed, acute graft-versus-host disease (GvHD) was observed in 5/14 cases (36%). There was no impact of RSA on graft failure/poor graft function; none of the patients with aGvHD had the RSA positive donor. RSA were observed more often in the TA-TMA group, however with no statistical significance. All data, including demographic characteristics and antibodies, are summarized in table 1. Conclusions: Our preliminary findings indicate that the incidence of anti-HLA-positive donors with RSA is relatively high despite the lack of transfusion history. The impact of RSA on transplant-related complications remains unknown, however, the association between RSA and TA-TMA requires further testing. Disclosure: Nothing to disclose
Background: The vast majority of allogeneic hematopoietic stem cell transplant (allo-HSCT) patients require red blood cell (RBC) and platelets transfusion during the preengraftment phase after HSCT. Both RBC and platelets transfusion had been associated with adverse effects, including transfusion reactions or transmissible diseases. It had been shown that allo-HSCT patients who received more RBC transfusions after transplantation had a higher risk severe acute graft-versus-host disease (aGvHD) and had worse overall survival. The principal aim of this study was to identify the principal clinical factors that are associated with RBC and platelets transfusions in order to identify possible interventions that can reduce transfusion requirements. Methods: We retrospectively reviewed a cohort of 72 adult patients who had undergone alloSCT at a single Spanish transplant center (Fundación Jiménez Díaz University Hospital) between January 2017 and December 2020. Patient demographics and transplant-related data were extracted from the clinical history of the patients. It was collected the number of RBC and platelets units transfused before the HSCT, between day +0 and +30 post-transplant and between day +30 and +100 post-transplant. All the statistical analyses were done with SPSS program 25.0. Results: A total of 13 patients (18%) did not require RBC transfusion in the first 30 after alloSCT. Median pretransplant Hb was 13.2 gr/dl in patients not transfused and 11.2 gr/dl in those transfused (p < 0.01). There were no differences between the underlying disease, the conditioning regimen or ABO groups and the amount of RBC or platelets transfusions required. Low pretransplant hemoglobin (Hb) level, the use of erythropoietin (EPO) prior to transplantation and the development of veno-occlusive disease (VOD/SOS) have been identified with increase RBC requirements in the first 30 days. Acute graft-versus-host disease (aGvHD) increased transfusion needs in the period from day +31 to +100. Prior autologous HSCT, the development of VOD/SOS and grade III-IV mucositis were associated with increased platelet requirements in the first 30 days after transplantation. GvHD was associated with higher platelets transfusions on days +31 to +100. Conclusions: Anemia, the use of EPO, VOD/SOS and aGvHD are factors associated with higher RBC requirements, while prior autologous HSCT, grade III/IV mucositis, VOD/SOS and aGvHD are associated with platelets transfusion. The identification of factors that avoid RBC and platelets transfusions may allow the development of strategies aimed at reducing RBC and platelets transfusions requirements and improving outcomes after allo-SCT. Clinical Trial Registry: Anemia, the use of EPO, VOD/SOS and aGvHD are factors associated with higher RBC requirements, while prior autologous HSCT, grade III/IV mucositis, VOD/SOS and aGvHD are associated with platelets transfusion. The identification of factors that avoid RBC and platelets transfusions may allow the development of strategies aimed at reducing RBC and platelets transfusions requirements and improving outcomes after allo-SCT. Disclosure: The authors declare no conflicts of interest
Background: Oral mucositis [OM] is a complications of high dose chemotherapy which is frequently observed in hematopoietic stem cell transplant settings. Antioxidants line N acetyl cysteine [NAC] proposed to prevent the OM. In the present observational study to evaluate the effects of NAC on OM incidence and severity. Methods: The current study includes both pediatric and adult patient who were enrolled for both autologous and allogenic SCT received high dose chemotherapy or Myeloablative chemotherapy. Total 10patients were enrolled as observational study. NAC was given as parenteral route until the conditioning therapy complete and there after prescribed oral NAC until the engraftment.OM score was monitored during transplant period until engraftment. Results: In this observational study total 10 patient were included, five were children less than 18years old and remaining five were adults. Four were autologous SCT and six were allogenic SCT[2 were haploidentical SCT;4 Matched related donor].underlying diagnosis- Multiple myeloma(n = 1)-High dose melphalan 200mg/m2],Relapsed Lymphoma(n = 2)-BEAM conditioning,Neuroblastoma(n = 1)- BuMel conditioning, Severe aplastic anemia(n = 3)-Fluderabine + Cyclophosphamide and ATG, Hyper IgE syndrome (n = 1)-Flu + CTx +Bu+ TBI with PT/Cy, Congenital Amegakaryocytic thrombocytopenia (n = 1) -Flu + CTx +Bu +TBI with PT/Cy, All patient received high dose chemotherapy.OM Maximum grade observed was grade 2 in 4 patient remaining 6 had only erythema as grade 1.All patient were engrafted, no mortality was seen until day +100 post transplant. Conclusions: The NAC significantly reduces the OM in high dose chemotherapy setting, which may reduces the bacterial translocation and therefore gram negative sepsis, NAC did not affect engraftment and transplant outcomes. We also observed reduced stool volume in case of Gut GVHD and faster recovery when used for gut GVHD, though numbers were small may need larger study to confirm this. Clinical Trial Registry: The NAC significantly reduces the OM in high dose chemotherapy setting, which may reduces the bacterial translocation and therefore gram negative sepsis, NAC did not affect engraftment and transplant outcomes. We also observed reduced stool volume in case of Gut GVHD and faster recovery when used for gut GVHD, though numbers were small may need larger study to confirm this. Disclosure: nothing to declare
Background: Hepatic venoocclusive disease (HVOD) is a potentially severe complication of high-intensity chemotherapy (HIC) for hematopoietic stem cell transplantation (HSCT). The development of HVOD can be rapid and unpredictable, thus the recognition of the main HVOD risk factors and close monitoring of them are essential for the optimal complication management. The HVOD diagnosis has traditionally been based on the Baltimore or modified Seattle criteria, which assess the common signs and symptoms of HVOD, that typically takes part during the first three weeks after transplantation. The symptoms of HVOD are dynamic, variable and can be progressive. This complication is relatively frequent after HIC used for allogeneic HSCT; however, it is unusual in an autologous HSCT. Methods: The first of the cases is a 43-years-old woman with a diagnosis of IgA multiple myeloma ISS IIIA/ISS-R 2, who received autologous tandem transplantation after induction chemotherapy with two cycles of Bortezomib + Cyclophosphamide + Dexamethasone and five cycles of Carfilzomib + Lenalidomide + Dexamethasone due to progression. In the first transplant she arrived with a partial response, BUMEL conditioning was used. On day +25 she presents signs compatible with HVOD: hepatomegaly, ascites with increase in weight and hyperbilirubinemia. Defibrotide® was administered during 23 days, and condition improves. Subsequently, a second autologous HSCT was carried out with disease progression. A conditioning with MEL200 was used and the patient did not present signs compatible with HVOD. Secondly, a 29-year-old man with acute promyelocytic leukemia diagnosis achieved complete remission (CR) after induction treatment. However, three years later he presented clinical and molecular relapse, for this, rescue chemotherapy treatment was administered reaching CR again. He was admitted to autologous HSCT, performing BEA regimen conditioning. On day +13, he presented worsening of liver analytical parameters with increased bilirubin and transamintases, severe hydropic decompensation with weight gain of more than five kilos, and painful hepatomegaly. Due to the high suspicion of HVOD and the clinical and analytical worsening with bilirubin 8.6 mg/dL, Defibrotide® is applied assuming hemorrhagic risk, because at that time he presents severe thrombopenia and coagulopathy, in consequence a prophylactic transfusion of platelets and antithrombin is prescribed during the first days; Ursodeoxycholic Ac is also associated. After these measures and 21 days of Defibrotide® treatment, the patient presented a favorable evolution. Results: In both cases It can be observed as risk factors for the HVOD development: Busulfan administration as conditioning and the performance of an autologous HSCT. Conclusions: Identifying patients with higher risk of HVOD could be the key for the early diagnosis and unanticipated treatment with Defibrotide®, which is also associated with better results. Some investigators have suggested that drug treatment should begin with the first possible HVOD signs/symptoms, even if patients do not already meet all the criteria for a complete diagnosis. Furthermore, although the review data indicate that this complication occurs more frequently after allogeneic transplantation, recent studies in adult and pediatric patients with autologous HSCT reported an increasing incidence of this complication. Disclosure: Conflict of Interest: None
Background: SARS-CoV-2 outbreak has challenged Spanish health system, with most of health care facilities exclusively dedicated to COVID-19 during the first wave. This situation forced Hematology departments to defer non-urgent procedures or visits to the hospital. In allogeneic stem cell transplant (AlloSCT) patients, this recommendation conflicts with the need of close follow-up due to potential complications, particularly related to graft versus host disease (GVHD). In the COVID-19 scenario, we had to balance optimal care of AlloSCT patients with minimization of their risk of exposure in the hospital. Telemedicine emerged then as a fundamental tool that could be implemented from now on to improve patients quality of life. Methods: We elaborated a Telemedicine protocol based on daily review of scheduled appointments to evaluate the need of presenting visit, phone consultations and home-delivery drugs system developed by Pharmacy Department. To evaluate our protocol, we prospectively collected data of medical consultations for March 16th to April 31st, both in-person or by phone, and compared it with the same period from 2019. To validate the protocol, we specifically checked: 1) percentage of presential visits after a phone evaluation; 2) rate of chronic GVHD diagnosed. Results: From March 16th to April 30th, 574 out of 635 scheduled appointments were implemented, with 90% of assistance maintained during the outbreak peak. A total of 211 patients were attended: 25 patients in the first 100 days after AlloSCT with 100% face-to-face assistance (175 visits) and 186 patients from long-term follow-up (LTFU) clinic (399 consultations) with 43% face-to-face assistance. Regarding these LTFU consultations, we observed that 19 patients with GVHD, cytopenias or relapse represented 61% of face-to-face evaluations. When comparing to our control group from 2019 (206 patients, 489 consultations), we observed a reduction in a 55% of face-to-face visits in LTFU patients, whereas those in <100 days after AlloSCT maintained 100% in-person assistance. A total of 218 scheduled appointments (117 patients) were managed by phone; face-to-face evaluation was recommended in 14 cases due to infection (n = 2), suspected GVHD (n = 10) or suspected progression (n = 2). Twenty-four emergency calls were managed with a recommendation of face-to-face evaluation in 20 of them due to infectious disease (n = 16), suspected GVHD (n = 3) or symptoms consistent with cytopenias (n = 2) or thrombosis (n = 1). Among those patients managed by phone and considered not candidates for a face-to-face consultation, no cases of later hospital admission have been documented after a follow-up period of 28 to 70 days Chronic GVHD rate was similar in patients managed by phone in 2020 and those with presential evaluation in 2019 (9.8% vs 8.5%; p = 0.44). Results are summarized in Figure 1. Conclusions: Our telemedicine based protocol allowed us to maintain 90% of activity during 1st wave of covid-19 pandemic, limiting visit to the hospital to those in real need. Telemedicine proved to be a feasible tool for AlloSCT patients follow-up and it allows to diagnose common complications when applied by a trained Hematologist. Telemedicine implementation for AlloSCT patients would improve their quality of life by decreasing the number of visits to the hospital. Disclosure: Nothing to declare.
Background: Secondary neoplasms (SN) represent serious late complications in long-term survivors after hematopoietic stem cell transplantion. With improved outcomes and better post-transplant care, the number of long-term survivors is continually increasing. The risk of developing SN is the result of a complex interaction of factors related to treatment, recipient and immunosuppression drugs. This study aims to describe the SN reported in patients who received umbilical cord blood transplantations (UCBT). Methods: We performed a retrospective analysis of cases of SN in recipients of UCBT reported to Eurocord and to the European Group for Blood and Marrow Transplantation (EBMT) registries. Results: From October 1988 to December 2018, 253 cases of SN (2,4%) were reported within the cohort of 10358 patients who received UCBT. Graft sources of the 253 UCBT which resulted in SN included single (n = 159), double (n = 80), expanded (n = 2) UCB and UCB associated with other stem cell sources (n = 12). Patients with a follow-up less than 2 years (y) were excluded. Three main subgroups of SN were identified : post-transplant lymphoproliferative disorders (PTLD, n = 153, 60%), secondary leukemia/myelodysplasia (SL/MDS, n = 26, 10%) and solid tumors (n = 75, 30%). One hundred fifty-two patients developed PTLD within a median time of 3 months (0.3-9.6) after UCBT and had a median age at UCBT of 27y (0.3-68). Primary disease was hematological malignancy in 120 patients (79%). Ninety patients (59%) received myeloablative conditioning (MAC), 68 patients (45%) received total body irradiation (TBI) and 120 patients (79%) received anti-thymocyte globulins (ATG). Eigty-five patients (56%) died of PTLD. At last follow-up, 31 patients (20%) were alive with a probability of 5y-survival after the diagnosis of PTLD of 21 ± 3%. Twenty-six patients developed SL/MDS within a median time of 27 months (15-50) after UCBT, including 16 acute myeloid leukemia (AML), 2 acute lymphoblastic leukemia (ALL), 4 donor-derived AML and 4 MDS. The median age at UCBT was 22y (1-62). Twenty-three patients (88%) were transplanted for malignant diseases [ALL (9); AML (4); Myeloproliferative disease (4); Lymphoma (6)], two patients for bone marrow failure and one patient for hemoglobinopathy. Thirteen patients (52%) received MAC regimen, 10 patients (40%) received TBI and 13 patients (52%) ATG. At last follow-up, 9 patients (35%) were alive with a probability 5y-survival after the diagnosis of SN of 19 ± 14%. Seventy-five patients (developed 83 solid tumors within a median of 57 months (32-99) after transplantation. The median age at UCBT was 45y (0.5-67). Thirty one patients (42%) received MAC, 47 (63%) received TBI and 36 (52%) received ATG. The most frequent tumor sites included lung (12), bone/soft tissue (11), thyroid (9), gastrointestinal (8), oral cavity (7) and skin (7 basal cell carcinoma BCC; 4 non-BCC), in addition to less common sites (20). Thirty-seven (49%) patients were still alive at last follow-up; the probability of 5y-survival after the diagnosis of solid tumors was 48 ± 6%. Conclusions: Recipients of UCBT are at risk to develop both early and late onset SN with poor outcome. Identification of risk factors and life-long screening for early detection of solid tumors are mandatory to improve overall survival. Clinical Trial Registry: Not applicable Disclosure: Nothing to declare.
Background: Insight into the long-term patientreported outcomes (PROs) after pediatric hematopoietic stem cell transplantation (HSCT) for non-malignant diseases is lacking but essential for optimal shared decision-making, counseling and improving care. Methods: In this single-center cohort study PROs were evaluated from December 2020 – March 2021 among patients ≥2 years after pediatric allogeneic HSCT for non-malignant disease from the Leiden University Medical Center. Validated age-appropriate PRO measures (PedsQLTM 4.0 and PROMIS item-banks) were selected based on availability in Dutch and optimal international reference. Patients aged ≥8 years completed self-report, if aged <8 years parents completed the proxy version. Mean PedsQLTM scores and PROMIS T-scores were compared to Dutch reference data using independent t-tests. Variables associated to PROMIS T-scores were determined using univariate regression analysis. Results: 119 of 174 patients participated in this study (68% response rate), median age was 15.8 years (range 2-49), of whom 72 males. Median follow-up duration after HSCT was 8.7 years (IQR 4.2-15.4). Indications for pediatric HSCT were inborn errors of immunity (IEI, N = 41), hemoglobinopathies (HB, N = 37), bone marrow failure (BMF, N = 41). Conditioning regimens were mainly treosulfan-based (41%), busulfan-based (34%), and cyclophosphamide-based (17%). Significant lower scores compared to the Dutch population were found on the following PedsQLTM 4.0 subscales: Physical Health (children (5-7 years) and adolescents), Psychosocial health (adolescents), School Functioning (children (5-12 years) and adolescents). Young adults (18-30 years) reported no significantly different scores. Table 1 presents mean delta PROMIS T-scores compared to Dutch reference data, if available, per age category. *p < 0.05; aN = 14; bN = 56; cN = 52; d N = 27 1Higher scores indicate more symptoms; 2Higher scores indicate better functioning. USA reference (mean T-score = 50; SD = 10) was used if Dutch reference was not available. Young adults (19-30 years) show the most favorable scores compared to Dutch reference in the PROMIS item-banks. Univariate regression analysis showed significantly worse scores for HB compared to IEI on Pain Interference, Physical Functioning, and Satisfaction with Social Roles and Activities. Significantly better scores were seen on Pain Interference, Fatigue, and Mobility in patients with busulfan-based compared to treosulfan-based regimen. Higher age at HSCT showed significantly worse scores on Pain Interference, Satisfaction with Social Roles and Activities, and Fatigue. Conclusions: This study showed normalization of long-term PROs in patients after pediatric HSCT for non-malignant diseases. More attention is needed for physical health, school, and cognitive functioning. Children and adolescents seem most vulnerable, indicating the need for early supportive care to prevent long-term impaired quality of life. Disclosure: Nothing to declare.
Background: Spending less inpatient time on transplant wards has been shown to improve quality of life and outcomes after allogeneic stem cell transplantation (aHSCT). One of the major obstacles for early discharge after aHSCT is aside from frailty the high risk for infections and the need for monitoring of graft versus host disease. This requires frequent hospital visits which is in times of Covid19 often a challenge and patients express reluctancy due to the higher risk of disease transmission in congested outpatient clinics. Furthermore, structured data acquisition is missing, especially in the home care setting. Methods: We have developed a mobile phone application for aHSCT patients that allows for specific symptom reporting and, in combination with a wearable device for online monitoring of major vital signs in real time. The application captures currently more than 50 data points relevant for aHSCT and infectious disease monitoring. It is based on REACT/NATIVE and JSON coding and enables specific designs based on requirements. Data captured by the wearable devices and manual symptom reporting are displayed in a close to real time fashion on a browser-based dashboard to the physician in a comprehensive fashion to allow for trend assessment of all transplant relevant information. It provides a photo function to capture skin images which are displayed on the physicians dashboard. Daily symptom reporting can be performed as often as necessary by the patient and data from wearable device are obtained multiple times every hour. The mobile phone application runs on android and iOS devices. The physicians dashboard includes several decision buttons that capture time stamped diagnosis or treatment decisions to enable machine learning and pattern recognition. Results: Data from symptom reporting and wearable device are displayed on a dashboard to the responsible health care provider with only a minor time delay. With this platform we capture and display in addition to vital signs (pulse, breathing rate, oxygen saturation of the peripheral blood, body weight) and simple GvHD related symptom reporting information on the patient’s physical activity, food intake, fluid intake, sleeping patterns, blood pressure and body temperature. The dashboard displays all transplant relevant information (HLA match, conditioning regimen, ABO incompatibility, CMV/EBV/Toxoplasmosis serology etc.) of the patient and of the donor and allows for a quick connection to the patient in case trends are reason for concern. It displays all data longitudinally over several days and therefore allows to observe trends and developments. Access to the dashboard is centre specific and secured by a dual verification process. Patient data are stored in HIPAA/PHIPA compliant fashion but storage can be adapted to country specific requirements. Conclusions: This application enables remote home monitoring of patients after aHSCT. It has been designed to improve quality of life by enabling more controlled home care and potentially earlier discharge. Both front ends are developed in a modular fashion and can easily be adapted to specific requirements for example after CD19 CAR T cell therapy or after autologous transplantation. Disclosure: Sudeep Takkar is founder of Reknowledge Inc. Armin Gerbitz is founder of Curetrax Inc.
Background: Prescence of comorbidity prior to allogenic hematopoietic cell transplantation (aHCT) impairs overall survival (OS) and increases the rate of non-relapse mortality (NRM). Therefore, predictive tools for assessing OS and NRM prior to transplant are much warranted. Methods: In this retrospective single-center study of 663 consecutive adult HCT recipients, we investigated the predictive value of pulmonary impairment prior to allogenic hematopoietic cell transplantation by stratifying patients by the three scores defined in the commonly used HCT-CI; (low-risk group, no pulmonary impairment (DLCO and/or FEV1 > 80%); intermediate-risk group, moderate pulmonary impairment (DLCO and/or FEV1 66-80%); high-risk group, severe pulmonary impairment (DLCO and/or FEV1 ≤ 65%)). The predictive value of this pulmonary impairment index (PI-I) was compared to HCT-CI (low-risk group, HCT-CI score 0; intermediate-risk group, HCT-CI score 1-2; high-risk group, HCT-CI score ≥3). Results: 241 patients (36%) had moderate pulmonary impairment and 142 patients (21%) had severe pulmonary impairment. In the group of myeloablative conditioning (MAC) patients, the frequencies were 107 patients (38%) and 46 patients (16%), respectively, and in the non-myeloablative (NMA) conditioning group, the frequencies were 134 patients (35%) and 96 patients (25%), respectively. In univariate analysis, both the HCT-CI and the PI-I were associated with OS after transplantation when comparing patients in high-risk groups with patients in low-risk groups. Using the PI-I, the HRs of the 2-year OS in the entire population and in the MAC group were 1,98 (P < 0,001) and 3,27 (P < 0,001), respectively, whereas the HRs using the HCT-CI were 1,83 (P < 0,001) and 2,57 (P = 0,002), respectively. None of the indexes showed significance in the NMA group. Using the HCT-CI without scores for pulmonary disease, we found that this index showed no significance separating risk groups within the groups of MAC and NMA patients. In multivariate analysis, stratified for age and conditioning regimens, comparing high-risk groups to low-risk groups, the HRs of 2-year OS using the PI-I and the HCT-CI were 1,88 (P < 0,001) and 1,72 (P = 0,002), respectively. The 2-year NRM incidence in the three risk-groups using the PI-I and the HCT-CI was 10% (95% CI 7%-14%), 13% (95% CI 9%-17%), and 24% (95% CI 17%-31%) (P < 0,001), respectively and 12% (95% confidence interval CI 8%-17%), 10% (95% CI 7%-14%), and 19% (95% CI 14%-24%) (P = 0,003), respectively. In the MAC group, the 2-year NRM was significant using the PI-I (P = 0,003), but not using the HCT-CI (P = 0,23). Conclusions: In conclusion, our single-center study suggest that pulmonary function alone is a strong predictor of 2-year OS and NRM after aHCT. A pulmonary function test is readily accessible in most centers, and the result is easily translated into a risk score. Clinical Trial Registry: In conclusion, our single-center study suggest that pulmonary function alone is a strong predictor of 2-year OS and NRM after aHCT. A pulmonary function test is readily accessible in most centers, and the result is easily translated into a risk score. Disclosure: Nothing to declare
Background: Graft-versus-Host Disease (GvHD) relapse free survival (GRFS) is increasingly used as composite endpoint to evaluate allogeneic haematopoietic stem cell transplantation (allo-HSCT). To our knowledge, GRFS is not systematically linked to (long-term) patient-reported outcomes (PROs). The aim of this study was therefore to investigate the impact of GvHD and/or Relapse according to the refined GRFS definition (i.e. being alive with neither grade III–IV acute GvHD nor severe cGvHD and without disease recurrence or death) on the prevalence of substantial symptoms and health-related quality of life (HRQoL) of patients three years after allo-HSCT. Methods: Since 2013, patients admitted for allo-HSCT in the UMC Utrecht were approached by their nurse to complete questionnaires at regular time-points after transplantation (yearly ± 50 patients). The Functional Assessment of Cancer Therapy General and Bone Marrow Transplantation (FACT-G and BMT) were used to assess HRQoL and symptoms. Patients graded items on a 5-point Likert scale:‘0: Not at all’, ‘1: A little bit’, ‘2: Somewhat’, ‘3: Quite a bit’, ‘4: Very much’. FACT-subscales were calculated. To evaluate long-term PROs, all patients alive at three years after allo-HSCT were selected who also completed a questionnaire. Questionnaire data were linked to clinical data as reported to the EBMT-registry. Prevalence rates of substantial symptoms were computed based on patients scores ≥3, and were compared between patients meeting and not meeting GRFS criteria. Multivariable linear regression models were performed to study differences on physical, emotional, social and functional well-being and HSCT-related issues, including demographic (age, sex) and clinical (donor type, conditioning) covariates. P < .05 was considered statistically significant and mean differences >0.5SD were considered clinically relevant based on Norman’s rule of thumb. Results: From 130 included patients the median age was 54 years at time of transplantation and 59% were male. Donor type was matched sibling (23%), matched unrelated (55%) or mismatched unrelated (22%). Conditioning consisted of ATG/busulfan/fludarabine with ex vivo T-cell depletion (48%) or fludarabine/TBI without ATG (9%) or with ATG (18%) or other (24%). Median time since allo-HSCT was 36.5 months. 60% of this selection reached GRFS. Reasons for failures (n = 52) were relapse (n = 18), grade III-IV aGVHD and/or cGvHD (n = 27), or both (n = 7). No statistically significantly differences were observed on these characteristics between patients with or without GRFS. Patients meeting criteria of GRFS less frequently reported substantial symptoms and consequences of emotional wellbeing (Figure 1). Statistically significant differences were observed for lack of energy (16.9% versus 34.6%), shortness of breath (5.2% versus 23.5%), being unable to enjoy life (4.0% versus 13.5%) and feeling distant from other people (1.3% versus 12.0%). Functional well-being (mean (SD): 20.6(5.1) versus 17.9(5.2)) and BMT-related well-being (29.5(5.9) versus 25.4(7.2)) as FACT-G total (87.7(12.0) versus 77.4 (14.5)) were statistically significantly and clinically relevantly higher for patients reaching GRFS, independent of the covariates. Conclusions: Patients reaching GRFS reported a higher HRQoL and lower symptom levels 3 years after allo-HSCT when compared to those who did not reach GRFS. These data underline that GRFS should be the primary long-term outcome to address the success of allo-HSCT. Disclosure: JK is shareholder of Gadeta (www.gadeta.nl), patent holder on multiple patents dealing with genetic engineering and received research grants from Novartis, Miltenyi Biotech and Gadeta. Other authors have nothing to declare.
Background: The optimal timing of rabbit anti-thymocyte globulin (rATG) for prevention of graft-versus host disease (GVHD) in matched sibling donor peripheral blood stem cell transplantation (MSD-PBSCT) remains to be elucidated. Methods: In this retrospective study, consecutive patients received MSD-PBSCT with rATG as GVHD prophylaxis in our hospital were enrolled. Twenty-nine patients were assigned to 2d-ATG group and forty-four to 4d-ATG group respectively according to the timing of rATG (2d-ATG regimen, a 5 mg/kg total dose divided from days −5 to −4; 4d-ATG regimen, a 5 mg/kg total dose divided from days −5 to −2). Results: There was no significant difference between the two groups in the cumulative incidences (CI) of grades 2-4 acute GVHD (aGVHD) at day 100 after transplantation (34.5% vs 40.9%, p = 0.587). The 2-year CI of severe chronic GVHD was higher in the 2d-ATG group than that in the 4d-ATG group without statistical significance (10.3% vs 2.6%, p = 0.311). The 2-year cumulative incidence of relapse (CIR) in the 2d-ATG group was lower than that in the 4d-ATG group, and 2-year disease-free survival (DFS) was better in 2d-ATG group (CIR: 13.8% vs 50.0%, p = 0.001; DFS: 72.4% vs 50.0%, p = 0.035). 4 patients in 2d-ATG group died of non-relapse causes, whereas non in 4d-ATG group. Multivariate analyses suggested that 2d-ATG regimen was an independent influence factor for lower CIR (HR: 0.212, p = 0.0008) and better DFS (HR: 0.421, p = 0.003). Conclusions: This study indicated that different timing of rATG affected the transplant outcomes of MSD-PBSCT patients, and 2d-ATG regimen had lower CIR and better DFS. Disclosure: Nothing to declare.
Background: Allogeneic hematopoietic stem cell transplantation (HSCT) recipients are at risk for various complications during their post-transplant follow-up. Acute events may lead to emergency department (ED) consultation in an unknown proportion of patients. Methods: We performed a retrospective analysis of all consecutive adult (≥18 years) patients who underwent allogeneic HSCT at the Medical University of Vienna between 01/2013 and 08/2021. The primary objective was to analyze the rate of ED admissions after allogeneic HSCT. Secondary objectives included the temporal distribution, reasons, and outcome of ED admissions. Data cut-off for individual follow-up was October 19, 2021. Results: 394 patients (median age: 51.58 [IQR: 40.44-59.63] years; f:m = 156:238) were included in the analysis. The median follow-up for the entire cohort was 1.57 [0.66-3.45] years. We recorded 142 ED admissions in 91/394 (23%) patients. 55 (60%), 21 (23%), and 15 (16%) patients presented once, twice, and ≥3 times to the ED during their follow-up. Time from HSCT to ED presentation was 0-6 months in 45 (32%), 6-12 months in 26 (18%), 12-24 months in 40 (28%), and >24 months in 31 (22%) patients, respectively. ED admissions were primarily recorded on Sundays (n = 27; 19%), Saturdays (n = 26; 18%) and Tuesdays (n = 23; 16%). Most ED consultations occurred between 12 p.m. and 6 p.m. (n = 57; 40%), followed by 6 p.m. to 12 a.m. (n = 38; 27%). The symptoms reported at ED consultation were pain (n = 89, 63%), reduced general condition (n = 72, 51%), fever (n = 63, 44%), diarrhea/vomiting (n = 41, 29%), and dermal symptoms (n = 31, 22%). An infection had been clinically or microbiologically documented in 64 (45%) ED visits. Hospitalization was required in 89 (63%) of 142 ED admissions, 10 (1%) patients were transferred to an ICU. The in-hospital mortality rate was 11/89 (12%). In univariate analysis, risk for ED admission was greater in patients with acute GVHD (34% vs. 19%, p < 0.01), chronic GVHD (42% vs. 18%, p < 0.01), and in males (27% vs. 17%, p = 0.02). Age, underlying disease, and donor type were not associated with ED consultation. Conclusions: Almost every fourth HSCT recipient in our cohort presented to the ED at least once during the individual post-transplant follow-up. Patients reported various symptoms and frequently required hospitalization, associated with a 12% mortality rate. Acute and chronic GVHD appear to be the major risk factors for requiring ED admission. Disclosure: Nothing to declare.
Background: Understanding the long-term psychosocial impact of pediatric hematological stem cell transplantation (HSCT) for non-malignant diseases is needed in order to optimize pre-HSCT counseling, supportive care, and long-term follow up programs after HSCT for this group of patients and caregivers. Methods: In this qualitative study 14 patients transplanted for a non-malignant disease during childhood at the Leiden University Medical Center, The Netherlands were included. Inclusion criteria were HSCT two or more years ago and ≥12 years of age. In-depth interviews were conducted online to explore patients’ perspectives on the long-term psychosocial impact of HSCT on their current lives. Using a Grounded Theory approach four main themes were identified. Results: Patients’ median age at HSCT was 10 years (range 0.5-17 years, date of HSCT 1987-2018) and median age at time of interview was 19 years (range 14-49 years). Half of the patients was male. Indications for HSCT were inborn errors of immunity (n = 4), hemoglobinopathies (n = 4), or bone-marrow failure syndromes (n = 6). The main themes of psychosocial impact of HSCT that were uncovered, were: ‘Doing okay’, ‘Experiencing persistent involvement of healthcare services’, ‘Influence on relationships with loved ones’, and ‘Impact on patient’s life course’. Almost all patients reported recovery and curation after HSCT, feeling good, and the current ability to pursue their ambitions. Furthermore, they had been able to put the HSCT event behind them at some point. Patients reported health limitations due to side effects of the HSCT (e.g., fatigue, compromised fertility, growth restrictions, epilepsy) leading to necessary adjustments in daily life. Many patients reported a sense of vulnerability, feeling more susceptible to diseases than their peers, and fear of new complications or disease recurrence. Frequent hospital visits and check-ups reinforced the sense of vulnerability. However, many patients also reported to have accepted the side-effects, hospital visits, and the HSCT itself. For most patients family-relations had been affected, both positively (more parent-child equality) and negatively (increased dependency in family relationships). The impact of a family donor ranged from experiencing a positive intense connection, to the feeling of being indebted. For most patients the HSCT did not affect friendships. Patients reported that the HSCT had influenced their social-emotional development. For some patients, the HSCT had impacted (school)career choices. Since patients had faced major life decisions at a young age, they experienced gratefulness and did not take life for granted. Most patients mentioned that the previous HSCT did not influence how they envisioned the future. Table 1. Illustrative quotations. Conclusions: This study is one of the first qualitative studies characterizing the long-term psychosocial impact experienced by patients after pediatric HSCT for non-malignant diseases. Patients showed active coping strategies and resilience after this intensive treatment. The key themes emerging from our data highlights the need of optimal patient-adjusted supportive care in the long-term outpatient clinic. Disclosure: Nothing to declare.
Background: Late endocrine disorders are among the most common complications in survivors after autologous and allogenic stem cell transplantation (HSCT). There is evidence to suggest that long-term cancer survivors may be at high risk for premature development of metabolic syndrome, have higher fasting plasma glucose and insulin levels, impaired glucose tolerance, hypertriglyceridemia, low HDL cholesterol level. survivors of allogeneic HCT were 3.65 times (95% confidence interval [CI], 1.82-7.32) more likely to report diabetes than healthy people but recipients of autologous HCTs were no more likely than healthy people to report diabetes in a few series. The aim of this study is to evaluate the incidence of endocrine complications after autologous HSCT, stratified by hematological neoplasms diagnosis. Methods: Observational, analytical, retrospective cohort study in patients undergoing autologous stem cell transplantation (ASCT) at the National Cancer Institute of Mexico (INCAN), with survival greater than 1 year and followed for 10 years. The data were analyzed using SPSS statistical software. Results: 249 autologous transplants were carried out between 2011 and 2020. Patients with a survival less than 1-year or with institutional follow-up lower than 12 months were excluded, at the end, 188 patients were analyzed. The cumulative incidence of endocrine complications was 57.4% (n = 108), occurring in 55.3% of men and 61.1% of women, without difference by gender, (p = 0.43). Stratifying by diagnosis, the incidence of endocrine complications was 60.5% (n = 49) of Non-Hodgkin’s Lymphoma (NHL), 55.4% (n = 36) of multiple myeloma (MM) and 54.8% (n = 23) of Hodgkin’s Lymphoma (HL) (p 0.762). Post-transplant diabetes was higher in patients with MM 52.3% (n = 34) p 0.001, the incidence of post-transplant dyslipidemia was higher in patients with NHL 30.3% (n = 23), p 0.003, the incidence of hypothyroidism post-transplantation was higher in patients with HL 12 (60%) 0.043. There was no difference in the incidence of osteopenia and osteoporosis stratified by diagnosis, but there was a trend towards more osteoporosis in the HL group. The overall survival of patients with endocrine complications was equal to patients without endocrine complications p: 0.538, with median survival not yet reached. 70.5% (n: 31 pct) had post-transplant endocrine and cardiovascular disease p: 0.046, with 44.4% of HL, 75.5% of NHL and 81% of multiple myelomas developing the 2 disorders, with patients with multiple myeloma those that developed the most cardiovascular and endocrine complications simultaneously p: 0.053. Conclusions: Results of this study suggest that patients undergoing autologous transplantation also have an increased risk of post-transplant endocrine complications (incidence of 57%) and, especially, patients with multiple myeloma are more likely to develop diabetes and post-transplant endocrine and cardiovascular complications. Disclosure: No conflict interest
Background: Prolonged thrombocytopenia and poor graft function are significant causes of transplant related morbidity and mortality based on a multifactorial etiological landscape. Although thrombopoietin receptor agonists (TPO-RA), particularly Eltrombopag, have been reported to be efficacious in these clinical conditions, potential long-term adverse effects still remain to be elucidated. Methods: This retrospective study was performed to analyse the efficacy and side-effect profile of TPO-RAs in allogeneic hematopoietic stem cell transplant (allo-HCT) recipients who developed post-transplant prolonged thrombocytopenia and/or poor graft function. A total of 27 patients [median age: 55(21-73) years; male/female: 15/12] were included. Results: Eltrombopag was started on day 110(33-670) after transplant. Median initial dose was 25(25-50) mg/day perorally and increased properly to a maximum dose of 75(50-100) mg/day. Duration of the treatment was median 120(31-377) days. Overall response rate was 59.3% in the study population. Time-to-treatment response was 42(3-170) days. A positive correlation was demonstrated between treatment response and pre-treatment platelet count (p = 0.007; r = 0.538) with an adverse association with pre-treatment bone marrow megakaryocyte reserve (p = 0.047, r = −0.428), respectively. Bone marrow biopsies were performed in 25 patients before treatment and post-treatment biopsies were repeated in 22 patients. Mild-to-moderate bone marrow fibrosis (BMF) [median grade 1.5(1-2)] was detected in the post-treatment biopsies of 12 patients (54.5%), 9 of whom did not represent any grade of myelofibrosis in their inital biopsies. Median 1(1-2) grade improvement was observed in BMF after the cessation of the drug in 7 patients. Pre-treatment existence of BMF had a significant impact on the severity of post-treatment fibrosis (p = 0.049). The grade of treatment-related fibrosis was significantly increased when time-to-treatment response was longer (p = 0.008). Conclusions: The therapeutic efficacy of TPO-RAs in prolonged thrombocytopenia and poor graft function was confirmed in the present study as previously published. Besides multiple underlying factors which may have an impact on BMF in allo-HCT recipients, long-term use of TPO-RAs should also be kept in mind to be considered as a potential cause of myelofibrosis in this particular group of patients. Clinical Trial Registry: N/A Disclosure: Authors have no conflict of interest to declare.
Background: Most deaths after Allogeneic haematopoietic stem cell transplantation (HSCT) occur within the first 2 years. In 2-year survivors, the long term survival is good, but life expectancy remains lower than expected. Outcomes of 2-year survivors undergoing allo-HSCT at Oslo University Hospital were assessed. Methods: We retrospectively studied the outcomes of 421 patients with malignant haematological disease, age 18-72 years, who underwent transplantation from 2005 to 2019 and were alive and free of disease after 2-years. Data were reported from The OUS-HSCT registry. Median follow-up was 6.2 years (2.0-16.1), and 232 patients (55%) were observed for 5 years or more. Results: The probability of being alive 5 and 10 years after HSCT was 86% and 76%. The primary risk factors for late death included initial diagnosis of lymphoma or chronic lymphocytic leukaemia (CLL), previous blood stream- or invasive fungal infection (BSI, IFI), and extensive chronic graft-versus-host disease (cGVHD). Transplant-related mortality (TRM) and relapse at 5 years was 9.0% and 7.7%, respectively. Two factors were associated with the latter: CMV seronegative donor and CLL. Compared with the age and gender matched Norwegian general population, life expectancy was lower for each disease, except for CML. Conclusions: The prospect for long-term survival is good for 2-year survivors of allogeneic hematopoietic stem cell transplantation. However, life expectancy remains inferior to the age- and gender matched general population. Optimizing prophylaxis and treatment for chronic GVHD, BSI and IFI is needed along with improved adherence to guidelines for early detection of secondary malignancies. Measures to improve immune reconstitution, possibly the microbiota, and the use of CMV seropositive donors regardless of recipient sero-status may be warranted and should be addressed in further studies. Table 2. Results from multivariate analyses of factors affecting outcome in patients alive without relapse two years after HSCT. Hazard Ratio (HR), 95% confidence interval (CI) and p-value are displayed for the various variables. MVA; multivariate analysis, OS; overall survival, TRM; transplant-related mortality, RI; relapse incidence, BSI; blood stream infection, IFI; invasive fungal infection, CLL; chronic lymphocytic leukaemia, GVHD; graft-versus-host disease, Ext; extensive. *analysed as a time-dependent variable. Disclosure: Nothing to declare
Background: Hematopoietic stem cell transplantation (HSCT) is an increasingly used therapeutic option to treat hematological and autoimmune diseases, making it important to have proper knowledge of its related comorbidities. Our objective was to evaluate thyroid function and the incidence of thyroid disorders associated with HSCT during a 10-year period within an institution. Methods: A secondary analysis of an anonymized database which had patients over 18 years old who underwent HSCT at FOSCAL clinic was performed (2009-2019), patients who had previous thyroid disease were excluded. The incidence of thyroid disorders was determined at 1 and 3 years of follow-up from the moment the HSCT was performed. Results: A total of 278 patients were included, 54.7% were male, with a mean age of 45.8 years, 33.9% were overweight or obese, a Karnofsky score average of 92.7 points and had an average Charlson comorbidity index of 2.64. Patients were taken to HSCT as a therapeutic option for multiple myeloma (30.6%), lymphoma (29.1%), acute leukemia (22.3%) and chronic leukemia (8.3%). Prior to HSCT, 11.2% of the patients underwent to the external radiotherapy, performed in abdomen and pelvis (41.9%) and in the chest area (38.7%). Two patients received thyroid ablation with radioactive iodine prior to HSCT. The patients received cyclophosphamide near the HSCT were 20.5% and 19.8% of them received a myeloablative regimen. Most of the HSCT were autologous (70.9%). The average time elapsed from diagnosis to HSCT was 1.7 years. Ciclosporin post-HSCT was used in 22.8% of the patients. The average TSH level at 1year post-HSCT was 3.2 mU/L, while at 3 years it rose to 4.7 mU/L. Regarding FT4, the average lab results were 1.2ng/dL at 1 year and 1.3 ng/dL at 3 years. Finally, the cumulative incidence of hypothyroidism at 1 and 3 years after HSCT was 18% and 40.3% respectively, while the cumulative incidence of hyperthyroidism at 1 and 3 years post-HSCT was 6.6% and 12.8% respectively. Conclusions: The high incidence of thyroid gland disorders in patients who underwent HSCT makes it necessary to systematically search and identify these alterations through timely clinical and biochemical follow-up. Clinical Trial Registry: N/A Disclosure: N/A
Background: Hematopoietic stem cell transplantation (HSCT) is a potentially curative option which is being increasingly used in multiple hematological and autoimmune diseases; Being of our interest the study of endocrinological morbidity, the objective of this study was to evaluate the gonadal function and the incidence of hypogonadism in males undergoing HSCT at a 1-year follow-up Methods: A secondary analysis of a database was used, which consisted of patients older than 18 years who underwent HSCT (2009-2019) at Clinica FOSCAL, without previous hypogonadism. In addition to the extraction of clinical variables, an analysis of total testosterone levels was made before transplantation (pre-HSCT) and 1 year after the of transplant during follow-up (post-HSCT). A single testosterone value less than 300ng/dL was considered hypogonadism. The general, and subgroup incidence was estimated according to the intensity of the conditioning regimen and other sociodemographic, clinical, and HSCT-related variables. Results: Thirty-six men with a mean age of 41.2 ± 14.6 years were included in the analysis 70.9% had received an autologous HSCT. The mean total testosterone in the study population was 520 ± 270 ng/dL pre-HSCT and 560 ± 350 ng/dL at 1-year post-HSCT. An incidence of hypogonadism of 28.6% was found at the 1-year mark and 8.6% the patients presented testosterone values <200ng/dL. No association was found between age or conditioning therapy with the development of hypogonadism. Conclusions: One out of every 3 to 4 men undergoing HSCT presented hypogonadism at the1-year follow-up. This incidence suggests a systematic and early search for this complication. Disclosure: N/A
Background: Hematopoietic stem cell transplantation (HSCT) is a potentially curative option that is on rise in multiple hematological and autoimmune diseases; being for our interest the study of endocrinological morbidity, the objective of this study was to evaluate the incidence of hypogonadism in women less than 40 years who underwent HSCT at one year follow-up. Methods: A secondary analysis from a database of women between 18 and 40 years old who underwent HSCT (2009-2019) was performed at the Clinica FOSCAL, without previous hypogonadism. In addition to the clinical variables, an analysis of levels of FSH was made one year after transplantation(posHSCT). Primary ovarian insufficiency was considered an FSH value greater than 25 mU/mL in the presence of amenorrhea. The general incidence and between subgroups were estimated according to the intensity of the conditioning regimen and other variables like sociodemographic, clinics and relationated with HSCT. Results: Twenty-four women under 40 years were included in the analysis, with 71% being autologous HSCT. An incidence of 62.5% of primary ovarian insufficiency was found at 1year posHSCT. There was not found association between the aged or the conditioning therapy with the development of hypogonadism. Conclusions: Six out of ten women undergoing HSCT had primary ovarian insufficiency at 1 year of follow-up. This incidence suggests a routine evaluation of gonadal function and timely handling. Clinical Trial Registry: N/A Disclosure: N/A
Background: Currently, more than 400 monogenetic inherited immune disorders (IID) have been identified. Overtime, outcome of hematopoietic stem cell transplantation (HSCT) for IID has significantly improved. In clinical practice, distinction of IID between Primary Immune Deficiency Disorders (PIDD) and Primary Immune Regulatory Disorders (PIRD) has been increasingly used. Methods: All children with IID who underwent allogeneic HSCT from 1989 to 2021 at IRCCS Istituto G. Gaslini were included in the study. HSCTs have been retrospectively described with the aim of reporting the outcome in terms of overall survival (OS), graft failure (GF) and Graft-versus-Host Disease (GvHD) Results: 65 patients, 30 affected by a PIDD and 35 by a PIRD, received 71 allogeneic HSCTs. In 7 patients, 8 (11.3%) HSCTs were complicated by GF (5 primary and 3 secondary); 4 patients underwent 2 HSCTs, 1 patient underwent 3 HCTs. According to the year of HSCT ( ≤2006 vs >2006), the number of transplants was 20 and 51, respectively. Median age at transplant was 2.5 years (IQR, 0.9-5.5), 42 (59.2%) HSCTs were performed at ≤3 years of age, 29 (40.8%) at >3 years of age. The donor was a matched-unrelated (MUD) in 34 (47.9%) HSCTs, matched-related (MRD) in 19 (26.8%), and haploidentical in 18 (25.3%). Among haploidentical HSCTs, 17 were performed according to T cell receptor (TCR) αβ/CD19 depletion platform and 4/17 (23.5%) were complicated by GF. Overall, 8/71 HSCTs were complicated by GF. Conditioning regimens (CR) were Busulfan-based in 24 (33.8%) HSCTs, Treosulfan-based in 39 (54.9%) and 8 (11.3%) received different CRs. The 1-, 3-, and 20-year OS was 89.2% [95%CI (78.6-94.7)], 83.4% [95%CI (71.1-90.7)] and 83.4% [95%CI (71.1-90.7)] respectively. OS was significantly higher in patients transplanted after 2006 (3 year OS: 65.0% ≤2006 and 92.5% >2006, p = 0.006). After a median follow-up of 4 years (max 20 years), 10 patients were dead: 6 died during the first 42 days after HSCT (4 before engraftment and 2 after primary GF) and 4 died within 3 years. OS was higher, but not significantly, among patients with PIRD (3-year OS: 79.7% for PIDD and 86.8% for PIRD, p = 0.418), for patients transplanted at > 3 years of age (3-year OS: 78.4% for ≤3 years and 90.9% for >3 years, p = 0.135) and in Treosulfan-based CR HSCTs (3-year OS: in Busulfan 72.9% and 90.8% in Treosulfan, p = 0.079). Overall, 31 HSCTs were complicated by acute GvHD (aGvHD), 12 (38.7%) by grade 3-4 aGvHD. Considering year of HSCT ( ≤ 2006 vs >2006), the distribution of grade 3-4 aGvHD was statistically different between ≤2006, 61.5%, and >2006, 22.2% (p = 0.027). In haploidentical TCR αβ/CD19 depleted HSCTs, grade 3-4 aGvHD was absent Conclusions: Our experience supports that HSCT represents an effective strategy in treatment of IID. In last years, outcome of HSCT for IID has significantly improved. Treosulfan and haploidentical TCR αβ/CD19 depleted HSCT represent promising strategies in this setting. Disclosure: Nothing to declare
Background: Infant acute leukemia is a rare but aggressive disease. Standard approaches are curative in a minority of patients. Most treatment failures are due to relapse, treatment-related mortality and life-limiting late effects in survivors are also problematic. Among such approaches, allogenic stem cell transplantation, may offer the greatest potential for improving cure rates. Methods: A total of 63 pts with infant leukemia (AML- 37, BP-ALL-26, 30 female, 33 male, median age at the moment of diagnosis 0,6 years (0-1), at the moment of HST 1,2 years (0,5-3,9), underwent allogeneic HSCT between May 2012 and July 2021. Forty pts received haploidentical graft, 12 a graft from matched unrelated donor, 11 from matched related. Disease status at transplant was CR1 in 45 pts, >CR1 in 10 pts and AD (all AML) in 8 pts. Thirty-three (52%) pts had MLL gene–rearranged (ALL n = 23, AML n = 10). Fourteen pts with ALL received target therapy before HSCT. All pts received treosulfan-based myeloablative preparative regimen either melphalan (n = 30), thiotepa (n = 26) or vepesid (n = 7) were added as a second agent. TCR αβ + /CD19 + depletion of HSCT with CliniMACS technology was implemented in 51 (80%) cases. The median dose of CD34 + cells was 10 x106/kg (range 4,3-21). Median time of follow-up for survivors was 4,5 years (range: 0.3 – 9,4). Results: Primary engraftment was achieved in 59 of 63 pts (1 pt died due septic event, 3 non-engraftments received successful 2nd HSCT), the median time to neutrophil and platelet recovery was 14 and 16 days, respectively. All engrafted pts had verified morphologic remission and achieved sustained complete donor chimerism by day +30. Transplant-related mortality was 3 % (95% CI: 0,8-12): one pt died due septic event before engraftment, one in CR due viral infection and GvHD. Cumulative incidence of aGVHD grade II-IV was 13% (95% CI, 8 - 24), grade III-IV was 3% (95% CI, 0,08 - 12). No correlation between donor type, serotherapy and GvHD was noted. The cumulative incidence (CI) of relapse at 4,5 years was 25% (95%CI:16-38 for the whole cohort. Among patients with AML CI of relapse was 22 % (95%CI:12-40), as compared to ALL group, with CI of relapse of 30 % (95%CI:16-57), p = 0,7. CI of relapse in MLL + group was lower 17% (95%CI:7-37), in contrast in MLL- group 33 %(95%CI:20-55), p = 0,1. All patients with MLL + AML (n = 10) are alive. EFS at 4,5 years was 67% (95%CI: 55-79), OS –76% (95%CI:65-88). Statistically there was no significant difference in event-free or overall survival probabilities between leukemia type, remission status, donor type or GvHD-prophylaxis regimens. EFS in MLL + group was high 77% (95% CI 63 - 92) vs in MLL- group 56 % (95% CI 39 - 74), p = 0,056 Conclusions: We confirm that allogenic stem cell transplantation ensures high engraftment rate and low TRM. All major outcomes were equivalent between transplantation from unrelated, haploidentical and related donor. Improvement of anti-leukemic activity will require further refinement of target therapy, preparative regimen and post-transplant strategy of disease control. Disclosure: no
Background: Neutrophil engraftment is essential for a successful outcome after allogeneic HSCT, but neutrophils are also thought to promote engraftment syndrome and aGvHD. Neutrophil status is assessed by neutrophil counts in peripheral blood, but the function of these circulating cells, including activation and migration to sites of injury or infection, requires further analyses. Myeloid-related protein (MRP)-8/14 is expressed in granulocytes during inflammatory conditions and secreted as a stress response, inducing production of pro-inflammatory cytokines and increasing leukocyte recruitment and activation. In this study, we investigated associations between MRP-8/14 levels, neutrophil recovery, and clinical outcomes after pediatric HSCT. Methods: We included 73 children undergoing allogeneic HSCT between 2010-2017 for ALL (n = 21), AML (n = 10), other malignancies (n = 10) and benign disorders (n = 32). Median age was 8.0 years (range: 1.1-17.2 years). Donors were either MSD (n = 22) or MUD (n = 51). BM (n = 69) and PB (n = 4) were used as stem cell source. All conditioning regimens were myeloablative and based on TBI (n = 17), busulfan (n = 32) or other chemotherapy (n = 20). ATG was administered to 75% of patients as GvHD-prophylaxis. Six blood donors with a median age of 20 years (range: 18-20) were included as healthy controls. MRP-8/14 was measured by ELISA in blood samples collected prior to conditioning, at the day of HSCT before graft infusion, and at day +7, +14, +21, +28, +90 and +180 post-transplant. Results: All patients engrafted at median 22 days (10-30 days) after HSCT. Plasma level of MRP-8/14 decreased from pre-conditioning levels to nadir at day +7, before rising significantly until day +28 and then gradually declining. The rise in MRP-8/14 levels preceded the rise in circulating neutrophil counts, reflected into an increased MRP-8/14 to neutrophil ratio early post-transplant, indicating increased activation of emerging neutrophils.MRP-8/14 levels were significantly higher at day +14 in patients with acute leukemias compared with other diagnoses (0.50 mg/mL vs. 0.20 mg/mL, p = 0.023) and lower in patients receiving ATG for MUD transplants (0.20 mg/mL vs. 0.92 mg/mL, p = 0.00019). We further investigated whether MRP-8/14 levels were predictive of neutrophil recovery. Indeed, reduced levels of MRP-8/14 levels were observed at day +14 and +21 in patients with delayed neutrophil engraftment occurring after day +21 and in patients receiving G-CSF treatment to promote engraftment (P = 0.030 and P < 0.0001, respectively). Engraftment syndrome occurred in 8 patients (11.0%) and was associated with elevated MRP-8/14 levels at day +7 and +21 and increased neutrophil counts from day +9 to +25 (P = 0.0005-0.016). Patients who developed bacterial blood stream infections in the early post-transplant period (n = 13) had significantly lower MRP-8/14 at day +14 and +21, but comparable neutrophil counts during this period. In general, MRP-8/14 levels and neutrophil counts were comparable in patients with and without aGvHD. Conclusions: A rise in MRP-8/14 levels precedes the appearance of neutrophils, and MRP-8/14 may serve as predictor for delayed neutrophil recovery, bacterial blood stream infections and engraftment syndrome after HSCT. Thus, MRP-8/14 measurement could be be a useful marker to distinguish bacterial infections from engraftment syndrome and to guide commencement of G-CSF therapy. Disclosure: Nothing to declare
Background: In allogeneic hematopoietic stem cell transplantation, T lymphocytes play a decisive role in promoting hematopoiesis, transferring immunity to pathogens, and acting as mediators of the graft-versus-leukemia effect (GVL). However, they are also responsible for graft-versus-host disease (GVHD), the main cause of post-transplant morbidity and mortality, especially naive T cells (CD45RA + ) that cause more severe GVHD than memory T cells. Our hypothesis is that using the new graft-engineering technology in which T naive cells are selectively depleted from the donor graft we would reduce or minimize severe acute and chronic GvHD following allogeneic transplantation. Methods: We design a prospective observational study in which the patients with high-risk hematologic malignancies receive an allogeneic T naive-cell depleted transplant from a HLA matched related or unrelated donor. A total of 58 children (median age 9 years, range 1-21) diagnosed of acute leukemia ALL (n = 20), AML (n = 22), MDS (n = 8), NHL (n = 4) and other (n = 4) were included in the study between 2016 and 2021. Twenty-one patients were in 1st CR, 19 patients in 2nd CR, 18 in >2nd CR (3th CR or active disease). Median donor age was 18 years (range; 1-51). There were 32 matched related donors (MRD) and 26 matched unrelated donors (MUD). Results: All patients received two donor cell products on day 0. The first product was given as a primary source of hematopoietic progenitor cell graft. A CD34 + enriched graft containing a median dose of 7,14x106/kg (range: 1,51-18). The second one was a CD45RA + depleted product. On day +1, +15 and +30, CD45RA + depleted DLIs were infused with a median cell number of 1 x 106/kg (range: 1-13,8). With a median follow up of 24 months (range; 3-60), 54 patients achieved neutrophil engraftment with a median time of 13 (range: 8–27) days. The median time to platelet engraftment was 11 (range: 6-34) days. The cumulative incidence of relapse was 33 ± 6% and the cumulative incidence of NRM was 9 ± 3%. Only 8 patients developed acute GVHD (13%) and 3 patients chronic GVHD (5%). DFS and OS were 58 ± 7% and 70 ± 6%, respectively. On univariate analysis, MDS (88 ± 11%, p = 0,03), CR at transplant (70 ± 7% vs 32 ± 12%, p = 0,015) and CD34 + cell infused (>7 x 106/kg, 41 ± 10% vs ≤7 x 106/kg, 73 ± 9%, p = 0,018) were associated with DFS. On multivariate analysis, complete remission at transplant (yes vs no, HR:5; 95%CI, 1,7-14-, p = 0,003) and the number of CD34 + cells infused/kg (≤7 vs >7, HR 5; 95% CI,1.7-12,5; p = 0.003) influenced on DFS. Conclusions: Our results strongly suggest that allogeneic transplant using “naïve” T-cell-depleted grafts from matched related and unrelated donors in children provide a good platform for DLI with very low incidence of GVHD. Disclosure: Nothing to declare
Background: Matched Sibling Donor Bone Marrow Transplantation (MSD-BMT) is the standard of care for pediatric patients with severe acquired Aplastic Anemia (sAAA) or single-lineage bone marrow failure (BMF) syndromes. At our center, standard of care (SOC) MSD-BMT using cyclophosphamide (200mg/kg) and Thymoglobulin (9mg/kg) for sAAA or cyclophosphamide (200mg/kg) and pharmacokinetic (PK) adjusted Busulfan for single-lineage failures yielded a 5-year overall survival of 100%. Given these data, we sought to reduce toxicity associated with conditioning by reducing conditioning intensity. Methods: CHP14BT057 (NCT02928991) is a prospective, single center, single arm trial for pediatric patients with BMF that utilizes fludarabine to minimize or eliminate cyclophosphamide. Pediatric patients with sAAA or single-lineage BMF with an available, unaffected MSD were eligible. For sAAA, conditioning included: fludarabine 150mg/m2, cyclophosphamide 120mg/kg and Thymoglobulin 9mg/kg. Calcineurin inhibitor +/− methotrexate was used for GVHD prophylaxis. For single-lineage BMF, conditioning included fludarabine 30mg/m2 with PK adjusted Busulfan and Thymoglobulin 9mg/kg with a calcineurin inhibitor and mycophenolate mofetil for GVHD prophylaxis. We used a historical cohort (n = 10) who received MSD-BMT with SOC conditioning regimens as a comparator. Results: 18 patients enrolled on or were treated per CHP14BT057. Demographics of CHP14BT057 and SOC cohorts are in table 1. Median follow up was 772 days (205-1543). 1 patient with COVID-related sAAA died of vasculopathy during conditioning. Of the 17 patients who received bone marrow infusion, the overall survival was 100%. There were no graft failures compared to 1 in SOC (patient with congenital sideroblastic anemia, successfully salvaged). Acute GVHD was minimal: 1 patient (6%) with grade 2-4 aGVHD compared to 1 patient (10%) in SOC. Limited chronic GVHD occurred in 4 (24%) patients compared to 2 (20%) in SOC. 1 patient on CHP14BT057 had residual PNH clone. Engraftment and immune reconstitution kinetics are shown in figure 1 and similar to SOC. Time to neutrophil and platelet engraftment per CIBMTR criteria was 21 and 30 days, respectively. This compared to 18 and 28 days in SOC. Viral reactivation on CHP14BT057 included: 3/17 CMV, 8/17 EBV, 1/17 BK and 1/17 Varicella compared to 2/10 CMV, 1/10 EBV 1/10 Adenovirus, 1/10 Varicella in SOC arm. No patients required viral specific CTLs or had end organ disease. Other BMT-related complications included: mild VOD (n = 1) and TA-TMA (n = 1) on CHP14BT057 compared to mild VOD (n = 1) and a rhizomucor infection requiring resection (n = 1) on SOC. Conclusions: Dose reduction of cyclophosphamide in MSD-BMT for pediatric BMF is a safe method to provide curative therapy without compromising efficacy. Patients with large PNH clones and/or other evidence of clonal hematopoiesis may require more intensive conditioning regimens. Clinical Trial Registry: NCT02928991 clinicaltrials.gov Disclosure: Ellen Levy is now an employee of Merck David Barrett is now an employee of Tmunity Therapeutics
Background: Immunosuppressive therapy (IST) had been the recommended treatment in paediatric patients with severe idiopathic aplastic anaemia (SAA) and refractory cytopenia of childhood (RCC) who lacked a matched related donor. IST’s response rate is fair, but many patients relapse and become candidates for a stem cell transplant (SCT). The results of matched unrelated donor (MUD) transplants after IST are inferior compared to matched related donor transplants in the first line. In 2015, Dufour et al. published their excellent results treating SAA patients with upfront SCT from MUD, attaining significantly better outcomes than a historical series of patients that had received MUD SCT after IST. Many paediatric centres in Spain have adopted the upfront MUD STC strategy for newly diagnosed SAA and RCC. Methods: We performed a multicentre retrospective review of children with SAA or RCC that received treatment with upfront MUD SCT in GETMON/GETH associated hospitals. Results: From 2014 to 2021, 8 patients (6 males/2 females) with SAA (6) and RCC (2) underwent an upfront MUD SCT. At diagnosis, the median age was 11 years old (range, 0.55-17.13). All received bone marrow from MUD 10/10. All had a good performance status (Lansky > 70). None of them had significant cytogenetic anomalies. 7/8 patients were CMV positive, and a mismatched CMV (donor negative and receptor positive) was used in 3 cases. The median time from diagnosis to SCT was 124.5 days (range, 79-210 days). Conditioning regimens were Flu+Cy in 6 patients with SAA and Treo/Bu +Flu+Thio in 2 patients with RCC. All patients received Serotherapy with ATG. GVHD prophylaxis consisted of cyclosporine/methotrexate in 7/8. The mean cell dose was 3.12 x 10e6 CD34 + /kg (SD 1.25). Median neutrophils engraftment was on day +20 and platelet engraftment on day +29. Acute GVHD grade ≥ 2 was seen in 4 (all responded to steroid treatment). Viral reactivation was common (7/8). All but one patient reached transfusion independence at a median time of 33 days (range, 19-398). No transplant-related mortality (TRM) was registered. Two patients presented increasing receptor’s lymphoid chimerism and were successfully treated with augmentation of immunosuppression. All patients are alive without chronic GVHD. One patient experienced poor graft and recovered with CD34 boost. One patient is still platelet dependent and is on treatment with romiplostim. Both had received CD34 dose under 2 x 10e6/kg. The median duration of post-transplant immunosuppressive treatment was 546 days (range, 48-679). EFS was 100% after a median follow up of 33.38 months (range, 2-84). Conclusions: In our experience, upfront MUD SCT offers excellent results with 100% overall survival without chronic GVHD and no graft failure. Many patients suffered viral reactivations and other infectious complications accordingly to immunosuppression required for SCT for these conditions. Management of mixed chimerism and poor graft remains the principal clinical concern for these patients. Higher cell doses could help to revert these problems. Longer follow-up is needed to assess late sequelae and QoL. Our data support upfront MUD SCT for newly diagnosed SAA and RCC paediatric patients. Disclosure: Nothing to declare
Background: Invasive fungal diseases (IFD) represent a significant cause of morbidity and mortality after allogeneic hematopoietic cell transplantation (allo-HCT) in children. The optimal strategy for prophylaxis and treatment is not universally accepted. In 2017, UK guidelines for antifungal management in children receiving allo-HCT were defined and a prospective audit was conducted. Methods: From March 2017 to December 2019, we prospectively collected data on allo-HCT performed in 7 UK pediatric centers to audit adherence to and effectiveness of national antifungals guidelines. Data on patients/transplants characteristics, antifungal prophylaxis, incidence of new IFD and treatment, prevalence of GvHD were included. Fungal infections were defined as possible, probable, or proven according to EORTC definitions. Results: During the study period, 368 children were enrolled, with a median follow-up of 361 days (95%CI 56-1000 days). Baseline characteristics are shown in Table I. Before allo-HCT, 64 patients (17%) had a history of IFD, that was probable/proven in 28 (7.5%). At the start of conditioning most patients (301/368, 81%) were on single agent prophylaxis with itraconazole (146, 39%) or liposomal amphotericin B (155, 42%). Twenty-three patients (6%) were on antifungal treatment due to previous IFD. Median length of anti-fungal prophylaxis was 191 days (range, 17-819). Incidence of new IFD was 18% (95%CI 11-26, 66 events in 61 patients), with 18/66 probable/proven infections (5%). Most IFDs occurred within the first 6 months after transplant (Figure 1a). Most patients (83%) with probable/proven IFD received double antifungal agents, while mostly a single drug (77%) was used with possible IFD. A univariate risk factors analysis showed that primary immunodeficiency (PID) diagnosis, second allo-HCT, HLA mismatched donors, myeloablative conditioning, previous IFD and acute graft versus host disease (aGvHD) were significantly associated with occurrence of new IFD. In multivariate analysis (MVA), only aGvHD was confirmed as a significant risk factor (OR: 2.1, 95%CI 1.1 – 4, p < 0.05). Choice of anti-fungal prophylaxis didn’t impact on incidence of IFD. Non-relapse mortality (NRM) accounted for 40 deaths. Although only 5 IFD-related deaths were reported, IFD had a significant impact on survival, as children with a new IFD had a higher NRM, compared to patients without IFD post allo-HCT (26 vs 11%, p < 0.001) (Figure 1b). Table I. Figure 1 Conclusions: This study provides a prospective assessment of incidence and risk factors for IFD in a large cohort of children receiving allo-HCT in the UK, after the adoption of national anti-fungal guidelines. In MVA, aGVHD was associated with increased risk of IFD, which contributed to increased NRM. Patients with GVHD might benefit from further optimization of anti-fungal prophylaxis and treatment. Disclosure: The audit received financial support from Gilead.
Background: Levofloxacin (LVX) prophylaxis and nutritional support in pediatric allo-HSCT patients are relevant to clinical outcomes but evidence on their impact on the gut microbiome (GM) is still limited. Here, we longitudinally evaluated GM network architecture and species-level dynamics of pediatric patients before, during and after allo-HSCT. Methods: A total of 90 stools from 30 pediatric patients underwent shotgun metagenomic sequencing on Illumina NextSeq platform. Patients were given: i) enteral nutrition (EN); ii) parenteral nutrition (PN); or iii) PN preceded by LVX prophylaxis (PN LVX ( + )). Species-level compositional insights were obtained to build correlation networks. Network evaluation (i.e., computing modularity and cohesion) was used to characterize the microbial consortia. Results:Three network parameters were adopted to describe the GM community: i) modularity, i.e., the measure of connections between and within modules, with high values determining a lower spread of any external stressor, meaning an improved resistance to it; ii) total cohesion (TC), i.e., the quantification of connectivity in terms of positive and negative interactions, with high values denoting a dense and plastic community; and iii) the ratio of negative to positive cohesion (N:P), informative on the reliance of the system on positive interactions, where low values stand for high stress conditions since negative interactions are prevented and disrupted by the pressure of the stressor. PN LVX (+) resulted in lower modularity, TC and N:P ratio compared to EN, indicative of a less plastic and structurally altered GM, more susceptible to external stressors and less predisposed to prompt recovery. In addition, PN and more markedly PN LVX (+) was associated with the appearance of a module containing bacterial species of concern such as what we named “Klebsiella spp. triad” (comprising K. pneumoniae, K. quasipneumoniae and K. variicola), [Ruminococcus] gnavus, Flavonifractor plautiiand Enterococcus faecium, which altered the network structure compared to EN-fed patients. Conclusions: By evaluating GM network properties longitudinally after LVX prophylaxis and PN in pediatric allo-HSCT patients, we found a less plastic community, less able to maintain a dense net of interactions, thus to withstand stress. In particular, we detected the emergence of network modules comprising several potential pathobionts, such as Klebsiella spp., R. gnavus, F. plautii and E. faecium that have previously been linked to the production of possibly harmful molecules. Our results shed light on the harmfulness of LVX prophylaxis before allo-HSCT and help support EN as the first-choice nutritional support after allo-HSCT. Disclosure: Nothing to declare
Background: Increasing evidences support feasibility and safety of sports in fragile patients, where the concept of inclusion substitutes for the concept of competition. Methods: Patients undergoing HSCT in a single Institution are offered to participate in the “Sport-Therapy" research project, consisting of precision exercise training by sports medicine doctors and exercise scientists, besides osteopathic treatment, in order to prevent toxicities, derived from bed-resting and treatment. Three training sessions per week (150’), consisting of aerobic, strength, balance and flexibility exercises, are planned with two operators per session for each patient. The training is held in the patient room, in the ward or in the clinic, in case of preventive isolation, in the gym-hall or in the garden, as soon as the patient may attend small groups (<5). The training is personalized for each patient, according to the parameters measured at the beginning of the program, by means of multiple performance tests (6-minute-walking, timed-up-and-downstairs, quick motor function, strength tests), and continuously reassessed on the basis of the patient exercise tolerance and vital signs (heart-rate and blood saturation) throughout the training session. Four sport techniques have been implemented: indoor climbing, no pedal bike, soccer, golf. Results: Out of 123 consecutive patients 3 years or older (44% ALL, 16% AML, 6% NHL, 3% HL, 32% non malignant disease) transplanted in the period April 2017-October 2021, 93 (76%) started the exercise training during the HSCT hospitalization, at a median of 15 days after HSCT. 30 additional patients, transplanted in the same period or earlier, were enrolled in the later post-HSCT course, mainly due to specific issues, such as osteonecrosis (18 cases). Both pre- and post-functional assessments are available for 72% of the 123 patients. An adapted Yo-Yo test was performed in the 33% of the patients. Adherence: 63% of the patients attended more and 22% less than one third of the training sessions, whereas 15% dropped-out, with main reasons being mainly logistical. No accidents occurred. Patient and parental satisfaction, as investigated by questionnaires, exceeded 90%. The program went beyond the expectations, as four unplanned goals were achieved: earlier diagnosis of system impairment, compared with what clinicians could diagnose after a medical visit in bed-rest. The body systems are critically challenged during exercise, which triggers structural weaknesses or damages as well as a reduced cardio-respiratory reserve; precise assessment of the range of motion of joints involved by chronic GVHD allowed basal and periodical measurements for treatment efficacy assessment; 8 pts with cGVHD have been longitudinally evaluated; timely diagnosis of lung impairment by GVHD by means of routine lung function test upon enrollment and periodical monitoring, with the possibility to anticipate diagnosis and proper GVHD treatment at an earlier stage, ultimately optimizing efficacy earlier detection and proper management of osteonecrotic lesions, based on the identification of gait or motion abnormalities or pain. Specifically tailored exercise protocols, upon suspected/diagnosed ON, pre-surgery preservation training, post-surgery rehabilitation. Conclusions: Personalized exercise programs are feasible and safe since the very early post-HSCT cohort. The project went far beyond the exercise training and improved patient clinical management. Clinical Trial Registry: Ethical Committee Università degli Studi di Milano Bicocca #284 Disclosure: Nothing to declare
Background: Pre-transplant risk assessment is an important tool to optimize allogeneic hematopoietic cell transplantation (HCT) outcomes. HCT-comorbidity index (HCT-CI) accounts for patient’s organ function, but does not incorporate disease characteristics or inherent resistance to therapy. Disease risk index (DRI) and refined-DRI based on disease-specific characteristics and remission status prior to HCT accurately assess risk of relapse post-HCT, but do not account for patient comorbidities. Two recent studies in adults combined the HCT-CI/Age and refined-DRI scores to develop the HCT-composite risk (HCT-CR) model that better predicts overall survival (OS) in HCT. Our goal was to design a combined pediatric-HCT-CR that can predict outcomes in children with acute leukemia undergoing allogeneic HCT. Methods: A retrospective chart review of children (<18 years) who underwent their first allogenic HCT for acute lymphoblastic leukemia (ALL) or acute myeloid leukemia (AML) between January 2017 and August 2020 at Texas Children’s Hospital was performed. Primary outcome was overall survival (OS) and secondary outcomes were relapse-free survival (RFS) and non-relapse mortality (NRM). Patients were stratified into three risk groups of a combined pediatric-HCT-CR based on pediatric-DRI and simplified-malignant HCT-CI: low-risk (low/intermediate DRI and simplified malignant HCT-CI < 3), intermediate-risk (low/intermediate DRI and simplified malignant HCT-CI > = 3), and high-risk (high DRI and any simplified malignant HCT-CI score), using classification and regression trees analysis for OS and RF. Kaplan-Meier method was used to estimate survival curves and log-rank test to compare curves. Results: The cohort included 103 children with ALL: 59(57)% and AML: 44(43%). Median age was 10 (range 0-17) years. Donor type included matched related donor: 25(24%), matched unrelated donor: 29 (28%), mismatched unrelated donor: 20(19%), haploidentical: 17(17%), and umbilical cord blood: 12(12%). 92(89%) received myeloablative conditioning. Pediatric-DRI was low in 25 (24%), intermediate in 63 (61%), and high in 15 (15%) patients. Simplified malignant-HCT-CI scores were 0, 1-2, and 3+ in 43 (42%), 24 (23%), and 36 (35%) patients, respectively.OS was 75% (95%CI: 67-83) at 1 year and 66% (95%CI: 56-76) at 3 years. RFS was 77% (95%CI: 69-85) at 1 year and 66% (95%CI: 55-77) at 3 years. NRM was 16% (95%CI: 9-23) at 1 year. Pediatric-DRI was prognostic for RFS, as low, intermediate, and high-risk groups translated to 3-year RFS of 84% (95%CI: 68-100), 63% (95%CI: 49-77), and 52% (95%CI: 37-78), respectively (p = 0.04). There was no correlation between pediatric-DRI score and OS (p = 0.95). Simplified malignant-HCT-CI > = 3 was suggestive of inferior survival: 3-year OS 72% (95%CI: 60-84) vs. 56% (95%CI: 37-75), p = 0.16. The combined pediatric HCT-CR model demonstrated a trend in predicting RFS (p = 0.09). The intermediate-risk group of pediatric HCT-CR indicated a lower likelihood of 3-year OS compared to other groups: 57% (95%CI: 38-76) vs. 71% (95%CI: 59-83), p = 0.19 (Figures 1a,b,c,d). Conclusions: The pilot data presented here suggest that combining disease and patient-related factors into a pediatric-HCT-CR may be an important and prognostic tool in pre-transplant risk stratification for children with acute leukemia undergoing allogeneic HCT. A larger analysis of children who received allogeneic HCT at our center is ongoing with a plan to internally refine and validate the model. Disclosure: Helen Heslop: Equity in Allovir and Marker Therapeutics (both publically traded). Advisory boards for Tessa Therapeutics, Gilead, Novartis, Kiadis, Fresh Wind Biotherapies, Takeda and GSK. Research support from Tessa Therapeutics and Kuur Therapeutics (now Athenex). Other authors report no conflicts.
Background: Treosulfan properties, as potent cytotoxic, myeloablative and immunosuppressive effect with low organ toxicity are the main strengths to incorporate it as conditioning regimen in vulnerable hematopoietic stem cell transplantion (HSCT) patients as pediatric cohort. To analyze the role of treosulfan-based conditioning in children, the Spanish Group of Hematopoietic Stem Cell Transplantation (GETH) performed a retrospective analysis from 11 centers between the period 2012-2021. Methods: A total of 160 children (median 5 years old, IQR: 2-10) diagnosed with non-malignant diseases (n = 117) or high risk malignancies (n = 43), including 36 (22.5%) patients undergoing second/third hematopoietic stem cell transplantation from matched related (MRD) (n = 74), matched unrelated (MUD) (n = 32), haploidentical (n = 39), mismatch unrelated (n = 13) and autologous (n = 1) donors. The source of progenitor cells included bone marrow (n = 88), mobilized peripheral blood (n = 65) and cord blood (n = 7). Preparative regimens were treosulfan-based according to body surface area in combination with fludarabine and thiotepa (n = 109), only fludarabine (n = 38), other drugs as clofarabine (n = 14), depending on the diagnosis (Table I). Results: Engraftment was achieved in 91.3%. The median (IQR) myeloid and platelet engraftment was 16 (13-21) and 18 (14-24) days, respectively. The probability of venooclussive disease (VOD) was 8,8 ± 2%. The cumulative incidence of acute graft versus host disease was 39,6 ± 4% for grades I-IV and 30 ± 8% for grades II-IV. The cumulative incidence of overall chronic graft versus host disease was 10,6 ± 3% and 3,6 ± 1% moderate/severe. The 2-year overall survival (OS) was 80 ± 4%, and was significantly lower in malignancies (61 ± 9%) vs no malignancies (87 ± 3%). The 2-year cumulative incidence of relapse and transplant related mortality was 24 ± 7% and 14 ± 3% respectively. In the complete series, the number of CD4+ T cells higher than 200 µL at 3 months impacted favorably in OS with a hazard ratio 0,19 (0,043-0,846). In the non-malignant cohort the VOD with a hazard ratio 5,53 (1,21-25,25), p = 0,005 and in the cohort of malignant disorders the number of CD34+ cells lower than the median in the graft with a hazard ratio 12,6 (1,5-106) p = 0,021, impacted in the OS. Conclusions: Treosulfan-based conditioning is a safe and effective approach for children with non-malignancies and high risk malignancies. Particularly favorable results were achieved in non-malignant disorders and patients with faster CD4+ T cell reconstitution. Venooclussive disease was not completely avoided and impacted negatively in OS in the non-malignant cohort. The high number of CD34+ cells in the graft impacted negatively in OS in the cohort of malignant diseases. Disclosure: Nothing to declare
Background: *Equally contributed as authors Prior to 2003, when imatinib was licensed for CML-treatment in minors, SCT as a curative approach was ideally performed soon after diagnosis. The study CML-paed I (1995-2004) recommended SCT from a sibling within 6 months after diagnosis and within 12 months from an HLA-matched unrelated donor (MUD) resulting in 5-year overall survival of 87 ± 11% for sibling SCT but inferior (52 ± 9%) and comparable to adult data for MUD-SCT [Suttorp M et al. 2009]. So far, no data exists on the long-term HRQOL of former pediatric CML patients. We here present patients’ self-reporting to a questionnaire sent out to adolescents and young adults (AYAs) formerly enrolled on pediatric CML-SCT trials. Methods: Eligible patients underwent SCT at German centers and were identified from the records of the Children’s Cancer Registry. Following approval by the Institutional Ethical Board and patients’ consent, the completed self-assessment questionnaires SF36 and FACT-BMT were mailed back as of Jan 2021. Results: 111 out of 171 (65%) patients survived long-term. Missing key data resulted in the exclusion of 25 survivors. 37/86 (43%) remaining patients responded to the questionnaire. In 37 responders, CML was diagnosed at a median time point in Aug 2001 (range 1993-2013, median patient age 11 years (range 1-17, gender N = 24 female (65%)). SCT was performed at a median age of 12 years (range 2-19 years, interval from diagnosis to SCT median 7 months, range 2-46). The questionnaire was filled in 4-27 years (median 19) after SCT at a median age of 29 years (range 18-43). 10 patients (27%) did not participate in regular medical follow-up examinations. Self-reported key findings in the total cohort comprised cGvHD-associated damages (N = 8, 22%) like lung problems, dry eyes (each N = 7, 19%), skin alterations (N = 6, 17%), and hair problems (N = 4, 11%). Conditioning regimen consequences reported frequently comprised hypothyroidism (N = 11, 30%), infertility (N = 9, 24%, 7 female), and sexual dysfunction (N = 3, 9%, 2 female). Following SCT, 10 patients (27%) experienced 13 CML-relapses at a median interval SCT to relapse of 34 months (range 2-83). Only one patient underwent 2nd SCT when relapse treatment with 2G-TKI failed. Six secondary malignancies (dysplastic melanocytic nevus and ALL, rhabdomyosarcoma, thyroid carcinoma, basal cell carcinoma (N = 2) occurred in five patients (13%). 18 patients (49%) considered the sequelae of SCT an education obstacle. When filling in the questionnaire, N = 20 (54%), N = 7 (19%), N = 5 (14%), and N = 4 (11%) patients worked full time, part- time, were unemployed or had not yet finalized their education, respectively. 20 unmarried patients (54%) lived as singles, 8 patients (22%) lived in a partnership, 6 patients (16%) were married, and 3 patients (8%) had been divorced. Four patients (11%) reported on a total number of 7 children. Conclusions: This assessment of HRQOL in AYAs transplanted because of CML two decades ago demonstrates self-reported satisfactory well-being only in the absence of cGvHD. Adherence of the whole group to regular (annual) physical examinations is insufficiently low. While relapse of CML nowadays is treated primarily with TKIs, secondary cancers due to conditioning require careful lifelong monitoring. Disclosure: Nothing to declare
Background: The pathogenesis of acute graft-versus-host disease (aGvHD) is initiated by innate immune activation due to conditioning-induced tissue damage, leading to activation of cytotoxic donor T lymphocytes and increased tissue damage. The soluble IL-33 receptor, known as suppressor of tumorigenesis 2 (ST2), is thought to be released from Th1 and Th17 cells and has been reported as a promising biomarker for aGvHD-related outcomes, although studies in children are sparse. In this time course study, we investigated the prognostic value of plasma ST2 levels in monitoring aGvHD development and treatment response after pediatric HSCT. Methods: We included 117 children undergoing HSCT between 2010-2020 in Denmark. Median age was 8.9 years (range: 1.1-17.9). Diagnoses included ALL (n = 29), AML (n = 18), other malignancies (n = 25), and benign disorders (n = 45). Donors were either MSD (n = 33) or MUD (n = 84). BM (n = 112) or PB (n = 5) was used as stem cell source. All patients received a myeloablative conditioning regimen based on TBI (n = 23) or chemotherapy alone (n = 94). GvHD prophylaxis consisted of cyclosporine A, either alone or in combination with methotrexate. ST2 was measured by ELISA in consecutive plasma samples collected before conditioning and at day 0, +7, +14, +21, +30, +60, +90 and +180 post-transplant. Plasma samples from 17 healthy young adults (aged 17-20 years) were included for comparison. Results: Plasma levels of ST2 in the patients were comparable to those in healthy controls (median: 16760 pg/mL vs. 16323 pg/mL) before conditioning but increased early after transplantation reaching a maximum around day +30 (P < 0.0001) (figure). Patients with malignant diagnoses had significantly higher ST2 levels before start of conditioning compared to patients with benign diseases (median: 18121 pg/mL vs. 14754 pg/mL, P = 0.007). Furthermore, busulfan-based conditioning was associated with elevated ST2 levels from day 0 to +21 (all P < 0.05). Thirty-eight patients (32.5%) developed aGvHD grade II-IV (MAGIC criteria) with median onset at day +17 (range: 5-35). These patients had significantly higher levels of ST2 from day +7 to +90 compared to patients with aGvHD grade 0-I, correlating with severity (figure). This was confirmed in a multivariable analysis adjusted for malignant diagnosis, donor type and busulfan-based conditioning (ST2 day +14: OR = 1.95 per doubling in ST2, P = 0.0003). Forty-nine patients (41.9%) received glucocorticoid treatment for aGvHD. The cumulative prednisolone equivalents dose for patients with aGvHD from diagnosis to day +365 (median 0.23 mg/kg/day (range: 0-10.7)) correlated significantly with the ST2 levels measured at the time of aGvHD diagnosis (rs = 0.27, P = 0.04). Moreover, ST2 levels tended to be elevated in patients with steroid resistant/ dependent aGvHD (n = 10) at all timepoints between day +14 and +180 compared to steroid responsive patients, reaching significance at day +90 (P = 0.01). ST2 levels were, however, similar in the two groups at the timepoint of aGvHD diagnosis. Conclusions: Our findings support the use of ST2 as a prognostic biomarker of aGvHD after pediatric HSCT. We demonstrate that ST2 has not only a prognostic value in terms of predicting aGvHD but may also predict aGvHD severity and treatment response. Disclosure: Nothing to declare
Background: Haematopoietic stem cell transplantation (HSCT) has a role to cure leukaemia that is refractory to chemotherapy. This is principally mediated by a graft versus leukaemia (GVL) effect of donor-derived T-cells, directed at the recipient leukaemia. Reduction of relapse has been reported after cord blood (CB) HSCT with augmented GVL since the graft is T-cell replete with frequent donor/recipient HLA mismatch. Although acute graft versus host disease (GVHD) can be significant, chronic GVHD is uncommon. Xenograft studies have reported that CB CD8 + T-cells can mediate GVL but T-cell reconstitution after CB transplant is CD4 + biased and CD8 + recovery is late. We have previously reported very early, unprecedented CD8 + T-cell expansion in a small number of T-cell replete, CB recipients receiving concomitant pooled granulocyte infusions in the early post-transplant period for refractory infection management. These CD8 + T-cells were polyclonal, activated, cytotoxic and had switched from an infused naïve to memory phenotype. These cells may mediate an enhanced GVL effect. We now report initial results of an investigator-led study of children with very high-risk leukaemia, receiving pooled granulocytes after unrelated donor CB HSCT. Methods: Patients aged <16 years with high-risk leukaemia were recruited following referral from the UK national leukaemia multidisciplinary team. Transplants were performed at Royal Manchester Children’s Hospital using T-replete, HLA-mismatched (5/8 – 7/8) unrelated CB donors. Patients received daily infusions of pooled granulocytes in the peri-transplantation period. 7 daily doses of granulocytes were transfused (each 10 ml/kg to a maximum of 200ml). We collected and assessed clinical data including tolerability of granulocytes, cytokine release syndrome (CRS), disease response, transplant related mortality and rates of acute and chronic GVHD. Assessment of T-cell immunophenotypic profile using fluorescence-activated cell sorting was performed during lymphocyte expansion to characterise cell populations. Results: We report data from 2 fully evaluable patients with 3 further patients currently scheduled to receive transplant shortly. All 5 patients had high-risk, chemo-refractory, relapsed AML and had received a previous transplant. In the evaluable patients, the pooled granulocyte product was well tolerated with grade 1-2 CRS. Both patients entered morphological, cytogenetic and flow MRD disease remission (molecular awaited). Acute GVHD was seen in both patients. 1 patient died of transplant related toxicity. Early lymphocyte expansion and contraction was seen, with both CD4 + and CD8 + T-cell populations following this pattern within days of receiving granulocyte infusions. The majority of CD8 + T-cells seen were activated with CD38+/HLA-DR+ expression and were cytotoxic with granzyme B and perforin expression. They produced interferon-gamma in response to stimulation and there was a notable switch in phenotype from CD45RA+/CCR7+ naïve to CD45RA-/CCR7- effector memory and CD45RA+/CCR7- TEMRA T-cells. Conclusions: CB transplant reduces leukaemia relapse after HSCT compared to other cell sources. This GVL effect is mediated by CB-derived T-cells, likely of CD8 + phenotype. We have initiated an investigator-led clinical trial of granulocytes to induce early, CB graft-derived T-cells to augment GVL effect in patients at the highest risk of leukaemia relapse. We report safety and early clinical response data in conjunction with T-cell data in this patient cohort. Disclosure: Nothing to declare
Background: We compared the outcomes of allogeneic hematopoietic cell transplantation (HSCT) from alternative donors between unrelated donor (URD) and haploidentical family donor (HFD). Methods: Between March 2003 and October 2021, 66 patients with acquired severe and very severe aplastic anemia (32 SAA, 34 vSAA) received upfront allogeneic HSCT from alternative donors (URD 30, HFD 36) at Asan Medical Center Children’s Hospital. For HSCT from HFD (HSCT-HFD), 5 patients received CD3-depleted PBSC and 31 received TCRαβ-depleted graft. Conditioning regimens consisted of cyclophosphamide, fludarabine and r-ATG for URD and TBI (4-6 Gy), fludarabine, cyclophosphamide and r-ATG for HFD. Results: Of 66 patients, 2 patients, who received HHCT-HFD, experienced primary graft failure (GF) and the remaining 34 achieved engraftment of neutrophil at a median of 11 days (range, 9-20 days). The median days of neutrophil engraftment was faster in HSCT-HFD compared to HSCT-URD at 10 days and 12 days, respectively. (P < 0.001). Additional 2 patients from HFD experienced graft rejection and one from URD developed poor graft function. The cumulative incidences (CI) of any graft failure were 11.1% for HFD and 3.8% for URD (P > 0.05). The CI of grades 2-3 and grades 3 acute GVHD were 33% and 11%, respectively, which were not different between HHCT-URD and HHCT-HFD. No patient developed grade 4 acute GVHD. Two patients from URD developed severe chronic GVHD with CI of 7.4%, while no patients from HFD developed moderate/severe chronic GVHD (P > 0.05). One patient from URD died of GF and three patients from HFD died of transplant-related causes (GF, CMV pneumonia and TMA 1 each), leading to TRM of 5.6% for URD and 9.0% for HFD, respectively (P > 0.05). All survived 62 patients were transfusion independent. At a median follow-up of 4.7 years (range,0.2-18.8 years), 3-year estimated overall survival, failure-free survival, and mod/severe cGVHD-free and failure-free survival were 94%, 96%, and 89% for URD and 91%, 83% and 83% for HFD. Table 1. Characteristics of patients with SAA. Figure 1. Outcomes of alternative donor HSCT for pediatric patients with SAA Conclusions: Our study is another emerging evidence of upfront haploidentical HSCT for pediatric patients with SAA in terms of low TRM and moderate/severe chronic GVHD with favorable failure-free survival. Clinical Trial Registry: NCT 01759732 Disclosure: Authors have no personal or financial interests to declare.
Background: Acute lymphoblastic leukemia (ALL) is the main indication for allogeneic transplantation in the pediatric age. An important proportion of these patients achieve long-term remission after transplant and can be considered cured. However, relapse after transplant still is the main cause of transplant failure and the leading cause of death. For many years, relapsed ALL patients have been considered candidates for a second transplant. This landscape has changed since advent of immunotherapy and CAR-T cell therapy. Methods: A total of 29 children (18 female) with ALL (median age 7 years; range; 1-15) who relapsed after an allogeneic transplant since 2013 to 2020 were included in this retrospective study. Median time to relapse was 4 months (range; 2-24). Twenty-three out of 29 patients were in CR and MRD negative at time of first allogeneic transplant. Previous transplants were from a MUD in 12 cases, Haploidentical Donor in 8, MSD in 7 and CBU in 2. Four out of 29 (14%) patients had previous chronic GvHD. Results: CAR-T cell therapy was used in 12 patients (10 preceded by bridge chemotherapy and 2 by monoclonal antibody). Second allogeneic transplant was performed in a total of 5 patients (using a different donor of first transplant), 2 with identical unrelated donors and 3 with haploidentical donors. The other patients progressed before they could receive consolidation therapy. CR was obtained in all 12 CAR-T patients (100%) and in 4 out of 5 (80%) patients after transplant. One patient died of TRM. With a median follow-up for survivors of 2 years (range; 1-8), 6 patients relapsed again after obtain CR: 5 after CAR-T cell therapy and 1 after transplant. Thirteen patients are alive, 3 of them with active disease, with an overall survival of 21 ± 5%. Ten patients are alive in CR (7/12: 58% after CAR-T and 3/5: 60% after transplant). There are no differences in terms of DFS between therapy groups: 7/12 (58%) after CAR-T and 3/5 (60%) after transplantation. Conclusions: Despite small sample size, our results suggest that treatment of relapsed ALL patients after allogeneic transplant is moving to cellular and immunotherapies rather a second allogeneic transplant. The role of transplantation as consolidation of the CAR-T cell therapy. must be established, since half of the patient relapse after CAR-T. Disclosure: Nothing to declare
Background: Non-osteopenic bone disease (BD) in children post-HSCT for IEI remain unrecognised. We present post-HSCT long term follow up (LTFU) data from large tertiary paediatric immunology centre. Methods: Between 2000-2018, 432 children received HSCT for IEI. 342 children were alive at last assessment and included for analysis. Patients’ records were checked for BD and potential risk factors; underlying IEI, steroid use/duration, hormonal replacement therapy (HRT); growth and gonadal hormones, weight-for-age (centiles) and donor engraftment at onset of BD. Exclusion criteria included decreased bone density, fractures, anomalies due to underlying IEI, short stature without other BD. BD was divided into 5 categories; Bone tumors; Congenital defect with late diagnosis; Avascular necrosis (AVN); Evolving bone deformities; Slipped upper femoral epiphysis (SUFE). Results: 27/342 (7%) children developed BD at a median of 7.7 years post-HSCT (29 HSCTs in 27 children). Conditioning regimen included Treosulfan- (n = 20), Fludarabine/Melphalan- (n = 7) and Busulfan- (n = 2) based conditioning. Donors were 10/10 HLA matched (n = 15), 7-9/10 mismatched (n = 13) and haplo-identical (n = 1). Underlying IEI included severe combined immune deficiency (SCID) (n = 9); Wiskott Aldrich Syndrome (WAS) (n = 7), other non-SCID (n = 11). Six patients have >1 category. Two patients had HRT, 17/27 (63%) had steroid therapy > 6 months. Of note, 4/8 patients with benign bone tumor had WAS. Eight developed AVN at a median of 7 years post-HSCT; 6/8 had steroid use >12 months. Seven had genu valgum at a median of 8.1 years post-HSCT; 6/7 had steroid use >6 months and 1/7 received HRT 11 months ahead of BD. Six patients developed SUFE; 5/6 boys; 1/6 overweight and all presented at 10 years or below. Conclusions: Non-osteopenic BD post-HSCT for IEI are not rare and should be actively looked for in LTFU clinic. Prolonged steroid use (>6 months) is associated with increased rates of BD including AVN, genu valgum and benign bone tumors. Increased rates of BD among WAS warrants investigation to understand potential mechanism in context of specific IEI. Disclosure: Nothing to declare
Background: Endothelial injury (EI) is the common trigger of numerous posttransplant complications. Little has been published about complications secondary to EI in the ex vivo T-cell-depleted haploidentical hematopoietic cell transplantation platform, and even less when considering pediatric population. The primary end point is to analyze incidence, outcome and survival rates of complications secondary to EI in pediatric patients, both in general terms and specifically for each pathology. As secondary outcome, the possible relation between the complications secondary to EI and graft versus host disease (GVHD) will be studied. Methods: A total of 159 patients (106 males) diagnosed from hematological malignancies that underwent allogenic HSCT from haploidentical donors using ex vivo T-cell depletion between 2005 and 2020 were included. Seventy-nine patients were diagnosed of AML and 80 patients of ALL. Forty-eight transplants were in 1stCR, 55 in 2ndCR and 56, beyond 2ndCR or with active disease at time of transplant.Donors mean age was 40 (range 2–54) years. All patients received myelo-ablative conditioning and total-body irradiation was not used in any case. A P-value ≤0.05 was considered statistically significant. Results: The cumulative incidence of EI was 45 ± 5%, with a follow-up median of 7 (1,7-17) years. Patients developing these complications have an incidence of transplant-associated mortality (TAM) of 40 ± 7% compared to 12 ± 4% in patients that did not develop them (p = 0,0001). Regarding overall survival rates (OS), clinical and statistically relevant differences were found (p = 0,05): patients that developed complications had 45 ± 6% OS compared to 63 ± 5% OS in patients that did not. OS for the whole group was 60 ± 4%. Descriptive statistics for each complication related (sinusoidal obstruction syndrome, engraftment syndrome, [ARL1] thrombotic microangiopathy, diffuse alveolar hemorrhage and posterior reversible encephalopathy syndrome) to are collected in Table 1. In the multivariate analysis of the results, it was found that having any complication related to EI increases up to 4 times the risk of developing acute GVHD (being EI present the hazard ratio (HR) is: 4,1, CI95%: 2,1-8,1, p = 0,0001). Conclusions: From the results obtained in the study, we can infer that complications related to EI affect the outcome of ex vivo T-cell-depleted haploidentical hematopoietic cell transplantation in pediatric patients with malignant hemopathies, resulting in an decreased of OS and increased TAM. Additionally, EI is directly related to other complications that associates high morbidity and mortality rates such as GVHD. Scientific and clinic efforts should be directed to recognize risk factors for developing EI, that will consequently allow to establish early diagnosis, early treatment and better prevention measures for EI. Disclosure: No disclosures.
Background: Hematopoietic stem cell transplantation (HSCT) is a curative modality for variable malignant and non-malignant conditions. The first comprehensive state of the art pediatric HSCT program in Amman-Jordan was established in 2003 at The King Hussein Cancer Center (KHCC). We describe the pediatric HSCTs activities and trends by The Pediatric HSCT Program at KHCC which performs a significant number of HSCTs reaching 100 per year. Methods: Data collected since the start of the program in 2003 to date, from medical files and electronic medical records, were retrospectively reviewed and analyzed, after obtaining Institutional Review Board Approval. Results: Between January, 2003 and October 2021, a total of 1051 HSCTs were performed for pediatric patients with a median age of 8 years (0.13-31), of which 58% were males and 72 % were Jordanian (n = 758). Median follow-up time was 2.9 years (0.087-12.5 years). Allografts accounted for the majority of HSCTs (77%; n = 811); whereas, autografts accounted for 23 %(n = 240). Allogeneic HSCTs included full-matched family/related donors in 79% (n = 641), haploidentical HSCTs in 16% (n = 132) and unrelated donors in 5% (n = 38). Stem cell sources included PBSCs in 75% (n = 788), BM 21% (n = 221) and CBUs 4% (n = 38) of the HSCTs. Myeloablative and reduced intensity (mainly for non-malignant disorders) conditioning regimens were employed in 71 % and 27% of the HSCTs, respectively; and no conditioning was given in 2%. Malignant conditions were the main indications for HSCTs (56%; n = 591), whereas, non-malignant conditions accounted for 44% (n = 460). The most common indication for allogeneic HSCTs were leukemia (37%; n = 297), followed by hemogloinopathies in 26% (n = 212), bone marrow failure syndromes in 17% (n = 137) and immune deficiencies in 11% (n = 88); while solid tumors (69%; n = 165), followed by lymphomas (41%; n = 99) were the main indications for autologous HSCTs. The 5-year OS and EFS for all HSCTs were 81% ±2.3 and 75%±3.4, respectively; and for allogeneic HSCTs 84%±3.1 and 77%±2.2. Cumulative incidence of TRM at 1 year for allogeneic HSCTs was 1.9%, compared to 5% in the previous treatment era (before 2010), p = 0.004). Whereas, that for autologous HSCT was 0.54%. Disease progression/ relapse of underlying condition was the main cause of mortality (76%). Conclusions: HSCTs have provided long-term disease-free survival and cure for a wide spectrum of malignant and non-malignant conditions in children. Referral to a dedicated pediatric HSCT center and implementing contemporary practices such as refinement of conditioning regimens, high resolution patient and donor typing, expertise of the treating teams and robust supportive care services have contributed to significant improvement in outcomes of pediatric HSCTs at KHCC. In recent years, a constant increment in the total number of pediatric HSCTs performed at KHCC is evident. In particular, haploidentical HSCTs are increasingly employed as they constitute a readily available alternative donor option, similar to international practices. Disclosure: no conflict of interest
Background: Patients undergoing autologous high dose chemotherapy with stem cell rescue (aHDC-SCR) commonly receive granulocyte colony-stimulating factor (GCSF) to reduce the duration of neutropenia. Timing of GCSF administration after aHDC-SCR is varied and has been analyzed in adult studies. These have shown mixed results when assessed for time to neutrophil recovery, duration of hospitalization, infection rates and cost. There is a paucity of data in pediatrics. Methods: A single-center, retrospective pediatric study was conducted at a tertiary care academic institution, The Hospital for Sick Children. Patients who received aHDC-SCR were reviewed to determine the primary outcome of days to neutrophil >500/µL, and secondary outcomes duration of hospitalization, proportion of patients with febrile neutropenia, infection, engraftment syndrome and cost of GCSF. Variation in neutrophil recovery was analyzed using the Mann-Whitney U test and other secondary endpoints used Chi-Squared test comparing cohorts of patients treated with varying approaches to GCSF administration. Results: This retrospective sequential cohort of 61 pts (102 transplants) who were aged 0.5-21 years and who underwent aHDC-SCR for treatment of neuroblastoma, brain tumors, lymphoma and other solid tumors. Three groups were identified based on the timing of GCSF administration: A) early GCSF, given on day 0/+1 post-transplant (N = 72), B) delayed GCSF (N = 7) where GCSF was started d 5-10 post HSCT, C) no GCSF (N = 13) and D) those that were not planned to receive GCSF, but received due to a clinical reason (N = 10). There was no difference in neutrophil recovery between group A and B (10.7 and 10.9 days respectively). However, neutrophil recovery was delayed (p < 0.001) in those not planned to receive GCSF (group C & D) (14.5 days). The mean neutrophil recovery was similar irrespective of primary diagnosis and CD34+ cell dose (mean 8.6, range 9 – 21 x106 /kg) infused. The proportion of patients with febrile neutropenia, infection and engraftment syndrome were similar regardless of administration and timing of GCSF. The duration of hospitalization was not different between groups who received and not received GCSF (36.1 days and 26.4 days, respectively). This resulted in no difference in the cost of hospitalization (per diem charges) between groups who received GCSF and no GCSF group. However, the cost difference per transplant was closed to $35,000 higher in those who received early GCSF. Conclusions: Time to neutrophil recovery was reduced with post-transplant GCSF administration. The duration of hospitalization was not shorter if GCSF was given. The episodes of fever and neutropenia, infection and engraftment syndrome were similar. The cost of hospitalization was higher due to the cost of GCSF. Further studies are needed to confirm findings and assess cost-benefit of GCSF in this patient population. Disclosure: Nothing to declare.
Background: The timing of allogeneic hematopoietic cell transplantation (HCT) for pediatric patients with chronic-phase chronic myeloid leukemia (CML-CP) is controversial in this tyrosine kinase inhibitor (TKI) era. Methods: A subanalysis was conducted on 14 patients (18%) from the nationwide, prospective, multicenter, observational study (CML-08, UMIN 00000258, n = 78) that was carried out by the Japanese Pediatric Leukemia/Lymphoma Study Group (JPLSG). These patients were under 18 years of age and diagnosed with BCR-ABL1-positive CML-CP between October 2009 and September 2014. All had been treated according to the modified European LeukemiaNet 2009 recommendations and opted for HCT during the 10-year observation period. Results: In total, there were 10 males and four females. The median diagnostic age was 12 (range: 3–16) years, with six patients younger than 10. The median observation period from diagnosis to the last follow-up was 66 (range: 26–110) months. All patients were initially treated with imatinib (IM), except for one who was administered nilotinib. As a pre-transplant therapy, two patients received IM, other patients received two to four different TKIs. Twelve patients received HCT for inadequate responses to TKIs, one did it for intolerance, and one did it for her young age. Two had progressed to the blast phase during treatment with TKIs but achieved a second CP (CP2) before HCT. The median duration from diagnosis to HCT was 24 (range: 8–86) months. HLA-matched sibling donors (MSD) were available for five patients. Among the first CP (CP1) patients, all but one received a reduced-intensity conditioning regimen. Five-year overall survival and event-free survival were 86% and 59%, respectively. One patient had an engraftment that initially failed, yet later succeeded upon the second HSCT. Graft-versus-host disease (GVHD), at a grade III to IV acute GVHD or an extensive/ severe chronic GVHD, was experienced by one (7%) and two (14%) patients, respectively. Two patients, a CP1 transplanted from an HLA mismatched related donor and a CP2 transplanted from an MSD, died from post-transplant complications within a year. Five patients received a TKI and/or donor lymphocyte infusions following HCT treatment, besides those who received a retransplantation for graft failure. Short stature was observed in a patient who suffered from severe chronic GVHD. On the other hand, HCT enabled some young patients to recover from impaired growth that had been induced by TKIs. At the last follow-up, all surviving patients were in deep molecular responses, and their performance status scores were zero, except the one with short stature. Conclusions: HCT is still an alternative for CML-CP patients who do not maintain an optimal response with TKIs. In this study, HCT was successful in most patients even without MSD, but by no means all. We should carefully consider each patient’s situation and circumstances before administering HCT. Disclosure: Nothing to declare
Background: The use of reduced intensity conditioning (RIC) regimens in patients with non-malignant diseases has allowed hematopoietic stem cell transplantation (HSCT) in children with moderate or severe comorbidities. However, this practice has been associated with a higher incidence of mixed chimerism (MC). The objective of this study is to analyze the incidence of MC in patients with non-malignant diseases who received a RIC regimen and to demonstrate that its presence does not have an impact on overall survival (OS) and disease-free survival (DFS). Methods: Retrospective study where children who received a first HSCT following a RIC regimen for non-malignant diseases between 2013 and 2019 were included. Primary endpoint: disease free survival. Secondary endpoint: engraftment, graft failure, incidence of MC, acute and chronic graft versus host disease (GvHD) and OS. Results: With a median follow up of 28 months, 78 patients were included (median 5 years old, IQR: 0.2-17), with primary immunodeficiency (n = 41,52.6%), severe aplastic anemia (n = 23,29.5%) and bone marrow failure (n = 14,17.9%). Fifty patients (64.1%) underwent a first HSCT from a matched donor (unrelated n = 32 /related n = 18) and twenty eight (35.9%) from a mismatched donor (unrelated n = 19/haploidentical n = 7/related n = 2). The source of stem cells included bone marrow (n = 57,73.1%), peripheral blood (n = 12,15.4%) and cord blood (n = 9,11.5%). Most used conditioning regimen included Cyclophosphamide and Fludarabine (n = 32, 42.3%), Busulfan and Fludarabine (n = 23, 29.5%), Treosulfan and Fludarabine (n = 9, 11.6%) and Fludarabine and Melphalan (n = 7, 9%). All patients except one received T cell depletion (98.7%): in vivo (n = 66, 84.6%) and ex vivo (n = 11, 14.1%). Median time for neutrophil engraftment was 19 days, IQR 14-22. Seven patients had primary graft failure (9%). A total of 40 patients (51%) presented MC, of whom 5 (12%) had secondary graft failure. The incidence of grade III-IV aGvHD at D + 100 was 19.5% (95% CI 11.39 - 32.33%), the incidence of cGvHD at 2 years was 15% (95% CI 8.31-26.19%). The OS at 1 and 3 years post HSCT were 83% (95% CI 72.6-89.8%) and 77.% (95% CI 66-85.2%). The EFS at 1 and 3 years post HSCT were 78.72% (95% CI 67.6-86.4%) and 70.5% (95% CI 58-79.8%). Overall, 18 patients (23%) died, of whom 4 after primary graft failure and 5 after a secondary graft failure. The causes of death were infections (n = 9), transplant-associated thrombotic microangiopathy (n = 3), diffuse alveolar hemorrhage (n = 1), liver (n = 1) and respiratory (n = 1) failure, multi organ failure (n = 1) and not documented/unknown (n = 1). There were no significant differences in terms of OS and EFS in patients with mixed chimerism and patients with full donor chimerism [OS 87% (95% CI 71.8-94.4%) vs 72% (95% CI 52-85.1%) p 0.25; EFS 82% (95% CI 66.5-90.9%) vs 72% (95% CI 52.0-85.1%) p 0.49]. Patients who received a matched donor, bone marrow as stem cell source and in vivo T cell depletion presented a better OS and DFS. Conclusions: Children with non-malignant diseases have a high incidence of MC when receiving a RIC regimen. The presence of MC does not have a negative impact on OS and DFS. Disclosure: Nothing to declare
Background: Primary immunodeficiency diseases (PID) are characterized by the occurrence of frequent infections and are caused by many genetic defects. Hematopoietic stem cell transplantation (HSCT) is a curative treatment option for the majority of PID. We want to present our experience about HCST in PID in a single pediatric center in Argentina. Methods: Retrospective study on HSCT in PID collected from clinical charges from January 1999 to June 2021.Donor type, demographic data, stem cells source, conditioning regimen (CR) and outcome were evaluated. Results: 16 patients were included in this study and 17 transplants were performed. 6 were female and 10 male.The median age was 4.98 years (range 2 months- 15.16 years) . The diagnosis were SCID 5 patients, CID 3 patients (1 non characterized; 1 CD40 ligand deficiency 1 and PNP deficiency) HLH 5 patients (2 grisceli, 1 chediak and 2 non characterized), 2 CGD and 1 IPEX. Related donor (RD) were used in 10 transplants(haplo 5 and 5 Match related donor) and Unrelated 7 transplants. The source used was BM 10, PB 6 and 1CB. Graft failures were presented in 5 patients. Primary were presented in 3 patients (17.64%), secondary ocurred in 2 patients, both of them caused by CMV reactivation. 3 of the 5 patients are dead. The GS was 70.6% and TRM was 11.76%. Acute GVHD happened in 4 patients (23.5 %) Grade III/IV 1 (5.8%) and Chronic GVHD none. Viral reactivation presented in 9 transplants (52%), CMV 5, VEB 4 and others 4. Some of them had coinfections . 3 of them used ATG and 3 Alemtuzumab for GVH prophylaxis. Conditioning regimen used were 12 MAC, 2 RIC and 2 without conditioning. 11 patients are alive (and 9 are cured of their PID). Conclusions: The transplantation procedures appear to have provided a permanent cure in nine PID patients. Early diagnosis and prompt performance of SCT with an optimal donor and conditioning regimen contributed to the favorable outcomes that were similar to reported in literature. Virus reactivation is a frequent complication and contributes to graft failure. New and better viral treatments and alternatives GVH prophilaxys strategies may contribute to improve outcomes. Disclosure: Nothing to declare
Background: Allogenic haematopoietic stem cell transplantation (HSCT) remains the only proven curative therapy for juvenile myelomonocytic leukaemia (JMML). Despite that, leukaemia relapse remains the major cause of treatment failure in 30-60% of children with JMML and tends to occur within the first year after the allograft. Methods: We report on 3 high risk JMML patients (age > 2 years, PLT < 30 x 109/L, somatic PTPN11 mutation) who all underwent haploidentical peripheral blood HSCT with CD3/CD45 RA T- cell depletion followed by post-transplant Decitabine as pre-emptive therapy to prevent relapse. All 3 patients were diagnosed in their home country and presented to our institution for a second opinion. Patient 3 in particular had been diagnosed as relapsed mixed phenotype acute leukaemia whilst receiving maintenance therapy as per COG protocol when her unique features of monocytosis, monosomy 7 and PTPN11 prompted the diagnosis of Acute JMML. All 3 patients had peripheral blasts >5%, BM blasts >15% and patient 1 and 2 also had an elevated HbF > 20% at diagnosis. They all had poor response to various initial therapy and achieved cCR only after combination therapy with Etoposide +AraC +Aza. All 3 patients underwent pre-HSCT splenectomy with the hope of reducing tumor burden at the time of HSCT to reduce the risk of recurrence. Diagnostic Features and Transplant conditioning/ All 3 patients had PTPN11 somatic mutation (samples sent to C Niemeyer, Uni Freiberg) ADE: Cytarabine, Daunorubicin, Etoposide (AML like therapy), Aza: Azacytidine 1cCR: clinical complete response based on criteria for evaluating response and outcome in clinical trials for children with JMML (C Niemeyer et al, 2015) 2Patient 2 failed his first haploidentical maternal stem cell transplant and required a second transplant Results: All 3 patients are alive and remain disease free at a median of 31.5 months from time of HSCT. There were no reported aGVHD or cGVHD and all 3 patients received 6 cycles of monthly decitabine from as early as 1 month post HSCT. Patient 2 required a second transplant due to early graft rejection with macrophage activation syndrome from suspected central line infection with acute decline in counts and fall of donor chimerism. He was successfully re-transplanted with a 2nd haploidentical HSCT despite multiple infections including Klebsiella and Sternotrophomonas maltophilia sepsis with a rapidly progressing thigh cellulitis which required debridement and skin grafting. All the patients achieved neutrophil engraftment (ANC > 0.5) between 9-15 days and platelet engraftment (PLT > 50) before 20 days and GVHD prophylaxis was only in the form of CD3/CD45RA T-cell depletion. Conclusions: Our small experience in treating this high risk group of patients has been encouraging and the use of post transplant Decitabine appears to be beneficial in preventing early relapses without additional GVHD. Splenectomy should also be considered in this high risk group. Clinical Trial Registry: NA Disclosure: Nothing to declare
Background: Iron-overload (IO) is a late complication of HSCTs, mainly investigated in chidren transplanted for hemoglobinopathies. We present factors associated with IO, with emphasis on the differences between disease groups: hemoglobinopathies, leukemia, and other nonmalignant disorders, and the trend of change over time before and after transplant. Methods: All children allo-transplanted between 2009-2019, with ≥2 years follow-up, were included. IO was assessed by blood-ferritin level at different time points. The values at 2-years post-HSCT were used to determine statistical significance (ANOVA & t-test) between different disease-groups and other factors. Statistical analysis was conducted by SPSS software (Chicago, IL); mean ferritin values were log-transformed. Study was approved by IRB. Results: Forty eight allo-transplanted children (52% males, median age at HSCT 9.1 years) were included. Indications for HSCT were non-malignant disorders in 28 children (58%), mainly Hemoglobinopathies (15 patients), and Leukemia in th e other 20 (42%). Median follow-up after HSCT was 4 years (range: 2-12.8); all but 3 patients (6%) were alive at last follow-up. The incidence of chronic GVHD was 31%. All but 2 patients were exposed to RBCs-transfusions, mostly (75%) before transplant. Number of transfused RBCs units was 22.6/patient (mean) for all cohort, and was significantly different between the disease-groups: 32 and 9, for the hemoglobinopathies & leukemia, versus other nonmalignant disorders, respectively (p < 0.001). After transplant, no chelation-therapy nor phlebothomy were given to any patient, including hemoglobinopathies. Ferritin levels before HSCT were 1145 ng/mL (mean); and after transplant, at the following time-points: 1-year post HSCT, 2-years post HSCT, and at last follow-up, were: 708, 474, and 268 ng/mL, respectively. The hemoglobinopathies & leukemia groups had significant higher levels vs. other nonmalignant disorders-group (p < 0.001). Higher age at HSCT, higher number of RBCs-units transfused, and disease-group: hemoglobinopathies & leukemia vs. other nonmalignant disorders (p < 0.05, p < 0.01, and p < 0.001, respectively), but not chronic GVHD (p = .982), were correlated with higher 2-year-ferritin level (Table 1). Table 1: Factors correlated with higher blood-ferritin level 2-years post-Allo-HSCT in children. † back transformed note: categories not sharing subscript are significantly different Conclusions: These retrospective data show that in children: 1. post-HSCT IO is correlated mainly with pre-transplant factors; 2. non-malignant indications for HSCT other than hemoglobinopathies are associated with lower level of IO, presumably due to lower burden of pre-HSCT blood-transfusions; 3. and spontaneous improvement without intervention is excpected. Collavorative prospective studies about the role of pos-transplant-celation would be valuable. Disclosure: Nothing to declare
Background: Children with acquired severe aplastic anaemia (SAA) or refractory cytopenia of childhood (RCC) who relapsed or are refractory after conventional therapy and lack a suitable donor urgently need novel therapies. Haploidentical stem cell transplant (Haplo) offers a curative option, but the experience is limited. We review our experience with Haplo for this indication. Methods: We carried on a multicentre retrospective review of paediatric patients treated with Haplo for relapsed or refractory SAA/RCC that lack suitable donors in Grupo Español de Trasplante de Medula Osea en Niños (GETMON)/ Grupo Español de Trasplante hematopoyetico (GETH) associated centres. Results: Between 2017 and 2020, 9 patients (4 females/5 males) with refractory SAA (7) and RCC (2) were treated with Haplo in five centres. Most of them were heavily pretreated patients, refractory to immunosuppressive treatment (8/9) and eltrombopag (7/9). Iron overload was common, and most of the patients had ferritin above 1500 ng/ml. The median age at Haplo was 11.20 years (range, 8.99-16.94). All had good performance status with Lansky > 70. The median time from diagnosis to Haplo was 10.54 months (range, 2.14-56.61). Lymphodepletion strategies were post-transplant cyclophosphamide (3), α/β CD19 depletion (2), and CD45 RA depletion (4). Conditioning was based on fludarabine+cyclophosphamide combination in all SAA patients and was myeloablative with busulfan/treosulfan + fludarabine+thiotepa in RRC patients. In all SAA (7/9), reduced dose TBI or nodal irradiation was part of the conditioning. Mean CD34 cells dose was 5.62 x 10e6/kg (SD 2). All patients engrafted (medians: neutrophils day + 13 and platelets day + 13) and all reached transfusion independence (median 15 days, range, 10-82). Viral reactivation was common (6/9), and three patients developed post-transplant microangiopathy. 5/9 developed acute GVHD ≥ 2 (3 were grade 4) that evolved to chronic in 2 (1 severe). All patients achieved sustained full chimerism. One patient developed progressive receptor chimerism that was reverted with donor lymphocyte infusions (DLI) but developed severe autoimmune cytopenia requiring intensive immunosuppressive treatment. Two patients died of transplant-related complications (microangiopathy and respiratory failure). Overall survival was 76.2% (SD 14.8) with a median follow up of 36 months (range, 10-50). There was no secondary graft failure. Conclusions: Haplo transplant offers a relatively successful therapeutic opportunity for refractory SAA/RIC, a subset of patients with limited curative alternatives. We observed excellent engrafting rates likely related to the addition of low dose radiotherapy to the conditioning. TRM was acceptable, and transplant-related microangiopathy was involved in the two cases. However, it is important to work forward to reducing transplant toxicity. We still face a significant rate of severe acute GVHD and, to a lesser degree, cGVHD that should be improved. The limited number of cases precludes us from assessing which Haplo platform is best suited for these patients. Longer follow-up is needed to assess QoL in patients treated with Haplo. Disclosure: Nothing to declare
Background: Survival at 100 days post-transplant is a critical point followed by transplant centers to assess the quality of their transplant program. Deaths within 100 days post-transplant that are not due to relapse or progression of disease are set to be transplant-related (TRM). TRM are presumed to be due to toxicities of pre-transplant conditioning (chemotherapy, radiation) and its associated complications. Methods: We retrospectively collected data of all first allogeneic HCT at KHCC between 2003 to 2019. Data were collected for both HLA-matched related (MRD) and haploidentical HCT. We calculated TRM at day-100 and at 1 year post HCT. Results: Over the period of 2003-2019, 629 first allogeneic HCTs were performed (547 MRD, 82 haploidentical). Referrals from surrounding countries account for 26% of all transplants (165/629). HCT was performed in 50% of patients for non-malignant disorders. Day 100 and 1 year TRM were very low at 2.2% (14/629) and 3.8% (24/629), respectively. We observed significant reduction in Day-100 and 1 year TRM over time (TRM at day-100 and 1 year decreased from 3% and 6% in 2003-2011 to 1.5% and 2.4% in 2012-2019). Causes of death were mostly sepsis (14/24), bleeding (4/24), GvHD (3/24) Conclusions: In a high-volume single center experience where referrals from surrounding countries account for one forth of transplants, there was a very low day-100 and 1 year TRM compared to benchmarks. Our data suggest that a specific transplant infrastructure with a highly experienced team, staff training and retention, strict infection control measures, better venous access care, excessive pre transplant screening, and great vigilance in detecting and treating early infections, including sepsis and CMV likely contributes to a better transplant outcomes Clinical Trial Registry: NA Disclosure: Nothing to declare
Background: CBL syndrome is caused by germline heterozygous mutations in the CBL gene. It is a rare and heterogeneous genetic disease that clinically overlaps with the phenotypic features of Noonan syndrome. It presents as dysmorphic features, congenital heart defects and increased risk of cerebral vasculopathy. Paediatric management and treatment can vary from a “wait and watch” strategy to allogeneic hematopoietic stem cell transplantation (HSCT). Methods: We report a paediatric patient diagnosed with CBL syndrome in 2020 from a Spanish tertiary University Hospital with cerebral vasculitis that underwent a HSCT. Clinically she presented with persistent splenomegaly, thrombocytopenia, progressive episcleritis and persistent monocytosis. The diagnosis of CBL syndrome was carried out by whole exome sequencing (WES). Cerebral vasculitis was treated with steroids, cyclophosphamide and required ventriculoperitoneal shunting. In order to prevent the progression of the central nervous system vasculitis and the risk of developing juvenile myelomonocytic leukemia (JMML) the patient was submitted to an allogeneic bone marrow transplantation from an unrelated donor with 9/10 HLA compatibility. She received a conditioning regimen based on treosulfan, fludarabine, thiotepa and rabbit anti-thymocyte globulin. Tacrolimus and methotrexate were used as graft versus host disease (GVHD) prophylaxis. Results: Amongst the multiple complications the patient presented severe hepatic veno-occlusive disease which led to a 10 day stay in the Intensive Care Unit. She also developed a Pseudomona septicaemia and GVHD grade III-IV with pulmonary, intestinal and hepatic involvement. Previous to the discharge she presented 100% chimerism in both lymphocytes and granulocytes. Four months after transplant, transfusion dependency and severe neutropenia were observed together with hypersplenism and severe splenomegaly that complicated oral feeding; so a splenectomy was performed. Despite that, the bicitopenia persisted and the patient lost the donor granulocyte chimerism. (See Figure 1). Immunosuppressive drugs were withdrawn. Bone marrow biopsy showed bilineal dysplasia and acquisition of t(1;11(q21;q23) with affection of KMT2A gene, but without monocytosis. Within the following month after immunosuppression stop and splenectomy, the patient showed a spontaneous hematologic recovery. Total donor chimerism in myeloid and lymphoid series was observed, and a repeated bone marrow biopsy showed normalization of the three series with loss of the CBL1 and KMT2A mutations. Figure 1 Conclusions: We would like to highlight the important role that has played in the haematological recovery of our patient both the immunospurression stop and the splenectomy. CBL is a rare syndrome and very few transplant cases have been reported, but data suggests that HSCT not only can prevent developing JMML but it also improves cerebral vasculitis. Further investigation should be conducted on this topic. Disclosure: Nothing to declare
Background: Infections are one of the main causes of morbidity and mortality after hematopoietic stem cell transplant (HSCT). Incidence of nontuberculous mycobacteria (NTM) infections has raised during the past few years, especially in immunosuppressed hosts such as HSCT recipients in which cell-mediated immunity is compromised until immune reconstitution after HSCT occurs. Nevertheless, there are still few studies on the incidence, severity and management of NTM infections in paediatric HSCT recipients. Methods: We performed a retrospective study of NTM infections in children with HSCT of all consecutive paediatric patients from 2013 to 2020 in a single-centre setting. We included HSCTs performed for both malignant and non-malignant diseases. Epidemiological and clinical data were recorded from the medical history. Results: A total of 242 HSCT were performed during the study period, with 1.65% of NTM infection. 4 patients were included, three females and one male. The median age at the time of transplantation was 11 years (range, 5-16 years). The median time to infection after HSCT was 7 months (range, 3.5-11 months). All patients had graft-versus-host disease (GvHD) at the time of infection and 3 out of 4 had CD4 + counts <200/mm3. The only patient with CD4 + above 200/mm3, was at that time admitted in the ICU with respiratory failure due to lung GvHD and severe infection caused by E. Coli and Klebsiella sp., and has received prolonged immunosuppressive therapy. All the infections in this series were caused by Mycobacterium avium complex (MAC): two by M. avium and two by M. intracellulare. Two patients developed a disseminated infection and the other two had a pulmonary disease. All patients received combined antimicrobial therapy without significant side effects. With a median follow-up of 4 months (range, 2-40 months), 50% of the patients are alive; none of the deaths was attributed to mycobacterial infection. aGvHD: acute graft vs host disease; AML: acute myeloid leukemia; BAL: bronchoalveolar lavage; cGvHD: chronic graft vs host disease; gi: gastro-intestinal; haplo: haploidentical; HSCT: hematopoietic stem cell transplant; IEI: inborn error of immunity MDS: myelodysplastic syndrome; MMUD: mismatched unrelated donor; MUD: matched unrelated donor. Conclusions: NTM infection is an infrequent but severe complication of HSCT in paediatric patients, affecting mainly those with impaired cell-mediated immunity. All patients in our series had active lung GvHD. The most frequent causative species is MAC. Disclosure: Nothing to declare.
Background: Allogeneic hematopoietic stem cell transplant (HSCT) is a curative approach for hematological malignancies and non-malignant diseases. Disease relapse is associated with poor prognosis and survival, therefore carefully post-HSCT monitoring is required. Donor lymphocyte infusions (DLI) are used preemptively for patients with mixed donor chimerism (MDC) to enhance the graft-versus-leukemia effect, for high-risk patients as a prophylactic measure to prevent relapse and in relapsed patients along/without salvage chemotherapy. Methods: We performed a retrospective study to analyse pediatric patients with allo-HSCT and DLI procedures between February 2016 and November 2021, in Fundeni Clinical Institute. Patients’ diagnosis was: acute myeloid leukemia (AML)–6/16, chronic juvenile myelomonocytic leukemia (JMML)-3/16, acute lymphoblastic leukemia (ALL)-5/16, non-malignant disease-2/16. Conditioning regimens used for HSCT were accordingly to the disease. All patients were monitored with chimerism evaluations at 1, 3, 6, 9, 12 months and before every DLI. Chimerism analysis was performed using short tandem repeats (STR) technique. MDC was considered if less than 100%. Patients were divided into 3 groups: preemptive (MDC), prophylactic (high-risk AML/ALL) and treatment for relapse. DLI was administered using escalating doses. Results: We identified 16 patients, 13M:3F, who received 55 DLI. Median age at HSCT was 8.4 years, 3/16 patients with matched sibling donor, 2/16 with haploidentical donor(father), 8/16 with matched unrelated donor(MUD) and 4*/16 with mismatched MUD (1* previous haplo-HSCT). 6/16 received preemptive DLI (1 patient with 2 HSCT, DLI in both cases), 2/16 patients with prophylactic DLI and 8/16 patients received DLI at relapse. Median doses: 1x10^5/kg, 5x10^5/kg, 5 x10^5/kg, 1x10^6/kg and >1 x10^7/kg subsequent doses. Median donor chimerism at DLI was 48%, median days from transplant to first DLI was 86 days, median DLI administration was 2, for patients with preemptive DLI. 2/6 patients received Azacytidine, 1/6 patient developed graft-versus host disease (GvHD) post-DLI; 3/6 patients died due to disease progression, 1/6 died due to severe infections following graft loss, 2/6 are alive, one with 82% chimerism, 100% respectively after haplo-HSCT from father. In the prophylactic group 2/2 patients received Azacytidine. One patient with 100% donor chimerism, the other 97% before DLI. Median time from transplant to DLI was 324 days, with a median of 3 DLI. No GvHD, both are alive, with 100% donor chimerism in first case, 98% respectively. In the treatment group, DLI was combined with Azacytidine in 3/8 cases, chemotherapy followed by DLI in 4/8 cases, 1/8 without any salvage treatment. Median time to DLI was 416.5 days, with a median of 3 DLI. GvHD occurred in 3/8 patients. 6/8 patients died due to disease progression, 2/8 patients are alive, with 100% donor chimerism. Conclusions: Overall survival (OS) was 37.5%. Patients with preemptive DLI had 33,33% OS, patients in treatment group had 25% OS, compared to the prophylactic group (OS 100%). GvHD appeared in 25% of patients. In our study, prophylactic DLI showed better results when compared to both preemptive and treatment DLI, though the number of patients is low. Prophylactic DLI should be considered in high-risk patients to prevent relapse after HSCT. Close monitoring is needed for these patients. Disclosure: No disclosures.
Background: The conditioning regimens in pediatric haploidentical transplants not uniform across the world, the results of various conditioning regimens were variable with significant number of toxicities and economic burden in the low and middle income countries, here we are presenting our two case scenarios with different diagnosis using similar conditioning regimen, achieved complete donor chimerism and without any viral reactivation which is a major concern and has financial implication. Methods: Here we are presenting two case scenarios using same conditioning protocol – Hyper IgE syndrome with homozygous DOCK 8 mutation and Congenital Amegakaryocytic thrombocytopenia[CAMT] with cMPL mutation. We used modified Johns hospkins protocol – using Fluderabine [150mg/m2], Cyclophosphamide [14.5mg/kg for 2days], Busulfan [3.2mg/kg/day for 3days] with TBI[2Gy],with Post transplant cyclophosphamide[PT/Cy] on day +3 and +4 along with sublingual Tacrolimus and Mycophenolate mofetil as GVHD prophylaxis. We also used N acetyl cysteine infusion during PT/Cy for reducing the risk of mucositis. Two children followed for minimum 100days [range 100days-270days]. Results: Two children attained engraftment neutrophil on Day+16 in hyper IgE syndrome and Day +12 in CAMT, Platelet engraftment on Day+19 in Hyper IgE syndrome and Day+13 in CAMT, Maximum mucositis observed was Grade 2,No microbiologically proven infections, we did not observed Cytokine release storm, two children attained complete donor chimerism on Day+30 of transplant. No viral reactivations were observed, monitored for Cytomegalovirus and Urine for BK virus weekly. Hyper IgE syndrome child IgE level came to normal by Day+180 [2600IU/ml to 340IU/ml].No GVHD was observed in the two children. Conclusions: In Low and middle income countries viral reactivation is a major challenging in haploidentical stem cell transplantation and it has lot of financial burden on the families, minor modification in the conditioning regimen results in reduction of toxicities and overall outcome would improve, ours was small observational study need to be proved in larger multicentre study. Disclosure: nothing to declare Solid Tumours
Background: Neuroblastoma (NB) is a sympathetic nervous system malignancy, which predominantly affects children. It has a high relapse rate and causes 15% of cancer mortality in pediatrics. The high-risk NB remains one of the most challenging pediatric solid tumors. Autologous hematopoietic stem cell transplantation (Auto-HSCT) has a significant improvement in the treatment of these patients. Metaiodobenzylguanidine (MIBG) is used as a targeted therapeutic agent, which currently is conducted as part of clinical trials with an increasing number of centers participating with different doses of 131I-MIBG. This study reports the Auto-HSCT outcomes in NB pediatrics, which undergo 18 mCi/Kg 131I-MIBG therapies at Children’s Medical Center, the largest pediatric Iranian hospital. Methods: We report on twenty-three relapsed or refractory NB patients referred to Children’s Medical Center; for Auto-HSCT from February 2017 to October 2021. All transplants utilized PBSC sources. All patients received 131I-MIBG (18 mCi/Kg) at day -21, besides a conditioning regimen consisting of Carboplatin (1500 mg/m2) and Vp16 (1200 mg/m2) for five consecutive days from day -7. Furthermore, Melphalan (210 mg/m2) was administered for three consecutive days from day -7. Results: Eight females and fifteen males NB patients with the mean age at the Auto-HSCT time was six years (range, 2 - 8 years). The mean number of harvested MNC and CD34 + was 6 × 108 cells/kg (range, 2.3 - 14) and 3.4 × 106 cells/kg (range, 0.3 – 8.5), respectively. The median time to neutrophil and platelet engraftment was 12 and 8 days, respectively. With an average 15 months’ follow-up (range, 0.5 - 49), 60% overall survival (OS) and 43.4% disease-free survival (DFS) rates were achieved. The death cause was relapsed and transplant-related; infections and VOD; in seven and two patients, respectively. Conclusions: Different studies have suggested an efficient role of MIBG in NB patients as a pre-transplant conditioning regimen. These patients might rescue with high-dose conditioning regimens alongside the therapeutic MIBG. Finally, our study demonstrates the 18 mCi/Kg 131I-MIBG dose has a similar survival rate without more transplant-related complications, compared with the previous one using a lower 131I-MIBG dosage. Disclosure: Nothing to declare.
Background: NKG2C is an activating receptor expressed on adaptive NK (ANK) cells, encoded by the KLRC2 gene on chr 12p13.2. Early reconstitution of ANK cells has been shown to reduce relapse following both cord blood and haploidentical HCT. In this study, we analysed the impact of donor KLRC2 genotype on transplant outcomes following PTCy-based haplo-HCT. Methods: 100 patients with malignant (n = 72) and non-malignant(n = 28) diseases were included in the study. The donors were categorised based on KLRC2 sequence as KLRC2 wildtype (wt/wt)[D-KLRC2wt group] and KLRC2 deletion homozygous (del/del) or heterozygous (del/wt) [D-KLRC2del group]. All donors and recipients were CMV seropositive. Gene expression profiling by RNA-seq was carried out on PBMC samples from both donor groups. Results: Twenty-eight out of 100 donors had KLRC2 deletion (del/wt-26; del/del-2). Even though both groups were similar in pre-transplant, graft characteristics and engraftment kinetics, D-KLRC2del group had increased CMV reactivation (85.7% Vs 49%), CMV viral load (25.8 vs 5.7 x 103/ml) as well as persistence (41 days Vs 25 days), [p < 0.001]. Acute GVHD gr 2-4 was witnessed only in the D-KLRC2del group (27% vs 0, p = 0.0001), with a trend towards higher chronic GVHD as well (25.4% vs 10.3%, p = 0.09). Overall non-relapse mortality was 9.6%, but this was 25% in the D-KLRC2del group vs 3.4% in D-KLRC2wt group (p = 0.0001), with a marked impact on overall survival (63.4% vs 92.8%, p = 0.0001). There was no impact of KLRC2 genotype of the patient on outcomes. Even though the median donor-ANK (D-ANK) level was lower in D-KLRC2del group (15.5 vs 23%, p = 0.03), the ANK levels varied widely (0-67%). D-ANK cells and not D-KLRC2del had the strongest impact on relapse rate (24.4 vs 8.6%), [HR-0.8(95%CI-0.7-0.9) p = 0.0001]. Based on recursive partitioning to determine the optimal cut point for absolute counts of ANK cells, relapse was 2.2% in those with D-ANK > 14.5% vs 72.6% in those below 14.5% (p = 0.0001). To understand this dichotomy in the impact of D-KLRC2del genotype on GVHD and NRM, but not relapse, we studied the differential gene expression (with a cut-off for p value of 0.05) based on transcriptome analysis between D-KLRC2wt with D-KLRC2del groups. KLRC1 (NKG2A), SYK, FCER1G, EAT2 and NKp30 were upregulated in the D-KLRC2del group with downregulation of BCL11B, KLRC2, KLRC3 & KLRC4, indicating downregulated ANK and ADCC pathways. However, there was significant upregulation of alloreactive and inflammatory pathways in D-KLRC2del group (NFKB, NLRP3, TLR, TNF-α, IL12, IL17, IL18, IL33, CD28, CD86 and T-BET, JAK2, MAPK), along with downregulation of the regulatory pathways (TGF-β, FOXP3, STAT5, IL-10, IL-12RA, CD73). Conclusions: Our findings suggest a non-redundant role for adequacy of ANK cells in maintaining homeostasis between pro-and anti-inflammatory alloreactive pathways, along with an anti-leukemia potential independent of T cell derived alloreactivity. While KLRC2 del donor strongly correlated with acute GVHD and NRM, but not relapse, KLRC2wt donor with high D-ANK was associated with low incidences of GVHD, NRM as well as relapse, suggesting that incorporation of KLRC2 genotype and ANK cell repertoire in the algorithm for selection of haploidentical donors, might improve transplant outcomes. Clinical Trial Registry: NA Disclosure: Nothing to declare
Background: HLA-haploidentical allogeneic hematopoietic stem cell transplantation (Haplo-HCT) is frequently used as treatment for patients with active acute myeloid leukemia (AML). Here, we investigated whether 9/10 HLA-mismatched unrelated donor transplantation (MMUD-HCT) with post-transplant cyclophosphamide (PTCy) is an adequate alternative. Methods: This is a retrospective study from the acute leukemia working party of the EBMT. Inclusion criteria consisted of adult patients, first HCT with a Haplo donor or MMUD between 2010 and 2020 using PTCy as graft-versus-host disease (GVHD) prophylaxis, and primary refractory or relapsed disease. MMUD patients were pair-matched 1 to 2 with Haplo-recipients. Matching criteria included status at transplantation, conditioning intensity, Karnofsky performance score, and age at transplantation. Results: A total of 73 MMUD patients met the inclusion criteria. Their data were compared to those of 146 Haplo patients (out of 762 patients meeting the inclusion criteria) in a matched-pair analysis. Median follow-up was 27 months in MMUD patients and 34 months in Haplo recipients. Two-year incidences of relapse and non-relapse mortality (NRM) were 40% and 18% in MMUD patients, respectively, versus 50% (P = 0.23) and 24% (P = 0.3) in Haplo recipients. Two-year leukemia-free survival (LFS) and overall survival (OS) was 42% and 46% in MMUD recipients, respectively, versus 26% (P = 0.1) and 28% (P = 0.061) in Haplo-patients. Conclusions: In AML patients with active disease at transplantation, MMUD-HCT results in at least comparable outcomes to Haplo-HCT when PTCy is applied. These observations could serve as a basis for a phase III trial comparing these two donor types in patients with active AML at transplantation who lack an HLA-matched related or unrelated donor. Disclosure: No COI to disclose
Background: The use of Post-Transplant Cyclophosphamide (PTCy) as Graft Versus Host Disease (GVHD) prophylaxis has resulted in reductions in GVHD and improved outcomes in allogeneic hematopoietic cell transplant (HCT) using mismatched related donors. Methods: We performed a multi-center phase II prospective clinical trial using PTCy in mismatched unrelated donors (MMUD). The study met its primary endpoint of >65% overall survival (OS) at 1 year, with an OS of 76% [Shaw et al, JCO, 2021]. Here we report the 3-year outcomes in the study cohort. Results: 80 patients (40 each receiving myeloablative (MAC) or reduced intensity conditioning (RIC)) were enrolled and transplanted between December 2016-March 2019. The median patient age was 52 (18-70), and 48% were from racial/ethnic minority groups. 95% of patients in the MAC cohort were transplanted for acute leukemia/MDS, compared to 53% in the RIC cohort (the remainder having lymphoma, CLL or non-hodgkins lymphoma). 34% of patients had a KPS < 90 and the HCT-CI was >2 in 54%. HLA match was 4-6/8 in 39% of transplants (43% in RIC, 39% in MAC) and 7/8 in 61%. Median follow up (set date: September 2021) is now 34 months (range 12-46) in RIC and 36 months (range 18-49) in MAC. Three-year outcomes are shown in Table 1. OS in the RIC cohort was particularly good at 70%. Non- relapse mortality was 15%. Rates of chronic GVHD (cGVHD) were low at 20% for all grades and 5% for severe cGVHD, with a relapse rate of 29%. GVHD-/relapse-free survival (GRFS) was 44%. Although 3-year survival in the MAC cohort remained acceptable at 62%, rates of relapse were high at 51%. Non-relapse mortality was 10%. cGVHD occurred in 38% of patients, with 13% reporting severe disease. GRFS in this cohort was correspondingly low at 17%. OS in the 7/8 cohort was 63% and 71% in the 4-6/8 cohort. Conclusions: Outcomes for patients receiving a MMUD HCT using PTCy-based GVHD prophylaxis remain very good with follow up at 3 years post-transplant. Patients receiving RIC in particular have excellent survival and very low rates of cGVHD. Use of more mismatched donors (<7/8) was not associated with worse outcomes, providing early reassurance that access to transplant can be safely expanded to patients with no 7-8/8 matched donors. While cGVHD and OS remained acceptable in the MAC cohort, the relapse rate was disappointingly high. The predominance of acute leukemia patients in the MAC cohort may explain some of the difference in relapse rate between cohorts, warranting further study and analysis. Clinical Trial Registry: ClinicalTrials.gov NCT02793544 Disclosure: Bronwen E. Shaw - OrcaBio, Mallindrodt. Antonio Martin Jimenez-Jimenez - Research funding from AbbVie. Stephen R. Spellman -NMDP/Be The Match Joseph Pidala - Compensation: Consulting and advisory board membership – Syndax, CTI Biopharma, Amgen Miguel-Angel Perales - Compensation: •Member, Scientific Advisory Board: NexImmune; Ad hoc Advisory Board: Abbvie, Astellas, Celgene, Bristol-Myers Squibb, Incyte, Karyopharm, Kite/Gilead, Miltenyi Biotec, MorphoSys, Nektar Therapeutics, Novartis, Takeda; •Consulting: Merck, Omeros; •Member, DSMB: Cidara Therapeutics, Medigene, Servier: •Research Funding: Incyte (clinical trial), Kite/Gilead (clinical trial), Miltenyi (clinical trial), Novartis (clinical trial) Payments: > $5,000 for the following; Ad hoc Advisory Board: Bristol-Myers Squibb, Incyte, Kite/Gilead, Novartis; •Consulting: Merck, Omeros; •Member, DSMB: Cidara Therapeutics; •Research Funding: Incyte (clinical trial), Kite/Gilead (clinical trial), Miltenyi (clinical trial), Novartis (clinical trial); Relationships: Member, Board of Directors NMDP Steve Devine - Full time employee of NMDP/Be The Match, Research support from Magenta, Vor Bio, Orca Bio, Advisor to Janssen, BMS, Sanofi, Orca, Vor Bio
Background: In response to COVID-19 pandemic WMDA, EBMT, BSBMTCT and National Institute of Clinical Excellence (NICE) published recommendations to test donors for SARS-CoV-2 1-4. The exact timing and frequency of testing remains in debate and practice varies between donor registries. This study aims to illustrate COVID-19 testing practice in donors from the Anthony Nolan (AN) register, determine the incidence of COVID-19 within actively donating AN donors, and analyse the delay in donor harvesting and HSC graft infusion. Methods: AN donors between the 1st October 2020 to 30th September 2021 who had a positive SARS-CoV-2 PCR test at any stage of their donation pathway were included in this retrospective analysis. PBSC, BM and DLI donations were included. Donor age, type of donation, estimated and actual collection, and graft infusion dates were recorded. Donors were routinely tested for SARS-CoV-2 at medical, and either pre-starting GCSF/pre-admission for BM harvest or before the recipient started conditioning if the donation was to be infused fresh. Donors were also tested for SARS-CoV-2 if they had symptoms or had a contact with a person with COVID-19 infection. AN initially recommended a 3-month deferral when a donor tested SARS-CoV-2 positive, which was reduced to 28 days. Results: During the study period, 30 donors tested positive for SARS-CoV-2. Six were at verification typing (VT), 16 at pre-medical, 1 at medical, 5 post-medical and 2 on the day of donation (Figure 1A). The median age of the SARS-CoV-2 positive donors were 22 years (range, 19-52). 83% (n = 25) of the SARS-CoV-2 positive donors had been requested for PBSC collections; 3 being subsequent requests. Three requests were for DLI donations and two for BM harvests. Transplant centres (TC) sought alternative donors in 12 cases and 3 donations were cancelled due to the recipient deterioration. 15 donors who tested positive for SARS-CoV-2 successfully donated after a deferral period (Figure 1B). In three cases, the collected cells were not infused: on two occasions recipients died and in one case the cells were discarded because the donor had COVID-19 on the day of donation. The median delay from the initial proposed cell harvest date was 10.5 days (range, 0-68) when alternative donor was requested and 40 days (range, 0-155) when the same donor donated after the deferral period. The median delay in graft infusion was 15 days (range, 0-75) with alternative donors versus 35.5 days (range, 0-169) days when proceeding with the same donor (Figure 1C). Figure 1. (A) Number or donors who tested COVID19 positive at different stages of donation process. VT – verification typing, DOD – day of donation. (B) Outcomes of the deferred donations due to positive SARS-CoV-2 donor test. TC – transplant centre, WU – work-up (C) Delay of the HSC graft collection and infusion in days when alternative and the same donors donated. Conclusions: The incidence of COVID-19 positive donors during active donation stages was 4.7%. Although overall numbers were small, when TC sought alternative donors there was a shorter delay to both HSC collection and infusion. This study highlights the need for robust back up donor. Clinical Trial Registry: Not applicable Disclosure: None of the authors have any conflict of interest to declare.
Background: For patients without matched donors, the use of haploidentical donors with post-transplant cyclophosphamide (PTCy) is often considered. Most studies evaluating this combination have used bone marrow (BM) grafts with reduced intensity conditioning (RIC). The safety and relative efficacy of haploidentical donor transplants performed using peripheral blood stem cells (PBSC) and myeloablative conditioning (MAC) regimens remains less clear. Methods: We conducted a prospective cohort study at three transplant centres in Canada using the Cell Therapy Transplant Canada registry as a data collection tool. Patients without a matched donor undergoing transplant for a hematologic malignancy were eligible. A CD34 dose of 3-8x106/kg was infused following a MAC regimen of fludarabine (200 mg/m2) and busulfan (12.8 mg/kg) (Flu/Bu). At the discretion of the investigator, low dose total body irradiation could be added (200 or 400 cGy). GVHD prophylaxis consisted of PTCy (50 mg/kg on days +3 and +4) in combination with MMF (days 5-35) and tacrolimus (days 5-100). To determine how outcomes compared to matched sibling donors (MSD) and matched unrelated donors (MUD), a comparison was done with controls from the CTTC registry. To provide a more homogenous cohort, this analysis was restricted to patients undergoing transplant for acute myeloid leukemia (AML). Results: Thirty-four patients were accrued in the prospective cohort, with diagnoses of acute myeloid leukemia (n = 24), acute lymphoblastic leukemia (n = 7), or myelodysplastic syndrome (n = 3). The median age was 55 years (range 10-70). Of these, 52% were men, with a median KPS of 90 (range 80-100). The median HCT-CI was 0, with 4 patients having a HCT-CI of greater than 3. The protocol was well tolerated, with a cumulative incidence of non-relapse mortality of 19% at 2-years and 1 case of veno-occlusive disease (which resolved with therapy). The cumulative incidence of relapse at 2-years was 22%. The incidence of grade III/IV acute GVHD was 6%, and chronic extensive GVHD was 29%. Overall survival at 2-years was 63%. Based on the success of this protocol, it was adapted as the standard for patients undergoing haploidentical transplant at these centres, and an additional 20 patients with AML were transplanted. These 44 patients with AML (24 on the original study, 20 in the expansion cohort) were compared to 128 MSD transplants and 267 MUD transplants done with similar transplant characteristics (AML, MAC Flu/Bu, and PBSC), with data obtained from the CTTC registry. In multivariate analysis, adjusting for age, HCT-CI, KPS, and disease risk (by ELN), no difference in overall survival (OS), disease free survival (DFS), relapse rate, or non-relapse mortality was found between donor sources (Figure 1). Older age was a predictor of inferior DFS and NRM, HCT-CI was associated with NRM, and high-risk disease was associated with a higher relapse rate. Conclusions: The use of PBSCs from haploidentical donors with MAC appears to be safe and effective, with outcomes comparable to MSD and MUDs, and low rates of acute and chronic GVHD. The feasibility of this combination provides further evidence supporting the use of haploidentical donors outside the previously studied RIC/BM setting. Clinical Trial Registry: NCT02504047 Disclosure: Nothing to declare.
Background: Developments of allogeneic hematopoietic stem cell transplantation (allo-HSCT) procedures such as reduced-intensity conditioning and use of alternative donor sources have expanded eligibility. On the other hand, it has become more complicated to plan the optimal patient-specific strategy among various allo-HSCT procedures. We retrospectively investigated the prognostic effects of a personalized allo-HSCT procedure recommended by machine learning models. Methods: We analyzed patients who first underwent allo-HSCT between 2009 and 2018 in Japan and used random survival forest (RSF) and DeepSurv as machine learning models. First, we classified ten allo-HSCT procedures based on conditioning intensity, donor source, and post-transplant cyclophosphamide (PTCY) (Table). Second, we split the entire cohort into training and test cohorts chronologically according to the year of allo-HSCT. Third, we trained machine learning predictive models for overall survival (OS) after allo-HSCT. Age, disease, disease status, performance status, hematopoietic cell transplantation-specific comorbidity index (HCT-CI), cytomegalovirus serostatus, and ten allo-HSCT procedures were used as prognostic variables. Fourth, we calculated the predictive probabilities of 1-year OS for ten allo-HSCT procedures in each test patient. Fifth, upon imposing restrictions on donor selection for the recommendation of machine learning because some donors were unavailable depending on the patients in clinical practice, we defined the procedure with the highest predictive probability of 1-year OS as the allo-HSCT procedure recommended by machine learning. Sixth, we divided the test cohort into recommendation and non-recommendation groups according to whether the actual allo-HSCT procedure was concordant with that recommended by machine learning. Finally, we compared OS between the recommendation and non-recommendation groups. Table. Classification of allo-HSCT procedures. Results: In total, 17,449 patients were analyzed (training cohort, 13,595; test cohort, 3,854). In a log-rank test, the recommendation groups showed higher OS than the non-recommendation groups in both machine learning (P < 0.001 for RSF, P = 0.01 for DeepSurv, Figure). In multivariate analysis, the recommendation group using RSF was an independent favorable prognostic factor for OS, but not using DeepSurv (HR: 0.84, P = 0.01 for RSF; HR: 0.92,P = 0.42 for DeepSurv). Furthermore, in the subgroup transplanted from alternative donors, the recommendation group using RSF was independently associated with a significantly better prognosis than the non-recommendation group. Conclusions: These findings suggest that the patient-specific recommendation of the allo-HSCT procedure using RSF may improve prognosis. A further prospective randomized controlled trial is required to confirm the clinical value of the intervention based on machine learning models in the allo-HSCT field. Disclosure: Ayumi Shintani received honorarium from Shionogi. Naoyuki Uchida received honorarium from Chugai, Astellas, Otsuka, Sumitomo Dainippon, and Novartis.
Background: In the setting of mismatched-hematopoietic stem cells transplantation (mmHSCT), the detection of antibodies directed against donor-specific HLA allele(s) or antigen(s) (DSA) represents a barrier for stem cell engraftment. Thus, it is necessary to plan an immunosuppressive strategy, or to select an alternative donor. This prospective study aimed at evaluating the efficacy of our strategy for testing DSAs and the efficacy of the desensitization strategy (DS) employed in patients candidates for a mmHSCT between November 2017 and November 2020 at Sapienza University of Rome. Methods: Anti-HLA Abs research was performed with Luminex bead assays (Lifecode ID and LSA I/II-Immucor). Results were expressed as MFI (MFI > 1000 positive). If the patient had DSAs and no alternative donors, a DS was employed, with Rituximab (day -15), 2 single-volume plasmapheresis (PP; days -9 and -8), intravenous immunoglobulin (day -7) and infusion of HLA selected platelets for DSA absorption, if persistent DSAs were directed against class I HLA. DS was scheduled with or without PP, according to DSAs MFI (>1000 or <5000) and FCXM (flow cytometry cross-match). Results: Twenty-two out of 126 patients (17.46%) showed anti-HLA Abs, 5 of them DSAs (22%, 3.9% of total). One out of 5 patients died before receiving HSCT, due to disease progression; 3 patients received DS obtaining engraftment; 1 patient showed no DSAs and negative FCXM before starting the conditioning regimen, and DS was not necessary. Female sex (p = 0.033) and a history of previous pregnancies or abortions (p = 0.009) showed a statistically significant impact on alloimmunization. Factors associated with delayed PMN engraftment were patient’s female gender (p = 0.039), bone marrow as stem cell source (p = 0.025), and a high HSCT-specific comorbidity index (p = 0.028). None of the analyzed variables, including the DSA’s detection, influenced engraftment. Conclusions: Our analyses confirm the importance to test DSAs in mmHSCT. Our DS proved successful in removing DSAs. Prospective multicenter studies are needed to better define and validate consensus strategies on DSAs management in HSCT. Disclosure: Nothing to declare
Background: Viral infections are a common complication after HSCT and significantly contribute to non-relapse mortality (NRM). While it has been recognized that HLA mismatching (MM) between donor and recipient increases the risk for viral infections or reactivation after HSCT, the impact of HLA class I versus II MM and their respective vectors (HVG versus GVH) is largely unknown. Methods: 2w?>We retrospectively evaluated 140 patients undergoing HLA mismatched (MM) HSCT from haploidentical family (n = 127) or MM unrelated (n = 13) donors using post-transplant cyclophosphamide based immunosuppression, between August 2014 and May 2021 at a single institution. To assess the impact of recipient-donor HLA-disparity on viral reactivation, individual GVH- and HVG-directed MHC class I (A, B, C) and class II (DR, DQ, DP) mismatches were quantified and dichotomized as 0-1 MM versus 2-3 MM per class and vector, and were assessed for their impact on the incidence of BK-virus associated hemorrhagic cystitis (BKV-HC) and significant CMV reactivation, and on other major HSCT outcomes. Results: By multivariable analysis, presence of 2-3 (versus 0-1) HVG-directed class II MM was significantly associated with occurrence of BKV-HC (sub-hazard ratio (sHR) 3.75; 95%CI 1.06-13.21; p = 0.04). In contrast, no individual class/vector MM was predictive for CMV reactivation. Interestingly, despite its impact on BKV-HC, HVG class II MM was associated with reduced NRM (sHR 0.35; 95%CI 0.12-0.97; p = 0.04). Class I MM GVH was associated with lower risk for moderate/severe cGVHD (sHR 0.17; 95%CI 0.04-0.61; p = 0.01). In contrast, HVG class I MM (sHR 4.03; 95%CI 1.24-13.03; p = 0.02) and GVH class II MM (sHR 12.24; 95%CI 4.14-36.2; p < 0.001) were associated with increased moderate/severe cGVHD. Finally, GVH class II MM also associated with a lower risk for overall mortality (sHR 0.38; 95%CI 0.18-0.82; p = 0.01), while for GVH class I, an opposite trend for overall mortality was observed (sHR 2.42; 95%CI 0.93-6.31; p = 0.07). Conclusions: The greater importance of HLA MM for BKV as compared to CMV replication may be due to the recipient origin of BKV harbouring cells (urinary tract), while CMV infects lymphocytes, which are predominantly of donor origin after engraftment. The latter may result in a diminished role of an HLA barrier for antiviral immunity in case of CMV. Furthermore, the differential effects of HLA-MM in class I vs II on various HSCT outcomes illustrate the complexity of cellular immunity after PTCy based HSCT across HLA barriers. Disclosure: Nothing to declare.
Background: Hematopoietic stem cell transplantation from HLA-mismatched donors leads to an increased risk of acute and chronic graft-versus-host disease (GvHD). There is no established algorithm for selection of mismatched unrelated donors and we can use a model in silico that predicts the numbers of peptides derived from mismatched HLA alleles that can be presented by shared HLA (PIRCHE- Predicted Indirectly ReCognizable HLA Epitopes). Direct recognition of HLA-DPB1 mismatches can be predicted using the TCE (T-cell epitope) model but we can also predict indirect recognition of HLA-DPB1 mismatches using the model of PIRCHE. Considering the important role of indirect T-cell alloreactivity after transplantation, PIRCHE model can be one of the methods to identify the permissibility of HLA mismatches. The aim of this study is to explore whether the PIRCHE algorithm including match in DPB1 locus can be used to identify permissive and non-permissive HLA-mismatches in 9/10 HLA matched stem cell transplantations. Methods: We performed a retrospective study of 18 adult patients transplanted with single HLA-mismatched unrelated donors (9/10 mMUD) in our Department. Unambiguous 4d high resolution HLA-A, B, C, DRB1, DQB1 and DPB1 typing data were available for all donors and recipients. High-resolution HLA typing was performed by reverse sequence-specific oligo, or sequence-specific primer methods (One Lambda, USA). We retrospectively scored PIRCHE numbers (version v3.3.27, available at https://pirche.com), including DPB1 match/mismatch not available at time of transplantation. Results: PIRCHE Score cut-off points were generated by receiver operating characteristic (ROC) curves with regard to OS. The one with the highest area under curve (AUC 0,958; SE 0,0589; 95%Cl 0,652 - 1,000, p = 0.5346) was used to define cut-off between low and high indicators. The cutoffs that yielded the highest area under the curve were used to define low PIRCHE-I/-II/-score and high PIRCHE-I/-II/-score [PIRCHE-I: 0–6 (low) vs. >6 (high); PIRCHE-II: 0–10 (low) vs. >10 (high); PIRCHE score: 0-18 (low) vs. >18 (high)].The detection of low score of PIRCHE was associated with a lower cumulative incidence of chronic GvHD (OR 0,0338 95% CI: 0,0014 – 0,7949; p = 0.035) but not with acute GVHD. Additionally in the group with 9/10 match we compared of survival according to number of epipoes [class I: 0-5 (low) vs. >5 (high) ; II: 0-2 (low) vs. >2 (high) and score: 0-10 (low) vs. >10 (high)], derived from mismatched allogeneic HLA peptides that are subsequently presented by HLA molecules shared between the donor and recipient. In this case we did not add any predictive value [(HR (95% CI): 1,6795 (0,3293 - 8,5646) P = 0,5328]. Conclusions: These preliminary data suggest that low PIRCHE scores including DPB1 locus may be used to identify permissible HLA mismatches within single HLA-mismatched hematopoietic stem-cell transplantations. Clinical Trial Registry: These preliminary data suggest that low PIRCHE scores including DPB1 locus may be used to identify permissible HLA mismatches within single HLA-mismatched hematopoietic stem-cell transplantations. Disclosure: Nothing to declare
Background: In retrospective studies, the association of donor age (DA) and hematopoietic cell transplantation (HCT) outcomes is equivocal. We sought to determine this association in adult acute myeloid leukemia (AML) patients. Methods: Data from a single-centre cohort of adult AML patients transplanted between 01/07/2015 and 30/06/2020 was analysed retrospectively. Baseline characteristics and outcomes were extracted, and Fine-Gray regression was used to determine the association of DA and cumulative incidence of non-relapse mortality (NRM), and graft-versus-host disease. DA groups using decades were studied in Fine-Gray multivariable models. Patient age-adjusted HCT-Comorbidity Index (aaHCT-CI), patient Karnofsky performance score (KPS), donor type (related matched, haploidentical, matched unrelated), conditioning regimen intensity and acute graft-versus-host disease (aGVHD, time-dependent) were used as covariates to adjust for confounding, and model selection techniques were applied. Results: In this cohort of 89 transplanted AML patients, median follow-up was 2.7 years (interquartile range: 0.9-3.2) and cumulative incidence of NRM was 16% at 5 years (95% confidence interval: 6-30). DA was independently associated with NRM incidence as a continuous variable (HR = 1.08, p = 0.0008). Decade-based cut-offs yielded ≥ 50 years as a statistically significant and independent predictor of NRM (HR 8.9, p = 0.004). Other independently associated variables were grade 3-4 aGVHD and KPS < 90 (HR 14.8, p = 0.01 and HR = 3.8, p = 0.07, respectively). No association was found between NRM incidence and aaHCT-CI, donor type and conditioning regimen intensity. Univariate graphical association between DA > 50 years and NRM incidence is shown in Figure 1. No association was found between the above-mentioned variables and incidences of acute and chronic GVHD. Conclusions: In adults with AML, higher donor age with a cut-off of ≥ 50 years is associated with a higher risk of NRM across donor types, conditioning regimen intensities and patient age, comorbidities, and performance status. The mechanisms behind the association of DA and NRM remain to be elucidated. Disclosure: Nothing to declare.
Background: Hematopoietic stem cell transplantation (HSCT) is the only way to cure hematological malignancies, but finding HLA matched hematopoietic stem cell donors has always been a difficult problem. With the development of the research on haploid hematopoietic stem cell transplantation, this problem has been effectively solved and has become the main way of HSCT. However, some donors are found carrying Clonal hematopoiesis of indeterminate (CHIP), and the patients have no other donors to choose from, so they can only transplant donor hematopoietic stem cells who are carrying chip. Methods: The healthy donors tested in the Institute of Hematology & Blood Diseases Hospital, Chinese Academy of Medical Sciences from August 2015 to August 2020 were screened for blood system tumor gene mutation, the situation of the donors carrying the mutant gene was analyzed, the graft implantation time, survival time and the incidence of acute graft-versus-host disease between chip recipients and non chip recipients were compared, and the data were analyzed by SPSS 22.0. Results: 1. From August 2015 to August 2020 in our hospital, 351 cases of blood tumor genetic testing were performed among all donors, including 223 males (63.53%) and 128 females (36.47%). 147 people (41.88%) were positive for gene mutations, and 204 (58.12%) were negative for gene mutations. Among the patients with gene mutation positive, 93 were males, which constituted 41.70%; 54 were females, which constituted 42.18%. 2. 10 people with FAT1 mutation (11.11%), 8 people with SETBP1 mutation (8.89%), 4 people with TET2 mutation (4.44%), 10 people with NOTCH1 mutation (11.11%), 5 people with DNMT3A mutation (5.56%), 8 people with RELN mutation People (8.89%), 5 people (5.56%) with JAK2 mutation, and 6 people (6.67%) with KMT2D mutation. 3. The distribution of gene mutations of donors in different age ranges is as follows: 66.67% for ≤10 years old; 25.86% for 11-20 years old; 21.59% for 21-30 years old; 24.62% for 31-40 years old; 27.08% for 41-50 years old; and over 51 years old 26.31%. 4. Survival of patients after hematopoietic stem cells with genetic mutations and without genetic mutations, the cumulative survival rates of the two groups were 79.70% vs 80.10% and 77.40% vs 70.90%, p = 0.609; the two groups The median survival time was 15.19 ± 11.93 months and 13.70 ± 10.98 months, p = 0.387; the median time of granulocyte implantation in the two groups was 13.21 ± 2.7 days vs 13.19 ± 2.65 days, p = 0.964; platelet implantation time, respectively It was 23.18 ± 25.32 days vs 19.64 ± 16.72 days, p = 0.275; input of hematopoietic stem cells carrying gene mutations had no significant effect on the patient’s aGVHD, p = 0.293. 5. Survival analysis was performed on the gene mutations carried by the donor, and the results were It shows that JAK2 and KMT2D have poor prognosis for patients (P values are 0.026 and 0.048, respectively); TET2 gene is associated with disease recurrence, p = 0.028. Conclusions: About 42% of healthy people in China; CHIP exists in all age groups, and donors carrying JAK2/KMT2D/TET2 mutations are associated with poor prognosis. Disclosure: NO conflict of interest
Background: Since SARS-CoV-2 outbreak, WMDA, EBMT and IBMDR (Italian Donor Registry) have invited centers involved in transplantions to try hard to ensure "treatment continuity" of patients at risk of disease relapse or progression. Every donation center had to create a protected process ensuring: a) fast and safe selection of donors; b) continuity of donation procedures; c) safe release of the collected products. Methods: The cell collection facility of the Transfusion Center of the Policlinico Sant’Orsola- Malpighi IRCCS, Bologna, Italy, has been JACIE, CNT/CNS and WMDA accredited to assess the eligibility to cell donation of related and unrelated donors, and to collect cell products for transplantation or immunotherapies. In the SARS-CoV-2 "era", our facility has adopted severe safety measures to implement the international, national and regional guidelines. In particular, donors are visited in protected spaces, blood and instrumental exams are performed concurrently, to expedite eligibility assessment and limit donor discomfort. Each donor is subjected to molecular SARS-CoV-2 swabs at least at the first visit, 48 hours before the G-CSF mobilization and on the day of cell collection: negative results are a prerequisite for continuing the donation program. To facilitate this pre-donation path, special agreements with the external facilities involved in this program have been reached. From March 2020 to December 2021, 65 CSE and 11 DLI donors have been referred to our facility. CSE donors are mobilised with Filgrastim, 10ug/BWKg for five days and subjected to collection if the CD34 + circulating cells are found above the 20/ul threshold by our Immunology Laboratory. The collected products are delivered to the Seragnoli Institute Processing Laboratory, the Tissue Establishment of the Bologna Transplantation Program, for internal use or external shipment. Results: Main donor demographics, donation type and mobilization data are shown in Table 1. Conclusions: The SARS-CoV-2 pandemia has not only significantly challenged the activity related to collection procedures, by requiring the adoption of a series of stringent safety measures, but also increased their need. In spite of the emergency situation, our center succeeded in positively responding to the approximate 10% increment of demand from national and international transplantation units. In particular, we would like to stress that: a) all requested products were collected, provided that the donor was found eligible; b) no SARS-CoV-2 product contaminations or donor infections have been reported. This positive rtesults was due to a series of factors: the collaboration of both related and unrelated donors, the flexibility of all the actors involved in the process, and the validity of the guidelines issued by WMDA, EBMT, IBMDR and by our Regione Emilia Romagna. Disclosure: Nothing to declare
Background: Haploidentical Hematopoietic Stem Cell Transplantation(HSCT) with Post-transplant cyclophosphamide(PTCy) is a curative option for several conditions in children. PTCy is used for in-vivo T-cell depletion in the graft. Higher CD3-lymphocytes in peripheral blood stem cell(PBSC) graft may cause more chronic graft versus host disease(GvHD). We aimed to study the impact of graft CD3-lymphocyte cell dose on clinical outcomes in this group of patients at our center. Methods: Medical records of all children who underwent unmanipulated haploidentical PBSC transplant with PTCy from May2016 to August2021 were retrospectively analyzed. Donor received subcutaneous G-CSF@10ug/kg/day from day -4 to -1. PBSC were harvested on day0 and transfused to the patient. CD34/CD3 counts were enumerated in the final product by Flowcytometry. All patients received PTCy@50mg/kg/day on day +3,+4 as GvHD prophylaxis. Patients were divided into 2groups based on median CD3count and were analyzed for duration for engraftment, GvHD, Cytomegalovirus(CMV) reactivation, rejection, survival. Also, data was analyzed for significant difference in median CD3count among those who did or didn’t develop GvHD, CMV-reactivation, rejection, malignancy relapse, treatment related mortality(TRM). Statistical analysis was conducted with IBM-SPSS version 21.0.p < 0.05 was taken to be significant. Results: Kaplan-Meire OS Analysis of the 2 groups. Status of post-transplant events/parameters amongst the groups Our cohort comprised of 50children(males-35,females-15), aged 5months-18years(median 7.5years). Transplant indications were: Hematological malignancies-21, solid tumors-2, Hemoglobinopathies-13, Bone marrow failure-10, primary immunodeficiency-3, others-1. Median CD3cell dose- 488million/Kg(range:117.8- 2588.2), CD34cell dose-8.37million/kg(range:3.5-29.8). The cohort was divided into 2groups based on CD3count. There was a statistically significant positive correlation between graft CD3 and CD34count (p = 0.001). On analysis, there was no statistically significant difference in the time to neutrophil/platelet engraftment, occurrence of acute/chronic GvHD, CMV-reactivation, graft rejection, overall-survival(OS), event-free survival(EFS) among the 2groups(Table1). Though not statistically significant, the OS was better in the group which had higher than the median CD3count(Figure1). When we divided the cohort based on presence or absence of GvHD, CMV-reactivation, rejection and malignancy relapse; the median CD3count difference was statistically significant for CMV-reactivation (p = 0.044), but not for the rest. Those who didn’t have CMV-reactivation had a higher median CD3count. At a median follow-up of 13-months(range:14days- 68months), 31/50 patients were alive. Causes of death were relapse of cancer-6 and treatment related mortality-13. Conclusions: Our data from a small cohort of children undergoing haploidentical PBSCT with PTCy showed that CD3-lymphocyte dose in the graft did not have significant impact on clinical outcomes except that those who didn’t have CMV-reactivation had received higher median CD3count. A bigger cohort is needed to confirm our findings. Disclosure: Nothing to declare
Background: The Ezer Mizion Bone Marrow Donor Registry, consists of more than 1 million volunteer adult donors, serving Israeli and international patients who need an allogeneic HSCT. Peripheral blood stem cells donation is performed after administration of Filgrastim (Granulocyte Colony-Stimulating Growth Factor) with well-described adverse events. The primary aim of this study was to explore whether there is any correlation between the occurrence and severity of donors’ adverse events during the days of injections and their physical and emotional status at the point of short and long-term follow-up. Moreover, we investigated if there is a correlation between injections’ adverse events and CD34 + and WBC counts before collection. Methods: The dataset included 880 donors with self-reported questionnaires completed on the donation day, up to 30 days after donation (short-term follow-up), and 1 year after donation (long-term follow-up). WBC and CD34 + cell counts were also analyzed. Results: The short term follow-up results have shown that young age was positively related to better emotional status (r = −.10, p < .05); Worse self-reported physical status correlated with high incidence of symptoms (r = −.11, p < .01), while high severity of symptoms correlated with poor emotional status (r = −.09, p < .05). These correlations were still significant at long-term follow up (−.14 < r < −.10, p < .01). Note: Physical and emotional status was measured by a three-point score and are presented in the table: 1- Worse than normal, 2- Normal, 3- Better than normal Conclusions: Filgrastim adverse events were shown to influence donors’ physical and emotional status both at the short and long term follow-up, affecting donor recovery and manifesting the long term influence of donors’ experience during injections days. Many symptoms during injections were related to worse physical status, while more severe symptoms were related to worse emotional status both at the short and the long-term follow-up. However, WBC count and CD34 + cells did not correlate significantly with symptoms. It is reassuring that on long-term follow up donors exhibit full recovery. Based on these data we have suggested possible interventions to improve overall donors’ physical and emotional experience such as better management of pain and closer attention during questionnaires compilation. Disclosure: The authors have no conflicts of interest to declare.
Background: Mexico’s hematopoietic stem cell transplantation (HSCT) activity remains well below other middle-income countries in Latin-America such as Brazil and Argentina. The effects of the COVID19 pandemic on transplant center (TC) activity in our country are unknown. Therefore, we developed a nationwide survey on behalf of the Mexican Transplant and Cell Therapy Working Group (TCTMX) to account for its effects and perform a situational analysis. Methods: We performed a comprehensive cross-sectional study including active TCs interrogating HSCT activity from 2019 through September 2021. An electronic survey was sent to TCs during October-December 2021 and consisted of items regarding the number and characteristics of procedures performed and were compared yearly. Changes to their institutions’ transplant policies and practices during the COVID19 pandemic were also documented. At the time of writing, of 50 centers invited to participate, 31 had responded (62%) including all high-volume TCs, 28 with complete data included in this analysis. Results: Most TCs belong to the public health system (64%) and have a mixed pediatric and adult population (46.4%) and almost half concentrated in Mexico City (42.9%). Five centers performed exclusively autologous transplants (17.8%). The number of HSCTs decreased from 767 in 2019 to 460 in 2020 (p < 0.001), representing a 40% reduction in transplant activity. In 2021, 462 HSCTs have been performed reaching a monthly transplant rate of 51.3 compared to 38 in 2020, and close to the 63.9 reference in 2019 (p = 0.002). All types of HSCTs were diminished with the notable exception of those from haploidentical donors which have surpassed matched sibling donors. On the other hand, only 15 unrelated donor and 2 umbilical cord blood grafts were performed during the 3-year period studied. Most institutions have treated patients with COVID19 (78.6%) and experienced some form of reconversion (67.9%), which was higher in public centers (64.3% vs, 40%: p = 0.03). HSCT activity stopped completely in 19 TCs (68%) with a median duration of closure of 10 months (range 1-19), higher in public vs. private centers (median of 13 vs. 6 months; p = 0.09); 10% of TCs remained closed, all in the public setting. The most frequent motives behind TC closures were hospital reconversion in 11 (57.9%), precautionary measure in 4 (21.1%) and lack of resources in 4 (21%). Follow-up outpatient visits were totally or partially suspended in 10.7% and 39.3% of TCs, respectively, and were modified in 71.4% most frequently by telemedicine through videocalls. Reported modifications in conditioning regimens, immunosuppression or maintenance strategies specifically due to the pandemic were not common (10.7%, 14.3% and 10.7%, respectively), and most centers continue to treat patients with COVID19 (88%). Conclusions: The limited transplant activity in Mexico decreased significantly during the pandemic but is recovering and nearly at pre-pandemic levels. A notable exception were haploidentical grafts which remained stable throughout this period and have become the most common donor source in allogeneic transplantation. Most TCs were severely affected with a higher impact in public centers reflecting the fragility of our healthcare system. Clinical Trial Registry: None Disclosure: The authors declare no conflict of interest.
Background: Older patients with high risk myeloid malignancies (AML/MDS) have dismal prognosis and the only curative option is alloHCT. HLA identical siblings (SIB) or a matched unrelated donors (MUD) are preferred, however, for some patients only so-called alternative donors – partially matched unrelated (PMUD) or haploidentical donors (HID) are available. However, these have historically been associated with higher transplant related mortality (TRM). We investigated whether those donors i.e. PMUD and HID are an adequate alternative. Methods: Methods: Retrospective single centre analysis of 140 consecutively transplanted patients ≥60 years with AML (n = 123) or MDS (n = 17) in the period 10/2001-07/2020. GRFS "event" defined as aGvHD ≥ gr.III, extensive cGvHD, relapse or death during follow-up (whichever occurred first). Results: Median age 64 (60-74), SIB donors (26, 19%), MUD (61, 44%), PMUD (24, 17%) and HID (29, 21%). 32 patients (35%) with advanced disease (>CR1/PR1). Disease risk index (DRI): low /medium 52% of patients, high 48%. There were no significant differences between the groups with different donors, except for older age in patients with HID (median 67 years, p = 0.0006) and older age of donors in SIB (p < 0.0001). With a median follow-up of 54 months (6-171), 46 (33%) patients are alive. The 1-year/5-year probabilities of OS/GRFS were 59%/48% and 33%/28%, respectively. The cumulative incidences of TRM and relapses were 47% and 23%, respectively. OS and GRFS in individual donor groups were comparable (p = 0.1392), Fig1. In the multivariate analysis for GRFS, advanced disease was the most significant (HR 2,445, 95% CI 1,454-4,113, p = 0.0008) followed by CMV match (HR 2,167, 95% CI 1,320-3,556, p = 0.0022). The same factors together with aGVHD ≥ gr.III (HR 2.546, 95% CI 1.049-6.179, p = 0.0388) were associated for adverse OS in the multivariate analysis. No impact of donor type was detected. Conclusions: A significant portion of selected elderly patients with AML/MDS achieve long-term OS/GFRS after AloHSCT regardless of donor type. The key factor for outcome is risk of the underlying disease. In these patients, therefore, transplantation in the early stages of the disease is crucial, and any best available donor should be accepted. Disclosure: Nothing to declare
Background: Hematopoietic stem cell transplantation (HSCT) is an imperative treatment modality for high-risk acute leukemia patients. Donor availability is one of the main challenges in transplantation. Only 30% of patients have an available matched sibling donor (MSD) and matched unrelated donor (MUD) is an alternative for some of the remaining patients; However, none of these options are available to almost 30% of patients. Over the last decade, haploidentical HSCT (HID-HSCT) has dramatically evolved as an alternative option, with the merit of being available to all patients. The aim of this study is to report the outcome of HID-HSCT in acute leukemia pediatric patients Methods: From October 2016 to September 2021, 109 pediatric acute leukemia patients underwent HSCT; Of which 59 were ALL patients (median age: 4.5years) and 50 AML patients (median age: 7 years). Overall, from the 26 patients (17 male and 9 female) who received HID-HSCT, 17 were ALL patients (65.38%) and 9 were AML (34.61%), 3 of which (33.3%) had secondary AML. The graft source in all patients was peripheral blood stem cells. The median doses of CD34 + and CD3 + cells, in AML, were 5.5 × 106/kg and 364 × 106/kg, respectively; while, in ALL, they were 6.1 × 106/kg and 345 × 106/kg, in the same order. The conditioning regimen consisted of intravenous Busulfan, cyclophosphamide and rabbit anti-thymocyte globulin (ATG). Cyclosporin, metotheroxate and post-transplant cyclophosphamide were also administrated as a GVHD prophylaxis. In patients without signs of acute graft versus host disease (aGvHD), one dose of donor lymphocyte infusion (107cells/kg) was administrated from the same donor as a means to decrease the probability of rejection. Results: Engraftment transpired in all patients, with two patients (one ALL and one AML) experiencing secondary graft failure; although, both underwent successful HID-HSCT for their second transplantation. From amongst all 26 HID patients, overall survival (OS) rate was 53.84% with 33.3% pertaining to AML and 64.7% to ALL patients. Disease-free survival (DFS) was 49.2%, the rate in AML and ALL patients was 30% and 59.3%, respectively. Acute GvHD occurred in 46.15% of patients, which included 44.4% of AML and 49.2% of ALL patients. The leading cause of death in HID cases were relapse and aGvHD, sequentially; the former occurred in 6 (35.2%) ALL and 4 (44.4%) AML patients. Conclusions: Studies have illustrated that Haploidentical transplantation demonstrates a stronger graft vs. leukemia effect in pediatric acute leukemia; thus, it may be considered as the optimal choice for high-risk acute leukemia patients. The high incidence of acute GvHD, however, has been the main obstacle in Haploidentical transplantation. Our results, in corroboration with the previous findings, indicate that ALL haploidentical recipients showed auspicious results; we speculate that the low OS and DFS rates in AML patients was probably pertinent to secondary AML. Clinical Trial Registry: - Disclosure: Nothing to declare
Background: The recent advancement of transplantation procedures allows allogeneic hematopoietic cell transplantation (HCT) for patients who are ineligible for standard myeloablative conditioning due to older age and comorbidities. Little information is available on the alternative donor selection strategy for these patients without HLA-matched related or unrelated donors. Methods: We retrospectively compared the outcomes of 50 consecutive patients with hematological malignancies who underwent first allogeneic HCT from 1- or 2-locus HLA-mismatched unrelated donors (MMUD) (n = 15) or single unrelated cord blood (CB) (n = 35) after reduced intensity/toxicity conditioning in our institute between 2007 and 2019. Results: The median age at HCT was 58 years (range, 33-65 years) in patients who underwent MMUD transplants and 60 years (range, 23-72 years) in those who underwent CB transplants. The common indications for HCT were acute leukemia or myelodysplastic syndrome in both the MMUD group and the CB group (60% vs. 71%). As a rule, we have used uniform transplantation procedures and supportive care with the manual of our institute, reducing the potential bias from patient heterogeneity in a retrospective study. All patients received fludarabine-based reduced intensity or reduced toxicity conditioning and graft-versus host disease (GVHD) prophylaxis with calcineurin inhibitors plus methotrexate or mycophenolate mofetil. In the MMUD group, the graft source was bone marrow in 14 of 15 (93%) patients and either anti-thymocyte globulin or bortezomib was added to the GVHD prophylaxis in 8 of 15 (53%) patients. Four (27%) of 15 patients received HLA 6/8 MMUD and 26 (74%) of 35 received HLA 4/6 CB. The patient characteristics of the two groups, including the disease risk index, HCT-CI score, performance status, and donor coordination times, did not differ to a statistically significant extent. With a median follow-up of 102 months, the MMUD group showed better overall survival in comparison to the CB group (5-year OS: MMUD group, 62%; CB group, 31%, p = 0.021). The relapse rates of the MMUD and CB groups were similar (at 5 years: MMUD group, 27%; CB group, 33%, p = 0.68). The MMUD group tended to show a lower rate of non-relapse mortality and a low incidence of grade III-IV acute GVHD in comparison to the CB group (at 2 years: MMUD group, 7%; CB group, 40%, p = 0.12 and, at day100: MMUD group, 7%; CB group, 29%, p = 0.079, respectively). The cumulative incidence of extensive chronic GVHD in the MMUD group was significantly higher than that in the CB group (at 5 years: MMUD group, 40%; CB group, 9%, p < 0.01). In a multivariable Cox model adjusted for disease risk and patient age, MMUD was associated with lower overall mortality than CB (hazard ratio 0.38, 95% CI 0.15-0.95, p = 0.038). Conclusions: From the viewpoint of long-term survival, in comparison to CB, MMUD may be a preferred donor source for patients who are ineligible for standard myeloablative conditioning and who do not have HLA-matched donors. Better management of chronic GVHD, including prophylaxis with post-transplantation cyclophosphamide, could improve the long-term outcomes of MMUD transplantation. Disclosure: Hideo Koh: research funds (Takeda Pharmaceutical and AstraZeneca) and honoraria (Sumitomo Dainippon Pharma, MSD and Novartis). Teruhito Takakuwa: research funds (AbbVie, Celgene, Incyte Biosciences Japan and Bristol-Myers Squibb) and honoraria (Bristol-Myers Squibb, Sanofi, Janssen Pharmaceutical, Novartis and ONO PHARMACEUTICAL). Hiroshi Okamura: honoraria (NIPPON SHINYAKU). Mitsutaka Nishimoto: research funds (Astellas Pharma and Zenyaku Kogyo) and honoraria (Otsuka Pharmaceutical and CSL Behring). Yasuhiro Nakashima: research funds (Astellas Pharma, Celgene, AbbVie, Novartis, Bristol-Myers Squibb and Chugai Pharmaceutical) and honoraria (Amgen, Novartis, Chugai Pharmaceutical and SymBio Pharmaceuticals). Mika Nakamae: research funds (VERITAS) and honoraria (which family member received, see Hirohisa Nakamae’s disclosure). Masayuki Hino: research funds (JCR Pharmaceuticals, Asahi Kasei, Abbott, TEIJIN PHARMA, Kyowa Kirin, Otsuka Pharmaceutical, TAIHO PHARMACEUTICAL, DAIICHI SANKYO, Chugai Pharmaceutical, Takeda Pharmaceutical, Celgene, Labcorp Drug Development and TOSOH) and honoraria (CSL Behring, Meiji Seika Pharma, MSD, Astellas Pharma, AstraZeneca, Otsuka Pharmaceutical, ONO PHARMACEUTICAL, Kyowa Kirin, Sanofi, Celgene, Takeda Pharmaceutical, Chugai Pharmaceutical, NIPPON SHINYAKU, Novartis, Pfizer Japan, Bristol-Myers Squibb, Janssen Pharmaceutical, AbbVie, and Sumitomo Dainippon Pharma). Hirohisa Nakamae: research funds (Alexion, Bristol-Myers Squibb, Novartis and CMIC HOLDINGS) and honoraria (Astellas Pharma, Otsuka Pharmaceutical, Sumitomo Dainippon Pharma, Novartis and Bristol-Myers Squibb). All other authors have no conflicts of interest to disclose.
Background: The effect on clinical outcome of ABO incompatibility in haploidentical hematopoietic stem cell transplant (HSCT) is not well defined in a decade in which this procedure has spread worldwide. In this single center retrospective study, we investigate the influence of ABO mismatch on haploidentical HSCT. Methods: Patients receiving an haploidentical HSCT at our center between January’13-October’21 were selected (N = 112). We retrospectively analysed these cases and collected information about leukocyte, platelet, and red cell (RC) engraftment (Hb > 8g/dL for at least 14 days without transfusion support), red blood cell (RBC) concentrates until RC engraftment, treatment with erythropoietin (EPO) and thrombopoietin receptor agonists, post-transplant haemolysis, acute graft-versus-host disease (GVHD) incidence and overall survival. Results: Our cohort included 78 ABO-matched patients and 34 ABO-mismatched patients (18 with major, 14 with minor and 2 with bidirectional ABO mismatching). The clinical and demographic characteristics of patients are presented in Table 1. There were no significant differences between average time to leucocyte, platelet, and RC engraftment between both groups: 20 (±4) days, 28 (±17) days and 42 (±20) days respectively in ABO-matched group; 19 (±4) days, 24 (±8) days and 34 (±19) days respectively in ABO-mismatched group. RBC concentrates until RC engraftment were comparable between groups: 8 (±7) vs 7 (±6). The percentage of patients receiving treatment with EPO or Eltrombopag was similar in ABO-matched and ABO-mismatched transplants. 5/112 patients (4.46%) experienced graft failure (3 from the ABO-matched group, 1 with major mismatch and the other with bidirectional mismatch). One patient in the ABO-mismatched group developed pure red cell aplasia. The prevalence of post-transplant haemolysis was significantly higher in ABO-mismatched patients (44.1% vs 18.9%, P = 0.006). No association was found between ABO compatibility and the development of acute GVHD or acute GVHD grade III-IV. There were no differences in overall survival between ABO-matched and mismatched patients: 50 (35-66) months vs 49 (37-60) months, respectively. Table 1. Clinical and demographic characteristics of ABO-matched and ABO-mismatched patients. All patients received GVHD prophylaxis with cyclosporine and mycophenolate mofetil. Conclusions: ABO incompatibility did not have an influence on engraftment or red cell requirements in the setting of haploidentical HSCT in our study. We found no significant difference in acute GVHD development and overall survival between ABO-matched and mismatched patients. ABO mismatch was associated with a higher incidence of post-transplant haemolysis. Disclosure: Nothing to declare
Background: Given that a significant proportion of hematopoietic stem cell transplants (HCT) are performed with ABO blood group mismatched donors and the impact of ABO mismatch on survival outcomes and engraftment remain controversial, we aimed to explore retrospectively the impact of ABO mismatch in haploidentical transplant (haplo-HCT) recipients at Mayo Clinic Florida. Methods: Using data from patients without prior history of allogeneic HCT who underwent haplo-HCT at Mayo Clinic Florida between 2012 and 2020, comparative analysis between patients who received ABO-matched versus ABO-mismatched transplant was conducted for common clinical endpoints, including survival outcomes, engraftment kinetics, and graft-versus-host disease (GVHD). Bone marrow and peripheral blood chimerism studies were obtained 30 days post-transplant when possible. Neutrophil engraftment was defined as absolute neutrophil count ≥ 0.5x109/L for 5 days without colony-stimulating factor administration. Platelet engraftment was defined as platelets ≥ 20x109/L for 3 days without transfusion. Time to red blood cell (RBC) transfusion independence was defined as hemoglobin ≥ 7.0 g/dL for 7 days without transfusion. Results: Our study cohort included 60 patients at Mayo Clinic Florida who underwent haplo-HCT with median follow-up duration of 413 days, including 41 ABO-matched and 19 ABO-mismatched patients (2 bi-directional mismatches, 3 major mismatches, and 14 minor mismatches). There was no significant difference in baseline demographics, clinical characteristics, or disease between ABO-matched and ABO-mismatched groups. A median overall survival of 412 days was observed in the ABO-matched group compared to 421 days in the ABO-mismatched group [hazard ratio (HR) 0.50; 95% confidence interval: 0.19-1.34; P = 0.17]. Non-relapse mortality, disease free survival, and time to progression were also comparable between ABO-matched and ABO-mismatched groups. There was no significant difference in grade II-IV acute GVHD [HR 0.74; 95% confidence interval: 0.14-3.83; P = 0.72] or chronic GVHD [HR 0.95; 95% confidence interval: 0.31-2.94; P = 0.93] between groups. Finally, there was no significant engraftment difference between groups, including donor chimerism 30 days post-transplant, time to platelet and neutrophil engraftment, and time to RBC transfusion independence (Table 1). Table 1. Engraftment per ABO incompatibility group. Conclusions: In conclusion, ABO incompatibility has no apparent clinical impact on survival or engraftment kinetics in haploidentical hematopoietic stem cell transplant recipients. This study is limited by the small sample size and low number of patients with major/bidirectional ABO mismatch. Disclosure: nothing to declare
Background: Haploidentical Hematopoietic Stem Cell Transplantation (HaploHSCT) has spread rapidly worldwide because HLA-haploidentical donors are highly available and cyclophosphamide is effective. Methods: We retrospectively analyzed 44 patients with hematological malignancies who received HaploHSCT between 2013-2020 in our center (patient’s characteristics on table 1). Most of them received myeloablative conditioning (93.2%), based on fludarabine and busulfan with association in some cases with cyclophosphamide or thiotepa. Three patients received bone marrow as source of stem cells. Fourteen patients (31.8%) had a previous transplant: 9 (64.3%) allogeneic, 5 (35.7%) autologous. All recipients were screened for donor-specific antibodies (DSA) and donors who had HLA alleles targeted by DSA were avoided. The standard GVHD prophylaxis consisted of cyclophosphamide (50 mg/kg/day on days 3 and 4) and cyclosporine plus mycophenolate mofetil starting on day 5. Results: Neutrophil engraftment was achieved in 82% patients with a median of 20 days. Six of 44 patients (13.6%) experienced delayed engraftment. For platelets, 61% achieved engraftment with a median of 28 days. The incidence of acute GVHD was 34% (grades I-II 73.3%; grades III-IV 26.7%), and for chronic 9% (all cases limited). Thirty-three patients (75%) had CMV reactivation, one of them developed CMV disease. With a median follow up of 32.1 months, median overall survival was 41%. In the group of AML, 54.2% of patients remain alive 6 months after transplantation. For patients with lymphoma, 6-months survival was 67%. For ALL, 6-months survival was 71.4%. Eight patients (18.2%) relapsed (4 AML, 2 ALL, 1 PCL, 1 LNH) at a median of 131.5 (98-301) days. Conclusions: These results demonstrate Haplo-HSCT in our center achieves comparable clinical outcomes compared to literature reports. This study has several limitations: heterogeneity between patients and indications for transplant, and reduced sample size. HaploHSCT is one treatment option for adults with hematological malignancies because of high availability of donors. However, prospective randomized studies are needed to compare donor type (haploidentical vs matched unrelated donor). Disclosure: Nothing to declare
Background: Transfer of Plasmodium spp. from hematopoietic stem cell (HSC) donor poses a serious threat to the recipient. The World Marrow Donor Association (WMDA) finds it unacceptable to recruit donors who report travel to a malaria-risk area within the past three years and do not present negative malarial antibodies to exclude the risk of sub-clinical malaria infection. The previously recruited donor, who before the procedure, is found to have a positive result, can be accepted at the discretion of the requesting transplant centre. Still, there is no suggestion of risk mitigation strategy other than analysis of the exposure history. Methods: Here we propose a diagnostic workup minimizing the risk of Plasmodium spp. transfer with hematopoietic stem cells based on the case of a transplant from a seropositive donor. Results: A 21-year old woman required allogeneic hematopoietic stem cell transplantation (alloHSCT) due to AML with myelodysplasia-related changes. At the initial presentation, she had hyperleukocytosis of 307 G/l. The cytogenetic risk was high: hyperdiploid complex karyotype with KMT2A rearrangement without KMT2A-MLLT3 fusion gene. After the DAC induction and after consolidation therapy, MRD was found (0.4% and 0.2%, respectively). Therefore, relapse risk was high despite obtaining complete remission, and alloHSCT was indicated. The patient’s 28-year old sister was fully matched and was available as a donor. Thirteen months before the planned procedure, the donor spent eight days at Zanzibar (malaria-endemic region). She took prophylactic treatment, applied repellents, and did not experience any febrile episodes during or soon after that trip. However, the antibody test against Plasmodium spp. was positive. Polymerase Chain Reaction (PCR) against Plasmodium spp., antigen test, and thick blood smear were performed to rule out ongoing sub-clinical malaria – all with negative results. The same three tests were repeated from peripheral blood on the day of apheresis to rule out potential malaria reactivation due to immune system changes during G-CSF mobilization. Samples for those three tests were also taken from apheresis product; the product was frozen and released when negative results were obtained. FluBu4 conditioning was used with cyclosporin A and methotrexate GvHD prophylaxis. The post-transplant period was complicated only with mild engraftment syndrome presenting with skin rash. In samples taken on days 7, 14, and 21 Plasmodium spp. DNA was not found. Consolidation with eight cycles of post-transplant azacitidine was used. After 365 days from transplant patient remains in complete remission and did not experience any infectious complications (except mild SARS-CoV-2 infection). Conclusions: The proposed testing procedure may help establish a systematic approach to Plasmodium spp. seropositive hematopoietic stem cell donors, provide a risk mitigation strategy, and therefore facilitate transplants from this donor group. Disclosure: Nothing to declare
Background: Haploidentical stem cell transplantation (haplo-SCT) is considered a clinical therapeutic option for patients who are indicated for allogeneic stem cell transplantation. The number of patients transplanted with a haplo-relative increases each year worldwide due to its feasibility and accessibility. The recent advances in the field of haplo-HSCT allow a large number of patients with high risk hematological diseases to benefit from this treatment despite not having a matched donor. Methods: We present 11 patients (female/male: 1/1.2; mean age 46 years (28-62), 6 with acute myeloid leukemia, 4 with acute lymphoblastic leukemia, and 1 with idiopathic aplastic anemia) who underwent SCT from a haploidentical donor for a period of 3 years. Results: In 10 of the patients it was a first transplantation, and in one - second. 54.5% of patients were transplanted in first complete remission (CR1), 27.3% - in second complete remission (CR2), and one - in progressive disease. The stem cell source in all patients was peripheral stem cells. In 81.8% we performed myeloablative conditioning regimen, in 18.2% - conditioning with reduced intensity. As part of prophylaxis of graft-versus-host disease (GvHD) we administered post-transplant cyclophosphamide to 10 patients and antithymocyte globulin to 1 patient. The mean number of transfused CD 34 (+) cells was 6.53 x 106/kg (3.76-9.73). All patients achieved engraftment – of the neutrophils on D + 19 (13-25) and of the platelets on D + 22 (14-35). 36.4% of the patients had acute GvHD grade II-IV, 18% - grade IV. At the time of the analysis, 72.2% of patients were alive. 45.5% achieved remission of the disease, 27.3% developed relapse. Graft failure was observed in one patient. Causes of death included acute myocardial infarction, BKV encephalitis and relapse. Conclusions: Haploidentical SCT is an acceptable option in absence of a compatible donor and necessity of well-timed treatment. With all available strategies, virtually no patient who needs an allogeneic transplant should be excluded by the absence of a donor. Disclosure: Nothing to declare
Background: Autologous stem cell transplant (ASCT) is a widely recognized essential therapeutic step in eligible patients with newly diagnosed multiple myeloma (NDMM), but the optimal strategies for stem cell mobilization are still a matter of debate. High-dose cyclophosphamide with filgrastim (HD-Cy+G-CSF) might be used for its greater apheresis yield of CD34+ peripheral blood progenitor cells (PBPC) and potential anti-tumour effect, while filgrastim alone (G-CSF) may be preferred for its greater predictability and safety profile. The aim of this study was to compare these regimens on mobilization efficacy, cell grafting and transplant outcomes on NDMM patients submitted to ASCT. Methods: We retrospectively analysed NDMM patients submitted to ASCT with melphalan conditioning, between 2011 and 2020, in first complete remission (CR) after induction with bortezomib, cyclophosphamide and dexamethasone (VCD) or bortezomib, thalidomide and dexamethasone (VTD). Plerixafor was used pre-emptively before apheresis if the CD34+ count was <10x106 cells/l. EBMT definitions for neutrophil and platelet grafting were used. Results: A total of 73 patients in CR were mobilized, 20 with HD-Cy+G-CSF and 53 with G-CSF. Patients in the first group were younger (56 vs 64 years; p = 0.003). The induction treatment regimen was VCD in 21% and VTD in 79%, while post-transplant maintenance therapy was used in 58%, with no significant differences between groups. During mobilization, 70% of patients in the HD-Cy+G-CSF group had at least one episode of fever requiring antibiotics, in contrast with 6% in the G-CSF group (p < 0,001). HD-Cy allowed greater PBPC collection (8.39 vs 4.66x106 CD34+ cells/kg; p < 0,001), less apheresis sessions (2 or less in 85% vs 51%; p = 0.004) and less frequent need for pre-emptive plerixafor administration (15% vs 40%; p = 0.046), compared to G-CSF. The number of CD34+ cells infused was significantly higher in the HD-Cy+G-CSF group (4.20 vs 2.52x106 CD34+ cells/kg) but no differences were seen regarding neutrophil (median 12 vs 12 days) or platelet (median 16 vs 17 days) engraftment. Likewise, febrile neutropenia episodes, grade III-IV mucositis or number of transfused units of platelets or erythrocytes was similar between groups. There were also no differences between HD-Cy+G-CSF and G-CSF-mobilized patients regarding CR rates on day +100 post-transplant (80% vs 85%, respectively) or on 3-year time to next treatment survival probability (67% vs 61%, respectively). Conclusions: In NDMM achieving CR after induction with a bortezomib-based treatment, mobilization with G-CSF alone, despite having a smaller PBPC apheresis yield and greater need for pre-emptive plerixafor, shows less toxicity than HD-Cy and grants equally effective grafting after ASCT. This suggests HD-Cy+G-CSF may compromise the marrow microenvironment or cause significant toxicity to the PBPC collected, preventing the enhanced number of cells infused to translate into a more advantageous graft with better post-transplant outcomes. Choosing G-CSF as the preferred mobilization regimen may avoid possible infectious complications seen with HD-Cy+G-CSF without compromising ASCT outcomes in this specific group of NDMM with a favourable response to induction therapy. Disclosure: Nothing to declare.
Background: Graft cell dose may play a crucial role affecting not only engraftment but also non-relapse mortality (NRM) and survival after allogeneic hematopoietic stem cell transplantation (allo-HSCT). Most studies performed so far, considered allo-HSCT using standard GVHD prophylaxis, while the high-dose post-transplant cyclophosphamide (PTCy) setting has not been extensively studied yet. Currently, PT-Cy is widely used in clinical practice not only for haploidentical allo-HSCT but also for related and unrelated transplants. The objective of this retrospective study was to assess the impact of CD34 + cell doses in peripheral blood stem cells (PBSC) grafts, on the outcome of allo-HSCT using PTCy-based GVHD prophylaxis. Methods: We included a total of 193 consecutive adult patients with hematological malignancies who underwent allo-HSCT at the Hospital Clinic of Barcelona between 2014-2021. T-cell repleted PBSC grafts were infused in all cases and the maximum CD34 + cell dose was capped at 8×106/kg. Based on the binary partitioning method, an optimal cut-off of CD34 + cell dose value was proposed to separate the cohort in two groups in terms of our main outcome variable (overall survival, OS). Results: Patient characteristics are shown in Table 1. Median CD34 + cell dose was 5.86×106/kg (IQR: 4.49-7.16). One-hundred and thirty-one patients received a high-dose defined as >5x106/kg and 62 a low-dose defined as <5x106/kg. A total of 12 patients experienced graft failure without differences between groups. Median time to neutrophil engraftment was 19 (IQR 16-23) for high-dose and 20 days (IQR 18-23) for low-dose (p = 0.04). Median time to platelets recovery was 16 (IQR 13-26) and 20 days (IQR 13-28) (p = 0.2), respectively. No differences between groups were observed in the cumulative incidence of day +100 aGVHD (grade II-IV 24% for high-dose vs. 23% for low-dose, p = 0.68; and grade III-IV 5% vs. 3%, respectively, p = 0.4), or 2-year cGVHD (moderate/severe 11% vs. 4%, respectively, p = 0.1). NRM was significantly lower for high-dose group (1-year: 9% vs. 21%, respectively, p = 0.04), with comparable relapse rate (1-year: 19% vs. 19%, respectively, p = 0.98). After a median follow-up of 34 months, patients receiving a high-dose had better 1-year OS (82% vs. 67%, p = 0.03), with a not significant trend towards a better disease free survival (72% vs. 59%, p = 0.09). GVHD-free/relapse-free survival was comparable between groups (64% vs. 56%, p = 0.4). The multivariate analysis confirmed the negative association between the infusion of a CD34 + low-dose in OS (HR 1.7, p = 0.049). Age > 60 years (HR 2.1, p = 0.04), KPS ≤ 80% (HR 2.4, p = 0.002), and HCT-CI score > 3 (HR 1.96, p = 0.02) were additionally found to be significant risk factors for OS. Conclusions: Our results show that the infusion of CD34 + cell PBSC dose ≥5 ×106/kg had prolonged survival, mainly due to a reduced NRM rate. No differences were observed in relapse rate, suggesting that the infusion of CD34 + cell doses ≥5 ×106/kg did not protect from disease relapse. High-CD34 + dose did not increase the risk of GVHD. Taken together, these results suggest that, in the setting of PTCy allo-HSCT, an infusion of a CD34 + dose ≥5 ×106/kg could be beneficial. Clinical Trial Registry: No Disclosure: Nothing to declare
Background: Cyclophosphamide (Cy) in combination with granulocyte-colony stimulating factor (G-CSF) is typically used for peripheral blood stem cells mobilization in myeloma patients. The aim of this study was to evaluate the mobilization efficacy and safety of two different infusion times of Cy plus G-CSF for peripheral blood stem cells mobilization in newly diagnosed multiple myeloma (NDMM) patients. Methods: We retrospectively analyzed the mobilization efficacy and safety of “24 hours continuous infusion” (24 hours group) versus “short time (4-6 hours) infusion” (control group) cyclophosphamide in 156 (68 vs. 88) NDMM patients who receive Cy plus G-CSF mobilization and auto-stem cell transplantation (ASCT) in our department between September 2008 and May 2020. Results: Among 156 patients, the demographic characteristics including sex, age, hemoglobin, plasma cells percentage, type of myeloma, cytogenetics risk, international staging system, and serum creatinine, serum calcium, lactate dehydrogenase level were similar between two groups. The median numbers of collected CD34 + cells in 24 hours group and control group were 6.78 and 4.48×106/kg (p < 0.0001). However, the mean number of apheresis was 2.26 in 24 hours group and 1.51 in control group (p < 0.0001). The median numbers of collected CD34 + cells of one apheresis in 24 hours group and control group (2vs.45) were 11.57 and 4.79×106/kg (p = 0.493). The median numbers of collected CD34 + cells of two apheresis in 24 hours group and control group (49vs.40) were 8.04 and 4.16×106/kg (p < 0.0001). The median numbers of collected CD34 + cells of three apheresis in 24 hours group and control group (14vs.2) were 4.53 and 3.23×106/kg (p = 0.427). Target number of CD34 + cells/kg (defined as ≥6 × 106/kg) was collected from 55.9% patients in 24 hours group vs. 29.9% in control group (p = 0.001). The rate of patients who need secondary mobilization and bone marrow transplant was 1.5% in 24 hours group and 14.8% in control group (p = 0.004). The day of neutrophil engraftment after transplantation was 10 in 24 hours group and 11 in control group (p < 0.0001), and the day of platelet engraftment was 11 in 24 hours group and 12 in control group (p = 0.272). No differences of hematologic toxicity, non-hematologic toxicity and the cost in hospital were observed between two groups. Conclusions: 24 hours continuous infusion of Cy is effective with equivalent toxicity for stem cell mobilization in patients with newly diagnosed myeloma and could be considered in MM patients with expected secondary or tertiary transplantations. Disclosure: Nothing to declare
Background: Sirtuins are members of the NAD + -dependent class III histone deacetylase family, involved in the post-translational modification of proteins. They participate in the fate of HSC, their metabolism, stress response, differentiation, aging and migration. Mobilization of hematopoietic stem cells (HSC) from the bone marrow niche into the peripheral blood is a crucial step in the treatment of lymphoproliferative neoplasms with autologous hematopoietic stem cells transplantation (auto-HSCT). The aim of this study was to explore SIRT1, SIRT2, SIRT3, SIRT4, SIRT5 SIRT6 and SIRT7 levels during HSC mobilization. Sirtuins were investigated in the context of CD34 + cells mobilization efficacy with different chemotherapy regimens. Methods: Fifty patients were enrolled in the study (24 F, 26 M). The median (Me) age was 60 years. The investigated group consisted of thirty-nine multiple myeloma (MM), seven non-Hodgkin lymphoma (NHL) and four Hodgkin lymphoma (HL) patients. Sirtuins expression was evaluated in peripheral blood (PB). The blood serum samples were collected at two time points: before hematopoietic stem cell mobilization chemotherapy (day 0) and on the day of the first apheresis (day A). Sirtuins expression was evaluated by ddPCR method. Results: Our study revealed positive correlation between SIRT5, SIRT6 and SIRT7 expression on day A and the number of CD34 + cells collected at the first apheresis (R = 0.34, p = 0.02), (R = 0.31, p = 0.03), (R = 0.47, p < 0.001), (Figure 1). To evaluate the influence of sirtuins expression on the number of CD34 + cells on day A, patients were divided into “high” and “low” expression groups according to median sirtuins levels on day of first apheresis (above and below median). The group of SIRT7 “high expressors” collected more CD34 + x106/kg cells on day A than “low expressors” (5.01 vs 1.68 CD34 + x106/kg, p = 0.003). To assess the effect of the administrated mobilization chemotherapy on the level of sirtuins on the day of the first apheresis, we divided the patients into two groups: the first group received mobilization chemotherapy containing alkylating agents (cyclophosphamide/ifosfamide): Cyclophosphamide, DCEP, ICE, R-ICE, (n = 39), (Alk group). The second group consisted of patients mobilized with Cytarabine, DHAP, R-DHAP or GCS-F in monotherapy, (n = 11), (non-Alk group). The Alk group had a lower SIRT1, SIRT2, SIRT3 and SIRT5 level on the day of first apheresis than non-Alk group (Me = 300.53 vs 621.94 copies/200µL, p = 0.002), (Me = 628.95 vs 1493.02 copies/200µL, p < 0.001), (Me = 137.84 vs 304.77 copies/200µL, p = 0.02) and (Me = 202.95 vs 482.01 copies/200µL, p = 0.02) respectively. Figure 1. Scatter plots illustrating the positive correlation between SIRT7 expression on day A and CD34 + peak in peripheral blood. Conclusions: In conclusion, we observed that sirtuins influence HSC migration and hematopoietic landscape in the bone marrow niche during CD34 + cell mobilization for autologous transplantation. SIRT5, SIRT6 and especially SIRT7 play an important role in this process. Moreover, we noticed that alteration in sirtuins expression during first apheresis depends on whether the chemotherapy contains alkylating agents. Disclosure: Nothing to declare.
Background: Accurate prediction of the stem cell yield is essential for planning and executing of the PBSC leucapheresis procedure. Calculation based on pre-apheresis peripheral CD34+ cell count usually shows a good correlation between predicted and actually collected cell count, however slight underestimation and overestimation occurs. Methods: We calculated the predicted CD34+ yield with the following method: Predicted CD34+x106/kg = (benchmark collection efficacy x processed blood volume x peripheral CD34+ count per μl)/(patient’s weight x metric conversion factor). Data were retrospectively analyzed and divided into 3 subgroups, based on their accuracy of the calculated CD34+ yield compared with the actually collected CD34+ count. The results of the 3 subgroups which were underestimated (UE, 15% below predicted CD34+ yield), overestimated (OE, 15% above predicted CD34+ yield) and good estimation (GE, within +/− 15%) were compared with an ANOVA analysis. Additionally, multiple clinical and laboratory data were evaluated. Results: We investigated 607 apheresis (569 donors) procedures during the years 2019-2020. The mean value of the prediction coefficient for all apheresis procedures was 1.06. The Spearman’s correlation coefficient (r) between calculated and actually collected CD34 cells per kg bodyweight recipient was 0.9126 (p < 0.01), which indicates a very reliable prediction over this 2 years. Most of the collections (66%) showed a very good correlation between predicted and finally collected stem cell count. The OE subgroup consisted of only 8% apheresis procedures and the UE subgroup of only 26%. The mean prediction coefficient of the UE was 1.29 and of the OE subgroup 0.81. The analysis of clinical and laboratory data revealed a significantly lower CD34+/μl pre apheresis count in the UE group, as well as a trend to lower ferritin blood levels. The underestimation leads to slightly higher results than estimated, so these collections resulted in more than sufficient products. Conclusions: Our method of calculating the stem cell yield is highly predictive of the number of CD34+ cells actually collected, making planning and adjusting of the apheresis procedure very reliable. For donors with low pre apheresis CD34 + count and who are critical to meet the threshold for a successful apheresis, the calculation tends to underestimate the CD34+ yield which leads to a better than calculated collection. On the contrary, the clinical more adverse overestimation occurred predominately in the range of more than sufficient apheresis. For clinical practice, in case the donors estimated stem cell yield is just below the threshold for a successful collection, we maximize the processed blood flow volume using higher inlet flow rates and longer procedure times. On the other hand, in the event of a more than sufficient stem cell yield calculation, reducing the procedure time turned out to be safe and reliable. Disclosure: Nothing to declare
Background: For nearly two years, the COVID-19 pandemic has had detrimental impact on medical practices. Because of its intrinsic complexity, the delivery of allogeneic haematopoietic cell transplantation (HCT) in this context represents an enormous challenge. Transplant centres have adapted promptly and developed strategies to enable safe HCT, by introducing significant changes in their procedures, e.g. through an unprecedented use of cryopreservation for allogeneic haematopoietic progenitor cell (HPC) grafts. Methods: To explore how centres modified their strategies in terms of donor selection and implementation of new procedures (e.g. cryopreservation), a 14-item survey was disseminated by the EBMT-IDWP & CTIWP between January 2020 and June 2021. The survey was sent to 509 EBMT-affiliated allogeneic HCT centres and open between September 20th and October 15th, 2021. Results: Eighty-four of 509 (17%) centres from 24 countries responded. Of these, 68% hold a JACIE accreditation. Seventy-four percent of centres introduced COVID-19 mitigation measures for their donors (e.g. physical and social distancing) to minimize the risk of infection. Forty-one percent of centres changed their donor search strategy, and favoured recruitment of related haplo-identical donors over unrelated donors (URD). In addition, 33% and 55% of centres searched for a backup donor in the related and URD settings, respectively. Only 10% of centres considered cord blood (CB) as an alternative source to URD. Before COVID-19, only six percent were routinely cryopreserving HPC products, whereas this percentage increased to 92% during the pandemic. In details, 53/77 (69%) of centres cryopreserved both, related and URD grafts, 21 (27%) only URD and 3 (4%) related grafts only. In the majority of centres, changes in policies were introduced based on EBMT recommendations (60/75; 80%) and/or national guidelines (45/75; 60%). Bone marrow (BM) grafts were avoided by 33 (39%) centres. In the study period, a total of 2,502 related and 2,680 URD transplants have been reported. Of these transplants, 60% (n = 1,491) related and 78% (n = 2,098) of URD grafts have been cryopreserved pre-emptively and infused in the vast majority of cases. Three percent (n = 50) related and 4% (n = 79) URD cryopreserved products have not been infused during the observation period. Release criteria for cryopreserved products in terms of COVID-19 were based on a negative SARS-CoV-2 test (not further specified) in 35/77 (46%), interview of the donor (medical history) in 3/77 (4%), and both in 20/77 (26%). Related donors were tested for SARS-CoV-2 during donor work-up in 27/56 (48%) and during mobilization in 35/56 (63%) centres. Test strategies for URD where not covered by the survey. Conclusions: These data are limited by the low number of respondents, but show a historical increase in cryopreservation of allogeneic HPC grafts during the first three waves of the SARS-CoV-2 pandemic, more in URD than in related donors grafts. The use of BM decreased, and CB grafts were not used as an alternative at most centres. A significant proportion of cryopreserved products (3-4%) were not infused. The clinical impact of allogeneic cryopreserved HPCs will be analysed in the ongoing second part of this study. Disclosure: Nothing to declare.
Background: Due to COVID-19 restrictions, many centres have rapidly changed practice regarding processing of stem cell products. We sought to examine the impact of graft cryopreservation on transplant outcomes at the Irish National Stem Cell Transplant programme. Methods: We reviewed 44 patients undergoing allogeneic transplantation with cryopreserved products from 2020 compared to 44 using fresh product immediately prior to the pandemic. The cohorts were matched for age, stem cell source/type, disease, disease risk index, HCT-CI, ABO compatibility, HLA antigen mismatch status and conditioning regimen/intensity. Release specification of stem cell products including TNC, CD34+ viability, CD3+, CD4+, CD8+ and CD4+/8+ content were examined. Clinical outcomes included time to engraftment, chimerism at 100 days, GVHD incidence, relapse and death within 1 year. Results: The majority of our cohort received PBSC grafts. The median TNC, CD34+ and CD3+ infusion dose was similar in both groups. T-cell content of grafts was similar, with no significant difference in T-cell subsets (CD3+, CD4+, CD8+, CD4/8+). In the cryopreserved group median CD34+ viability at the time of thawing was 90.66% (53.37-97.36). In vitro progenitor assays showed the median CFU-GM in the fresh cohort was 36x104/kg and 48x104/kg post-thaw in cryopreserved products(Table1). Neutrophil engraftment was slightly delayed with cryopreservation (18.8 vs. 16.6 days, p = 0.089). There were no cases of primary graft failure. Time to platelet engraftment was similar in both groups (p = 0.29). Platelet engraftment failed in 3 patients with cryopreservation and 4 without. D100 chimerism did not differ between groups. Acute and chronic GVHD incidence was similar between groups. Interestingly, we identified an increased risk of relapse at 1-year post-transplant in the cryopreserved cohort despite matching for age, disease/risk, conditioning intensity and stem cell source. 10 patients relapsed within 1 year compared to 2 in the non-cryopreserved group (p = 0.014). Overall survival at 1 year was similar in both groups (p = 0.517) (Graph1). Conclusions: Large-scale adoption of cryopreservation of allogeneic stem cell products due to COVID-19 provided an opportunity to examine the impact of this practice in our patient cohort. Our data show that graft content is reliably maintained compared to fresh infusion in terms of CD34+ viability, T-cell counts and engraftment times. Although overall survival was not affected, we identified an increased rate of relapse within 1 year in patients with cryopreserved products, despite similar graft characteristics and matching for categorical variables. Considering similar T-cell quantity in both graft cohorts, functional rather than quantitative T-cell defects may explain this outcome. Indeed, the GVL effect could also be compromised by other variables, such as NK and monocyte alterations. Further and longitudinal review to explore this finding is required, in particular as global logistical restrictions continue. Clinical Trial Registry: N/A Disclosure: Nothing to declare
Background: Extracorporeal photopheresis (ECP) is an effective treatment for graft versus host disease (GVHD) and cutaneous T cell lymphoma (PTCL). Two types of systems are currently possible for this therapy: in-line or off-line. The advantage of the in-line system includes a shorter procedure time, a lower risk for contamination, and a reduced risk of improper infusion. The off-line system benefits from the lower extracorporeal volume and the higher amount of processed whole blood. However, it is a multistep procedure for which the laminar airflow cabinet and sterility testing are needed. A new type of in-line system has been developed to combine the benefits of both types of systems. Methods: The objective of this study was to compare two methods of ECP. Twelve patients (7 women and 5 men, median age 53.5 years) with PTCL (3 patients) or GVHD (9 patients) were treated with ECP 2 consecutive days in one cycle. A procedure was performed in standard off-line system - combination of Spectra Optia for collection and photoactivation by Macogenic G2 (Macopharma, Mouvaux, France). The second procedure in the same patient in this cycle was performed using a new system. Spectra Optia (Terumo BCT, Lakewood, U.S.) was used for mononuclear cell (MNC) collection and an accessory device UVA PIT System (PIT Medical Systems GmbH, Cadolzburg, Germany) for photoactivation as a functionally closed in-line multistep system. Apheresis procedure was in both systems the same with using of continuous MNC (CMNC) collection protocol. In the off-line system 8-methoxypsoralen (8-MOP) was added to the MNC bag in the laminar airflow cabinet and after photoactivation the cells were reinfused. In the in-line system 8-MOP was added to MNC bag using sterile tubing connections between the two systems, after photoactivation cells were reinfused due to the connected system to the patient infusion line. The patient remained connected to the Spectra Optia throughout the entire procedure. Sterility of all products was examined before administration. Results: 48 procedures (24 off-line/24 in-line) were performed in 12 patients. The processed total blood volume (TBV) was similar in each pair, 1.4 - 1.8fold of TBV (7093 ml vs. 7105 ml). No contamination was detected. The time of the entire procedure (from the beginning of MNC collection to the end of reinfusion) was measured. The average time in the off-line group was 280.9 (median 275) minutes and 201.2 (median 200) minutes in the in-line group. We did not observe any severe side effects except mild ACD-A toxicity (hypocalcemia), all procedures were finished completely except one (insufficient peripheral vein in the off-line group). No worsening of the disease was detected. Conclusions: The new in-line system provides a functionally closed procedure enabling treatment at the bedside with a low risk of cross-contamination, infection and infusion errors. It allows processing the same TBV as in off-line methods. The time of the entire procedure was considerably shorter. The procedure was safe and well-tolerated without bacterial contamination or worsening of the disease. Disclosure: The authors have nothing to declare.
Background: Characteristics of the collection material are the first to predict the end-product evaluation of anti-CD-19 CAR-T cells bag. This process starts when patient’s cells are collected in the apheresis unit, transferred to the cell processing lab and cryopreserved. Manufacturing eligibility requires viable total nucleated cells (TNC) ≥ 2x10^9, total CD3 + cell ≥ 1x10^9 and %T cells ≥ 3. Pre-collection peripheral blood analysis of %CD3 by flow cytometry is recommended to calculate the minimum total blood volume (TBV) to be processed and to achieve the required number of CD3 + cells. The analysis is both time consuming and costly. Thus, we aimed to further improve calculation of sufficient TBVs based on pre-apheresis blood samples. Methods: We retrospectively analyzed data from all consecutive patients that underwent leukapheresis and processing of collection material for the production of tisagenlecleucel between March, 2019 and December, 2021 in the Tel Aviv Sourasky Medical Center. We compared the percentage of lymphocytes in the cell count to the %CD3 + cells by Flow Cytometer analysis using Pearson correlation. Based on the correlation to precisely determine the minimum TBV needed for successful collection, we developed a novel equation based exclusively on peripheral blood cell count aiming to minimize TBV processed by the apheresis machine. Results: We analyzed data of 75 patients with DLBCL (standard group, n = 56 and early collection group, n = 19). Median age was 69 (range, 21-85) years with 46 patients (61%) having ECOG > = 2. median number of prior lines in the non-early collection group was 2 (range 2-5). All patients, except 2 were collected in 1 day. Median number of collection cycles was 2 (range, 2-6). All collected products were eligible for manufacturing according to NOVARTIS recommendations. Six (8%) products were manufacturing out-of-specification and 2 (2.7%) were not infused. Pre-collection, there was a statistically significant correlation between %CD3 and %lymphocytes among both patients in the standard group (r = 0.86) and those in the early-collection group (r = 0.95) with a linear equation of Y = 0.76X-1.2 and, Y = 0.76X + 2.08, respectively. While there were no correlations between both the pre-collection peripheral blood %CD3 or WBC, and the total collected CD3 + cells, when we applied the linear equation based on both pre-collection %CD3 and WBC, we identified that patients with > 800 lymphocytes/µl required at most 2 TBVs, Figure. Receiver operating curve utilizing pre-collection lymphocyte number of 800/µl predicted TBVs with an area under the curve of 0.978 (p < 0.001) with sensitivity of 81.6% and specificity of 100%, while those with less than 800, required more than 2 TBVs and to precise calculate the number of TBVs, addition parameters were required. These parameters included – the maximum number of cryobags in the vapor ship and logistics domains. Conclusions: We identified a pre-collection peripheral blood lymphocyte level of 800/µl as sufficient for THE collection with 2 TBVs. In cases of less than 800, additional parameters are required to calculate the required TBVs. This enables a high degree of collection-effectiveness. Disclosure: R.R received honoraria from Novartis
Background: The safety of autologous hematopoietic cell transplantation (auto-HCT) depends on the number of infused CD34( + ) cells. The optimal mobilization schema should be outpatient and result in the collection of a sufficient number of CD34( + ) cells within the shortest time. Standard mobilization protocol: Cytosine arabinoside (Ara-C) total 1600mg/m^2 given 400mg/m^2 iv bid for two days requires hospitalization. Here we launched a prospective study aimed at modifying it for ambulatory conditions. Methods: The design of the study was based on a 3 + 3 protocol performed in the reversed (de-escalation) direction. Standard mobilization schema: (Control Group) was modified and/or de-escalated to 4 schedules (table 1). G-CSF (5ucg/kg bid) was given 72 hours after the last dose of Ara-C until the first apheresis (day + 14). The minimal target dose (MTD) was 5x10^6 of CD34( + ) cells/kg. If the MTD was not achieved in one apheresis in 1 patient at least 3 more patients were treated using the same schema, if in more than 2 the group was closed. The effectiveness of each schema was scored as follows: achievement of MTD in 1 apheresis 3 points, in 2 aphereses - 2, in 3 and more - 1, 0 - if MTD was not achieved. The group with the best scoring will be expanded. The primary endpoint is the total (highest) number of CD34( + ) cells/kg collected in one leukapheresis. The secondary endpoints are the number of apheresis days, the total number of CD34( + ) cells in the 2 best aphereses. Results: Between 04/21-11/21, 42 patients were randomly enrolled in scheduled groups. The patient clinical characteristics and results are shown in Table 1. In the Control Group and Groups 1, 2, 3 the MTD was achieved by all except 1 patient each in Control, 1 and 2 group. In group 4 none of the patients achieved MTD in 1 leukapheresis therefore the study wasn’t continued with this schema. The highest score (3) per patient was achieved in Group 3 which is not different from the Control Group (2,67). Similarly, the highest number of CD34( + ) cells in one apheresis, as well as the highest total number of collected CD34( + ) cells in 2 aphereses were documented in the Control Group and Group 3. Also in these groups, cells were collected in one apheresis in most patients. Conclusions: The dosage of Ara-C at 800 mg/m^2 once a day for two days followed by standard G-CSF seems to be sufficient for collecting MTD in 1 leukapheresis. However, for the target dose of more than 10 x10^6/kg of CD34 cells a standard schedule of Ara-C dosing might be preferred. Disclosure: Nothing to declare
Background: The use of plerixafor can rescue about 70-75% of poor mobilizer patients(1). This drug comes in 24 mg vials, the standard dose is 0.24 mg/kg, equivalent to 18 mg for a 75 kg patient, leaving a remnant of 6 mg per vial. Plerixafor has a stability of up to 84 days (2) allowing the remnants to be used during that time. We report the results of emergency and compassionate use of a fixed dose of 12 mg to rescue poor mobilizers. Methods: We reviewed the records of all patients with myeloma or lymphoma who received an ASCT at our center (Jan 2020 - Nov 2021, were included those who needed emergency use of plerixafor, and in whom, for logistical or administrative reasons, the standard dose was not available and received 12 mg using remnants stored aseptically at room temperature. The definition of poor mobilizer used was that recommended by EBMT consensus (1): CD34 blood count on day 4 of mobilization less than 10 per uL or failure to obtain, after one apheresis, half the number of CD34 expected for transplantation. Mobilization and harvest consisted of filgrastim 7.5 mg/kg BID for 5 days, and one to three large volume apheresis. All patients consented to plerixafor application. Results: I- Sixteen patients met the inclusion criteria, 7 were female, median age was 54.5 years (R: 31-68), mean weight; 66.5 kg (R: 48-80). Six had myeloma, 5 of them were exposed to lenalidomide, 2 were heavily treated; 10 had lymphoma; 3 of them received HyperCVAD, five had two or more lines of treatment, and 3 had radiotherapy to pelvis or abdomen. Thirteen out of 16 cases had a blood CD34 count on day 4 of mobilization less than 10/uL, X: 3.23 (R: 2-8), three had an insufficient CD34/kg count after the first harvest. Twelve received one dose of plerixafor and four received two. The main adverse events were grade I-II diarrhea and myalgias (65%), there were not any cases of infection associated with the use of reminder of plerixafor. II- The mean number of apheresis was 1.6 (R: 1-3), the mean CD34 obtained was 2.28 million/kg (R: 0.5-3.42). Twelve out of 16 patients reached a CD34 number of 2.0 million/kg or more and another two: 1,6 and 1,75 million/kg respectively, all these 14 were transplanted. Two did not reach enough number of CD34 positive cells after three apheresis All patients were given myeloablative conditioning and filgrastim post-transplantation. All cases had a hematopoietic recovery, the average days for autonomous neutrophil and platelet production was 11.14 (R 10-12) and 14.0 (R 12-25) respectively. Transplant-related mortality was zero, and after a median follow-up of 7 months (R: 2-17), all are alive without loss of hematopoiesis. Conclusions: A fixed-dose of 12 mg of plerixafor, using the remnants of it, was effective (87%), and safe in rescuing poor mobilizers Considering the stability of the product and its cost, it is worth carrying out a dose optimization study. 1. Bone Marrow Transplantation (2014) 49: 865 2. Pharm. Technol. Hosp. Pharm. 2016; 1(2): 73 Disclosure: The authors did not have disclosures
Background: Failure rate of hematopoietic stem cell mobilization (HSC) with traditional protocols described in literature ranges between 10-25%. Current evidence suggests a beneficial use of plerixafor in selected cases, with a reduction in mobilization failure rate to 3%. However, its high cost is the major limiting factor for routine use, with a scarcity of studies in Latin America evaluating the cost-benefit of this drug. Thus, the need to assess cost-effectiveness of using plerixafor in public health services in an emerging country is meaningful. Methods: Retrospective observational study with patients over 18 years of age who underwent hematopoietic stem cell mobilization between January 2014 and December 2020. Clinical and pharmacoeconomic effectiveness of HSC mobilization protocols adopted over 03 different periods were evaluated: pre-Plerixafor protocol (Period 1), use of Plerixafor in mobilization failure (Period 2) and preemptive use of Plerixafor (Period 3). HSC mobilization failure was defined as collection of less than 2.0 x 106 CD34+ cells/kg after a mobilization protocol. The cost values used were based on SIGTAP (Brazilian public health cost chart) and on hospital cost of the drugs used. Results: Of the 623 patients evaluated, 51.7% (322) were diagnosed with Monoclonal Gammopathies and 42.5% (265) with Lymphomas. 49.9% (311) of this cohort underwent mobilization with G-CSF while 50.1% (312) received G-CSF and chemotherapy. HSC mobilization failure rate was 13.43%. 96.5% (517) of patients who successfully mobilized needed only 1 apheresis session to collect HSC. Success rate of HSC mobilization with only 1 collection was of 89% in Period 1, 82.7% in Period 2 and 85.5% in Period 3. Main factors associated with failure rate of HSC mobilization was age older than 32 years (p = 0.013), pre-mobilization white blood count less than 5,800 (p = 0.007) and previous use of Fludarabine, Lenalidomide or alkylating agents (p = 0.017). Such factors did not show statistical significance after analysis by multivariate logistic regression. Cost per patient was of R$1,556.83 in Period 1; R$3,820.77 in Period 2; R$4,278.09 in Period 3. Incremental cost-effectiveness ratio for a 10% benefit with the use of Plerixafor was of R$24,160.63. Conclusions: The incorporation of plerixafor in a Brazilian public health service did not reduce the rate of HSC mobilization failure in the analyzed periods, although it significantly increased the cost per patient. Evaluation of variables related to time until transplantation and associated morbidity can contribute to define the best use of new drugs. Disclosure: Nothing to declare.
Background: Before the March 2020, cryopreservation of allo-HSCT grafts was restricted only to exceptional situations. Since the begining of the COVID-19 pandemic, it is strongly recommended, to have secured stem cell cryopreserved product before the start of patient conditioning. The aim of the study was to evaluate the impact of cryopreservation of PBSC on clinical outcome after allo-HSCT. Methods: Study group: The study group consisted of 131 patients who underwent allo-HSCT at the Department of Bone Marrow Transplantation and Oncohematology, National Research Institute of Oncology, Gliwice Branch, Poland. We retrospectively investigated the clinical outcomes of 66 allo-HSCT performed with cryopreserved PBSC (from 03.2020 to 02.2021) and compared to 64 patients transplanted with unfrozen PBSC (from 01.2019 to 03.2020). The study groups did not statistically differ with regard to the diagnosis, age, conditioning regimen, type of donor and the number of transplanted cells. Methods: The PBSCs were cryopreserved with 5% DMSO solution within 72 hours after collection and stored in liquid nitrogen (median storage time 22 days; min 5, max 152). In the control group PBSC were infused within 72 hours after leukapheresis. The time to leukocyte and neutrophil recovery was defined as the first of 3 consecutive days, on which absolute cell count in peripheral blood was higher than 1×10^9/L and 0.5×10^9/L, respectively. We have compared two points in the time to platelet recovery, defined as the first of 3 consecutive days, on which platelet count in peripheral blood was higher than 50×10^9 and 100×10^9 respectively. The frequency of GvHD (grade 0 and I–III), CMV reactivation and donor chimerism level were also compared between groups. Statisitics: The differences between groups with regard to numerical variables were evaluated using Mann-Whitney U-test, chi-squared test was used in case of categorical variables. Time to hematopoietic recovery was estimated using the Kaplan–Meier method (observations were censored in case of death). The groups were compared with log-rank test (p < 0.05 was considered as statistically significant). Results: There were no statistically significant differences in the median days of leukocyte, neutrophil and platelet recovery between groups. The groups do not differ significantly in terms of GvHD, CMV reactivation and chimerism level. The results are presented in Table 1. Conclusions: Cryopreservation of unrelated donor products does not appear to negatively affect early clinical outcomes after allo-HSCT. On the other hand, the safety of transplant procedure is increased by the guaranteed availability of PBSC when recipient conditioning is started. Disclosure: Authors declare no conflict of interest
Background: Poor graft function (PGF) is a rare, life threatening complication after allogeneic hematopoietic stem cell transplantation (allo-HSCT), characterized by protracted multilineage cytopenia with dominant (>95%) allogeneic chimerism. We analyzed the outcomes of CD34-enriched or T-cell depleted (TCD) stem cell boost therapy (SCB) without preceding chemotherapy for PGF in 16 patients transplanted in Wroclaw Medical University stem cell transplant centers in years 2002-2021. Methods: The median age at SCB was 15.24 years (range 0.8-66 years). Patients were transplanted for malignant (14 patients: ALL-5, MDS-EB/AML- 4, other - 5) or nonmalignant (2 patients) diseases. One of the treated patients was after 6 months of unsuccessful eltrombopag therapy for PGF. Donor types were matched unrelated (7/16), and haploidentical donors (9/16). The median time from allo-HSCT to SCB was 126 days (range: 19 to 1085 days). Immunomagnetic selection was performed with Miltenyi CliniMacs Plus or Prodigy: CD34 enrichment in 11 patients, alpha-beta T-cell depletion in 4 patients, and combination of both procedures in 1 patient. The median SCB dose was 5.88 x 10^6 CD34 cells/kg (range, 1.34-32 x 10^6 CD34 cells/kg). The median T-cell dose/kg was 0.33 x 10^4 in CD-34 enrichment (range 0.04-13.1 x 10^4 cells/kg), and median alpha-beta T-cell/kg 1.31 x 10^4 in alpha-beta TCD (range 0.88-2.65 x 10^4 cells/kg). The post-SCB immunosuppressive treatment consisted of mofetil mycophenolate (MMF, 3 patients), MMF + tacrolimus (4 patients), none (6 patients), or other (3 patients). Filgrastim was administered in 9/16 patient for a median of 15 days (10-22). Results: Three lineage hematopoietic recovery was achieved in 14/16 patients. Absolute neutrophil count >500/uL was reached at median of 11 days after SCB (range 4-17 days). After a median follow-up of 17 months (range 2-84.5 months), the probability of OS was 70.6% (figure A.). The incidence of acute and chronic GVHD after SCB was 22.6% and 16.7% (figure B.), respectively. Non-relapse mortality was reported in 3/16 patients, and death due to relapse was observed in 1 patient. Conclusions: The SCB represent effective treatment for PGF with manageable complications and rapid neutrophil recovery, as reported in standard allo-HSCT. The major limitation is the need for cell processing facility equipped with specialistic equipment and availability of the allo-HSCT donor. Of interest, the efficacy and safety of TCD SCB product is worth further studies. Disclosure: Nothing to declare
Background: Harvesting of peripheral blood haematopoietic stem cells (PBSC) in multiple myeloma entails mobilisation with granulocyte colony stimulating factor alone (G-CSF) or in combination with chemotherapy, typically cyclophosphamide (Cyclo-G). Whilst historically Cyclo-G has been the technique of choice, more recently there has been a shift toward chemotherapy-free mobilisation. This retrospective study evaluates the mobilisation strategies utilised by a single regional comprehensive cancer centre. Methods: 83 patients underwent a total of 86 mobilisation procedures between January 2016 and September 2021. While a minimum of 2x106 CD34+/kg per autologous stem cell transplant (ASCT) is required for safe engraftment, higher doses of 3-5x106/kg are associated with optimal engraftment. It is common practice to collect sufficient PBSC for two ASCTs. Therefore our CD34+ minimum target was >4x106/kg, with an optimal target of >8x106/kg. The outcomes measured were stem cell yield, days of harvesting, rescue plerixafor use and mobilisation complications. Groups were compared using the Mann-Whitney or Chi-squared test. Results: 66 harvests used Cyclo-G mobilisation (Cyclophosphamide 2g/m2 then G-CSF 5mcg/kg for 10 days), and 20 used G-CSF (10mcg/kg for 5 days). 93.02% of harvests collected the minimum dose. The failure rate of Cyclo-G was 4.5% in comparison to G-CSF which was 15%. Patients receiving Cyclo-G yielded higher CD34+ doses (8.94 vs. 4.88 x 106/kg, p = <0.0001) and collected with fewer days of apheresis (1.6 vs. 2.4 days, p = 0.007). Harvests attaining the optimal collection target were more frequently seen with Cyclo-G (62% vs. 11%, p = 0.0001). Mobilisation with G-CSF resulted in a higher percentage of patients requiring salvage plerixafor (35% vs.13.6%, p = 0.0407). CD34+ yields were lower in patients who received IMiD containing induction regimens prior to mobilisation (5.18 vs. 8.98 x106/kg, p = 0.00003) and the use of Cyclo-G mobilisation did not overcome this negative impact (5.8 vs. 4.8x106/kg, p = 0.34). There were no recorded infective complications relating to mobilisation with G-CSF. Five patients receiving Cyclo-G were hospitalised, including one who required treatment for neutropenic sepsis. Conclusions: Patients receiving Cyclo-G mobilisation achieved larger stem cell harvests in fewer days, and required fewer doses of salvage plerixafor than G-CSF-only mobilisation. IMiD containing induction negatively impacted on CD34+ yield. Infectious complications rates were low. In the era of IMiD based induction and continuous IMiD maintenance, these data highlight the negative impact of IMiD treatment on CD34+ mobilisation and the importance of optimising the stem cell collection in the first line setting to ensure sufficient cells are collected for subsequent ASCTs. Incorporation of additional novel agents (e.g. daratumumab) into induction regimens has been shown to further compromise stem cell harvest yields, and consideration should be given to re-adoption of Cyclo-G as a standard of care for stem cell mobilisation. Disclosure: Nothing to declare
Background: Pandemic SARS-CoV-2 has increased the use of cryopreserved hematopoietic-stem-cells(HSC) in MUD transplants. The impact of unrelated donor graft freezing on outcome of allo-HSCT in terms of hematological recovery, acute graft-versus-host-disease(GVHD) and survival is still controversial. Methods: we evaluate the impact of variables related to cryopreservation on the viability of HSCs and engraftment in all MUD transplants performed at our Center using cryopreserved PBSC (CryoPBSC) from January 2020 to July 2021. The variables considered were: time between apheresis and freezing(t1), buffy composition, viability on satellite tube (48 hours after freezing) and bag (Trypan Blue and 7-AAD). We also compare graft composition, clinical characteristics, and outcome of 23 allo-HSCT from Cryo-PBSC (Cryo-Group) with 23 from fresh-PBSC (Fresh-Group) performed at the same period. Results: Tab.1 resume characteristics of 31Cryo-PBSC-MUD-transplants performed. Mean t1 was 43.5hours(23.8-53.5). Leukocytes on buffy were 245x103uL(128x103-484x103) with 24%(4-75) neutrophils(PMN). Median TB viability was 76(54-97) on freeze and 61%(27-89) on satellite tube. Mean time from freezing to reinfusion was 12.6 days(5.5-26.6). The post thawing 7-AADviable CD34s were 66%(29-89). Univariate analysis showed a tendency to inverse correlation between t1 and post thawing TB(p = 0.057, Spearman’s rho -0.3509). t1 correlated with PLT engraftment(p = 0.0036) while t1 didn’t correlate with neutrophil recovery (p = 0.2416). Buffy composition didn’t impact on post thawing viability. Hematological recovery occurred after 14days(10-17) for PMN > 500uL and 16days(11-30) for PLT > 20.000uL without cases of graft failure. The comparing sub-analysis between Cryo(complete data aviable only for 23 transplants) and Fresh group(23 transplants) shows no significant differences in clinical characteristics of patients, donors and transplants. In Cryo-Group median time from apheresis to cryopreservation was 1.78 days(0.99-2.23) while median time from cells collection and reinfusion was 15.04 days(7.66-25.45). In the Fresh Group median time from apheresis to reinfusion was 1.57 days (0.89-2.4). Viable CD34 + cells infused were significantly lower in Cryo-Group(4.98x106/kg vs 7.02x106/kg; p = 0.001). All patients engrafted with no statistical differences in neutrophils and platelets recovery. No differences in transfusion needs and acute GVHD ≥ 2 incidence(36%Cryo-Group vs 39% Fresh-Group;p = 0.463) were recorded. All patients were alive 100-days after transplant in Cryo-Group. Two out 23 patients in Fresh-Group died due to infections. Conclusions: In our series, TB on freezing seems to be influenced by t1 even though it doesn’t reach statistical significance. Viability 48hours after cryopreservation is a good indicator of post thawing viability. No differences between Cryo and Fresh groups were found in engraftment, acute GVHD ≥ 2 incidence and 100-days survival, despite a lower CD34 + infused dose in Cryo-Group. Disclosure: Nothing to declare
Background: Successful autologous hematopoietic stem cell transplantation depends on safe cryopreservation and storage of the cellular product. Any problems during these steps could result in cellular material loss. Methods: Clinical case: 35-year-old man diagnosed with Hodgkin’s lymphoma, initially treated with six cycles of BEACOPP and three cycles of BGD (Bendamustine, Gemcytabine, Dexamethasone) chemotherapy, was referred for autologous HSC transplantation. The patient was in CR phase. His blood type was A, RhD positive. Mobilization of HSCs was performed using ID-ARA C (total dose 3200 mg) on days 1 and 2, combined with G-CSF at a dose of 10μg/kg/day for 5 days, starting at +9 day. As a result of the apheresis, 22.5×106 per kg CD34-positive cells was obtained. According to the local protocol, cellular product was stored in 4°C, with shaking, until cryopreservation. Next day, the appearance of cell suspension has changed – the red blood cells were clumped. When the product has been transferred into temperature room, the aggregates disappeared, without hemolysis. After cooling, the cells clumped again. Despite this situation, the cells were frozen in 4 cryopreserved bags, in a mixture containing 5% DMSO, and stored in liquid nitrogen vapor. If the aggregates had not dissolve after thawing of the transplant material, the infusion of HSCs would be impossible. To check for clumping post-thaw, one bag was thawed and heated to room temperature 1 week after freezing. No aggregates of cells were seen, and the 200µm filter was not blocked during the transfer of the cell suspension into the transfer bag. Based on this result, the material was considered suitable for transplantation. Results: The patient underwent conditioning chemotherapy before autologous transplantation with BeEAM (bendamustine, etoposide, cytarabine, melphalan), and the transplant material was successfully infused. The viability of cells was low (65%). They were no adverse effects during the infusion, and no post-transplant complications occured. WBC engraftment (WBC > 1.0G/L) and ANC engraftment (WBC > 0.5 G/L) was achieved on day 9, and platelet engraftment (Ptl > 20G/L, without platelet transfusion for 7 days during seven preceding days) on day 13. Conclusions: This case report presents safe autologous transplantation in patient with reversible agglutination. This phenomenon could be associated with cold agglutinin disease (CAD), which may occur in lymphomas. CAD is often not clinically important, however, there is limited information about cryopreservation of cell suspension in this kind of situtation. We have found only two case reports concerning the successful transplantation of HSCs in patient with cold agglutinins, however, in both patients the red cells clumped in room temperature [Crowther et al, 2006; Badami et al., 2017]. HSCs from patient with CAD may be used for transplantation, however, checking the quality of the material by thawing one cryopreserved bag before conditioning is recommended to assure its viability. References: Badami et al. Autologous peripheral blood stem cell harvest and transplant in a patient with cold agglutinin disease secondary to lymphoma. Transfus Med. 2017;27:222-224. Crowther t al. Successful autologous peripheral blood stem cell harvest and transplant in a patient with cold agglutinins. Bone Marrow Transplant. 2006;37:329-30 Disclosure: N/A
Background: Autologous hematopoietic stem cell transplantation (ASCT) is widely used as a consolidation therapy in non-Hodgkin’s lymphoma (NHL), recurrent or refractory classic Hodgkin’s lymphoma (HL), Multiple Myeloma (MM) and other malignant diseases. However, in Mexico and other countries in Latin America, about 26% of general population do not have health insurance and there is a limited number of centers which performing ASCT; these factors hinder the access to this treatment in patients with hematological cancer. Considering the financial and infrastructure limitations, it is imperative to create strategies to carefully select patients for the transplant program, to reduce the risk of exclusion during the process and optimize the resources. This study aims to identify those factors that directly exclude patients with hematological and non-hematological malignancies for ASCT in a single center in Mexico. Methods: A retrospective study was conducted in patients with hematological and non-hematological diseases, candidates. for ASCT as consolidate therapy, at the Instituto Nacional de Cancerología (INCan), in Mexico, between January 2010 and. December 2020. The data was analyzed using. SPSS statistical software v23. Results: A total of 334 patients with hematological and non-hematological neoplasm who were candidates for an ASCT were analyzed. 284 (84%) received an ASCT (Multiple Myeloma n = 131, HL n = 64, NHL n = 133 and Germ cell tumor n = 5), while 50 patients (15%) failed to complete ASCT process. Causes that directly led the exclusion of this patients were identified and are summarized in table 1. The characteristics of patients not being able to continue to an ASCT were a median age 52 years (range 18-70), male predominance (54%). Most of the patients excluded had the diagnosis of Multiple Myeloma n = 21 (42%), followed by Non Hodgkin’s lymphoma n = 20 (40%) and Hodgkin’s lymphoma n = 9 (18%). The most common causes of exclusion was the relapse of the disease and peripheral blood stem cell mobilization failure. There were non-statistical significant differences results between both groups. Table 1. Exclusion cause for ASCT. Conclusions: The Instituto Nacional de Cancerología (INCan) is one of the few centers in Mexico performing HSCT. While 85% of the patients who are candidates for an ASCT, 15% of the patients fail to proceed with the transplant program. The main causes of failure to reach an ASCT are the relapse of the disease and peripheral blood stem cell mobilization failure, both constitute more than half of the situations that excluded these patients. Strategies to prevent the identified causes of exclusion from the transplant program have to focus on an earliest referral of the patients, reducing the waiting time to perform the transplant, and to have access to better mobilization therapies. Disclosure: Nothing to declare.
Background: Current guideline suggest using filgrastim or tbo-filgrastim for mobilization of hematopoietic progenitor cells in autologous setting. However, previous studies have suggested other forms of granulocyte colony stimulating factor (G-CSF) are equally efficacious, possibly with fewer aphereses required. Thus, we prospectively studied the efficacy of lenograstim, a glycosylated recombinant form of G-CSF, in multiple myeloma (MM) patients. Methods: From November 2011 to January 2020, 98 MM patients undergoing autologous stem cell transplant (ASCT) from 8 academic centers in Korea were enrolled. Donors were mobilized with subcutaneous lenograstim (Neutrogin®) with fixed doses of 10 ug/kg for four days. There was no dose adjustment of G-CSF during mobilization. The collection was performed on day 5, with goal of collecting 5 x 106 CD34 + cells/kg body weight. This study was carried out according to the Helsinki Declaration, and was approved by Institutional Review Board of each participating study center. All patients gave their informed consent. Results: Most of patients (N = 90, 91.8%) achieved targets of ≥ targets of 2 x 106 CD34 + cells/kg body weight and more than half of MM patients (N = 57, 58.2%) reached target of 5 x 106 CD34 + cells/kg body weight. Among those attaining of 2 x 106 CD34 + cells/kg, 48.0% (N = 47) patients met target requirement in a single leukapheresis. The median number of apheresis required for optimal collection of 5 x 106 CD34 + cells/kg was 2 (range 1-5). Half of patients (N = 49, 50.0%) showed optimal collection success within third apheresis. The mobilization failure rate was 8.2% (N = 8). The median number of CD34 + cell/kg using G-CSF only was 5.25 x 106/kg (range 0.49-13.47). There were ten patients (10.2%) with adverse events during the mobilization. The most frequently reported adverse event was bone pain (N = 6, 6.1%). Other side effects included diarrhea (N = 1, 1.0%), fever (N = 2, 2.0%), and abdominal pain (N = 1). Out of 98 patients, 93 were able to undergo ASCT. The median infused dose of CD34 + cell/kg body weight was 3.42 (range 1.80-11.00). Neutrophil engraftment was observed in all patients, and platelet engraftment was observed in 89 (95.7%) patients. The median time to neutrophil and platelet engraftment was 10 days (range 1-21 days) and 10 days (range 1-37 days), respectively. Table. Baseline characteristics and collection outcomes. Figure. Change of daily and total collected CD34 + cell count after starting apheresis Conclusions: Lenograsim can safely and effectively mobilize stem cells in MM autologous setting. Disclosure: Neutrogin® was provided by JW Pharmaceutical
Background: The worldwide pandemic caused by SARS-CoV-2 virus has brought significative burden to the Health Care system, including programs performing allogeneic hematopoietic stem cell transplantation (HSCT). The true impact on the whole procedure is still to be determined. We present the case of a safe bone marrow collection procedure from a SARS-CoV-2 positive donor. Methods: A 54 year-old woman, who came to our attention in September 2020 for pancytopenia, was diagnosed with severe aplastic anemia. The 55 year-old patient’s brother was selected as a potential HLA haploidentical donor. His past medical history was not significant. Pre-donation screening exams were permissive. Despite a first negative molecular test on nasopharyngeal swab, a new pre-operative test, performed on the basis of hospital policy, revealed positivity for SARS-CoV-2. The donor was completely asymptomatic. Considering the urgent need to treat our patient, as the general clinical conditions were rapidly worsening due to infectious complications, we decided not to stop the transplant procedure. Conditioning regimen was cyclophosphamide 300 mg/mq + fludarabine 30 mg/mq (days -6 to-3), GvHD prophylaxis was antithymocyte globulin 3,75 mg/Kg (days -1 and 0), total body irradiation (TBI) 400 cGy (day -1). Results: Bone marrow harvesting was performed in an operating room which provided airborne infection isolation routine. The involved medical team wore enhanced personal protection equipment. The procedure was free from clinical and anesthesiological complications. SARS-CoV-2 RNA on the product was found negative in real-time PCR. In the absence of any symptom, the donor was discharged the day after the procedure and was put in home isolation. Our patient received the processed product, which consisted in CD34+ cells 3.54 x 10^6/kg, total nucleated cells (TNC) 3.54 x 10^6/kg, CD3+ cells 0.285 x 10^8/kg, in 270 ml. The recipient was negative for SARS-CoV-2 on nasopharyngeal swab after stem cell infusion. Conclusions: Our case enlightens a successful bone marrow harvesting procedure from a SARS-CoV-2 positive donor, which was organised in urgent clinical need. It demonstrates that transplantation from asymptomatic positive donors is feasible and safe, as viral transmission did not happen, since the stem cell product was RT-PCR negative for SARS-CoV-2. Although EBMT does not recommend stem cell donation from positive individuals, donor care and selection in this pandemic era should be revisited, as SARS-CoV-2 swab positivity itself might not be an exclusion criteria for bone marrow donation. Further data are needed to assess whether harvested marrow could transmit SARS-CoV-2. Specific measures are also needed to provide the safety of the bone marrow collection medical team. Disclosure: Nothing to declare.
Background: Following the GRIFFIN trial data publication, we reviewed the use of D-VRd induction (4 cycles) and consolidation (2 cycles) as the new standard of care in transplant eligible newly diagnosed multiple myeloma (NDMM) patients and our ability to successfully mobilise stem cells compared with a historic cohort of patients who were treated with RVd and having SCM as part of planned first line treatment for Myeloma. GRIFFIN trial data showed a median CD34+ cell yield of 8.2 x 106/kg with the common requirement of plerixafor and the time to engraftment was indifferent in both arms. Methods: All patients were treated with D-VRd Induction (4 cycles) prior to the stem cell collection, and all patients who were mobilised were all given Cyclophosphamide 1.5g/m2 and GCSF. Historic data of patients who were treated with RVd Induction was collected in order to compare the SCM yield time to neutrophil engraftment and times. Results: 7 patients have been successfully mobilised. 1 patient is currently undergoing mobilisation and 4 patients are still on induction therapy. Of this 1 patient had to be mobilised twice due to failed first harvest and only 1 patient required plerixafor to successfully mobilise stem cells. The median CD34+x106/kg yield was 4.07 (1.4-6.94) and the median day to neutrophil engraftment was 12.4 (range 12-14). Of the above patients, 5 have completed their AHSCT and median CD34+ x 106/kg yield was 4.64, median day to neutrophil engraftment was 12.4 and the median CD34+ x 106/kg dose reinfused was 2.68. When compared to our historic data of patients who completed induction (4 cycles) with RVd the median CD34+x106/kg yield was 5.69, median day to neutrophil engraftment was 11.2 and median CD34+ x 106/kg dose reinfused was 4.10. Conclusions: Quadruplet therapy is now becoming the standard of care in NDMM. Patients can be successfully mobilised after DVRd 4 cycles with good engraftment kinetics. However, there was a trend to lower yields (18.4%), 1 day longer median time to engraftment and a lower quantity of cells being reinfused (34.6% less). It is important to schedule in stem cell collection after no more than 4 cycles of induction therapy assuming the disease is responding to reduce the risk of sub optimal collection in particular if a tandem procedure is being planned. Disclosure: Nothing to declare
Background: Plerixafor is a valuable stem-cell mobilizer for use in combination with G-CSF in patients with lymphoma or multiple myeloma, particularly those who are poor mobilizers. This study aimed was to evaluate the real-life impact of plerixafor+G-CSF mobilization regimen in cases where mobilization failed with at least two regimens for autologous stem-cell mobilization for patients MM and lymphoma. Methods: Between August 2009 and December 2021, 513 stem-cell transplants were performed in our center. It could be collected with a plerixafor+G-CSF regimen in 21 of 373 cases. Patients received G-CSF 10 µg/kg in the morning (at 06:00) for four consecutive days, then a single subcutaneous injection of plerixafor 0.24 mg/kg in the evening of day 4 (at 23:00). Aphereses was begun in all patients on the 5th day. Accepted the target CD34 + cells yield was ≥ 4 x 106/kg. Continue G-CSF and plerixafor up to 4 aphereses until 4x106 CD34 + cells /kg were collected. We can perform all apheresis collections with central venous access. Results: The patient’s characteristics are summarized in Table 1. 62% of the cases requiring plerixafor+G-CSF were due to lymphoma. The number of cases diagnosed with MM was six, 29%. A total of 75% of plerixafor+G-CSF patients reached target after two aphereses; only one patient required three procedures. Neutrophil engraftment of 16 autologous transplants was +12 to > 500 median day was reached (69 %). Platelet engraftment was +17.day arrived (50%). Plerixafor+G-CSF was well tolerated. The most common plerixafor-related adverse events were diarrhea (4.7%) and nausea (4.7 %). In this study, the addition of the results of plerixafor+G-CSF leads to increased stem cell collection in a shorter with no concomitant increase in adverse events. Table 1. Patient Characteristics. MM: multipl myeloma, NHL: non-Hodgkin lymphoma, HD: Hodgkin Disease Conclusions: In conclusion, our data suggest that patients who were failing an initial mobilization attempt, in particular, can use those with lymphoma, and MM or ıt can be used at an earlier stage in severely treated patients. Disclosure: No conflict of interest
Background: South Africa reported its first two SARS CoV-2 positive cases on 2 March 2020. To date there have been over 250 million infections and three periods of increased transmission: wave 1 (June-July 2020), wave 2 (December 2020 to February 2021) and wave 3 (June 2021 to September 2021). The South African National Blood Service (SANBS) provides a haematopoietic stem cell transplant (HSCT) service to 18 public and private clinical facilities across South Africa. In 2019 we performed a total of 329 HSCT collections, averaging 27 per month. Due to the pandemic, HSCT scheduling was disrupted. Reasons for this include governmental lockdown levels during specific periods of heightened infections, restricted availability of hospital beds for non-Covid-19 diseases and local and international recommendations on HSCT during the pandemic. Methods: All paediatric and adult HSCT collections performed by SANBS from March 2020 to October 2021 were analysed. The HSCT collections during and between the three Covid-19 waves were analysed in conjunction with demographic data including gender, age, diagnosis, type of transplant and type of facility. Results: During the 15 month period, 1st March 2020 to 31st October 2021, 344 HSCT collections were performed on 201 patients. The median age was 48 years, females accounted for 44.5% (n = 153) of HSCT collections and 95.9% (n = 330) were autologous HSCT collections. Indications for HSCT collections include multiple myeloma (n = 185), Non Hodgkin Lymphoma (n = 48), acute leukaemia (n = 39), Hodgkin Lymphoma (n = 34), neuroblastoma (n = 28), multiple sclerosis (n = 6), amyloidosis (n = 2), retinoblastoma (n = 1) and meduloblastoma (n = 1). 40.4% (n = 139) of HSCT collections were performed in public facilities. The number of HSCT collections per month are shown in Figure 1. The orange colour variants express waves 1, 2 and 3 respectively. The average(range) number of HSCT collections per month was 17(3-36). The average(range) number of HSCT collections in wave 1, 2 and 3 were 19(12-26); 7.6(4-12); 17.8(4-28) respectively, compared to 19.3(3-36) during ‘non-wave’ months. Conclusions: Covid-19 resulted in an overall decrease in HSCT collections at SANBS. Despite many challenges, HSCT collections at SANBS were performed during the three Covid-19 waves however they were decreased in comparison to ‘non-wave’ months. Wave 2 showed a marked decrease in HSCT collections with a rebound directly after. Clinical Trial Registry: N/A Disclosure: Nothing to declare
Background: High dose chemotherapy followed by autologous stem cell transplantation (ASCT) is the standart treatment approach for multiple myeloma (MM) patients. Peripheral blood stem cells (PBSCs) have become the most common prefered source for ASCT however nearly 15% of MM patients experienced poor PBSCs mobilization due to many factors such as higher age, previous chemotherapies and radiation. Our aim is define the factors of poor mobilization in patients with MM. Methods: In total, 110 MM patients who underwent autologous PBSC collection at Ege University Department of Hematology between january 2017 and December 2020 were evaluated retrospectively in our study. The patients were divided into two groups (patients with successful PBSC mobilization and patients who had poor PBSC mobilization) and risk factors were compared between these groups. Results: The median age was 58 (range, 31-71 years) years at the time of diagnosis and 59.1% (65/110) of patients were male. The median time from diagnosis to mobilization date was 7 months (range, 1-137 months). Number of patients who received G-CSF or cyclophosphamide plus G-CSF before peripheral stem cell harvesting were 73 (66.4%) and 37 (33.6%) respectively. In 98 of 110 patients (89.1%, ≥2 × 106/kg CD34 + PBSCs were collected at first apheresis. Twelve patients (10.9%) had poor mobilization and most of them (11/12) of them were male (p < 0. 0001). Three of 12 patients were previously treated with ASCT. Immunomodulatory drugs (thalidomide or lenalidomide) were most commonly used in patients with poor mobilization (p < 0.012). Conclusions: Mobilization failure was observed more frequently in males, patients with previous ASCT and treated with immunomodulatory drugs. Disclosure: no conflict of interest
Background: The collection of hematopoietic progenitor cells (HPC) by apheresis is a routine procedure for hematopoietic cell transplant. Although usually performed with peripheral venal access, a central venous catheter (CVC) is sometimes required, particularly in the autologous setting. Placing a CVC not only carries risk for the patient but also increases the cost of the procedure, as it requires the patient to be admitted and the use of ultrasonic guidance. The main risks are venous thrombosis and catheter-related blood infections. Predicting the success of the harvest is thus extremely helpful in avoiding health risk and cost overruns. Measuring circulating CD34 + cells/ µl in the mobilized peripheral blood (PBCD34) is the most widely used method to predict the success of the harvest, defined as a minimum of 2x106 CD34 + /kg of the patient. A minimum count of 10 to 20 PBCD34 is usually recommended. The usefulness of total leukocytes counts in the PB (PBWBC) for this purpose has also been investigated, but it appears not to be a good indicator of successful harvesting. Our goal was to determine a threshold of CD34 + and total leukocytes in the peripheral blood (PB), capable of predicting a successful HPC collection by apheresis, prior to a CVC placement. Methods: We retrospectively analysed all apheresis collections performed via CVC in our Institution from 2017 to August 2021, in a total of 176 patients. PBWBC and PBCD34 counts on the first day of collection and the total dose of HPC/kg of the patient collected were registered. Individuals mobilized with Plerixafor were excluded from this study. HPC collection yelding more than 2x106 CD34 + cells/kg were considered as good efficacy. Results: We observed a high percentage of good efficacy harvests (93%) with a minimum of 5 PB CD34. On the other hand, on patients with less than 5, we only observed 5% of good efficacy collections. We found no threshold of total leukocyte counts capable of predicting collection success, in accordance with the literature. In Portugal, our Institution included, the use of ultrasound-guided peripheral vein cannulation is not usually performed, so a CVC placement is the available alternative, with a placing cost of 700€. Using this threshold, we identified 45 patients who did not achieved a good successful collection, which corresponds to an increase of 31.500€ to the cost of mobilization, collection, processing and cryopreservation of the grafts. Conclusions: Considering that a PBCD34 count can be obtained in less than an hour, that the placement of a CVC carries risks to the patient and increases the cost of the procedure, we recommend that every patient proposed to apheresis HPC collection via CVC should perform a PBPD34 count before the CVC placement, and the collection cancelled if less than 5 PBCD34 is obtained. The frequently suggested threshold of 10-20 PBCD34 excludes many good efficacy collections, in patients that might miss a future chance for collection. This strategy allows the identification of patients not likely to achieve a good harvest and that should not proceed to catheterization, avoiding cost overruns and exposer to unnecessary health risk. Disclosure: Nothing to declare
Background: Haploidentical transplantation (Haplo-SCT) and cord blood transplantation (CB-SCT) are both effective alternative treatments in patients suffering from Acute Myeloid Leukemia (AML) and lacking an identical HLA donor. In the last years, many centers have abandoned CB-SCT mostly due to concern about poorer immune recovery. In this multicenter study we compared the results using both alternative approaches in AML. Methods: We included data from 12 spanish centers. A total of 128 consecutive cases (92 Haplo-SCT and 36 CB-SCT) from 2009 to 2019 were collected. Median age at HSCT was 6.7 (0.4-20.3) years. Positive MRD was detected at HSCT in 44 cases and a previous HSCT was performed in 41 cases. In 75 Haplo-SCT patients, some kind of ex-vivo lymphocyte depletion (CD34 + enrichment or CD3/CD19 + , alfa/beta/CD19 + or CD45RA + selection) was performed. Cy-post was used in the other Haplo-SCT. The median infused cellularity was 8.0 x10^6/kg CD34 and 71.4 x10^7/Kg TNC for haplo-SCT and 1.5 x10^5/Kg CD34 and 5.7 x10^7/Kg TNC for CB-SCT. Results: At median follow up of 13 months (0.26-140) 79 patients (61.7%) are alive. The overall survival was 57% at 8 years of follow up for both procedures. The TRM at day +100 was 10.9% for haplo and 16.7% for CBT. Relapse was observed in 22 cases (22.8%) for Haplo-SCT and in 7 cases (19.4%) for CB-SCT. Graft rejection was reported in 14 cases (15.2%) for haplo and in 5 cases (13.9%) for CBT. Severe acute GvHD (grade III-IV) was observed in 22.8% and 13.9% for haplo and CB respectively. Chronic GvHD was reported in 20.7% and 8.3% in haplo and cord respectively. Relapse and chronic GvHD free survival were 52.7% for haplo and 56.7%, at 8 years of follow up, for CBT with no significant difference. Immune recovery was faster for Haplo-SCT in the first 3 months with a median of 178 CD4/mm3 for haplo compared with a median of 83 CD4/mm3 for CBT at day +90, although the median of NK cells count was higher in CB (501 cell/mm3 vs 198 cells/mm3). At 6 months the median CD4/mm3 was 280 and 431 for haplo and CB respectively. Conclusions: Our study supports that both haploidentical transplantation and cord transplantation show similar results in pediatric AML patients. We obtained comparable survival rates, although CB-SCT shows a trend to decrease rates of relapse and chronic GVHD, demonstrating that it should still be considered a valuable option, particularly for pediatric patients. Disclosure: No conflict of interest to declare
Background: With less stringent HLA-matching requirements, rapid graft acquisition, and excellent anti-tumour effects, UCB is in many ways an ideal graft source; but slow engraftment and high early TRM have historically limited its use. Methods: We conducted a retrospective single centre analysis to determine the outcomes of UCBTs between 1/1/05 and 1/11/20 in 81 adults with haematological malignancies. Data from 10 further patients transplanted 1/11/20-1/8/21 were included in analyses for engraftment and aGVHD. Cords were selected with the Anthony Nolan Graft Identification and Advisory Service and followed UK (Shaw, 2009; Hough, 2016) and international cord selection guidelines, prioritising FACT-accredited banks. Results: The median age at transplant was 43 years (range 18-70). 46 patients had AML, 16 ALL, five MDS, 10 lymphoma, 3 MPDs and 1 MM. 45 patients received RIC (fludarabine/cyclophosphamide /2GyTBI), 32 patients received MAC (fludarabine /cyclophosphamide/14.4 GyTBI) and 4 had “Midi” conditioning (fludarabine /cyclophosphamide/thiotepa/4Gy TBI). Median age in MAC and RIC groups were 29.5 and 52.5 respectively. GVHD prophylaxis consisted of ciclosporin and mycophenolate and 2 patients received ATG. Median weight was 75kg (range 47-139). 29 patients received a single UCBT, and 62 double UCBT. Pre-thaw median TNC infused per patient was 4.39x10^7/kg (range 2.0-7.13) and median CD34 dose per patient was 2.35x10^5/kg (range 0.12-6.32). 8 of 9 patients with a CD34 dose <1x10^5/kg were transplanted prior to publication of 2016 UK recommendations including CD34 dose as a selection criterion. All patients received mismatched cords with HLA matching 3/8, 4/8, 5/8,6/8, 7/8 (in 2, 13, 60, 48, 19 cords respectively (data unavailable in 46 patients)). Neutrophil engraftment occurred at median 20 days in the whole cohort (22 days in UCBTs pre-2017 and 18 days in UCBTs 2017-2021, (p = 0.19). 3 patients had delayed engraftment and 5 patients (5.4%) failed to engraft. In 81 patients eligible for survival analyses the median follow-up was 24 months (range 12-135). OS at 2 and 5 years was 59.8% and 53.1% respectively and was superior in recipients of MAC UCBT compared to RIC (5-year OS 72.9% vs 40.0%, p = 0.02). This was due to a reduction in relapse in the MA group (5-year relapse 8.5% vs 26.3%; p = 0.035) with no difference in NRM (2-year NRM 18.2% vs 30.6%;p = 0.18). 5-year OS in patients aged 60 or older was equivalent to younger patients (50.4% vs 53.9%; p = 0.71) and MRD positive acute leukaemia patients had no survival deficit compared to those who were MRD-negative (52% vs 60% p = 0.88). aGVHD grade I-IV and III-IV occurred in 53% and 13% of those alive at day 100 respectively. cGVHD and severe cGVHD occurred in 12% and 4.6% of those alive at one year respectively. Conclusions: Our data show that optimal CBU selection leads to excellent engraftment, and OS equivalent or superior to many matched donor analyses, but importantly with very low incidence of severe cGVHD. Patients receiving MAC derive particular benefit, and increasing the intensity of RIC for fitter older patients using midi regimens where possible may further improve outcomes. In the context of the COVID19 pandemic CBU also offers secure and rapid donor access. Disclosure: The authors declare no conflicts of interest
Background: Cryopreservation has become a risk-mitigation strategy for allogeneic transplant during the COVID-19 pandemic. Nevertheless, cryopreservation may adversely impact transplant outcomes. Previous experiences are non-conclusive, with some suggesting a detrimental impact on engraftment, chimerism, and GVHD (Maurer 2021; Hsu 2021), and others, especially in the haploidentical setting, which do not identify differences (Hamadani 2020). Methods: Here we report a retrospective analysis on 112 patients transplanted from allogeneic peripheral blood stem cells (PBSC) from 1° January 2016 to 1° September 2021. Forty-five patients received cryopreserved grafts and 67 fresh PBSCs. Both the groups were balanced in terms of age, diagnosis, disease status at transplant, conditioning regimen intensity, and donor source (Tab.1). Median follow-up was 504 days from transplant (range: 21 - 1530). GVHD prophylaxis consisted of high-dose post-transplant cyclophosphamide, cyclosporine, and mycophenolate-mofetil in the familiar haploidentical setting; pre-transplant Thymoglobulin®, cyclosporin and a short course of methotrexate for HLA-identical sibling, matched (10/10) and mismatched (9/10) unrelated donor (MUD and MMUD). Engraftment was defined as a ≥ 95% donor chimerism at day +30 in the presence of neutrophil-engraftment. Results: Despite a superimposable time to neutrophil-engraftment (defined as 500/mcl for 3 consecutive days) for cryopreserved vs fresh (median 18 days vs 16 days, p = 0.1), graft failure was significantly higher for cryopreserved products (13% vs 3%, p = 0.03). No differences could be depicted for grade 2-4 aGVHD (26% vs 15%, p = 0.1), cGVHD (15% vs 16%, p = 0.9), TRM (17% vs 12%, p = 0.4), relapse (11% vs 21%, p = 0.2), 2-years OS (73% vs 75%, p = 0.1). However, when data were stratified for the donor-source, incidence of grade 2-4 aGVHD resulted significantly higher for cryopreserved vs fresh in the HLA-identical setting (HLA-identical sibling and unrelated donor) (42% vs 3%, p < 0.001). Chronic GVHD, relapse, NRM and OS were similar in these two groups of patients. No significative differences emerged in patients receiving graft cryopreserved or fresh from haploidentical donor. Conclusions: Taken together, our data suggest a donor-independent, increased risk of graft failure with cryopreserved cells and a higher probability of aGVHD, which is limited to the HLA-identical donor. Cryopreservation may adversely impact engraftment due to a suboptimal post-thawing cell recovery, while the impact on lymphocytes’ fitness and the hypothetical selection of specific subpopulations is currently unknown. Further investigations in a larger multicentre setting, to explain the biological assumptions are urgently needed. Disclosure: Nothing to declare
Background: Granulocyte transfusion has been shown to reduce infection-related mortality in neutropenic patients. Objective: We detected an erroneous change in the HLA tissue type of the patient with granulocyte transfusion (GTX) given to patients diagnosed with Acute Myeloblastic Leukemia (AML) in the neutropenic period. Methods: Results: Case Report 1: A 26-year-old female patient was diagnosed with AML. The HLA tissue group of the patient was examined. The patient was given 7 + 3 (ARA-C + Dounoromisin) chemotherapy with the diagnosis of AML. In the bone marrow examination performed on the patient, myeloblast was detected with a rate of 70% compatible with AML in the flow cytometry. Gemtuzumab + FLAG (Fludarabine, ARA-C, G-CSF) were given. On the 8th day of this treatment, due to neutropenic fever and deep neutropenia (WBC: 0.02x103/µL neutrophil: zero), GTX (4x10 10) was given to the patient. HLA tissue group was studied again from the patient 2 hours after GTX. It was determined that the HLA tissue group studied for the second time was completely different from the HLA tissue group of the patient, which was studied for the first time. In the blood control, it was determined that the HLA tissue group of the patient had transformed into the HLA tissue group of the granulocyte donor. Case Report 2: A 32-year-old male patient was diagnosed with AML. The HLA tissue group of the patient was examined. The patient was given 7 + 3 chemotherapy with the diagnosis of AML. The leukocytes returned to normal, and the bone marrow flow cytometric examination revealed 4% myeloid blast, and the patient was considered in remission. Then, High dose ARA-C was given for the 1st and 2nd times. GTX (5.8x10 10)was given to the patient who had deep neutropenia (WBC: 0.1x103 µL/neutrophil 0.01x103/µL) on the 11th day after high dose ARA-C given for the second time. At the 2nd hour after GTX, blood was drawn again for HLA tissue group control. In the HLA tissue typing examined, there was no agreement with the HLA tissue group detected before the treatment. Tissue group similarity could not be determined exactly. Conclusions: HLA compatibility is the main criterion for donor selection in hematopoietic stem cell transplantation. Peripheral blood is frequently used for HLA tissue group determination. We checked whether the HLA tissue type from the peripheral blood changes with these foreign DNA containing cells from the peripheral blood after the GTX application to our patients who had deep leukopenia due to chemotherapy. We determined that this could change with the blood sample we took at the 2nd hour after GTX. In conclusion, HLA tissue typing from peripheral blood should be performed 24 hours after cellular transfusions containing DNA in deeply neutropenic patients given GTX. In this period, if tissue typing is to be examined, sampling should be preferred from regions such as buccal mucosa other than peripheral blood. Disclosure: Nothing to declare | true | true | true |
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PMC9628696 | 36319063 | Mengmeng Jiang,Yang Yang,Liling Niu,Ping Li,Yibo Chen,Ping Liao,Yifei Wang,Jingbin Zheng,Fengyang Chen,Huanhuan He,Hui Li,Xin Chen | MiR-125b-5p modulates the function of regulatory T cells in tumor microenvironment by targeting TNFR2 | 01-11-2022 | Tumor Microenvironment,CD4-Positive T-Lymphocytes,Immunotherapy,Lymphocytes, Tumor-Infiltrating | Background Tumor necrosis factor receptor type 2 (TNFR2) is primarily expressed by CD4+FoxP3+ regulatory T cells (Tregs), especially those present in tumor microenvironment. There is compelling evidence that TNFR2 plays a crucial role in the activation, expansion, and phenotypic stability of Tregs and promotes tumor immune evasion. Understanding of epigenetic regulation of TNFR2 expression in Tregs may help device a novel strategy in cancer immunotherapy. Methods MiR-125b-5p-overexpressing or knockdown murine CD4 T cells and Tregs were constructed, and the effect of miR-125b-5p on Tregs proliferation, suppressive function and TNFR2 expression were examined. In vivo antitumor efficacy of Ago-125b-5p (miR-125b-5p agomir) was evaluated in MC38 tumor bearing mice, and tumor-infiltrating Tregs and CD8+ cytotoxic T lymphocytes (CTLs) were analyzed. RNA-seq analysis was applied to reveal the genes and signaling pathways regulated by miR-125b-5p in Tregs. Results In this study, we found that TNFR2 was a direct target of miR-125b-5p. Overexpression of miR-125b-5p decreased the proportion of Tregs and their expression of TNFR2 and consequently inhibited its proliferation and suppressive function by regulating the metabolism-related signaling pathways. Moreover, in colon cancer bearing mice, the administration of Ago-125b-5p markedly inhibited the tumor growth, which was associated with reduction of Tregs and increase of IFNγ+CD8+ T cells in tumor environment. Furthermore, in human colon adenocarcinoma patients, we verified that miR-125b-5p expression was downregulated, and low levels of miR-125b-5p were associated with poor prognosis. Interestingly, the expression of miR-125b-5p and TNFR2 were negatively correlated. Conclusions Our study for the first time found that the expression of TNFR2 by Tregs was regulated by miR-125b-5p. Our results showed that miR-125b-5p had the capacity to inhibit the expression of TNFR2 and immunosuppressive activity of Tregs and consequently enhanced the antitumor efficacy. This property of miR-125b-5p may be therapeutically harnessed in the treatment of human cancers. | MiR-125b-5p modulates the function of regulatory T cells in tumor microenvironment by targeting TNFR2
Tumor necrosis factor receptor type 2 (TNFR2) is primarily expressed by CD4+FoxP3+ regulatory T cells (Tregs), especially those present in tumor microenvironment. There is compelling evidence that TNFR2 plays a crucial role in the activation, expansion, and phenotypic stability of Tregs and promotes tumor immune evasion. Understanding of epigenetic regulation of TNFR2 expression in Tregs may help device a novel strategy in cancer immunotherapy.
MiR-125b-5p-overexpressing or knockdown murine CD4 T cells and Tregs were constructed, and the effect of miR-125b-5p on Tregs proliferation, suppressive function and TNFR2 expression were examined. In vivo antitumor efficacy of Ago-125b-5p (miR-125b-5p agomir) was evaluated in MC38 tumor bearing mice, and tumor-infiltrating Tregs and CD8+ cytotoxic T lymphocytes (CTLs) were analyzed. RNA-seq analysis was applied to reveal the genes and signaling pathways regulated by miR-125b-5p in Tregs.
In this study, we found that TNFR2 was a direct target of miR-125b-5p. Overexpression of miR-125b-5p decreased the proportion of Tregs and their expression of TNFR2 and consequently inhibited its proliferation and suppressive function by regulating the metabolism-related signaling pathways. Moreover, in colon cancer bearing mice, the administration of Ago-125b-5p markedly inhibited the tumor growth, which was associated with reduction of Tregs and increase of IFNγ+CD8+ T cells in tumor environment. Furthermore, in human colon adenocarcinoma patients, we verified that miR-125b-5p expression was downregulated, and low levels of miR-125b-5p were associated with poor prognosis. Interestingly, the expression of miR-125b-5p and TNFR2 were negatively correlated.
Our study for the first time found that the expression of TNFR2 by Tregs was regulated by miR-125b-5p. Our results showed that miR-125b-5p had the capacity to inhibit the expression of TNFR2 and immunosuppressive activity of Tregs and consequently enhanced the antitumor efficacy. This property of miR-125b-5p may be therapeutically harnessed in the treatment of human cancers.
Tumor necrosis factor receptor type 2 (TNFR2), one of two receptors transducing TNF biological function, preferentially expressed by the most highly suppressive CD4+FoxP3+ Tregs, including those in the tumor environment. TNFR2 signals play decisive role in the activation, expansion, function and phenotypical stability of Tregs. There is compelling evidence that targeting of TNFR2 represents a novel strategy in enhancing the efficacy of tumor immunotherapy by eliminating Treg activity.
We found TNFR2 was a direct target of miR-125b-5p in Tregs. Overexpression of miR-125b-5p markedly inhibited TNFR2 expression and suppressive function of Tregs. Furthermore, in vivo treatment with miR-125b-5p agomir showed the capacity to inhibit tumor growth in mouse models, accompanied by the decrease of Tregs and increase of CD8 cytotoxic T lymphocytes (CTLs) in tumor tissues.
These findings for the first time provide experimental evidence that miR-125b-5p could directly regulate TNFR2 expression and suppressive function of Tregs, and this property of miR-125b-5p may be harnessed to design novel cancer immunotherapy.
Overcoming the immunosuppressive tumor microenvironment (TME) is a prerequisite for an effective immunotherapy against tumor.1 The accumulation of CD4+FoxP3+ regulatory T cells (Tregs) is largely attributable to the maintenance of immunosuppressive environment in tumor tissue,2–4 and consequently, the abundance of Tregs is associated with poor prognosis of broad spectrum of human cancers, including colorectal cancer,5 lung cancer,6 and breast cancer.7 Targeting of Tregs has become a strategy to enhance the efficacy of cancer immunotherapy. MicroRNAs (MiRNAs) are a series of highly conserved single-stranded nucleotide (20–25 nt) non-coding RNAs. The function of miRNAs is mediated by binding of RNA-induced silencing complex to complementary sequences of 3′ untranslated region (3′UTR) in the target mRNA, then inhibit the gene expression at post-transcriptional level.8–10 Through its regulation, miRNAs have been shown to act as oncogenes or tumor suppressors in cancer development and play critical roles in the regulation of immune responses.11 12 Increasing evidence demonstrated that miRNAs could regulate the development and suppressive function of Tregs. For example, miR-142-3p could impair the differentiation of Tregs and reduce Foxp3 stability in models of type 1 diabetes by directly downregulating TET2 expression.13 miR-21, a key regulator of Treg stability, is more highly expressed by Treg cells and can regulate Foxp3 expression.14 15 Our group for the first time found and reported that TNF potently promotes the activation and expansion of Tregs by interacting with TNFR2, one of TNF receptors that expressed by highly suppressive subset of Tregs.16 17 We also reported that tumor-infiltrating Tregs express markedly higher levels of TNFR2 as compared with peripheral Tregs.18 A recent single-cell RNA-sequencing study showed that in patients with metastatic melanoma, TNFR2 is one of the most upregulated genes in Tregs.19 Upregulation or downregulation of TNFR2, therefore, can modulate Treg activity for therapeutic purpose. Recent studies showed that TNFR2 could be regulated by miRNAs. For example, protumor TNFR2 signal in gastric cancer tissue may be regulated by miR-19a and miR-103a.20 Downregulation of TNFR2 by Let-7f-5p was reported to be the molecular mechanism underlying the osteoprotective effects of plastrum testudinis extracts,21 while miR-125a-5p was purported to be a positive regulator of osteoclast formation by targeting TNFR2.22 However, the epigenetic regulation of TNFR2 expression in Tregs remains elusive. In this study, we first predicted several potential miRNAs with pseudo binding sites in the 3′UTR of TNFR2 and found that TNFR2 was a direct target of miR-125b-5p. Through transfection of miR-125b-5p mimic or inhibitor (anti-miR-125b-5p), the effects of miR-125b-5p on TNFR2 expression by Tregs and on the proliferation and inhibitory function of Tregs were examined. Moreover, the antitumor efficacy of chemically synthesized miR-125b-5p analog (Ago-125b-5p) was determined in murine tumor models. The results of our study indicate that miR-125b-5p reduced the function of Treg cells by downregulation of TNFR2. This property of miR-125b-5p may be therapeutically harnessed in the treatment of human cancers.
A total of 31 pairs of colon adenocarcinoma (COAD) tissues and matched adjacent normal tissues were acquired from Tianjin Medical University Cancer Institute and Hospital (Tianjin, China). The patients were clinically and histopathologically diagnosed as COAD according to WHO criteria. None of the patients have received radiotherapy and chemotherapy before enterectomy.
Female wide type C57BL/6J mice (6–8 weeks old) were provided by the Animal Facility of University of Macau. The mouse colon cancer cell lines of CT26 and MC38 were purchased from American Type Culture Collection (ATCC). The flow cytometry fluorescent conjugated antibodies of FITC anti-mouse CD45 (30-F11), and PE-Cyanine7 anti-mouse CD4 (GK1.5) were purchased from eBioscience; PerCP-Cy5.5 anti-mouse TCRβ/CD3 (H57-597), PE anti-mouse IFN-γ (XMG1.2), and APC anti-mouse CD8a (53–6.7) were purchased from BD Pharmingen; PE anti-mouse CD120b/TNFR2 (TR75-89) was purchased from BioLegend, and APC anti-mouse Foxp3 (FJK-16s) for intracellular staining of Foxp3 was purchased from Invitrogen. Recombinant mouse IL-2 (200 µg/mL) and TNF (200 µg/mL) were obtained from BD Pharmingen. The miRNeasy Mini kit (Cat# 217004) was purchased from QIAGEN, and the PrimeScript RT reagent kit (Cat# RR047A.) was purchased from TAKARA.
The online software of Targetscan (http://www.targetscan.org/), miRDB (http://mirdb.org/), miRWalk (http://mirwalk.umm.uni-heidelberg.de/) and Starbase V.3.0 (http://starbase.sysu.edu.cn/) were used to predict potential miRNAs that target TNFR2 and the binding sites of miRNA in TNFR2 mRNA.
Mouse CD4+ T cells were purified from spleen and lymph nodes using CD4 (L3T4) microbeads. CD4+CD25+ T cells were purified using the mouse CD4+CD25+ Regulatory T Cell Isolation Kit (Cat# 130091041, Miltenyi Biotec), yielding a purity of ~90% for CD4+FoxP3+ Treg cells. The purified cells were cultured in a U-bottom 96 well plate in a medium of RPMI1640 supplemented with 10% FBS, 2 mM L-glutamine, 10 mM HEPES buffer, 0.1 mM non-essential amino acids, 1 mM sodium pyruvate, 1% penicillin (100 U/mL)/streptomycin (100 mg/mL), and 50 µM 2-Methylmercaptoethanol, at 37℃ in a humidified incubator with 5% CO2. For T cells culture, IL-2 (10 ng/mL, BD Pharmingen) was added into the culture medium to maintain the survival of CD4+ T cells, and TNF (10 ng/mL, BD Pharmingen) was added to activate the Tregs expansion and proliferation.
The mimics of miR-125b-5p, let-7a-5p, let-7b-5p, let-7c-5p, let-7d-5p, let-7k, and anti-miR-125b-5p and their relative negative control (NC) were chemically synthesized by RiboBio (Guangzhou, China). MiR-125b-5p, anti-miR-125b-5p or five other miRNAs and their negative controls (miR-NC, anti-miR-NC) were transfected into CD4+ T cells or CD4+CD25+ T cells by using riboFECT transfection reagent at a working concentration of 100 nM, following the manufacturer’s instructions. The lentiviral vector GV369 expressing mouse miR-125b-5p (LV-miR-125b-5p) and its empty vector (LV-miR-NC) were generated by Genechem (Shanghai, China). The CD4+CD25+ T cells were transfected with lentivirus at a multiplicity of infection (MOI) of 30 according to the manual protocol. All cells were prepared for analysis after 48 to 72 hours of transfection.
Total RNA and miRNA were extracted and purified with miRNeasy Kit (Qiagen), and 0.5 µg of RNA was transcribed into cDNA using PrimeScript RT reagent kit with DNA erase, according to the manufacturer’s instructions. The Bulge-loop miRNA qRT-PCR primer sets for the quantification of miR-125b-5p and other miRNAs were designed and synthesized by RiboBio. The quantitative RT-PCR detection for mRNA and miRNA using TB Green Premix Ex Taq II (Takara). β-actin and U6 were used as the normalization control for mRNA and miRNA. Primers used in this study were as follows: β-actin (forward: 5′-GCTTCTTTGCAGCTCCTTCGT-3′ and reverse: 5′- CCTTCTGACCCATTCCCACC-3′); TNFR2 (forward: 5′-ACAGTGCCCGCCCAGGTTGTCTTG-3′ and reverse: 5′-GAAATGTTTCACATATTGGCCAGGAGG-3′); Foxp3 (forward: 5′- CTTATCCGATGGGCCATCCTG-3′ and reverse: 5′- GTGGAAACCTCACTTCTTGGTC-3′). Data analysis were performed using the 2-ΔΔCт methods.
293T cells (2×105) were seeded in a 24 well plate, then transfected with wildtype pGL3-TNFR2 3′-UTR (WT) or mutant pGL3-TNFR2 3′-UTR (MUT) and pRL-TK renilla luciferase plasmid with miR-125b-5p mimic, or miR-NC using the lipofectamine 3000 reagent (Invitrogen) according to the manufacturer’s instructions. After 48-hour culture, the relative luciferase activity was determined by the dual-luciferase reporter assay system (Promega); the renilla luciferase value was used as the normalization for firefly luciferase value.
For in vitro assays of suppression of proliferation by Tregs, CD4+FoxP3+ Tregs (0.5~1×105 cells/well) were seeded in a U-bottom 96-well plate, cultured in complete RPMI-1640 medium with 10 ng/mL IL-2 and 10 ng/mL TNF, then transfected with lentivirus of miR-125b-5p (LV-miR-125b-5p) or scramble control (LV-miR-NC) for 2 days. CFSE-labeled CD4+CD25− effector T cells (Teff, 5×104 cells/well) were cocultured with 2×105 irradiated (3000 Rad) APCs (Antigen-presenting cells, T lymphocytes depleted splenocytes) and 0.5 µg/mL of soluble antimouse CD3 antibody, then Tregs were added to the wells at a desired ratio (Teff: Treg=1:1, 2:1, 4:1, 8:1). After 48～72 hours, CFSE dilution was determined by flow cytometry. The percentage of suppression by Teff/ Treg ratio was calculated by the following formula: [Y (Teffs alone) − Y (Teffs+Tregs)] / Y (Teffs alone) × 100. The Y value represents the average number of divided Teff cells.
Purified CD4+CD25+ Treg cells were cultured in RMPI1640 complete medium containing 10 ng/mL IL2 and 10 ng/mL TNF, then infected with GFP labeled lentivirus of miR-125b-5p (LV-miR-125b-5p) or scramble control (LV-miR-NC), respectively. After 2 days, FACS-sorted CD4+FoxP3+GFP+ Tregs and total RNA were also isolated. RNA sequencing libraries preparation and sequencing were conducted at Shanghai Genechem Co, Ltd. RNA libraries were generated using NEBNext UltraTM RNA Library Prep Kit for Illumina (NEB, USA) following manufacturer’s recommendations, and sequencing were performed on an Illumina Novaseq platform. For transcriptome sequencing, FPKM can be used as gene expression data.
C57BL/6J mice (female, 6~8 weeks old) were subcutaneously implanted with 0.1 mL PBS containing 5×105 MC38 tumor cells in the right flank. When tumor size reached 50~100 mm3, the tumor-bearing mice were randomly divided into two groups (n=5 mice/group), then intratumorally injected with chemical synthetic analogs of miR-125b-5p (Ago-125b-5p) or its negative control Ago-NC (2.5 nmol/mice, respectively) twice a week for a total of five doses. The tumor size (length and width) and bodyweight of tumor-bearing mice were monitored every 3 days. The tumor size was calculated by the formula: (length×width2 /2. After indicated treatments, the tumors were excised, minced and digested in RPMI1640 medium containing collagenase IV (100 U/mL) and 0.1 mg/mL DNase, and draining lymph nodes (dLNs) or spleen were minced and pushed into a 70 µm cell strainer to create single cell suspensions. Finally, cells were washed using PBS and stained with specific diluted antibodies to determine the contribution of lymphocytes to the development of antitumor immune defense.
Mouse inflammation cytokines were detected using the mouse inflammation kit by BD Cytometric Bead Array, including mouse IL-6, IL-10, MCP-1, IFN-γ, TNF and IL-12p70. For cytokines staining, 10 µL of mouse serum from treated mice were added 10 µL of each mouse inflammation capture bead, 10 µL PE detection reagent and 20 µL PBS, then incubated at room temperature and prevented light for 2 hours. Reconstitute mouse inflammation standards by serial dilutions were used to make a standard curve. Finally, flow cytometry analysis was performed to detect the cytokines level, then convert to concentrations according to the calibration curve.
A mouse TNFR2/TNFRSF1B ELISA kit was obtained from SinoBiological Inc (Cat# KIT50128, Beijing, China) for the quantitative detection of mouse soluble TNFR2 in peripheral blood serum from mice treated with Ago-125b-5p or Ago-NC. It is based on a Sandwich assay principle and carried out according to the manufacturer’s specifications. The concentration of mouse TNFR2 was calibrated according to the standard curve. All samples and standards were assayed in duplicate.
After blocking non-specific FcR, cells were incubated with appropriately diluted antibodies according to the manufacturer’s instructions. For intracellular staining of Foxp3, the cells were resuspended in fixation/permeabilization buffer; for IFN-γ staining, the cells were cultured and stimulated by PMA (phorbol 12-myristate 13-acetate) and ionomycin, then resuspended in fixation/permeabilization buffer. All cells acquisition was performed using a BD Fortessa cytometer (BD Biosciences), and data were analyzed by FlowJo software (V.11.0). FACS analysis was gated on the live cells only by using a LIVE/DEAD Fixable Dead Cell Stain kit.
GraphPad Prism software (V.8.3.0) was used for data analysis. The statistical significance between two groups was analyzed by Student’s t-test, and the multiple groups’ comparison was analyzed by one-way analysis of variance. The results data were represented as mean±SD (SEM) of multiple experiments. Statistical significance was determined by *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.
To determine the effect of miR-125b-5p on TNFR2, online resource in websites including Targetscan, miRDB, miRWalk and Starbase V.3.0 were used to predict the putative binding of miRNAs to 3′ untranslated region (3′UTR) of TNFR2. There are six common potential miRNAs obtained from the analysis of four databases, as summarized in figure 1A. The results suggested that several miRNAs might regulate TNFR2 expression. To verify the indeed miRNA that responsible for targeting TNFR2 expression, we examined the expression of miRNAs in CD4+ T cells. As shown in figure 1B, the expression of miR-125b-5p by CD4+ T cells was significantly lower as compared with five other miRNAs. Then we synthesized and transfected these six miRNAs mimic into CD4+ T cells and found that the proportion of TNFR2+ Tregs were markedly reduced. Interestingly, the overexpression of miR-125b-5p was much more potent in the inhibition of TNFR2 expression, in comparison with overexpression of five others (figure 1C, p<0.01). The 3′UTR of TNFR2 containing the complementary binding sites within miR-125b-5p was shown in figure 1D. In addition, the luciferase activities were tested in 293 T cells transfected with miR-125b-5p mimic, including wildtype (WT) or Mutant (MUT) version of predicted TNFR2 3′UTR binding sites of miR-125b-5p. We observed that the overexpression of miR-125b-5p reduced the luciferase activity in WT 3′UTR of TNFR2 but not mutated type (figure 1E). These data suggested that TNFR2 could be directly targeted by miR-125b-5p.
Previously, we and others showed that TNFR2 plays a crucial role in the phenotypical and functional stability of Tregs, and TNF-TNFR2 signal stimulates the activation of Tregs and enhances their suppressive activity.23 24 Thus, miR-125b-5p may reduce the stability of Tregs through downregulation of TNFR2 expression. To test this idea, CD4+ T cells were cultured in the medium containing IL-2 with or without TNF (10 ng/mL each). The result showed that TNF could markedly increase the proportion of FoxP3+ Tregs and TNFR2 expression by Tregs (online supplemental figure S1A, B p<0.001), as expected. Then, miR-125b-5p expression in CD4 T cells was overexpressed or suppressed by transfecting with miR-125b-5p mimic or anti-miR-125b-5p, respectively (the transfection efficiency was shown in online supplemental figure S1C). The result showed that the overexpression of miR-125b-5p in CD4 T cells markedly reduced the proportion of FoxP3+ Treg cells by 40%, and downregulated their TNFR2 expression by ~40% (figure 2A, B, p<0.001). In sharp contrast, silence of miR-125b-5p expression resulted in a 30% increase in the proportion of Tregs in CD4 T cells and an increase of ~25% TNFR2 expression by Tregs (figure 2C, D, p<0.01), which might be explained by the low levels of miR-125b-5b expression. Interestingly, the transcriptional levels of TNFR2 and FoxP3 expression were also markedly reduced in miR-125b-5p overexpressing Tregs (figure 2E, p<0.0001), while their expression were increased by the treatment with anti-miR-125b-5p (figure 2F, p<0.01). 10.1136/jitc-2022-005241.supp2 Furthermore, overexpression of miR-125b-5p also markedly reduced the proportion of FoxP3+ Tregs in TNFR1−/− CD4+ T cells by 37%, but it only resulted in a 25% reduction of Tregs in TNFR2−/− CD4+ T cells (online supplemental figure S2A, B p<0.01), which suggesting that miR-125b-5p may also downregulate FoxP3 expression in a TNFR2-independent manners. Moreover, overexpression of miR-125b-5p markedly decreased the expression of TNFR2 by TNFR1−/− Treg cells (online supplemental figure S2C, D p<0.001). Therefore, our data clearly indicated that miR-125b-5p could significantly reduce TNFR2 and FoxP3 expression in Tregs. 10.1136/jitc-2022-005241.supp3
There is compelling evidence that TNFR2 expression by Tregs is required for their immunosuppressive function.25 26 Thus, decreased expression of TNFR2 by miR125b-5p may attenuate Treg function. To test this possibility, Treg cells from normal C57BL/6J mice were MACS-purified based on surface expression of CD4 and CD25 and the purity of FoxP3+ Tregs >90% (figure 3A). The proliferation of Tregs was induced by stimulating with IL-2 and TNF as we previously reported.27 Results showed that the overexpression of miR-125b-5p reduced the proliferation of Tregs by 40% (figure 3B, C, p<0.01). In contrast, the proportion of proliferating Tregs was increased by 30% after the silence of miR-125b-5p (figure 3D, E, p<0.01). However, there was no difference in cellular viability between miR-125b-5p or anti-miR-125b-5p treatment and its controls (online supplemental figure S3). Therefore, we assumed that altered proliferative expansion may result in an increase or decrease in the number of Tregs, which will affect its function. Besides, transfection of miR-125b-5p or anti-miR-125b-5p did not alter the activation and proliferation of CD4+CD25− Teff cells (online supplemental figure S4A–E) and even CD8 T cells (data not shown). 10.1136/jitc-2022-005241.supp4 10.1136/jitc-2022-005241.supp5 To examine the effect of miR-125b-5p on the suppressive function of Tregs, CD4+CD25+ Treg cells were infected with LV-miR-125b-5p and cocultured with CFSE-labeled Teff cells. The result showed that the suppressive activity of miR-125b-5p-overexpressing Treg cells was markedly reduced (figure 3F, G, p<0.05), indicating that miR-125b-5p could regulate the function of Tregs which is at least partially resulted from the downregulation of TNFR2. Moreover, consistent with previous report, TNF treatment markedly reduced PD-1 expression but increased CTLA4 expression in Tregs (online supplemental figure S5A p<0.05). However, overexpression of miR-125b-5p markedly reduced CTLA4 expression, but increased PD-1 expression in Tregs (online supplemental figure S5B, p<0.01). In contrast, silence of miR-125b-5p showed an opposite effect (online supplemental figure S5C, p<0.05). Therefore, miR-125b-5p may preferentially regulate the proliferation and function of Treg cells. 10.1136/jitc-2022-005241.supp6
To further understand the molecular basis of regulatory effect of miR-125b-5p on Tregs, transcriptome sequence of LV-miR-125b-5p transfected Tregs showed a differentially expressed genes pattern (figure 4A), including downregulation of 112 (fold change <0.5), and upregulation of 218 genes (fold change >2) (figure 4B). As shown in figure 4C, the most upregulated genes include Tnfsf4, Bmp1, Hs3st1, Cd80, Gpr162, and the downregulated genes include Lag3, Ccl1, Pgk1, Cxcl9, Itga1, etc. The GO and KEGG pathway analysis revealed that the differentially expressed genes mostly involved in glycolysis, carbon metabolism, and HIF-1 signaling pathways, which are known regulators of cell metabolism (figure 4D). In addition, GSEA analysis showed that the expression of miR-125b-5p affect immune responses to tumor cell progression (figure 4E). Taken together, these findings further shed light on the important roles of miR-125b-5p in regulating the function of Tregs.
To further determine the effect of overexpression of miR-125b-5p on tumor growth, C57BL/6 J mice bearing established subcutaneous MC38 tumors were intratumorally injected with Ago-125b-5p, the growth of tumors was monitored. The result showed that Ago-125b-5p treatment significantly reduced the tumor growth (figure 5A, B, p<0.05) and potently elongated the tumor-bearing mice survival (figure 5C, p<0.05). The treatment with Ago-125b-5p resulted in a marked increase in the infiltration of CD3+ T lymphocytes into the tumor and draining LNs (figure 5D, p<0.01), while the ratio of CD8 versus CD4 T cells was increased in the tumor tissue as well as draining LNs (figure 5E, p<0.01). Furthermore, the treatment with Ago-125b-5p markedly reduced the serum levels of IL-6, IL-10, TNF and MCP-1, while levels of IFNγ were increased in tumor bearing mice (figure 5F, p<0.05). We further analyze Tregs and their TNFR2 expression in tumor environment and found that the proportion of Foxp3+ Treg cells in tumor-infiltrating CD4+ T cells was significantly decreased in mice treated with Ago-125b-5p (figure 6A, C, p<0.001), accompanied by a marked decrease of TNFR2 expression by Treg cells (figure 6B, D, p<0.001). Intriguingly, the soluble levels of TNFR2 were reduced in serum in mice treated with Ago-125b-5p (figure 6E, p<0.01). The expression of TNFR2 was also downregulated in tumor-infiltrating CD8+ T cells by Ago-125b-5p treatment (figure 6F, p<0.01), while the treatment increased the expression of IFN-γ in CD8+ T cells in tumor tissue (figure 6G, H, p<0.01) and in dLNs (data not shown). These data suggest that the reduction of TNFR2-expressing Treg cells and mobilization of CD8+ T cells were attributable at least in part to the antitumor effect of miR-125b-5p.
Through the bioinformatics analysis, we found that the mature sequence of miR-125b-5p is highly conserved among most vertebrate species, including human and mouse (figure 7A). To predict putative miRNAs that have binding sites in 3′UTR of human TNFR2, we analyzed the online database (Targetscan, miRDB and miRWalk). The result indicated that human TNFR2 may also be directly targeted by miR-125b-5p (figure 7B). We also found that human COAD tumor tissues expressed higher levels of TNFR2 and lower levels of miR-125b-5p, as compared with adjacent normal tissues according to the data from TCGA cohort (figure 7C). Furthermore, qRT-PCR analysis on 31 pairs of colon cancer tumor tissues, we could confirm that miR-125b-5p levels were significantly decreased in tumor tissues (figure 7D, p<0.05), and the relationship between TNFR2 expression and miR-125b-5p in human cancer was shown in figure 7E. χ2 test showed that low miR-125b-5p was associated with more advanced TNM stage and lymphatic metastasis (online supplemental figure S1). Furthermore, Kaplan-Meier survival analysis indicated that poor prognosis of patients with cancer was associated with low miR-125b-5p expression (figure 7F, p=0.0008). Taken together, these results suggested that miR-125b-5p was negative correlated to TNFR2 expression in human tumor tissues, aligning well with the results of our in vitro and in vivo laboratory mouse studies.
Disruption of the synthesis of miRNA in Tregs results in the complete dysfunction of Tregs and induces the occurrence of inflammatory disease and autoimmunity,28–30 indicating that miRNAs are critical in the development and function of Treg cells. It was proposed that miR-155 plays a role in regulating the activity of induced Treg (iTreg) and natural Treg (nTreg) by suppression of cytokine signaling 1 (a negative regulator of IL-2/STAT5 signaling pathway).31 Acting as an immuno-metabolic regulator, miR-142–5 p was shown to be essential for controlling Treg suppressive function through the maintenance of high intracellular concentration of cAMP.32 It was also shown that miR-21 was a positive regulator, while miR-31 acting as a negative regulator of human natural Tregs by modulating Foxp3 expression.15 Moreover, miR-17 was found to inhibit the function of Tregs by downregulating the coregulators of the Foxp3 transcriptional factor,33 and NF-kB-driven miR-34a could disrupt Treg/Th17 balance via targeting Foxp3.34 In this study, we for the first time found that miR-125b-5p could negatively control Treg activity via, at least partially, downregulating TNFR2 expression. The mature sequence of miR-125b-5p is evolutionarily conserved in diverse vertebrate species, making it an attractive target for translation research since the findings based on mouse study is likely to extrapolate to humans. Previously, the study on miR-125b-5p main focused on human cancer cells, such as hepatocellular carcinoma.35 It was found that the expression of miR-125b-5p was markedly downregulated in tumor cells and acted as a tumor suppressor.36 37 Nevertheless, miR-125b-5p was also reported to play a role in the regulation of immune responses. For example, it inhibits the activation and induces the apoptosis of human γδ T cells.38 In this study, through bioinformatics analysis, we first predicted six miRNAs that may bind with 3′UTR of TNFR2, including miR-125b-5p and five let-7 family members. Among them, miR-125b-5p most potently inhibited TNFR2 expression by CD4 T cells. In view of the potential role of miR-125b-5p in suppressing tumor growth and regulating immune cells, we further investigated the effect of miR-125b-5p on TNFR2 expression by Tregs. The results of our study suggest that miR-125b-5p directly acts on and negatively regulates TNFR2 expression in Tregs, consequently reduces the number of Tregs and attenuates their suppressive activity. The fact that miR-125b-5p has a similar effect on Tregs in WT and TNFR1-KO T cells, while a minor but a clear effect was observed in TNFR2-KO T cells (online supplemental figure S2A, B), suggesting that miR-125b-5p can regulate Tregs through TNFR2-dependent and TNFR2-independent mechanism. Indeed, it was reported that miR-125b could directly act on Foxp3 and promote autophagy.39 TNFR2 can also be expressed by effector CD8+ T cells, which drives a cytotoxic ability to CD8+ Teff cells during the early immune response, as well as an apoptosis signal to terminate the immune response.40 Besides, the high expression of TNFR2 on cytotoxic CD8 T cells may attenuate their antitumor effector function.19 Therefore, miR-125b-5p as a direct target on TNFR2 may mitigate the negative effect of TNFR2 expression by CD8 CTLs. This notion is supported by the fact that Ago-125b-5p treatment reduced TNFR2 expression by CD8 T cells in tumor bearing mice, while promoting the effector function of CD8 CTLs as evidenced by the upregulation of IFNγ (figure 6F, G, H). Further study is needed to clarify if the activation of CD8 CTLs in Ago-125b-5p-treated mice is resulted from the down regulation of TNFR2 on CD8 T cells, or from the elimination of Tregs. In TME, myeloid-derived suppressor cells (MDSCs) with potent immunosuppressive function can also express TNFR2 and the signal of TNFR2 can stimulate the accumulation and activation of MDSCs.41 42 Whether miR-125b-5p also regulate the activity of MDSCs through decreasing TNFR2 expression should be further clarified in future study. The soluble TNFR2 (sTNFR2) is produced by proteolytic cleavage of its membrane bound counterpart. Its level in serum is a potent immunosuppressive mediator and acts as a predictor of treatment response to inflammatory disease.43 44 Increased levels of sTNFR2 were reported to be associated with enhanced colorectal cancer risk and significantly accelerated their all-cause mortality.45 46 Therefore, serum sTNFR2 might be a powerful predictive factor for cancer treatment. This notion is also supported by the fact that, in our study, sTNFR2 were reduced remarkably by the treatment of Ago-125b-5p in tumor bearing mice, suggesting the reduction of sTNFR2 could be attributable to the mobilization of antitumor immune response. It was reported that intracellular metabolism played crucial roles in modulating the activation and expansion of Treg cells.47 48 The activation of Tregs by TNF-TNFR2 signaling were attributable to stabilize human regulatory T cells switch to glycolysis, with increased intracellular lactate levels and glucose consumption.49 To demonstrate the potential molecular mechanism of miR-125b-5p on the activity of Tregs, we performed an RNA-seq analysis in miR-125b-5p overexpressed Tregs and found that miR-125b-5p exerted its roles on Tregs by regulating the glucose and lipid metabolism related signaling pathways, including HIF-1 signaling pathway, carbon metabolism and glycolysis (figure 4). Therefore, the overexpression of miR-125b-5p partially disrupt TNF-TNFR2 signaling on suppressing Treg cells by interfering cellular metabolism related signaling. To date, several miRNAs-based therapeutics have reached clinical trials, including a miR-34 mimic for the treatment of cancer,50 and anti-miR-122 for the treatment of hepatitis.51 Since miRNAs are small, water soluble and can be injected subcutaneously or intravascularly, targeted delivery of miRNA with nanoparticles, plasmids, viral vectors or lipid delivery vectors into desirable site appears to be feasible and safe.52 Our future study will focus on the development of appropriate delivery methods for miR-125b-5p or its modified analogs in a preclinical and then clinical settings.
In summary, we for the first time found that miR-125b-5p could directly act on TNFR2. Our data favor the notion that downregulation of TNFR2 expression is at least partially attributable to the negative regulation of miR-125b-5p on Treg activity. Therefore, in addition to the known antitumor effect by directly acting on tumor cells, targeting of miR-125b-5p may represent a novel strategy in cancer immunotherapy by eliminating tumor infiltrating TNFR2-expressing Tregs. 10.1136/jitc-2022-005241.supp1 | true | true | true |
PMC9628902 | 35722711 | Yanghui Huang,Guangyu Zheng | Circ_UBE2D2 Attenuates the Progression of Septic Acute Kidney Injury in Rats by Targeting miR-370-3p/NR4A3 Axis | 11-05-2022 | Circ_UBE2D2,MiR-370-3p,NR4A3,septic acute kidney injury,apoptosis | As circ_UBE2D2 has been confirmed to have targeted binding sites with multiple miRNAs involved in septic acute kidney injury (SAKI), efforts in this study are directed to unveiling the specific role and relevant mechanism of circ_UBE2D2 in SAKI. HK-2 cells were treated with lipopolysaccharide (LPS) to construct SAKI model in vitro. After sh-circ_UBE2D2 was transfected into cells, the transfection efficiency was detected by qRT-PCR, cell viability and apoptosis were determined by MTT assay and flow cytometry, and expressions of Bcl-2, Bax and Cleaved-caspase 3 were quantified by western blot. Target genes associated with circ_UBE2D2 were predicted using bioinformatics analysis. After the establishment of SAKI rat model, HE staining and TUNEL staining were exploited to observe the effect of circ_UBE2D2 on tissue damage and cell apoptosis. The expression of circ_UBE2D2 was overtly elevated in LPS-induced HK-2 cells. Sh-circ_UBE2D2 can offset the inhibition of cell viability and the promotion of cell apoptosis induced by LPS. Circ_UBE2D2 and miR-370-3p as well as miR-370-3p and NR4A3 have targeted binding sites. MiR-370-3p inhibitor reversed the promoting effect of circ_UB2D2 silencing on viability of LPS-treated cells, but shNR4A3 neutralized the above inhibitory effect of miR-370-3p inhibitor. MiR-370-3p inhibitor weakened the down-regulation of NR4A3, Bax and Cleaved caspase-3 and the up-regulation of Bcl-2 induced by circ_UB2D2 silencing, but these trends were reversed by shNR4A3. In addition, sh-circ_UBE2D2 could alleviate the damage of rat kidney tissue. Circ_UBE2D2 mitigates the progression of SAKI in rats by targeting miR-370-3p/NR4A3 axis. | Circ_UBE2D2 Attenuates the Progression of Septic Acute Kidney Injury in Rats by Targeting miR-370-3p/NR4A3 Axis
As circ_UBE2D2 has been confirmed to have targeted binding sites with multiple miRNAs involved in septic acute kidney injury (SAKI), efforts in this study are directed to unveiling the specific role and relevant mechanism of circ_UBE2D2 in SAKI. HK-2 cells were treated with lipopolysaccharide (LPS) to construct SAKI model in vitro. After sh-circ_UBE2D2 was transfected into cells, the transfection efficiency was detected by qRT-PCR, cell viability and apoptosis were determined by MTT assay and flow cytometry, and expressions of Bcl-2, Bax and Cleaved-caspase 3 were quantified by western blot. Target genes associated with circ_UBE2D2 were predicted using bioinformatics analysis. After the establishment of SAKI rat model, HE staining and TUNEL staining were exploited to observe the effect of circ_UBE2D2 on tissue damage and cell apoptosis. The expression of circ_UBE2D2 was overtly elevated in LPS-induced HK-2 cells. Sh-circ_UBE2D2 can offset the inhibition of cell viability and the promotion of cell apoptosis induced by LPS. Circ_UBE2D2 and miR-370-3p as well as miR-370-3p and NR4A3 have targeted binding sites. MiR-370-3p inhibitor reversed the promoting effect of circ_UB2D2 silencing on viability of LPS-treated cells, but shNR4A3 neutralized the above inhibitory effect of miR-370-3p inhibitor. MiR-370-3p inhibitor weakened the down-regulation of NR4A3, Bax and Cleaved caspase-3 and the up-regulation of Bcl-2 induced by circ_UB2D2 silencing, but these trends were reversed by shNR4A3. In addition, sh-circ_UBE2D2 could alleviate the damage of rat kidney tissue. Circ_UBE2D2 mitigates the progression of SAKI in rats by targeting miR-370-3p/NR4A3 axis.
Sepsis is a disease characterized by systemic inflammatory response syndrome (SIRS) resulted from bacterial, viral or fungal infections [1, 2]. The host response to infection causes multiple organ failure, with the kidney being one of the most commonly affected organs [3, 4]. One of the most serious and common complications of sepsis is sepsis acute kidney injury (SAKI) [5, 6]. Sepsis is associated with up to 50% of acute kidney injury (AKI), and over 60% of patients with sepsis have AKI [6] whose mortality rate exceeds that of sepsis patients without AKI [7]. Recent studies have verified that the pathogenesis of sepsis-induced AKI includes a series of complex interactions between vascular endothelial cell dysfunction, inflammation and renal tubular cell apoptosis [8, 9]. However, efforts to translate these findings from the laboratory to the clinic in clinical trials have proven to be a failure. Therefore, it is necessary to comprehensively interpret the pathogenesis of septic renal injury in order to develop more effective treatment strategies. Circular ribonucleic acid (circRNA) is a kind of widely existing non-coding ribonucleic acid, which has a closed-loop structure and high stability [10, 11]. There is increasing evidence showing that many circRNAs are closely related to a variety of human diseases, including sepsis [12, 13]. However, limited attention has been paid to the role of circRNA in SAKI, which needs to be supplemented and improved. According to the relevant studies about the impacts of circRNA on sepsis-associated diseases, circ_0114428 was confirmed to regulate sepsis-induced kidney injury by targeting the miR-495-3p/CRBN axis [14], and circular RNA TLK1 promotes sepsis-associated AKI by modulating inflammation and oxidative stress through miR-106a-5p/HMGB1 axis [15]. CircRNA ubiquitin-conjugating enzyme E2 D2 (circ_UBE2D2) is a newly identified RNA molecule [16]. In retrospect of existing literature, we found that miR-942-5p, miR-122-3p, miR-370-3p and miR-337 are implicated in SAKI process [17-20], and circ_UBE2D2 has targeted binding sites with these miRNAs . However, the role of circ_UBE2D2 in SAKI has not been discussed. Whether it can affect the SAKI process through endogenous competitive binding with miRNAs is worth further exploring. NR4A3, a member of nuclear receptor subfamily 4, is an important regulator of cellular function and inflammation [21]. Studies have evidenced that NR4A3 is a pro-apoptotic gene which is strongly induced and expressed in AKI [22, 23]. Through predicting target miRNAs of circ_UBE2D2 and NR4A3 by StarBase and miRDB respectively, we uncovered that they can bind to miR-370-3p together. Thus, we speculated that circ_UBE2D2 may affect the process of septic kidney injury by targeting miR-370-3p to promote the expression of NR4A3.
The animal experiment in this study was approved by the Animal Ethics Committee of Nanfang Hospital.
Renal tubular epithelial cells HK-2 were purchased from the Cell bank of Chinese Academy of Sciences (China). All cells were cultured in DMEM/F12 medium containing 10% fetal bovine serum and cultured in a humidified atmosphere at 37°C with 5% CO2. To establish a SAKI model in vitro, HK-2 cells were treated with 10 μg/ml lipopolysaccharide (LPS, L2630, Sigma, USA) [24, 25].
HK-2 cells were inoculated into a 24-well plate and cultured at 37°C in a 5% CO2 incubator. MiR-370-3p inhibitor (miR20000722-1-5) and miR-370-3p inhibitor negative control (NC, miR2N0000001-1-5) were obtained from RiboBio (China). When the cell confluence reached 80% under an inverted microscope, miR-370-3p inhibitor, negative control, as well as short hairpin RNA against circ_UBE2D2 (shRNA-circ_UBE2D2) and shNR4A3 lentiviruses were transfected into cells that were cultured with DMEM containing 10% fetal bovine serum in a 24-well plate. After the culture medium was refreshed, the culture was continued for 3 days and cells were collected for subsequent experiments.
Targeted binding sites of circ_UBE2D2 or NR4A3 to miR-370-3p were predicted by StarBase (http://starbase.sysu.edu.cn/) and miRDB. The 3'UTR sequences of circ_UBE2D2 or NR4A3 were obtained from NCBI (https://www.ncbi.nlm.nih.gov/). Then, the 3'UTR of wild-type or mutant circ_UBE2D2 or NR4A3 (circ_UBE2D2-WT/NR4A3-WT, circ_UBE2D2-MUT/NR4A3-MUT) was separately cloned into pmirGLO vector (E1330, Promega, China). HK-2 cells were co-transfected with circ_UBE2D2-WT/NR4A3-WT/circ_UBE2D2-MUT/NR4A3-MUT and miR-370-3p mimic/mimic control under the help of Lipo3000. The transfection process was referred to cell transfection above. Dual-luciferase reporter detection system (E1910, Promega, China) was used to detect luciferase activity of each group 48 h after transfection.
Cell suspension containing about 2 × 103 HK-2 cells was transferred to the 96-well plate at 100 μl/well. 10 μl MTT reagent (ChemicalBook, China) was added to each well, and incubated at 37°C for 1 h. The absorbance value at 450 nm was determined by a microplate reader (BIO-RAD550, Bio-Rad, USA).
2× 103 HK-2 cells were seeded into each well of the 6-well plate, and then the cells were digested by trypsin to prepare single-cell suspension for cell apoptosis detection. The single-cell suspension was reacted with Annexin V-FITC staining solution and propidium iodide (PI) staining solution for 5 minutes (min) in the dark. Ultimately, a flow cytometer was exploited to detect cell apoptosis.
Twenty Wistar rats (weight: 200-220 g) purchased from SLAC Laboratory Animal Co., Ltd, were reared in cages under standard temperature and humidity with a 12 h light/dark cycle. Animal experiments were carried out in accordance with the Regulations of the Peoplés Republic of China on the Administration of Experimental Animals and the Guiding Opinions on the Ethical Treatment of Experimental Animals. The rats were randomly divided into 4 groups with 5 rats in each group, which were named as control group (rats were treated with sham surgery only), cecal ligation and puncture (CLP) group (rats were subjected to AKI surgery), CLP+shNC group (rats were treated with AKI surgery and injected with shNC) and CLP+ sh-cirC_UBE2D2 group (rats underwent AKI surgery and were injected with sh-cirC_UBE2D2).
For the establishment of SAKI rat model, the operation involved exposing the cecum, followed by ligation and perforation. Thereafter, the rats were placed in a warm environment to recover. Before sample collection (rats in the control group were intraperitoneally injected with pentobarbital 60 mg/kg immediately after intramuscular injection of normal saline), rats in each group were deeply anesthetized. The abdominal cavity was opened through a median incision, and blood samples were taken from the inferior vena cava, and then centrifuged at 3,000 r/min for 10 min to separate the serum. The levels of blood urea nitrogen (BUN) and serum creatinine (Scr) in serum were measured using a MedLab automated biomedical analyzer (Nanjing medease science and technology corporation, China). The rats were sacrificed by cervical dislocation. Both kidneys were extracted quickly, and the right kidney was stored at -80°C for western blot experiment. The renal tissue was fixed with 4%paraformaldehyde for subsequent hematoxylin-eosin (HE) staining.
The tissue fixed by fixative solution was cut into sections with a thickness of 5 µm. After routine dewaxation and hydration, the tissue sections were stained with hematoxylin and eosin, and finally sealed with neutral gum. The tissue sections were imaged under a Motic-6.0 Image acquisition system, and tissue damage was analyzed.
Other tissue sections were used for TUNEL staining. The number of cells in the kidney tissue and that of positive cells were counted under the microscope. The ratio of positive cell number to cell number in the kidney tissue was calculated, which was defined as the cell apoptosis rate.
Total RNA was extracted from cells and tissues by Trizol reagent (12183555, Thermo Fisher Scientific, USA) and then reverse-transcribed into complementary (c)DNA (cDNA synthesis kit, Takara) according to the instructions of kit. Power SYBR Green PCR Master Mix (Takara) was used for real-time PCR with an ABI7500 System (Applied Biosystems) as per the manufacturer's specification. The gene expression was quantified using 2-ΔΔCT method [26]. The RNA primer sequences are listed in Table 1. β-actin was used as an internal reference.
Cells and tissues were collected and lysed with Cell Lysis Buffer (R0278, Sigma-Aldrich, USA) to obtain total protein. The protein content of the sample was determined by BCA kit (55R-1544, Fitzgerald, USA) at 562 nm. Then, total protein was isolated with 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), transferred to PVDF membrane (24937, Sigma-Aldrich, China), and sealed with 5% skim milk powder at room temperature for 2 h. The sealed membrane was washed with Tris Buffered Saline with Tween (TBST) for 3 times. Subsequently, the membrane was reacted with the primary antibodies at 4°C overnight. On the next day, the membrane was washed with TBST for 3 times, followed by the incubation with secondary antibodies at room temperature for 2 h. The protein bands were detected by chemiluminescence (Sinopharm Chemical Reagent Co. Ltd, China) and photographed by gel imaging system. The gray value of the target protein is divided by the gray value of the internal reference β-actin to correct the error, and the obtained result represents the relative content of the target protein in a sample. The primary antibodies used are those against NR4A3 (ab259939, Abcam), Bcl-2 (ab32124, Abcam), Bax (ab32503, Abcam), Cleaved caspase-3 (ab32042, Abcam) and β-actin (ab8226, Abcam).
All measurement data were described by mean ± standard deviation. Data in Fig. 1A, Figs. 2D-2E were analyzed by independent sample t test, and one-way analysis of variance (ANOVA) was adopted for inter-group comparisons. All statistical analyses were implemented by Graphpad 8.0 software, and p < 0.05 was considered statistically significant.
In order to explore the effect of circ_UBE2D2 on LPS-induced renal tubular epithelial cell damage, we first detected the expression of circ_UBE2D2 in LPS-induced HK-2 cell damage. It was found that the expression level of circ_UBE2D2 in LPS-induced HK-2 cells overtly exceeded that in untreated cells (Fig. 1A). Besides, the expression of circ_UBE2D2 in cells transfected with circ_UBE2D2 silencing vector was detected to be signally reduced in LPS-induced HK-2 cells (Fig. 1B), which confirmed that our transfection experiment was successful. Therefore, follow-up biological function research can be carried out. Initially, through MTT assay, the effect of circ_UBE2D2 on LPS-induced cell viability was determined. The data revealed that circ_UBE2D2 silencing could impair the effect of LPS-induced on declining HK-2 cell viability (Fig. 1C). According to the findings of flow cytometry, circ_UBE2D2 silencing was discovered to inhibit LPS-induced apoptosis (Fig. 1D). Moreover, western blot was utilized to assess the effect of circ_UBE2D2 on LPS-induced HK-2 cell apoptosis-related protein and NR4A3 expressions. It was noticed that circ_UBE2D2 silencing could inhibit NR4A3, Bax and Cleaved caspase-3 expressions that had been up-regulated by LPS, and promote Bcl-2 expression that had been down-regulated by LPS (Fig. 1E).
StarBase and miRDB were introduced to predict the binding miRNAs of circ_UBE2D2 and NR4A3, respectively, and the co-targeting miRNA with the highest score (miR-370-3p) was selected as the research object (Fig. 2A). Then, StarBase was used to predict the targeted binding relationship between circ_UBE2D2 and miR-370-3p, and the genes that have targeted binding sites with miR-370-3p. The results indicated that circ_UBE2D2 and miR-370-3p had a targeted binding relationship, and NR4A3 was the target gene of miR-370-3p (Figs. 2B and 2C). Furthermore, the dual-luciferase reporter assay was conducted to verify the targeted binding relationship between circ_UBE2D2 and miR-370-3p and between miR-370-3p and NR4A3. After co-transfection of miR-370-3p mimic with circ_UBE2D2-WT or NR4A3-WT into cells, the luciferase activity of cells was observed to be lessened; however, co-transfection of miR-370-3p mimic with circ_UBE2D2-MUT or NR4A3-MUT barely affected the luciferase activity of cells (Figs. 2D and 2E). Next, the effect of circ_UBE2D2 on the expression of miR-370-3p was detected by qRT-PCR, uncovering that circ_UBE2D2 silencing could attenuate the inhibitory effect of LPS on miR-370-3p expression (Fig. 2F).
To fathom out the effect of circ_UBE2D2 targeting miR-370/NR4A3 on the biological functions of LPS-induced HK-2 cells, sh-circ_UBE2D2, shNR4A3 and miR-370 inhibitor were transfected into the cells. Firstly, the expressions of NR4A3 and miR-370 in the transfected cells were detected by qRT-PCR, confirming the success of transfection experiment where miR-370-3p inhibitor diminished miR-370-3p expression, and shNR4A3 reduced NR4A3 expression (Figs. 3A and 3B). Furthermore, cell viability was evaluated through MTT assay, the results of which affirmed that miR-370-3p inhibitor reversed the promoting role of circ_UB2D2 silencing in cell viability, and shNR4A3 offset such effect of miR-370-3p inhibitor (Fig. 3C). Thereafter, cell apoptosis was gauged via flow cytometry. The findings revealed that miR-370-3p inhibitor neutralized the suppressing effect of circ_UB2D2 silencing on cell apoptosis, and shNR4A3 weakened the facilitating effect of miR-370-3p inhibitor on cell apoptosis (Fig. 3D). In line with the data of western blot, miR-370-3p inhibitor reversed the suppression of Bax and Cleaved caspase-3 expressions by circ_UB2D2 silencing and the promotion of Bcl-2 expression, while the reversing effect of miR-370-3p inhibitor was impaired by shNR4A3 (Fig. 3E).
To further confirm the results obtained through in vitro experiments, the CLP rat model was constructed to conduct in vivo experiments and verify the above results. First of all, sh-circ_UBE2D2 lentivirus was injected into CLP rats, and then qRT-PCR was implemented to quantitate the expressions of circ_UBE2D2, NR4A3 and miR-370-3p in the rat kidney tissue of each group. It could be noted that circ_UBE2D2 expression in CLP rats was elevated, and then diminished under the injection of sh-circ_UBE2D2 lentivirus (Fig. 4A). Moreover, sh-circ_UBE2D2 also inhibited the up-regulation of NR4A3 in the CLP rats (Fig. 4B). Moreover, sh-circ_UBE2D2 promoted miR-370-3p expression in the CLP rats (Fig. 4C). The damage of the rat kidney tissue of each group was observed through HE staining. In the control rats, the kidney tissue was normal. However, the kidney tissue of CLP rats presented vacuolar degeneration and bleeding, while that of CLP rats injected with sh-circ_UBE2D2 lentivirus exhibited less vacuolar degeneration (Fig. 4D). Through TUNEL staining, the positive cells in the CLP rats were observed to be markedly increased compared to those in control rats, while the positive cells in CLP rats injected with sh-circ_UBE2D2 lentivirus were notably reduced. In addition, the kidney injury marker (BUN and Scr) levels were measured, and the results showed that BUN and Scr levels were higher in the serum of CLP rats than in the serum of control rats, but circ_UBE2D2 silencing dwindled the levels of BUN and Scr in the serum of CLP rats (Figs. 4F and 4G).
CircRNAs are a new type of endogenous non-coding RNA that can competitively bind to miRNA as competitive endogenous RNA (ceRNA) to regulate gene expression [27-29]. Previous studies have revealed the role of some circRNAs in SAKI [30-32]. For instance, Garcia et al. described the role of circRNA in sepsis specifically and discussed the feasibility of circRNA as a biomarker for sepsis diagnosis [32]. More and more evidence also suggested that the misregulation of circRNA is an early event of sepsis [33]. Circ_UBE2D2 has targeted binding sites with miRNAs involved in SAKI process; however, the role of circ_UBE2D2 in SAKI has not been clarified, and whether circ_UBE2D2 can affect SAKI process through endogenous competitive binding with miRNAs deserves further investigation. In our study, we detected abnormal up-regulation of circ_UBE2D2 in LPS-induced HK-2 cells, which rendered us more skeptical about the possibility of circ_UBE2D2 playing a biological role in the progression of SAKI. Then, we constructed circ_UBE2D2 silencing vector and transfected it into SAKI cells, and unveiled that sh-circ_UBE2D2 could enhance SAKI cell viability and inhibit apoptosis, initially revealing the role of circ_UBE2D2 in SAKI. In order to further expound the influence of circ_UBE2D2 on SAKI process by targeting downstream genes, bioinformatics analyses were applied and predicted that miR-370-3p and circ_UBE2D2 had a strong targeted binding relationship. Chen et al. identified four pairs of miRNA-mRNAs associated with sepsis, including miR-370-3p, through bioinformatics analysis [34]. In addition, it has also been confirmed that paclitaxel-dominated gene regulatory axis makes impacts upon promoting the chemotherapy effect of paclitaxel in alleviating SAKI [18]. Furthermore, long chain genes lncRNA NEAT1 and Lnc-MALAT1 can modulate the progression of sepsis through sponging miR-370-3p [35-37]. In this study, miR-370-3p inhibitor reversed the effects of circ_UB2D2 silencing on promoting cell viability and inhibiting cell apoptosis. Based on the above findings, circ_UBE2D2 was demonstrated to be capable of impacting SAKI progression via targeting miR-370-3p. Furthermore, studies have proven that NR4A3 is a pro-apoptotic gene that is strongly expressed in AKI [22, 23]. Based on StarBase prediction results, we found that circ_UBE2D2 and NR4A3 both can bind to miR-370-3p. Hence, we speculated that circ_UBE2D2 may influence the process of renal injury in sepsis by targeting miR-370-3p to promote NR4A3 expression. In accordance with our conjecture, shNR4A3 offset the impacts of miR-370-3p inhibitor on inhibiting cell viability and promoting apoptosis. In addition, to verify the effects of circ_UBE2D2/miR-370-3p/NR4A3 regulatory axis on the cellular level, we established the SAKI rat model, and the results corroborated that circ_UBE2D2 could still affect the expressions of miR-370-3p and NR4A3 in vivo. Circ_UBE2D2 silencing ameliorated renal tissue injury and apoptosis in SAKI rats. All in all, the study authenticates the positive role of circ_UBE2D2/miR-370-3p/NR4A3 regulatory axis in SAKI progression and demonstrates that silencing circ_UBE2D2 can promote cell viability while inhibiting cell apoptosis in SAKI by targeting miR-370-3p/NR4A3 axis, which provides a new possible gene target for SAKI treatment. | true | true | true |
PMC9628928 | 35791074 | Hang Li,Lixin Chai,Zujun Ding,Huabo He | CircCOL1A2 Sponges MiR-1286 to Promote Cell Invasion and Migration of Gastric Cancer by Elevating Expression of USP10 to Downregulate RFC2 Ubiquitination Level | 11-05-2022 | Gastric cancer,circCOL1A2,miR-1286,USP10,RFC2 | Gastric cancers (GC) are generally malignant tumors, occurring with high incidence and threatening public health around the world. Circular RNAs (circRNAs) play crucial roles in modulating various cancers, including GC. However, the functions of circRNAs and their regulatory mechanism in colorectal cancer (CRC) remain largely unknown. This study focuses on both the role of circCOL1A2 in CRC progression as well as its downstream molecular mechanism. Quantitative polymerase chain reaction (qPCR) and western blot were adopted for gene expression analysis. Functional experiments were performed to study the biological functions. Fluorescence in situ hybridization (FISH) and subcellular fraction assays were employed to detect the subcellular distribution. Luciferase reporter, RNA-binding protein immunoprecipitation (RIP), co-immunoprecipitation (Co-IP), RNA pull-down, and immunofluorescence (IF) and immunoprecipitation (IP) assays were used to explore the underlying mechanisms. Our results found circCOL1A2 to be not only upregulated in GC cells, but that it also propels the migration and invasion of GC cells. CircCOL1A2 functions as a competing endogenous RNA (ceRNA) by sequestering microRNA-1286 (miR-1286) to modulate ubiquitin-specific peptidase 10 (USP10), which in turn spurs the migration and invasion of GC cells by regulating RFC2. In sum, CircCOL1A2 sponges miR-1286 to promote cell invasion and migration of GC by elevating the expression of USP10 to downregulate the level of RFC2 ubiquitination. Our study offers a potential novel target for the early diagnosis and treatment of GC. | CircCOL1A2 Sponges MiR-1286 to Promote Cell Invasion and Migration of Gastric Cancer by Elevating Expression of USP10 to Downregulate RFC2 Ubiquitination Level
Gastric cancers (GC) are generally malignant tumors, occurring with high incidence and threatening public health around the world. Circular RNAs (circRNAs) play crucial roles in modulating various cancers, including GC. However, the functions of circRNAs and their regulatory mechanism in colorectal cancer (CRC) remain largely unknown. This study focuses on both the role of circCOL1A2 in CRC progression as well as its downstream molecular mechanism. Quantitative polymerase chain reaction (qPCR) and western blot were adopted for gene expression analysis. Functional experiments were performed to study the biological functions. Fluorescence in situ hybridization (FISH) and subcellular fraction assays were employed to detect the subcellular distribution. Luciferase reporter, RNA-binding protein immunoprecipitation (RIP), co-immunoprecipitation (Co-IP), RNA pull-down, and immunofluorescence (IF) and immunoprecipitation (IP) assays were used to explore the underlying mechanisms. Our results found circCOL1A2 to be not only upregulated in GC cells, but that it also propels the migration and invasion of GC cells. CircCOL1A2 functions as a competing endogenous RNA (ceRNA) by sequestering microRNA-1286 (miR-1286) to modulate ubiquitin-specific peptidase 10 (USP10), which in turn spurs the migration and invasion of GC cells by regulating RFC2. In sum, CircCOL1A2 sponges miR-1286 to promote cell invasion and migration of GC by elevating the expression of USP10 to downregulate the level of RFC2 ubiquitination. Our study offers a potential novel target for the early diagnosis and treatment of GC.
Gastric cancers (GC) develop as malignant tumors in the digestive tract and are reported to be the fifth most common carcinoma and the third-leading cause of cancer-associated mortality across the globe [1, 2]. Risk factors for the incidence of GC include helicobacter pylori infection, tobacco intake, age, sex, and diets high in salty and smoked foods [3]. Endoscopic resection and perioperative chemotherapy have been developed to treat GC according to the stage. Combination chemotherapy, such as platinum salt plus fluoropyrimidine, has been regarded as the standard of care [4]. Despite the progress made in treatment options for GC, the mortality rate and prognosis remain unsatisfactory due to the difficulty in timely diagnosis [5]. Most early-stage GC is asymptomatic or features only mild symptoms. Moreover, most GC patients are diagnosed at the advanced stage after the cancer cells have already metastasized to seriously threaten their life and health [2]. Therefore, it is important to study the underlying mechanisms of GC cell migration and invasion. Circular RNAs (circRNAs), characterized by their covalently closed structure, are associated with the progression of multiple cancers [6]. A large number of reports concerning the roles of circRNAs in GC have emerged. For instance, circFAM73A facilitates the cancer stem cell-like properties of GC via regulating the miR-490-3p/HMGA2 axis and β-catenin stabilization mediated by HNRNPK [7]; circLMO7 sponges miR-30a-3p to propel GC progression through the WNT2/β-catenin pathway [8]; and circFGD4 has been found to attenuate GC cell progression through regulation of the miR-532-3p/APC axis [9]. Exploring the roles of circRNAs in the incidence and development of GC is crucial to finding potential targets for the early diagnosis and effective treatment of GC patients. As the circRNA of our study, circCOL1A2 has been reported to propel the migratory and invasive abilities of tumor cells in tongue squamous cell carcinoma [10]. Its host gene, collagen type I alpha 2 (COL1A2), is recognized as a new biomarker for GC and promotes GC cell progression by targeting the PI3k-Akt signaling pathway [11, 12]. However, the effects of circCOL1A2 on GC cell migration and invasion have never before been studied. High-throughput sequencing technology has been widely used for cancer study and in the analyses of differentially expressed genes and cancer biomarkers [13]. ‘Bioinformatics’ refers to the utilization of mathematical, statistical and computational approaches to process and analyze biological data; it can be used to profile cancer-specific genes [14]. In our study, we utilized high-throughput sequencing technology and bioinformatics to screen out circCOL1A2 before performing experiments to probe into its role and mechanisms. Finally, by researching the regulatory mechanism underlying migration and invasion of gastric cancer, we are able to offer insight into novel, targeted therapies for this disease.
GES-1, AGS, HGC-27, MKN-45 and 293T cells were commercially acquired from ATCC (USA). All the cells were cultured in DMEM (MD207-050, Gibco-BRL/Invitrogen, USA), along with 10% FBS (16000-044, Gibco, USA) and 1% Penicillin-Streptomycin Solution under the condition of 5% CO2 at 37°C.
Full-length sequences of RFC2 were inserted into pcDNA3.1 vector for the construction of overexpression vectors, with vector itself as negative control (NC). Short hairpin RNA (shRNA) targeting circCOL1A2 and USP10 as well as sh-NC were constructed for interference. The overexpression and knockdown of miR-1286 were conducted using miR-1286 mimics and inhibitor.
qPCR was carried out as described before [15]. The total RNAs were extracted from GES-1, AGS, HGC-27 and MKN-45 cells using TRIzol reagent (T9108, Takara, Japan). After the removal of genomic DNA, the RNAs were subjected to reverse-transcription by Hifair III 1st Strand cDNA Synthesis SuperMix (11141ES10, Takara) for qPCR. The samples were measured using a qRT-PCR kit (QR0100-1KT, Sigma-Aldrich, USA). The results were calculated on a basis of 2-ΔΔCt. Bio-repeats were implemented in triplicate.
Western blot was carried out as described previously [16]. The total proteins of AGS, MKN-45 and 293T cells were extracted by using RIPA lysis buffer (KeyGEN BioTECH, China). Afterwards, the total proteins were treated with 10% SDS/PAGE Resolving Gel Master Mix (P0670-250ml, Beyotime, China) for separation, followed by transfer onto polyvinylidene fluoride (PVDF) membranes (T2234, Thermo Fisher, USA). Then, 5% skim milk was adopted to block the membranes, which were then subjected to incubation with primary antibodies overnight at 4°C. After the washes, the membranes were incubated with secondary antibodies for 1 h at room temperature. The results were then visualized and recorded. β-Actin was used as an internal reference. Bio-repeats were implemented in triplicate. The primary antibodies used in this assay include Anti-β-actin, Anti-MMP2 (ab181286, Abcam, UK), Anti-MMP9 (ab228402, Abcam), Anti-USP10 (ab109219, Abcam) and Anti-RFC2 (ab174271, Abcam).
Transwell assay was implemented as described previously [16]. The transwell assay, with or without Matrigel, was designed to assess GC cell invasion and migration respectively. The transfected cells were put into the upper chamber with serum-free medium, with complete medium supplemented to the lower chamber. After 24 h, the cells in the upper chamber were abraded. Following the fixation, the cells in the lower chambers were stained by 0.2% crystal violet solution before counting under the microscope. Bio-repeats were implemented in triplicate.
Wound healing assay was conducted as described previously [16]. GC cells were prepared in the 6-well plates. The wounds were scraped by pipette tips, followed by washing. The wound widths were examined at 0 h and 24 h by Image J.
These two assays were implemented as reported previously [17]. FISH was performed using the RiboTM Fluorescent In Situ Hybridization Kit (RiboBio, China). GC cells were treated with 4% PFA for fixation. We then used 0.5% Triton X-100 to permeabilize the cells for 15 min at 4°C. Subsequently, digoxigenin-labeled (DIG-labeled) target gene probe or control probe mix was used to incubate the cells for 4 h at 55°C. Hoechst-conjugated Anti-DIG antibodies were adopted to detect signals. A laser confocal microscope was employed for image obtaining. DAPI (D9542, Sigma-Aldrich) was used to counterstain nuclei. Bio-repeats were implemented in triplicate. A Nuclear and Cytoplasmic Extraction Reagent Kit (Thermo Scientific) was employed to perform the subcellular fractionation, followed by measurement of the extracted RNAs by qPCR. U6 or β-actin was utilized as the nuclear or cytoplasmic control.
RIP assay was carried out as reported previously [17]. Cells were lysed with RIPA lysis buffer. Cell lysate was subjected to incubation with magnetic beads conjugated with Anti-Ago2 (ab186733, Abcam) and Anti-IgG (ab6789, Abcam). The precipitated RNAs were measured by qPCR. Bio-repeats were implemented in triplicate.
RNA pull-down assay was carried out as reported previously [17]. The structure buffer was added into 1 μg of biotin-labeled circCOL1A2 or USP10. Then, biotinylated circCOL1A2/USP10 was heated and ice-bathed for 3 min for denaturing. Following that, 15 μl of streptavidin beads were added into biotin-labeled and denatured RNA for a 2h-incubation at 4°C. The magnetic bead-probe complex was then mixed with the cell lysate and incubated overnight at 4°C. After incubation, the RNA was extracted by Trizol and measured by qPCR for miR-1286 enrichment. Bio-repeats were implemented in triplicate.
Dual-luciferase reporter assay was carried out as reported previously [17]. For this assay, pmirGLO-circCOL1A2/USP10-Wt was created by subcloning the sequences of circCOL1A2/USP10 into pmirGLO vectors. Likewise, circCOL1A2/USP10 with mutated binding sites to miR-1286 seed region was subcloned into vectors for the creation of pmirGLO-circCOL1A2/USP10-Mut. Next, after culturing 293T cells in 96-well plates, we transfected them with the reporter vectors and mimics of miR-1286, or NC mimics. The empty vectors themselves were used as NC. The relative activity of luciferase was analyzed. Renilla Luciferase was used as the internal reference. Bio-repeats were implemented in triplicate.
Co-IP assay was carried out as reported previously [18]. Flag-RFC2 was constructed for 48 h-transfection with 293T cells, which were then lysed using RIPA lysis buffer, followed by the addition of Anti-Flag-RFC2 and Anti-USP10, as well as Anti-IgG. Then, A/G beads were employed to capture the complexes containing the proteins and antibodies. After the elution in PBS, the bound proteins were denatured for western blot. Bio-repeats were implemented in triplicate.
IF assay was carried out as described previously [18]. GC cells were inoculated in 96-well plates. Subsequently, the cells were fixed by 4% formaldehyde for 20 min, followed by permeabilization using 0.1% Triton X-100. Next, the cell samples were incubated with Anti-USP10 and Anti-RFC2 at 4°C overnight, followed by incubation with the secondary antibodies at room temperature for 1 h. DAPI staining was adopted to counterstain the cell nuclei. A fluorescence microscope (XSP-63B, Shanghai Optical Instrument Factory, China) was utilized to observe immunofluorescence. Bio-repeats were implemented in triplicate.
IP assay was carried out as reported previously [19]. Flag-RFC2 was constructed, followed by transfection into 293T cells for 48 h. The 293T cells were then lysed and incubated with Anti-Flag-RFC2 overnight at 4°C. Protein A/G-beads were added to the complex for 4 h-cultivation at 4°C. Lastly, the precipitated proteins were transferred to SDS-PAGE gel for western blot.
These assays were conducted as reported previously [20]. GC cells were cultured to detect RNA and protein stability. To assess the stability of circCOL1A2 and COL1A2, RNase R (RNR07250, Epicentre, USA) was used to treat the cells. After treatment with RNase R, qPCR was used to compare the expressions of circCOL1A2 and COL1A2. For analysis of protein stability, western blot was used to detect the level of RFC2 at 0, 3, 6, 9 h, respectively, after treatment with 50 μm cycloheximide (CHX). Bio-repeats were implemented in triplicate.
Student’s t-test and one-way/two-way analysis of variance (ANOVA) were adopted to compare differences between groups. A p-value of under 0.05 was considered to indicate statistical significance.
We utilized GeoChip to analyze circRNAs differentially overexpressed in GC tissues. We identified circRNA_105040 and circRNA_081069 based on GSE141977, which includes 3 GC tumors and 3 adjacent non-tumor tissues (Fig. 1A). CircRNA_105040, which has the highest fold change, has been studied in GC previously [21]. Therefore, we selected circRNA_081069, with the second highest fold change, for our study. CircRNA_081069 was termed as circCOL1A2, as its host gene is COL1A2. Subsequently, qPCR was used to detect the expression of circCOL1A2 in GC cell lines (HGC-27, AGS and MKN-45) and human gastric mucosa epithelial cell line (GSE-1). As a result, circCOL1A2 was found to be upregulated in the GC cell lines (Fig. 1B). Due to their higher expressions of circCOL1A2, AGS and MKN-45 cells were selected for follow-up experiments. Next, PCR-agarose gel electrophoresis showed that circCOL1A2 was amplified in complementary DNA (cDNA) by both convergent primer and divergent primer, while it could not be amplified in genomic DNA (gDNA) by divergent primer, which verified the circular structure of circCOL1A2 (Fig. 1C). We then performed qPCR to detect the expressions of circCOL1A2 and COL1A2 mRNA after the treatment with RNase R. The expression of circCOL1A2 was revealed as remaining almost unchanged, while that of COL1A2 mRNA was significantly reduced (Fig. 1D). As circRNAs feature relatively stable structures, we further validated the circular structure of circCOL1A2. To sum up, circCOL1A2 with a circular structure is upregulated in GC tissues.
Next, we detected the effects of circCOL1A2 on GC cell progression. First, qPCR was used to detect the expression of circCOL1A2 after the transfection of sh-circCOL1A2-1/2/3, and showed that the plasmids inhibited the expression of circCOL1A2 (Fig. S1A). Due to the higher efficiency, sh-circCOL1A2-1 and sh-circCOL1A2-2 were used for follow-up experiments. Afterwards, migration and invasion of AGS and MKN-45 cells were evaluated by wound healing and transwell assays after the knockdown of circCOL1A2. The results showed that circCOL1A2 ablation inhibited the migratory and invasive capacities of GC cells, as evidenced by the increased wound width and decreased cell number (Figs. 2A and 2B). MMP2 and MMP9 are migration marker proteins [22]. Hence, we used western blot to detect the effects of circCOL1A2 on MMP2 and MMP9 levels in AGS and MKN-45 cells. After the interference with circCOL1A2, the levels of MMP2 and MMP9 were decreased, indicating the suppression of cell migration (Fig. 2C). Taken together, circCOL1A2 promotes cell migration and invasion of GC.
We proved that circCOL1A2 can promote the migration and invasion of GC cells. As circCOL1A2 cannot encode proteins, we next probed into the underlying mechanisms of circCOL1A2 in GC cells. FISH and subcellular fraction assays were used to detect the subcellular location of circCOL1A2 in AGS and MKN-45 cells. The results showed that circCOL1A2 was located both in the nuclei and cytoplasm of GC cells, but mainly in the cytoplasm (Fig. 3A). Based on the subcellular location of circCOL1A2, we conjectured that circCOL1A2 might regulate GC cells via ceRNA mode. For verification, we conducted the RIP assay and found the enrichment of circCOL1A2 in the Anti-AGO2 group, indicating the existence of circCOL1A2 in RNA-induced silencing complex (RISC). The results proved the ceRNA mode of circCOL1A2 (Fig. 3B). Next, we utilized bioinformatics to screen the downstream target of circCOL1A2 (Fig. 3C). We identified miR-1286 based on the prediction of starBase (http://starbase.sysu.edu.cn/) and Circular RNA Interactome (https://circinteractome.nia.nih.gov/). Subsequently, miR-1286 mimics and inhibitors led to the increase and reduction in miR-1286 expression respectively, as verified by qPCR (Figs. S1B and S1C). RNA pull-down and luciferase reporter assays were performed to detect the interaction between miR-1286 and circCOL1A2. According to the results, miR-1286 binds to circCOL1A2 in GC cells, as evidenced by the enrichment of miR-1286 in the Bio-circCOL1A2-Wt group, and decreased luciferase activity in the pmirGLO-circCOL1A2-Wt group (Figs. 3D and 3E). We then performed qPCR to detect miR-1286 expression after the ablation of circCOL1A2 and found that circCOL1A2 could not influence the expression of miR-1286 (Fig. 3F). However, the RIP assay using miR-1286 inhibitor showed that the enrichment of circCOL1A2 in Anti-AGO2 group was inhibited by miR-1286 ablation (Fig. 3G). The abovementioned results indicated that circCOL1A2 acts as a sponge of miR-1286.
As previous results proved that circCOL1A2 binds to miR-1286, we then explored the target mRNA of miR-1286. The downstream mRNAs of miR-1286 were predicted by starBase under certain conditions (CLIP data >=18; Degradome-Data >=2; pan-Cancer >=2). We next used UALCAN (http://ualcan.path.uab.edu/) to predict the expressions of candidate mRNAs in GC tumors. To ensure stringency, we only selected genes with AgoExpNum>=15. Five candidate genes were selected. KDM6B [23] and MAP1B [24] have been studied thoroughly and therefore lack research value. ACTN4 has been proved to be a novel therapeutic target for GC [25]. There is a dearth of reliable research reports on phospholipase PNPLA6. The role of deubiquitinase USP10 in GC has been investigated in a previous study [26], which was consistent with our expectations. In addition, USP10 is highly expressed in GC tumors according to the data from bioinformatics (Fig. 4A). For further validation, qPCR was performed to detect the expressions of PNPLA6, USP10 and ACTN4 after the knockdown of circCOL1A2 in AGS cells. The results showed that only USP10 expression decreased significantly (Fig. 4B). Afterwards, we detected USP10 expression in AGS, MKN-45 and GES-1 cells and found that USP10 was high-expressed in GC cells (Fig. 4C). Thus, we selected USP10 as the focus of our study. The binding sites between USP10 3’UTR and miR-1286 were predicted. RNA pull-down and luciferase reporter assays validated the interaction between miR-1286 and USP10 3’UTR (Figs. 4D and 4E). Rescue experiments were conducted to explore the relationships among circCOL1A2, miR-1286 and USP10. The qPCR and western blot results revealed detection of the mRNA and protein levels of USP10 after the transfection of sh-NC, sh-circCOL1A2-1, sh-circCOL1A2-1+inhibitor NC or sh-circCOL1A2-1+miR-1286 inhibitor. The miR-1286 inhibitor was found to counteract the inhibitory effect of circCOL1A ablation on USP10 at protein and mRNA levels (Fig. 4F). Taken together, circCOL1A2 regulates USP10 via competitively binding to miR-1286.
It has been reported that USP10 can be used as a biomarker of GC [27], but its specific regulatory mechanisms in GC remain to be explored. Therefore, we probed into its role in GC. We first analyzed the proteins interacting with USP10. STRING database (https://string-db.org/) was used to predict the proteins binding to USP10. USP10, as an important deubiquitinase, interacts with a considerable number of proteins, but most of them are deubiquitinases of the same family and are key factors of other pathways. Comparatively, RFC2 has a high possibility of interacting with USP10 and belongs to the replication factor C family, which has been reported to promote cell invasion and migration of various cancers [28]. UALCAN was used to predict the expression of RFC2 in GC, showing its association with GC (Fig. 5A). We then conducted qPCR and found that RFC2 expression was upregulated after the transfection of pcDNA3.1-RFC2, and USP10 expression was depleted after the transfection of sh-USP10-1/2/3 (Figs. S1D and S1E). Because of higher efficiency, sh-USP10-1 and sh-USP10-2 were selected for assays. Western blot and qPCR were used to detect the mRNA and protein levels of RFC2 after interference with USP10. As a result, USP10 ablation was shown to inhibit the protein level of RFC2 in AGS cells, instead of mRNA level (Fig. 5B). The IF assay validated the co-localization of USP10 and RFC2 in the nuclei of GC cells. The prediction of Hum-mPLoc 2.0 (http://www.csbio.sjtu.edu.cn/bioinf/hum-multi-2/) also proved the same (Fig. 5C). The interaction between USP10 and RFC2 was proved by Co-IP assays in 293T cells (Fig. 5D). IP and western blot were used to detect the ubiquitination level of RFC2 in 293T cells. The results showed that the ubiquitination level of RFC2 was increased after interference with USP10, but was reversed by the addition of MG132 (Fig. 5E). As MG132 is a proteasome inhibitor, this indicates that USP10 affects RFC2 protein level through the ubiquitin-proteasome mechanism. Afterward, we performed A western blot to detect the degradation of RFC2 after CHX (a transcription inhibitor) treatment and USP10 interference. The results showed that in CHX-treated AGS cells, the degradation of RFC2 was accelerated after USP10 interference, which further verified that USP10 mediates the ubiquitination level of RFC2 to stabilize RFC2 protein (Fig. 5F). Taken together, USP10 downregulates the RFC2 ubiquitination level in GC cells.
Next, we used rescue experiments to verify that USP10 promotes GC cell progression by regulating RFC2. We performed wound healing and transwell assays in GC cells transfected with sh-NC, sh-USP10-1 or sh-USP10-1+pcDNA3.1-RFC2. The results showed that RFC2 enhancement reversed the increased wound width and decreased cell number by USP10 ablation (Figs. 6A and 6B). The western blot results showcased that MMP2 and MMP9 levels were reduced by USP10 ablation, but were then countervailed by RFC2 overexpression (Fig. 6C). In conclusion, USP10 promotes migration and invasion of GC cells by regulating RFC2 expression.
GC is one of the most common cancers and is characterized by high mortality, due to the difficulty of diagnosis and treatment. Advanced-stage GC patients suffer from the risk of metastasis. Therefore, it is vitally important to study the mechanisms underlying GC cell migration and invasion. Various studies have proven that circRNAs participate in the regulation of GC, including circFAM73A [7], circLMO7 [8] and circFGD4 [9]. We identified circCOL1A2 using the GEO database. CircCOL1A2 has been reported to facilitate the migratory and invasive capacities of cancer cells in tongue squamous cell carcinoma [10]. However, the effects of circCOL1A2 on cell migration and invasion in GC have never been reported. We verified the circular structure of circCOL1A2 using electrophoresis and stability analysis. Moreover, qPCR showed that circCOL1A2 was overexpressed in GC cells, indicating their interrelationship. Wound healing, transwell, and western blot assays proved that circCOL1A2 facilitates cell migration and invasion of GC. Furthermore, FISH results showed that circCOL1A2 was mainly located in the cytoplasm of GC cells, suggesting the ceRNA mode. We then utilized the database to screen potential miRNAs. Next, luciferase reporter, RIP and RNA pull-down assays were conducted to ensure that miR-1286 was the downstream target of circCOL1A2. USP10 was identified as the potential target of miR-1286 using bioinformatics and qPCR. Again we employed the luciferase reporter, RIP and RNA pull-down assays to verify that miR-1286 interacts with USP10. USP10 has been reported to promote cell proliferation of hepatocellular carcinoma via deubiquitinating and stabilizing YAP/TAZ [29]; USP10 suppresses the growth and invasive capacity of lung cancer cells by overexpressing PTEN [30]; USP10 modulates oncogene-induced senescence through deubiquitination and stabilization of p14ARF [31]; USP10 propels cell proliferation in colon cancer through deubiquitinating and stabilizing Musashi-2 [32]. However, its role in GC cells has rarely been reported. We used bioinformatics, qPCR and western blot to screen out the protein interacting with USP10, namely, RFC2. RFC2 has been reported to be correlated with various malignancies. For instance, RFC2, targeted by miR-744, regulates the cell cycle and proliferation of colorectal cancer cells [33]. Additionally, RFC2 was found to modulate the cell cycle and DNA replication of liver cancer [34]. However, RFC2 ubiquitination has never been studied in GC. We performed gene expression analysis and IF assay and found that USP10 affects the protein level of RFC2 and is co-localized in the nucleus with RFC2. The results of Co-IP validated the interaction between RFC2 and USP10. IP and a western blot showed that USP10 downregulates RFC2 ubiquitination level in GC cells. The results of western blot using CHX showed that USP10 affects the stability of RFC2. Rescue experiments demonstrated that USP10 promotes migration and invasion of GC cells by regulating RFC2. To sum up, our study demonstrated that circCOL1A2 sponges miR-1286 to promote cell invasion and migration of GC by elevating the expression of USP10 to downregulate RFC2 ubiquitination level. The present study is the first to prove that circCOL1A2 regulates USP10 via sequestering miR-1286, thereby promoting GC cell migration and invasion. In addition, we have verified for the first time that USP10 facilitates GC cell migration and invasion by regulating RFC2 expression. Exploring the mechanisms of circCOL1A2 in GC cells offers insight into therapeutic targets for GC diagnosis and treatment. That said, our study has room for improvement in some respects. For instance, no in vivo verification was conducted in this study. Nude mice can be adopted to assess the effect of circCOL1A2 on GC cells. Furthermore, the clinicopathological relevance of circCOL1A2 remains to be explored. In the future study, we will conduct in vivo experiments and clinicopathological analysis to further our understanding of circCOL1A2 in GC.
Supplementary data for this paper are available on-line only at http://jmb.or.kr. | true | true | true |
PMC9629064 | 35427207 | Jinjin Zhang,Haixia Wang,Hongbin Chen,Hongbo Li,Pan Xu,Bo Liu,Qian Zhang,Changjun Lv,Xiaodong Song | ATF3 -activated accelerating effect of LINC00941/lncIAPF on fibroblast-to-myofibroblast differentiation by blocking autophagy depending on ELAVL1/HuR in pulmonary fibrosis AUTOPHAGY | 15-04-2022 | Autophagy,EZH2,fibroblast-to-myofibroblast differentiation,FOXK1,ELAVL1,lncRNA,myofibroblast proliferation and migration,pulmonary fibrosis,STAT1 | ABSTRACT Idiopathic pulmonary fibrosis (IPF) is characterized by lung scarring and has no effective treatment. Fibroblast-to-myofibroblast differentiation and myofibroblast proliferation and migration are major clinical manifestations of this disease; hence, blocking these processes is a practical treatment strategy. Here, highly upregulated LINC00941/lncIAPF was found to accelerate pulmonary fibrosis by promoting fibroblast-to-myofibroblast differentiation and myofibroblast proliferation and migration. Assay for transposase-accessible chromatin using sequencing and chromatin immunoprecipitation experiments elucidated that histone 3 lysine 27 acetylation (H3K27ac) activated the chromosome region opening in the LINC00941 promoter. As a consequence, the transcription factor ATF3 (activating transcription factor 3) bound to this region, and LINC00941 transcription was enhanced. RNA affinity isolation, RNA immunoprecipitation (RIP), RNase-RIP, half-life analysis, and ubiquitination experiments unveiled that LINC00941 formed a RNA-protein complex with ELAVL1/HuR (ELAV like RNA binding protein 1) to exert its pro-fibrotic function. Dual-fluorescence mRFP-GFP-MAP1LC3/LC3 (microtubule associated protein 1 light chain 3) adenovirus monitoring technology, human autophagy RT2 profiler PCR array, and autophagic flux revealed that the LINC00941-ELAVL1 axis inhibited autophagosome fusion with a lysosome. ELAVL1 RIP-seq, RIP-PCR, mRNA stability, and rescue experiments showed that the LINC00941-ELAVL1 complex inhibited autophagy by controlling the stability of the target genes EZH2 (enhancer of zeste 2 polycomb repressive complex 2 subunit), STAT1 (signal transducer and activators of transcription 1) and FOXK1 (forkhead box K1). Finally, the therapeutic effect of LINC00941 was confirmed in a mouse model and patients with IPF. This work provides a therapeutic target and a new effective therapeutic strategy related to autophagy for IPF. Abbreviations: ACTA2/a-SMA: actin alpha 2, smooth muscle; ATF3: activating transcription factor 3; ATG: autophagy related; Baf-A1: bafilomycin A1; BLM: bleomycin; CDKN: cyclin dependent kinase inhibitor; CLN3: CLN3 lysosomal/endosomal transmembrane protein, battenin; COL1A: collagen type I alpha; COL3A: collagen type III alpha; CXCR4: C-X-C motif chemokine receptor 4; DRAM2: DNA damage regulated autophagy modulator 2; ELAVL1/HuR: ELAV like RNA binding protein 1; EZH2: enhancer of zeste 2 polycomb repressive complex 2 subunit; FADD: Fas associated via death domain; FAP/FAPα: fibroblast activation protein alpha; FOXK1: forkhead box K1; FVC: forced vital capacity; GABARAP: GABA type A receptor-associated protein; GABARAPL2: GABA type A receptor associated protein like 2; IGF1: insulin like growth factor 1; IPF: idiopathic pulmonary fibrosis; LAMP: lysosomal associated membrane protein; lncRNA: long noncoding RNA; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; NPC1: NPC intracellular cholesterol transporter 1; RGS: regulator of G protein signaling; RPLP0: ribosomal protein lateral stalk subunit P0; ROC: receiver operating characteristic; S100A4: S100 calcium binding protein A4; SQSTM1/p62: sequestosome 1; STAT1: signal transducers and activators of transcription 1; TGFB1/TGF-β1: transforming growth factor beta 1; TNF: tumor necrosis factor; UIP: usual interstitial pneumonia; ULK1: unc-51 like autophagy activating kinase 1; VIM: vimentin. | ATF3 -activated accelerating effect of LINC00941/lncIAPF on fibroblast-to-myofibroblast differentiation by blocking autophagy depending on ELAVL1/HuR in pulmonary fibrosis AUTOPHAGY
Idiopathic pulmonary fibrosis (IPF) is characterized by lung scarring and has no effective treatment. Fibroblast-to-myofibroblast differentiation and myofibroblast proliferation and migration are major clinical manifestations of this disease; hence, blocking these processes is a practical treatment strategy. Here, highly upregulated LINC00941/lncIAPF was found to accelerate pulmonary fibrosis by promoting fibroblast-to-myofibroblast differentiation and myofibroblast proliferation and migration. Assay for transposase-accessible chromatin using sequencing and chromatin immunoprecipitation experiments elucidated that histone 3 lysine 27 acetylation (H3K27ac) activated the chromosome region opening in the LINC00941 promoter. As a consequence, the transcription factor ATF3 (activating transcription factor 3) bound to this region, and LINC00941 transcription was enhanced. RNA affinity isolation, RNA immunoprecipitation (RIP), RNase-RIP, half-life analysis, and ubiquitination experiments unveiled that LINC00941 formed a RNA-protein complex with ELAVL1/HuR (ELAV like RNA binding protein 1) to exert its pro-fibrotic function. Dual-fluorescence mRFP-GFP-MAP1LC3/LC3 (microtubule associated protein 1 light chain 3) adenovirus monitoring technology, human autophagy RT2 profiler PCR array, and autophagic flux revealed that the LINC00941-ELAVL1 axis inhibited autophagosome fusion with a lysosome. ELAVL1 RIP-seq, RIP-PCR, mRNA stability, and rescue experiments showed that the LINC00941-ELAVL1 complex inhibited autophagy by controlling the stability of the target genes EZH2 (enhancer of zeste 2 polycomb repressive complex 2 subunit), STAT1 (signal transducer and activators of transcription 1) and FOXK1 (forkhead box K1). Finally, the therapeutic effect of LINC00941 was confirmed in a mouse model and patients with IPF. This work provides a therapeutic target and a new effective therapeutic strategy related to autophagy for IPF. Abbreviations: ACTA2/a-SMA: actin alpha 2, smooth muscle; ATF3: activating transcription factor 3; ATG: autophagy related; Baf-A1: bafilomycin A1; BLM: bleomycin; CDKN: cyclin dependent kinase inhibitor; CLN3: CLN3 lysosomal/endosomal transmembrane protein, battenin; COL1A: collagen type I alpha; COL3A: collagen type III alpha; CXCR4: C-X-C motif chemokine receptor 4; DRAM2: DNA damage regulated autophagy modulator 2; ELAVL1/HuR: ELAV like RNA binding protein 1; EZH2: enhancer of zeste 2 polycomb repressive complex 2 subunit; FADD: Fas associated via death domain; FAP/FAPα: fibroblast activation protein alpha; FOXK1: forkhead box K1; FVC: forced vital capacity; GABARAP: GABA type A receptor-associated protein; GABARAPL2: GABA type A receptor associated protein like 2; IGF1: insulin like growth factor 1; IPF: idiopathic pulmonary fibrosis; LAMP: lysosomal associated membrane protein; lncRNA: long noncoding RNA; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; NPC1: NPC intracellular cholesterol transporter 1; RGS: regulator of G protein signaling; RPLP0: ribosomal protein lateral stalk subunit P0; ROC: receiver operating characteristic; S100A4: S100 calcium binding protein A4; SQSTM1/p62: sequestosome 1; STAT1: signal transducers and activators of transcription 1; TGFB1/TGF-β1: transforming growth factor beta 1; TNF: tumor necrosis factor; UIP: usual interstitial pneumonia; ULK1: unc-51 like autophagy activating kinase 1; VIM: vimentin.
Idiopathic pulmonary fibrosis (IPF) is generally defined as a specific form of chronic, progressive, fibrosing interstitial pneumonia of unknown cause with the histopathological and/or radiological pattern of usual interstitial pneumonia (UIP) [1]. Raghu proposed the acceptance of IPF as a manifestation of irreversible pulmonary fibrosis of many entities that is caused by several occult or overt causative factors, including genetics, sex, age, intrinsic and extrinsic environmental factors, and systemic factors. This proposal is in contrast to the notion that IPF is a specific entity characterized by UIP of unknown cause [2]. The renewal of this concept may change the clinical diagnosis and treatment strategy of IPF and highlight the process of pulmonary fibrosis. Thus, understanding how fibroblast-to-myofibroblast differentiation causes uncontrolled proliferation and high myofibroblast migration and extracellular matrix deposition leading to pulmonary fibrogenesis is necessary. Long noncoding RNAs (lncRNAs) belong to a class of regulatory noncoding RNAs that have a length more than 200 nt and are usually unable to code protein but can control multiple biological processes [3]. Their regulatory functions mainly depend on distinct processing and different subcellular localizations. For example, lncRNA FAST orthologs exhibit different subcellular localization in human embryonic stem cells (HsESCs) and murine ESCs (MmESCs) because of differential RNA processing. In HsESCs, cytoplasm-localized HsFAST binds to the WD40 domain of the E3 ubiquitin ligase BTRC/b-TrCP and blocks its interaction with phosphorylated CTNNB1/b-catenin to prevent degradation. As a result, WNT signaling, which is required for pluripotency, is activated. By contrast, MmFAST is retained in the nucleus of MmESCs, and its processing is suppressed by the splicing factor PPIE, which is highly expressed in MmESCs but not HsESCs [4]. Further study on these differentially expressed lncRNAs may provide a novel approach for the diagnosis and treatment of certain diseases. The prostate-specific lncRNAs of PCA3 (prostate cancer associated 3) and PCGEM1 (PCGEM prostate-specific transcript) detected in urine or blood have been used to differentiate prostate tumor and normal tissues and have diagnostic specificity and sensitivity of 83% and 67%, respectively, relative to those from the biopsy data [5]. The fat-specific lncRNA Lep/Ob controls the quantitative expression of the Lep (leptin) gene, and its defects can lead to a hypoleptinemic form of obesity that is responsive to LEP treatment [6]. In fibrosis, many lncRNAs are involved in various organs fibrosis, such as the liver, lung, heart and kidney. The pro-fibrotic lncRNA NONMMUT022555/lncRNA PFL contributes to cardiac fibrosis by functioning as a competing endogenous RNA of MIRLET7D/let-7d [7]. LncRNA H19-mediated M2 polarization of macrophages promotes myofibroblast differentiation in pulmonary fibrosis induced by arsenic exposure [8]. LncRNA Erbb4-IR (intron region) contributes to TGFB1/TGF-β1 (transforming growth factor beta 1)-SMAD3 (SMAD family member 3)-mediated renal fibrosis via downregulating SMAD7 (SMAD family member 7) [9]. In general, the study of lncRNAs associated with fibrosis, particularly pulmonary fibrosis, is only in the preliminary stage. Our previous studies revealed that different lncRNAs regulate pulmonary fibrogenesis in different cell types. LncITPF (a novel lncRNA renamed by its function) forms an RNA-protein complex with HNRNPL (heterogeneous nuclear ribonucleoprotein L) to control its host gene ITGBL1 in fibroblasts, thus accelerating lung fibrosis. LncRNA BC158825/LncPCF promotes pulmonary fibrosis by sponging microRNA (miRNA) Mir344-5p to target MAP3K11 in alveolar epithelial cells []. However, the regulatory mechanism of lncRNA on cellular differentiation remains largely unknown. In the present work, whole-transcriptome analysis was performed to screen differentially expressed lncRNAs in human lung fibroblasts. The upregulated RNA transcript of LINC00941/lncIAPF was further investigated. Recent research showed that LINC00941 promotes proliferation and invasion of colon cancer by acting on the MIR205-5p-MYC axis as a ceRNA mechanism [13]. However, its upregulation mechanism and the underlying mechanism by sequestering RNA-binding protein (RBP) remain largely unknown. Here, highly expressed LINC00941 regulation on fibroblast-to-myofibroblast differentiation through sequestering RBP to target macroautophagy/autophagy process and its potential clinical relevance in patients with IPF was further explored. Due to its inhibitory function on autophagy, we renamed LINC00941 as lncIAPF (Inhibit Autophagy in Pulmonary Fibrogenesis). The findings can provide a diagnostic biomarker in blood, or a potential therapeutic target related to autophagy for IPF.
TGFB1 was used to activate fibroblast differentiation into myofibroblast in human fetal lung fibroblast MRC-5 cell. Whole-transcriptome sequencing was first conducted to test the differentially expressed lncRNAs in normal MRC-5 cells and MRC-5 cells activated with TGFB1 for 72 h. LINC00941 RNA transcript had markedly higher expression than other RNAs (Figure 1A,B). With the use of Affymetrix Human Transcriptome Array 2.0 microarray, LINC00941 was found to have the highest expression among lncRNAs and the highest co-expression degree with the mRNAs of fibrotic markers [14] and thus selected for further investigation. For sequencing data confirmation, lncIAPF expression was assessed in MRC-5 cells activated with TGFB1 for 0, 6, 12, 24, 48 and 72 h. qRT-PCR results reflected that lncIAPF was upregulated (Figure 1C). Afterward, 5′- and 3′-rapid amplification of complementary DNA ends (RACE) experiment was performed to analyze the full-length sequence of lncIAPF. The results uncovered that lncIAPF has 1967 base pairs in length (Fig. S1A). The protein-coding ability scores of lncIAPF were evaluated using the following tools: Coding Potential Calculator (http://cpc.cbi.pku.edu.cn/) and Coding Potential Assignment Tool (http://lilab.research.bcm.edu/cpat/index.php). The scores were 0.187173 and 0.1735537, respectively, indicating that lncIAPF was devoid of protein-coding potential. In the UCSC and Ensemble databases, lncIAPF was also annotated as a lncRNA. Subsequently, the factor underlying the considerable increase in lncIAPF expression in lung fibrosis was investigated. Chromatin undergoes dynamic, organizational changes in normal or disease state; hence, an assay for transposase-accessible chromatin using sequencing (ATAC-seq) was performed to test chromosome-region opening during pulmonary fibrosis [15]. Considering that chromosome-region opening occurs prior to gene expression, normal cells and those treated with TGFB1 for 6 h were tested. ATAC-seq findings revealed that TGFB1 considerably increased the chromosome opening peak in lncIAPF promoter compared with that in the normal group (Figure 1D). This result indicated that lncIAPF transcription is more active in the TGFB1-treated group than in the normal group. Chromatin immunoprecipitation (ChIP)-seq was then performed to study histone modification. Significant histone H3 lysine 4 trimethylation (H3K4me3) and H3K27ac modification peaks were found near the transcription start sites of lncIAPF in each group. Significant difference was found in the H3K27ac modification peak (Figure 1E) but not in the peak value of H3K4me3 (Figure 1F) between the TGFB1-treated and normal groups. Therefore, the differential expression of lncIAPF was related to H3K27ac modification but not to H3K4me3 modification. Further analysis was performed on the transcription factors that specifically bound to lncIAPF promoter. ATF3 ranked first according to p values based on ATAC-seq and hence was selected for further study. After the motif analysis of the ATF3 binding site, ChIP was performed to confirm the binding between ATF3 and lncIAPF promoter (Figure 1G). qRT-PCR results verified that ATF3 knockdown markedly reduced lncIAPF expression and vice-versa (Figure 1H). These findings indicated that H3K27ac modification contributes to chromosome-region opening in lncIAPF promoter. After chromosome-region opening, ATF3 binds to lncIAPF promoter and initiates lncIAPF transcription. It may be one of the reasons that lncIAPF upregulates in pulmonary fibrosis.
The smart silencer consisting of six RNA interference sites (si-lncIAPF) and overexpression vector (recombinant plasmid, RP) of lncIAPF were designed and transfected into MRC-5 cells to investigate lncIAPF function. The effects of si-lncIAPF and RP on fibroblast-to-myofibroblast differentiation were assessed by cell shape, differentiation-related proteins, fibrotic protein markers, and myofibroblast proliferation and migration. Real-time cellular analysis system manifested that myofibroblast proliferation and migration was considerably increased by lncIAPF overexpression but blocked by lncIAPF smart silencer compared with those in the TGFB1-treated groups (Figure 2A, B). These results were further confirmed by scratch wound-healing assay (Figure 2C). Western blot analysis revealed that TGFB1 rapidly increased the amount of fibrotic markers, including ACTA2/a-SMA (actin alpha 2, smooth muscle), COL1A (collagen type I alpha), COL3A (collagen type III alpha), and VIM (vimentin), and differentiation-related proteins, including FAP/FAPα (fibroblast activation protein alpha) and S100A4 (S100 calcium binding protein A4). However, their expression was reduced by lncIAPF smart silencer and promoted by lncIAPF overexpression (Figure 2D). Expression of COL1A and FN1 (fibronectin 1) in the media of the cultured cells also reduced by lncIAPF smart silencer and promoted by lncIAPF overexpression (Figure 2E). Immunofluorescence staining images depicted that the TGFB1-treated fibroblasts appeared in a spindle shape and had strong proliferation and migration abilities and rapidly increased ACTA2. LncIAPF overexpression had the same effect as TGFB1. Meanwhile, lncIAPF smart silencer weakened the proliferation and migration abilities and decreased ACTA2 (Figure 2F). All these results suggest that lncIAPF is a pro-fibrotic factor that can accelerate pulmonary fibrosis by promoting fibroblast differentiation into myofibroblasts and myofibroblast proliferation and migration.
LncRNA can form an RNA-protein complex with its binding protein to exert certain functions. Hence, RNA affinity isolation, protein mass spectrometry, and western blot analyses were performed to search for the binding protein of lncIAPF by dividing its full-length sequence into two sections, namely, 1–1017 and 1005–1895 nt sections. After segmentation, high-quality RNA transcriptions were obtained (Figure 3A). Silver staining was performed to detect lncIAPF-bound proteins from RNA pull-down. Staining images depicted that the active functional domain was in the 1005–1895 nt section, and the 1–1017 nt section had no differently expressed protein compared with the control (Figure 3B). The lncIAPF-bound proteins were further analyzed by mass spectrometry. Considering the varying protein bands found in the 1005–1895 nt section, it was divided into a, b, and c groups for further mass spectrometry. The results preliminarily exhibited that ELAVL1 was the major lncIAPF-binding protein. Mass spectrometry and western blot findings confirmed their binding relation (Figure 3C,D). RNA immunoprecipitation (RIP) results also revealed that ELAVL1 was specifically enriched by lncIAPF to prove their binding relationship. LncRNA overexpressed in colon carcinoma-1 (OCC-1) serves as positive control because it is upregulated in a subset of colon carcinomas (Figure 3E) [16]. The 1005–1895 sequence of lncIAPF was further divided into six sections: 1005–1047, 1048–1895, 1048–1613, 1330–1895, 1614–1895, and 1048–1329 to identify the binding domain. RNA pull-down analysis showed that ELAVL1 blot appeared in three sections: 1048–1895, 1048–1613, and 1330–1895, indicating that the binding domain was approximately in the 1330–1613 section of lncIAPF (Figure 3F). For confirmation, 13 pairs of primers for lncIAPF and RNase-RIP experiment were designed (Table S1). The RNase-RIP results showed that ELAVL1 anchored at the 9#, 10#, and 11# primers in the amplification region of lncIAPF (Figure 3G). This region was also located in 1330–1613 nt in lncIAPF. ELAVL1 expression was then assessed by western blot. When the duration of TGFB1 treatment was increased from 0 to 72 h, its expression was higher compared with that in normal cells (Fig. S1B). Western blot and immunofluorescence results showed that ELAVL1 expression was increased by lncIAPF overexpression but reduced by lncIAPF knockdown (Figure 3H,I). Hence, ELAVL1 was selected for further study. The underlying mechanism between lncIAPF and ELAVL1 was further explored. LncRNA location determines its regulatory mechanisms, such as the transcriptional or posttranscriptional modification of mRNA and protein activity modulation. Thus, RNA fluorescence in situ hybridization (RNA FISH) and nuclear-cytosol fractionation were performed to detect lncIAPF location. RNA FISH analysis revealed that lncIAPF was visible in the nucleus and cytoplasm (Figure 3J). Nuclear-cytosol fractionation revealed that after TGFB1 treatment, only a small amount of lncIAPF translocated from the nucleus to cytoplasm (Figure 3K). ELAVL1 translocation was also detected. Primarily located in the nucleus in normal cells, a small amount of ELAVL1 translocated from the nucleus to cytoplasm after TGFB1 or overexpressed lncIAPF treatment. LncIAPF knockdown reduced the total amount of ELAVL1 protein but did not trigger its nucleocytoplasmic translocation (Figure 3L). ELAVL1 regulates numerous genes in the nucleus and cytoplasm through different mechanisms. We inferred that lncIAPF and ELAVL1 expression are affected by their stabilities, and not by their translocations. Hence, stability experiments were performed to explore the underlying mechanism. Gain- and loss-of-function studies revealed that ELAVL1 overexpression increased lncIAPF level, and ELAVL1 knockdown decreased lncIAPF expression (Figure 4A). Half-life of lncIAPF analysis indicated that lncIAPF stability was enhanced by ELAVL1 overexpression but reduced by ELAVL1 knockdown (Figure 4B). The stability of ELAVL1 protein was also measured by using cycloheximide experiments, and the results showed that ELAVL1 stability was enhanced by lncIAPF overexpression but reduced by lncIAPF knockdown (Figure 4C). Ubiquitination experiments also elucidated that lncIAPF overexpression decreased ELAVL1 ubiquitination, which led to ELAVL1 stability. LncIAPF knockdown promoted ELAVL1 ubiquitination, which resulted in ELAVL1 degradation (Figure 4D). The above results suggest that lncIAPF and ELAVL1 affect each other’s stability. The rescue experiment showed that the levels of fibrotic markers such as ACTA2, VIM, COL1A and COL3A were increased by lncIAPF overexpression, but this effect was reversed by the interference of ELAVL1 (Figure 4E). Myofibroblast proliferation and migration were enhanced by lncIAPF overexpression. However, these trends reversed by interference of ELAVL1 expression (Figure 4F). Thus, the effect of lncIAPF on fibrosis depends on ELAVL1.
Which signaling pathway is regulated by lncIAPF? Differentially expressed mRNAs were obtained in TGFB1+ knockdown lncIAPF NC and TGFB1+ knockdown lncIAPF groups by using RNA sequencing. Go analysis and functional enrichment of differential mRNA showed that lncIAPF regulation mainly enriched in autophagy signaling pathway. Some autophagy-related genes (ATGs) were up-regulated and some were down-regulated by knockdown lncIAPF (Figure 5A). Autophagy can function as a cytoprotector or deleterious effector to participate in the pathogenesis of pulmonary fibrosis [17]. Hence, the autophagy role was first studied in bleomycin (BLM)-treated mice model. The in vivo results indicated that excess collagen was deposited and the alveolar wall became thicker in BLM group. Rapamycin is known as autophagy inducer. H&E and Masson images revealed that rapamycin alleviated fibrosis (Figure 5B). The findings showed that autophagy can function as a cytoprotector in pulmonary fibrosis. For further illustration which stage of autophagy is blocked, real-time detection of tandem dual-fluorescence mRFP-GFP-LC3 adenovirus was performed to investigate the effects on autophagy by the knockdown and overexpression of lncIAPF. If autophagy is activated, then GFP green fluorescence will be quenched due to the decrease in pH value. Red fluorescence is still emitted even when the pH stability of mRFP red fluorescent is higher than that of GFP. Hence, red fluorescence indicates autolysosomes. Yellow fluorescence represents the co-localization of both GFP and mRFP fluorescence indicating the autophagosomes. This color indicates that the fusion proteins failed to completely fuse with lysosome, and autophagy is partially blocked. The percentage of yellow fluorescence spots reflects the autophagosome-lysosome fusion rate. So we used the IncuCyte S3 live-cell analysis system to observe the autophagy processes under various conditions. Videos of the whole autophagy process within 20 h in normal, TGFB1, overexpressed lncIAPF/NC and knockdown lncIAPF/NC groups were recorded as Supplementary Video. These videos vividly displayed the autophagy processes at different times for different groups. The videos showed that autophagosome formed normally and the fusion between autophagosome and lysosome was blocked in TGFB1 group. Overexpressed lncIAPF inhibited autophagy by blocking the fusion between autophagosome and lysosome, not blockade of autophagosome formation. Knockdown lncIAPF promoted the fusion between autophagosome and lysosome. Baf-A1 (Bafilomycin A1) is an autophagy inhibitor, which can inhibit the fusion between autophagosome and lysosome and enhanced yellow fluorescence [18]. Therefore, Baf-A1 was added to confirm blockade of the fusion between autophagosome and lysosome, not autophagosome formation in TGFB1 and overexpressed lncIAPF groups, whereas knockdown of lncIAPF promoted the fusion between autophagosomes and lysosomes (Video S1–S4). Meanwhile, the images of fluorescence changes were detected with the laser confocal microscope. Dual-fluorescence images revealed that the normal group emitted red and yellow fluorescence. The numbers of yellow fluorescence spots were markedly increasing in TGFB1 group compared with normal group. The yellow fluorescence was enhanced in the overexpressed lncIAPF-treated group but weakened in the lncIAPF knockdown-treated group. After adding Baf-A1, we observed a constant increase in the number of cells accumulating yellow fluorescence spots compared with every group without Baf-A1, which is suggestive of defective fusion of autophagosomes with lysosomes (Figure 5C). On the basis of the above findings, overexpressed lncIAPF inhibits autophagy by blocking autophagosome fusion with lysosome, not blockade of autophagosome formation. Knockdown lncIAPF promotes autophagosome fusion with lysosome. Subsequently, the 84 ATGs were analyzed using human autophagy RT2 profiler PCR array. According to the expressed profiles of ATGs, 32 and 8 were upregulated and downregulated, respectively, in the lncIAPF overexpressed group compared with the normal group (Figure 5D). These changed genes include two parts: autophagy machinery components and autophagy regulators. Autophagy machinery components contain genes encoding proteins that are involved in autophagic vacuole formation (ATG4B, ATG4C, ATG4D, ATG9A, ATG9B, ATG10, ATG16 L1, ULK1, RGS19, AMBRA1, and GABARAPL2) and those linking autophagosome to lysosome (GABARAP, LAMP1 and NPC1). Autophagy regulators include the co-regulators of autophagy and apoptosis (IFNG, BCL2, BCL2L1, CDKN1B, CDKN2A, CXCR4, FADD, FAS, CLN3, CASP3, IGF1, SNCA, SQSTM1/P62, TGFB1, TNF, and TNFSF10) and those responsible for the response to other intracellular signals (EIF4G1, HGS, CRPK1, CTSS, DRAM2 [DNA damage regulated autophagy modulator 2], PIK3C3, RAB24, ACTB, TMEM74, and RPLP0) (Table 1). Given that autophagy function is activated by autophagic flux, western blot was used to detect the expression of autophagic flux marker proteins, such as LC3-II:LC3-I and SQSTM1. The expression of SQSTM1 and LC3-II:LC3-I decreased in the normal group. Gain- and loss-of-function studies indicated that LC3-II:LC3-I and SQSTM1 levels were decreased by lncIAPF knockdown but increased by lncIAPF overexpression (Figure 5E). Inhibition of ELAVL1 in autophagy was confirmed by ELAVL1 effect on autophagic flux. The results identified that LC3-II:LC3-I and SQSTM1 levels were decreased by si-ELAVL1 but increased by ELAVL1 overexpression (Figure 5F). The rescue experiment elucidated that interference on ELAVL1 expression reversed the increasing effect of lncIAPF overexpression for autophagic proteins including LC3-II:LC3-I, SQSTM1 (Figure 5G). Therefore, the effect of lncIAPF on autophagy depends on ELAVL1.
Potential interactions between ELAVL1 and other genes were analyzed using GeneMANIA to investigate the effect of lncIAPF-ELAVL1 axis on autophagy. The results showed that ELAVL1 directly bound to and regulated the EZH2 (enhancer of zeste 2 polycomb repressive complex 2 subunit), STAT1 (signal transducers and activators of transcription 1) and FOXK1 (forkhead box K1) (Fig. S1C). Emerging evidence has confirmed that both nuclear and cytosolic events can regulate the induction or repression of autophagy [19]. FOXK1, STAT1 and EZH2 can repress autophagy by controlling the genes associated with autophagy such as LC3-I, LC3-II and SQSTM1 via the MTOR pathway in muscle development [20], breast cancer [21] and tumorigenesis [22]. However, this function has not been reported in pulmonary fibrosis. Given their roles in autophagic regulation, EZH2, STAT1 and FOXK1 were selected for subsequent studies. First, RNA binding protein immunoprecipitation sequencing (RIP-seq) was performed to identify the target mRNAs to ELAVL1. ELAVL1 RIP-seq data revealed that ELAVL1 bound to the mRNAs of EZH2, STAT1 and FOXK1 (Figure 6A, B). RIP-PCR results further confirmed that ELAVL1 bound to EZH2, STAT1 and FOXK1 (Figure 6C). RIP-PCR was then used to examine the effects of lncIAPF on the binding between ELAVL1 and its target genes EZH2, STAT1 and FOXK1 at the mRNA level. The binding degree of ELAVL1 with EZH2, STAT1 and FOXK1 was inhibited by lncIAPF knockdown but intensified by lncIAPF overexpression (Figure 6D). In general, ELAVL1 can influence mRNA stability. Hence, the mRNA stabilities of EZH2, STAT1 and FOXK1 were detected by using half-life experiments. The data reflected that the stabilities of EZH2, STAT1 and FOXK1 were promoted by lncIAPF overexpression but reduced by lncIAPF knockdown (Figure 6E). Western blot results further confirmed that the protein expression of EZH2, STAT1 and FOXK1 was decreased by lncIAPF knockdown but increased by lncIAPF overexpression (Figure 6F). Whether the controlling effect of lncIAPF on EZH2, STAT1 and FOXK1 depends on ELAVL1 was confirmed by rescue experiments (Figure 6G). EZH2, STAT1 and FOXK1 expression levels were enhanced by lncIAPF overexpression. However, these trends reversed by interference of ELAVL1 expression, suggesting that the regulating ability of lncIAPF for EZH2, STAT1 and FOXK1 depends on ELAVL1. The effects of EZH2, STAT1 and FOXK1 on fibrotic proteins and autophagic flux were tested by gain- and loss-of-function studies. The results demonstrated that overexpressed EZH2, STAT1 and FOXK1 promoted the expression of fibrotic proteins and inhibited autophagic flux (Figure 6H,I). Also, Baf-A1 was added to confirm blockade of the fusion between autophagosome and lysosome in overexpressed EZH2, STAT1 and FOXK1 groups, not autophagosome formation. Overexpressed EZH2, STAT1 and FOXK1 inhibited the fusion of autophagosome and lysosome (Figure 6J). Knockdown EZH2, STAT1 and FOXK1 inhibited the expression of fibrotic proteins and promoted autophagic flux (Figure 6K). The rescue experiments elucidated that overexpressed EZH2, STAT1 and FOXK1 reversed the downward trend of fibrotic and autophagic proteins caused by si-lncIAPF (Figure 6L), whereas knockdown of EZH2, STAT1 and FOXK1 reversed the upward trend of these proteins caused by overexpressed lncIAPF (Figure 6M). The effects of EZH2, STAT1 and FOXK1 on autophagy were further studied by using human autophagy RT2 profiler PCR array (Figure 7A). The overexpression of EZH2, STAT1 and FOXK1 co-regulated 30 upregulated and 13 downregulated ATGs (Table 2–4). Among these genes, 21 upregulated ATGs, namely, ATG4B, ATG4D, ATG9B, ATG9A, ATG10, ATG16L1, ULK1, RGS19, AMBRA1, LAMP1, NPC1, BCL2L1, CDKN1B, FADD, CLN3, IGF1, SQSTM1, TGFB1, TNFSF10, HGS, and ACTB, overlapped with those regulated by lncIAPF overexpression. Two downregulated ATGs, namely, DRAM2 and GABARAP (GABA type A receptor-associated protein), overlapped with those regulated by lncIAPF overexpression (Figure 7B). Western blot analysis further confirmed that DRAM2 and GABARAP levels were increased by lncIAPF knockdown but decreased by lncIAPF overexpression. ULK1 (unc-51-like autophagy activating kinase 1) and TGFB1 were decreased by lncIAPF knockdown but increased by lncIAPF overexpression (Figure 7C). DRAM2 and GABARAP contribute to autophagy via autophagosome-lysosome fusion [23,24]. ULK1 mainly help for the early stage of autophagy. This finding supports the conclusion that lncIAPF overexpression inhibits autophagy by blocking autophagosome fusion with lysosome. Gain- and loss-of-function studies were performed to measure the effect of GABARAP on fibrosis. Overexpressed GABARAP reduced fibrotic protein expression and knockdown GABARAP promoted their expression (Figure 7D). The rescue experiments elucidated that si-GABARAP reversed the decreasing trend of fibrosis and the increasing trend of autophagic flux caused by lncIAPF knockdown. Overexpressed GABARAP reversed the increasing trend of fibrosis and the decreasing trend of autophagic flux caused by overexpressed lncIAPF (Figure 7E). To further clarify the correlation between fibrosis and autophagy, si-GABARAP packaged into the adenovirus vector was transfected into MRC-5 cell. Western blot demonstrated that overexpressed EZH2, STAT1, FOXK1 and ELAVL1 promoted fibrotic proteins expression (Figure 7F). These results reflected that the regulatory signals of lncIAPF on autophagy are mainly exhibited through EZH2, STAT1 and FOXK1.
Animal experiments and clinical studies were performed to evaluate the potential of lncIAPF as a therapeutic target. Given that lncIAPF was screened from human fibroblast, it has a relatively low degree of homology to that in mice. Therefore, we first test whether overexpressed humanized lncIAPF was effective in mice to validate the mouse model is relevant to the proposed mechanism. In mouse fibroblast L929 cells, qRT-PCR results indicated that overexpressed lncIAPF promoted lncIAPF expression (Fig. S2A). Western blot results illustrated that overexpressed lncIAPF promoted fibrogenesis and inhibited autophagy (Fig. S2B,C). In BLM-treated mice model, bleomycin also promoted these proteins expression (Fig. S2D). The findings corroborated the human fibroblast results, which suggest that the mouse model is relevant to the proposed mechanism. Hence, we can use mice model to further confirm the proposed mechanism. Due to poor homology, perhaps lncIAPF interference does not work effectively in mice model. According to the similar study [25], overexpressed lncIAPF was selected for mice experiments. Overexpressed lncIAPF was packaged into the adenovirus vector marked with green fluorescent protein and sprayed into the mouse lung from the trachea (Figure 8A). The efficiency of overexpressed lncIAPF was detected by qRT-PCR. The data proved the high expression of lncIAPF in the overexpressed lncIAPF-treated mice, indicating that the overexpressed lncIAPF adenovirus vector is highly efficacious (Figure 8B). Lung-function assessment revealed that lncIAPF overexpression affected the forced vital capacity (FVC) of mice (Figure 8C). H&E and Masson staining results presented massive collagen deposits, fibrosis lesions, and distorted lung architecture in the overexpressed lncIAPF group compared with those in the sham group (Figure 8D, E). Western blot results further confirmed that lncIAPF overexpression increased the levels of fibrotic and differentiation-related proteins including COL1A, VIM, ACTA2, FAP, S100A4 and decreased that of epithelial marker CDH1/E-cadherin (Figure 8F). Meanwhile, the expression of enhanced autophagic proteins including SQSTM1, LC3II:LC3I, ELAVL1, EZH2, STAT1 and FOXK1 (Figure 8G) was increased, suggesting that lncIAPF promotes pulmonary fibrosis by blocking autophagy in vivo. To improve the convincing mechanism and overcome the low degree homology of lncIAPF in mice, we synthesized si-ELAVL1 packaged into the adenovirus vector to spray into the mouse lung. Body weight monitoring revealed that BLM-treated mice lost substantial body mass compared with sham mice. However, si-ELAVL1 effectively blocked this loss compared with the control group (Figure 8H). The results of MicroCT imaging system for small animal demonstrated that fibrosis changes occurred in both lungs in BLM group, and the degree of fibrosis was significantly reduced in si-ELAVL1 group (Figure 8I). Lung function assessment demonstrated that the FVC was improved in si-ELAVL1 group (Figure 8J). H&E and Masson images revealed that excess collagen was deposited and the alveolar wall became thicker in BLM group, and si-ELAVL1 attenuated collagen deposition (Figure 8K). Western blot results further confirmed that the levels of fibrotic, differentiation-related and autophagic proteins reduced in si-ELAVL1 group (Figure 8L). The findings indicated that si-ELAVL1 had significant therapeutic effect. Meanwhile, the findings also suggested that lncIAPF promotes pulmonary fibrosis by blocking autophagy in vivo through the proposed mechanism. Finally, to assess the clinical application value, lncIAPF and the related gene expression were explored in patients with IPF. The characteristics and physiologies of patients with IPF were shown in Table 5. LncIAPF was highly upregulated in the blood collected from patients with IPF (Figure 9A). The receiver operating characteristic curve (ROC) between lncIAPF and FVC showed that the sensitivity and specificity values are 87.5% and 75.0% in IPF patients, respectively. The area under the ROC curve was 0.879 (Figure 9B). ROC curve between lncIAPF and DLco showed that the sensitivity and specificity values are 42.9% and 78.6%, respectively. The area under the ROC curve was 0.579 (Figure 9C). ROC curve between lncIAPF and PaO2 showed that the sensitivity and specificity values are 92.9% and 92.0%, respectively. The area under the ROC curve was 0.949 (Figure 9D). ROC curve between lncIAPF and PaCO2 showed that the sensitivity and specificity values are 50.0% and 87.5%, respectively. The area under the ROC curve was 0.520 (Figure 9E). Immunofluorescence staining of lung tissues revealed that LC3 and SQSTM1 increased in the patients with IPF. These findings imply that autophagy was blocked. The levels of ELAVL1 and its target genes EZH2, STAT1 and FOXK1 also increased in the patients with IPF (Figure 9F). These clinical findings corroborated the in vivo and in vitro results, indicating that interference in lncIAPF expression may offer a novel biomarker or therapeutic approach against IPF.
LncRNAs are crucial to epigenetic regulation and participate in the pathogenesis of various diseases [26,27]. Their location determines their regulatory mechanisms. Nuclear lncRNA mainly regulates chromatin, transcription, or alternative splicing; and cytoplasmic lncRNA mainly affects mRNA stability, protein translation, or posttranslational modification. For example, the nuclear lncRNA MALAT1 suppresses antiviral innate responses by directly binding to TARDBP/TDP43 (TAR DNA binding protein) in the nucleus and prevents the activation of TARDBP43 by blocking the activated CASP3 (caspase 3)-mediated TARDBP43 cleavage to TARDBP35. The cleaved TARDBP35 increases the nuclear IRF3 protein level by binding and degrading Rbck1 pre-mRNA to prevent IRF3 proteasomal degradation upon viral infection, thus selectively promoting antiviral IFN-I production [28]. The cytoplasmic lncRNA H19-DMD (dystrophin) interactions inhibit E3-ligase-dependent polyubiquitination at Lys 3584 in Duchenne muscular dystrophy, thus preventing protein degradation [29]. The pan-cancer lncRNA MILIP mainly located in cytoplasm is critical for cancer cell survival, division, and tumorigenicity. Yu et al. reported that MYC/c-Myc can alternatively inactivate TP53/p53 through the lncRNA MILIP, which restrains TP53 SUMOylation by suppressing the SUMO E3 ligase TRIML2 (tripartite motif family like 2) and consequently facilitates TP53 polyubiquitination and turnover [30]. In the present study, lncIAPF was found in the nucleus and cytoplasm and functioned as a pro-fibrotic factor to facilitate pulmonary fibrosis by promoting fibroblast-to-myofibroblast differentiation and myofibroblast proliferation and migration. Mechanistic studies revealed that H3K27ac and ATF3 bind to the chromatin open region of lncIAPF promoter and thereby enhance lncIAPF transcription. The highly expressed lncIAPF forms an RNA-protein complex with ELAVL1 to inhibit autophagosome fusion with lysosome by controlling the stability of the target genes EZH2, STAT1 and FOXK1 (Figure 10). Moreover, the clinical value of lncIAPF as a therapeutic target for IPF was verified. Many factors can cause lncRNA expression changes. For example, 8p12 loci loss of LINCTSLNC8 reduces LINCTSLNC8 expression, leading to hepatocellular carcinoma malignancies [31]. H3K27 acetylation-activated lncCCAT1 affects cell proliferation and migration by regulating SPRY4 and HOXB13 expression in esophageal squamous cell carcinoma [32]. The transcription factor TP63 binds to the super enhancer at the LINC01503 locus and activates its transcription, leading to squamous cell carcinoma cell proliferation, migration, invasion, and xenograft tumor growth [33]. However, no direct factors that induce lncRNA change in pulmonary fibrosis have been reported. Here, H3K27ac was found to activate the chromosome region of lncIAPF promoter opening during fibrogenesis. As a regulator, the transcription factor ATF3 bound to lncIAPF promoter and enhanced the lncIAPF expression. LncRNAs are closely associated with the development of pulmonary fibrosis, particularly by acting as pro- or anti-fibrotic factors in several process such as alveolar epithelial cell injury [34], fibroblast-to-myofibroblast transition, extracellular-matrix deposition [35], and macrophage activation [36]. LncRNA participation in fibroblast differentiation into myofibroblasts is one of the research focus on pulmonary fibrogenesis. For example, lncRNA DNM3OS contributes to fibroblast differentiation into myofibroblast function as a reservoir of three miRNAs, namely, MIR199A-5p, MIR199A-3p and MIR214-3p, through the TGFB signaling pathway [37,38]. LncPFAR enhances lung fibroblast activation by sponging MIR138 to regulate the YAP1-Twist axis [39]. LncFENDRR [40], lncCDKN2B-AS1 [41] and lncPFAL [42] can accelerate lung fibrosis by accelerating fibroblast differentiation into myofibroblast. Various kinds of ncRNAs, such as lncRNA, miRNA, circRNA and mRNA, can even form a fine regulatory network to control fibroblast differentiation into myofibroblast. Lnc865, lnc556, circ949, circ057, Mir29b-2-5p and STAT3 are typical examples. Among which, lnc865 and lnc556 can crosstalk with circ949 and circ057 by sponging Mir29b-2-5p to target STAT3 translocation. After phosphorylation, p-STAT3 as a transcription factor can be transported into the nucleus from the cytoplasm and activate gene expression [43]. However, the lncRNA regulation of fibroblast-to-myofibroblast differentiation through the autophagy signal pathway in pulmonary fibrosis is poorly investigated. Our data reported that the highly upregulated lncIAPF promotes fibroblast-to-myofibroblast differentiation and myofibroblast proliferation and migration by blocking autophagy in pulmonary fibrosis. Autophagy is a type II programmed cell death in which cellular components are sequestered in an autophagosome, which then fuses with a lysosome to degrade those contents. Dysregulated autophagy has implications in health and diseases [44,45]. In general, autophagy is a protective mechanism in the lung of patients with IPF [46]. Autophagy deficiency in macrophages exacerbates inflammation and fibrosis following silica exposure [47] and bleomycin challenge [48]. Targeting autophagy in IPF, including the use of IL17A (interleukin 17A) neutralizing antibodies [49], Mir449a [50], or PDGFRB (platelet derived growth factor receptor beta) inhibitors, may offer a therapeutic potential [51]. Recently, more and more lncRNAs are revealed to be involved in the regulation of autophagy. In-vitro and in-vivo experiments uncover that lncRNA EIF3J-DT activates autophagy and induced drug resistance in gastric cancer cells by targeting ATG14 [52]. LncRNA SNHG11 aggravates oncogenic autophagy to facilitate cell proliferation, stemness, migration, invasion and epithelial-to-mesenchymal transition in gastric cancer [53]. LncRNA BCYRN1-induced autophagy enhances asparaginase resistance in extranodal NK/T-cell lymphoma [54]. In pulmonary fibrosis, Ni’s group found that silica-induced pulmonary fibrosis is facilitated by lncHOTAIR through the sponging of MIR326 and attenuated by the increased MIR326 expression through the promotion of the autophagy activity of fibroblasts by targeting PTBP1 (polypyrimidine tract binding protein 1). However, the direct regulation between lncHOTAIR and autophagy has not yet been clarified [55]. The present study revealed that lncIAPF directly controls autophagy by blocking autophagosome fusion with lysosome depending on ELAVL1 in pulmonary fibrosis; knockin lncIAPF blocked autophagy, whereas lncIAPF knockdown enhanced this process. We reasoned that lncIAPF binds to ELAVL1 protein and blocks ELAVL1 susceptibility to ubiquitination and degradation, thereby increasing the levels of ELAVL1 and its target autophagy-related mRNAs such as EZH2, STAT1 and FOXK1 that are negatively associated with autophagy. These changes prevent autophagy. As an RNA-binding protein, ELAVL1 participates in disease occurrence and progression by binding to lncRNAs. The macrophage-specific lncMAARS regulates apoptosis and atherosclerosis by tethering ELAVL1 in the nucleus [56]. LncOCC-1 suppresses cell growth by destabilizing ELAVL1 protein in colorectal cancer [16]. However, ELAVL1 binding to lncRNA in pulmonary fibrosis has not yet been reported. In this work, lncIAPF bound to ELAVL1 in pulmonary fibrosis and that the lncIAPF-ELAVL1 complex regulated autophagy flux through the target genes EZH2, STAT1 and FOXK1. EZH2 functions as a histone methyltransferase by catalyzing histone methylation at H3 Lys27. STAT1 and FOXK1 function as transcription factors and play important roles in cell growth, differentiation and death []. As cytosolic genes, these three genes can control autophagy []; however, this function has not been reported in pulmonary fibrosis. The findings indicated that together with the lncIAPF-ELAVL1 complex, these three genes negatively regulate autophagy in pulmonary fibrosis. Although lncRNAs have been found in humans, only a small number of lncRNAs have been functionally characterized in pulmonary fibrosis, and most of their mechanisms are largely unknown. Our observations confirmed that lncIAPF overexpression accelerates fibroblast-to-myofibroblast differentiation by blocking autophagy in pulmonary fibrosis, and this effect is dependent on ELAVL1. This finding provides a novel treatment strategy related to autophagy for IPF.
IPF patients were diagnosed in accordance with the American Thoracic Society and European Respiratory Society criteria for IPF, and age-matched healthy controls at Binzhou Medical University Hospital were included. All patients provided written informed consent, and ethical consent was obtained from the Committee of Binzhou Medical University.
Experiments on the studied animals were approved by the Committee on the Ethics of Animal Experiments of Binzhou Medical University. Eight-week-old C57BL/6 mice were obtained from the Model Animal Research Center of Nanjing University (Nanjing, China). All mice were bred and maintained in a 12 h light/dark cycle and allowed free access to food and water. The mice were divided into different groups (10 mice in each group) according to the experimental requirements. The BLM-treated mouse was administered 5 mg/kg bleomycin, which was sprayed into the lungs using a Penn-Century MicroSprayer (Penn-Century Inc., Wyndmoor, PA, USA). The experimental mice were administered 1.0 × 1012 vg/mL adenoviral-lncIAPF/NC or adenoviral-si-ELAVL1/NC, which was also sprayed into the lungs using a Penn-Century MicroSprayer. The sham group only received the same amount of normal saline as the control group. Lung tissues were harvested on the 28th day following treatment.
Human fetal lung fibroblast (MRC-5 cell line) and mouse fibroblast (L929 cell line) were obtained from American Type Culture Collection (CCL-171™ and CCL-1™) and cultured at 37°C in an atmosphere containing 5% CO2 and in advanced minimum essential medium (MEM; Gibco, 11,090,081), supplemented with 10% fetal bovine serum. The cells were treated with 5 ng/mL TGFB1 (Gibco, PHG9202) for different times according to the requirement of experiments. Western-blotWestern blot were performed using standard protocols established in our laboratory, specific steps were referred to published articles [11]. The item number and brand of the antibody were listed in Table 6.
Fifty μL pre-cooled RSB (containing 0.1% NP40 [Thermo Scientific, 85,124], 0.1% Tween-20 [Thermo Scientific, 28,320], 0.01% digitalis saponin [MedChemExpress, HY-N4000]) was added to 5 × 104 living cells, which were incubated on ice for 3 min. Then, 1 mL pre-cooled RSB only contained 0.1% Tween-20 was added and centrifuged at 500 g for 5 min. The supernatant was removed. The precipitate was resuspended with 50 μL transposition mix (TD, 2× reaction buffer from Nextera kit [Illumina, FC-121-1030], 25 µL; TDE1, Nextera Tn5 Transposase from Nextera kit, 2.5 µL; and nuclease-free H2O 22.5 µl; total volume 50 µL), and placed into a mixometer (Youning, Hangzhou China) at 177 × g for 30 min. The DNA was first purified with Qiagen MinElute PCR Purification Kit (QIAGEN, 28,004), and was further purified with Zymo DNA Clean and Concentrator-5 Kit (ZYMO RESEARCH, D4004). Then PCR was performed in a 50 μL final volume as follows: 7 cycles of 98°C for 3 min, 98°C for 15s, and 63°C for 30s, finally, 72°C for 30s, 72°C for 5 min. The PCR product was sequenced by illuminaHiSeq/NextSeq platform. Subsequently, the data was analyzed using unique mapped reads.
Cells (5 × 104) were cultured in E-plate (proliferation plate; Agilent, 5,469,830,001) and CIM-plate (migration plate; Agilent, 05665817001), respectively, in an RTCA DPlus instrument system. The proliferation and migration curves were automatically recorded by the system. Cell index representing the amount of proliferating or migrating cells was calculated by using RTCA from ACEA Biosciences (ACEA Biosciences, China), as previously described in a study [11].
Cells (5 × 105) were seeded in a 96-well plate and incubated in an incubator. After overnight incubation, the cells were wounded with cell scratcher, washed with PBS (SparkJade, CR0013-500ML), replaced with complete medium culture, and placed in IncuCyte S3 live-cell analysis system (Essen BioScience, USA) for real-time dynamic observation. Images were taken on IncuCyte S3 software.
RNA affinity-isolation assay was carried out as previously described [11]. Briefly, lncIAPF transcripts were transcribed using T7 and SP6 RNA polymerase in vitro, then by using the RNeasy Plus Mini Kit (QIAGEN, 74,134) and treated with TURBO DNase I. Purified RNAs were biotin-labeled using Biotin RNA Labeling Mix (Roche Diagnostics, 11,685,597,910). The biotinylated lncIAPF and its antisense RNAs were mixed and incubated with cell lysates. Then, avidin magnetic beads were added to each binding reaction, and the mixtures were incubated. The binding proteins were separated by SDS-PAGE, visualized by silver staining and identified by regular liquid chromatography-mass spectrometry and western blot analysis.
Cells were collected and extracted RNA. Complementary DNA synthesis was performed according to the RT2 First Strand kit (QIAGEN, 330,404). After cDNA obtainment, the PCR reaction was carried out in a 25 μL final volume using 40 ng cDNA as follows: initial denaturation at 95°C for 10 min, followed by 40 cycles of 95°C for 15s, 55°C for 35s and 72°C for 30 min.
RNA FISH was performed with the FISH kit according to the manufacturer’s protocol (Ribo Bio Technology, C10910). Cells were seeded in a 24-well plate. When the cell density reached 50–60%, the medium was discarded and 4% paraformaldehyde was added. After washing with PBS, 500 μL of pre-cooled permeate solution was added into cell samples for 3 min. Then permeate solution was washed away with PBS, 200 μL pre-hybridization buffer was added to each well and incubated for 30 min. Subsequently, 100 μL 20 μM lncIAPF, RNA18S and RNU6 FISH Probe Mix were added to each well and incubated overnight. Cells were washed with SSC (Solarbio, S1030) and PBS solution, and stained with DAPI staining solution for 10 min. Finally, the images were observed with a confocal microscope.
RIP assays were performed using an EZ-Magna RIP™ RNA-binding protein immunoprecipitation kit (Millipore, 17–701) according to the manufacturer’s instructions. Cells at approximately 90% confluence was lysed using complete RIP lysis buffer containing RNase inhibitor and protease inhibitor and then 100 μL of whole cell extract was incubated with RIP buffer containing magnetic beads conjugated to specific antibody (Cell Signaling Technology, 12,582). The nonspecific control was normal rabbit anti-IgG antibody (Cell Signaling Technology, 2729). Purified RNA was subjected to qRT-PCR analysis.
ChIP-PCR was performed following the SimpleChIP® Plus Sonication Chromatin IP Kit protocol (Cell Signaling Technology, 56,383). Briefly, cells were crosslinked with 1% formaldehyde in culture medium for 10 min at room temperature, and chromatin from lysed nuclei was sheared to 200–600 bp fragments. Chromatin was immunoprecipitated with antibody of ATF3 (Cell Signaling Technology, 18,665) or normal rabbit IgG (Cell Signaling Technology, 2729) overnight. Antibody/antigen complexes were recovered with Protein G agarose beads (Cell signaling technology, 9007) for 2 h at 4°C. Two sequential elutions were performed. Eluted chromatin was diluted 1:3 with lysis buffer, supplemented with 1% Triton X-100 (Sigma-Aldrich, 9036–19-5) and incubated with 3 mg of normal rabbit IgG or anti-ATF3 antibody, overnight at 4°C. The immunoprecipitated DNA was collected. Purified DNA was performed with ChIP-PCR. The amount of immunoprecipitated DNA in each sample was determined as the fraction of the input and normalized to the IgG control.
1 × 106/mL MRC-5 cells were seeded in a six-well plate including the control group, TGFB1-induced group, si-ELAVL1-treated group. Cells in control group were treated with 5 μg/mL actinomycin D (Aladdin, A13142-5 mg) alone for 4 h. Cells in TGFB1-induced group were first treated with 5 μg/mL TGFB1 for 24 h, then co-treated with 5 μg/mL actinomycin D for 4 h. Cells in si-ELAVL1-treated group were first treated with 5 μg/mL TGFB1 for 24 h, then co-treated with 10 μg/mL si-ELAVL1 for 24 h, finally co-treated with 5 μg/mL actinomycin D for 4 h. EZH2, STAT1 and FOXK1 levels were measured by qRT-PCR at actinomycin D treatment for different times. GAPDH was used as an internal standard.
MRC-5 cells were seeded in a 6-cm cell culture plate. Cycloheximide (HX-R, T181074-1 g) was added to the medium at a final concentration of 10 μg/mL, thereby inhibited the synthesis of ELAVL1 protein. After cycloheximide treatment for different times, MRC-5 cell was harvested. The levels of ELAVL1 and GAPDH (Affinity, AF7021) proteins were detected by western blot.
Cell samples were lysed in NP-40 lysis buffer (Thermo Scientific, 85,124) at 4°C for 30 min. The cell lysates were first incubated with antibodies of ELAVL1 and control IgG at 4°C overnight, and then were incubated with protein A/G (Absin, F08D04) beads for 3 h at 4°C. The beads were washed for three times with NP-40 lysis buffer. Western blotting analysis was carried out against anti-ubiquitin antibody (Abcam, ab134953).
MRC-5 cell samples were seeded in a 24-well plate. 3 μL Dual-fluorescence mRFP-GFP-LC3 virus stock solution (Hanbio Inc, Shanghai, China) was diluted in 250 μL 10% FBS MEM medium and added into each well. After hatching for 2 h, the MEM medium contained virus was removed and the fresh MEM medium without FBS was added. After hatching for 12 h, real time dynamic changes of cells were monitored under the IncuCyte S3 (Essen BioScience, USA) instrument and images were collected using a confocal microscope.
Statistical analysis were performed using SPSS version 19.0 software. Data were presented as the mean ± SD of at least three independent experiments. Unpaired Student’s t test was used for experiments comparing two groups, whereas one-way ANOVA with Student-Newman-Keuls post hoc test was applied for experiments comparing three or more groups. Statistical significance was considered at p < 0.05.
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PMC9629112 | 36310384 | Hairun Gan,Luting Li,Xinyan Hu,Jianxun Cai,Xiaojun Hu,Haopei Zhang,Ni Zhao,Xiwei Xu,Hui Guo,Pengfei Pang | DDX24 regulates the chemosensitivity of hepatocellular carcinoma to sorafenib via mediating the expression of SNORA18 CANCER BIOLOGY & THERAPY | 30-10-2022 | Hepatocellular carcinoma (HCC),sorafenib (SFN),DDX24,SNORA18,chemosensitivity | ABSTRACT Sorafenib (SFN) is a multi-kinase inhibitor drug for the treatment of advanced hepatocellular carcinoma (HCC), but its limited efficacy is a major obstacle to the clinical outcomes of patients with HCC. We aimed to explore a novel molecular mechanism underlying the chemosensitivity of HCC to SFN, and to identify a promising therapeutic target for HCC treatment. In this study, bioinformatic analysis revealed that DDX24 was associated with poor survival in HCC cases, and significantly related to the pathways modulating tumor development. DDX24 regulated HCC cell proliferation and migration potentials. Moreover, reduction of DDX24 promoted the sorafenib-mediated inhibition of HCC cell growth and migration, the elevation of sorafenib-induced HCC cell apoptosis. DDX24 overexpression suppressed the inhibitory effect of SFN on cell proliferation and migration and reduced the apoptosis induced by SFN. Further, DDX24, combined with SFN treatment, presented a synergistic enhancement of the sensitivity of SFN to the growth and migration of HCC cells via AKT/ERK and the epithelial-mesenchymal transition (EMT) pathways, and that it modulated apoptosis via the caspase/PARP pathway. Mechanistically, SNORA18 served as a target gene for DDX24, regulating the chemosensitivity of sorafenib-treated HCC cells. Furthermore, SNORA18 knockdown or overexpression could partially reverse the inhibition or elevation of cell viability, colony formation and migration induced by DDX24 in sorafenib-treated HCC cells, respectively. Collectively, our results suggest that DDX24 regulates the chemosensitivity of HCC to SFN by mediating the expression of SNORA18, which may act as an effective therapeutic target for improving SFN efficiency in HCC treatment. | DDX24 regulates the chemosensitivity of hepatocellular carcinoma to sorafenib via mediating the expression of SNORA18 CANCER BIOLOGY & THERAPY
Sorafenib (SFN) is a multi-kinase inhibitor drug for the treatment of advanced hepatocellular carcinoma (HCC), but its limited efficacy is a major obstacle to the clinical outcomes of patients with HCC. We aimed to explore a novel molecular mechanism underlying the chemosensitivity of HCC to SFN, and to identify a promising therapeutic target for HCC treatment. In this study, bioinformatic analysis revealed that DDX24 was associated with poor survival in HCC cases, and significantly related to the pathways modulating tumor development. DDX24 regulated HCC cell proliferation and migration potentials. Moreover, reduction of DDX24 promoted the sorafenib-mediated inhibition of HCC cell growth and migration, the elevation of sorafenib-induced HCC cell apoptosis. DDX24 overexpression suppressed the inhibitory effect of SFN on cell proliferation and migration and reduced the apoptosis induced by SFN. Further, DDX24, combined with SFN treatment, presented a synergistic enhancement of the sensitivity of SFN to the growth and migration of HCC cells via AKT/ERK and the epithelial-mesenchymal transition (EMT) pathways, and that it modulated apoptosis via the caspase/PARP pathway. Mechanistically, SNORA18 served as a target gene for DDX24, regulating the chemosensitivity of sorafenib-treated HCC cells. Furthermore, SNORA18 knockdown or overexpression could partially reverse the inhibition or elevation of cell viability, colony formation and migration induced by DDX24 in sorafenib-treated HCC cells, respectively. Collectively, our results suggest that DDX24 regulates the chemosensitivity of HCC to SFN by mediating the expression of SNORA18, which may act as an effective therapeutic target for improving SFN efficiency in HCC treatment.
Hepatocellular carcinoma (HCC) is the sixth most common cancer worldwide and ranks third as a leading cause of cancer-related death, with a mortality rate of 82%. HCC is often diagnosed late due to the asymptomatic nature of this disease in the early stage, and it is not feasible to achieve curative treatment in patients with advanced HCC. The asymptomatic onset, rapid tumor progression, therapy resistance, cancer recurrence and distant metastasis are the principal causes accounting for the poor prognosis of HCC, resulting in a severe burden for healthcare systems. Sorafenib (SFN), a tyrosine kinase inhibitor (TKI), is the first-line FDA-approved drug for the treatment of unresectable advanced HCC, providing a 44% improved overall survival (OS) rate in patients. Despite the encouraging efficacy of SFN, its effectiveness is mainly compromised by a decline in sensitivity, and acquired chemoresistance. The specific mechanisms of the distinct sensitivities of patients with HCC administered SFN are complicated. This is ascribed to the complex interaction of multiple factors, including the activation or dysregulation of various signaling pathways, hypoxia-inducible responses, enrichment of the liver cancer stem cells (LCSC) and mitophagy of reactive oxygen species. Therefore, enhancing SFN sensitivity in HCC treatment is of great value for improving the OS of patients with HCC, and elucidating the molecular regulation underlying drug sensitivity is vital. DEAD-box proteins are the largest family of RNA helicases with ATPase activity and function in numerous aspects of eukaryotic RNA metabolism. DDX24 is an important member of the DEAD-box RNA helicases carrying a conserved Asp-Glu-Ala-Asp (D-E-A-D) motif. Based on our recent findings, a mutated DDX24 gene could cause vascular malformations, and previous studies have revealed that DDX24 is upregulated in multiple human cancer cells, inhibiting cell growth by inducing cell cycle arrest and senescence in osteosarcoma cells. Moreover, DDX24 also regulates the proliferation of colon cancer and gastric cancer cells. Most published research associated DDX24 strongly with the development of carcinoma, however, the specific role of DDX24 in HCC is still obscure. Small nucleolar RNA (snoRNA) is a massive subgroup of non-coding RNA (ncRNA) with a length of 60–300 nucleotides predominately found in the eukaryotic nucleolus. Based on the structural elements of snoRNAs, they are categorized into three types containing the C/D box, ribonuclease 7–2/MRP and H/ACA box snoRNAs, which directly bind to their substrate via base pairing for inducing 2′O-methylation and pseudoacylation, respectively. Since the snoRNAs are crucial regulatory factors responsible for post-transcriptional modification of ribosomal RNAs (rRNAs), spliceosomal RNAs (snRNAs) and transfer RNAs (tRNAs), they can modulate various physiological and pathological processes, such as cell proliferation, differentiation and angiogenesis. From recent studies, an elevated snoRNAs expression could significantly induce oncogenesis of different carcinoma due to a disordered synthesis of ribosomes. Additionally, snoRNAs have been proven to be closely related to the prognosis of patients with HCC and a potential molecular therapeutic target in HCC. Small nucleolar RNA H/ACA box 18 (SNORA18) transcribed from the chromosome 11q21 genomic region was identified as a regulator of tumor growth in pancreatic cancers. Currently, evidence assessing the particular effect of SNORA18 in the progression of HCC, and mediating the chemosensitivity of SFN, remains elusive. In this study, we examined the functional role of DDX24 on the development of HCC and on the modulation of the sensitivity to SFN in HCC treatment. Furthermore, we elucidated a potential mechanism by which DDX24 regulated SFN sensitivity in HCC therapy by mediating the expression of its target SNORA18.
To explore the specific function of DDX24 in HCC, we first analyzed 371 liver cancer samples and 50 adjacent samples from The Cancer Genome Atlas (TCGA) database. We divided patients with liver cancer from the TCGA dataset into two groups based on their DDX24 expression. A Kaplan-Meier analysis indicated that the DDX24 level was strongly correlated with patient’s overall survival time (p < .05, Figure 1a). Subsequently, we explored the potential biological pathways associated with DDX24 to evaluate its molecular mechanism in HCC. The gene network map using the GeneMANIA tool demonstrated that DDX24 was surrounded by 20 nodes representing genes with close correlations (Figure 1b), and the protein-protein interaction (PPI) network using the STRING tool revealed that DDX24 was connected with 10 proteins (Figure 1c). We found DDX24 was correlated with DKC1, CEP250, PSMD14, INO80, G3BP2, SIRT7, KDM1A, CCND1, FBL, IDO1 and DDX27, functioning as an oncogene in the development of HCC. Furthermore, we identified the top 50 co-expressed genes of DDX24 in liver cancer samples from the TCGA database and performed an interaction network analysis via the Reactome Pathway (p < .05, Figure 1d). Next, the top 18 co-expressed genes (|Spearman correlation coefficient| >0.4 and p < .01, Supplementary Table 1) were subjected to the Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis. A KEGG pathway enrichment analysis revealed that DDX24 was likely involved in crucial biological signaling relating to promotion of HCC progression, including PI3K-Akt-mTOR, apoptosis, JAK-STAT, Notch and the Necroptosis signaling pathways (Figure 1e). In summary, a high expression of DDX24 is significantly related to patient overall survival, and DDX24 may be involved in the pathways regulating HCC development.
As the KEGG pathway enrichment analysis showed that DDX24 was correlated with HCC development, we, therefore, reduced and elevated the expression of DDX24 in Hep3B and Bel-7402 cells to further investigate the effect of DDX24 on HCC tumorigenesis. Real-time quantitative polymerase chain reaction (RT-qPCR) and western blot analysis were used to confirm the alteration of DDX24 levels (Figure 2a,). We found that DDX24 knockdown and overexpression significantly inhibited and promoted the cell proliferation and migration potentials of HCC cells, respectively (Figure 2c-f). Consistent with these results, the suppression of DDX24 in Hep3B and Bel-7402 cells also remarkably decreased the phosphorylation of AKT and ERK, and the expression of epithelial-mesenchymal transition (EMT) associated proteins ZO-1, N-cadherin and β-catenin (Figure 2g,). However, DDX24 overexpression had a converse effect on protein levels (Figure 2h,). These data indicate that DDX24 regulates the proliferation and migration of HCC cells in vitro.
Sorafenib (SFN) is a multi-kinase inhibitor approved as the standard first-line treatment for advanced HCC cases. However, the decline in sensitivity to SFN limits its efficacy in improving survival time. Recent studies have reported that the AKT/ERK pathway is involved in HCC sensitivity to SFN, and our data demonstrated that DDX24 could regulate the expression of phospho-AKT and phospho-ERK. We speculated that DDX24 might regulate the sensitivity of HCC cells to SFN treatment. To verify this hypothesis, HCC cells with DDX24 silencing were administrated to a medium containing different concentrations of SFN. We found that reduction of DDX24 increased the sensitivity of HCC cells to SFN via cell viability analysis, and the IC50 values of SFN in DDX24 knockdown Hep3B and Bel-7402 cells were lower (Hep3B shDDX24-1: 4.23 ± 0.19 μmol/l, Hep3B shDDX24-2: 4.82 ± 0.18 μmol/l, Bel-7402 shDDX24-1: 4.93 ± 0.50 μmol/l, Bel-7402 shDDX24-2: 4.08 ± 0.40 μmol/l) in contrast to the corresponding control cells (Hep3B shNC: 5.58 ± 0.08 μmol/l, Bel-7402 shNC: 7.04 ± 0.18 μmol/l) (Figure 3a). However, the inhibitory effect of SFN on DDX24 overexpressing cells was weaker than that of the vector group in cell viability (Figure 3c). As shown in Figure 3b, CCK-8 assays showed that downregulation of DDX24 dramatically increased the suppression of SFN on Hep3B and Bel-7402 proliferation. Moreover, we also found that the inhibitory effect of SFN on DDX24 knockdown cells was stronger than a nonspecific shRNA control using colony formation assays (Figure 3e). Meanwhile, overexpression of DDX24 reduced the inhibitory effect of SFN on cell proliferation and colony formation (Figure 3d,). We next measured the pivotal protein expression of the AKT/ERK signaling pathway. The results revealed that SFN treatment decreased the phosphorylation of AKT and ERK, but had no effect on DDX24, total AKT and total ERK expression. Additionally, silencing DDX24 was shown to further enhance the SFN suppression of phospho-AKT and phosphor-ERK proteins (Figure 3g). These findings suggest that DDX24 regulates sorafenib-mediated inhibition of HCC cell proliferation via the AKT/ERK pathway in vitro.
We next explored whether apoptosis mediated the anti-proliferation activity induced by DDX24 and SFN treatment in HCC cells. The flow cytometry assays using Annexin V-FITC/PI double staining revealed that the combined treatment of DDX24 knockdown and SFN exerted a synergistic effect on elevating cell apoptosis in Hep3B and Bel-7402 cells, compared with SFN treatment alone (Figure 4a). Conversely, we found DDX24 overexpression decreased the cell apoptosis induced by SFN (Figure 4b). Additionally, we measured the downstream apoptosis-related protein expression using western blot analysis. The results showed that SFN administration increased the expression of cleaved-PARP and cleaved caspase-7, and this trend was further promoted following DDX24 inhibition (Figure 4g). Together, these data show that DDX24 regulates sorafenib-induced apoptosis in HCC cells via the caspase/PARP pathway in vitro.
Metastasis is a crucial determinant of cancer-related prognosis and OS time, and mounting evidence has suggested that the EMT signaling pathway plays a significant role in HCC metastasis and its sensitivity to SFN. As our results demonstrated that DDX24 could modulate the expression of EMT related proteins, we aimed to detect whether DDX24 combined with SFN treatment performed a synergistic inhibition on HCC cell migration. In our study, migration assays indicated that the inhibition of cell migration rate caused by SFN was remarkably increased by DDX24 knockdown in Hep3B and Bel-7402 cells (Figure 4c). However, the inhibition of migration mediated by SFN could be reversed via DDX24 overexpression (Figure 4d). We next investigated the cytoskeleton using IF assays and found that SFN treatment reduced the number of spike-like filopodia at the edges of HCC cells, while this phenomenon was also more prominent following DDX24 silencing (Figure 4e,). Furthermore, western blot analysis revealed that the expression of EMT associated proteins ZO-1, N-cadherin and β-catenin, was suppressed in Hep3B and Bel-7402 cells treated with SFN, and that this was more dramatic in DDX24 knockdown cells (Figure 4h). Therefore, we conclude that DDX24 regulates the sorafenib-mediated inhibition of HCC cell migration via the EMT pathway in vitro.
To investigate whether DDX24 regulated the anti-tumor effect of SFN against HCC growth in vivo, we used a subcutaneous mouse HCC xenograft model. Hep3B cells showing stable DDX24-shRNAs or control shRNA were injected subcutaneously into BALB/c nude mice aged four to six weeks. Tumor-bearing mice were administered with vehicle or SFN orally, once daily. All the mice tolerated the treatment well without observable signs of toxicity and had stable body weights throughout the study. As shown in Figure 5a-c, the in vivo anti-tumor assays revealed that the size and weight of xenografts in the control shRNA group receiving SFN treatment were decreased in contrast to the control shRNA group receiving PBS. Furthermore, a significant inhibitory effect on the tumor size and weight was exhibited in the DDX24-shRNAs group, compared to the control shRNA group receiving SFN. Consistent with the in vivo findings, immunohistochemistry (IHC) assays of xenografts generated from DDX24-shRNAs+SFN tumor tissues demonstrated an obvious reduction in Ki67, p-EKT, β-catenin and ZO-1 levels (Figure 5d). The western blot analysis also showed a declined expression of p-EKT, β-catenin and ZO-1 (Figure 5e). Collectively, these results confirm that DDX24 knockdown regulates the sorafenib-mediated inhibition of HCC growth in vivo.
To explore the underlying mechanism of DDX24 in regulating the chemosensitivity of HCC cells exposed to SFN, we performed RNA-seq comparing HCC cells treated with SFN to controls in order to determine aberrantly-expressed downstream genes. After the screening of differentially expressed RNAs by fold change (FC) filtering (log2FC ≥3.0) and student’s t testing (p < .01), there were 46 upregulated RNAs in Bel-7402 cells administrated with SFN versus the controls (Figure 6a). We then applied the result of RNA-seq on DDX24 knockdown Hep3B cells and a negative control from the online Gene Expression Omnibus (GEO) database (GEO Submission: GSE145635). As shown in Figure 6b, C15orf38-AP3S2, SLC6A6, SNORA18 and RPL12P38 emerged as candidates as they were both upregulated in HCC cells with SFN treatment and DDX24 knockdown. Furthermore, RT-qPCR analysis confirmed that SNORA18 was significantly increased when HCC cells were treated with SFN, and in HCC cells after DDX24 silencing (Figure 6c,). In addition, the expression of SNORA18 was remarkably elevated or reduced in DDX24 knockdown combined with SFN, or overexpression combined with SFN, respectively, in contrast to the group treated with SFN alone (Figure 6e,). We next sought to investigate the function of SNORA18 in HCC cells, and RT-qPCR was performed to measure the reduction and elevation of SNORA18 levels (Figure 6g,). The findings revealed that downregulation or upregulation of SNORA18 enhanced or suppressed the cell proliferation and migration abilities of Hep3B and Bel-7402 cells, respectively (Figure 6h, 6-l). We also found that the inhibition of cell viability, colony formation and migration induced by silencing DDX24 and SFN treatment was partially reversed through SNORA18 knockdown (Figure 7a-d). However, SNORA18 overexpression could partially reverse the DDX24-induced inhibitory effect of SFN on cell viability, colony formation and migration (Figure 7e-h). Moreover, reduction of SNORA18 in Hep3B cells treated with DDX24-specific shRNA and SFN could increase the expression of phosphorylation of AKT and ERK, ZO-1, N-cadherin and β-catenin, while decreasing the expression of cleaved-PARP and cleaved caspase-7 (Figure 7i). These findings indicate that DDX24 modulates the chemosensitivity of HCC cells to SFN via the SNORA18 pathway.
The most common malignant solid tumor of the liver, HCC accounts for 85% of all primary liver carcinoma cases. SFN is the first FDA approved multi-kinase inhibitor drug for the treatment of advanced HCC. However, the declined sensitivity and developed accompanying extension of the medication time, limit its improvement of the clinical outcomes of patients with HCC. Hence, it is urgent to explore novel therapeutic strategies to enhance the sensitivity of SFN in HCC treatment. In this study, we found that DDX24 acted as an oncogene in HCC development via bioinformatic analysis, and DDX24 regulated HCC cell proliferation and migration by mediating the phosphorylation of AKT and ERK, and EMT protein levels. As SFN contributes mainly to the blockade of PI3K/AKT, MEK/ERK and the EMT signaling pathways, we further confirmed that a combination of DDX24 knockdown and SFN administration exerted a synergistic effect on suppressing HCC growth through modulating the AKT/ERK pathway in vitro and in vivo. Meanwhile, the results also revealed that downregulation or upregulation of DDX24 elevated or suppressed sorafenib-mediated inhibition of migration ability in HCC cells via the EMT pathway, respectively. Previous studies reported that SFN could induce caspase-mediated apoptosis in several human cancers. Our data showed that silencing DDX24 combined with SFN treatment presented a synergistic enhancement on HCC cell apoptosis through the caspase/PARP pathway. However, overexpression of DDX24 reduced the cell apoptosis induced by SFN treatment. To the best of our knowledge, this is the first study to systematically investigate the possible association between DDX24 and SFN treatment of HCC. DDX24 is vital in plenty of cellular processes, for instance, DDX24 is implicated in the ribosome biogenesis. The defect of ribosomal maturation and function can result in the dysregulation of essential procedures, which transforms diseased and normal cells into cancer cells. Our previous research revealed that DDX24 mutations were pathogenic factors of vascular deformities and played a significant role in the migration ability of endothelial cells. Based on published reports, DDX24 functions as a modulator of the p300-p53 axis by suppressing the p300-mediated acetylation of p53, which promotes the proliferation of osteosarcoma cells and lung carcinoma cells. Additionally, other studies revealed that a perturbation of DDX24 inhibited colon cancer and gastric cancer growth. Collectively, DDX24 functions as a new hallmark and therapeutic target for carcinoma, and our results, consistent with other published research, suggest that DDX24 can modulate the proliferation and migration potentials of HCC cells. Moreover, our findings also indicate that DDX24 may serve as a novel regulator of SFN sensitivity in HCC treatment. Furthermore, understanding the specific molecular modulation on chemosensitivity of HCC to SFN can help identify potential targets and develop more effective therapeutic strategies against HCC. Mechanistically, we uncovered that DDX24 regulated the chemosensitivity of HCC cells to SFN by a small nucleolar RNA H/ACA box 18 (SNORA18) dependent pathway. Small nucleolar RNA (snoRNA) is a critical modulator participating primarily in the processing, folding and modification of pre-ribosomal RNAs (pre-rRNAs). This implies that its function may be correlated with the DDX24-mediated biogenesis of ribosomes. Recently, there has been an accumulation of interest in illustrating the malfunctioning roles of snoRNAs on the molecular mechanism of HCC development. For instance, SNORD76 enhanced the invasion of HCC cells through elevating the EMT pathway, while the snoU2_19 abrogation inhibited the HCC cell proliferation via Wnt/β-catenin signaling. Other reports have revealed that ACA11 knockdown represses the proliferation, migration and invasion of HCC cells, and SNORD52 highly expressed in HCC, could promote the development of HCC cells. However, the complicated relationship between snoRNAs and HCC is not clearly known, and there are few studies identifying the efficacy of snoRNAs on SFN sensitivity. SNORA18 belonging to the H/ACA box snoRNAs is located at chromosome 11q21 within an intron of the small nucleolar RNA host gene TAF1D, which participates in the package of preinitiation complex (PIC) during RNA polymerase I dependent transcription. In a recent research, SNORA18 regulated the cell invasion and tumor metastasis of pancreatic cancers via binding to the KHSRP protein. Another study indicated that SNORA18, packed in intracellular vesicular endosomes, could be used as a diagnostic marker for differentiating patients with pancreatic dual adenocarcinoma (PDCA). All these reported findings suggest a possible role of SNORA18 in oncogenesis. The results of our study indicate that the combined treatment of DDX24 knockdown and SFN administration cause a synergistic increase in SNORA18 expression. The expression of SNORA18 was reduced in DDX24 overexpression HCC cells incubated with SFN, in contrast to the group treated with SFN alone. Moreover, silencing SNORA18 partially reversed the reduction of cell proliferation, colony formation and migration abilities induced by DDX24 knockdown and SFN treatment. Based on our data, we also found that SNORA18 knockdown could reverse the alteration of biological function in HCC cells treated with DDX24-specific shRNA and SFN by regulating the AKT/ERK, EMT and caspase/PARP pathways. As DDX24 and snoRNAs can both mediate ribosome biogenesis, we speculate that the DDX24/SNORA18 axis modulates the sensitivity of HCC cells administered with SFN by affecting the ribosomal synthesis. Details about the specific mechanism of this hypothesis will, however, need to be explored in future studies. In conclusion, by means of a bioinformatics analysis, the highlighted findings of our research identified DDX24 as an adverse indicator of HCC prognosis and that it was significantly related to the pathways modulating tumor development. Furthermore, we identified that DDX24 regulated the sensitivity of SFN in HCC treatment by mediating SNORA18 (Figure 8). Modulating the expression of DDX24 and its target SNORA18 may serve as effective and specific therapeutic strategies for HCC treatment in future.
Hep3B human HCC cells were acquired from American Type Culture Collection (ATCC, Manassas, VA, USA), and Bel-7402 human HCC cells were obtained from Cell Bank of the Chinese Academy of Sciences (Shanghai, China). These cells were incubated in Dulbecco’s Modified Eagle Medium (DMEM; GIBCO, Catalog #C11995500BT, NY, USA) supplemented with 10% fetal bovine serum (FBS; ExCell Bio, Catalog #FSP500, Shanghai, China) and 0.1% penicillin-streptomycin in a humidified atmosphere containing 5% CO2 at 37 °C. Sorafenib (SFN; Selleck, Catalog #S7397, Texas, USA) was dissolved in DMSO and diluted at the indicated concentration before it was added to the complete medium for treatment. The cells were authenticated by short tandem repeat (STR) profiling and were confirmed to be mycoplasma free before use.
DDX24 shRNAs (shDDX24-1, 5’-GCUGCUAGAGAUGCUCAAUTT-3’; shDDX24-2, 5’-CCGUUUAGCUCGACAGAUUTT-3’), SNORA18 antisense oligonucleotide (SNORA18 ASO, 5’-UUACUCTATGAGGCGUUUCC-3’) and their corresponding negative control were synthesized by GenePharma Company (Shanghai, China). GV658-DDX24, GV658-SNORA18 and control vector plasmids were synthesized by Genechem Company (Shanghai, China). Transfection was performed with Lipofectamine 3000 (Invitrogen, Catalog #L3000015, California, USA) when cells in a 6-well plate had grown to the appropriate density (80%-90%) according to the manufacturer’s instruction. The cells were harvested for subsequent experiments after 48–72 hr of transfection.
To establish HCC cell lines with DDX24 stable knockdown, Hep3B and Bel-7402 cells were infected with DDX24 knockdown lentivirus or empty vector (U6-sh-DDX24-EGFP-IRES-puromycin; GeneCopoeia, Rockville, Maryland, USA) according to the manufacturer’s instruction. After 72 hr, cells were grown in a medium containing 1.0 μg/ml of puromycin (Invitrogen, Catalog #P8230, California, USA) for antibiotic selection. The efficiency of RNA interference was evaluated by quantitative real-time PCR (RT-qPCR) and western blot analysis.
Total RNAs were extracted using E.Z.N.A.® Total RNA Kit I (OMEGA, Catalog #R6834-02, Georgia, USA) or TRIzol reagent (Invitrogen, Catalog #abs60154, California, USA), and subsequently converted to cDNA using HiScript II One Step RT-PCR Kit (Vazyme, Catalog #R323-01, Nanjing, China) according to the manufacturer’s protocol. qPCR was employed to detect the expression of genes using All-in-OneTM qPCR Mix (GeneCopoeiaTM, Catalog #P222-02, Rockville, Maryland, USA) on Bio-Rad CFX96 following the manufacturer’s instruction, and analysis using Bio-Rad Manager software (Bio-Rad, Hercules, CA). GAPDH or U6 was used as internal controls, and the 2−∆∆Ct method was used to calculate the relative expression of genes. Primers for DDX24 (catalog no. Hs-QRP-23370), GAPDH (catalog no. Hs-QRP-20169), C15orf38-AP3S2 (catalog no. HQP055868), SLC6A6 (catalog no. HQP109841), RPL12P38 (catalog no. CS-QP0062) and SNORA18 (catalog no. CS-HmiR0023) were purchased from GeneCopoeia Inc (GeneCopoeiaTM, Rockville, Maryland, USA). The sequence of U6 primer was forward: 5’-CGCTTCGGCAGCACATATAC-3’ and reverse: 5’-AAATATGGAACGCTTCACGA-3’.
Whole cells were lysated in a RIPA lysis buffer (Beyotime Biotechnology, Catalog #P0013C, Shanghai, China) containing a protease/phosphatase Inhibitor Cocktail. BCA assays (Beyotime Biotechnology, Catalog #P0010, Shanghai, China) were used to detect the concentration of proteins in each sample. Proteins were separated by 5–20% SDS-PAGE gels (Genescript, Catalog #M42012C, Nanjing, China) and transferred onto PVDF membranes (Roche, Catalog #03010040001, Basel, Switzerland). After blocking with 5% milk for 1 hr at room temperature, primary antibodies were incubated overnight at 4 °C. After washing 3 times with TBST, a HRP-conjugated anti-rabbit or anti-mouse secondary antibody was incubated for 1 hr at room temperature. The protein bands were visualized with the use of a chemiluminescence kit (Thermo, Catalog #34580, Carlsbad, USA). The antibodies used are listed in Supplementary Table 2.
Cell Counting Kit-8 (CCK-8; KeyGEN BioTECH, Catalog #KGA317, Nanjing, China) was used to assess cell viability. In brief, cells were plated in a 96-well plate (Corning, Catalog #3599, NY, USA) at a density of 3 × 103 cells per well (48 hr after transfection) and incubated overnight at 37 °C. Cells were then treated with DMSO or indicated concentrations of SFN for 48 hr. Subsequently, the medium was removed and replaced with 100 μl 10% CCK-8 solution. The absorbance was detected at 450 nm using a microplate reader. The concentration of SFN causing 50% inhibition of HCC cell activity was defined as IC50. All experiments were repeated independently three times.
Briefly, cells were plated in a 6-well plate (Corning, Catalog #3516-50, NY, USA) at a density of 2 × 105 cells per well (48 hr after transfection) and incubated overnight at 37 °C. Then cells were treated with DMSO or SFN medium for 14 days, and the medium was changed once every three days. At the end of incubation, colonies were fixed with ethanol for 30 min and stained with 5% crystal violet (Beyotime Biotechnology, Catalog #C0121, Shanghai, China) for 1 hr. The number of colonies with >10 cells was calculated using ImageJ software. Each experiment was conducted at least three times.
To detect the percentage of cell apoptosis, flow cytometry assays were used. Briefly, HCC cells were seeded into a 6-well plate at a density of 2 × 105 cells (48 hr after transfection). Cells were then treated with indicated concentrations of SFN for 48 hr. Next, cells were resuspended in a binding buffer containing Annexin V-FITC and propidium iodide (PI) (KeyGEN, Catalog #KGA108, Nanjing, China) in accordance with the manufacturer’s guideline, after washing with PBS three times. Finally, apoptotic cells were detected by flow cytometry and analyzed through FlowJo 7.6 software. Each experiment was conducted at least three times.
The ability of cell migration was detected by trans-well assays. Briefly, HCC cells (8x104 cells per well, 48 hr after transfection) suspended in serum-free medium were placed into the upper chambers of a trans-well filter (Corning, Catalog #353097, NY, USA), while the lower chambers were supplemented with DMEM containing 10% FBS, placed in a 24-well plate (NEST, Catalog #702001, Jiangsu, China). Indicated concentrations of SFN were added to the medium both in the upper and lower chambers. After 24 hr, the migration cells located on the lower membrane were immersed with methanol for 30 min and stained with 5% crystal violet for 1 hr. Migration cells from three fields were selected randomly under a bright field microscope and counted using ImageJ software. All experiments were repeated independently three times.
Briefly, HCC cells were seeded into chamber slides (NEST, Catalog #801002, Jiangsu, China) at a density of 1 × 103 cells (48 hr after transfection) and cultured for 24 hr. The cells were then exposed to indicated concentrations of SFN for 48 hr. Next, the cells were fixed with 4% paraformaldehyde for 30 min at 4°C, permeabilized in 1% Triton X-100 (Solarbio Life Science, Catalog #T8200, Beijing, China) for 30 min at room temperature, and incubated in a blocking buffer for 1 hr at room temperature. This was followed by staining with Phalloidin-iFluor 555 in the dark for 1 hr at room temperature so as to bind them to F-actin (1:50 dilution, Abcam, Catalog #ab176756, Cambridge, UK). They were washed again three times in PBST. Finally, Fluoroshield Mounting Medium with DAPI (Abcam, Catalog #ab104139, Cambridge, UK) was applied to the chamber slides and incubated for 20 min in the dark. The images of samples were pictured under a confocal microscope.
Tumor tissues excised from mice were fixed overnight in 10% neutral buffered formalin, dehydrated at a gradient concentration, and embedded in paraffin. The tissues were cut into 4 μm thick and fixed on the silicified glass slide. Subsequently, the IHC was carried out using a streptavidin–peroxidase-conjugated method. Briefly, the slides were deparaffinized, rehydrated, immersed in antigen retrieval solution, boiled at 100°C for 10 min, and incubated with a peroxidase inhibitor for 10 min at room temperature. Next, nonspecific binding was blocked with normal goat serum at room temperature for 1 hr, and incubated overnight at 4°C with primary antibodies. After secondary antibodies were incubated at room temperature for 1 hr, 3,3’-Diaminobenzidine tetrahydrochloride (DAB; ZSGB-BIO, Catalog #ZLI-9017, Beijing, China) and Mayer’ Hematoxylin solution (Solarbio Life Science, Catalog #G1080, Beijing, China) were followed. Microscopic images were obtained under a bright field microscope, and the protein expression was visualized by IHC staining and evaluated using the CellProfiler software. The information relating to the antibodies are summarized in Supplementary Table 2.
Total RNAs were extracted using the TRIzol reagent. Then strand-specific RNA-seq libraries were constructed using each group of samples and sequenced using a Illumina NovaSeq 6000 RNA-Seq System (Illumina, USA). Read counts for each gene were normalized into Fragments Per Kilobase of transcript per Million mapped reads (FPKM) values. Differentially expressed genes (DEGs) with log2FC ≥1.5 or ≤-1.5 fold change, p < .01 were identified, and normalized using respective negative and vector controls. Differentially expressed genes (log2FC ≥1.5, p < .01) from the results of RNA-seq on Bel-7402 cells administrated with SFN and DDX24 knockdown Hep3B cells (GEO Submission: GSE145635) were included in the venn diagram, and the diagram of simultaneous upregulated gene lists were created using a free online tool (http://bioinformatics.psb.ugent.be/webtools/Venn/).
Animals were purchased from the Vital River Laboratory Animal Technology Company (Beijing, China), and maintained in a pathogen-free condition in Guangdong Provincial Key Laboratory of Biomedical Imaging under standard conditions at the animal care facility. BALB/c female nude mice aged four to six weeks were randomly divided into four groups (n = 5): shNC+PBS group, shNC+SFN group, shDDX24-1+ SFN group and shDDX24-2+ SFN group. Hep3B cells (5x106 per mouse) with or without DDX24 stable knockdown were suspended in 100 μl PBS and injected subcutaneously into the right flanks of the animals. After five days, the mice were treated with SFN (60 mg/kg body weight) or parallel PBS as a control, orally once daily by gastric infusion, and the length, width, and height of each tumor were monitored every four days until the end of this study (tumor volume was calculated as follows: V = πLWH/6). Finally, the mice were humanely sacrificed by anesthesia, and xenografts were harvested for weighing and photographing. The relative expression of proteins in tumor tissues was detected by IHC assays. Animal protocols were approved by the Institutional Animal Care and Use Committee of the Fifth Affiliated Hospital of Sun Yat-sen University (No. 00087). All animals procedures were performed in accordance with the principles of the Declaration of Helsinki.
Gene expression and clinical data for 371 liver cancer samples and 50 adjacent samples were obtained from The Cancer Genome Atlas (TCGA) database of UCSC Xena (https://xenabrowser.net/datapages/). This dataset showed the gene-level transcription estimates, as in log2(x + 1), and transformed the RSEM normalized count. Kaplan-Meier analysis was conducted for the high and low DDX24 expression groups based on the median mRNA level of DDX24 in the TCGA cohort to assess the ability to predict patient survival. The relationships between DDX24 and its neighboring genes were investigated via the GeneMANIA tool (http://genemania.org/), then these neighboring genes were used to construct a gene network map. The protein-protein interaction (PPI) network for DDX24 was constructed via the STRING database (https://string-db.org/cgi/). Co-expressed genes of DDX24 (p < .05) were screened from the TCGA database. The interaction network analysis of the top 50 co-expressed genes was analyzed via the Reactome Pathway database (https://www.reactome.org/), and the results of 34 interacting genes were visualized using Cytoscape tool (V 3.7.2). Then the top 18 co-expressed genes (|Spearman correlation coefficient| >0.4 and p < .01) were integrated into the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis (https://david-d.ncifcrf.gov/). The results of the KEGG analysis were visualized using the R package (V 3. 6. 2).
All data were presented in terms of the mean ± SD from at least three independent experiments. Statistical analysis was performed using GraphPad Prism software (version 7.0). The differences between groups were analyzed using two student’s t test, or by one-way ANOVA. Statistical significance was indicated as follows: ****p < .0001; ***p < .001; **p < .01; *p < .05; n. s. represented not significant.
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PMC9629447 | 36338541 | Roshan Dutta,Praveen Guruvaiah,Kiran Kumar Reddi,Suresh Bugide,Dhana Sekhar Reddy Bandi,Yvonne J K Edwards,Kamaljeet Singh,Romi Gupta | UBE2T promotes breast cancer tumor growth by suppressing DNA replication stress | 02-11-2022 | Abstract Breast cancer is a leading cause of cancer-related deaths among women, and current therapies benefit only a subset of these patients. Here, we show that ubiquitin-conjugating enzyme E2T (UBE2T) is overexpressed in patient-derived breast cancer samples, and UBE2T overexpression predicts poor prognosis. We demonstrate that the transcription factor AP-2 alpha (TFAP2A) is necessary for the overexpression of UBE2T in breast cancer cells, and UBE2T inhibition suppresses breast cancer tumor growth in cell culture and in mice. RNA sequencing analysis identified interferon alpha–inducible protein 6 (IFI6) as a key downstream mediator of UBE2T function in breast cancer cells. Consistently, UBE2T inhibition downregulated IFI6 expression, promoting DNA replication stress, cell cycle arrest, and apoptosis and suppressing breast cancer cell growth. Breast cancer cells with IFI6 inhibition displayed similar phenotypes as those with UBE2T inhibition, and ectopic IFI6 expression in UBE2T-knockdown breast cancer cells prevented DNA replication stress and apoptosis and partly restored breast cancer cell growth. Furthermore, UBE2T inhibition enhanced the growth-suppressive effects of DNA replication stress inducers. Taken together, our study identifies UBE2T as a facilitator of breast cancer tumor growth and provide a rationale for targeting UBE2T for breast cancer therapies. | UBE2T promotes breast cancer tumor growth by suppressing DNA replication stress
Breast cancer is a leading cause of cancer-related deaths among women, and current therapies benefit only a subset of these patients. Here, we show that ubiquitin-conjugating enzyme E2T (UBE2T) is overexpressed in patient-derived breast cancer samples, and UBE2T overexpression predicts poor prognosis. We demonstrate that the transcription factor AP-2 alpha (TFAP2A) is necessary for the overexpression of UBE2T in breast cancer cells, and UBE2T inhibition suppresses breast cancer tumor growth in cell culture and in mice. RNA sequencing analysis identified interferon alpha–inducible protein 6 (IFI6) as a key downstream mediator of UBE2T function in breast cancer cells. Consistently, UBE2T inhibition downregulated IFI6 expression, promoting DNA replication stress, cell cycle arrest, and apoptosis and suppressing breast cancer cell growth. Breast cancer cells with IFI6 inhibition displayed similar phenotypes as those with UBE2T inhibition, and ectopic IFI6 expression in UBE2T-knockdown breast cancer cells prevented DNA replication stress and apoptosis and partly restored breast cancer cell growth. Furthermore, UBE2T inhibition enhanced the growth-suppressive effects of DNA replication stress inducers. Taken together, our study identifies UBE2T as a facilitator of breast cancer tumor growth and provide a rationale for targeting UBE2T for breast cancer therapies.
Breast cancer is the most commonly diagnosed cancer type and the second-leading cause of cancer-related deaths among women (1,2). The current 5-year overall survival (OS) rate is estimated at 27% for locally advanced, unresectable disease (3). Breast cancer is categorized based on appearance as tubular, mucinous, medullary, or papillary (4,5). Breast cancer subtypes are also classified based on the presence or absence of hormone receptors (HRs), including estrogen receptor (ER) and progesterone receptor (PR) (6). In addition to differences in HR expression, breast cancers are classified based on the overexpression of human epidermal growth factor receptor 2 (HER2), which is detected in 10–15% of breast cancer cases (6). Approximately 15%–20% of breast cancers are classified as triple-negative breast cancer (TNBC), characterized by the absence of ER, PR and HER2 expression (6). Therapeutic options for breast cancer include surgery combined with chemotherapy (7), although locally advanced, unresectable, or metastatic disease is typically treated using chemotherapy combined with radiotherapy, hormonal therapy, targeted therapy, or immunotherapy (8,9). However, existing treatment options do not provide long-term survival benefits in patients with metastatic disease, and in many cases, the disease becomes resistant to therapy or relapses after an initial response (10–12). Because the TNBC subtype does not express any HRs, patients with TNBC do not respond to hormonal therapies, making these cases difficult to treat and leading to a generally poor prognosis (6,13). To achieve better clinical outcomes, an improved understanding of the underlying breast cancer pathogenesis remains necessary to identify novel breast cancer drivers for the development of targeted therapies. Diverse cellular pathways are regulated by the covalent conjugation of ubiquitin to proteins through the concerted actions of a ubiquitin-activating enzyme (E1), a ubiquitin-conjugating enzyme (E2), and a ubiquitin ligase (E3). Previous studies have shown that E2 enzymes play important roles in the ubiquitination of cellular proteins, with effects on the genesis and progression of various cancer types (14,15). Ubiquitin-conjugating enzyme E2T (UBE2T) is an essential E2 enzyme (16,17) involved in the efficient repair of damaged DNA in the Fanconi anemia pathway (18), and the regulation of this pathway may involve a UBE2T self-inactivation mechanism (18). In addition, UBE2T-mediated ubiquitination regulates several cancer-relevant pathways, including the ubiquitination of AKT, which activates the AKT/β-catenin pathway (16), and the ubiquitination of β-catenin, which induces its nuclear translocation (19). UBE2T also promotes autophagy via the p53/AMP-activated protein kinase (AMPK)/mammalian target of rapamycin (mTOR) signaling pathway in lung adenocarcinoma (20). In this study, we found that UBE2T is overexpressed in breast cancer and that UBE2T overexpression predicts poor prognosis. UBE2T expression is regulated by transcription factor AP-2 alpha (TFAP2A), and UBE2T inhibition suppresses breast cancer tumor growth. Mechanistically, UBE2T inhibition in breast cancer cells suppresses interferon alpha–inducible protein 6 (IFI6) expression, resulting in DNA replication stress, cell cycle arrest, and apoptosis induction. However, the ectopic expression of IFI6 in UBE2T-knockdown cells prevented DNA replication stress and apoptosis and partly restored breast cancer cell growth. Furthermore, UBE2T inhibition enhanced the growth-suppressive effects of DNA replication stress inducers. Therefore, our studies indicate that UBE2T acts as a facilitator of breast cancer growth and may represent a novel target for breast cancer therapy.
The MCF7, T47D and HEK293T cell lines were obtained from American Type Culture Collection (Manassas, VA, USA) and maintained in a humidified atmosphere containing 5% CO2 at 37°C in Dulbecco's modified Eagle's medium (Life Technologies, Carlsbad, CA, USA) or Roswell Park Memorial Institute (RPMI)-1640 medium (Life Technologies), as recommended. All media were supplemented with 10% fetal bovine serum (Life Technologies) and 1% penicillin/streptomycin (Life Technologies).
Female NSG mice (Jackson Laboratory, Stock No. 005557, Bar Harbor, ME, USA), 5–6 weeks of age, were subcutaneously injected in the flank with 5 × 106 breast cancer cells (MCF7 and T47D) expressing either non-specific (NS) or UBE2T small hairpin RNAs (shRNAs). Tumor volumes were measured every week. Tumor sizes were calculated using the following formula: length × width2 × 0.5. All protocols for mouse experiments were approved by the Institutional Animal Care and Use Committee of the University of Alabama at Birmingham.
pLKO.1 lentiviral vector–based shRNAs targeting specific candidate genes and NS control shRNAs were obtained from Horizon Discovery. Details regarding the shRNAs are provided in Supplementary Table S5. Lentiviral particles were prepared by transfecting HEK293T cells with either gene-specific shRNA plasmids or NS shRNA plasmids, together with lentiviral packaging plasmids (described in detail at https://portals.broadinstitute.org/gpp/public/resources/protocols). All lentiviral transfections were performed using Effectene Transfection Reagent (Qiagen, Hilden, Germany). Stable cell lines were generated by infecting breast cancer cells seeded in 12-well plates with shRNA lentiviral particles, followed by selection with appropriate concentrations of puromycin (0.5–1.5 μg/ml) to enrich infected cells. For the pLX304-Blast-V5–based lentivirus, infected breast cancer cells were selected using 2 μg/ml blasticidin (ThermoFisher Scientific, Waltham, MA, USA).
V5-tagged IFI6 lentiviral expression construct (plasmid pLX304-Blast-V5) was purchased from Horizon Discovery (Waterbeach, UK) and is listed in Supplementary Table S5.
Total RNA was extracted with TRIzol Reagent (Invitrogen, Carlsbad, CA, USA) and purified using the RNeasy Mini Kit (Qiagen). cDNA was generated using the M-MuLV First Strand cDNA Synthesis Kit (New England Biolabs, Ipswich, MA, USA), according to the manufacturer's instructions. qPCR was performed using gene-specific primers with the Power SYBR-Green Master Mix (Applied Biosystems, Foster City, CA, USA), according to the manufacturer's instructions. The beta-actin (ACTB) gene was used as a normalization control. The primer sequences for all genes analyzed in this study are provided in Supplementary Table S5.
Whole-cell protein extracts were prepared using IP Lysis Buffer (Pierce Chemical, Rockford, IL, USA) containing Protease Inhibitor Cocktail (Roche, Basel, Switzerland) and Phosphatase Inhibitor Cocktail (Sigma-Aldrich, St. Louis, MO, USA). Lysed samples were centrifuged at 12 000 rpm for 40 min, and the clarified supernatants were stored at −80°C. Protein concentrations were determined using Bradford Protein Assay Reagent (Bio-Rad Laboratories, Hercules, CA, USA). Equal amounts of protein samples were electrophoresed on 10% or 12% sodium dodecyl sulfate (SDS)-polyacrylamide gels and transferred onto polyvinylidene difluoride (PVDF) membranes (Millipore, Burlington, MA, USA) using a wet transfer apparatus (Bio-Rad). Membranes were blocked in 5% skim milk prepared in Tris-buffered saline containing 0.1% Tween-20 (TBST) and probed with primary antibodies. After washing, the membranes were incubated with the appropriate horseradish peroxidase (HRP)-conjugated secondary antibodies (1:2000; GE Healthcare Life Sciences, Marlborough, MA, USA). The blots were developed using SuperSignal West Pico or Femto Chemiluminescent Substrate (ThermoFisher Scientific). All antibodies used for immunoblotting are listed in Supplementary Table S5.
MCF7 and T47D cells expressing either NS or UBE2T shRNA were analyzed for cell cycle phases using fluorescence-activated cell sorting (FACS). Cells were collected, washed twice with ice-cold 1 × phosphate-buffered saline (PBS), and fixed in 70% ethanol overnight. The cells were then washed three times with 1× PBS and resuspended in 400 μl propidium iodide/Triton X-100 staining solution [0.1% (v/v) Triton X-100 in PBS with 2 mg DNAse-free RNase and 0.40 ml 500 μg/ml propidium iodide]. The samples were incubated for 15 min at 37°C. Flow cytometry analysis was performed using a BD LSRFortessa (BD Biosciences, San Jose, CA, USA), and samples were analyzed using FlowJo software (Ashland, OR, USA).
DNA fiber assays were performed as described previously (21). Briefly, cells were plated in the appropriate medium until they reached 30–40% confluency. After 48 h, iododeoxyuridine (IdU; Sigma-Aldrich: I7125) was added to exponentially growing cells (final concentration: 25 μM), and the cells were incubated for 30 min at 37°C in 5% CO2. Then, cells were washed with PBS and incubated with a second label, chlorodeoxyuridine (CldU; Sigma-Aldrich: C6891), at a final concentration of 250 μM for an additional 30 min at 37°C. The cells were then trypsinized and counted. A 3-μl cell suspension containing 2 × 103 cells were applied to the end of a glass slide and air-dried for 5 min. The cells were lysed by adding 7 μl lysis solution (50 mM ethylenediaminetetraacetic acid and 0.5% SDS in 200 mM Tris–HCl, pH 7.6). The glass slides were placed at a 15° angle to allow the DNA fibers to spread across the length of the slide and then placed horizontally to air dry. The slides were fixed with methanol:acetic acid (3:1) for 10 min, washed with double-distilled water, and treated with 2.5 M HCl for 30 min. The fixed cells were blocked with 5% bovine serum albumin (BSA) for 30 min at room temperature and incubated with primary antibodies (anti-BrdU [mouse antibody, BD Biosciences #347580] for IdU at a 1:25 dilution and anti-BrdU [rat antibody, Abcam, Cambridge, UK #ab6326] for CldU at a 1:400 dilution, each in 5% BSA) for 1 h at room temperature in a humidified chamber. The slides were then washed three times with 1× PBS for 5 min and incubated with secondary antibodies (1:500 sheep anti-mouse Cy3, Sigma, Cat# C218-M for IdU; and 1:400 goat anti-rat Alexa Fluor 488, Invitrogen, cat A11006 for CldU) in 5% BSA for 1 h at room temperature in the dark. The glass slides were then washed and visualized at 60× magnification to locate the fibers. Pictures were captured with one color channel, and data were analyzed with ImageJ software.
Formalin-fixed, paraffin-embedded tissue microarray (TMA) slides containing breast cancer samples and matched normal breast tissues were obtained from US Biomax (#BC081120f). Briefly, following slide deparaffinization, antigen retrieval was performed in citrate buffer (pH 6.0) at 97°C for 20 min, using the Lab Vision PT Module (ThermoFisher Scientific). Endogenous peroxides were blocked by incubation in hydrogen peroxide for 30 min, followed by washing with 1× Tris-buffered saline, and proteins were blocked by incubation with 0.3% BSA for 30 min. Slides were incubated in anti-UBE2T antibody (dilution 1:1000) or anti-IFI6 antibody (dilution 1:1000), followed by secondary anti-rabbit HRP-conjugated antibody (Dako, Jena, Germany). Slides were then stained using the Dako Liquid DAB+ Substrate Chromogen System and counterstained with Dako Automation Hematoxylin Histological Staining Reagent. UBE2T and IFI6 staining was scored by Dr Kamaljeet Singh, who was blinded to the identity of each slide. All antibodies used for IHC analyses are listed in Supplementary Table S5.
Datasets comparing gene expression between breast cancer and normal breast tissues were identified by searching the Oncomine cancer profiling database. The Cancer Genome Atlas (TCGA) breast (22), Curtis breast (23) and Zhao breast (24) datasets were analyzed. The TCGA breast dataset includes 389 invasive ductal breast carcinoma samples and 61 normal breast duct samples. The Curtis breast dataset includes 114 normal breast samples and 1556 invasive ductal breast carcinoma samples. The Zhao breast dataset includes three normal breast samples and 36 invasive ductal breast carcinoma samples. All three datasets were analyzed for UBE2T gene expression. The Curtis breast and TCGA breast datasets were used to analyze the co-expression of various transcription factors and UBE2T at the mRNA level. The TCGA breast invasive carcinoma PanCancer dataset (1084 samples) was analyzed for UBE2T alterations (https://www.cbioportal.org). Gene Expression Profiling Interactive Analysis (GEPIA), a web server for cancer and normal gene expression profiling and interactive analyses, was used to analyze IFI6 and UBE2T expression and identify associations between overall survival (OS) and high and low UBE2T expression levels (25). Breast invasive carcinoma samples were analyzed for TFAP2A transcript levels using the UALCAN bioinformatics tool (http://ualcan.path.uab.edu/index.html). Km plotter was used to plot OS and relapse-free survival (RFS) among patients with high and low UBE2T expression levels (https://kmplot.com/analysis/). DepMap portal was used to analyze the transcript levels of various genes in breast cancer samples (https://depmap.org/portal/).
Transcription factors binding with 100% sequence identity on the promoter region of UBE2T were identified using the PROMO tool (http://alggen.lsi.upc.es/cgi-bin/promo_v3/promo/promoinit.cgi?dirDB=TF_8.3) (26). We selected a 2-kB upstream promoter region of the UBE2T gene to identify human transcription factors able to bind with 0% dissimilarity using PROMO.
MCF7 and T47D cells expressing NS or UBE2T shRNA were used to prepare total RNA, which was then used for gene expression analysis on an Illumina HiSeq 2500 system. Total RNA was extracted using TRIzol® reagent (Invitrogen), according to the manufacturer's instructions, and purified on RNeasy mini columns (Qiagen), according to the manufacturer's instructions. mRNA was purified from ∼500 ng total RNA using oligo-dT beads and sheared by incubation at 94°C. Following first-strand synthesis with random primers, second-strand synthesis was performed with dUTP to generate strand-specific sequencing libraries. The cDNA libraries were then end-repaired and A-tailed. Adapters were ligated, and second-strand digestion was performed using uracil–DNA–glycosylase. Indexed libraries that met appropriate cutoffs for both were quantified by qPCR using a commercially available kit (KAPA Biosystems, Wilmington, MA, USA). The insert size distribution was determined using LabChip GX or an Agilent Bioanalyzer. Samples with a yield ≥0.5 ng/μl were used for sequencing on the Illumina HiSeq 2500 system. Images generated by the sequencers were converted into nucleotide sequences by the base-calling pipeline RTA 1.18.64.0 and stored in fastq format.
RNA sequencing was performed for two cell lines (MCF7 and T47D) expressing UBE2T-specific shRNAs or NS shRNA. RNA sequencing was carried out for 15 samples. The MCF7 samples (n = 9) comprise three biological replicates for three groups (control, shRNA1, shRNA2). The T47D samples (n = 6) consist of two biological replicates for three groups (control, shRNA1, shRNA2). Single-end 75-bp reads were generated using the Illumina NextSeq500 sequencing instrument. Pre-alignment quality assessments of the raw fastq sequences were conducted using FastQC (version 0.11.7) (27). The raw fastq sequences were aligned to the human hg38 reference genome (GenBank assembly accession: GCA_000001405.28) using STAR (version 2.7.1a) (28) with default parameters. Post-alignment quality assessments were performed using RSeQC (version 2.6.3) (29). Samtools (version 0.0.19) (30) and IGV (version 2.6.2) (31) were used to index and view the alignments, respectively. Gene expression was quantified as gene-level counts using the htseq-count function (version 0.12.3) and the UCSC gene annotations for the human genome. The htseq-count default parameters were used, except for the strand parameter, which was set to ‘reverse’ to account for the strandedness of the library. Differentially expressed genes were identified using DESeq2 (version 1.28) with default parameters (32). Genes with p-values less than 0.05 were considered differentially expressed. Interactivenn was used to generate Venn diagrams (33). The normalized gene expression data were used for downstream analyses. The complex heatmap package version 1.12.0 (34) was used to generate heatmaps. To determine altered cellular functions in both cell lines under different treatment conditions, over-representation enrichment analysis was performed using the WEB-based GEne SeT AnaLysis Toolkit (Webestalt) (35), with the genome as the reference set and the community-contributed_Hallmark50 database as the functional database. A hypergeometric test was used to test for the over-representation of functions among the differentially expressed genes common to both cell lines. The Benjamini and Hochberg method was used to calculate adjusted P-values (q) with the significance cutoff filter set to q < 0.05.
Soft-agar assay was performed by seeding 5 × 103 breast cancer cells stably expressing the indicated shRNA or cDNA constructs onto 0.4% low-melting-temperature agarose (Sigma-Aldrich) layered on top of 0.8% agarose. After 3–4 weeks of incubation, colonies were stained with 0.005% crystal violet and imaged using a microscope. Colony sizes were measured using ImageJ software (https://imagej.nih.gov/ij/) and plotted. Statistical analyses were performed using Student's t-test in GraphPad Prism 7 software (GraphPad, San Diego, CA, USA).
CUT&RUN assays were performed in MCF7 cells using the CUT&RUN Assay Kit (Cat#86652; Cell Signaling Technology Danvers, MA, USA), according to the manufacturer's instructions. Briefly, 2 × 105 cells were harvested, washed, bound to activated concanavalin A–coated magnetic beads, and permeabilized. The bead–cell complexes were incubated overnight with the appropriate antibody at 4°C. The complexes were washed three times, and the cells were resuspended in 100 μl protein A and G/micrococcal nuclease (pAG/MNase) and incubated for 1 h at room temperature. The samples were then washed three times with digitonin buffer containing protease inhibitors, resuspended in 150 μl digitonin buffer, and incubated for 5 min on ice. MNase was activated by adding calcium chloride, and the samples were incubated at 4°C for 30 min. The reaction was stopped by adding 150 μl stop buffer, and the samples were incubated at 37°C for 10 min to release the DNA fragments. DNA was extracted using the DNA purification columns included in the CUT&RUN Assay Kit. qPCR was performed using UBE2T promoter–specific primers, and relative fold change was calculated as the ratio of immunoprecipitated DNA to IgG-precipitated DNA. The primer sequences and antibodies used for the CUT&RUN assays are listed in Supplementary Table S5.
Annexin V binding to cells (MCF7 and T47D cells expressing either NS, UBE2T, or IFI6 shRNA) was measured using an Annexin V staining kit (BD Pharmingen™ #556547, BD Pharmingen, San Diego, CA, USA), according to the manufacturer's protocol. In brief, after 24 h of plating, cells were collected, washed twice with 1 × PBS, resuspended in 1× binding buffer, and stained with 5 μl FITC-Annexin V and 5 μl PI. After incubation for 15 min in the dark, cells were analyzed with FACS using LSR Fortessa (BD Biosciences, Franklin Lakes, NJ, USA).
To perform the MTT assay, 5 × 103 MCF7 cells expressing either NS or UBE2T shRNA were plated in a final volume of 100 μl in 96-well plates. After 24 h, hydroxyurea and aphidicolin were added to 100 μl media at a range of concentrations as shown in figure and were then added to the cells. After 72 h of inhibitor treatment, cell viability was evaluated by adding 20 μl of 5 mg/ml MTT solution dissolved in 1 × PBS to each well, followed by incubation for 1 h in a 37°C incubator. The MTT solution was gently removed, and 100 μl dimethyl sulfoxide was added to each well. After mixing by pipetting, absorbance was measured at 590 nm and 630 nm using the Biotek Synergy MX Multi-Format Microplate Reader (Winooski, VT, USA). The average measurement at 630 nm was subtracted from the average measurement at 590 nm, and the relative cell viability at each concentration was plotted with respect to vehicle-treated cells.
All experiments were conducted in at least three biological replicates. The results for individual experiments are expressed as the mean ± standard error of the mean (SEM). To assess measurements of tumor progression in mice and MTT assays, statistical analyses were performed by analyzing the area under the curve using GraphPad Prism software, version 7.0, for Macintosh. In all other experiments, P-values were calculated using a two-tailed unpaired Student's t-test in GraphPad Prism software, version 7.0, for Macintosh.
UBE2T has been implicated as an important mediator of cancer growth and metastasis in several cancer types (16,36–38). Based on these findings, we examined whether UBE2T is overexpressed in breast cancer and whether its overexpression predicts poor prognosis. We analyzed publicly available breast cancer datasets and found that UBE2T mRNA was significantly overexpressed in breast cancer samples due to both gene amplification and transcriptional upregulation [TCGA breast (22), Curtis breast (23), and Zhao breast (24) datasets] (Figure 1A–E). To confirm UBE2T overexpression at the protein level, we analyzed a breast cancer TMA (#BC081120f), which included 100 cases of invasive breast cancer tissue and 10 adjacent normal breast tissues (Supplementary Table S1). We performed IHC analysis, which showed the significant elevation of UBE2T protein expression in the large majority of patient-derived breast cancer samples relative to normal breast tissue samples, consistent with gene amplification and transcriptional upregulation (Figure 1F, G). We next examined whether UBE2T overexpression was associated with prognosis and found that breast cancer samples with higher UBE2T expression levels were associated with shorter on relapse-free survival and (RFS) and shorter overall survival (OS) (Figure 1H, I) among breast cancer patients. Collectively, these results demonstrate that UBE2T is overexpressed in breast cancer and predicts poor prognosis.
Breast cancer can be classified into several different subtypes based on the presence or absence of HRs (6). The luminal A breast cancer subtype, which expresses ER or PR but not HER2, accounts for 73% of all breast cancer cases and is the most common and slowest growing subtype. Because UBE2T is overexpressed in breast cancer, we rationalized that UBE2T might be involved in breast cancer tumor growth. Therefore, we examined whether the inhibition of UBE2T expression prevents tumor growth in the highly prevalent luminal A breast cancer subtype. We used two sequence-independent shRNAs to knock down UBE2T expression in the luminal A breast cancer cell lines MCF7 and T47D (Figure 2A, B). After UBE2T knockdown was validated in these breast cancer cell lines via qPCR and immunoblotting, these cells were tested for their abilities to grow in an anchorage-independent manner using soft-agar assays. Breast cancer cells expressing an NS shRNA were used as a negative control. Anchorage-independent growth in soft agar serves as a reliable surrogate assay for in vivo tumorigenesis (39). We found that UBE2T knockdown in breast cancer cells significantly reduced their abilities to form colonies in soft agar (Figure 2C, D). Based on these results, we examined whether UBE2T knockdown also inhibits breast cancer tumor growth using an in vivo xenograft mouse model of breast cancer. We subcutaneously injected breast cancer cells (MCF7 and T47D) expressing either UBE2T or NS shRNA into the flanks of immunodeficient female NSG mice. Consistent with the cell culture studies, UBE2T knockdown inhibited breast cancer tumor growth in vivo (Figure 2E). Collectively, these results demonstrate that UBE2T inhibition prevents breast cancer tumor growth in both cell culture and mice.
We next attempted to determine the mechanism driving UBE2T overexpression in breast cancer cells to promote cell growth. Because UBE2T was overexpressed at the transcriptional level, we performed experiments to identify transcriptional regulators of UBE2T. We first analyzed the DNA sequence of the UBE2T promoter (∼2 kb) to identify transcription factor consensus DNA-binding sites using the PROMO search tool for putative transcription factor identification (26,40). This analysis identified 25 transcription factors that displayed perfect matches for the consensus DNA-binding sequence (Supplementary Figure S1). To prioritize transcription factors for further analysis, we examined whether any of these potential transcription factors were also overexpressed in patient-derived breast cancer samples using publicly available breast cancer datasets. We identified eight transcription factors that were overexpressed in both the TCGA breast cancer dataset and the Curtis breast cancer dataset (Figure 3A, B). However, only four of these transcription factors were significantly co-overexpressed with UBE2T in patient-derived breast cancer samples (Figure 3C). Based on these analyses, we used shRNAs targeting all four shortlisted transcription factors and tested whether their knockdown had any effects on UBE2T expression. We used breast cancer cells expressing NS shRNA as controls. Our results showed that TFAP2A knockdown reduced UBE2T expression at the mRNA and protein levels in breast cancer cells (Figure 3D, E). However, the knockdown of the other three transcription factors (ELK1, GTF2I and FOXP3) did not reduce UBE2T expression levels in breast cancer cells (Supplementary Figure S2). TFAP2A knockdown also inhibited the abilities of breast cancer cells to grow in an anchorage-independent manner in soft-agar assays, mimicking the growth inhibitory phenotype observed with UBE2T inhibition in breast cancer cells (Figure 3F, G). To determine whether UBE2T is a direct target of TFAP2A, we performed CUT&RUN assays and observed the significant enrichment of TFAP2A on the UBE2T promoter (Figure 3H). We also found that TFAP2A mRNA was significantly overexpressed in breast cancer tumor tissue, similar to the overexpression of UBE2T mRNA (Figure 3I). Collectively, these results demonstrated that the transcription factor TFAP2A is overexpressed in breast cancer cells and is necessary for the transcriptional overexpression of UBE2T.
To more comprehensively determine the mechanisms through which UBE2T contributes to breast cancer growth and identify new mediators of UBE2T function, we performed transcriptome-wide mRNA expression profiling of breast cancer cell lines (MCF7 and T47D) expressing either UBE2T or NS shRNA. We identified 186 commonly downregulated genes (≥−1.0-fold) and 24 commonly upregulated genes (≥1.0-fold) in MCF7 cells expressing either UBE2T shRNA relative to NS shRNA expressing cells (Supplementary Table S2). We also identified 14 commonly downregulated genes (≥−1.0-fold) and 31 commonly upregulated genes (≥1.0-fold) in T47D cells expressing either UBE2T shRNA relative to NS shRNA expressing cells (Supplementary Table S3 and Figure 4A, B). We analyzed these significantly altered genes in MCF7 and T47D cells for biological pathway enrichment and found that UBE2T knockdown resulted in the significant upregulation of pathways associated with cell cycle progression, including the p53 pathway (Figure 4C). Based on these results, we performed a cell cycle analysis of MCF7 and T47D cells expressing UBE2T shRNA. Compared with NS shRNA expressing control cells, UBE2T shRNA expressing cells showed an increased proportion of cells in the G1 phase and a decreased proportion of cells in the G2/M phase (Figure 4D and Supplementary Figure S3), which led to increased apoptosis (Figure 4E and Supplementary Figure S4). We also observed several significantly downregulated oncogenic pathways in UBE2T-knockdown cells compared with control cells, including the mTOR complex 1 (mTORC1) and tumor necrosis factor-alpha (TNFA) signaling pathways (Supplementary Figure S5A). Collectively, these results show that UBE2T knockdown leads to cell cycle arrest and apoptosis induction, which inhibit breast cancer cell growth.
We identified 118 commonly downregulated genes and 144 commonly upregulated genes in UBE2T shRNA expressing cells as compared to control cells expressing NS shRNA (Supplementary Table S4) in both MCF7 and T47D cells. The downregulated genes included several interferon-stimulated genes (Figure 5A), among which IFI6 was the most significantly repressed gene identified in UBE2T shRNA expressing cells (Figure 5B, C). We also found that IFI6 was one of the most significantly expressed genes next to UBE2T in breast cancer cell lines (Supplementary Figure S5B). Based on these results, we next investigated the role of UBE2T-regulated IFI6 expression in breast cancer cell growth. To explore whether IFI6 plays a role in breast cancer cell growth downstream of UBE2T, we first examined whether IFI6 is overexpressed in patient-derived breast cancer samples compared with normal breast tissues, similar to UBE2T overexpression. We analyzed publicly available breast cancer datasets (25) and found that, similar to UBE2T mRNA, IFI6 mRNA was significantly overexpressed in breast cancer samples relative to normal breast samples (Figure 5D). Similarly, IFI6 protein expression assessed by IHC using a breast cancer TMA (#BC081120f) demonstrated the significant elevation of IFI6 expression in a large majority of patient-derived breast cancer samples compared with normal breast tissue samples (Figure 5E, F). To study the role of IFI6, we knocked down IFI6 expression using two shRNAs with independent sequences in MCF7 and T47D cells (Figure 5G–H). After IFI6 knockdown was validated, these breast cancer cell lines were tested for their abilities to grow in an anchorage-independent manner using soft-agar assays. Breast cancer cells expressing NS shRNA were used as a negative control. We found that IFI6 knockdown in breast cancer cells resulted in significant reductions in the abilities of these cells to form colonies in soft agar (Figure 5I–J), mimicking the growth inhibitory phenotype observed in UBE2T-knockdown cells. Our previous study in melanoma cells showed that IFI6 regulates DNA replication stress pathways to promote melanoma growth (41). Based on these findings, we examined whether IFI6 knockdown resulted in DNA replication stress in breast cancer cells, leading to growth inhibition. We found that the loss of IFI6 expression resulted in dysregulated DNA replication, as indicated by the detection of significantly fewer ongoing DNA replication forks and more stalled forks in IFI6 shRNA expressing breast cancer cells than in NS shRNA expressing cells (Figure 5K and Supplementary Figure S6). In addition, increased DNA replication stress in IFI6 knockdown cells compared with control cells was associated with increased apoptosis (Figure 5L and Supplementary Figure S7). Guided by these results, we also examined DNA replication stress in UBE2T shRNA expressing breast cancer cells and found that inhibition of UBE2T expression also led to the activation of DNA replication stress (Figure 6A and Supplementary Figure S8A). Based on these results, we performed experiments to determine whether IFI6 acts as a downstream mediator of UBE2T-induced tumor growth in breast cancer. We ectopically expressed IFI6 in breast cancer cells expressing UBE2T shRNA (Figure 6B) and tested whether IFI6 overexpression restored normal DNA replication by preventing DNA replication stress and apoptosis. We found that the ectopic expression of IFI6 in UBE2T-knockdown cells partially rescued UBE2T knockdown–induced DNA replication stress and apoptosis (Figure 6C, D and Supplementary Figure S8B). Consistent with this finding, the ectopic expression of IFI6 in UBE2T shRNA expressing breast cancer cells also rescued their abilities to grow in soft-agar assays (Figure 6E, F). These results demonstrate that UBE2T activates IFI6 expression to suppress DNA replication stress and prevent apoptosis induction, facilitating breast cancer cell growth. However, we would also like to note that it is also possible that IFI6 may also influence cell growth and other tumor phenotypes by regulating alternative UBE2T-independent pathways to promote breast cancer cell growth.
Finally, we asked whether our results were clinically significant. We first tested the effects of DNA replication stress inducers on the growth of MCF7 and T47D cells expressing UBE2T shRNA using cell-based assays. We treated NS shRNA expressing and UBE2T shRNA expressing breast cancer cells with the DNA replication stress–inducing agents’ hydroxyurea and aphidicolin (42–44) and measured cell viability using the MTT assay. We found that hydroxyurea and aphidicolin induced more potent inhibitory effects on the growth of breast cancer cells expressing UBE2T shRNA than on the growth of cells expressing NS shRNA, and these effects increased in a concentration-dependent manner (Figure 6G). Similar results were obtained in the soft-agar assay (Figure 6H, I). These results demonstrate that UBE2T suppression might enhance the therapeutic benefits of drugs that function by inducing DNA replication stress, such as hydroxyurea and aphidicolin.
UBE2T belongs to the family of E2 ubiquitin-conjugating enzymes and has previously been implicated in DNA repair and carcinogenesis (18,45–47). Previous studies have reported that UBE2T is overexpressed in several cancer types, such as melanoma, ovarian cancer, renal cancer and hepatocellular carcinoma, and its overexpression correlates with cancer progression and poor prognosis (16,37,38). UBE2T expression has also been considered to serve as an early biomarker for cancer prognosis (36). In this study (Figure 7), we found that UBE2T is overexpressed at both the mRNA and protein levels in patient-derived breast cancer samples, and UBE2T overexpression predicted poor survival among patients with breast cancer. The transcription factor TFAP2A regulates UBE2T expression in breast cancer cells, and previous study has shown that TFAP2A regulates the expression of several genes that promote cancer growth and contribute to determining anticancer therapy resistance and sensitivity (48). Deregulation of cell cycle and apoptotic pathways has been associated with aberrant cell proliferation and cancer development (49,50). In our study, we found that UBE2T inhibition leads to upregulated cell cycle progression as observed in MCF7 cell expressing UBE2T shRNA and further G1 cell cycle arrest and apoptosis induction. During the G1 phase of the cell cycle, major regulatory events occur, including cell growth, increasing protein contents, and organelle doubling, which serve as signals for the cell to enter the cell division stage (51). Therefore, cell cycle arrest in the G1 phase, such as that observed in UBE2T shRNA expressing breast cancer cells, prevents progression to the S and G2 phases and inhibits cell division. Moreover, prolonged cell cycle arrest leads to cell death, further inhibiting cancer cell proliferation (52,53). Upregulated cell cycle progression observed in MCF7 cell expressing UBE2T shRNA was associated with genes such as transcription factor E2F targets CKS1B, KPNA2, LDLR, minichromosome maintenance proteins (MCM2, MCM7) etc. that leads to DNA hyper-replication induced cycle arrest stress and apoptosis induction (41,54). UBE2T is also involved in activating oncogenic signaling pathways, such as the mTOR and TNFA signaling pathways, which are known to modulate cancer growth and progression (55–57). Additionally, we found that UBE2T inhibits DNA replication stress pathways and is essential for the Fanconi anemia pathway, which is involved in the maintenance of genome stability through DNA damage repair, replication fork stabilization, and the alleviation of oxidative and mitotic stress (58). We identified that UBE2T regulates DNA replication stress in breast cancer cells by stimulating the expression of IFI6, suppressing DNA replication stress, cell cycle arrest, and apoptosis induction. IFI6 is an interferon-stimulated gene that belongs to the FAM14 gene family (59). IFI6 is overexpressed in many cancer types and is involved in stabilizing mitochondrial function, resulting in apoptosis inhibition and tumor growth promotion (60). Our previous study also showed that IFI6 is a target of NRAS and promotes tumor growth by suppressing DNA replication stress in melanoma cells (41). The DNA replication stress pathway also regulates the cell cycle transition (61,62). The inhibition of the DNA replication stress pathway prevents the progression from G1 to S phase, resulting in the accumulation of cells in the G1 phase (63). In this study, we showed that the inhibition of UBE2T expression correlated with the repression of IFI6 expression, the activation of the DNA replication stress pathway, G1 cell cycle arrest, and apoptosis induction, leading to the inhibition of breast cancer cell growth and proliferation. We further showed that IFI6 overexpression effectively reverses DNA replication stress and the inhibition of colony-forming growth induced by UBE2T knockdown in breast cancer cells, partially restoring breast cancer cell proliferation. The DNA replication stress pathway can be pharmacologically activated by a group of small-molecule DNA replication stress inducers, such as hydroxyurea and aphidicolin (43), which have been used for cancer treatment (64,65). We show that UBE2T knockdown increases the sensitivity of breast cancer cells to DNA replication stress inducers. Thus, the UBE2T→IFI6→DNA replication stress→apoptosis pathway represents a potential new therapeutic target for breast cancer cases associated with high UBE2T expression levels. A previous study reported the identification of UBE2T inhibitors, including M435-1279 (66), which may potently inhibit breast cancer growth when combined with DNA replication stress inducers. Collectively, these studies identify UBE2T as a facilitator of breast cancer tumor growth through the suppression of IFI6 expression, DNA replication stress, and apoptosis induction. These studies indicate that UBE2T may serve as a potential therapeutic target for breast cancer, either alone or in combination with other anticancer agents, such as DNA replication stress inducers.
Although, in our study we find that UBE2T mediates its function through IFI6 in UBE2T-IFI6-DNA replication-apoptosis pathway dependent manner in breast cancer cells, it is possible that IFI6 could influence cell growth and other phenotypes by alternative UBE2T-independent mechanisms. Another observation in our study was that UBE2T regulates mRNA expression level of IFI6. However, it is possible that UBE2T might affect ubiquitination of IFI6 and other proteins including transcription factors that may be involved in regulating IFI6 transcription. Future studies are required to address both these aspects more comprehensively.
All correspondence and materials should be requested from Dr Romi Gupta.
These data were submitted to and are available from the Gene Expression Omnibus (Accession# GSE156603 for UBE2T-knockdown RNA-seq). Additional data related to any experiments presented in this article are available from the corresponding author upon request.
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PMC9629490 | Marco Preussner,Karine F. Santos,Jonathan Alles,Christina Heroven,Florian Heyd,Markus C. Wahl,Gert Weber | Structural and functional investigation of the human snRNP assembly factor AAR2 in complex with the RNase H-like domain of PRPF8 | 27-10-2022 | spliceosomal assembly,U5 snRNP,AAR2,PRPF8 | The crystal structure of human AAR2 bound to the central spliceosomal factor PRPF8 and in vitro functional data yield insights into the structural basis of snRNP assembly in humans. | Structural and functional investigation of the human snRNP assembly factor AAR2 in complex with the RNase H-like domain of PRPF8
The crystal structure of human AAR2 bound to the central spliceosomal factor PRPF8 and in vitro functional data yield insights into the structural basis of snRNP assembly in humans.
Precursor messenger RNA (pre-mRNA) splicing is catalyzed by a highly dynamic, multi-megadalton ribonucleoprotein (RNP) machinery, the spliceosome (Will & Lührmann, 2011 ▸; Wahl et al., 2009 ▸). The small nuclear RNPs (snRNPs) U1, U2, U4, U5 and U6 are the main subunits of the major, U2-type spliceosome. Each of these snRNPs contains a particle-specific snRNA, seven common Sm proteins [or like Sm (LSm) proteins in the case of U6] and a set of particle-specific proteins (Will & Lührmann, 2001 ▸). The U5 snRNP is the only snRNP subunit that is also employed by the minor spliceosome (Will & Lührmann, 2005 ▸). Apart from the snRNPs, a multitude of proteins and protein complexes that are not stably associated with an snRNP also join the spliceosome to facilitate and regulate pre-mRNA splicing (Agafonov et al., 2011 ▸). For each round of splicing, the spliceosome assembles anew on a pre-mRNA by the stepwise association of snRNPs and non-snRNP proteins. Its catalytic center is not preformed but only emerges during assembly by repeated, extensive remodeling of the specific RNA–protein interaction network of each assembly stage to eventually elicit intron excision and exon ligation via two transesterification reactions, referred to as step 1 and step 2 (Wahl et al., 2009 ▸; Will & Lührmann, 2011 ▸). SnRNPs themselves are assembled via complex pathways, which in the cases of U1, U2, U4 and U5 include cytoplasmic and nuclear phases (Will & Lührmann, 2001 ▸; Matera & Wang, 2014 ▸; Gruss et al., 2017 ▸). The corresponding snRNAs are synthesized by RNA polymerase II (Pol II), modified with an m7G cap, processed by the integrator complex and exported to the cytoplasm. Here, the Sm proteins are assembled stepwise to form a ring-like structure around a U-rich Sm site in the snRNAs via the protein arginine methyltransferase 5 complex and the survival motor neuron (SMN) complex. Trimethylguanosine synthase 1 then catalyzes hypermethylation of the m7G cap to generate an m2,2,7G cap. The hypermethylated cap and the assembled Sm core domain act as a composite nuclear localization signal that facilitates re-entry of the Sm core RNPs into the nucleus. The integration of particle-specific proteins into snRNPs also requires specific assembly factors and chaperones. For example, in human cells, the adaptor protein, nuclear FMR1-interacting protein 1, and the heat-shock protein 90 (HSP90)/Rvb1–Rvb2–Tah1–Pih1 (RT2P) chaperone machinery in collaboration with a nuclear-localized SMN complex facilitate integration of the U4/U6-specific proteins NHP2-like protein 1 and pre-mRNA processing factor (PRPF) 31 into the U4/U6 di-snRNP (Bizarro et al., 2015 ▸). The HSP90/RT2P chaperone machinery also supports the assembly of a U5 snRNP module composed of the PRPF8 protein (Prp8p in yeast), the 116 kDa U5 small nuclear ribonucleoprotein component (EFTUD2; Snu114p in yeast), the U5 small nuclear ribonucleoprotein 200 kDa helicase (SNRNP200; Brr2p in yeast) and the SNRNP40 protein in the cytoplasm, thereby promoting formation of the mature U5 snRNP (Malinová et al., 2017 ▸). Several additional proteins have been implicated in U5 snRNP or U4/U6-U5 tri-snRNP assembly in metazoans (Malinová et al., 2017 ▸; Klimešová et al., 2021 ▸; Erkelenz et al., 2021 ▸; Bergfort et al., 2022 ▸). Some snRNPs are profoundly remodeled during pre-mRNA splicing, necessitating specific recycling mechanisms to reassemble the particles for further rounds of splicing. For example, during spliceosome activation the U4 and U6 snRNAs, which are extensively base-paired in the U4/U6 di-snRNP, are unwound by the SNRNP200 helicase, and U4 snRNA and all U4/U6-associated proteins are displaced (Laggerbauer et al., 1998 ▸; Raghunathan & Guthrie, 1998 ▸; Agafonov et al., 2011 ▸). Furthermore, in human cells the U5 snRNP enters the spliceosome as a 20S particle but is released as a 35S particle after splicing due to incorporation of the PRPF19 complex and additional factors (Makarov et al., 2002 ▸). Late de novo snRNP biogenesis steps and recycling of snRNPs after pre-mRNA splicing take place in nuclear Cajal bodies (Staněk & Neugebauer, 2004 ▸, 2006 ▸; Sleeman et al., 2001 ▸; Sleeman & Lamond, 1999 ▸). The U5-specific PRPF8 protein is one of the most conserved nuclear proteins and coordinates proteins, snRNAs and the pre-mRNA at the catalytic center of the spliceosome (Grainger & Beggs, 2005 ▸). PRPF8 harbors two regulatory pseudo-enzyme domains at its very C-terminus, comprising an RNase H-like (RH) and a Jab1/MPN-like (JM) fold (Pena et al., 2007 ▸, 2008 ▸; Zhang et al., 2007 ▸; Yang et al., 2008 ▸; Ritchie et al., 2008 ▸). In yeast, the A1 cistron-splicing factor (Aar2p) has been characterized as a U5 snRNP assembly and recycling factor that mediates the formation of pre-U5 snRNPs lacking the Brr2p helicase in the cytoplasm (Gottschalk et al., 2001 ▸; Boon et al., 2007 ▸). Aar2p concomitantly binds the RH and JM domains of Prp8p (Weber et al., 2011 ▸, 2013 ▸; Galej et al., 2013 ▸). By sequestering the Prp8p JM domain, which is a major binding site of the Brr2p RNA helicase, Aar2p initially prevents the integration of Brr2p into U5 snRNP (Weber et al., 2013 ▸; Galej et al., 2013 ▸). Aar2p accompanies the Brr2p-deficient pre-U5 snRNP particle into the nucleus, where phosphorylation of its Ser253 residue triggers refolding and release of Aar2p, allowing Brr2p entry to complete U5 snRNP biogenesis (Boon et al., 2007 ▸; Weber et al., 2013 ▸). In a previous study, we demonstrated that the human Aar2p homolog AAR2 is produced from the c20orf4 gene in HeLa cells and that it stably binds the PRPF8 RH domain in vitro (Santos et al., 2015 ▸). However, human AAR2 and yeast Aar2p exhibit only 24% sequence identity, questioning the extent to which their structures and molecular mechanisms are conserved. While proteomics studies and pull-down experiments have suggested that human AAR2 also participates in U5 snRNP assembly (Malinová et al., 2017 ▸; Klimešová et al., 2021 ▸), human AAR2 has been identified in a complex with PRPF8, EFTUD2, SNRNP200 and SNRNP40 (Malinová et al., 2017 ▸), indeed indicating potential differences in the Aar2p/AAR2-mediated U5 snRNP assembly steps in yeast and humans. Here, we report a co-crystal structure of human AAR2 in complex with the PRPF8 RH domain (PRPF8RH) and present further interaction studies of AAR2 with PRPF8 fragments and the SNRNP200 helicase in vitro. In contrast to the situation in yeast, we find that a human AAR2–PRPF8RH complex does not bind the PRPF8 JM domain and thus permits the formation of a trimeric AAR2–PRPF8–SNRNP200 complex. As in yeast, the human AAR2–PRPF8RH interaction is abrogated in vitro by a phosphomimetic S284E (S253E in yeast) mutation, indicating highly conserved regulation of AAR2 by phosphorylation. Furthermore, AAR2 seems to lock PRPF8RH in its first-step conformation and block the conformational switch to a step 2-like, Mg2+-coordinated conformation during U5 snRNP biogenesis. Our results shed the first light on the human AAR2–PRPF8RH interface and imply a different role of AAR2 in spliceosomal assembly than in yeast.
We employed a modified pFL vector encoding a truncated version of human SNRNP200 lacking the first 394 residues (residues 395–2136) and containing an N-terminal, TEV-cleavable His10 tag (Santos et al., 2012 ▸), a pET-M11 plasmid encoding human PRPF8RH (residues 1747–2016) and containing an N-terminal, TEV-cleavable His6 tag (Pena et al., 2008 ▸; Weber et al., 2011 ▸), a pET-M11 plasmid encoding human PRPF8JM (residues 2064–2335) and containing an N-terminal TEV-cleavable GST tag (Mozaffari-Jovin et al., 2013 ▸), and pFL vectors encoding human AAR2 or AAR2Δloop (AAR2 with residues 170–200 replaced by three serines) with both AAR2 constructs containing an N-terminal, TEV-cleavable His10 tag (Santos et al., 2015 ▸), which have been described previously. The inserts were derived from codon-optimized synthetic genes. A DNA fragment encoding the C-terminal fragment of PRPF8 (encompassing the RH and JM domains; PRPF8RH-JM; residues 1760–2335) was PCR-amplified from a codon-optimized prpf8 synthetic gene and cloned into the pIDK donor vector by restriction-enzyme cloning using XhoI and NdeI (NEB). Snrnp200395–2136 -pFL and prpf81760–2335 -pIDK were fused by Cre-Lox recombination and used for bacmid preparation. A codon-optimized DNA fragment encoding human AAR2 residues 1–364, lacking the C-terminal 20 residues of AAR2, was cloned into a modified pFL vector by restriction-enzyme cloning using EcoRI and HindIII (NEB) to guide the production of AAR21–364 containing a C-terminal His6 tag. A codon-optimized DNA fragment encoding human AAR2 was cloned into pcDNA3.1(+) using BamHI and XhoI restriction enzymes (NEB) to guide the production of AAR2 containing an N-terminal FLAG tag. Mutations were introduced with the QuikChange site-directed mutagenesis kit (Stratagene) according to the manufacturer’s instructions. All constructs were verified by dye-terminator sequencing (Seqlab). Purified human SNRNP200395–2136 (8 mg ml−1; Santos et al., 2012 ▸), PRPF8RH (30 mg ml−1; Pena et al., 2008 ▸), PRPF8JM (10 mg ml−1; Mozaffari-Jovin et al., 2013 ▸), AAR2 (12 mg ml−1; Santos et al., 2015 ▸), AAR2Δloop (12 mg ml−1; Santos et al., 2015 ▸), an SNRNP200395–2136–PRPF8JM complex (Mozaffari-Jovin et al., 2013 ▸; 8 mg ml−1) and an AAR2–PRPF8RH complex (20 mg ml−1; Santos et al., 2015 ▸) were obtained as described previously. AAR21–364 (11 mg ml−1) was produced and purified as described for AAR2 but with omission of the TEV protease cleavage step, leaving the His6 tag intact. SNRNP200395–2136 and PRPF8RH-JM were co-produced based on a recombinant baculovirus derived from the recombined bacmid in Sf9 cells. For purification of the SNRNP200395–2136–PRPF8RH-JM complex, cells were resuspended in resuspension buffer [50 mM Tris–HCl pH 8.0, 300 mM NaCl, 5%(v/v) glycerol, 1 mM DTT, 0.05%(v/v) NP40] supplemented with EDTA-free protease inhibitors (Roche) and DNase I (NEB) and lysed via sonification. After centrifugation, the lysate of about 50 column volumes was filtered and passed through Ni–NTA beads (Qiagen). The beads were washed twice with ten column volumes of resuspension buffer containing 15 mM imidazole. The captured complex was eluted with two column volumes of resuspension buffer containing 500 mM imidazole, TEV protease (0.5 mg per millitre of protein solution) was added and the mixture was dialyzed against 20 mM HEPES–NaOH pH 7.5, 200 mM NaCl, 1 mM DTT overnight at 4°C. Five column volumes of the buffer-exchanged sample were again passed through Ni–NTA beads and the flowthrough was collected. The complex was concentrated to a final concentration of 7 mg ml−1 and further purified by size-exclusion chromatography (SEC) on a Superdex S200 10/300 column (GE Healthcare) in 20 mM HEPES–NaOH pH 7.5, 200 mM NaCl, 1 mM DTT.
Individual proteins and protein mixtures were analyzed by analytical SEC on a Superdex 200 Increase PC3.2/30 column (GE Healthcare) in 20 mM Tris–HCl pH 7.5, 250 mM NaCl, 0.5 mM DTT at a flow rate of 50–70 µl min−1. For analysis of complex formation, proteins (at the concentrations stated in Section 2.1) were mixed in equimolar ratios in 60 µl size-exclusion buffer and incubated for 30 min on ice. Elution fractions were supplemented with SDS–PAGE loading buffer and analyzed by SDS–PAGE.
Crystallization of the human AAR2Δloop–PRPF8RH complex has been described previously (Santos et al., 2015 ▸). Briefly, 1 µl purified human AAR2Δloop–PRPF8RH complex concentrated to 14 mg ml−1 in 20 mM Tris–HCl pH 7.5, 150 mM NaCl, 0.5 mM DTT was crystallized employing an equal volume of reservoir solution consisting of 0.1 M HEPES pH 7.0, 10%(w/v) PEG 6000, 5%(v/v) 2-methyl-2,4-pentanediol. Crystals were transferred to reservoir solution supplemented with 10%(v/v) 2-methyl-2,4-pentanediol and flash-cooled in liquid nitrogen. Diffraction data were collected on beamlines BL14.1, BL14.2 and BL14.3 of the BESSY II storage ring, Berlin, Germany and on beamline P14 of the PETRA III storage ring, Hamburg, Germany at 100 K and were processed with XDS (Kabsch, 2010 ▸). The structure was solved by molecular replacement with Phaser (McCoy et al., 2007 ▸) using chain A of the structural coordinates of human PRPF8RH (PDB entry 3e9l; Pena et al., 2008 ▸), omitting the water molecules. An initial model of the AAR2Δloop subunit was obtained by automated model building with phenix.autobuild (Adams et al., 2010 ▸). The model was completed through alternating rounds of automated refinement using phenix.refine (Afonine et al., 2012 ▸) and manual model building using Coot (Emsley et al., 2010 ▸). The quality of the final model was assessed with MolProbity (Chen et al., 2010 ▸). Of note, a relatively large number of difference density peaks were observed in the F o − F c map. Structural figures were prepared with PyMOL (Schrödinger).
Structures of yeast Aar2p in complex with the Prp8p RH and JM domains or with full-length Prp8p have been reported (Weber et al., 2011 ▸, 2013 ▸; Galej et al., 2013 ▸). In contrast, the structure of human AAR2, which exhibits only 24% sequence identity to the yeast ortholog (Supplementary Fig. S1), and the structural basis for its interaction with PRPF8 remain unknown. We previously reported the crystallization of a human AAR2 variant in which an internal loop (residues 170–200) was replaced by three serine residues (AAR2Δloop) in complex with PRPF8RH (Santos et al., 2015 ▸). In yeast Aar2p, the corresponding internal loop was shown to hinder crystallization, to be protease-cleavable and to be irrelevant for the interaction with Prp8p C-terminal domains (Weber et al., 2011 ▸, 2013 ▸; Santos et al., 2015 ▸). Here, we report the crystal structure of the human AAR2Δloop–PRPF8RH complex at 2.35 Å resolution. The structure was solved by molecular replacement with Phaser using the structural coordinates of PRPF8RH (PDB entry 3e9l; Pena et al., 2008 ▸) as a molecular-replacement search model (Table 1 ▸). Apart from ten N-terminal residues, the region spanning residues 159–201 including the three serines connecting residues 170–200 of the internal loop, a loop connecting helices α9 and α10 (residues 313–321) and six C-terminal residues of AAR2Δloop (Supplementary Fig. S1), all residues of AAR2Δloop and PRPF8RH could be reliably modeled into well defined electron density (Supplementary Fig. S2). Residues 65–71 of AAR2 and residues 2001–2008 of PRPF8RH were modeled with low confidence due to weaker electron density. Despite the low sequence identity, the overall structure of human AAR2Δloop in the AAR2Δloop–PRPF8RH complex is very similar to that of yeast Aar2pΔloop in complex with the Prp8p RH and JM domains (Weber et al., 2013 ▸; Galej et al., 2013 ▸; root-mean-square deviation of 2.13 Å for 236 pairs from 330 AAR2 and 342 Aar2p Cα atoms; Supplementary Fig. S3). As previously observed for Aar2p (Weber et al., 2011 ▸, 2013 ▸; Galej et al., 2013 ▸), human AAR2Δloop exhibits an N-terminal domain (NTD; residues 10–158) mainly composed of β-strands, an α-helical C-terminal domain (CTD; residues 202–364) and a C-terminal, irregularly structured tail (residues 365–384) (Fig. 1 ▸ a). In the full-length yeast Aar2p–Prp8p structure (PDB entry 4i43; Galej et al., 2013 ▸), the C-terminal peptide of Aar2p is fully structured and contacts several other Prp8p domains. Superposition with the present human AAR2–RH complex structure suggests that due to the shorter AAR2 C-terminal peptide in humans, contacts with other PRPF8 domains may be limited. Hence, the differences in the functionally important C-terminal peptide of yeast and human Aar2p/AAR2 may hint at a somewhat different mode of action of AAR2 in U5 snRNP or U4/U6-U5 tri-snRNP assembly in humans. Despite the overall structural similarity of both individual components, the protein interfaces between yeast and human Prp8pRH/PRPF8RH and Aar2p/AAR2 are markedly different. As in yeast, the NTD lacks direct interactions with PRPF8RH, while the CTD and the C-terminal tail of AAR2 establish two interfaces with PRPF8RH (interfaces I and II, respectively; Figs. 1 ▸ a–1 ▸ c). In interface I, an edge of the AAR2Δloop CTD laterally contacts PRPF8RH (Figs. 1 ▸ d and 1 ▸ e). Interface II is built by the C-terminal residues 366–377 of AAR2 extending across the PRPF8 RH domain below the protruding β-finger module (Figs. 1 ▸ f and 1 ▸ g). Both interfaces bury a comparable surface area in the yeast and human systems (interface I, 399 and 412 Å2, respectively; interface II, 733 and 511 Å2, respectively). Interface I is dominated by hydrophobic contacts, with only four of 12 PRPF8RH-interacting residues conserved between yeast Aar2p and human AAR2, underlining the different organization of the interactions. The conserved core of interface I includes interactions between Ile225 of AAR2 (Ile189 in Aar2p) and Val1874 of PRPF8RH (Val1946 in Aar2p) as well as between Met230 of AAR2 (Met195 in Aar2p) and Trp1839 of PRPF8RH (Trp1911 in Aar2p) (Figs. 1 ▸ d and 1 ▸ e; Supplementary Fig. S1). Compared with yeast Aar2p, the AAR2 CTD harbors two extended helices (α11 and α12; Supplementary Fig. S3). In addition, Ile225 and Met230 are shifted by four residues (about one helical turn) along the α5 helix compared with the equivalent residues in yeast Aar2p (Figs. 1 ▸ d and 1 ▸ e), giving rise to a markedly different angle with which human AAR2Δloop contacts the PRPF8 RH domain compared with yeast Aar2pΔloop in the Aar2pΔloop–Prp8pRH–Prp8pJM complex (Fig. 1 ▸ c). Also, the AAR2Δloop residues participating in interface II are only partially conserved between yeast and humans (two of eight residues; Supplementary Fig. S1).
The low degree of conservation of AAR2 and observed marked differences in the interface with PRPF8RH have apparent consequences for AAR2 function and likely for interactions within the spliceosome. To test the importance of the specific contacts between AAR2Δloop and PRPF8RH that are observed in our co-crystal structure, we conducted analytical SEC runs with wild-type (WT) proteins and variants. To this end, we investigated the binding of WT AAR2 to WT PRPF8RH in previous work, which is only shown here for comparison (Figs. 2 ▸ a–2 ▸ c; Santos et al., 2015 ▸). In yeast, the C-terminal tail of Aar2p is dispensable for Prp8RH binding (Weber et al., 2011 ▸). Conversely, in the human system, AAR21–364, which lacks the C-terminal tail, no longer binds stably to PRPF8RH (Figs. 2 ▸ a–2 ▸ d). Likewise, converting Trp1839 of PRPF8RH or Met230 of AAR2, which are part of the conserved core of interface I, individually to alanine residues abrogated complex formation (Fig. 2 ▸ e and 2 ▸ f). Again, the situation differs in yeast, where only Trp1911 of Prp8pRH (equivalent to Trp1839 in human PRPF8RH), but not Met195 of Aar2p (equivalent to Met230 in human AAR2), is essential for the interaction (Weber et al., 2013 ▸). The low sequence conservation and the resulting structural differences in the AAR2–PRP8RH interface might also have consequences for the wider protein interaction network around AAR2. Concomitant binding of the Prp8p RH and JM domains by Aar2p in yeast sequesters the JM domain, preventing binding of the Brr2p RNA helicase to Aar2p–pre-U5 snRNP (Weber et al., 2013 ▸; Galej et al., 2013 ▸). In yeast Aar2p–Prp8p complexes (Weber et al., 2011 ▸, 2013 ▸; Galej et al., 2013 ▸), the C-terminal tail of Aar2p runs along the protruding Prp8pRH β-finger module, stringing the β-finger and the central Prp8pJM β-sheet into an extended, intermolecular β-structure (Figs. 1 ▸ b and 1 ▸ c). While the beginning of the C-terminal tail in human AAR2Δloop maintains similar interactions with PRPF8RH as in yeast, for example employing Val373–Val375 to form a short β-sheet of three hydrogen bonds to PRPF8RH, distal parts of the C-terminal tail (beyond Val374) deviate from the direction of the Aar2p C-terminal tail (Figs. 1 ▸ b and 1 ▸ c). In yeast, the formation of the penultimate β-strand of Aar2p and the concomitant sequestration of JM from Brr2p is mediated exclusively by a series of hydrophobic residues at the very C-terminus of Aar2p, which are complementary to hydrophobic residues of the neighboring β-strands of RH and JM (Weber et al., 2013 ▸; Galej et al., 2013 ▸). A structure-based alignment revealed that the respective very C-terminal residues of AAR2, Pro378, Glu379, Gly380 and Glu382, are unlikely to support β-sheet formation with the corresponding highly conserved residues of the PRPF8RH β-finger and PRPF8JM due to their steric or polar properties (Supplementary Fig. S1; compare Figs. 1 ▸ f and 1 ▸ g). However, we cannot exclude that in the context of the full-length proteins the very C-terminus of hAAR2 may engage in a yeast-like interaction with the PRPF8 JM domain. The C-terminal tail of human AAR2Δloop in the observed conformation would not be able to concomitantly bind the PRPF8JM domain as observed in yeast. Indeed, also confirming a prior study (Malinová et al., 2017 ▸), AAR2–PRPF8RH did not stably bind PRPF8JM in analytical SEC (Figs. 2 ▸ g and 2 ▸ h) and failed to sequester PRPF8JM from a pre-formed SNRNP200395–2136–PRPF8JM complex (Fig. 3 ▸ a and 3 ▸ b). AAR2 alone or in complex with PRPF8RH did not bind stably to SNRNP200395–2136 or to a SNRNP200395–2136–PRPF8JM complex (Figs. 3 ▸ c and 3 ▸ d). Instead, a stable AAR2–PRPF8RM-JM–SNRNP200395–2136 ternary complex was formed upon mixing the components (Fig. 3 ▸ b).
Aar2p can be phosphorylated at five positions in vivo (Ser253, Thr274, Tyr328, Ser331 and Thr345) and phosphomimetic S253D or S253E variants of Aar2p interfered with Aar2p–Prp8p interaction in yeast extracts (Weber et al., 2011 ▸). Structural analysis of a phosphomimetic Aar2pS253E variant suggested that phosphorylation leads to a local conformational rearrangement of the Aar2p CTD and thereby to disruption of the Prp8pRH binding site (Weber et al., 2011 ▸). A structure-based sequence alignment revealed that Ser284 in human AAR2 corresponds to Ser253 in yeast Aar2p (Supplementary Fig. S1; Fig. 3 ▸ e), and AAR2 has been found to be phosphorylated at Ser284 in human liver cancer cells (Hornbeck et al., 2012 ▸). Recapitulating the situation in yeast, an AAR2S284E phosphomimetic variant failed to stably bind PRPF8RH in analytical SEC (Fig. 3 ▸ f). Taken together, our interaction studies reveal differences in the relative importance of AAR2/Aar2p regions in maintaining a stable interaction with the PRPF8/Prp8p RH domain in the human and yeast systems. Furthermore, AAR2 does not sequester the PRPF8 JM domain to intermittently prevent SNRNP200 association with the U5 snRNP. AAR2 displacement from PRPF8 may involve reversible phosphorylation of AAR2 at Ser284. Thus, the U5 snRNP assembly steps apparently differ in detail in yeast and humans.
Mutations in the prpf8 gene can lead to retinitis pigmentosa (RP; Růžičková & Staněk, 2017 ▸), a disease that causes blindness in humans, and the corresponding PRPF8/Prp8p variants cause defects in U5 snRNP assembly (Malinová et al., 2017 ▸) and splicing (Mayerle & Guthrie, 2016 ▸; Mozaffari-Jovin et al., 2013 ▸) in humans and yeast. In baker’s yeast, two sets of prp8 mutant alleles, corresponding to RP-related mutations in humans that disrupt either the first or the second step of splicing, cluster in the Prp8p RH domain (Grainger & Beggs, 2005 ▸). Furthermore, the human PRPF8 RH domain can undergo a conformational switch in a protruding β-finger module, with one conformation promoting the first step and an alternative, Mg2+-bound conformation supporting the second step of splicing (Schellenberg et al., 2013 ▸). Despite the biochemical and structural evidence reported previously, which supports this switch, a caveat of our AAR2–PRPF8RH structure may be that the RH β-finger module makes crystal contacts with a neighboring symmetry-related RH β-finger module. However, recent cryogenic electron-microscopy structures of spliceosomes also confirm this conformational switch, rationalize some of the effects of PRPF8 RH domain variants and demonstrate repeated, long-range repositioning of the PRPF8/Prp8p RH domain during the splicing reaction in yeast and humans (Wan et al., 2016 ▸; Bertram, Agafonov, Liu et al., 2017 ▸; Yan et al., 2015 ▸, 2017 ▸; Zhang et al., 2017 ▸; Bertram, Agafonov, Dybkov et al., 2017 ▸; Rauhut et al., 2016 ▸; Galej et al., 2016 ▸; Plaschka et al., 2017 ▸; Fica et al., 2017 ▸; Wilkinson et al., 2021 ▸). Comparison of our AAR2Δloop–PRPF8RH structure with the PRPF8RH step 1 and step 2 conformations revealed that AAR2 binding is compatible with the PRPF8RH step 1 conformation but that steric clashes ensue between the AAR2 C-terminal tail and the PRPF8 RH domain in the Mg2+-bound step 2 conformation (Figs. 4 ▸ a–4 ▸ d). To test whether AAR2 is likely to prevent a switch of PRPF8RH into the step 2 conformation, we explored whether AAR2 binds a PRPF8RH variant that is stabilized in the step 2 conformation (T1789P; Schellenberg et al., 2013 ▸). Indeed, unlike WT PRPF8RH, PRPF8RH,T1789P partly dissociated from AAR2 upon increasing the Mg2+ concentration in analytical SEC (Figs. 4 ▸ e–4 ▸ h), suggesting that a step 2 conformation in PRPF8RH is incompatible with AAR2 binding.
We have elucidated similarities and differences in the structures and interaction profiles of yeast Aar2p and human AAR2 and have identified a putative, conserved phosphorylation event that is most likely to be involved in the functional cycle of AAR2 as a U5 snRNP assembly factor. Based on our findings, we conclude that the precise roles of Aar2p and AAR2 in U5 snRNP biogenesis differ. In yeast, an Aar2p–pre-U5 snRNP, from which the Brr2p RNA helicase is excluded, seems to constitute an important U5 snRNP assembly intermediate (Boon et al., 2007 ▸; Weber et al., 2013 ▸). In contrast, our observations of (i) human AAR2 failing to sequester the PRPF8 JM domain from SNRNP200395–2136 and (ii) AAR2 concomitantly binding to a PRPF8 fragment encompassing the RH and JM domains and SNRNP200395–2136 suggest that an equivalent, long-lived intermediate is not formed in the human system. Association of AAR2 with the PRPF8 RH domain as in our AAR2Δloop–PRPF8RH structure would prevent the PRPF8 RH domain from engaging with other regions of PRPF8, the N-terminal region of SNRNP200, the C-terminal region of PRPF31, PRPF6, U4/U6 di-snRNAs and U5 snRNA as observed in the human U4/U6-U5 tri-snRNP (Agafonov et al., 2016 ▸; Charenton et al., 2019 ▸). This finding suggests that prevention of the premature association of U4/U6 di-snRNP components with pre-U5 particles may be an important function of AAR2 in the human system. In addition, transient blocking of binding sites on the PRPF8 RH domain, possibly supported by allosteric effects due to the selective stabilization of a step 1-like conformation in the PRPF8 RH domain by AAR2, may help to order assembly steps during U5 snRNP biogenesis. The above findings and suggestions are in agreement with the previous observation of the interaction of human AAR2 with a PRPF8–EFTUD2–SNRNP200–SNRNP40 U5 submodule (Malinová et al., 2017 ▸). As most protein-coding genes in humans contain multiple introns (Lee & Rio, 2015 ▸), pre-mRNA splicing is an inherent step in their expression. Moreover, pre-mRNA splicing predominantly occurs co-transcriptionally (Alpert et al., 2017 ▸) and splicing is physically and functionally coupled to transcription, other pre-mRNA processing steps and mRNA export (Carrocci & Neugebauer, 2019 ▸; Tellier et al., 2020 ▸). Thus, efficient splicing is a prerequisite for efficient gene expression and, due to its stabiliziation of the step 1 configuration of RH, a potential role of human AAR2 in pre-mRNA splicing cannot be ruled out. AAR2 may have a moonlighting function during pre-mRNA splicing independent of its role as a U5 snRNP assembly factor. By binding the PRPF8 RH domain during a stage of splicing when it is available, for example, in the pre-catalytic B complex (PDB entry 7abg; Townsend et al., 2020 ▸), AAR2 may hinder the transition to subsequent stages, thus impeding splicing and, as a consequence, gene expression. As in the case of U5 snRNP assembly, direct blocking of binding sites on PRPF8RH and allosteric effects due to the stabilization of a step 1 conformation in PRPF8RH may support such a splicing-inhibitory role of AAR2. Again, the observed high nuclear levels of AAR2 might ensure that sufficient AAR2 is available to serve multiple functions, as moonlighting is known for some splicing factors that are in excess over other splicing machinery. For example, U1 snRNP has additional roles in 3′-end processing of Pol II transcripts (telescripting; Di et al., 2019 ▸). However, AAR2 has never been found to be associated with the spliceosome at any stage of splicing (Agafonov et al., 2011 ▸), arguing against a direct effect of AAR2 on splicing. Further studies on human Aar2 in a spliceosomal context will hopefully resolve these remaining questions.
Structure coordinates and diffraction data have been deposited in the Protein Data Bank (http://www.pdb.org) under accession code 7pjh. All other data supporting the findings of this study are described in the manuscript or in the supporting information or are available from the corresponding authors on request.
The following references are cited in the supporting information for this article: Barton (1993 ▸), Kabsch & Sander (1983 ▸) and Pettersen et al. (2004 ▸).
PDB reference: AAR2 bound to PRPF8 RNaseH domain, 7pjh Supplementary Figures. DOI: 10.1107/S2059798322009755/cb5141sup1.pdf | true | true | true |
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PMC9629937 | Man Zhang,Xiao-Long Wang,Hui Shi,Lan-Qing Meng,Hong-Feng Quan,Lin Yan,Hui-Fang Yang,Xiao-Dong Peng | Betaine Inhibits NLRP3 Inflammasome Hyperactivation and Regulates Microglial M1/M2 Phenotypic Differentiation, Thereby Attenuating Lipopolysaccharide-Induced Depression-Like Behavior | 26-10-2022 | Depression is one of the most important mental illnesses and is closely related to inflammation. Betaine is a natural product with an anti-inflammatory and antioxidant activities. However, the mechanism by which betaine ameliorates depression-like behaviors induced by lipopolysaccharide (LPS) is poorly understood. The purpose of this study was to investigate the neuroprotective effect of betaine on LPS-induced depression-like behavior in mice and its mechanism of action. ICR mice were randomly divided into four groups: the control group, the LPS model group (0.83 mg/kg), the positive drug group (MIDO, 50 mg/kg), and the betaine group (5% and 1% in drinking water). The betaine group was administered for 21 days, and on the 22nd day, except for the blank group, LPS (0.83 mg/kg) was intraperitoneally injected to establish a lipopolysaccharide-induced mice depression-like model. Twenty-four hours after LPS injection, the tail suspension test (TST), open field test (OFT), and sucrose preference test (SPT) were performed to evaluate the effect of betaine on LPS-induced depressive behavior in mice. After the behavioral study, the mouse brain, hippocampus, and serum were taken for detection. The expressions of cytokines and inflammatory mediators were detected by ELISA, HE staining, immunofluorescence, immunohistochemistry, and western blotting. Western blotting was used to detect the protein expression levels of the nucleotide-binding oligomerization domain-like receptor protein 3 (NLRP3), caspase-1, and ASC, the protein expression levels of the microglial polarization markers COX-2, inducible nitric oxide synthase (iNOS), and CD206. The results showed that betaine significantly ameliorated the depression-like behavior in LPS-induced mice, significantly attenuated the production of proinflammatory cytokines and increased the release of an anti-inflammatory cytokines. Betaine decreased the expression of the NLRP3 inflammasome, decreased the expression of M1 polarization markers, tumor necrosis factor-alpha (TNF-α), interleukin-1β (IL-1β), COX-2, and iNOS and promoted the expression of M2 polarization marker CD206. Our study suggests that betaine may promote the transition of microglia from the M1 to the M2 phenotype by inhibiting NLRP3 inflammasome activation, thereby attenuating lipopolysaccharide-induced depression-like behavior. | Betaine Inhibits NLRP3 Inflammasome Hyperactivation and Regulates Microglial M1/M2 Phenotypic Differentiation, Thereby Attenuating Lipopolysaccharide-Induced Depression-Like Behavior
Depression is one of the most important mental illnesses and is closely related to inflammation. Betaine is a natural product with an anti-inflammatory and antioxidant activities. However, the mechanism by which betaine ameliorates depression-like behaviors induced by lipopolysaccharide (LPS) is poorly understood. The purpose of this study was to investigate the neuroprotective effect of betaine on LPS-induced depression-like behavior in mice and its mechanism of action. ICR mice were randomly divided into four groups: the control group, the LPS model group (0.83 mg/kg), the positive drug group (MIDO, 50 mg/kg), and the betaine group (5% and 1% in drinking water). The betaine group was administered for 21 days, and on the 22nd day, except for the blank group, LPS (0.83 mg/kg) was intraperitoneally injected to establish a lipopolysaccharide-induced mice depression-like model. Twenty-four hours after LPS injection, the tail suspension test (TST), open field test (OFT), and sucrose preference test (SPT) were performed to evaluate the effect of betaine on LPS-induced depressive behavior in mice. After the behavioral study, the mouse brain, hippocampus, and serum were taken for detection. The expressions of cytokines and inflammatory mediators were detected by ELISA, HE staining, immunofluorescence, immunohistochemistry, and western blotting. Western blotting was used to detect the protein expression levels of the nucleotide-binding oligomerization domain-like receptor protein 3 (NLRP3), caspase-1, and ASC, the protein expression levels of the microglial polarization markers COX-2, inducible nitric oxide synthase (iNOS), and CD206. The results showed that betaine significantly ameliorated the depression-like behavior in LPS-induced mice, significantly attenuated the production of proinflammatory cytokines and increased the release of an anti-inflammatory cytokines. Betaine decreased the expression of the NLRP3 inflammasome, decreased the expression of M1 polarization markers, tumor necrosis factor-alpha (TNF-α), interleukin-1β (IL-1β), COX-2, and iNOS and promoted the expression of M2 polarization marker CD206. Our study suggests that betaine may promote the transition of microglia from the M1 to the M2 phenotype by inhibiting NLRP3 inflammasome activation, thereby attenuating lipopolysaccharide-induced depression-like behavior.
Depression, as a mental illness, has many symptoms, such as cognitive impairment, loss of appetite, pessimism, and suicidal tendencies. The inflammatory hypothesis of depression was proposed in 1991 and attracted widespread attention [1], and the neuroimmunoinflammatory hypothesis of depression was proposed in 1995 [2]. Since then, the relationship between inflammatory cytokines and depression has been increasingly studied. Exploring the innate immune function of glial cells is critical to understanding the role of the brain's immune system in fighting inflammation and psychiatric disorders. Excessive production of inflammatory cytokines and microglial activation in the brain signals depression and dysfunction in brain signaling. Microglia belong to the nonglial system and are resident mononuclear phagocytes in the central nervous system [3]. During the central nervous system injury from infection, traumatic brain injury, or ischemic injury, microglia change from a quiescent state to an amoeba-activated state. Activated microglia can be divided into two main types: classical- (proinflammatory, M1) and alternative- (anti-inflammatory, M2) activated states. Proinflammatory cytokines, interferons, tumor necrosis factor (TNF), and lipopolysaccharide (LPS) can often activate microglia [4]. Depression can induce the polarization of microglia into the M1 to secrete proinflammatory factors. Various antidepressant drugs have an anti-inflammatory effects and can reverse the polarization of microglia to the M1 type. The M2 type can improve symptoms related to depression [5]. However, few pharmacological compounds have been shown to modulate the transformation of microglia to the M2 phenotype [6, 7]. Therefore, the drug-induced polarization of microglia from the M1 phenotype to the M2 phenotype may provide a new strategy for the treatment of depression. Environmental stress before the onset of microglia-dependent depression increases the steady-state concentration of the activated NLRP3 inflammasome, and the activity of the NLRP3 inflammasome and its tight regulation largely determines the morphology and the regulation of microglia. Studies have found that NLPR3 gene knockout (NLRP3−/−) [8] or Caspase-1 gene knockout (CASP1−/−) [9] can alleviate depressive-like behaviors in mice due to chronic stress and NLRP3−/−; the microglial morphology in mice is biased toward an M2-like state [10]. The study found that NLRP3 inhibitors had inhibitory effects on LPS-induced depression in a mouse model, showing the key role of the NLPR3 inflammasome in linking stress and neuroinflammatory states. The NLRP3 inflammasome has been shown to be activated in patients with depression [11]. Knockout of NLRP3 in mice alleviated depression-like behaviors and suppressed proinflammatory processes [12]. Studies have shown that in CUMS-induced depression, NLRP3 polarizes microglia toward the M1 phenotype [13]. It has recently been reported that patients with major depressive disorder have higher levels of NLRP3, caspase-1, and IL-1β in serum or peripheral blood mononuclear cells [14]. Furthermore, when caspase-1 was specifically inhibited, this led to a decrease in depression-like behaviors triggered by multiple stimuli, such as estrogen deficiency, chronic mild stress, and LPS injection [14]. To be precise, NLRP3 and caspase-1 are closely related to depression, which lays the foundation for further research. Therefore, modulating the NLRP3 signaling pathway and further inhibiting neuroinflammation may be a therapeutic strategy to improve depression. Minocycline (MIDO), a broad-spectrum tetracycline antibiotic, has various beneficial effects on the central nervous system, including anti-inflammatory, antioxidant, and neuroprotective effects [15, 16]. Various studies have demonstrated that minocycline is considered a novel treatment for several psychiatric disorders associated with inflammation, including depression [16]. Various antidepressant drugs have an anti-inflammatory effects and can reverse the polarization of microglia to the M1 type. Therefore, we used minocycline as a positive control. The long-term use of western medicine in the treatment of depression may cause adverse reactions such as dependence, toxicity, memory loss, organ dysfunction, and delayed clinical effects, while herbal medicine for depression has made great progress in delaying the course of the disease, improving the efficacy of western medicine, and reducing side effects. Herbal medicines with antidepressant effects (herbal antidepressants), including prescriptions, individual herbs, and phytochemicals, are also widely used to treat depression. [17, 18]. Betaine (Figure 1), also known as trimethyl glycine, is a water-soluble compound that penetrates the blood-brain barrier and is highly distributed in the hippocampus in vitro [19, 20]. Betaine is mainly used as an osmotic regulator and a methyl donor to participate in the dual physiological role of metabolism in vivo [21]. Betaine has various pharmacological effects, such as antiepileptic, neuroprotective, and memory-improving effects [19, 22, 23]. In particular, it acts as a methyl donor to interfere with depression caused by the hyperhomocysteinemia [24]. However, whether betaine inhibits lipopolysaccharide-induced microglial phenotype polarization remains unclear. In our previous study, it was found that betaine can regulate the phenotypic changes of microglia, thereby reducing the inflammatory response of microglial cells induced by LPS stimulation, and the effect of betaine is related to the regulation of the nuclear factor-κB (NF-κB) signaling pathway [25]. However, the effect of betaine on LPS-induced depression-like behavior has not been fully elucidated. Importantly, the polarization of the microglial phenotype and the role of inflammatory markers in response to betaine treatment have not been well-studied. Therefore, the purpose of this study was not only to investigate the role of betaine in response to the LPS model of depression but also to explore the mechanism of its role in microglial polarization.
Sixty male, six-week-old ICR mice, weighing 18–22 g, were purchased from the Animal Center of Ningxia Medical University. The batch number is SCXK (Ning) 2021-0001. All animals were housed under standard conditions with a temperature of 23 ± 5°C, a dark/light cycle of 12 h/12 h, and a humidity of 40–60%. These animals had free access to food and water. This study was approved by the Animal Experiment Ethics Committee of Ningxia Medical University.
Mice were randomly divided into the control group (n = 12), the LPS model group (0.83 mg/kg, n = 12), the positive drug group (MIDO, 50 mg/kg, n = 12), the 5% betaine group (n = 12), and the 1% betaine group (n = 12). Betaine (purity >99%, B2629, Sigma-Aldrich) at concentrations of 5% and 1% was added to the drinking water of mice, and minocycline (MIDO, 50 mg/kg, i.p.) was used as a positive control. LPS (L2630, Sigma-Aldrich) and MIDO (M9511, Sigma-Aldrich) were dissolved in 0.9% (w/v) saline. The dose that gave the best efficacy in the previous preexperiment was 5% betaine. Each group was given the corresponding vehicle and drugs for 21 consecutive days. There was no difference in drinking water consumption between the animals exposed to betaine and the tap water controls. On the 15th day, the positive drug group was given an intraperitoneal injection of MIDO. At 9:00 in the morning of the 22nd day, except for the normal group, LPS (0.83 mg/kg) was intraperitoneally injected, and the body weight and food intake were monitored in the following two hours, six hours, ten hours, and twenty-four hours, and voluntary activities were observed at two hours and twenty-four hours. After the injection of LPS, the sugar water experiment was performed, and 24 hours after the injection, the behavioral test was performed. Blood was taken after the behavioral test, and the whole brain and hippocampus were collected. Brains were fixed with 4% paraformaldehyde (P0099, Beyotime Biotechnology, China) or store in -80°C low temperature freezer (Thermo Scientific Forma902, Thermo Fisher Instruments Co., Ltd.) until use.
Before the injection of LPS, each group of mice was tested for body weight, given food, and weighed. At 24 hours after the injection of LPS, the body weight and food intake of each group of mice were tested, and the difference in weight was calculated.
The mice were trained to adapt to two bottles of 1% sucrose solution (w/v) for 24 h, followed by two bottles of pure water for 24 h. After the deprivation of food and water for 12 h, every mouse was individually faced with two bottles filled with 1% sucrose solution or pure water for 24 h. The feeder locations were switched at 12 h intervals to avoid possible site preference. Consequently, the consumption of sucrose solution and pure water was assayed. Sucrose preference was calculated according to the formula: sucrose preference (%) = sucrose consumption (g)/[sucrose consumption (g) + pure water consumption (g)] × 100%.
In a quiet, dark room, the mice were vertically suspended in a 50 cm × 50 cm × 50 cm open box without lids, with the mouse nose tip at about 5 cm from the bottom of the carton box and connected to a JH-2 tension transducer. After the start of suspension, the force changes of the tension transducer were recorded by the BL-420E+ biological function experimental system for 6 minutes, and the immobility time of the mice was counted after 4 minutes.
The OFT was carried out with a square arena apparatus (50 × 50 × 40 cm) with 16 equal squares. Each mouse was individually placed in the central area facing the wall. The behavior of the mice was observed over the next 5 minutes. The distance traveled and time spent in the central area were monitored.
TNF-α, IL-6, IL-1β, IL-18, and IL-10 in the mice hippocampus were detected with kits (Jiang Lai Biological Co., Ltd.). Tissue corticosterone and C-reactive protein levels were detected with kits (Jiang Lai Biological Co., Ltd.). The hippocampi were weighed and chopped. The samples were treated with ice-cold PBS containing protease inhibitor and then centrifuged at 4000 g at 4°C (refrigerated high-speed centrifuge, Fresco 17, Thermo Fisher). The supernatant was subjected to ELISA measurement. All ELISA experiments were conducted in accordance with the manufacturer's protocol. The absorbance was calculated by standard curves.
Intact brain tissue was removed and dehydrated with different concentrations of ethanol (dehydrator, JJ-12J, Wuhan Junjie Electronics Co., Ltd.). Immerse the dehydrated brain tissue in paraffin at 60°C for sectioning and take coronal sections (microtome, RM2016, Shanghai Leica Instrument Co., Ltd.). Sections were then incubated with hematoxylin and eosin staining solution for 5 min. Dehydrate with xylene (10023418, Sinopharm Chemical Reagent Co., Ltd.) and add neutral glue to seal the slices. The pathological changes of brain tissue were observed under light microscope (XSP-C204, CIC).
The brain tissue of each group of mice was perfused with normal saline, perfused with paraformaldehyde, and placed into a fixative. The primary antibodies used were COX-2 (1 : 500, GB11077-1, Servicebio, China) and CD206 (1 : 500, GB113497, Servicebio, China). Brain coronal sections (microtome, RM2016, Shanghai Leica Instrument Co., Ltd.) were washed 3 times with PBS for 5 min each, placed in citrate buffer (pH 6.0, G1202, Servicebio, China), and then placed in a microwave oven to start antigen retrieval. After antigen retrieval, equilibrate to room temperature and wash 3 times with PBS for 5 min each. Incubate in 3% hydrogen peroxide solution for 20 minutes, then begin a total of 3 washes of 5 minutes each. After blocking with 5% goat serum (G1209, Servicebio, China) for 1 h, the blocking solution was discarded. The corresponding primary antibody solution was added and incubated with the samples at 4°C overnight. The next day, after equilibrating at room temperature, the primary antibody solution was aspirated, and the samples were washed three times with PBS for 5 min each time. The required secondary antibody (G1213, Servicebio, China) was added dropwise and the samples were placed at room temperature for approximately 2 h. The secondary antibody solution was then poured off, and the samples were washed three times in PBS for 5 min each time and incubated with avidin-coupled horseradish peroxidase complex solution at room temperature for 1 h. The avidin-coupled horseradish peroxidase complex solution was discarded, and samples were washed with PBS three times for 5 min each time. DBA (G1211, Servicebio, China) chromogenic solution was added, dropwise, color was developed for 20-60 s, and the samples were washed with PBS. They were then dehydrated with gradient ethanol, cleared with xylene, sealed with neutral gum, and observed under a microscope (XSP-C204, CIC).
After the behavioral test, the brains were fixed with 4% paraformaldehyde (P0099, Beyotime Biotechnology, China) and dehydrated. Brain coronal slices of 10 μm were made with a microtome (RM2016, Shanghai Leica Instruments Co., Ltd.). Sections were washed and blocked with 0.3% Triton and 5% calf serum (F8687, Beyotime Biotechnology, China) for 1.5 hours at room temperature and then incubated with primary antibodies detecting Iba1 (1 : 6000, GB13105-1, Servicebio, Wuhan, China) and NLRP3 (1 : 3000, DF7438, Servicebio, Wuhan, China) in the dark at 4°C for 24 hours in medium, followed by incubation with secondary antibody (G1213, Servicebio, China) for 1 hour at room temperature. The nuclei were counterstained with DAPI (G1012, Servicebio, China), and the sections were slightly dried and mounted with an antifluorescence quenching mounting medium. Sections were observed under a fluorescence microscope, and images were collected. Images were taken under a microscope (Nikon Eclipse C1, Nikon, Japan). To quantify phenotypic changes in microglia, the proportional areas were analyzed with Iba1 labeling and NLRP3 staining as previously described. The proportional area is shown as the average area of the positive labels for all the representative pictures.
Mice were sacrificed after behavioral testing. Under sterile conditions, hippocampal tissue was rapidly removed from each mouse brain and placed in liquid nitrogen. Tissue samples were homogenized in radioimmunoprecipitation assay lysis buffer (RIPA lysis buffer, G2002, Servicebio), then centrifuged at 12,000× g for 15 min (grinder, KZ-II, Shanghai Jingxin Industrial Development Co., Ltd.). The supernatant was then collected at 4°C. The BCA (KGPBCA, KeyGEN BioTECH, China) protein assay and western blot were performed to determine protein concentration. Western blot analysis was used to detect protein expression in the cells, using antibodies against the following proteins: CD206 (1 : 1000), iNOS (1 : 1000), COX-2 (1 : 1000), NLRP3 (1 : 1000), P-NF-κB (1 : 500), and β-actin (1 : 2000, Proteintech, Wuhan, China); TLR4 (1 : 500), Myd88 (1 : 1000), ASC (1 : 1000), and Caspase-1 (1 : 1000) (Abcam, Cambridge, MA, USA). This was followed by treatment with HRP-anti-rabbit secondary antibody (1 : 10,000; ProteinTech, Wuhan, China) for 1.5 h at 20°C. An ECL agent (Thermo Fisher Scientific, Inc., Waltham, MA, USA) was used to analyze the protein bands, and the ImageJ software (NIH, Bethesda, MD, USA) was used to analyze protein expression.
All values were expressed as mean ± SEM and analyzed using SPSS Statistics V22.0 (IBM, Armonk, NY, USA). The data is normally distributed (normality test). Data were statistically analyzed using one-way analysis, followed by Student's t-test (comparing two groups) or LSD post hoc test (comparing more than two groups). P < 0.05 was considered statistically significant.
Behavioral assays to evaluate the effects of betaine on the LPS-induced mice (Figure 2(a)). The results showed that betaine could significantly improve the feeding behavior and decreased food intake (Figure 2(c)) and weight loss (Figure 2(b)) of LPS-induced model mice. Betaine significantly improved the autonomous behavior of LPS-induced model mice (Figure 2(f)), significantly shortened the immobility time of mice in the tail suspension experiment (Figure 2(e)), and significantly increased the preference of LPS-induced model mice for sucrose drinking (Figure 2(d)).
Betaine significantly improved the integrity of cellular morphological features in the hippocampal brain region of mice with LPS-induced depression-like behavior. The cells in the hippocampus of mice in the normal group were neatly arranged; in the model group, the cells in the hippocampus were irregularly arranged; some neurons had pyknotic nuclei and a small number of microglia phagocytosed necrotic neurons. Even light red plaques can be seen in some areas (Figure 3). Both betaine and minocycline improved the damage to hippocampal neurons to different degrees. The results suggest that betaine has a protective effect on the histomorphological damage of neurons in the hippocampus of mice with acute LPS-induced depression-like behavior.
LPS stimulation significantly increased the levels of M1 proinflammatory cytokines, including TNF-α, IL-1β, and IL-6 and decreased the level of the M2 anti-inflammatory cytokine IL-10. Compared with the model group, betaine significantly reduced the levels of the IL-1β, IL-6, IL-18, and TNF-α inflammatory factors and increased the amount of IL-10 in the hippocampus of LPS-treated mice (Figures 4(a)–4(e)). The results suggest that betaine can inhibit the production of proinflammatory factors in the brains of mice induced by LPS while promoting the production of anti-inflammatory factors.
The level of corticosterone in the hippocampus of mice was determined by ELISA, and the content of C-reactive protein in the serum and hippocampus was determined (Figures 5(a)–5(c)). The results showed that the levels of corticosterone and C-reactive protein in LPS-induced mice increased, and betaine significantly reduced the levels of serum C-reactive protein (CRP) and corticosterone (CORT) in LPS-induced mice.
To determine whether the protective effect of betaine on lipopolysaccharide-stimulated microglia is related to polarization, we performed an immunohistochemistry. The M1 polarization marker COX-2 and the M2 polarization marker CD206 were labeled by immunohistochemistry in mouse brain regions (Figures 6(a) and 6(c)), and it was found that LPS could induce an increase in M1 polarization markers in mice (Figures 6(b) and 6(d)). Betaine and minocycline can reduce the expression of COX-2 to varying degrees and increase the expression of CD206, so it is suggested that betaine can promote the transformation of microglia from the M1 phenotype to the M2 phenotype.
To determine whether the protective effect of betaine on lipopolysaccharide-stimulated microglia is related to NLRP3, we used an immunofluorescence assay. Iba1 is a microglial marker whose expression is positively correlated with microglial activation (Figure 7(a)), and the fluorescence intensity of Iba1 indicates the degree of microglial activation. The results showed that the expression of NLRP3 in the brain region of mice induced by LPS increased, the degree of activation of microglia increased, and the expression of NLRP3 in the brain region of mice treated with betaine decreased to varying degrees (Figures 7(b)–7(d)).
TLR4 is the recognition receptor of LPS, while Myd88 is the junction protein of the LPS-induced inflammatory process. We found that both TLR4 and Myd88 levels were elevated in LPS-induced mice, and betaine administration reduced the expression of TLR4 and Myd88. NF-κB induces NLRP3 mRNA expression, which is required for the formation of the inflammasome complex, so we determined the expression of NF-κB and NLRP3 related proteins by western blotting (Figures 8(b) and 8(c)). The results showed that the expression of NF-κB and NLRP3 related proteins was upregulated in LPS-induced mice, and the related protein expression of NF-κB and NLRP3 was decreased by the administration of betaine. To observe the polarization of microglial cells, we detected INOS and COX-2 (Figure 8(a)), the polarization markers of microglial M1 and CD206, and the polarization marker of M2. The level of microglial polarization was observed to be biased toward the M2 phenotype.
Depression is one of the most important mental illnesses and is closely related to inflammation. Under stress or pathological conditions, microglia secrete inflammatory mediators that disrupt the neuronal function and impair neurogenesis, increasing the vulnerability to stress and promoting the occurrence and development of depression [26, 27]. Betaine is a natural product with an anti-inflammatory and antioxidant activities. Previous studies have shown that betaine is mainly involved in the synthesis and metabolism of central monoamine transmitters as an osmotic regulator and methyl donor, thus playing an auxiliary role in the clinical treatment of depression [19, 28]. Betaine alone also has antidepressant-like effects [29, 30]. However, the mechanism by which betaine improves lipopolysaccharide-induced depression-like behavior is unclear. Therefore, we investigated the neuroprotective effect of betaine on LPS-induced depression-like behavior in mice and its mechanism of action. With research advances in recent years, the important role of immune inflammation in the pathology of depression has gradually emerged [31–33]. The LPS-induced depression model is one of the classic acute depression models. As an immune stimulator, LPS can initiate the body's innate immune response, induce the secretion of various inflammatory cytokines, and further induce the central nervous system's immune response. This extensive immune inflammation can eventually lead to a series of depressive-like symptoms, such as anhedonia and reduced mobility [34]. Therefore, in this study, a model of acute inflammation induced by LPS was constructed. In our study, it was found that compared with the LPS model group, the betaine group could alleviate LPS-induced depression-like behaviors, such as weight loss and decreased food intake, 24 hours after LPS injection. In our previous preliminary experiments, it was found that the dose of 5% betaine was the best effective dose. It was also confirmed in behavioral experiments, and it was found that high-dose betaine had a more effective inhibitory effect on LPS-induced depression-like behavior. In many experimental animal models, minocycline has significant efficacy in attenuating central nervous system- (CNS-) induced neuroinflammation and depressive behavior and preventing lipopolysaccharide-induced cognitive impairment in mice [35]. Previous studies have reported that minocycline is a second-generation tetracycline that selectively inhibits microglial activation, and M1ibit activation of LPS regulates the polarization of microglia to the M2 phenotype and exert an anti-inflammatory effect. In the present experiment, minocycline had a protective effect on LPS-induced depression-like behavior in mice, and minocycline inhibited M1 microglial activation to protect against inflammation, which was in line with numerous studies. The mechanism by which inflammation causes depressive-like behaviors likely involves one or more inflammatory molecules, such as C-reactive protein (CRP) or prostaglandin E2 (PGE2) and the hypothalamus–pituitary–adrenal (HPA) axis [36]. CRP is an acute phase response protein produced by the body after being stimulated by stress, and it is the most sensitive marker of the systemic inflammatory response. Under normal circumstances, the content is very small, and its blood concentration rises sharply during acute trauma and infection. We measured the content of CRP in the serum and hippocampus of different groups of mice 24 hours after LPS injection and found that betaine can reduce the expression level of CRP in mice and alleviate the inflammatory response of mice induced by LPS injection. In terms of signaling, it is now recognized that the two most important systems involved in the stress response are the sympathetic branch of the autonomic nervous system and the hypothalamic–pituitary–adrenal (HPA) axis [37]. LPS induces oxidative stress and the release of proinflammatory factors, leading to corticotropin-releasing hormone (CCH), elevated serum corticosterone, hypothalamic–pituitary–adrenal axis dysfunction, and depression-like behaviors in animals. In this study, it was found that the CORT concentration in the hippocampus of the LPS model group mice increased, depression behaviors were increased, and these behaviors were reversed by betaine, which is consistent with the positive drug minocycline study results. Thus, these findings and the results of this experiment suggest that betaine reduces CORT concentrations and improves depressive-like behavior. These results further confirm that betaine has an anti-inflammatory activity, possibly related to antidepressant-like effects. Neuroinflammation is a crucial pathological basis for depression, and various classical antidepressant treatments can improve neuroinflammation and exert antidepressant effects. Persistent inflammation accompanied by elevated levels of proinflammatory cytokines can lead to depressive symptoms [38], so the overproduction of proinflammatory cytokines plays a key role in the development and progression of psychiatric disorders. Proinflammatory cytokines, including interleukin-6 (IL-6) and tumor necrosis factor-alpha (TNF-α), have been found to be higher in patients with major depressive disorder [39], while their antioxidant capacity is lower [40]. Previous in vivo studies have reported that the inhibition of proinflammatory cytokines can alleviate depressive symptoms [41–43]. Furthermore, in mice, administration of IL-10 reduced the depression-like behaviors [44]. Therefore, modulating cytokine expression can alleviate depressive symptoms. In this experiment, betaine reduced the production of proinflammatory cytokines, including IL-6, IL-18, TNF-α, and IL-1β and increased the level of anti-inflammatory IL-10 compared with the LPS model group. Therefore, betaine can reduce LPS-induced systemic inflammation in mice. This is consistent with our expected results. Overproduction of inflammatory cytokines and activation of microglia in the brain is implicated in depression and dysfunction in brain signaling. The hippocampus is a key brain region responsible for learning and memory and is closely related to the occurrence and development of depression. Microglia have both resting and activated states; in a healthy state, MGs are resting, and during CNS injury due to infection, brain injury, or ischemic injury, the microglial transition from a resting state amoeba is activated [45] and releases a large number of neurotoxic substances, such as oxygen free radicals and proinflammatory factors, causing an inflammatory response in the central nervous system. In addition to the resting state, there are two functionally distinct activated states, M1 and M2 [46]. The M1 microglial phenotype exerts a proinflammatory effect, while the M2 phenotype is involved in the anti-inflammatory processes in the brain. The M1 phenotype can be induced by LPS with increased production of proinflammatory cytokines. Most compounds that reduce neuroinflammation inhibit only microglia of the M1 phenotype. Few compounds promote microglial polarization to the M2 phenotype [7]. Therefore, how to inhibit the excessive activation of microglia to reduce the damage caused by the inflammatory response is particularly important. In our previous study, it was demonstrated that betaine induces polarization of microglia toward the M2 phenotype and inhibits LPS-induced inflammatory responses by inhibiting the TLR4/NF-κB pathway. In our experiment, betaine significantly reduced the LPS-induced protein expression of iNOS and COX-2, which are related to M1 markers, in mouse hippocampal microglia and upregulated the protein expression of CD206, a M2 marker, in mouse hippocampal microglia. Therefore, betaine may modulate the polarized phenotype of microglia in LPS-induced depression model mice and induce the transformation of the microglial phenotype from M1 to M2 type. Recent evidence suggests that the causative agent of depression may be associated with NLRP3 [3, 14]. NLRP3 inflammasome is activated by signals from the TLR4/MyD88/NF-κB pathway that upregulates NLRP3 and pro-IL-1β, and from DAMPs/PAMPs like ROS and ATP that recruit ASC and caspase-1 to promote inflammasome assembly. The complex then synthesizes and secretes IL-1β, which triggers an inflammatory cascade [47–49]. Zhang et al. found that CUMS activated the NLRP3 inflammasome and increased IL-1β content in rat hippocampus, and administering the NLRP3 inhibitor VX765 significantly alleviated the neuroinflammation and depression-like behavior in the animals [50]. This is consistent with our findings. We demonstrate that betaine can significantly reduce the expression of TLR4/MyD88/NF-κ pathway signaling, and betaine can reduce the expression of NLRP3 and the levels of IL-1β and caspase-1 compared with the LPS model group. Therefore, betaine may attenuate LPS-induced depression-like behavior by inhibiting M1 microglia polarization and promote M2 microglia polarization by inhibiting NLRP3 signaling. Our findings confirmed that betaine can reduce the expression of TLR4/MyD88/NF-κB and polarize microglia toward the M2 phenotype by modulating the NLRP3 signaling pathway, thereby attenuating depression-like behaviors in LPS model mice. These results reveal the important role of the NLRP3 inflammasome in microglial activation and provide new ideas for the clinical application of betaine.
Betaine can effectively alleviate LPS-induced depression-like behavior in mice, which may regulate the transition from the M1 to M2 phenotype of microglial cells by inhibiting the overactivation of NLRP3 inflammasome. | true | true | true |
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PMC9630107 | 36217026 | Sara G. Pelaz,Arantxa Tabernero | Src: coordinating metabolism in cancer | 10-10-2022 | Cancer metabolism,Oncogenes | Metabolism must be tightly regulated to fulfil the dynamic requirements of cancer cells during proliferation, migration, stemness and differentiation. Src is a node of several signals involved in many of these biological processes, and it is also an important regulator of cell metabolism. Glucose uptake, glycolysis, the pentose-phosphate pathway and oxidative phosphorylation are among the metabolic pathways that can be regulated by Src. Therefore, this oncoprotein is in an excellent position to coordinate and finely tune cell metabolism to fuel the different cancer cell activities. Here, we provide an up-to-date summary of recent progress made in determining the role of Src in glucose metabolism as well as the link of this role with cancer cell metabolic plasticity and tumour progression. We also discuss the opportunities and challenges facing this field. | Src: coordinating metabolism in cancer
Metabolism must be tightly regulated to fulfil the dynamic requirements of cancer cells during proliferation, migration, stemness and differentiation. Src is a node of several signals involved in many of these biological processes, and it is also an important regulator of cell metabolism. Glucose uptake, glycolysis, the pentose-phosphate pathway and oxidative phosphorylation are among the metabolic pathways that can be regulated by Src. Therefore, this oncoprotein is in an excellent position to coordinate and finely tune cell metabolism to fuel the different cancer cell activities. Here, we provide an up-to-date summary of recent progress made in determining the role of Src in glucose metabolism as well as the link of this role with cancer cell metabolic plasticity and tumour progression. We also discuss the opportunities and challenges facing this field.
The hallmarks of cancer—shared commonalities that unite all types of cancer cells at the level of cellular phenotype—have been recently updated [1]. They comprise the acquired capabilities for sustaining proliferative signalling, evading growth suppressors, resisting cell death, enabling replicative immortality, inducing/accessing vasculature, activating invasion and metastasis, avoiding immune destruction and deregulating cellular metabolism [1]. Src, one of the best studied oncoproteins, has been shown to regulate these hallmarks that ultimately control the behaviour of transformed cells and contribute to tumour progression and metastasis [2]. Excellent reviews with comprehensive information about Src substrates and the broad spectrum of cellular events regulated by this kinase were published during those years in which an exponential growth of the research in the field took place [3, 4]. However, the effects of Src on metabolism and their relevance on cell transformation was not yet uncovered at the time. Importantly, “deregulating cellular metabolism” has been recently included as a core hallmark of cancer [1]. Because of the extensive number of studies showing a role of Src in metabolism, in this review we will summarise the effects of Src on glucose metabolism, and discuss their contribution to Src oncogenic activity and the related therapeutic opportunities. Before addressing this topic, we will introduce Src family kinases, their structural properties, as well as the regulation of their activity and oncogenic properties. We will focus on Src, the best studied and prototypical member of the family and we will refer to other Src family members when necessary.
The seminal discovery that the transforming element of the Rous sarcoma virus (v-src) in chickens was a transduced form of the cellular gene c-src gave rise to the identification of the first oncogene and proto-oncogene [5]. c-Src (herein termed Src) is the founding member of the Src family of non-receptor protein tyrosine kinases (SFKs), which are key regulators of signal transduction implicated in fundamental cellular processes, many of them related to human cancers [6]. SFKs include Src, Fyn, Yes, Lck, Hck, Blk, Lyn, Fgr and Frk. Src, Fyn and Yes are ubiquitously expressed, while Lyn, Hck, Fgr, Blk and Lck are predominantly and differentially expressed in the various cell types of the haematopoietic lineage. Although some unique functions have been reported for some SFK members, extensive functional redundancy among SFKs exits in different cell types [7, 8]. SFKs share a conserved domain structure (Fig. 1A–C) composed of the SH4 region, which contains the lipidation site (mainly myristylation site) for membrane localisation; a unique domain characteristic of each individual kinase; the SH3 domain, which binds proline-rich sequences; the SH2 domain, which binds phosphotyrosine-containing sequences; the SH1 domain, which is the catalytic kinase domain and contains the substrate- and the ATP- binding site, as well as the autophosphorylation site (Tyr416 in chicken Src, the most used terminology because of its discovery and Tyr419 in human Src); and a short C-terminal tail, which contains the negative-regulatory tyrosine residue (Tyr527 in chicken Src and Tyr530 in human Src). All family members show extensive sequence homology in the SH1, SH2, and SH3 domains and in the SH4 region, but diverge in the unique domain (reviewed in [9]). SFKs have multiple regulatory mechanisms, which converge on tyrosine phosphorylation at two sites – residues Tyr416 and Tyr527 for Src and nearby for other SFK members—with opposing effects. Because most of the regulatory mechanisms are shared by SFKs, we will refer to Src as the prototypical member of this family. Phosphorylation of Tyr416, located within the activation loop of the kinase domain (SH1), activates the enzyme while phosphorylation of Tyr527, located within the C-terminal tail, inhibits enzymatic activity (Fig. 1D). In resting cells, Src is maintained in an inactive conformation, in which intramolecular contacts of the SH2 domain with phosphorylated Tyr527 and that of the SH3 domain with the proline rich region (Fig. 1A) cause an assembled or closed state (Fig. 1D). The oncogenic v-Src protein lacks the inhibitory Tyr527 site and that is why it is constitutively active and highly transforming. Thus, although v-Src and c-Src are proposed to have the same substrates, the lack of regulation in v-Src causes the permanent activation and the subsequent neoplastic transformation, while c-Src is not transforming unless mutated, overexpressed or overactivated. Src is activated in response to a great diversity of extracellular signals via integrins [10], G-protein-linked receptors [11], steroid receptors [12], and receptor tyrosine kinases (RTKs) [13], such as platelet-derived growth factor receptor (PDGFR), epidermal growth factor receptor (EGF-R) family, fibroblast growth factor receptor (FGF-R), insulin-like growth factor-1 receptor (IGF-1R), c-Met, colony-stimulating factor-1 receptor (CSF-1R) and stem cell factor receptor (SCF-R), among others [13]. Although the participation of some intermediates, such as Ral-GTPase, has been reported [14], the aforementioned receptors compete for binding to Src SH2 or SH3 domains and disrupt the intramolecular interactions, allowing for Src kinase activation. For instance, PDGFR binds to Src SH2 domain while ß3 subunit of integrins binds to Src SH3 domains (reviewed in [15]). The disassembly of intramolecular contacts allows autophosphorylation at Tyr416 [16] and dephosphorylation at Tyr527, which can be catalysed by the receptor protein tyrosine phosphatase α (PTPRA) [17], the nonreceptor tyrosine phosphatase SHP-1 (PTPN6) [18] or PTP1B [19]. Src adopts an open conformation in which the Src SH3 domain interacts with the proline rich region and the Src SH2 domain with the phosphotyrosine site, both at the target substrate. The Src kinase domain is then in the competent position for the phosphorylation of the specific tyrosine within the substrate (Fig. 1D). The inhibition of Src requires the activity of the C-terminal Src kinase (CSK) [20] or CSK homolog kinase (Chk/MATK) [21], which phosphorylate Src at Tyr527. In addition, several phosphatases, such as phosphatase and tensin homolog (PTEN), have been shown to dephosphorylate Src at Tyr416 [22, 23], a step required to complete the inactivation and intramolecular assembly of Src (Fig. 1D). Whereas Src is a membrane-associated protein, CSK does not contain a membrane-binding motif and requires a membrane-anchor protein, such as the CSK binding protein or phosphoprotein associated with glycosphingolipid-enriched microdomains (Cbp/PAG). The interaction of CSK to Cbp/PAG is required for CSK to catalyse the phosphorylation of Src at Tyr527 causing Src inhibition. Interestingly, other membrane-associated proteins, such as caveolin-1 [24, 25], paxillin [26] and connexin43 [27] have the ability to recruit CSK and Src, favouring Src inhibition. Connexin43 can recruit CSK together with PTEN, which allows a cooperative and complete inhibition of Src [27]. This cooperative mechanism is also found in haematopoietic cells, where CSK interacts with the phosphatase encoded by PTPN22, which dephosphorylates Tyr394 of Lck and Tyr417 of Fyn (the equivalent to Tyr416 in Src) to inhibit Lck and Fyn activity concomitantly with CSK [28]. Furthermore, phosphorylation of Cbp/PAG facilitates the binding and activity of CSK. The protein-tyrosine phosphatase SHP-2 (PTPN-11) prevents Src inhibition by removing the phosphorylation of Cbp/PAG, thereby inhibiting CSK recruitment and access to Src [29]. On top of that, other mechanisms, such as the oxidation of key cysteine residues within the Src protein, can contribute to the regulation of Src activity [30, 31]. In brief, the regulation of Src activity is a complex process that involves a dynamic conformational transition in which the crosstalk of multiple signalling molecules takes part.
The relevance of Src activity can be inferred by the presence of src genes across the whole range of metazoan evolution [3]. Src catalyses tyrosine phosphorylation at specific positions in a wide variety of proteins, regulating their activity. Among the proteins that have been found proposed to be Src substrates are those receptors mentioned previously that activate Src activity, as they can also be reciprocally activated by Src; transcription factors, such as Stat3 [32]; adaptor proteins, such as Shc [33], which leads to the subsequent activation of the Ras/Raf/Erk signalling cascade; other kinases, such as phosphoinositide 3-kinases (PI3Ks) [34], MAPK [34] or Akt [35]; channel proteins involved in cell communication, such as connexin43 [36] or pannexin1 [37]; cytoskeleton components such as FAK, p130 CAS, cortactin, paxillin or p190 Rho-GTPase-activating protein (GAP) (for a review, see [38]), and key metabolic enzymes that will be described in detail the following sections. As a consequence of these direct phosphorylations, extensive proteins and signalling pathways can be secondarily affected, including cyclin D1 or HIF-1ɑ [39]. Because Src functions as both effector and regulator of a plethora of receptors, this kinase facilitates the crosstalk between different signalling pathways. Src is, therefore, a node of communication in a complex network of interacting proteins [40], which can regulate many cellular events, including proliferation, differentiation, survival, migration, cytoskeletal organisation, adhesion, cell communication, stemness and metabolism. Despite the high diversity of Src effectors, it is important to keep in mind that Src activity can be used differently by individual extracellular stimuli, contributing to their ability to generate unique cellular responses in a context-dependent manner [14].
Since the seminal discovery of the transforming ability of v-Src, the role of Src in cancer has been extensively studied: Src is the SFK that is most often implicated in cancer. Indeed, although mutations in Src are a rare event, both overexpression and overactivation of Src have been observed in numerous cancer types, including those of the brain, mainly glioblastoma (GBM), as well as cancer of the liver, lung, colon, breast, bladder and pancreas, contributing to their malignancy grade (Fig. 2, reviewed in [41]). The increased Src activity found in cancer cells can be caused by multiple factors, including an enhanced expression of Src activators, frequently found in cancer, such as integrins [10], EGFR [14, 42], the constitutively active mutant form EGFRvIII [43], HER2 or ErbB2 [44] or other RTKs. Alternatively or concomitantly, downregulation of CSK [45], upregulation of SHP-2 or alterations in other Src regulatory molecules can contribute to the increased Src activity found in many cancers [46]. As described previously, Src integrates and regulates receptor signalling and directly transduces it to downstream effectors affecting many cellular events related to cell transformation, including metabolism, proliferation, differentiation, apoptosis, cell adhesion, migration, invasion, stemness and metastasis. Src may also play a prominent role in the tumour microenvironment, by inducing angiogenesis [43] or immune evasion [47]. Definitely, the study of Src activity and its target proteins will help to understand the biology of cancer, as well as its diagnosis and prognosis. For instance, the detection of site-specific phosphorylation levels of Src target proteins in peripheral circulating exosomes might be informative in cancer diagnosis and/or prognosis.
Cancer stem cells (CSCs) or tumour-initiating cells are a subpopulation of undifferentiated tumour cells with distinct stem cell-like features, such as self-renewal and phenotypic plasticity, an emerging cancer hallmark [1]. CSCs are involved in metastasis, tumour recurrence and are highly resistant to conventional therapies [48]. Although multiple signalling pathways participate in stemness, Src activity appears as a key contributor [49]. Indeed, the cancer stem cell marker, CD133, can interact and activate Src, which through the phosphorylation of FAK contributes to cancer stem cell migration [50]. At least two signalling pathways responsible for maintaining the stemness are related to Src activity in non-small cell lung cancer cells. Tescalin mediates the mutual activation of Src and IGF1R, which results in Stat3 activation and stemness [51], while EGFR/Src/Akt signalling modulates Sox2 expression and self-renewal [52]. In fact, the inhibition of Src activity with Dasatinib and PP2 reduces the clonogenic, self-renewal, and tumour-initiating capacity of pancreatic cancer stem cells [53]. Similarly, the inhibition of Src with Dasatinib, Saracatinib, PP2 or TAT-Cx43266-283 promotes a reduction in the expression of the inhibitor of differentiation-1 (Id1) and Sox2, with the subsequent reversion of stemness in human glioblastoma stem cells [54]. An important Src-regulated feature of CSCs is their metabolic plasticity, which allows them to survive in the ever-changing tumour microenvironment by conveniently shifting between different metabolic pathways used in energy production and catabolism [55]. These data, together with many more studies on this field, suggest that Src participates in stemness through several mechanisms triggered by a variety of signals present in different types of tumours. Therefore, maintaining the stemness should be considered as an important outcome of the high activity of Src frequently found in cancer.
The transformed metabolic phenotype found in many tumours described in the late 1920s by Getty and Carl Cori [56, 57] and Otto Warburg [58, 59] included increased glucose uptake and metabolism. Glucose metabolism can be anabolic as well as catabolic, providing fuel and precursors for many if not most cell activities. To allow for the necessarily flexible and fine-tuned regulation of glucose metabolism, the implicated enzymes exhibit many levels of regulation, such as selective tissue expression, substrate affinity and specificity, modulable enzyme kinetics, subcellular localisation, and post-transcriptional modifications. Although beautiful, this intricacy can complicate the interpretation and generalisation of experimental results. Sound evidence of Src-mediated modification of glycolysis arose very early in the history of Src research. A series of seminal works in the 1970s showed that cell transformation by Rous sarcoma virus (i.e., Src activity) induced a marked increase in glycolytic activity [60–62], mimicking the transformed metabolic phenotype found in many tumours. Since then, Src has been found to modulate glycolysis via different mechanisms, including regulation of master glycolytic transcription factors (HIF-1ɑ [63, 64] and MYC [65]), insulin secretion [66, 67], modulation of glycolytic enzyme activity by phosphorylation (HK, PFKFB3, G6PD), and through well-known Src substrates lying at the heart of energy metabolism and cancer, such as the PI3K-AKT-mTOR axis [68–70] and EGFR [71]. This section discusses many instances of mostly direct regulation of glycolysis by Src, one glycolytic protein at a time (see Fig. 3 for an overview).
Glucose is imported into the cell by glucose transporters (GLUTs), of which there are 14 isoforms in humans [72]. Two isoforms are the most relevant and frequently overexpressed in cancer [73]. GLUT-1 (SLC2A1), the most ubiquitous and abundant isoform [74], and GLUT-3 (SLC2A3), the ‘neuronal’ isoform, which shows the highest affinity for glucose [75]. In the 1980s, early work linked higher glycolysis rates induced by v-Src transformation (the constitutively active form of Src) to increased levels of GLUT-1 mRNA and protein, although the underlying mechanism(s) remained unknown [76, 77]. Since then, Src activity has been shown to regulate GLUT expression through two transcription factors: in astrocytes (nervous system cells) and GBM cells (their ‘tumoral counterpart’), Src activity can modulate the levels of GLUT-1 and GLUT-3 through HIF-1α protein levels [55, 78]; in breast cancer cells, Src inhibition can decrease MYC expression resulting in decreased GLUT-1 mRNA and protein levels [79].
Intracellular glucose is phosphorylated by hexokinases (HKs) to glucose 6-phosphate (G6P, which is trapped into the cell), the first rate-limiting step of glycolysis (Fig. 3). Functionally, Src activity is known to regulate the expression and activity of hexokinases, most notably the major 1 and 2 isoforms (HK-1 and HK-2), in healthy as well as cancerous cells [55, 78–80], yet it was not until recently that a direct activation of HK-1 and HK-2 by Src phosphorylation was reported [81]. Phosphorylation of HK-1 and HK-2 by Src on residues Y732 and Y686, respectively, enhanced glycolysis and efficiently stimulated their catalytic activities, in the case of HK-1 by enhanced enzyme kinetics (including initial velocity [VO], maximum velocity [Vm] and substrate affinity [Km]) and changes in oligomerization status. In the context of cancer, the authors showed that Src-stimulated tumorigenesis and metastasis was dependent on HK activity using different approaches, such as mutations in Src phosphorylation site of either HK1 or HK2 and hexokinase silencing in xenograft mouse models [81]. In agreement with these results, decreased levels of GLUT-3, HK-2 and tumorigenicity in GBM mouse models have been found upon Src inhibition [55, 82]. Finally, hexokinase IV (better known as glucokinase) has also been proven to change its activity and subcellular localisation in response to Src downregulation in insulin-producing cells [67].
The resulting product of glucose phosphorylation, G6P, is isomerised to yield fructose-6-phosphate (F6P), but the specific rate-limiting step in the glycolytic pathway is the irreversible phosphorylation of F6P to fructose-1,6-bisphosphate (F-1,6-BP) by phosphofructokinase-1 (PFK-1). PFK-1 is allosterically activated by a different derivative of F6P, namely fructose-2,6-bisphosphate (F-2,6-BP), produced by 6-phosphofructo-2-kinase/fructose 2,6-bisphosphatase (PFK-2 or PFKFB). Among the four currently identified PFKFB isoforms (termed PFKFB1 to 4), PFKFB3 is the strongest glycolysis-inducer and is upregulated in many human cancers [83]. Intriguingly, PFKFB activity was found to be modulated by Src more than 35 years ago [84]. Although the authors noted the possibility that PFKFB phosphorylation by a tyrosine kinase (e.g., Src) could be the underlying mechanism, they eventually proposed an indirect mechanism involving PKC and transcriptional activity [85]. It has been reported thereafter that the absence of bisphosphatase activity strongly suggests that PFKFB3 was the predominant isoform in these samples [86]. Recently, the same group that established the HK regulation by Src identified PFKFB3 as a potential Src interaction candidate by mass-spectrometry screening [87]. They subsequently found that the N-terminal domain of PFKFB3 interacts with the SH1 domain of Src, resulting in Src phosphorylation of PFKFB3 at Tyr194. This modification induced increased cell glucose uptake, glycolysis and pentose phosphate pathway activity compared to baseline (inactive Src) and to PFKFB3-Tyr194Phe phospho-deficient cells. As expected, the PFKFB3-Tyr194Phe mutation impaired proliferation, migration, and xenograft formation. In vivo, PFKFB3-Tyr194Phe knock-in mice exhibited decreased glycolysis and attenuated spontaneous tumorigenicity. Furthermore, the levels of active (i.e., phosphorylated) PFKFB3 and Src were found to correlate in clinical tumour samples [87].
Pyruvate kinase (PK), contrary to what its name suggests, catalyses the conversion of phosphoenolpyruvate (PEP) into pyruvate. As with other glycolytic enzymes, the PK isoenzyme PKM2 (the ‘muscle’ isoform, also expressed in many other cell types) was promptly identified as a Src substrate in the 1980s, but, in contrast to most other glycolytic enzymes, PKM2 phosphorylation by v-Src led to decreased affinity for PEP and more rapid ATP-mediated inactivation; in other words, Src phosphorylation inhibited PKM2 activity [88, 89]. Indeed, PKM2 is found mainly as a tetramer in healthy cells, which is the active PKM2 conformation and funnels glucose to pyruvate for energy production via mitochondrial oxidation. However, in cancer cells most PKM2 is found as a dimer—the so-called ‘tumour’ PKM2—due to (among other mechanisms) [90] phosphorylation mediated by oncoprotein kinases such as Src [88, 89, 91]. The tumour PKM2 dimer exhibits inhibited PKM2 enzyme activity, shunting glycolytic intermediaries towards anabolic processes to support rapid cell proliferation by a process that can be induced by Src phosphorylation of PKM2 [92, 93].
Lactate dehydrogenase (LDH) catalyses both the reduction of pyruvate into lactate (LDH isoform A, LDHA), and the oxidation of lactate into pyruvate (LDH isoform B, LDHB). LDH, as well as enolase (discussed below) and phosphoglycerate mutase (PMu), was identified as a v-Src substrate in the early 1980s [94, 95]. These authors established that LDH was phosphorylated at Tyr238 by Src (RSV infection) in chick embryo cells (a common system at the time), although its functional impact remains to be explored. In addition, a 2017 report found that Src – as well as HER2 – phosphorylated LDHA at Tyr10 inducing a more active tetramer conformation that provided anti-anoikis (a form of anchorage-dependent cell death) and pro-invasive and metastatic advantages to breast cancer cells [96].
Two other glycolytic enzymes have been found to be Src substrates, namely glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and enolase. Although Src can phosphorylate GAPDH at Tyr41 [97], the outcome of this modification has been linked to vesicle trafficking [98] and the DNA damage response [99], not (yet) to glycolysis. As for enolase, in the early 1980s two different groups reported enolase phosphorylation by v-Src [94, 95, 100] and the mild enzyme kinetic effects ensuing this modification [100]. In spite of the prominent role of enolase in glycolysis and in several other cellular processes (such as tissue remodelling [101]), most of the literature linking Src and enolase refers to the use of the latter as a substrate in Src kinase activity assays [102]. Given the relevance of both GAPDH [103] and enolase activity in cancer [104], we are left to wait and wonder about whether and how Src phosphorylation might influence their role in cancer biology, as it has been clearly established for other glycolytic enzymes.
An alternative fate for G6P is the pentose phosphate pathway (PPP). This route metabolises G6P in two stages: first, an oxidative phase which produces nicotinamide adenine dinucleotide phosphate (NADPH), and second, a non-oxidative phase that yields carbohydrates. Therefore, the PPP produces NADPH, an intracellular antioxidant necessary for reductive biosynthesis, ribose-5-phosphate, for nucleic acid synthesis, and erythrose 4-phosphate, for aromatic amino acid synthesis. Several studies have shown a link between Src or Fyn activity and the regulation of PPP in contexts other than cancer [105–108]. In endothelial cells, an initial report proposed that G6PD phosphorylation by Src at residues Tyr428 and Tyr507 might modulate G6PD activity [107]. In cancer cells, the group of Li et al., who discovered the direct regulation of HK and PFKFB3 by Src, found that Src phosphorylation of G6PD at Tyr112 induces kinetic changes that increase G6PD catalytic activity and PPP flux [109]. As a result, mice injected with mutant Tyr112Phe-G6PD colorectal cancer cells developed significantly smaller tumours than their G6PD wild-type counterpart. Importantly, in clinical colorectal samples, Src and G6PD abundance and activity (probed by phosphorylation levels) were found to correlate [109]. In glioma cells, upon EGFR activation, Fyn is activated causing the phosphorylation of G6PD at Tyr481, leading to enhanced PPP activity, tumour growth and radiation resistance. Indeed, in human glioblastoma patients, the phosphorylation of 6-phosphogluconate dehydrogenase, another PPP enzyme frequently upregulated in cancer cells, at Tyr481 by Fyn is associated with increased Fyn expression and with reduced survival and worse prognosis [110]. Overall, these studies indicate that at least Src and Fyn can phosphorylate PPP enzymes at different tyrosine residues subsequently increasing PPP activity (Fig. 3), which fuels DNA replication and cell death resistance contributing to tumour malignancy.
In the early 2000s, accruing evidence prompted the notion that mitochondrial function was regulated by protein phosphorylation, as it was being found for other organelles. Soon, attention was directed towards tyrosine phosphorylation specifically, and Abl and Src were the first tyrosine kinases demonstrated to exhibit mitochondrial localisation and activity [111–113], despite lacking canonical mitochondrial targeting sequences. Physiological regulators of Src were also promptly detected in mitochondria, including CSK [112], SHP-2 [114, 115] and PTP1B [115, 116], and even a new activator of Src was discovered, PTPD1 [117] (Fig. 4). Reported Src substrates in the mitochondria include, most notably, several complexes of the electron transport chain (ETC) [118], such as complexes I, III, IV and V [113, 115, 119–121]. Importantly, pyruvate dehydrogenase (PDH) was also described as a Src substrate [122] (Fig. 4). PDH is part of the pyruvate dehydrogenase complex, which converts pyruvate to acetyl-CoA in the mitochondria and therefore is the first step in the mitochondrial metabolism of glucose. PDH activity can be regulated through serine phosphorylation by pyruvate dehydrogenase kinases and, as reported, through tyrosine phosphorylation by Src [122]. More recently, a proximity-dependent biotin-tagging system was used to study the endogenous interactome of mitochondrial Src (mtSrc) [123]. This has led to the identification of over 50 mtSrc-interacting candidate proteins involved in different aspects of mitochondrial biology. Initially, studies showed that an increase in mtSrc activity can lead to an increase in complex I, III and IV activity along with a decrease in complex V activity [115], and, accordingly, that a reduction in mtSrc activity can lead to a reduction in mitochondrial respiration through a decrease in complex I activity [119, 120]. Interestingly, later reports showed opposite effects of Src and mtSrc activity in mitochondrial respiration. When Src was overexpressed – in a cell-wide manner – in mouse embryonic fibroblasts (MEFs), increased phosphorylated Src levels and decreased levels of ETC complexes I and IV were found compared to wild-type MEFs, with unchanged cell proliferation [124]. Conversely, when Src was silenced in wild-type MEFs, or when Src activity was inhibited in Src overexpressing MEFs with PP2 or siRNA, the levels of ETC complexes I and IV increased compared to control conditions. Moreover, MEFs harbouring null mutations in both alleles of Src, Yes and Fyn showed decreased cell proliferation, increased ETC complex I and IV levels and increased ETC complex I/III and IV activity [124]. In the same report, metastatic liver cancer samples showed higher Src phosphorylation and decreased ETC complex levels (except for complex V) when compared to healthy liver samples, and Src inhibition with PP2 or siRNA in a liver cancer cell line led to increased ETC complex I and IV levels [124]. In triple negative breast cancer (TNBC), Park et al., using established cell lines as well as patient-derived xenografts, showed that mitochondrial fatty acid oxidase (FAO) activates mtSrc, which stimulates ETC complex activity, thereby providing ATP to maintain the Src activating phosphorylation [125]. However, this pro-tumoral mtSrc activity might be restricted to a ‘Goldilocks’ zone, as Djeungoue-Petga et al. reported that overexpression of mtSrc led to decreased mitochondrial membrane potential and respiration and cell death in several TNBC cell lines [126]. As previously mentioned, Src can regulate PDH activity, the initial step in the mitochondrial metabolism of glucose. Indeed, Src directly phosphorylates PDH on Tyr289 causing decreased PDH activity and ROS production in vitro and decreasing metastatic burden in vivo [122]. Importantly, these authors showed that combinatorial therapy consisting of Src inhibition and pro-oxidative agents had a synergistic anti-proliferative effect on breast cancer cells [122]. Mitophagy, or mitochondrial autophagy, is a process by which defective or unwanted mitochondria are selectively targeted for autophagy and degraded [127]. Several proteins, many of them susceptible to regulation by phosphorylation, finely regulate mitophagy in response to environmental as well as cell-intrinsic cues, including FUNDC1, which does so in response to cell stress such as hypoxia or mitochondrial depolarisation [127]. Under unstressed physiological conditions, FUNDC1-mediated mitophagy is inhibited by phosphorylation at Tyr18 by Src. Under hypoxia, FUNDC1 is dephosphorylated resulting in mitophagy induction. However, when Src is present, FUNDC1 phosphorylation is preserved, inhibiting FUNDC1-mediated mitophagy in response to hypoxia [128]. Nonetheless, Src does not work alone in this duty. FUNDC1 has two phosphorylation sites, Ser13 (phosphorylated by casein kinase-2) and Tyr18 (phosphorylated by Src), that functionally cooperate to regulate mitophagy. Indeed, inactivation of either Src or casein kinase-2 alone is not sufficient to activate mitophagy, while inhibition of both kinases strongly activates FUNDC1-mediated mitophagy [129]. In brief, several reports positively correlate Src activity with mitochondrial metabolism [115, 119, 120, 125] while others show the opposite trend [122, 124, 126]. Interestingly, in all cases Src activity is related with a pro-tumoral role. Several processes could underlie these a priori contradictory reports. Beyond experimental differences, such as different subcellular localisation of Src overexpression, physiological explanations across cell types might include differences in metabolic plasticity – the ability to switch metabolic pathways in response to intrinsic or extrinsic changes to maximise survival and proliferation – or differential expression of other SFKs leading to compensatory activity upon Src inhibition. Importantly, the presence and activity of different Src interacting partners, a factor often overlooked and/or understudied, might contribute greatly to the outcome of Src inhibition. In the 2020 s, the group of E. Hébert-Chatelain has added extensive and crucial information to understand the role of Src in mitochondrial and cell metabolism using omics technologies [123, 130]. They examined the mitochondrial phosphoproteome and metabolome in Src + /+ and Src−/− mice either fed ad libitum or after fasting for 24 h. Src deletion led to impaired ETC activity and mitochondrial metabolism in several organs, along with accumulation of glucose and mitochondrial metabolites. These authors also found changes in the mitochondrial phosphoproteome and heterogenous ETC activity, oxygen consumption and metabolite abundance depending on Src phenotype and feeding state [130]. Another important contribution from this group, that supports further research in this field, is the identification of 51 candidate proteins of the mtSrc interactome involved in the tricarboxylic acid cycle, OXPHOS, cristae biology, fatty acid and amino acid metabolism and mitochondrial organisation and transport [123], highlighting the role of Src activity in the regulation of mitochondrial metabolism.
Because of the prominent role of Src in cancer, several Src inhibitors have been studied in preclinical models and some of them have successfully reached clinical use. Among them, ATP-competitive Src inhibitors – Dasatinib, Saracatinib or Bosutinib – are the most extensively studied in preclinical models and clinical trials and the use of some of them, such as Dasatinib, has been approved for hematologic tumours (recently reviewed in [131]). These inhibitors bind to the ATP-binding site in Src, which is highly conserved among tyrosine kinases. The lack of kinase specificity has been exploited to target simultaneously several oncogenic kinases, but it has also been associated with undesired side effects. Despite the good results in preclinical models, the results from clinical trials with ATP-competitive Src inhibitors, such as Dasatinib, Saracatinib or Bosutinib, alone or in combination, have been discouraging so far [131]. Most evidence suggests that higher specificity and ability to reach tumour cells, reduction in side effects and drug resistance mechanisms as well as finding predictive response biomarkers is required for successful clinical results [131]. Interestingly, new appealing strategies in the development of Src inhibitors are emerging. For instance, targeting the peptide substrate binding site instead of the ATP binding site within the kinase domain of Src (Fig. 1). Potential advantages include higher kinase inhibition selectivity due to the unique sequence of the peptide substrate site, and greater binding efficacy since the inhibitor will not need to compete with mM intracellular concentrations of ATP [132]. Indeed, Src inhibitors KX2-391 and KX2-361 target the peptide substrate site at nM potencies and have selectivity among tyrosine kinases. These peptide substrate-competitive Src inhibitors are progressing in clinical trials for advanced malignancies refractory to conventional treatments (KX2-361; https://clinicaltrials.gov/ct2/show/NCT02326441) and for topical treatment against actinic keratosis (KX2-391; https://clinicaltrials.gov/ct2/show/NCT02838628). Drug-resistance is another obstacle for a successful clinical outcome with Src inhibitors. In a recent study an interesting drug-resistance mechanism developed by ATP-competitive inhibitors has been revealed [133]. The binding of ATP-competitive inhibitors to Src allosterically disassembles Src to the open state with higher propensity to form a complex with Src substrates. If inhibitor concentration is reduced or if cells acquire a drug-resistant mutation, Src substrates will be readily phosphorylated, activating the Src signalling pathway. Consequently, the activation of Src and its downstream phosphorylation cascade can be paradoxically induced by ATP-competitive inhibitors. To prevent the relief of Src autoinhibition promoted by classical Src inhibitors, Temps et al. have developed the Src inhibitor, eCF506, which binds and locks the closed inactive conformation of Src [134]. This mechanism has the advantage of inhibiting the kinase activity as well as the protein binding to SH3 and SH2 domains in Src. Indeed, eCF506 decreases Src activity and the phosphorylation of the Src-binding protein FAK, which contrasts with some studies reporting that ATP-competitive Src inhibitors may facilitate FAK binding to Src and the subsequent phosphorylation [133, 134]. Another approach employed to design Src inhibitors is the recapitulation of the cellular mechanism for Src inhibition. As described in Fig. 1, some proteins such as CBP or Cx43 recruit Src together with its endogenous inhibitors, which causes Src inhibition. The Cx43 mimetic peptide, TAT-Cx43266-283, acts as a docking platform for Src, CSK and PTEN and consequently inhibits Src activity [27]. TAT-Cx43266-283 inhibits the oncogenic activity of Src and exerts important anti-tumoral effects in several preclinical models of glioblastoma in vitro, ex vivo, and in vivo, including freshly removed surgical specimens from patients [54, 135]. Tumour cell proliferation, survival, migration, invasion, metabolic plasticity and autophagy are impaired by TAT-Cx43266-283 enhancing the survival of GBM-bearing mice [55, 82, 136]. One of the main advantages of the Src inhibitor TAT-Cx43266-283 is that its effects are specific for the glioma stem cell subpopulation, with no effects on healthy brain cells. The cell specificity may depend on the levels of Src activity in each cell type, since TAT-Cx43266-283 recruits the open and active conformation of Src. Indeed, the toxicity of TAT-Cx43266-283 for neurons and astrocytes is much lower than that exerted by the ATP-competitive Src inhibitor Dasatinib [82]. In addition, because TAT-Cx43266-283 promotes the transition from the open to the closed Src conformation, both the activity and the scaffolding properties of Src are impaired, as judged by the reduction in Src activity and FAK phosphorylation promoted by TAT-Cx43266-283 [135]. One important conclusion to be drawn from the studies performed with Src inhibitors is that the inhibition of Src, even when using the same inhibitor and resulting in a reduction in Src activity, can impact different signalling pathways in different cell types [137]. Because Src-mediated pathways can act both in co-operation or crosstalk with other signalling pathways, these results suggest that ultimately, the effects of Src inhibition will depend on the level and activity of the repertoire of Src partners present in each cell type. Therefore, a profound study of the effect of each Src inhibitor on the main Src related signalling pathways – FAK, Akt, EGFR, Erk, Stat, etc – and cellular processes – proliferation, survival, migration, invasion, stemness and metabolism – should be carried out in the specific tumour model. We would like to highlight the relevance of studying the effects of Src inhibitors on metabolism because of the relevance of metabolic plasticity for drug resistance [138] as well as the possible side-effects due to metabolic alterations in non-cancer cells [130]. In addition, an intense metabolic cooperation has been described between tumoral cells and their microenvironment: disrupting this metabolic crosstalk might be critical to impair tumour growth. The study of the effect of Src inhibitors on different metabolic pathways in tumour cells as well as cells from the microenvironment would enhance the possibilities to achieve clinical benefits from Src inhibitors in solid tumours.
Numerous studies carried out over the last years have demonstrated that key proteins and enzymes involved in glucose uptake, glycolysis, PPP and mitochondrial metabolic pathways are Src substrates (Figs. 3, 4). Consequently, the activity of Src can modulate specific metabolic pathways required to fuel diverse cancer cell activities. Not surprisingly, the inhibition of Src activity affects cancer cell metabolism with a concomitant reduction in tumour progression [55]. As a relevant proof of the link between the effects of Src on metabolism and its oncogenic activity, mutations of key metabolic enzymes, such as HK1, HK2, PFKFB3 or G6PDH at specific Src-phosphorylation sites reduce Src-evoked tumour progression [81, 87, 109]. Moreover, emerging reports are showing that Src has roles in the metabolism of several other biomolecules beyond glucose, including lipids [139, 140] and amino acids [141, 142]. Together, these data clearly indicate that the effects of Src on cell metabolism contribute to its oncogenic effect and therefore, an integrated perspective on the role of Src on these cellular functions should be considered. Cancer progression implies heterogenous metabolic requirements to sustain diverse cellular processes, including stemness, proliferation, migration or differentiation. As described in previous sections, Src activity is not associated to an increase or decrease in a specific metabolic pathway. Overall, Src activity can regulate diverse metabolic pathways and hence we propose that this oncoprotein is in a good position to coordinate metabolism with each specific tumour cell process. For instance, Src activity can regulate mitochondrial metabolism in cancer stem cells [55], which are highly dependent on OXPHOS [143], while it activates glucose uptake and glycolysis in proliferative cells (see ‘Src regulates glucose uptake and glycolysis’), highlighting the contribution of Src activity to the metabolic plasticity required by cancer cells during tumour progression. The increased Src enzymatic activity found in many tumours (Fig. 2) indicates that Src should be further studied as a drug development target, despite the lack of Src mutations or gene amplification found in cancer. As mentioned in the previous section, an intense study of the effects of Src inhibitors in cancer cell metabolism as well as in the metabolism of cells from the tumour microenvironment will help to elucidate the potential benefits, side effects or resistance mechanisms caused by Src inhibitors in a clinical setting. Given the demonstrated accuracy of artificial intelligence for protein structure prediction [144] (Fig. 1), a speed up in the development of specific Src inhibitors is expected once this computational tool reaches the same benchmark for the prediction of interaction affinities and the structure of small peptides, disordered regions and specific amino acid mutations. Although the effects of Src on metabolism were discovered many years ago, several questions remain to be answered: How do different signals that converge in Src activation result in changes in distinct cellular and metabolic processes? Do different Src-binding partners affect Src structure differentially to accommodate a specific substrate? How is Src modulation of metabolism integrated with that of other metabolic sensors and regulators, such as AMPK or mTOR? This review is focused on Src, however, the effect of other SFK members (such as Yes, Fyn or Fgr) on metabolism has been reported, as we have discussed. Indeed, several interesting questions are still unanswered: What is the contribution to metabolism of each SFK member (e.g., is there functional redundancy, as sometimes found for other SFK functions)? Is it important to design specific inhibitors for some SFK members? The answers to these and other questions will give a more complete view of Src and SFK biology, which is required to develop new and more successful therapies that target Src in cancer. In brief, the studies summarised in this review—and many others that we were unable to cite due to space limitations—indicate that Src can orchestrate glucose metabolism to fuel a great variety of signalling pathways and cellular processes, including the adaptation of cellular metabolism required for each cell activity during tumour progression. Hence, similarly to other well-known signalling molecules, including AKT, AMPK, mTOR or HIF-1α, Src might be regarded as a master regulator of glucose metabolism and coordinator of metabolism with different cancer cell processes. | true | true | true |
PMC9630201 | 35849202 | Hai B. Tran,Rachel Jakobczak,Adrian Abdo,Patrick Asare,Paul Reynolds,John Beltrame,Sandra Hodge,Peter Zalewski | Immunolocalization of zinc transporters and metallothioneins reveals links to microvascular morphology and functions | 18-07-2022 | Vascular dysfunction,Pulmonary arterial hypertension,SLC39A/ZIPs zinc transporters,ZIP12,Metallothioneins,Multifluorescence quantitative confocal microscopy | Zinc homeostasis is vital to immune and other organ system functions, yet over a quarter of the world’s population is zinc deficient. Abnormal zinc transport or storage protein expression has been linked to diseases, such as cancer and chronic obstructive pulmonary disorder. Although recent studies indicate a role for zinc regulation in vascular functions and diseases, detailed knowledge of the mechanisms involved remains unknown. This study aimed to assess protein expression and localization of zinc transporters of the SLC39A/ZIP family (ZIPs) and metallothioneins (MTs) in human subcutaneous microvessels and to relate them to morphological features and expression of function-related molecules in the microvasculature. Microvessels in paraffin biopsies of subcutaneous adipose tissues from 14 patients undergoing hernia reconstruction surgery were analysed for 9 ZIPs and 3 MT proteins by MQCM (multifluorescence quantitative confocal microscopy). Zinc regulation proteins detected in human microvasculature included ZIP1, ZIP2, ZIP8, ZIP10, ZIP12, ZIP14 and MT1-3, which showed differential localization among endothelial and smooth muscle cells. ZIP1, ZIP2, ZIP12 and MT3 showed significantly (p < 0.05) increased immunoreactivities, in association with increased microvascular muscularization, and upregulated ET-1, α-SMA and the active form of p38 MAPK (Thr180/Tyr182 phosphorylated, p38 MAPK-P). These findings support roles of the zinc regulation system in microvascular physiology and diseases. Supplementary Information The online version contains supplementary material available at 10.1007/s00418-022-02138-5. | Immunolocalization of zinc transporters and metallothioneins reveals links to microvascular morphology and functions
Zinc homeostasis is vital to immune and other organ system functions, yet over a quarter of the world’s population is zinc deficient. Abnormal zinc transport or storage protein expression has been linked to diseases, such as cancer and chronic obstructive pulmonary disorder. Although recent studies indicate a role for zinc regulation in vascular functions and diseases, detailed knowledge of the mechanisms involved remains unknown. This study aimed to assess protein expression and localization of zinc transporters of the SLC39A/ZIP family (ZIPs) and metallothioneins (MTs) in human subcutaneous microvessels and to relate them to morphological features and expression of function-related molecules in the microvasculature. Microvessels in paraffin biopsies of subcutaneous adipose tissues from 14 patients undergoing hernia reconstruction surgery were analysed for 9 ZIPs and 3 MT proteins by MQCM (multifluorescence quantitative confocal microscopy). Zinc regulation proteins detected in human microvasculature included ZIP1, ZIP2, ZIP8, ZIP10, ZIP12, ZIP14 and MT1-3, which showed differential localization among endothelial and smooth muscle cells. ZIP1, ZIP2, ZIP12 and MT3 showed significantly (p < 0.05) increased immunoreactivities, in association with increased microvascular muscularization, and upregulated ET-1, α-SMA and the active form of p38 MAPK (Thr180/Tyr182 phosphorylated, p38 MAPK-P). These findings support roles of the zinc regulation system in microvascular physiology and diseases.
The online version contains supplementary material available at 10.1007/s00418-022-02138-5.
The complex system of small blood vessels, namely arterioles, capillaries and venules, collectively called microvessels, is central in life-threatening conditions such as pulmonary arterial hypertension (PAH), coronary microvascular disease and microvascular brain disease. The microvascular wall consists of a few cell types, of which endothelial and smooth muscle cells are the two major populations in arterioles (diameter 10–100 µm) and venules (12–400 µm). In healthy adult microvessels, both these cell types are relatively quiescent, not proliferating, but sensitive to chemical or mechanical stimuli for activation (Ricard et al. 2021; Ourne et al. 2021). This activation may lead to increased cell proliferation and even switching of the smooth muscle cell phenotype (Ourne et al. 2021; Bkaily et al. 2021), resulting in a broad range of morphological and physiological changes known as “vascular remodelling” (Mulvany 1999). In animal models of diabetes and metabolic syndrome, hypertrophic remodelling of microvessels was identified in early stages when coronary arteries remained normal by angiography, indicating a key role for remodelling of micro- rather than macrovessels in initiation of haemodynamic disorders in these disease (Lambazi & Trask 2017). Multiple cellular and molecular events are ascribed to mechanisms of microvascular remodelling in pulmonary arterial hypertension, including proliferation and hypertrophy of smooth muscle cells, instigated in particular by signalling cascades with calcium (Masson et al. 2021), hypoxia-induced factor (Liu et al. 2019), sphingosine-1-phosphate (Ranasinghe et al. 2020), etc. Zinc homeostasis is vital for functioning of the immune and other organs and systems. Cellular zinc homeostasis is regulated by three major families of proteins: (1) Solute Carrier 39 family/Zrt- and Irt-like proteins (SLC39A/ZIPs), which import zinc ions into the cytosolic compartment from the extracellular space or intracellular vesicles; (2) Solute Carrier 30 family/Zinc transporters (SLC30/ZnTs), which export zinc ions from the cytosolic compartment to the extracellular space, or intracellular vesicles; (3) metallothioneins (MTs), which have a high zinc binding capacity, thus playing key roles in intracellular zinc storage and buffering. To date, 14 ZIPs, 10 ZnTs and 4 isoforms of MTs with multiple subtypes/variants have been described in mammals (Kambe et al. 2021). ZIPs have been identified as playing major roles in a broad array of vital functions and diseases (Takagishi et al. 2017). Their expression and functional roles in vascular physiology and diseases had been paid little attention (Zalewski et al. 2019) until the recent ground-breaking finding that ZIP12 is at least partly responsible for hypoxia-induced PAH in both human and rats (Zhao et al. 2015), inspiring other studies into this field (Tran et al. 2021; Xiao et al. 2021; Zhu et al. 2022). Data on vascular expression and functions of other members of the zinc regulation system remain scant. ZIP14 was shown to mediate influx of Zn2+ in sheep pulmonary artery endothelial cells, which may act together with MT to protect against LPS-induced apoptosis (Thambiayya et al., 2012). MT expression and anti-oxidative stress functions in vasculature have been implicated in a number of studies using models of cultured endothelial cells (Kaji et al. 1993; Conway et al. 2010; Thambiayya et al. 2012; Fujie et al. 2020). Thus, despite a growing interest into the zinc regulation system in vascular health and diseases, the understanding of vascular expression and functions of ZIPs, ZnTs and MTs in vivo remains a large gap in our knowledge. A systematic background analysis of the zinc regulation system in human vasculature would benefit further investigations in this field. Following on from our previous study (Abdo et al. 2021) aiming to characterize the zinc regulation system in human vasculature, in this study we employed multifluorescence quantitative confocal microscopy (MQCM) to investigate immunoreactivities of human microvessels in paraffin tissue sections for multiple ZIPs and MTs. Their detailed distribution among the major cell types of microvessels and association with microvascular morphology and expression of vascular function-related molecules was investigated.
A panel of 13 primary antibodies to zinc transporters and metallothioneins and 6 to other molecules was used in this study. Their source, animal species, class/type, dilutions, immunogens and published details relating to specificity are summarized in Supporting Information Table S1. To minimize cross reactivity in multifluorescence labelling, all secondary antibodies were donkey IgG F(ab’)2 fragments, absorbed against cross-species reactivities. They were obtained from Jackson ImmunoResearch, including anti-rabbit IgG-AF594, anti-goat IgG-AF488 and anti-mouse IgG-AF647, used at 1:200. For alternative combinations of primary antibodies in some experiments conjugates switched colours (anti-rabbit IgG-AF488, anti-goat IgG-AF594, anti-goat IgG-AF647 and anti-mouse IgG-AF488). All primary and secondary antibodies were diluted in PBS with 0.5% Tween-20 and 10% serum-free protein blocker (DakoCytomation Inc., Carpinteria, CA, USA) added.
Subcutaneous tissue biopsies were collected from 14 patients undergoing hernia reconstructive surgeries at The Queen Elizabeth Hospital in Adelaide, Australia. Informed consent was obtained from each donor, utilising protocols approved by the Central Adelaide Local Health Network Human Research Ethics Committee at The Queen Elizabeth Hospital (approval number 2009012). According to the ethics approval, individual patient demographic data and their disease status were known only to three authors, RJ, JB and PZ.
To minimize variations that could have resulted from tissue processing, the pre-fixation time when tissue samples were preserved in RPMI medium on ice for transport was kept < 2 h; the fixation protocol was kept uniformly for 24 h at room temperature in phosphate saline-buffered 10% formalin, a protocol accepted by most authors (Engel and Moore 2011). For quantitative analysis, sections from multiple paraffin blocks were cut to 5 µm thickness and mounted onto tissue arrays for batch analysis of all samples. Tissue sections were stained with H&E in a standard protocol at the Histopathology service at Adelaide Health and Medical Sciences and then scanned at a 40× objective with a Nanozoomer digital slide scanner (Hamamatsu Photonics, Hamamatsu, Shizuoka, Japan).
Immunofluorescence of human paraffin tissue sections was performed following a protocol described in our previous study (Tran et al. 2021). MQCM was carried out using an Olympus confocal microscopy system (Olympus FV3000, Tokyo, Japan) and ImageJ morphometric software (NIH, MA, USA) as previously described (Tran et al. 2021). Briefly, ten optical fields containing microvessels were serially captured from each biopsy under a 60× silicone-immersed objective simultaneously in four fluorescence channels set for AF488, AF594, AF647 and DAPI. All microvessels of 20–100 µm diameter in each frame were then selected and their areas in monochromatic images measured for mean fluorescence intensities (MFI) by ImageJ. Analogous to the flow cytometry mean fluorescence intensity (MFI), which is a quantitative measurement of fluorescence brightness averaged from all events counted in a gate, in immunofluorescence MFI is averaged from all pixels of the region of interest (ROI) of an image. MFI values were measured using MQCM as follows. From a merged multi-colour confocal image, the area of a microvessel section as the ROI was delineated first using the ImageJ software drawing tools. Using the CTR-SHIFT-E keys, the ROI was then applied to monochromatic channel images which had previously been converted to greyscale by menu Image/Type/16-bit. Then, the menu Image/Adjust/Threshold was applied, allowing the software to automatically predict a threshold. Next, in the menu Analyze/Analyze Particles, the mean greyvalue was selected from Set Measurement to measure the ROI MFI. The MFIs of ten ROIs captured from each sample were averaged and corrected for autofluorescent background. The latter were measured for each channel in a similar way as for the samples stained with conjugate alone (negative controls, which were included in every batch analysis). For punctate immunofluorescence of ET-1, particle counting function was carried out in a predetermined threshold band (70, with maximum intensities varying between 44 and 154 in the AF488 channel) for uniform gating in only bright particle sizing of > 10 square pixels (> 3.13 µm2). Numbers of particles counted in a vascular area were then normalized by numbers of nuclei counted in the DAPI channel in the same area. Microvessels were subdivided into two subpopulations, ‘muscularized’ when having walls consisting of at least two cell layers in the whole perimeter and ‘non-muscularized’ for walls consisting of fewer than two layers. The percentage of ‘muscularized’ microvessels varied between 0 and ~ 60%. After obtaining quantitative results of all included samples, representative confocal images were selected for those most closely reflecting the final quantitative results, without referring to the donor age and disease status.
Statistical analysis was undertaken using Prism 9 software (GraphPad Software, CA, USA). For difference between subgroups of microvessels, a paired two-tailed Wilcoxon test was used. Changes were considered statistically significant at p < 0.05.
Tissues donated from 14 patients (10 males, age range: 23–91 years) undergoing hernia reconstructive surgeries were analysed. Their demographic characteristics, body/mass index, usage of vitamin or zinc supplements, cigarette smoking status and history of pathological conditions known at the time of surgeries are summarized in Supporting Information Table S1. Cardiovascular diseases were reported in four cases (infarct, 1; deep thrombosis of lower extremity, 1). Conditions that may be risk factors for cardiovascular diseases included hypertension (2), diabetes (3), asthma (2) and COPD (1). No history was reported for four.
In H&E staining subcutaneous biopsies consisted of mostly adipocytes, scattered with islands of dense irregular connective tissue, nerve fibres and microvessels. By applying the inclusion criterion of diameter between 20 to 100 µm, the selected microvessels included both venules and arterioles but excluded much smaller capillaries (< 10 µm by definition). The microvessels varied in their wall thickness and relative degree of muscularization (Fig. 1). Muscularized microvessels showed increased wall-to-lumen projection, often having rough endothelial surfaces, increased nuclear density and fibrotic staining in the tunica intima (Fig. 1a, b). At high-resolution confocal microscopy, microvascular cell layers of tunica intima (endothelium) and tunica media (smooth muscle) could be clearly demarcated, allowing for sub-classification by degree of muscularization (Fig. 1c).
Preliminary experiments were carried out to titrate primary antibodies and define optimal dilutions, as described in the Methods. In the conditions of our protocols, similar patterns of ZIP1 immunofluorescence in the endothelium and smooth muscle were detected by a homemade sheep antibody (Michalczyk and Ackland 2013) and a commercial goat antibody (Fig. 2a). For ZIP2, similar patterns of endothelium and smooth muscle staining were detected using the two rabbit polyclonal antibodies (Abcam and Novus, Fig. 2a). Other ZIPs detected in microvessels were ZIP8, ZIP10, ZIP12 and ZIP14 (Figs. 1b, 2b). In the described protocol the Abcam antibodies to ZIP6 and ZIP9 did not give immunofluorescence exceeding the level of background fluorescence in the vasculature (Fig. 2c). Next, all three tested antibodies to MTs revealed positive immunoreactivities in human microvessels (Fig. 3). High resolution by confocal microscopy allowed detailed localization of ZIPs and MTs, roughly equally in both endothelium and smooth muscle (ZIP10, ZIP14, MT1, MT1/2) or relatively more abundantly in endothelium (ZIP1, ZIP2, ZIP8, ZIP12) or smooth muscle (MT3) (Figs. 1b, 2, 3). Immunofluorescence of ZIPs showed both diffused cytoplasmic and thin cytoplasmic patterns; the latter was particularly distinctive for ZIP1 and ZIP12. Immunofluorescence of MT1/2 and MT1 with monoclonal antibodies detected bright spots overlapping the nucleus and weaker cytoplasmic staining in both endothelium and smooth muscle. MT3 immunofluorescence with a polyclonal antibody revealed bright cytoplasmic staining in smooth muscle and weaker staining in endothelium (Fig. 3).
The limited number of patients and heterogeneity of their history would not allow for a quantitative analysis of potential changes of ZIP/MT immunoreactivities by disease or by age/gender subgroups. However, a distinctive variation of ZIP1, ZIP2, ZIP12 and MT3 among microvessels of the same donor led us to postulate that there are changes associated with microvascular morphology. For the purpose of quantitative analysis, the relatively “muscularized” were discerned from “non-muscularized” microvessels, arbitrarily based on whether their wall contained at least two muscle cell layers throughout their perimeters. The microvascular immunoreactivities of ZIP1, ZIP2, ZIP12 and MT3 quantified by their mean fluorescence intensities confirmed statistically significant increases in muscularized vs. non-muscularized microvessels (Figs. 4, 5). Of note, while upregulated ZIP1, ZIP2 and ZIP12 were recorded mostly in the endothelium, increased MT3 was mostly seen in the smooth muscle compartment.
Next, we tested whether muscularized and non-muscularized subpopulations of microvessels would display any difference regarding expression and localization of molecules that have previously recognized roles in vascular functions. A panel of function-related markers was investigated including eNOS, ET-1, HIF-1α, α-SMA and p38 MAPK. As expected, while the α-SMA immunoreactivity was localized specifically to the smooth muscle (Fig. 5a), most of ET-1 (Fig. 4b) and eNOS (Fig. 6a) were localized to the endothelium. Notably, punctate ET-1 immunofluorescence could be visualized in endothelial-smooth muscle junctions on the endothelial side (Fig. 4b). HIF-1α was mostly localized to the endothelium (Fig. 6a). Interestingly, while staining with an antibody to the total p38 MAPK detected bright immunoreactivity in both smooth muscle and endothelium, the active form of p38 MAPK-P was mostly localized to the latter (Supporting Information Figure S1). Importantly, in quantitative analysis ET-1, p38 MAPK-P and α-SMA revealed significant increases in muscularized microvessels. Although eNOS and HIF-α also showed a strong variation among microvessels of the same donor, their overall change among all donors was not statistically significant, increasing with muscularization in some donors but decreasing in others (Fig. 6b).
To our best knowledge this work is the first in vivo description of protein expression of multiple ZIPs and MTs in human microvasculature. A complete list analysis of the ZIPs and MTs and the third family of zinc regulation proteins, SLC30As/ZnTs, remains however a task for future investigations. Despite a limitation that expression at the gene level was not analysed, protein expression of multiple members of ZIPs and MTs supports the hypothesis that there is a redundancy in the zinc regulation system which may be required for a tight control of zinc homeostasis in vascular functions. As another limitation, immunofluorescence results should be interpreted with caution in relation to antibodies’ specificity. To minimize this, most of the primary antibodies used in this study were tested independently by Western blots or immunogen/antibody competition in our previous publications, or published by the manufacturers; furthermore, fragment secondary antibodies pre-absorbed against cross-species reactivities were employed. Results of this study are mostly in line with our previous data on gene expression in primary cell culture (Abdo et al. 2021), with exceptions as discussed below. Thus, while abundant mRNA expression was found in primary cell cultures for ZIP6 and ZIP9, their protein expression in this study could not be detected in microvessels from subcutaneous biopsies. Furthermore, while the relative abundance of gene expression in cell cultures was low for ZIP2, ZIP12 and MT3, protein immunoreactivities in microvascular tissues were varying and brighter in subpopulation(s) of microvessels. Apart from a potential sensitivity issue that could not be ruled out, one possible cause was that while mRNA data were obtained from primary cells of aorta and pulmonary artery, protein expression data were from in vivo sampling of microvessels. Furthermore, while primary cell culture data reflected a normal state in which vascular cells expressed minimal levels of ZIP2, ZIP12 and MT3, biopsies included pathological conditions that could induce these proteins. In accordance with this, previous data provide multiple evidence that ZIP12 could be induced in vivo in vasculature in human patients and rat models of PAH (Zhao et al. 2015; Tran et al. 2021; Xiao et al. 2021). In vitro, both ZIP2 and ZIP12 were shown to be induced at mRNA and protein levels in vascular cells by depletion of zinc (Abdo et al. 2021). The tissue localization data in this study were in line with previous studies (Zhao et al. 2015; Abdo et al. 2021; Tran et al. 2021); ZIP12 was localized to both the endothelial and smooth muscle cell types of the vascular wall. Microvascular expression of ZIP14 was consistent with previous data in that influx of labile zinc in cultured sheep pulmonary artery endothelial cells was sensitive to ZIP14 siRNA indicating the presence of functional ZIP14 in this cell type (Thambiayya et al. 2012). Our findings of highly expressed ZIP1 in microvessels were in line with a notion that ZIP1 is expressed ubiquitously across cell types (Schweigel-Röntgen 2014). Regarding other ZIPs, to our knowledge ours is the first study to examine their localization in vascular walls. Known as free radical scavengers, MTs are surrogate markers of oxidative stress and indicators of labile intracellular zinc levels (Mareiro et al. 2017). MTs have been studied in various cell culture models of vascular endothelial cells in oxidative stress associated with exposure to heavy metals or other stress stimuli (Kaji et al. 1993; Conway et al. 2010; Thambiayya et al. 2012; Fujie et al. 2020; Rubiolo et al. 2021). Metallothioneins have also been commonly reported to be elevated in PAH patients as well as in experimental models of PAH (Maarman 2018). In vitro studies showed that MT can respond to nitride oxide (free radical and vasodilator mediator) by releasing Zn2 + ions (Kroncke et al. 1994; Thambiayya et al. 2012), which could be relevant to a mechanism of vasodilation. Surprisingly MT expression and functions in the other vascular cell type, namely smooth muscle, have been paid little attention. In a rare report, using immunohistochemical staining and immunogold electron microscopy, it was noted that most of the MT induced in human atherosclerotic lesions was localized to the vascular smooth muscle cells (Göbel et al. 2000). Our finding of smooth muscle as the major harbour of MTs in the microvascular wall further puts this cell type in the spotlight of future investigations into vascular zinc biology. By the selected projection size (20−100 µm), microvessels analysed in this study fall into categories of arterioles and venules, which can be roughly distinguished from each other histologically by the level of their muscularization and wall to lumen ratio. A precise differentiation between arterioles and venules requires their localization prior to or after capillary circulation by serial sections (Bonner-Weir and Orci 1982), or at least according to more detailed morphological characteristics, for example, by transmission electron microscopy (Sharp et al 2019) or photoacoustic imaging (Matsumoto et al. 2018), which were not available within this study. Increased proliferation of smooth muscle layer is considered a key process in vascular pathology, for example, in atherosclerosis (Sedding et al. 2018) and diabetic vascular restenosis (Moshapa et al. 2019). In studies of PAH, increased levels of lung microvascular muscularization serve as an indicator of pathological changes leading to resistance and increased pulmonary blood pressure (Zhao et al. 2015; Harper et al. 2019; Maietta et al. 2021). The data presented here indicate that the level of ZIP1, ZIP2, ZIP12 and MT3 expression is higher in those microvessels having increased muscularity, which are likely to be arterioles rather than venules. However, this needs to be confirmed in further studies. The differences between the two microvessel subpopulations in their expression of vascular-active molecules, however, give a notable indication on functional differentiation, in addition to morphological features. Known primarily as a potent vasoconstrictor, ET-1 has broad effects on various pathways critical for vascular functions and diseases, for example, induction of VCAM-1 (Ishizuka 1999), pro-inflammatory activation of leucocytes including neutrophils (Kaszaki et al. 2008) and macrophages (Zhang et al. 2021). Relevant to the vascular pathology, ET-1 was reported to activate smooth muscle by stimulating protein synthesis, promoting proliferation and hypertrophy of pulmonary arterial smooth muscle cells (Chua et al. 1992; Zamora et al. 1993). In this study increased particulate immunofluorescence of ET-1 in muscularized microvessels was localized to endothelial-smooth muscle junctions, supporting the hypothesis that paracrine ET-1-mediated endothelial-smooth muscle crosstalk may be required for not only vasoconstriction, but also proliferation of vascular smooth muscle. As a marker of both endothelial-mesenchymal transition and vascular remodelling, increased α-SMA expression is known to be associated with exposure to mechanical stress, which could also induce activation of p38 MAPK (Wang et al. 2006). In a study of mechanisms leading to PAH, activation of p38 MAPK was found to be associated with oxidative stress and inflammation (Church et al. 2015). Thus, data presented here argue that upregulation of zinc regulation proteins ZIP1, ZIP2, ZIP2 and MT3 is associated with a functionally activated state compared to a relatively quiescent state of microvessels.
In conclusion, this study provides background data of protein expression and localization of multiple ZIPs and MTs in endothelial and smooth muscle layers of human microvascular walls. The presented data support the hypothesis that the zinc regulation system in the human microvasculature, in particular the ZIP1, ZIP2, ZIP12 and MT3 proteins, plays an important role in microvascular physiology and could be a therapeutic target for diseases that involve microvascular remodelling.
Below is the link to the electronic supplementary material.Supplementary file1 (PDF 3251 KB) | true | true | true |
PMC9630288 | Fangbing Chen,Meng Lian,Bingxiu Ma,Shixue Gou,Xian Luo,Kaiming Yang,Hui Shi,Jingke Xie,Weika Ge,Zhen Ouyang,Chengdan Lai,Nan Li,Quanjun Zhang,Qin Jin,Yanhui Liang,Tao Chen,Jiaowei Wang,Xiaozhu Zhao,Lei Li,Manya Yu,Yinghua Ye,Kepin Wang,Han Wu,Liangxue Lai | Multiplexed base editing through Cas12a variant-mediated cytosine and adenine base editors | 02-11-2022 | Genetic engineering,CRISPR-Cas9 genome editing | Cas12a can process multiple sgRNAs from a single transcript of CRISPR array, conferring advantages in multiplexed base editing when incorporated into base editor systems, which is extremely helpful given that phenotypes commonly involve multiple genes or single-nucleotide variants. However, multiplexed base editing through Cas12a-derived base editors has been barely reported, mainly due to the compromised efficiencies and restricted protospacer-adjacent motif (PAM) of TTTV for wild-type Cas12a. Here, we develop Cas12a-mediated cytosine base editor (CBE) and adenine base editor (ABE) systems with elevated efficiencies and expanded targeting scope, by combining highly active deaminases with Lachnospiraceae bacterium Cas12a (LbCas12a) variants. We confirm that these CBEs and ABEs can perform efficient C-to-T and A-to-G conversions, respectively, on targets with PAMs of NTTN, TYCN, and TRTN. Notably, multiplexed base editing can be conducted using the developed CBEs and ABEs in somatic cells and embryos. These Cas12a variant-mediated base editors will serve as versatile tools for multiplexed point mutation, which is notably important in genetic improvement, disease modeling, and gene therapy. | Multiplexed base editing through Cas12a variant-mediated cytosine and adenine base editors
Cas12a can process multiple sgRNAs from a single transcript of CRISPR array, conferring advantages in multiplexed base editing when incorporated into base editor systems, which is extremely helpful given that phenotypes commonly involve multiple genes or single-nucleotide variants. However, multiplexed base editing through Cas12a-derived base editors has been barely reported, mainly due to the compromised efficiencies and restricted protospacer-adjacent motif (PAM) of TTTV for wild-type Cas12a. Here, we develop Cas12a-mediated cytosine base editor (CBE) and adenine base editor (ABE) systems with elevated efficiencies and expanded targeting scope, by combining highly active deaminases with Lachnospiraceae bacterium Cas12a (LbCas12a) variants. We confirm that these CBEs and ABEs can perform efficient C-to-T and A-to-G conversions, respectively, on targets with PAMs of NTTN, TYCN, and TRTN. Notably, multiplexed base editing can be conducted using the developed CBEs and ABEs in somatic cells and embryos. These Cas12a variant-mediated base editors will serve as versatile tools for multiplexed point mutation, which is notably important in genetic improvement, disease modeling, and gene therapy.
Base editors, including cytosine base editors (CBEs) and adenine base editors (ABEs), are newly developed CRISPR/Cas-based genome modified tools that combine catalytically impaired Cas nucleases with different kinds of deaminases. CBEs and ABEs can precisely and efficiently convert C-to-T and A-to-G, respectively, at single-base resolution. Since their development, base editors have been widely adopted in various organisms to induce point mutations to mimic disease-causative mutations and agronomically important variations. Given that numerous biological traits and diseases involve multiple single-nucleotide variants (SNVs) at different positions or multiple genes, simultaneous base editing of multiple loci is urgently needed for disease modeling or crop improvement. Cas12a, a member of the type V CRISPR family formerly called Cpf1, can process multiple sgRNAs (also CRISPR RNAs (crRNAs)) from a single array transcript through its intrinsic RNase activity, thus conferring the flexible capacity of multi-gene targeting. In virtue of the recognition of thymidine (T)-rich protospacer-adjacent motif (PAM) sequences, the catalytically inactivated dead Cas12a (dCas12a) has been adopted for the development of base editors to expand the targeting scope. However, studies rarely reported Cas12a-mediated multiplexed base editing systems. The use of Cas12a for multiple base editing is impeded by poor efficiencies and strict PAM requirement, as the common Lachnospiraceae bacterium Cas12a (LbCas12a) and Acidaminococcus sp. Cas12a (AsCas12a) recognize canonical TTTV PAM sequences (where V is A, C, or G). Cas12a variants developed recently by structure-guided protein engineering, including RR, RVR, enAsCas12a, and impLbCas12a, enable the recognition of alternative non-canonical PAMs, whereas extremely low base editing efficiencies hinder their practical applications, especially for multiple loci editing. Thus, versatile Cas12a-mediated base editor systems that can execute efficient editing of targets with non-canonical PAMs in addition to TTTV are urgently needed, which will be favorable for multiplexed base editing. Although the compromised efficiencies of Cas12a-mediated base editors are likely in part due to the incapability to generate Cas12a nickase, we and several other groups have demonstrated recently that the efficiencies of dCas12a-mediated CBEs and ABEs can be improved substantially with highly active deaminases on targets with TTTV PAM sequences. In this study, we sought to develop LbCas12a (hereafter abbreviated as Cas12a) variant-mediated robust CBEs and ABEs with expanded target scope, which are capable of multiplexed base editing efficiently. By fusing highly active deaminases with engineered dCas12a variants, including RR, RVR, and enCas12a, we confirmed that these Cas12a variant-mediated CBEs and ABEs can act robustly at endogenous sites with canonical TTTV PAM and non-canonical PAMs, such as VTTV, TYCN, and TRTV (where Y is C or T, and R is A or G). Importantly, these base editors can performed multiplexed base editing in HEK293T cells and porcine embryos. With large improvements in efficiencies and targeting ranges, these newly developed Cas12a variant-derived CBEs and ABEs will serve as potent base editing tools and pave the way for multiplexed base editing to introduce or correct point mutations at several loci simultaneously, which is valuable in basic research, agriculture, and medicine.
We initially attempted to screen for optimal cytosine deaminases that confer powerful C-to-T editing for Cas12a-mediated CBEs. Seven dCas12a-CBEs fused with individual cytosine deaminases were compared in HEK293T cells and porcine embryos at sites with TTTV PAM sequences. The dCas12a-A3A harboring human APOBEC3A (hA3A) exhibited superior efficiencies at most sites and different sequence contexts compared with other dCas12a-CBEs (Supplementary Fig. 1). Importantly, the introduction of the hA3A-Y130F or hA3A-Y132D variant to dCas12a-A3A could improve the accuracy of base editing by narrowing the extremely wide activity windows, from 10 nt (positions 6–15) to 6 nt (positions 7–12), and alleviate the potential toxic effect on embryo development due to the exceedingly high deaminase activity of hA3A while maintaining considerable C-to-T editing efficiencies (Supplementary Figs. 2, 3a). The introduction of the hA3A-N57G variant to dCas12a-A3A, on the other hand, conferred TCR or TCCR preference (Supplementary Fig. 3b), which can attain excellent accuracy when several Cs are located in the activity window. Therefore, the hA3A-Y130F and hA3A-N57G were selected for the construction of robust Cas12a-CBEs with expanded PAM compatibility. Several AsCas2a variants have been reported to recognize alternative non-canonical PAMs. To relieve the intrinsic limit of LbCas12a for TTTV PAM sequences to develop versatile Cas12a-mediated base editors with expanded target scope, we engineered LbCas12a by introducing certain amino acid mutations, based on its homology to AsCas12a, resulting in the generation of three LbCas12a variants, including RR (G532R/K595R), RVR (G532R/K538V/Y542R), and enCas12a (D156R/G532R/K538R) (Supplementary Fig. 4). Subsequently, we constructed PAM-expanded CBEs by fusing hA3A-Y130F with the three dCas12a variants and tested them at sites with canonical or non-canonical PAMs in HEK293T cells (Fig. 1a; Supplementary Fig. 4a, b). All four CBEs, including dCas12a-A3A-Y130F, enCas12a-A3A-Y130F, RR-A3A-Y130F, and RVR-A3A-Y130F, can convert C-to-T at TTTV PAM sequences with comparable efficiencies (18.3–50.0%), although the efficiencies of enCas12a-A3A-Y130F were ~1.6-fold higher than those of the other three ones at the ADAM6 site (Fig. 1b, d; Supplementary Data 1; Supplementary Fig. 5a). However, among the 14 tested sites with non-canonical PAMs, dCas12a-A3A-Y130F showed a poor performance except at two sites, KLF4-6 and DNMT1-1, with VTTV PAM sequences (Fig. 1c, d; Supplementary Data 1; Supplementary Figs. 5a, b), consistent with previous reports. By contrast, three engineered Cas12a-variant versions of CBEs showed a robust editing effect when targeting sites with non-canonical PAMs, while the editing efficiencies differed for individual sites. In general, enCas12a-A3A-Y130F exhibited robust editing for most tested sites with VTTV, TTCN, TCCV, TATV, and TTTT PAMs, with editing efficiencies 1.6–2.3-fold that of dCas12a-A3A-Y130F (Fig. 1c, d; Supplementary Data 1; Supplementary Fig. 5a, b). Notably, RR-A3A-Y130F and RVR-A3A-Y130F showed preference in editing sites with TTCN/TCCV (2.4-fold of dCas12a-A3A-Y130F) and TATV (2.3-fold of dCas12a-A3A-Y130F) PAMs, respectively (Fig. 1c, d; Supplementary Data 1; Supplementary Fig. 5a, b). Despite the differences in Cas12a variants adopted, the editing windows of these CBEs spanned ~7 nt (positions 6–12), consistent with that of dCas12a-A3A-Y130F (Supplementary Fig. 5c). These results indicated that these engineered Cas12a variant-derived CBEs retained their capacities for non-canonical PAM recognition. We thus further tested these CBEs in porcine embryos and observed that dCas12a-A3A-Y130F and its three variants (enCas12a-, RR-, and RVR-A3A-Y130F) can function efficiently at sites with TTTV PAM sequences, with efficiencies of 36.0% to 73.3% (Fig. 1e, g; Supplementary Data 1; Supplementary Fig. 6a). Meanwhile, enCas12a-, RR-, and RVR-A3A-Y130F can recognize and edit sites with the corresponding non-canonical PAMs, where dCas12a-A3A-Y130F showed extremely low efficiencies (Fig. 1f, g; Supplementary Data 1; Supplementary Fig. 6a), consistent with the findings in HEK293T. We observed that enCas12a-A3A-Y130F outperformed the other two engineered variants (RR- and RVR-A3A-Y130F) when targeting sites with VTTV PAM sequences in embryos, with efficiencies of 23.8% vs 4.6 and 10.3%, respectively (Fig. 1f, g; Supplementary Data 1). Although RR- and enCas12a-A3A-Y130F can both edit sites with TTCN/TCCV PAMs, the former showed a higher efficiency for TCCV PAM sequences (Fig. 1f, g; Supplementary Data 1). Similarly, RVR-A3A-Y130F showed the highest efficiencies (21.5%) among four CBEs for TATV/TGTV PAMs (Fig. 1f, g; Supplementary Data 1). The activity window (positions 7–12) in embryos was slightly narrower than that in HEK293T cells (Supplementary Figs. 5c and 6b). We also fused another hA3A variant (hA3A-N57G) to the dCas12a variants (Supplementary Fig. 4a, b). The results indicated enCas12a-, RR-, and RVR-A3A-N57G retained their preference for TCR and TCCR motifs at sites with canonical TTTV PAM or non-canonical PAMs in HEK293T cells and porcine embryos (Supplementary Fig. 7), which was beneficial to minimizing bystander activities. These results indicated that CBEs consisting of fused hA3A-Y130F and Cas12a variants, including enCas12a, RR, and RVR, can conduct C-to-T conversion at sites with canonical and non-canonical PAMs efficiently, including NTTN, TYCN, and TRTV, collectively. In addition, the hA3A-N57G variant in these Cas12a variant-mediated CBEs conferred similar TCR/TCCR specificity to the original dCas12a-A3A-N57G.
To perform A-to-G conversion, we firstly compared two dCas12a-ABEs fused with individual adenine deaminases, including TadA-TadA*7.103 and TadA*8e respectively, in HEK293T cells and porcine embryos at sites with TTTV PAM sequences (Supplementary Fig. 8a). The results showed that dCas12a-ABE8e with TadA*8e monomer showed considerably more robust efficiency than dCas12a-ABE7, which fused with TadA-TadA*7.10 dimer, although both ABEs showed similar sequence preferences for TA motif (Supplementary Figs. 8, 9), consistent with the findings of a previous study on plants. In addition, a V106W mutation was introduced into TadA*8e, which was reported to reduce RNA off-target, and the resulting dCas12a-ABE8e-V106W retained a high efficiency for A-to-G conversion (Supplementary Figs. 8, 9), although the RNA off-target was not evaluated in this study. A similar editing window (positions 8–12) for dCas12a-ABE7, dCas12a-ABE8e, and dCas12a-ABE8e-V106W was observed between the HEK293T cells and porcine embryos (Supplementary Figs. 8e, 9c). We thus attempted to construct new ABEs with expanded target scope by fusing engineered Cas12a variants with TadA*8e-V106W, resulting enCas12a-ABE8e-V106W, RR-ABE8e-V106W, and RVR-ABE8e-V106W, which were expected to convert A-to-G at sites with non-canonical PAMs (Fig. 2a; Supplementary Figs. 4a, c). We then tested the base editing efficiencies and preferences at endogenous sites with TTTV PAM or other non-canonical PAMs in HEK293T cells. These Cas12a variant-derived ABEs performed efficient A-to-G editing at sites with non-canonical PAMs, in addition to sites with TTTV PAM sequences (Fig. 2b–d; Supplementary Data 1; Supplementary Fig. 10). Among the three ABE variants, enCas12a-ABE8e-V106W showed robust editing activities at sites with TTCC (23.7%) and ATTG (16.3%) PAMs, which were 2.2-fold and 1.6-fold that of dCas12a-ABE8e-V106W, respectively (Fig. 2c; Supplementary Data 1). RR-ABE8e-V106W exhibited efficient A-to-G editing for TTCC/TTCT/TCCA/TTTT PAMs (20.1% on average), whereas RVR-ABE8e-V106W can edit sites with TATA PAM sequences, with frequencies increasing by twofold compared with those of other ABEs (Fig. 2c; Supplementary Data 1). For several tested non-canonical PAMs, the efficiency differences were not as drastic as expected among individual Cas12a variants (Supplementary Fig. 10b), which disagrees with previous reports, and the cause was unknown. We also confirmed the capacities of these new ABEs for alternative PAM recognition in porcine embryos at three sites with canonical or non-canonical PAMs. All four ABEs showed comparable efficiencies for TTTG PAMs (24.3–33.0%), whereas RR-ABE8e-V106W and enCas12a-ABE8e-V106W exhibited the highest efficiencies for TCCA (35.4%) and CTTC (41.7%) PAMs, respectively (Fig. 2e; Supplementary Data 1). These results revealed that the Cas12a variant-derived ABEs fused with TadA*8e-V106W can alleviate the limit of wild-type Cas12a for TTTV PAM recognition and execute A-to-G conversions at TTTV and alternative PAMs in mammalian cells and embryos.
To take advantage of the unique feature of Cas12a in multiplexed genome editing, we harnessed Cas12a variant-mediated CBEs and ABEs to perform multiplexed base editing, simultaneously, at several loci with a single CRISPR array (termed as multiplex-sgRNA) (Fig. 3a; Supplementary Fig. 11). We initially tested in HEK293T cells with a multiplex-sgRNA containing three tandem sgRNAs (named hcr3) to edit three sites with TTTV PAM sequences simultaneously (Fig. 3b). For the control, we targeted three sites with pooled single sgRNAs and a single site with the corresponding sgRNA in parallel (Supplementary Fig. 11). All three sites, including CFTR, KLF4, and TET1, can be edited effectively by these CBEs and ABEs with hcr3, and the efficiencies were comparable or superior to those with pooled sgRNAs (Fig. 3c, d; Supplementary Data 1). For example, dCas12a-A3A-Y130F showed an efficiency of 30.0% at CFTR with hcr3 compared with 25.0% observed with pooled sgRNAs (Fig. 3c; Supplementary Data 1). Similarly, RVR-ABE8e-V106W exhibited an improved efficiency with hcr3 compared with pooled sgRNAs at CFTR (37.3% vs 26.0%) (Fig. 3d; Supplementary Data 1). Both the tandem multiplex-sgRNA and pooled sgRNAs contributed compromised efficiencies of C-to-T and A-to-G editing, compared with single sites edited alone. The average efficiencies of four CBEs for CFTR, KLF4, and TET1 with hcr3 were 28.5, 36.2, and 15.5%, respectively (Fig. 3c; Supplementary Data 1). By contrast, the corresponding efficiencies were 35.7, 45.2, and 19.3%, respectively, when a single site was targeted with a single sgRNA (Fig. 3c; Supplementary Data 1). Similar results were observed in multiplexed editing using Cas12a-ABEs (Fig. 3d; Supplementary Data 1). The relative inefficiency of multiple editing, when compared with a single locus, was consistent with the previous observations. This finding may be due to the unsaturated base editor proteins at individual sites. As in our transfection experiments, the amount of expression plasmids for the base editor proteins remained constant although the number of target sites increased. Further, we attempted to extend the number of simultaneous base editing loci to five with tandem multiplex-sgRNA (named hcr5) (Fig. 3b), and observed that all the five endogenous sites, including DNMT3B, KLF4, TET1, PRR5L, and CFTR, can be edited by these Cas12a variant-mediated CBEs and ABEs (Fig. 3e, f; Supplementary Data 1). The efficiencies of C-to-T editing for enCas12a-A3A-Y130F at the five sites were 40.3, 41.0, 15.0, 27.0, and 27.3%, respectively (Fig. 3e; Supplementary Data 1). Accordingly, the efficiencies of A-to-G editing for enCas12a-ABE8e-V106W were 8.3, 38.0, 23.0, 12.0, and 37.0%, respectively (Fig. 3f; Supplementary Data 1). For the DNMT3B site, a low efficiency for ABEs was due to all As located outside the main activity window. We observed comparable efficiencies for the CFTR site between CRISPR array hcr3 and hcr5, where the CFTR-sgRNA is located at the first and fifth positions, respectively (Fig. 3c–f; Supplementary Data 1; Supplementary Fig. 12). This result indicated, to a certain extent, that positioning within the array is not crucial for efficiency variation but likely the target itself, consistent with those of previous reports. Given the positive results from HEK293T cells, we examined the efficiencies of multi-loci base editing with a single CRISPR array in embryos, especially for sites with non-canonical PAMs. Four genes, including PUM1, GHR, HMGA2, and PUM2, in porcine genome were thus selected as targets (Fig. 4a), which are all associated with body sizes. Premature stop codons were expected to be generated through C-to-T conversion by Cas12a-CBE. Particularly, PUM1 and PUM2 possessed canonical TTTV PAM, whereas non-canonical TTCA and TCCA for GHR and HMGA2, respectively (Fig. 4a). In view of the inclusion of TTCA/TCCA PAMs, we employed RR-A3A-Y130F, a variant that recognize TYCV PAM sequence (Y = C or T), along with tandem multiplex-sgRNA (named pcr4) or pooled sgRNAs in porcine parthenogenetic (PA) embryos by microinjection (Fig. 4b). The results showed that the pcr4 can attain high efficiencies of 70.2, 76.2, 84.1, and 78.8% on average for PUM1 (C7), GHR (C8), HMGA2 (C9), and PUM2 (C12), respectively (Fig. 4c, d; Supplementary Data 1). Pooled sgRNAs resulted in considerable editing efficiencies although the values were slightly lower for PUM2. However, the pooled sgRNAs showed more variability among individual embryos for the four sites compared with the multiplex-sgRNA (Supplementary Fig. 13). Importantly, the pooled sgRNAs probably impaired the embryonic development to a certain extent (Fig. 4e, f; Supplementary Data 1), presumably due to the increased amount of the injected RNAs. These results indicated that multiplexed base editing with up to five and four loci in HEK293T cells and embryos, respectively, can be realized by a single CRISPR array along with these Cas12a variant-derived CBEs and ABEs. Compared with pooled sgRNAs, a single transcript of CRISPR array can potentially maintain a considerable transfection efficiency in mammalian cells and minimize the adverse effect of microinjection when multiple loci need to be modified.
Finally, we evaluated the editing precision of these Cas12a variant-mediated base editors, including the product purity, level of indels, and off-target activity. Sites with canonical TTTV PAM sequences in human genome were selected to avoid the preference of Cas12a variants for distinct PAMs (Supplementary Data 2 and 3). Also, a non-targeting sgRNA (scrambled sgRNA), which contains the same nucleotide composition as CFTR while does not target the human genome, was used as a negative control (Supplementary Data 2). Targeted deep sequencing of the selected targets showed that Cas12a and its variants, including enCas12a, RR, and RVR, resulted in a high product purity, with slightly unintended C-to-non-T editing for CBEs and A-to-non-G for ABEs, respectively (Fig. 5a, b; Supplementary Data 1; Supplementary Figs. 14, 15a, b). Notably, indels can only be detected at the background level for all these Cas12a variant-mediated base editors (Fig. 5c; Supplementary Data 1; Supplementary Fig. 15c), which can be attributed to the use of catalytically dead Cas12a instead of nickase ones, consistent with the previous study. To evaluate the off-target activity of the developed base editors, we considered Cas12a-dependent off-target from the tolerance for mismatch, which represented the most dominant part of the off-target effect. Potential off-target sites were predicted using the Cas-OFFinder tool (http://www.rgenome.net/cas-offinder/), with up to three nucleotide mismatches and PAMs restricted to NTTN, TYCN and TRTN (Supplementary Data 3). Among these potential off-target sites, most showed no evident off-target editing (Fig. 5d, e; Supplementary Data 1; Supplementary Figs. 16–18), except for PRR5L-OT1 and PRR5L-OT3, which shared one and two mismatches with the PRR5L target, respectively (Fig. 5d; Supplementary Data 1; Supplementary Fig. 16). Consistent with the relaxed PAM compatibility, off-target editing for PRR5L-OT1 and PRR5L-OT3, both with TTCT PAM sequences, occurred only for base editors derived from enCas12a and RR but not for those from dCas12a and RVR (Fig. 5d; Supplementary Data 1; Supplementary Fig. 16). Cas12a-independent DNA and RNA off-target effect, which was mainly attributed to the nature of deaminases, has not been detected in this study. We speculated a substantial mitigation for these CBEs and ABEs when Y130F or N57G in hA3A and V106W in TadA*8e were introduced, respectively, as demonstrated by several previous studies in detail. These results indicated that the established Cas12a variant-mediated CBEs and ABEs displayed a considerably adequate precision and specificity at the tested sites with high product purity, negligible indels, and low off-target activity (Table 1; Supplementary Data 1).
Base editors have been developed to perform precise genome modification with high efficiency and specificity, and they are powerful for the precise base substitution of single or multiple loci. Without inducing double-stranded DNA breaks (DSBs), base editors also show significant advantages in multi-gene disruption by introducing premature stop codons at multiple loci, whereas traditional nucleases mediate DSBs at multiple loci, possibly increasing the risk of genomic instability and chromosomal variation. Despite their superiority, base editors have only been harnessed for multiplexed editing by pooling multiple sgRNAs, which is potentially compromised by the transfection efficiencies in cultured cells and toxic effects in embryos with the increase in sgRNAs. Transfer RNA (tRNA) was recently adopted in the tandem sgRNAs of Cas9 system to achieve multiplexed base editing. However, this strategy complicates its construction and application and may also be compromised by the insufficient transcription of sgRNAs. By contrast, Cas12a enables multiplexed targeting with a single CRISPR array, which is also promising in multiplexed base editing. In this study, we have engineered new Cas12a-mediated CBEs and ABEs with high efficiencies and alleviative PAM limitations by combining highly active hA3A or TadA*8e with the indicated dCas12a variants (Table 1; Supplementary Data 1) and verified that these versatile Cas12a variant-derived base editors can engage in multiplexed base editing with a single transcript of CRISPR array in mammalian cells and embryos. We have only tested multiplexed editing up to five loci with the tandem CRISPR array. However, extending to a greater number of loci can be reasonably expected as shown in previous report. The reduction in the number of plasmids or RNA will potentially improve the transfection efficiencies in mammalian cells and minimize the adverse effects of microinjection on embryos while retaining intrinsic efficiencies of base editing. Meanwhile, the protein-coding sequence of base editors and tandem CRISPR array can be encoded in a single transcript. This all-in-one strategy shows as a unique superiority for Cas12a while it is difficult to be used in the Cas9 system, which highlights the irreplaceability of Cas12a-mediated base editor systems for multiplexed base editing. Our limited results showed no evident position effect of sgRNAs within the array, which is supported by previous studies. However, this statement has not been validated in this research and the expression of individual sgRNAs from CRISPR array has not been detected. Thus, more comprehensive pieces of evidence are needed. In addition to the enCas12a, RR, and RVR variants adopted in this study that collectively recognize NTTN, TYCN, and TRTV PAMs, the LbCas12a variant, that is, impLbCas12a, has recently been reported through the combination of the mutations of RR, RVR, and enCas12a variants, which showed a preference for TNTN PAM and partly overlapped with enCas12a. However, existing Cas12a variants are insufficient for researchers to target almost all interested sites, especially for non-canonical PAMs. Therefore, more efforts and strategies are essential to further loosen the PAM constraint of Cas12a to exploit fully its advantages in multiplexed base editing. Notably, Cas12a-mediated base editors may be less efficient than Cas9 in certain genome loci although a significant improvement has been achieved compared with the original version. This relative inefficiency is most likely due to the employment of dCas12a, instead of Cas12a nickase, which is not available at the present, given that previous studies have indicated nicking the unedited strand can improve the base conversion efficiencies. Alternatively, the efficiencies of Cas12a-mediated base editors can potentially be elevated further through strategies other than the deaminase activity, such as employing dCas9 to auxiliarily bind the proximity location of the target, exploiting more robust Cas12a variants, or covalent conjugating crRNA to Cas12a. The adoption of dCas12a in base editors, on the other hand, offers application benefits, such as low DNA damage response and reduced indels. We have only analyzed Cas12a-depedent off-target effects of these base editors but not Cas12a-independent off-target effects on the genome and transcriptome. Nevertheless, a comprehensive assessment of the off-target effects is valuable in the future to boost their application. In conclusion, we have engineered a series of Cas12a-mediated CBEs and ABEs with improved efficiencies and targeting scope by adopting highly active deaminases and Cas12a variants. These base editors have shown dramatically elevated efficiencies at sites with non-canonical PAMs. Notably, leveraging the dual RNase/DNase function of Cas12a, simultaneous base editing of multiple loci with a single CRISPR array can be realized flexibly through these CBEs and ABEs. The established base editors will serve as valuable tools for multiplexed base editing and substantially promote their wide applications in agriculture and biomedicine.
Vector dCas12a-A3A was constructed previously by replacing rAPOBEC1 with an hAPOBEC3A fragment in restriction endonuclease-linearized dCas12a-BE expression vector using the ClonExpress MultiS kit (Vazyme). dCas12a-A3B, dCas12a-A3G, dCas12a-CDA1, dCas12a-AID, and dCas12a-eAID expression vectors were constructed using a similar strategy. To generate fragments of enCas12a, RR, and RVR, we used dLbCas12a to introduce D156R/G532R/K538R, G532R/K595R, and G532R/K538V/Y542R mutations, respectively, by polymerase chain reaction (PCR) amplification with the corresponding primers. These fragments of Cas12a variants were then cloned into restriction endonuclease-linearized dCas12a-A3A-Y130F or dCas12a-A3A-N57G to replace the original dLbCas12a, resulting in the construction of enCas12a-A3A-Y130F, RR-A3A-Y130F, RVR-A3A-Y130F, enCas12a-A3A-N57G, RR-A3A-N57G, and RVR-A3A-N57G expression vectors. dCas12a-ABE7 expression vector was constructed by cloning dCas12a fragment into the linearized Cas9-ABE using the ClonExpress MultiS kit (Vazyme). The TadA*8e fragment was amplified from TadA*7.10 in Cas9-ABE by several pair of primers to introduce eight mutations (A109S/T111R/D119N/H122N/Y147D/F149Y/T166I/D167N), and an additional V106W mutation was introduced to generate the TadA*8e-V106W fragment. Then, dCas12a-ABE8e and dCas12a-ABE8e-V106W expression vectors were constructed by cloning TadA*8e and TadA*8e-V106W fragments into the linearized dCas12a-ABE7 vector to replace the TadA-TadA*7.10 fragment, respectively. Expression vectors enCas12a-ABE8e-V106W, RR-ABE8e-V106W, and RVR-ABE8e-V106W were constructed by cloning three Cas12a variants into linearized dCas12a-ABE8e-V106W to replace dCas12a. Target-specific oligonucleotides were annealed and ligated into BpiI-linearized U6-sgRNA plasmid to generate corresponding sgRNA expression vectors. Target sequences were listed in Supplementary Data 2.
HEK293T cells (obtained from ATCC and preserved by our laboratory) were maintained in Dulbecco’s Modified Eagle Medium high glucose (HyClone) supplemented with 10% fetal bovine serum (Gibco). Cells were seeded in a 24-well plate at a density of approximately 1.6 × 105 cells per well to reach 70–90% confluence and transfected with 50 µL serum-free Opti-MEM (Gibco), which contained 3 µg polyethylenimine (PEI) (Sigma-Aldrich), 0.3 µg sgRNA expression vector and 0.7 µg base editor expression vector. Cells transfected with 50 µL serum-free Opti-MEM, which only contained 3 µg PEI (without vector), were served as untreated control. About 60 h after transfection, EGFP+ cells were sorted by FACS AriaIIU (Becton Dickinson), given that all base editor expression vectors contain EGFP fluorescence labels, and subjected to lysis in 10 µL lysis buffer with 1% NP40 (MP Biomedicals) and 50 µg mL−1 proteinase K (Tiangen).
MssI-linearized base editor vectors, including the T7 promoter, were used as templates to transcribe mRNA in vitro using the HiScribe T7 ARCA mRNA Kit (with tailing) (New England Biolabs), and mRNA was purified using the RNeasy MiniElute Cleanup Kit (Qiagen) in accordance with the manufacturer’s instructions. To produce sgRNAs, we amplified templates from the corresponding U6-sgRNA vectors, accompanied by the introduction of T7 promoter sequence, and then transcribed them using the HiScribe T7 Quick High Yield RNA Synthesis Kit (New England Biolabs). Transcribed sgRNAs were purified using the RNeasy MiniElute Cleanup Kit (Qiagen).
Porcine PA embryos were used in our microinjection, and oocyte collection, in vitro maturation, and parthenogenetic activation were conducted as previously described. Briefly, immature porcine oocytes, which were derived from ovaries obtained from a slaughterhouse, were matured in vitro. Mature oocytes with visible first polar bodies were then subjected to electrical activation to initiate PA development. Six hours after activation, the one-cell embryos were injected with RNA mixture of the base editor mRNA (150 ng µL−1) and sgRNA (50 ng µL−1) into the cytoplasm by the FemtoJet5247 microinjector (Eppendorf). The embryos were then cultured in vitro with PZM-3 medium for 6 days, followed by collection of single embryos for lysis in 5 µL lysis buffer.
Target genomic sites were PCR amplified using the lysate of HEK293T cells and porcine embryos by the Rapid Taq Master Mix (Vazyme), with primers flanking each examined target site. Sanger sequencing was then performed by IGE Biotechnology (Guangzhou, China), and the ratios of C-to-T and A-to-G conversions were calculated by the EditR software (https://moriaritylab.shinyapps.io/editr_v10/). The primers used to amplify the target genomic sequences are listed in Supplementary Data 2.
For target deep sequencing, primers containing Illumina forward and reverse adapters were used to amplify genomic regions spanning the target sites for 30 cycles of the first round of PCR using the Phanta Max Super-Fidelity DNA Polymerase (Vazyme), followed a second round with 12 cycles of PCR amplification with barcode-containing Illumina primers. The purified products of the second round of PCR amplicons with different tags were pooled and subjected to Annoroad Biotechnology (Beijing, China) for deep sequencing on the Illumina NovaSeq 6000 platform. The deep sequencing data were analyzed through the CRISPResso2 tool. Potential off-targets of selected target sites were predicted using the previous reported Cas-OFFinder (http://www.rgenome.net/cas-offinder/), with up to three mismatches and no bulge, and the PAM sequences were restricted to NTTN, TYCN, and TRTN. Once more than ten potential off-targets for one site were predicted, only ten were analyzed. The amplicons spanning the predicted off-targets with unique barcodes were pooled and used for deep sequencing as described above. Potential off-target sequences and primers used to amplify the on-target and off-target sites are listed in Supplementary Data 3.
Statistics and graphs were prepared using Prism v8 (GraphPad), and error bars indicated the mean with standard error of the mean (SEM). Statistical significance was determined using the corresponding methods described in figure legends, with p values of <0.05 considered statistically significant. Data represented the results of three independent replicate experiments unless mentioned specially, and sample sizes were indicated in figure legends.
Further information on research design is available in the Nature Research Reporting Summary linked to this article.
Supplementary Information Description of Additional Supplementary Data Supplementary Data 1 Supplementary Data 2 Supplementary Data 3 Reporting Summary-New | true | true | true |
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PMC9630337 | Sharif Rahmy,Sanket J. Mishra,Sean Murphy,Brian S. J. Blagg,Xin Lu | Hsp90β inhibition upregulates interferon response and enhances immune checkpoint blockade therapy in murine tumors | 20-10-2022 | heat shock protein 90 (hsp90),isoform-selective inhibitor,immune checkpoint blockade (ICB),prostate cancer,breast cancer,CDK4/6,interferon response,endogenous retrovirus | Response resistance to the immune checkpoint blockade (ICB) immunotherapy remains a major clinical challenge that may be overcome through the rational combination of ICB and specific targeted therapeutics. One emerging combination strategy is based on sensitizing ICB-refractory tumors with antagonists of 90kD heat shock protein (Hsp90) that target all four isoforms. However, pan-Hsp90 inhibitors are limited by the modest efficacy, on-target and off-tumor toxicities, and induction of the heat shock response (HSR) that overrides the effect of Hsp90 inhibition. Recently, we developed Hsp90β-selective inhibitors that were cytotoxic to cancer cells but did not induce HSR in vitro. Here, we report that the Hsp90β inhibitor NDNB1182 downregulated CDK4 (an Hsp90β-dependent client protein) and induced the expression of endogenous retroviral elements and interferon-stimulated genes. In syngeneic mouse models of prostate cancer and breast cancer, NDNB1182 significantly augmented the efficacy of ICB therapy. Furthermore, NDNB1182 showed superior tolerability to the pan-Hsp90 inhibitor Ganetespib in mice. Our findings provide evidence that Hsp90β inhibition is a potentially effective and safe regimen to combine with ICB to treat immunotherapy-refractory solid tumors. | Hsp90β inhibition upregulates interferon response and enhances immune checkpoint blockade therapy in murine tumors
Response resistance to the immune checkpoint blockade (ICB) immunotherapy remains a major clinical challenge that may be overcome through the rational combination of ICB and specific targeted therapeutics. One emerging combination strategy is based on sensitizing ICB-refractory tumors with antagonists of 90kD heat shock protein (Hsp90) that target all four isoforms. However, pan-Hsp90 inhibitors are limited by the modest efficacy, on-target and off-tumor toxicities, and induction of the heat shock response (HSR) that overrides the effect of Hsp90 inhibition. Recently, we developed Hsp90β-selective inhibitors that were cytotoxic to cancer cells but did not induce HSR in vitro. Here, we report that the Hsp90β inhibitor NDNB1182 downregulated CDK4 (an Hsp90β-dependent client protein) and induced the expression of endogenous retroviral elements and interferon-stimulated genes. In syngeneic mouse models of prostate cancer and breast cancer, NDNB1182 significantly augmented the efficacy of ICB therapy. Furthermore, NDNB1182 showed superior tolerability to the pan-Hsp90 inhibitor Ganetespib in mice. Our findings provide evidence that Hsp90β inhibition is a potentially effective and safe regimen to combine with ICB to treat immunotherapy-refractory solid tumors.
Novel immunotherapies have revolutionized the treatment of cancer patients in recent years. The broadest impact comes from immune checkpoint blockade (ICB) that reinvigorates anti-tumor cytotoxic T lymphocytes (CTLs) using antibodies against CTLA4 or PD1/PD-L1 and generates therapeutic responses across a variety of cancer types (1). However, some solid tumor types remain largely resistant to ICB therapy, including breast cancer and prostate cancer which represent the first and third most frequently diagnosed malignancies worldwide, respectively (2). Advanced prostate cancer (metastatic and castration-resistant) shows overwhelming de novo resistance to anti-CTLA4 or anti-PD1 therapies (3–6). When ipilimumab and nivolumab were combined in a preliminary phase II clinical trial (CheckMate 650), improved objective response rates were reported (25% and 10% for pre- and post-chemotherapy), yet inadequate response rate and strong adverse effect remain frustrating (7). For metastatic triple-negative breast cancer, two ICB drugs, atezolizumab and pembrolizumab, in combination with chemotherapy, have been approved by the FDA to treat PD-L1+ cases (8, 9). However, the majority of the patients remain nonresponsive to ICB therapy. In order to bring broad benefits to patients with advanced prostate cancer and breast cancer, therapeutic approaches to overcome the resistance and sensitize the disease to ICB are urgently needed. The 90kD heat shock protein (Hsp90) family functions as an evolutionarily conserved molecular chaperone to regulate protein homeostasis under physiological and stress conditions (10–13). Hsp90 family consists of four members: Hsp90α (inducible) and Hsp90β (constitutive) in the cytoplasm, glucose-regulated protein 94 (Grp94) in endoplasmic reticulum, and tumor necrosis factor receptor-associated protein-1 (Trap1) in mitochondria. Hsp90 proteins are responsible for the proper folding, disaggregation and intracellular trafficking of over 400 client proteins, including protein kinases, steroid hormone receptors, transcription factors, E3 ubiquitin ligases and more. Many of these clients control the hallmarks of cancer, therefore inhibition of Hsp90 may offer a unique benefit of co-targeting many oncogenic pathways (13). Various reports suggest that mutated and overexpressed oncoproteins rely more on the Hsp90 chaperone activity for proper folding, thus neoplastic cells may be more dependent on the function of Hsp90 chaperone for survival and proliferation than normal cells are, which could create a therapeutic window (14–17). Many cell types express Hsp90 on the cell surface or secrete Hsp90 into the extracellular space (18, 19), and often Hsp90 expressed on the cell surface is more abundant in cancer cells than normal cells (20). Tumor cells appear to be more sensitive to Hsp90 inhibition, because the Hsp90 complex in cancer cells is distinct from normal cells with its higher affinity binding state (14). Moreover, in approximately 50% of cancers especially those fueled by MYC, Hsp90 acts as a nucleating site to form functionally integrated complexes termed ‘epichaperome’, which render tumor cells more sensitive to Hsp90 inhibitors (Hsp90i) (17). Lastly, Hsp90i preferentially accumulate in tumor cells as compared with normal cells (21–23). Therefore, Hsp90 is an attractive target for developing drugs to treat malignancies, including prostate cancer and breast cancer (24, 25). Following the discovery of natural product inhibitors of Hsp90, geldanamycin (26) and radicicol (27), avid investment in the design, synthesis and evaluation of drug-like Hsp90i ensued (16, 28). So far, 18 small molecule drugs as pan-Hsp90 inhibitors (pan-Hsp90i) have entered clinical trials, but none has demonstrated satisfactory benefit-risk profile to be approved by the Food and Drug Administration (FDA) (29, 30). Pan-Hsp90i target the N-terminal domain (NTD) and bind competitively to the ATP binding site of all four Hsp90 isoforms. Challenges with current Hsp90i include limited efficacy, dose-limiting toxicities (DLTs), and various on-target and off-tumor toxicities. A major cause of the limited efficacy and DLTs is the induction of the pro-survival heat shock response (HSR) by pan-Hsp90 inhibition, because pan-Hsp90i trigger dissociation of heat shock factor 1 (Hsf1) from Hsp90 complex and Hsf1 subsequently enters nucleus and activates the transcription of Hsp27, Hsp40, Hsp70 and Hsp90 (31, 32), which counteracts the effect from the inhibitor. On the other hand, Hsp90i-associated hepatic, cardio and ocular toxicities may result from the disruption of clients. For example, the cardiac potassium channel human ether-a-go-go related gene (hERG) depends on Hsp90 for functional maturation, thus Hsp90i can cause deleterious effects on the hERG-related membrane potential (33). Interestingly, hERG is solely dependent on Hsp90α (34), suggesting that inhibition of Hsp90α (but not other isoforms) is likely to account more for the cardio and ocular toxicities. Moreover, Hsp90α and Hsp90β govern clientele and exert biological functions in non-redundant manners despite highly similar structures (35, 36), emphasizing the value of developing isoform-selective inhibitors. Using a structure-based molecular design and optimization approach, Blagg and colleagues developed various Grp94 inhibitors (37–40), Hsp90α inhibitors (Hsp90α-i) (41, 42), and Hsp90β inhibitors (Hsp90β-i) (43, 44). Among them, Hsp90β-i induce degradation of Hsp90β-dependent clients without concomitant degradation of Hsp90α clients or induction of the HSR (43, 44). Therefore, Hsp90β-i may overcome the obstacles encountered by pan-Hsp90i that have struggled in clinical trials. Emerging evidence shows the involvement of Hsp90 in tumor immunity and the potential of enhancing immunotherapy with Hsp90i, although the mechanisms remain inadequately characterized and all these studies used pan-Hsp90i. For example, Ganetespib induced type I interferon response genes, such as interferon-induced protein with tetratricopeptide repeats (IFIT), through an unknown mechanism and promoted tumor cell killing by autologous T cells in vitro and anti-CTLA4 immunotherapy in mouse model of colorectal cancer (45). In addition, Ganetespib and 17-AAG decreased PD-L1 transcription through destabilizing Hsp90 clients MYC and STAT3 in monocytes and tumor cells at the sub-cytotoxic concentration, and Ganetespib diminished PD-L1 level on MC38 tumor cells in vivo (46). It is not yet explored whether isoform-selective Hsp90i, especially Hsp90β-i, can sensitize prostate cancer and breast cancer to immunotherapy, which is the central question for the current study to address.
We first conducted in silico analysis to support the proposition of inhibiting Hsp90β to enhance ICB therapy. From a phase 2 clinical trial of ipilimumab (anti-CTLA4) in 30 patients with metastatic castration-resistant prostate cancer (47), we retrieved the expression values of HSP90AB1 (encoding Hsp90β) from the published RNA-seq data of the patient samples in the study (n=18) and separated the cohort to HSP90AB1high and HSP90AB1low groups. We compared the two groups for PSA progression free survival (PFS) and overall survival (OS). HSP90AB1high group showed significantly worse outcome than the HSP90AB1low group under ipilimumab therapy ( Figure 1A ). Next, we noticed that the KMplot tool recently gathered the survival data for patients across various cancer types treated with ICB therapies (48), so we used KMplot to compare the OS of HSP90AB1high and HSP90AB1low patients for either anti-PD1 treatment or anti-PD-L1 treatment. In both regimens, patients with HSP90AB1high tumors showed significantly shorter OS ( Figure 1B ). The Blagg laboratory recently reported the structure-based rational development of the 2H-isoquinolin-1-one based series of Hsp90β-i (44). To enhance the solubility of these Hsp90β-i compounds for in vivo application, Blagg’s group replaced the solvent exposed fragment (cyclohexanolamine) to reduce intermolecular pi-stacking, and synthesized the newest Hsp90β-i, NDNB1182 ( Figure 1C ) (Mishra et al, manuscript under preparation). Like other 2H-isoquinolin-1-one series of Hsp90β-i, NDNB1182 was evaluated for the binding affinities against the four Hsp90 isoforms by measuring the ability to competitively displace FITC-labeled geldanamycin (a pan-Hsp90i) in a fluorescence polarization (FP) assay (44, 49). NDNB1182 exhibited improved selectivity (over 150-fold) for the Hsp90β isoform over the highly identical Hsp90α isoform ( Figure 1C ). While pan-HSP90i luminespib reduced expression of the HSP90α-selecitve client protein Src (42, 50), NDNB1182 did not alter Src levels ( Figure 1D ). The purity of the compound was confirmed to be >95% by high-performance liquid chromatography (HPLC) ( Figure 1E ). We performed the resazurin assay to evaluate the cytotoxicity of NDNB1182 against murine colorectal cancer cell line MC38, murine prostate cancer cell lines Pten-CaP8 and RM1, and murine Lewis lung carcinoma LLC. The IC50 values for all the lines fell below 100nM and did not show significant differences among the lines (P = 0.0889, Figure 1F ). As comparison, the effect of NDNB1182 was tested on two normal cell types, mouse prostate primary epithelial cells (grown from the dissociated prostate glands of wild type C57BL/6 mice) and the mouse fibroblast cell line L cells (ATCC, CRL-2648). IC50 of NDNB1182 was 430nM for normal prostate cells and 1255nM for L cells ( Figure 1G ), which were significantly higher than the IC50 values of the four mouse prostate cancer cell lines. Next, we plotted HSP90AB1 expression levels in various human prostate lineage cell lines at the Depmap portal. The result showed that various prostate cancer tissue-derived cell lines (MDAPCA2B, VCAP, NCIH660, LNCAP, 22Rv1, PC3) expressed HSP90AB1 at higher levels than the normal-like or ectopically transformed prostate cell lines (PRECLH, WPE1NA22, P4E6, SHMAC4, SHMAC5) ( Figure 1H ). Overall, these results support that Hsp90β is a promising target for prostate cancer and suggest that NDNB1182 selectively targets Hsp90β.
The pan-Hsp90i Ganetespib was reported to induce IFIT1 expression at 125nM and higher (45). To examine whether isoform-selective Hsp90α-i or Hsp90β-i has this activity, we treated MC38 with Ganetespib, Hsp90α-i 12h (42) and NDNB1182 for 6 hours. Hsp90α inhibition induced no increase in Ifit1 expression, whilst Ganetespib and NDNB1182 both significantly increased Ifit1 expression with NDNB1182 inducing Ifit1 by a higher magnitude ( Figure 2A ). We further confirmed the dose-dependent induction of IFIT1 expression by NDNB1182 in human prostate cancer cell line DU145 ( Figure 2B ). Mbofung et al. did not identify the mechanism for Ganetespib to induce interferon-stimulated genes (ISGs), which is addressed in our study. We previously showed that Hsp90β-i compounds led to degradation of Hsp90β-dependent clients including CDK4 but did not trigger the undesired HSR such as HSF1 upregulation (43, 44). NDNB1182 was confirmed to induce a dose-dependent decline of CDK4 expression, but no increase of HSF1 ( Figure 2C ). Our result reinforced that CDK4/6 are Hsp90β-selective clients (51, 52). Interestingly, CDK4/6 inhibition was shown to reduce the expression of DNMT1 (encoding DNA methyltransferase 1), an E2F target gene, resulting in hypomethylation of the genome and activation of the expression of endogenous retroviral (ERV) elements and ISGs to enhance tumor antigen presentation and ultimately anti-tumor immunity (53). Taking these together, we hypothesized that NDNB1182 could achieve an equivalent effect as CDK4/6 inhibitor (like Palbociclib) to stimulate ERV and anti-viral ISG expression. To test this, we treated MC38 with Ganetespib and NDNB1182 and examined the expression of a list of murine ERV elements (54). Both inhibitors stimulated the levels of Ifit1 and ERV genes AblMLV1 and EndoPP1 ( Figure 2D ). Interestingly, we observed that lower confluence of MC38 cells corresponded to higher Ifit1 induction by both Hsp90 inhibitors ( Figures 2A, D ). This observation may be related to the DNA methylation dynamics during cell cycle (see Discussion). We expanded the treatment and detection to more syngeneic cell lines: the prostate cancer cell line PPS that we developed from the PB-Cre+ PtenL/L p53L/L Smad4L/L transgenic mouse model (55), and a mammary cancer cell line PyMT-7160 that we established in this study from the MMTV-PyMT (mouse mammary tumor virus promoter driven polyoma middle T-antigen) transgenic mice with autochthonous mammary adenocarcinoma (56). In both cell lines, NDNB1182 induced the expression of Ifit genes and various ERV elements (AblMLV1/2, EcoMLV, EndoPP1/2, MLV), although the exact fold changes differed ( Figures 2E, F ). Furthermore, both NDNB1182 and Palbociclib induced Ifit1 expression in PPS ( Figure 2G ). Ifnb1 (encoding interferon β1, a major type I interferon) was upregulated significantly in MC38 cells treated with Ganetespib and NDNB1182 ( Figure 2H ). These results demonstrate that NDNB1182, by inhibiting Hsp90β but not other isoforms, can downregulate CDK4/6 expression, activate ERV elements and stimulate the interferon response in cancer cells. This trait prompted us to test whether NDNB1182 could enhance ICB therapy.
We tested the anti-tumor effect of NDNB1182 in two prostate cancer syngeneic models, Myc-CaP and PPS. Myc-CaP was derived from c-myc transgenic mice in the FVB/N background (57) and responded poorly to ICB therapy (58). We confirmed that Myc-CaP failed to shrink under anti-PD1 plus anti-CTLA4 ICB treatment ( Figure 3A ). In this model, neither NDNB1182 (dosed at 50mg/kg, daily) nor Ganetespib (dosed at 25mg/kg, daily) had a significant impact on tumor growth ( Figure 3B ). Nevertheless, this model provided promising results for the toxicity profile of NDNB1182, because in the same experiment while Ganetespib showed significant toxicity leading to 80% (4 out of 5) mortality within 13 days of treatment, mice treated with NDNB1182 at two-fold of the dose showed no significant change of survival ( Figure 3C ). PPS tumors grown in C57BL/6 background responded partially to ICB monotherapy (60). We treated PPS-carrying animals with vehicle control, NDNB1182, ICB (anti-PD1 + anti-CTLA4), or combination of NDNB1182 and ICB. Therapy with NDNB1182 or ICB each showed partial response, but the combination achieved remarkably enhanced efficacy ( Figure 3D ). Immunohistochemistry (IHC) staining of CD8α demonstrated that NDNB1182 alone induced a moderate increase of CD8+ T cell infiltration to the tumors, ICB alone had no effect, but the NDNB1182 plus ICB combination dramatically augmented the infiltration of CD8+ T cells ( Figure 3E ). Importantly, the animal body weight was not affected by NDNB1182 or NDNB1182 plus ICB treatments, showing low toxicity by NDNB1182 monotherapy or the combination treatment ( Figure 3F ). Furthermore, vehicle and NDNB1182 treated mice showed no discernable histological differences in the liver ( Figure 3G ) or the spleen (data not shown). NDNB1182 also caused no increase in apoptosis (as indicated by positive cleaved caspase 3 staining) in the liver or spleen ( Figure 3H ). These results provide further support for the safety of NDNB1182 at the dosage used. The results from Myc-CaP and PPS models connect the anti-tumor activity of NDNB1182 with the ICB response of the models and illustrate the potential of enhancing ICB therapy with Hsp90β inhibition.
To test the activity of NDNB1182 to enhance immunotherapy in another tumor type, we orthotopically injected FVB/N female mice with PyMT-7160 mammary cancer cells and implemented a similar design of single and combination treatments. NDNB1182 and ICB each had moderate impact on tumor growth, but the NDNB1182 and ICB combination showed dramatic anti-tumor activity ( Figure 4A ). We dissociated the tumors at the endpoint and quantified the CTLs (CD45+ CD8+) and antigen-presenting dendritic cells (DCs, CD11c+ MHC-II+) with flow cytometry. We quantified CD11c+ MHC-II+ DCs because these cells may play an important role in presenting the tumor antigens to activate the T cell immunity. Consistent with the anti-tumor efficacy, only the NDNB1182 plus ICB treatment stimulated the tumor infiltration of both CTLs ( Figures 4B, C ) and DCs ( Figures 4D, E ) significantly compared with the control. We further confirmed that tumors treated with ICB plus NDNB1182 expressed Ifit genes and ERV elements MLV and EcoMLV at the highest level compared with other treatment regimens ( Figure 4F ). These results support that the combination of Hsp90β inhibition and ICB offers the most potent control on breast cancer progression through ERV and interferon response activation and anti-tumor immunity reprograming.
Most previous studies regarded Hsp90 chaperone as one machinery and inhibited Hsp90 activities in cancer models and clinical trials with pan-Hsp90i, which may largely account for the disappointing clinical performance of Hsp90 inhibition in cancer treatment so far. Because different Hsp90 isoforms have non-redundant functions executed by recognizing different sets of client proteins, targeting all isoforms is neither necessary nor beneficial. Here, we have challenged this paradigm by reporting, for the first time, the in vivo anti-tumor activity of Hsp90β-i and its potential as an immunotherapy sensitizer. Specifically, we made the following discoveries: (1) the new Hsp90β-i NDNB1182 demonstrated high selectivity toward Hsp90β over the other three Hsp90 isoforms and killed cancer cells at the lower 100nM IC50 level; (2) NDNB1182 induced the expression of various ERV elements and Ifit genes across different cancer cell lines; (3) NDNB1182 significantly augmented the efficacy from ICB therapy in the PPS prostate cancer and PyMT-7160 breast cancer models; (4) NDNB1182 displayed better tolerability than the pan-Hsp90i Ganetespib and did not cause mouse body condition deterioration or rapid weight loss when dosed at tumor-restricting levels. Pan-Hsp90i as cancer monotherapy in clinical trials has encountered many challenges of limited efficacy and DLTs, leading to the dampened interest in oncological targeting of Hsp90 (29, 30). For example, a phase II trial using AUY922 in patients with metastatic gastrointestinal stromal tumor only showed modest anti-tumor effect but caused significant ocular toxicity (61). However, recent publications linking Hsp90 function with cancer immune modulation and showing how Hsp90 inhibition may enhance cancer immunotherapy in preclinical models have revived the interest in the combination of Hsp90i and ICB in the clinic (62, 63). For example, there are at least two ongoing Phase I trials that combine Hsp90i (XL888, TAS-116) and anti-PD1 in advanced gastrointestinal cancers (NCT03095781, NCT04999761). Nevertheless, the on-target toxicities from inhibiting all Hsp90 isoforms remain a concern and may lead to the difficulty in dose management of the combination treatment (63). This reasoning further highlights the significance of developing isoform-selective Hsp90i, which may still achieve the desired efficacy by targeting the isoform-dependent oncoprotein clients while avoiding the toxicities associated with the disruption of other clients, such as Hsp90α client hERG (34). To this end, we believe that Hsp90β emerges as the most promising isoform for the combinatorial targeting, because Hsp90β-i can induce degradation of Hsp90β-dependent clients that have well-established oncogenic functions (e.g. CDK4, CDK6, CXCR4, BRAF, HER2) without concomitant degradation of Hsp90α clients or induction of the HSR (43, 44). Our study confirms that NDNB1182 is effective in vitro and in vivo without causing discernable toxicities in mice (by contrast, Ganetespib was lethal even when administered at the half dose), electing NDNB1182 as a promising candidate for further preclinical validation and clinical development. Our result showing that NDNB1182 activated the expression of ERV and IFIT genes is consistent with the previous finding with Ganetespib (45), suggesting the inhibition of Hsp90β among all four isoforms likely contributed the most to the activity of Ganetespib in the antiviral-like response in cancer cells. The CDK4 downregulation by NDNB1182 provides a logical mechanistic connection from Hsp90β inhibition to ERV and IFIT overexpression, because CDK4 is a client for Hsp90β and it is known that CDK4/6 inhibition can reactivate ERV expression and interferon response in cancer cells through downregulating DNMT1 expression and DNA methylation (53). CDK4/6 promotes Rb phosphorylation, leading to the release of E2F and subsequent transcriptional activation of E2F targets, including DNMT1. CDK4/6 inhibitors hinder the G1/S transition by inhibiting Rb phosphorylation and E2F release. Hsp90i may achieve the same effect by diminishing CDK4/6 protein level in the cells. Therefore, either by preventing Rb phosphorylation through CDK4/6 activity inhibition or by reducing the available pool of CDK4/6 through Hsp90 inhibition, the end result is a reduction in DNMT1 protein and subsequent DNA hypomethylation. We noticed that the effect of NDNB1182 on Ifit1 induction was inversely related to cell confluence during the inhibitor treatment ( Figures 2A, D ). Because DNMT1 as the maintenance methyltransferase is responsible for the preservation of 5-methylcytosine in the genome during DNA replication (64), we speculate that a less confluent cell culture has higher portion of dividing cells than a much more confluent cell culture, therefore, the effect of NDNB1182 on DNA hypomethylation and Ifit1 upregulation became more manifested in less confluent cells. Further experiments will help verify this proposition. In addition, we notice that the specific ERV elements activated by NDNB1182 differed among the cell lines in our study, and it is probably caused by the distinct epigenetic status of these ERV elements in these cell lines. Nonetheless, all the cell lines showed IFIT upregulation, thus producing equivalent interferon response signaling output to cooperate with immune checkpoint inhibition to reinvigorate T cell immunity. Indeed, efficacious immunotherapy depends on a potentiated type I interferon response (65, 66). There are a few limitations in our study. First, the in vivo results were generated only using syngeneic models. While these models have served well as preclinical platforms for immunotherapy discovery and development, other types of cancer models such as genetically engineered mouse models and humanized mouse models with patient-derived tumors and reconstituted human immune system will provide important confirmative evidence. Second, the immunotherapy-sensitizer activity of NDNB1182 was only tested together with ICB therapy. Other immunotherapy modalities such as oncolytic virus and CAR-T therapy may also benefit from the combination with Hsp90β inhibition, especially in solid tumors. Third, our study does not exclude other immune modulatory mechanisms by Hsp90β inhibition besides the CDK4-ERV-IFIT axis that may also contribute to the combinatorial efficacy from NDNB1182 plus ICB treatment, which clearly warrants further investigations. Fourth, NDNB1182 was administered via intraperitoneal injection in our study, which would be inconvenient for the clinical application. Our teams are working on formulations that will allow oral delivery of NDNB1182 (or its improved analog). Lastly, given that hypomethylation and ERV may contribute to genomic instability in cancer (67, 68), the application of Hsp90β inhibition in clinical cancer therapy may not be suitable for certain patients (for example, young patients). In conclusion, our results establish the preclinical evidence to support the rational combination of Hsp90β antagonists and immunotherapy in the treatment of intractable solid tumors, illuminating a clinical path for the better outcome of many cancer patients.
Murine cell lines Pten-CaP8, RM1, LLC, and Myc-CaP and human cell line DU145 were purchased from ATCC and cultured in medium types recommended by ATCC. MC38 was purchased from kerafast and cultured in the recommended medium. PPS was developed from a spontaneous prostate tumor of PB-Cre+ PtenL/L p53L/L Smad4L/L transgenic mice (55). PyMT-7160 was developed from a spontaneous mammary tumor of the FVB/N-Tg(MMTV-PyVT)634Mul/J mice (Jackson Laboratory, 002374). PPS and PyMT-7160 were cultured in DMEM (GE Healthcare, SH30243.FS) supplemented with 10% fetal bovine serum (GE Healthcare, SH30396.03) and 100U/ml penicillin-streptomycin (Caisson Labs, PSL01). All the cell lines were cultured at 37°C in a humidified incubator with 5% CO2. All cells were tested for mycoplasma-free status using a Mycoplasma Assay Kit (Agilent Technologies, 302109).
Hsp90α-i 12h (42) and Hsp90β-i NDNB1182 (Mishra et al, manuscript under preparation) were synthesized by the Blagg laboratory using the cited protocols. The identity of the chemicals was confirmed by high resolution mass spectrometry and nuclear magnetic resonance, while the purity of the compounds was confirmed to be >95% by high-performance liquid chromatography (HPLC). Ganetespib (MedChem Express, HY-15205) and Palbociclib (LC Laboratories, P-7788) were purchased.
C57BL/6J males (Jackson Laboratory, 000664) and FVB/NJ males and females (Jackson Laboratory, 001800) were purchased at 5 weeks of age and used for experiments after one week of acclimation. All animals were maintained under pathogen-free conditions and cared for in accordance with the International Association for Assessment and Accreditation of Laboratory Animal Care policies and certification.
RNA was isolated from cells and tissues using the EZ-10 Spin Column Total RNA Miniprep Kit (Bio Basic, BS1361) according to the manufacturer protocol. After RNA extraction, cDNA was synthesized using the All-in-one 5x RT MasterMix (ABM, G592). qPCR reactions were performed with the 2X SYBR Green Master mix (Bimake, B21203) and run on the CFX Connect Real-Time PCR Detection System (Bio-Rad, 1855201). Gapdh was used for normalization. Student’s t-test was performed based on the ΔΔCT values. Unless otherwise specified, n=3 biological replicates per group were used for all qRT-PCR experiments. Primers were purchased from Eurofins Genomics. Primer sequences are listed in Supplementary Table 1 .
Cell and tissue samples were lysed in RIPA buffer containing protease inhibitor (Bimake, B14012) and phosphatase inhibitor (Bimake, B15002). Protein concentration was determined using the Pierce BCA Protein Assay Kit (Thermo Scientific, 23225). After determining concentration, 30µg of protein lysate was boiled at 95°C for 5 minutes in Laemmli buffer (Bio-Rad, 161-0747), and subsequently run on SDS-PAGE. Gels were transferred to PDVF membranes (Bio-Rad, 1620177) using the Bio-Rad Trans-Blot Turbo Transfer System. After transfer, membranes were blocked for 1 hour in 5% fat free milk in TBS-T. After blocking, membranes were incubated in primary antibodies at concentrations according to manufacturer specifications for either 2 hours at room temperature or 16 hours at 4°C, washed three times in TBS-T, incubated for 1 hour at room temperature with HRP-linked secondary antibody (Cell Signaling Technologies, αRabbit cat#7074, αMouse cat#7076), and then washed again three times in TBS-T. Subsequently membranes were imaged using Clarity ECL (Bio-Rad, 1705060) on the ChemiDoc XRS+ imager (Bio-Rad, 1708265). The antibodies used are listed in Supplementary Table 2 .
Tumors were minced and digested in DMEM with 10% FBS and 1 mg/ml collagenase IV (STEMCELL Technologies, 07427) at 37°C for 1 h, followed by passing through 40μm strainers. Erythrocytes were depleted via hypotonic lysis. Cells were treated with mouse Fc-shield anti-CD16/CD32 (Tonbo Biosciences, 70-0161) for 15 minutes, and stained with primary fluorophore-conjugated antibodies for 30 minutes. Cells were washed twice and resuspended in a buffer containing DAPI (viability dye) and analyzed on the Beckman Coulter Cytoflex S cytometer. The antibodies used are listed in Supplementary Table 2 .
Tumors and livers/spleens were fixed in 10% neutral buffered formalin (VWR, 16004-128) for 24 hours and prepared as paraffin-embedded 5 µm sections. Antigen retrieval was performed using sodium citrate buffer (pH 6.0) at 95°C for 30 minutes followed by 115°C for 10 seconds. After antigen retrieval, endogenous peroxidase activity was blocked using 3% hydrogen peroxide for 10 minutes. Subsequently samples were blocked in 5% normal goat serum in TBS-T for 30 minutes and incubated with primary antibody anti-mouse CD8 (Cell Signaling Technology, 98941) or cleaved caspase 3 (Cell Signaling Technology, 9661) in a humidified chamber at 4°C for 16 hours. After washing, the VECTASTAIN Elite ABC-HRP Kit (Vector Laboratories, PK-6101) was used as secondary antibody and signal detection. Counterstain was performed using Mayer’s hematoxylin for 30 seconds. Imaging was performed using a Leica Aperio scanscope with 20× objective. At least 6 biological replicates were counted to quantify tumor-infiltrating CD8+ T cells or cleaved caspase 3 staining. Hematoxilin and eosin stain (H&E) Livers were fixed in 10% neutral buffered formalin for 24 hours and prepared as paraffin-embedded 5 µm sections. After rehydration, samples were stained for 8 minutes in Mayer’s hematoxilin. Excess dye was washed off in water for 8 minutes. Samples were immersed in 95% ethanol and stained in Eosin Y 1% (VWR, 101432-132) for 3 minutes. Excess dye was washed off in 95% ethanol after which slides were dehydrated and coverslipped and imaged using the Aperio scanscope.
Syngeneic prostate tumor cell lines Myc-CaP or PPS was injected subcutaneously into 6-week-old FVB/NJ males or C57BL/6J males, respectively. Syngeneic mammary tumor cell line PyMT-7160 was injected to the mammary fat pads of 6-week-old FVB/NJ females. Tumors were measured with calipers and volumes were calculated using the formula length × width2 ÷ 2. Mice with tumors reaching the pre-specified volume range were randomized to receive the following therapies: anti-PD-1 (clone RMP1-14, Leinco Technologies, P362) and anti-CTLA4 (clone 9H10, Leinco Technologies, C1614) were injected intraperitoneally at 10mg/kg, twice per week; NDNB1182 or Ganetespib was dissolved in 10% DMSO, 40% polyethylene glycol 300, 5% Tween-80 and 55% ddH2O and injected intraperitoneally at 50mg/kg and 25mg/kg daily, respectively. To select the dosage of NDNB1182 for animal experiments, a maximum tolerated dose (MTD) pilot experiment was conducted in five 10-week old FVB/NJ mice with escalated dosages. After 10 days of daily treatment, the dose of 50mg/kg was the highest dose that caused no significant body weight loss or body condition deterioration, thus this dose was defined in our study as the desired dose to administer NDNB1182 in mice. For Ganetespib, 25mg/kg daily was used based on previous reports (69, 70). All treatments were continued until the specified experimental endpoints were reached.
Statistical analyses were performed using GraphPad Prism v8.0. Unless otherwise mentioned, all data were presented as mean ± SEM (standard error of the mean). Sample sizes, error bars, P values, and statistical methods were denoted in the figures or figure legends. Statistical significance was defined as P < 0.05.
The original contributions presented in the study are included in the article/ Supplementary Material . Further inquiries can be directed to the corresponding author.
The animal study was reviewed and approved by Institutional Animal Care and Use Committee at University of Notre Dame.
SR: Conceptualization, investigation, methodology, data curation, formal analysis, validation, visualization, funding acquisition, writing – original draft. SJM: Resources. SM: Investigation. BB: Supervision. XL: Conceptualization, formal analysis, visualization, supervision, project administration, funding acquisition, writing – original draft, writing – review & editing. All authors contributed to the article and approved the submitted version.
SR was supported by an Interdisciplinary Interface Training Program Grant from the Walther Cancer Foundation and Harper Cancer Research Institute at University of Notre Dame. XL was supported by National Institutes of Health grant R01CA248033, Department of Defense CDMRP PCRP grants W81XWH2010312 and W81XWH2010332, and Boler Family Foundation endowment at University of Notre Dame.
We would like to thank the Lu lab members for constructive suggestions. We are grateful for the support from core facilities used in this study, especially Freimann Life Science Center and Tissue Repository at Harper Cancer Research Institute.
The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher. | true | true | true |
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PMC9630352 | Aleksandra Boikova,Megan J. Bywater,Gregory A. Quaife-Ryan,Jasmin Straube,Lucy Thompson,Camilla Ascanelli,Trevor D. Littlewood,Gerard I. Evan,James E. Hudson,Catherine H. Wilson | HRas and Myc synergistically induce cell cycle progression and apoptosis of murine cardiomyocytes | 20-10-2022 | Myc (c-Myc),HRas gene,cardiomyocyte,proliferation,cell-cycle | Aim Adult mammalian cardiomyocytes are incapable of significant proliferation, limiting regeneration after myocardial injury. Overexpression of the transcription factor Myc has been shown to drive proliferation in the adult mouse heart, but only when combined with Cyclin T1. As constitutive HRas activity has been shown to stabilise Cyclin T1 in vivo, we aimed to establish whether Myc and HRas could also act cooperatively to induce proliferation in adult mammalian cardiomyocytes in vivo. Methods and results Using a genetically modified mouse model, we confirmed that constitutive HRas activity (HRasG12V) increased Cyclin T1 expression. HRasG12V and constitutive Myc expression together co-operate to drive cell-cycle progression of adult mammalian cardiomyocytes. However, stimulation of endogenous cardiac proliferation by the ectopic expression of HRasG12V and Myc also induced cardiomyocyte death, while Myc and Cyclin T1 expression did not. Conclusion Co-expression of Cyclin T1 and Myc may be a therapeutically tractable approach for cardiomyocyte neo-genesis post injury, while cell death induced by HRasG12V and Myc expression likely limits this option as a regenerative therapeutic target. | HRas and Myc synergistically induce cell cycle progression and apoptosis of murine cardiomyocytes
Adult mammalian cardiomyocytes are incapable of significant proliferation, limiting regeneration after myocardial injury. Overexpression of the transcription factor Myc has been shown to drive proliferation in the adult mouse heart, but only when combined with Cyclin T1. As constitutive HRas activity has been shown to stabilise Cyclin T1 in vivo, we aimed to establish whether Myc and HRas could also act cooperatively to induce proliferation in adult mammalian cardiomyocytes in vivo.
Using a genetically modified mouse model, we confirmed that constitutive HRas activity (HRasG12V) increased Cyclin T1 expression. HRasG12V and constitutive Myc expression together co-operate to drive cell-cycle progression of adult mammalian cardiomyocytes. However, stimulation of endogenous cardiac proliferation by the ectopic expression of HRasG12V and Myc also induced cardiomyocyte death, while Myc and Cyclin T1 expression did not.
Co-expression of Cyclin T1 and Myc may be a therapeutically tractable approach for cardiomyocyte neo-genesis post injury, while cell death induced by HRasG12V and Myc expression likely limits this option as a regenerative therapeutic target.
The adult heart is one of the least regenerative organs of the human body. Shortly after birth of a mammal, cardiomyocytes exit the cell cycle and are subsequently characterised by a reduced rate of turnover (<1% per year in humans) (1). Consequently, if the adult mammalian heart is damaged (e.g., myocardial infarction), the default response is to replace the lost cardiomyocytes with non-contractile fibrotic scar tissue and cardiomyocyte hypertrophy which frequently lead to heart failure. A number of strategies have been proposed to promote myocardial regeneration post damage, including the direct injection of stem cells or stem cell-derived cardiomyocytes, direct reprogramming of non-myocytes into cardiomyocytes and endogenous cardiomyocyte proliferation (2). Endogenous cardiomyocyte proliferation requires driving the resident quiescent cardiomyocytes into a productive mitogenic cell cycle. Genetic lineage tracing studies in the regenerative neonatal mouse and zebrafish hearts indicate that the majority of newly generated cardiomyocytes are derived from endogenous cardiomyocyte proliferation rather than differentiation from a mesenchymal progenitor (3–5). These studies suggest that forced cardiomyocyte proliferation is a viable strategy for myocardial regeneration and several potential factors capable of reactivating endogenous proliferation of cardiomyocytes in adult hearts have been identified, for example, inhibition of Hippo protein kinase signalling, enforced expression of cell cycle regulators, inactivation of thyroid hormone signalling, and hypoxia, all induce regeneration (6–10). We have recently shown that combined ectopic expression of Myc and Cyclin T1 can lead to extensive cardiomyocyte proliferation in the mouse heart (11), although exploitation of the therapeutic potential requires further exploration. Myc is a pleiotropic transcription factor that, amongst other activities, regulates cell growth and proliferation in mammalian cells (12). Consequently, Myc expression in normal cells is tightly regulated. Following damage in a regenerative tissue, the release of mitogens stimulates a transient increase in the short-lived Myc protein followed by a transient proliferative response (13). In contrast, Myc transcriptional programmes decrease during postnatal cardiac maturation and fail to reactivate post-infarction (14). Moreover, even when Myc expression is driven ectopically in the adult mammalian heart, it is unable to activate transcription of many target genes and cardiomyocytes remain almost entirely resistant to proliferation (11, 15). We previously established that Myc-driven transcription, and consequently cell proliferation, are critically dependent on the level of Cyclin T1 in cardiomyocytes (11). Cyclin T1 strongly associates with CDK9 to form the positive transcription elongation factor b (P-TEFb) (16) that phosphorylates paused RNA PolII and the elongation factors DSIF and NELF, leading to productive transcriptional elongation (16, 17). Both components of P-TEFb, Cyclin T1 and CDK9 are tightly regulated by various transcriptional and post-transcriptional mechanisms (18, 19). P-TEFb is also dynamically controlled by an association with an inactivation complex (7SK snRNA, Larp7, MEPCE, and HEXIM) (20, 21). Unlike other cyclin-dependent kinases, CDK9 protein stability is not cell cycle dependent (22), but is primarily determined by binding to Cyclin T1 (23, 24). The level of the Cyclin T1 protein is thus the key factor in controlling the amount of the P-TEFb complex in a cell (23, 24). During postnatal cardiac maturation in mice the level of P-TEFb and phosphorylated RNA PolII steadily decline (11). Transgenic overexpression of Cyclin T1 in the heart leads to increased levels of both CDK9 and phosphorylated RNA PolII and, when expressed throughout development, leads to cardiac hypertrophy (11, 25, 26). The RAS proteins (K-RAS, N-RAS, and H-RAS in humans) are small GTPases that function as molecular switches, cycling between their “off” GDP-bound and “on” GTP-bound states in response to mitogenic signalling (27). Depending on the cellular context, Ras can activate several downstream pathways that regulate protein synthesis, cell growth, survival, and cell motility (28), in the heart, Ras activation leads to reversible cardiac hypertrophy (29, 30). Hypertrophy, be it transgenically induced by constitutively active RAS or secondary to pressure overload or other hypertrophic stimuli, is accompanied by an increase in total RNA content. This is a result of increased P-TEFb activity and phosphorylation of RNA Pol II that increases total RNA and protein synthesis (25, 31). Using a genetically modified mouse model that combines the elevated expression of HRasG12V and an ectopically switchable Myc allele (MycERT2), we show that expression of constitutively active HRasG12V in the heart leads to increased P-TEFb levels that, when combined with constitutive Myc activity, drives signs of cardiomyocytes proliferation in vivo.
All animals were kept under SPF conditions. Mice were maintained on regular diet in a pathogen-free facility on a 12 h light/dark cycle with continuous access to food and water. All mice were euthanised under the schedule 1 method of cervical dislocation. Mouse strain Tg(Myh6-tTA)6Smbf/J was obtained from the Jackson Laboratory, and mouse strain Tg(tetO-HRAS)65Lc/Nci was obtained from the NCI Mouse Repository. Mouse strain R26CAG–c–MycER was produced in house. 1 mg of (Z)-4-hydroxytamoxifen (4-OHT; Sigma, H7904) was i.p injected into adult mice in 10% ethanol and vegetable oil (5 mg/ml), and tissues were collected 4 h post injection. 1 mg of tamoxifen (Sigma, T5648) was i.p injected into adult mice in 10% ethanol and vegetable oil (10 mg/ml) twice over a 24-h period and tissues collected at 24 h post initial i.p. injection. Animals requiring doxycycline treatment were supplied with drinking water containing doxycycline hyclate 100 mg/L (Sigma D9891) in water containing 3% sucrose to increase palatability, this was replenished two times per week in light-protected bottles. Ear biopsies were collected from 2 to 5 weeks old mice and genotyped by PCR with the following oligonucleotide primers: for Rosa26CAG; Universal forward: 5′-CTCTGCTGCCTCCTGGCTTCT-3′ Wild-type reverse: 5′-CGAGGCGGATCACAAGCAATA-3′ and CAG reverse: 5′-TCAATGGGCGGGGGTCGTT-3′. Primers for the TetO-HRas allele were; H003: 5′-TGAAAGTCGAGCTCGGTA-3′ and H004: 5′-CCCGGTGTCTTCTATGGA-3′. Primers for the Myh6-tTA allele were; oIMR8746: 5′-CGCTGTGGG GCATTTTACTTTAG-3′ and oIMR8747: 5′-CATGTCCAGAT CGAAATCGTC-3′.
Snap-frozen animal tissues were ground on liquid nitrogen and proteins extracted in buffer containing 1% SDS, 50 mM Tris pH 6.8, and 10% glycerol on ice for 10 min. Lysates were boiled for 10 min, followed by sonication for 15 min at room temperature. Total protein (50 μg) was electrophoresed on an SDS–PAGE gel and transferred onto immobilon-P (Millipore) membranes. These were then blocked in 5% non-fat milk and incubated with primary antibodies overnight at 4°C. Secondary antibodies were applied for 1 h followed by chemiluminescent visualisation (Thermo Scientific, 32106 or Millipore, WBKLS0500). Immunoblots were either developed on Fuji RX X-ray film and scanned on an Epson Perfection V500 Photo flatbed scanner or visualisation was performed on LiCOR Odyssey Fc. Protein quantifications were performed on the LiCOR Odyssey Fc. Primary antibodies: GAPDH (D16H11) XP® (Cell Signaling Technology, 5174, used at 1:5,000), phospho-Rpb1 CTD (Ser2; E1Z3G; Cell Signaling Technology, 13499, used at 1:2,500), CDK9 (C12F7; Cell Signaling Technology, 2316, used at 1:1,000), Cyclin T1 (D1B6G; Cell Signaling Technology, 81464, used at 1:1,000), p-ERK is phospho-p44/42 MAPK (Thr202/Tyr204, Cell Signaling Technology, 9101, used at 1:500), anti-rabbit IgG HRP (Sigma, A0545, used at 1:10,000). Sample loading was normalised for equal protein content. Expression of GAPDH is included as a confirmation of efficiency of protein isolation and comparable loading between individual tissue samples.
Immunohistochemistry was performed on formalin-fixed paraffin-embedded 4 μm sections. Sections were de-paraffinized and rehydrated, followed by antigen retrieval by boiling in 10 mM citrate buffer (pH 6.0) for 10 min. Endogenous peroxidase activity was blocked with 0.3% hydrogen peroxide for 30 min. Sections were then treated with rabbit VECTASTAIN Elite ABC horseradish peroxidase kit (Vector Laboratories, PK-6101) following the manufacturer’s protocols. Sections were blocked in normal goat serum for 20 min, followed by 1 h incubation in the primary antibody at room temperature. Sections were then washed three times in PBST and incubated for 1h in secondary antibody, sections were then washed again in PBST and then incubated in ABC complex for 30 min. Sections were developed in DAB (3,3’-diaminobenzidine) for 5 min, counterstained in haematoxylin, dehydrated, and mounted in DPX. Staining was imaged on a Zeiss Axio Imager using the Zeiss ZEN software using the AutoLive setting and interactive white balance. Primary antibodies: p-ERK is phospho-p44/42 MAPK (Thr202/Tyr204, Cell Signalling Technology, 9101, used at 1:500), and anti-Cyclin T1 (AbCam, ab238940, use at 1:500).
Sections were pre-processed followed by antigen retrieval in the same way as for immunohistochemistry. Sections were blocked in 2.5% goat serum, and 1% BSA in PBST for 20 mins at room temperature. Primary antibody, made up of blocking buffer, was added for 1 h at room temperature, followed by three 5 min PBST washes, and secondary antibodies were added for 1 h at room temperature. Nuclei were visualised using Hoechst (Sigma, 861405) and sections mounted in ProLong™ Gold Antifade Mountant (Thermo Fisher, P36930). Antibodies: anti-phospho-Histone H3 (Ser10; Merck Millipore, 06-570, use at 1:500), cardiac troponin T (13-11; Thermo Fisher, MA5-12969, use at 1:100) and (CT3, Santa Cruz Biotechnology, sc-20025, use at 1:50), anti-Aurora B antibody (Abcam, ab2254, use at 1:200), anti-Mklp1 (Abcam, ab174304, use at 1:400), anti-CD206 (R&D Systems, AF2535, use at 1:100), anti-PCM1 (Sigma-Aldrich, HPA023370, use at 1:100), anti-Ki67 (SolA15; Thermo Fisher, use at 1:100) and (Atlas Antibodies, HPA023374, use at 1:100), Alexa Fluor 555 goat anti-rabbit IgG (H+L; Life Technologies, A21428), Alexa Fluor 555 goat anti-mouse IgG (H+L; Life Technologies, A21422), Alexa Fluor 555 goat anti-rat IgG (H+L; Life Technologies, A21434), Alexa Fluor 488 goat anti-rabbit IgG (H+L; Life Technologies, A11008), Alexa Fluor 488 goat anti-rat IgG (H+L; Life Technologies, A11006), Alexa Fluor 350 goat anti-mouse (H+L; Life Technologies A11045), wheat germ agglutinin, Alexa Fluor™ 488 conjugate (Thermo Fisher, W11261). TUNEL staining we performed following the manufactures instructions using the ApopTag® Fluorescein In Situ Apoptosis Detection Kit (S7110 Sigma-Aldrich). Staining was imaged on a Zeiss Axio Imager using the Zeiss ZEN software using the AutoLive setting or Leica Stellaris 5 confocal microscope. Quantification was performed by counting the number of positive cardiomyocytes for at least five images per organ/mouse, the mean of five raw counts was calculated and is represented by each data point per graph. Cardiomyocyte cell areas were quantified by encircling individual cardiomyocytes stained with cardiac troponin and wheat germ agglutinin.
Total RNA was isolated using TRIzol Reagent (Thermo Fisher, 15596-018) following manufacturer’s instructions. cDNA was synthesised from 1 μg of RNA using the High-Capacity cDNA Reverse Transcription Kit with RNase Inhibitor (Thermo Fisher, 4374966) following manufacturer’s instructions. qRT-PCR reactions were performed on an Applied Biosystems QuantStudio 5 Real-Time PCR System using Fast SYBR Green Master Mix (Thermo Fisher, 4385612), following manufacturer’s instructions. Primers: Ccnd1; forward 5′- GCGTACCCTGACACCAATCTC-3′, reverse 5′-CTCCTCTTCGCACTTCTGCTC-3. Cdk4; forward 5′-ATGGCTGCCACTCGATATGAA-3′, reverse 5′-TC CTCCATTAGGAACTCTCACAC-3′. Cdk1; forward 5′- AGAA GGTACTTACGGTGTGGT-3′, reverse 5′-GAGAGATTTCCC GAATTGCAGT-3′. Ccnb1; forward 5′-AAGGTGCCTGT GTGTGAACC-3′, reverse 5′-GTCAGCCCCATCATCTGCG-3′. Cad; forward 5′- CTGCCCGGATTGATTGATGTC-3′, reverse 5′-GGTATTAGGCATAGCACAAACCA-3′. Pinx1; forward 5′-AGCAAGGAGCCACAGAACATA-3′, reverse 5′-GGTGAGCAATCCAGTTGTCTT-3′. Bzw2; forward 5′-AGC GACTGTCTCAGGAATGC-3′, reverse 5′-CTGTTTCCGGAA GGTCGTT-3′. Polr3d; forward 5′-AAAAGCGTGAACGGGAC AGG-3′, reverse 5′-AATGGGACTGGATCACTTCCG-3′. St6galnac4; forward 5′-TGGTCTACGGGGATGGTCA-3′, reverse 5′-CTGCTCATGCAAACGGTACAT-3′. Actin; forward 5′-GACGATATCGCTGCGCTGGT-3′, reverse 5′-CCACGATG GAGGGGAATA-3′. Gapdh; forward 5′-AGGTCGGTGTGA ACGGATTTG-3′, reverse 5′-TGTAGACCATGTAGTTGAGG TCA-3′. Fosl1; forward 5′-ATGTACCGAGACTACGGGGAA-3′, reverse 5′-CTGCTGCTGTCGATGCTTG-3′. Egr3; forward 5′-CCGGTGACCATGAGCAGTTT-3′, reverse 5′-TAATGGG CTACCGAGTCGCT-3′.
Total RNA was purified using PureLink RNA Mini Kit (Thermo Scientific). On-column DNA digestion was performed using PureLink DNase (Thermo Scientific). 1 μg of purified RNA was treated with Ribozero rRNA removal kit (Illumina). RNA quality and removal of rRNA were checked with the Agilent 2100 Bioanalyser (Agilent Technologies). Libraries for RNA-seq were then prepared with the NEBNext® Ultra™ II DNA Library Prep Kit for Illumina (NEB) following the manufacturer’s instructions starting from the RNA fragmentation step.
Sequence reads were adapter trimmed using Cutadapt (32) (version 1.11) and aligned using BWA aln for short reads (version 2.5.2a) to the GRCm38 (mm10) assembly with the gene, transcript, and exon features of the Ensembl (release 70) gene model. Expression was estimated using RSEM (33) (version 1.2.30). Transcripts with zero read counts across all samples were removed prior to analysis. Normalisation of RSEM expected read counts was performed by dividing by million reads mapped to generate counts per million (CPM), followed by the trimmed mean of M-values (TMM) method from the edgeR package (34). Differential expression analysis was performed using edgeR.
RNA-seq files from E-MTAB-7595, E-MTAB-7636, and GSE95755 were combined into a single count matrix. Before remapping, poor quality sequences (<30 phred score) and adapter sequences were trimmed with Trimmomatic (35). Reads were mapped with STAR (36) to the reference mouse genome sequence GCRm38.p4/mm10 using default settings. The combined count matrix was generated using HTSeq-count (37) on union mode. Differential expression analysis was performed with EdgeR(v3.20.8) (34) using the glmLRT function. Differential expression comparisons were considered significant if FDR p < 0.05. Gene ontological analysis was performed using DAVID and Heatmaps were assembled using GENE-E (Broad Institute).
Bioinformatic and statistical analyses were performed using R with Bioconductor packages and comEpiTools packages (38, 39). Gene lists from RNA and ChIP sequencing were analysed in Enrichr or GSEA/MSigDB (40). Statistical analyses for IHC and q-RT-PCR were performed using GraphPad Prism v9.0d (GraphPad Software, Inc., San Diego, CA, USA) as indicated with p ≤ 0.05 considered to be statistically significant. Venn diagrams were drawn in the Lucidchart. Heatmaps were drawn in R and Morpheus.
We employed the cardiac-specific, doxycycline-controlled transgenic line TetO-HRas [Tg(tetO-HRAS)65Lc/Nci]; Myh6tTA [Tg(Myh6-tTA)6Smbf/Jm] (30) to overexpress HRasG12V specifically in cardiomyocytes. Mice were generated and removed from doxycycline treatment at 4 weeks of age and the subsequent expression of constitutively active HRasG12V led to an increase in hypertrophic cardiomyocytes (∼10%, Supplementary Figure 1A) interspersed with CD206 positive macrophages and cardiomyocytes of normal volume (Figure 1A), as previously described (30). Hypertrophic cardiomyocytes displayed elevated pERK immunohistochemical staining (Figure 1A) and qRT-PCR analysis confirmed increased expression of the ERK-AP1 target genes Egr3 and Fos1L (Figure 1B), confirming enhanced HRas signalling. Despite the heterogeneity of the model, whole hearts expressing HRasG12V displayed a moderate increase in Cyclin T1 and Phosphorylated RNA Pol II (Figures 1A,C and Supplementary Figure 1B).
To test the hypothesis that persistent HRasG12V expression renders heart tissue Myc responsive, TetO-HRas; Myh6tTA mice were crossed to the R26CMER/+ (R26CAG–c–MycERT2) allele to generate TetO-HRas; Myh6-tTA; R26CMER/+ mice. In these mice, the MycERT2 fusion protein is constitutively expressed from a CAG-enhanced Rosa26 promoter. MycERT2 is inert in the absence of a ligand but rapidly activated following administration of the ERT2 ligand 4OHT (4-hydroxytamoxifen), a primary metabolite of tamoxifen. In these mice, HRasG12V and MycERT2 proteins can be independently activated by the withdrawal of doxycycline or administration of 4OHT (or tamoxifen), respectively (Figure 2A). Doxycycline was withdrawn from TetO-HRas; Myh6-tTA; R26CMER/+ animals at weaning to induce expression of HRasG12V and, once mice reached 8 weeks of age, MycERT2 was activated by injection of 4OHT for 4 h. We confirmed the elevated expression of Cyclin T1, CDK9, and phosphorylated RNA Pol II in the heart tissue of these animals compared to controls (Figure 2B and Supplementary Figure 1C). We have previously determined that Myc-driven transcription is limited by the low level of endogenous P-TEFb within the adult heart. To establish if an HRasG12V-driven increase in P-TEFb levels could enhance Myc-driven transcription, we quantified the expression of a panel of Myc target genes post MycERT2 activation. Cad, a Myc target gene previously shown to be transcribed following MycERT2 activation in all tissues, including the heart, showed increased expression in the heart tissue of both Myc activated (R26CMER/) and HRasG12V/Myc activated (TetO-HRas; Myh6-tTA; R26CMER/+) tissues, confirming the functionality of the MycERT2 fusion in both conditions (Figure 2C). We then determined the expression of a panel of Myc target genes that we have previously observed as unchanged in the adult heart in response to MycERT2 activation alone. The co-expression of HRasG12V sensitised the heart tissue to Myc-dependent expression of these targets over controls (Figure 2C). We also compared changes in global gene expression in heart of HRasG12V (TetO-HRas; Myh6-tTA; R26+/+) mice and Myc/HRasG12V (TetO-HRas; Myh6-tTA; R26 CMER/+) mice 4 h after MycERT2 activation. We have previously observed marked induction of a Myc-driven transcriptional programme in the liver of MycERT2 expressing (R26 CMER/+) mice in the absence of co-expressed HRasG12V (11) (Figure 2D). In contrast to the weak transcriptional response elicited by MycERT2 activation alone, in the presence of HRasG12V, MycERT2 resulted in a marked transcriptional response (Figure 2D). MycERT2 and HRasG12V expressing hearts displayed 1198 up-regulated DEGs and 693 down regulated DEGs (Supplementary Table 1) in comparison to HRasG12V expression alone. Myc target gene identities overlapped with genes induced by MycERT2 in the adult liver, and in the adult heart in the presence of elevated Cyclin T1 expression (AAV9-driven cardiomyocyte-specific) (11) (Figure 2E). These genes are Myc targets, involved in direct Myc-regulated processes such as ribosomal biogenesis and RNA metabolic processes (Figure 2E, Supplementary Figure 1D, and Supplementary Table 2). In contrast, there was little overlap between downregulated genes observed following MycERT2 and HRasG12V activation in the heart and genes downregulated by MycERT2 alone in the liver, suggesting a level of tissue specificity for Myc-dependent transcriptional repression. Consistent with this, genes downregulated in response to HRasG12V and MycERT2 overlapped with those downregulated in the MycERT2 and Cyclin T1 expressing heart – these genes were characteristic of a homeostatic heart transcriptional programme, such as HCN4, MYOT (Figure 2E and Supplementary Table 3). To determine if the transcriptional changes observed in response to combined activation of MycERT2 and HRasG12V in the adult heart were similar to that observed in a regenerating neonatal heart, we analysed previous data to compare the transcriptional profiles of cardiomyocytes isolated from surgically infarcted hearts at different stages of development (14). Our unbiased comparison revealed that the P1 “neonatal regeneration gene expression signature” correlated most closely with the expression changes within the TetO-HRas; Myh6-tTA; R26CMER/+ hearts (Figure 3A). Upregulated genes that overlapped with those also elevated in the “neonatal regeneration gene expression signature” enriched for GO terms including cell cycle control (Figure 3B). Interestingly, downregulated genes that overlapped with those also reduced in the “neonatal regeneration gene expression signature” contained genes involved in the negative regulation of mTOR signalling and we have previously observed a broad reprogramming of metabolism with a transition from neonatal to adult cardiac maturation (41). Furthermore, when regulated metabolic genes from our analysis are overlaid on the KEGG metabolic pathways TetO-HRas; Myh6-tTA; R26CMER/+ hearts show broad reversion and reprogramming to neonatal metabolic pathways (Supplementary Figure 2A). Consistent with this reversion, gene set enrichment analysis (GSEA) indicated MycERT2 and HRasG12V expressing hearts display enrichment of genes involved in glycolysis, characteristic of neonatal cardiomyocyte metabolism, and a reduction in genes involved with oxidative phosphorylation and fatty acid metabolism characteristic of adult cardiomyocyte metabolism (Figure 3C).
To determine if this Myc-dependent “regenerative transcriptional response” results in productive proliferation, MycERT2/HRasG12V expressing mice (TetO-HRas; Myh6-tTA; R26CMER/+) or mice expressing either HRasG12V or MycERT2 alone were administered tamoxifen via intraperitoneal injection at 8 weeks of age to activate MycERT2 and hearts collected after 24 h. Transcriptional analysis indicated that activation of MycERT2 in the presence of HRasG12V induced robust expression of multiple cell cycle genes (Cdk4, Ccnd1, Cdk1, Ccnb1) that were not induced by HRasG12V or Myc activation alone (except Cdk4 which is a known Myc target, Figure 4A). Furthermore, markers of cytokinesis were upregulated in Myc and HRasG12V expressing hearts (Figure 4B) and GSEA indicated that these hearts displayed significant enrichment for genes involved in cell cycle-related pathways such as E2F targets and G2/M checkpoints (Supplementary Figure 2B). These transcriptional events also underpinned significant markers of cell-cycle progression indicated by Ki67 (general cell cycle) and p-H3 (mitotic) positive cardiomyocytes specifically marked by PCM1 and cardiac troponin T, respectively (Figures 4C,D). In addition, Aurora B kinase displayed staining representative of cell cycle progression (prophase, metaphase, late anaphase/telophase, and mid body localisation) combined with features of disassembled sarcomeres that become marginalised to the cell periphery (4, 42) (Figure 4E and Supplementary Figure 3). Furthermore, Mklp1 expression was observed in cardiomyocytes at the cleavage furrow (Figure 4F).
It has been previously noted that HRasG12V hypertrophic hearts display increased levels of cell death, mononuclear cell infiltration, and damage that ultimately lead to heart failure (30). We observed an increase in the presence of mononuclear cell infiltration such as CD206 positive macrophages (Figure 1A) and the apoptosis marker cleaved caspase 3 (Figure 5A). We also observed increased cleaved caspase 3 when elevated HRasG12V expression was combined with MycERT2 activation (Figure 5A) and an apoptotic gene expression signature was present (Supplementary Figure 1D). Therefore, longer-term analysis and cardiomyocyte dissociation and counting of these mice hearts were not possible. Although elevated levels of Myc expression are associated with apoptosis in some tissues (43) we did not observe apoptosis in hearts expressing activated MycERT2 in the absence of HRasG12V (Figure 5A). Using a different system, we next determined if the cell death observed with HRasG12V and Myc together was recapitulated by co-activation of MycERT2 and Cyclin T1. We infected, R26CMER/+, and R26LSL–CMER/+;Myh6Cre mice with cardiac-specific (cTnT promotor-dependent) overexpression of Cyclin T1 using AAV9-cTnT-Ccnt1 or a control β-galactosidase virus (AAV9-cTnT-LacZ). In contrast, to control mice, MycERT2 activation in the presence of Cyclin T1 increased cardiomyocyte proliferation as previously described (11), increased heart weight to tibia size ratio, increased number of Ki67 and p-H3 positive cardiomyocyte nuclei (Supplementary Figure 4) (11). However, we found no evidence of apoptosis (cleaved caspase 3) in cardiomyocytes in hearts overexpressing Myc and Cyclin T1 (Figure 5B). To confirm negativity we also performed Terminal deoxynucleotidyl transferase dUTP nick end labelling (TUNEL) for cell death which also indicated that elevated Myc and Cyclin T1 activity does not induce cardiomyocyte cell death within 48 h (Figure 5B). While the method for overexpressing Cyclin T1 differed, we compared the upregulated gene expression profile of Myc/HRas expressing hearts and Myc/Cyclin T1 expressing hearts and found the GSEA hallmark signatures vastly overlapped. Myc/Ras expressing hearts showed additional gene set enrichment for apoptosis (including the pro-apoptotic proteins Bak and Bok), epithelial mesenchymal transition and inflammatory response (Figure 5C and Supplementary Table 4). The only GSEA pathway that was enriched in Myc/Cyclin T1 expressing hearts was Wnt/Beta Catenin. These data indicate that cell death is specific to HRasG12V activation, not simply an inevitable consequence of enforced endogenous cardiomyocyte proliferation.
Myc is a transcription factor that serves as a pivotal instructor of tissue regeneration following injury in a regenerative organ (13), however, in the heart both Myc transcription and Myc-driven transcription are attenuated. When Myc is acutely overexpressed in the heart it competently binds to DNA but only drives a limited transcriptional programme, and cell-cycle progression is not observed within 48 h (11). Protracted Myc expression in cardiomyocytes eventually leads to DNA synthesis, and myocyte hypertrophy but not to cardiomyocyte cytokinesis (15). We have previously shown that the ability of Myc to drive all the transcriptional programmes necessary for cell division in cardiomyocytes depends on the level of P-TEFb (11). We and others have shown that the level of P-TEFb is dependent on the level of Cyclin T1 and elevated Cyclin T1 leads to a corresponding increase in CDK9 and phosphorylated RNA PolII (11, 25, 26). Since HRasG12V upregulates P-TEFb in cardiomyocytes (31) resulting in cardiac muscle hypertrophy, we hypothesised that Myc and Ras would co-operate to drive cardiomyocyte proliferation. We employed a previously described mouse model of HRasG12V overexpression in which oncogenic HRasG12V is under the control of a tetracycline response element (TetO-HRas) harbouring a cardiomyocyte-specific reverse tetracycline transactivator (Myh6-tTA). Withdrawal of doxycycline at 4 weeks of age leads to cardiomyocyte-restricted HRasG12V overexpression and pathogenic myocardial hypertrophy. Cessation of HRasG12V activity after induction of hypertrophy leads to hypertrophy resolution (30). We confirmed that long-term overexpression of HRasG12V in cardiomyocytes led to increased HRas signalling and cardiomyocyte hypertrophy as previously reported (29, 30, 44–46). Four weeks after HRasG12V expression higher levels of CDK9, Cyclin T1, and phosphorylated RNA Pol II in HRasG12V were observed compared to control animals, confirming that long-term HRasG12V expression upregulates the transcriptional elongation machinery. The exact mechanism of increasing P-TEFb activity by Ras is not known, however, recently the downstream Ras effector AP-1 (heterodimeric transcription factors comprising members of the Fos and Jun families) has been shown to be critical for both Zebrafish and Xenopus tropicalis heart regeneration (47, 48). Fosl1 plays an essential role in cardiomyocyte proliferation by interacting with JunB and binding the Ccnt1 promoter, increasing the expression of Cyclin T1 (48). Here we show that HRasG12V overexpression in cardiomyocytes leads to increased Fosl1 and an increase in Cyclin T1 protein levels. We have previously demonstrated that Myc co-operates with elevated Cyclin T1 expression to drive cardiomyocyte proliferation in vivo (10), therefore, we hypothesised that Myc and HRas would also co-operate in cardiomyocyte proliferation. In the presence, but not the absence, of HRasG12V, Myc activation competently elicited transcriptional programmes involved in cell growth, biogenesis, and metabolism that led to a cardiomyocyte proliferative response. Overlap of the genes altered in Myc and HRas activated hearts with those altered during regeneration of P1 neonatal cardiomyocytes indicated a strong correlation, suggesting that common regenerative transcriptional pathways are activated. Quantification of cardiomyocyte division is notoriously challenging as cell-cycle re-entry does not necessarily lead to cell division (49–51), and the cell death observed in the HRas model prohibited long-term experimentation and cardiomyocyte number estimation by dissociation techniques. Ki67 is expressed during all active phases of the cell cycle while p-H3 expression begins in G2 and is present throughout mitosis. These markers relay no information regarding cytokinesis and hence they are technically markers of cell cycling and not necessarily proliferation. Here, we observe both Ki67 and p-H3 expression in cardiomyocytes (Figure 4D) demonstrating that combined Myc and HRas overexpression drive cardiomyocytes into the cell cycle. Aurora B kinase is expressed during G2, anaphase, metaphase, telophase, and cytokinesis, where it is detected within the mid-body and can be used as a marker of cardiomyocyte division. However, it is present in an asymmetrical location within cardiomyocytes undergoing bi-nucleation (49) and caution is needed when interpreting staining patterns (50). Here we observed Aurora B kinase localisation in cardiomyocytes in prophase, metaphase, anaphase/telophase and symmetrical mid-bodies. Mitotic kinesin-like protein 1 (Mklp1) is an additional marker of cardiomyocyte cytokinesis where it accumulates at the cleavage furrow (52, 53). Here we observed Mklp1 staining at the cleavage furrow of cardiomyocytes in Myc/HRas overexpressing hearts. While these data provide some evidence that Myc and HRas together drive cardiomyocytes’ entry into the cell cycle with positivity for markers of cytokinesis, productive cell division cannot be formally confirmed without direct cardiomyocyte counting. In healthy adult mammalian heart apoptosis is rare with only 0.01–0.001% TUNEL-positive cardiomyocytes observed. This rises to 2–12% apoptotic cardiomyocytes in MI ischaemia and reperfusion injury (54–56). Apoptotic death is also a common feature of cardiomyocytes driven into the cycle by overexpression of some cell cycle regulators such as E2F1, CDK1, and CCNB (8, 57). Paradoxically oncogenes, particularly Myc and Ras, promote both pro-proliferative and pro-apoptotic signals, and the cell fate outcome depends very much on the cell type and context (58). In cardiomyocytes, PI3K and ERK activation downstream of HRas has strong anti-apoptotic effects, promoting cardiomyocyte survival in pressure-overload hypertrophy and heart failure (59). However, hypertrophic cardiomyopathy resulting from HRas mutation associated with chronic constitutively activated Ras/Raf/MEK/ERK pathway and pathological hypertrophy leads to apoptosis (60), similar to the phenotype seen in TetO-HRas; Myh6-tTA mice used in this study. We show that MycER expression alone did not lead to cell death in agreement with similar models (15). However, prolonged/persistent ectopic HRasG12V signalling leads to apoptotic cell death, which was exacerbated by transient Myc signalling. Transcriptional profiling indicated Myc/HRas hearts expressed increased levels of the pro-apototic factors Bak, Bok and enhanced inflammatory response, which may be promoting apoptosis. These data indicate endogenous cardiac regeneration is unlikely to be effective with the combination of Myc and HRas. More importantly, the data suggest patients with elevation in Ras signalling in hypertrophic hearts may not benefit or may be harmed, from driving endogenous cardiomyocyte regeneration via activation of cell cycle genes such as Myc. Here, we activated HRas for 4 weeks, further studies are required to establish whether a shorter period of HRas expression results in similar transcriptional changes and proliferation without hypertrophy and apoptosis. More encouragingly, elevated Cyclin T1 expression, absent of HRas activation, co-operated with activated Myc to drive cardiomyocyte proliferation in the absence of apoptosis, suggesting that co-expression of Cyclin T1 and Myc may be a therapeutically tractable approach for heart regeneration after injury. Myc/Cyclin T1 hearts expressed an increased Wnt/Beta catenin signature, which has recently been shown to be cardioprotective in an adult mammalian setting (61), deciphering the key protective and destructive pathways will be key to translating these findings. As with all genes able to drive proliferation in cardiomyocytes the expression must be tightly regulated and localised. Promising modes of delivery are being developed including cell-specific viral expression vectors and transient modified mRNA technologies.
HRasG12V is restricted to the cardiomyocytes in the TetO-HRas; Myh6-tTA; R26CMER/+ mice, but Myc is expressed across the whole animal, so care must be taken to draw conclusions from the bulk transcriptional data because the interaction between Myc activated non-myocytes and cardiomyocytes are possible. The different systems used to overexpress HRas (TetO-HRas; Myh6-tTA; R26CMER/+ mice) and Cyclin T1 (AAV9-cTnT-Ccnt1) mean the apoptosis and proliferation efficiencies between these groups cannot be directly compared.
All datasets generated and used in this study, have been deposited in ArrayExpress (www.ebi.ac.uk/arrayexpress) under accession codes: E-MTAB-7595, E-MTAB-8462, and E-MTAB-7636. Further information and requests for resources and reagents should be directed to, and will be fulfilled, by CW, [email protected].
All experimental procedures received ethical approval and were conducted in accordance with the Home Office UK guidelines, under project licences 70/7586 and 80/2396 (GE) and PP2054013 (CW) that were evaluated and approved by the Animal Welfare and Ethical Review Body at the University of Cambridge.
MB and CW conceptualised the study. AB, MB, GQ-R, CA, and LT performed the experimental work. GQ-R and JS performed the sequencing analysis. AB, MB, and CW wrote the manuscript. GE and TL edited the manuscript. GE, JH, and CW obtained the funding. All authors contributed to the article and approved the submitted version. | true | true | true |
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PMC9630489 | Peng Sun,Xuan Yang | Hsa_circ_0097271 Knockdown Attenuates Osteosarcoma Progression via Regulating miR-640/MCAM Pathway | 26-10-2022 | Background The dysregulation of circular RNAs (circRNAs) participates in the malignant progression of multiple cancers, including osteosarcoma (OS). However, the role of circ_0097271 in OS development remains unclear. We thus aimed at unveiling the functional role and mechanism of circ_0097271 in OS. Methods The expressions of circ_0097271, miR-640, and MCAM in OS were analyzed by qPCR. Cell proliferation and migration were inspected by CCK-8 assay, colony formation assay, and Transwell assay. Circ_0097271's role in vivo was assayed by establishing animal models. The predicted binding relationship between miR-640 and circ_0097271 or MCAM was verified by dual-luciferase reporter or RIP assay. Results Circ_0097271's expression was enhanced in OS samples and cells. The knockdown of circ_0097271 restrained OS cell growth and migration, and its downregulation also blocked solid tumor growth in vivo. Circ_0097271 targeted miR-640 and negatively modulated miR-640 expression. MiR-640 was poorly expressed in OS, and its depletion recovered OS cell growth and migration that were repressed by circ_0097271 knockdown. MiR-640 bound to MCAM 3'UTR and thus suppressed MCAM expression. MCAM knockdown repressed OS cell growth and migration, while additional miR-640 depletion partially abolished the anticancer effects of MCAM knockdown in OS cells. Conclusion Circ_0097271 is an oncogenic driver and contributes to OS development via targeting the miR-640/MCAM pathway, which provides a potential opinion for OS treatment. | Hsa_circ_0097271 Knockdown Attenuates Osteosarcoma Progression via Regulating miR-640/MCAM Pathway
The dysregulation of circular RNAs (circRNAs) participates in the malignant progression of multiple cancers, including osteosarcoma (OS). However, the role of circ_0097271 in OS development remains unclear. We thus aimed at unveiling the functional role and mechanism of circ_0097271 in OS.
The expressions of circ_0097271, miR-640, and MCAM in OS were analyzed by qPCR. Cell proliferation and migration were inspected by CCK-8 assay, colony formation assay, and Transwell assay. Circ_0097271's role in vivo was assayed by establishing animal models. The predicted binding relationship between miR-640 and circ_0097271 or MCAM was verified by dual-luciferase reporter or RIP assay.
Circ_0097271's expression was enhanced in OS samples and cells. The knockdown of circ_0097271 restrained OS cell growth and migration, and its downregulation also blocked solid tumor growth in vivo. Circ_0097271 targeted miR-640 and negatively modulated miR-640 expression. MiR-640 was poorly expressed in OS, and its depletion recovered OS cell growth and migration that were repressed by circ_0097271 knockdown. MiR-640 bound to MCAM 3'UTR and thus suppressed MCAM expression. MCAM knockdown repressed OS cell growth and migration, while additional miR-640 depletion partially abolished the anticancer effects of MCAM knockdown in OS cells.
Circ_0097271 is an oncogenic driver and contributes to OS development via targeting the miR-640/MCAM pathway, which provides a potential opinion for OS treatment.
Osteosarcoma (OS) is the most common primary malignant bone tumor, most often occurring in adolescence [1]. Due to its highly aggressive biological behavior, amputation is considered the primary surgical treatment [2]. With a better understanding of the OS development and tremendous advances in treatment measures, most patients are treated with limb-preserving surgical resection combined with neoadjuvant chemotherapy whenever possible [2, 3]. Accordingly, 5-year survival rates have increased from no more than 30% to more than 70% [4, 5]. As molecular pathogenesis continues to advance, gene therapy has become a controllable, targeted, and specific treatment for OS [6]. Therefore, novel oncogenic factors should be identified to further provide treatment strategies for OS. CircRNAs are a new class of regulatory factors that play specific roles in tumor progression and are classified as noncoding RNAs. CircRNAs originate from precursor mRNA via a “back-splicing” mechanism and featured by covalently closed structure and high stability in eukaryotic organisms [7, 8]. Growing studies unveil that circRNAs have huge potencies to mediate chemoresistance, tumor initiation, and aggressive progression [9]. For example, the circRNA expression profile exhibited the upregulation of circ_0001564 in OS, and circ_0001564 contributed to OS cell growth and survival via acting as miRNA sponges [10]. Circ_0081001 expression was enhanced in OS with methotrexate resistance, and depletion of circ_0081001 strengthened methotrexate chemosensitivity to repress OS development [11]. Unfortunately, numerous circRNAs have not been functionally exploited in OS. Circ_0097271 is derived from ATPase sarcoplasmic/endoplasmic reticulum Ca2+ transporting 2 (ATP2A2) genes by back-splicing. In view of the blank of circ_0097271' role in OS, we focus on circ_0097271 and investigate its functional role and mechanism in OS progression. The bioinformatics tool circInteractome presents that miR-640 was a putative target of circ_0097271 because circ_0097271 sequence fragment contains miR-640 binding sites [12]. MiR-640 was shown as a tumor suppressor in breast cancer [13], whereas, the role of miR-640 in multiple cancers, including OS, remains unclear. The bioinformatics tool TargetScan displays that miR-640 has binding site on the 3'UTR of MCAM [14], implying that MCAM was a potential target gene of miR-640. MCAM, also known as CD146, is a cell surface adhesion molecule that is widely declaimed to facilitate cancer progression and metastasis [15]. MCAM dysregulation was also involved in OS development [16, 17]. However, the interplays between miR-640 and MCAM in OS development are unclear, and the upstream regulators of MCAM have not been fully identified. Our present work for the first time investigated the expression pattern and functional role of circ_0097271 in OS development. In addition, we validated the binding relationship between miR-640 and circ_0097271 or MCAM in OS development to unveil the regulatory mechanism of circ_0097271. Our aim was to further understand OS pathogenesis from the insight of a novel circRNA, circ_0097271.
Human OS samples and matched noncancer normal control tissues were removed from 38 OS cases at our hospital. Before biopsy, all patients were firstly diagnosed with OS and had never received therapeutic measures against OS, such as chemotherapy and radiotherapy. Patients with other bone diseases or systemic diseases were excluded. The informed consent was acquired from each case. The procedures of human sample use were approved by our hospital.
Human OS cells, Saos-2 (RRID: CVCL_0548) (Bena, Beijing, China), SW1353 (RRID: CVCL_0543) (Bena), SOSP-9607 (RRID: CVCL_4V80) (FenghBio, Changsha, China), and HOS (RRID: CVCL_0312) (Procell, Wuhan, China) were cultured in 90% DMEM (Sigma-Aldrich, USA) added with 10% FBS (Sigma-Aldrich) in an incubator (37°C, with 5% CO2). We customized circ_0097271-specific or MCAM-specific small interference RNA (si-circ or si-MCAM) and their matched control (si-NC) from FenghBio. MiR-640 mimic, miR-NC, miR-640 inhibitor, and inhibitor-NC were directly bought from Ribobio (Guangzhou, China). When 70-80% cells were fused, the experimental cells were transfected with different oligonucleotides using Lipofectamine™ 2000 (11668019, Invitrogen, USA). 24 hours later, cells were collected and used for transfection efficiency detection. The sequence of siRNA is shown in Supplementary Table 1.
Total RNA was acquired from samples using the TRIzol reagent (15596-018, Invitrogen). Next, two commercial reverse transcription kits, including First-Strand cDNA Synthesis Kit (C0210A, GeneCopoeia, Shanghai, China) and miRNA First-Strand cDNA Synthesis Kit (QP013, GeneCopoeia), were utilized for cDNA synthesis. The diluted cDNA was reacted with SYBR Mixture (CW3008M, Cwbio, Beijing, China) for qPCR detection through a LightCycler 96 thermocycler (Roche, Switzerland). The expression of circ_0097271 and MCAM was normalized by GAPDH, and miR-640's expression was normalized by U6. The expression level was evaluated using the 2−ΔΔCt method. We displayed primer sequences in Table 1.
Total RNA was acquired and experienced with RNase R (R0301, Geneseed, Guangzhou, China) for 15 minutes at 37°C. After treatment, total RNA was reverse-transcribed and used for qPCR as abovementioned.
For circRNA subcellular location analysis, we briefly isolated cytoplasmic RNA and nuclear RNA from OS cells using the commercial PARIS kit (AM1921, Invitrogen) as the protocol suggested. The abundance of circ_0097271 in different locations was analyzed by qPCR assay, using GAPDH as an internal reference in the cytoplasm and U6 as an internal reference in the nucleus.
We prepared the transfected cells into 96-well plates at a density of 1500 cells/well in 100 μL culture medium. Every 24 h interval (24, 48, 72, and 96 hours), CCK-8 (96992, Sigma-Aldrich) was added into each corresponding test well with 10 μL per well, and cells were next incubated for additional 4 hours in incubators. The optical density (OD) was evaluated by measuring the 450 nm absorbance using a microplate reader (Biotek, USA).
We prepared the transfected cells into cell culture dishes at a density of 300 cells/well. Cells in complete culture medium were cultured in incubators for 12 days. The medium was refreshed every two days to induce colony formation. At last, cell debris was removed by PBS washing, and cell colonies (over 50 cells) after methanol fixation and 0.1% crystal violet (Sangon Biotech, Shanghai, China) staining were observed by light microscopy (Nikon, Japan).
For migration analysis, 5 × 105 cells were placed into the upper chamber (8-μm; Corning, USA) and maintained in 500 μL serum-free medium. The bottom of chambers was supplemented with matched culture medium (800 μL) containing 20% FBS. After 24 h incubation, cells migrated to the low surface were subjected to methanol fixation and 0.1% crystal violet staining. Representative pictures were captured by light microscopy in random 5 fields.
Nude mice (female, 6-week-old) used in this study were bought from Vital River (Beijing, China) and then acclimated for one week in pathogen-free animal room. Circ_0097271-specific short-hairpin-RNA (sh-circ) and its matched control (sh-NC) were synthesized by Geneseed and packaged into lentiviral vectors. The lentiviral particles of sh-circ or sh-NC were used to infect Saos-2 cells to mediate circ_0097271 stable downregulation. To construct transplanted tumor models, the infected Saos-2 cells (2 × 106 cells per mouse) were hypodermically injected into the groin of nude mice, with 5 mice in each group. Tumor volumes (length × width2 × 0.5) were weekly recorded by a vernier caliper. After 5 weeks, all mice were euthanized by intraperitoneal injection with pentobarbital. Then, tumor tissues were excised from animal body and used for further analysis. The procedures of animal use were approved by our hospital.
According to the predicted multiple binding sites between circ_0097271 and miR-640, circ_0097271 reporter vectors, including wild-type (WT), mutation at binding site 1 (Mut1), mutation at binding site 2 (Mut2), and common mutation (Co-Mut), were all constructed into pmirGLO vector to verify these putative binding sites. Similarly, the WT and Mut reporter vectors of MCAM were also constructed. The WT/MUT reporter vector was transfected with miR-640 mimic or mimic-NC into Saos-2 and SW1353 cells using Lipofectamine™ 2000.48 hours later, we examined luciferase activities in line with the protocol of a dual luciferase reporter assay system (Promega, USA).
Using a commercial RIP assay kit from Merck Millipore (17-704, USA), RIP assay was implemented to ensure the involvement of circ_0097271 with miR-640. In brief, Saos-2 and SW1353 cells were lysed, and cell lysates were incubated with magnetic beads coated with Ago2 antibody (Anti-Ago2) or IgG antibody (Anti-IgG). RNA complexes could be captured by antibody-coated beads and then eluted for qPCR analysis.
Utilizing the RIPA Lysis Buffer (P0013E, Beyotime, Shanghai, China), total proteins were acquired from samples. Then, proteins were quantified using the BCA Protein Assay Kit (P0011, Beyotime). After denaturing by boiling for 10 minutes, equal amounts of protein lysates (40 μg per lane) were separated by 10% SDS-PAGE and electron transferred to nitrocellulose membranes (Invitrogen). After blocking with 5% skim milk at room temperature for 1 hour, the membranes were probed with the primary antibody against MCAM (ab75769; 1/1000 dilution; Abcam, USA) or GAPDH (ab181602; 1/10000 dilution; Abcam) overnight at 4°C. After the incubation with the matched secondary antibody (ab205718; 1/20000 dilution; Abcam) for 2 hours at room temperature, the membranes were exposed to the ECL reagent (P0018AS, Beyotime) to display protein bands.
GraphPad Prism 8 (GraphPad Software, USA) was used for figure drawing and statistical analysis of collected data from three independent experiments. Measurement data were displayed as mean ± standard deviation. Student's t-test was used for the comparison of the difference between two groups, and analysis of variance was used for the comparison among groups. The expression correlation between miR-640 and circ_0097271 or MCAM in OS samples was analyzed by Pearson's analysis. P < 0.05 was considered a statistically significant difference.
To realize circ_0097271's expression pattern in OS, we conducted qPCR analysis and monitored that circ_0097271's expression was greatly reinforced in OS tumor samples in contrast to normal controls (Figure 1(a)). Diagnostic potentials of levels of circ_0097271 for OS were evaluated by ROC curve analysis with patients with OS tissues as true positive cases and normal tissues as true negative cases. Area under the curve was 0.9744, with standard error of 0.02340 and 95% confidence interval of 0.9285-1.020 (Supplementary Figure 1(a)). Circ_0097271's expression was also markedly enhanced in OS cell lines (Saos-2, SW1353, SOSP-9607, and HOS) in comparison to noncancer cell line (hFOB1.19) (Figure 1(b)). We selected Saos-2 and SW1353 cells in the following experiments because these two cell lines harbored relatively high expression of circ_0097271. Further analyses monitored that circ_0097271 was primarily distributed in the cytoplasmic fraction of Saos-2 and SW1353 cells but not in the nucleus (Figure 1(c)). Circ_0097271 was more resistant to RNase R digestion compared to its linear transcript, ATP2A2, because RNase R largely degraded ATP2A2 expression but rarely decreased circ_0097271 expression (Figure 1(d)). In summary, circ_0097271 was richly expressed in OS, with high stability.
We ensured that circ_0097271 expression was remarkably declined in si-circ-transfected Saos-2 and SW1353 cells in contrast to si-NC-transfected cells (Figure 2(a)). The OD450 value at 96 h of Saos-2 and SW1353 cells after si-circ transfection and the colony-forming ability of Saos-2 and SW1353 cells after si-circ transfection were greatly impaired, suggesting that circ_0097271 downregulation blocked OS cell growth (Figures 2(b) and 2(c)). In addition, we observed from Transwell assay that Saos-2 and SW1353 cells with circ_0097271 downregulation had the repressive migratory ability (Figure 2(d)). The data viewed that circ_0097271 knockdown restrained OS cell growth and migration.
We constructed the transplanted tumor models by injecting Saos-2 cells (with sh-circ or sh-NC infection) into nude mice. Then, we observed that the infection of Saos-2 cells infected with sh-circ resulted in tumor tissues with smaller tumor size, while the infection of Saos-2 cells infected with sh-NC resulted in tumor tissues with bigger tumor size (Figure 3(a)). The detailed data showed that circ_0097271 downregulation in tumor tissues reduced tumor volumes and tumor weights (Figures 3(b) and 3(c)). Overall, circ_0097271 knockdown impeded tumor development in animal models.
We exploited the downstream miRNAs targeted by circ_0097271, aiming to unveil circ_0097271's functional mechanism. Circ_0097271 was observed to have binding sites with miR-640 by circInteractome tool (Figure 4(a)). Then, we assembled multiple reporter vectors including WT or Mut sequences fragment of circ_0097271 to confirm the predicted binding sites between them. As a result, we found that miR-640 mimic largely lessened luciferase activities of WT reporter vector of circ_0097271 and partially reduced luciferase activities of Mut1 or Mut2 reporter vector of circ_0097271, while miR-640 mimic hardly weakened luciferase activities of Co-Mut reporter vector of circ_0097271 (Figure 4(b)), verifying that circ_0097271 had multiple binding sites with miR-640. Also, both circ_0097271 and miR-640 could be largely enriched by Anti-Ago2 in the RIP assay, in comparison to Anti-IgG, verifying the binding between circ_0097271 and miR-640 (Figure 4(c)). MiR-640 showed a low expression level in OS tumor samples in contrast to normal samples, as well as in OS cells (Saos-2 and SW1353) in contrast to hFOB1.19 cells (Figures 4(d) and 4(e)). Diagnostic potentials of levels of miR-640 for OS were evaluated by ROC curve analysis with patients with OS tissues as true positive cases and normal tissues as true negative cases. Area under the curve was 0.9204, with standard error of 0.03055 and 95% confidence interval of 0.8605-0.9803 (Supplementary Figure 1(b)). Moreover, miR-640 expression and circ_0097271 expression were inversely correlated in OS tumor samples (Figure 4(f)). In summary, miR-640 was targeted by circ_0097271 and showed the opposite expression pattern with circ_0097271 in OS.
In view of the binding between circ_0097271 and miR-640, we further reduced miR-640 expression in circ_0097271-depleted OS cell to observe functional effects. At first, we ensured that miR-640 expression was greatly strengthened in Saos-2 and SW1353 cells transfected with si-circ but greatly declined in OS cells transfected with miR-640 inhibitor; In comparison to alone si-circ transfection, si-circ+inhibitor cotransfection partly impaired the expression of miR-640 (Figure 5(a)). In terms of function, inhibition of miR-640 largely strengthened the OD450 values of Saos-2 and SW1353 cells at 72 and 96 h posttransfection, enhanced OS cells' colony-forming ability, and cell migratory ability (Figures 5(b)–5(d)). Besides, these malignant cell behaviors suppressed by circ_0097271 knockdown, including cell viability, colony-forming ability, and migration, were all effectively restored by further miR-640 depletion (Figures 5(b)–5(d)). The data deemed that circ_0097271 inversely regulated miR-640 expression to promote OS cell development.
There are numerous potential functional genes targeted by miR-640. We thus utilized TargetScan tool to predict miR-640's target genes, and miR-640 was shown to possess binding site on MCAM 3′UTR (Figure 6(a)). MiR-640 mimic effectively diminished luciferase activities of WT reporter vector of MCAM but rarely changed luciferase activities of Mut reporter vector of MCAM, verifying this special binding site between miR-640 and MCAM 3′UTR (Figure 6(b)). MCAM expression was markedly reinforced in OS tumor samples in comparison to normal samples, as well as in OS cells (Saos-2 and SW1353) in comparison to hFOB1.19 cells (Figures 6(c) and 6(d)). Diagnostic potentials of levels of MCAM for OS were evaluated by ROC curve analysis with patients with OS tissues as true positive cases and normal tissues as true negative cases. Area under the curve was 0.9834, with standard error of 0.01543 and 95% confidence interval of 0.9531-1.014 (Supplementary Figure 1(c)). Additionally, we identified that MCAM expression was inversely linked to miR-640 expression, and positively linked to circ_0097271 expression in OS tumor samples (Figures 6(e) and 6(f)). Moreover, circ_0097271 knockdown inhibited MCAM mRNA and protein expression levels (Figures 6(g) and 6(h)). In summary, MCAM was a target of miR-640, and positively regulated by circ_0097271.
We then performed rescue experiments to test the interactions between miR-640 and MCAM. At first, we observed that MCAM protein level was greatly reduced in Saos-2 and SW1353 cells transfected with si-MCAM but notably strengthened in OS cells transfected with miR-640 inhibitor; in comparison to alone si-MCAM transfection, si-MCAM+inhibitor cotransfection partially recovered MCAM expression (Figure 7(a)). In functional assays, MCAM downregulation strikingly weakened OS cell viability, colony-forming ability, and migratory capacity, which was completely opposite to the role of miR-640 depletion. Besides, MCAM downregulation-blocked cell viability, colony-forming ability, and migratory capacity were substantially restored by further miR-640 inhibition in OS cells (Figures 7(b)–7(d)). We concluded that miR-640 inhibition enhanced MCAM expression and thus attenuated the anti-cancer effects of MCAM knockdown.
Our study mainly discovered that circ_0097271's expression was greatly reinforced in OS samples and cells. Knockdown of circ_0097271 impeded OS cell growth and migration, and also repressed tumorigenesis in animal models. We further found that miR-640 was targeted by circ_0097271, and MCAM was a downstream target of circ_0097271/miR-640. Accordingly, we proposed that circ_0097271 accelerated OS malignant progression at least in part by modulating the miR-640/MCAM pathway (Figure 8). CircRNAs exerting diverse functional effects in OS have been widely stated. For instance, circ_0001721 was overexpressed in OS, and circ_0001721 downregulation restrained OS development via repressing glycolysis metabolism, cell growth, migration, and invasion [18]. Circ_0078767 was also forcefully expressed in OS, and its ectopic expression largely aggravated OS cell growth, migratory ability and invasiveness and thus accelerated the growth of transplanted tumors [19]. Inversely, circ_0001105 was lowly expressed in OS and positively associated with survival rate of OS patients, and circ_0001105 overexpression largely restrained OS cell growth and invasion [20]. In view of the important and diverse role of circRNAs in OS, we characterized circ_0097271 and assayed its aberrant high expression in OS tissues and cells. Loss-of-function assays viewed that silencing circ_0097271 repressed OS cell growth and migration, and also hindered the development of transplanted tumors, hinting that circ_0097271 was a potential oncogenic driver in OS. Therefore, we speculated that circ_0097271 holds huge promise to be a therapeutic target for OS. Regarding the regulatory mechanism of circRNAs, it is widely reported that circRNAs serve as competing endogenous RNAs (ceRNAs) to thus modulate the miRNA/mRNA networks [21]. For instance, circ_0001105 was shown to mediate OS proliferation and metastasis through miR-766-governed YTHDF2 axis [20], showing that circRNAs can function as miRNA sponges to regulate the expression of downstream mRNAs. Accordingly, we characterized the target miRNAs of circ_0001105 and discovered that circ_0097271 possessed binding sites with miR-640. MiR-640 was previously noted to be a suppressor in breast cancer, attributed to its inhibitory effects on Wnt7b/β-catenin oncogenic pathway [13]. Zhai et al. utilized meta-analysis to analyze microarray data in GEO database and discovered that miR-640 expression was markedly declined in hepatocellular carcinoma; besides, miR-640 enrichment restrained the proliferation of hepatocellular carcinoma cells [22]. Moreover, miR-640 overexpression could block Angiopoietin-1-induced endothelial cell angiogenesis, hinting that miR-640 had the potency to repress tumorigenesis [23], whereas, no studies reported the role of miR-640 in OS. We thus focused on miR-640 and identified its downregulation in OS tissues and cells. MiR-640 deficiency in OS cell aggravated cell growth and migration, and the suppression of circ_0097271 knockdown on OS cell growth and migration was partially attenuated by further miR-640 deficiency, suggesting that circ_0097271 targeted miR-640 to promote OS progression. We further validated that MCAM was a target gene of miR-640. MCAM, widely known as CD146, has been regarded as an oncogenic biomarker and therapeutic target for multiple cancers [15, 24]. For instance, MCAM was highly regulated in breast cancer, and its downregulation was linked to the suppression of epithelial-mesenchymal transition and chemoresistance in breast cancer cells [25]. MCAM was strikingly elevated in small cell lung cancer with chemoresistance, and the suppression of MCAM largely repressed cancer cell proliferation and drug resistance [26]. MCAM was also mentioned to be overexpressed in OS, and its high expression was related to OS metastasis and poor prognosis [27, 28]. Overall, the oncogenic effect of MCAM in diverse cancers has been verified. Consistent with these findings, we displayed the high expression of MCAM in OS samples and cells. Knockdown of MCAM restrained OS cell growth and migration, while these anticancer effects caused by MCAM knockdown were largely abolished by miR-640 deficiency because miR-640 deficiency strengthened the expression level of MCAM in OS cells. MCAM was a functional molecule downstream of the circ_0097271/miR-640 pathway. There are limitations in our present work. For example, the role of circ_0097271 on energy metabolism, cell cycle, and other cell behaviors is unclear, and more efforts should be done to address the detailed role of circ_0097271. Besides, the clinical practice and implication of circ_0097271 should be further summarized to enrich its potency as a biomarker for OS. These issues will be addressed in future work.
Circ_0097271 was overexpressed in OS and exerted oncogenic effects to enhance OS cell growth and migration. Circ_0097271 drove its oncogenic role in OS development at least in part by controlling the miR-640/MCAM axis. The management of circ_0097271-governed miR-640/MCAM axis might be a promising strategy for OS treatment. | true | true | true |
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PMC9630764 | Xinluan Wang | Commentary: “Baicalein mediates the anti-tumor activity in Osteosarcoma through lncRNA-NEF driven Wnt/β-catenin signaling regulatory axis” | 28-10-2022 | Commentary: “Baicalein mediates the anti-tumor activity in Osteosarcoma through lncRNA-NEF driven Wnt/β-catenin signaling regulatory axis”
Osteosarcoma (OS) is a common type of malignant bone tumor in adolescents with high risk of metastasis. The use of chemotherapy may result in marginal improvement in survival over surgery alone. However, owing to its high malignant and metastatic potential, metastatic OS has an especially poor prognosis, with only 30% overall survival. Some chemotherapy induced migration and stemness in OS cells might be the primary reason of recurrence and drug resistance, so an effective agent for suppressing cancer cells growth and metastasis is desirable in treating OS [1]. Natural products or derivatives from Traditional Chinese Medicine (TCM), such as taxol, topotecan, vinorelbine, etc., have been successfully approved for clinical use and showed effectiveness in improvement of cancer survival. In recent years, there have been many studies on the treatment of OS with natural products, which demonstrated considerable efficacy in OS [2,3]. In a recent issue of J Orthop Translat, Dr. JF Zhang and co-workers reported that baicalein, an active pharmaceutical ingredient from Rhizoma coptidis, was able to suppress tumor growth and metastasis both in vitro and in vivo through a lncRNA-NEF driven Wnt/β-catenin regulatory axis, in which lncRNA-NEF was upregulated by baicalein, and thus induced the inactivation of Wnt/β-catenin signaling [4]. The findings not only validate the anti-OS activity of a TCM-derived natural product and suggest it as a promising candidate, but also provide a novel underlying mechanism to develop effective drugs to treat relevant clinical problems. Given the extraordinary lack of progress seen in OS clinical trials, more efforts are needed to investigate these natural products by elucidating their mechanisms of action, chemically or biologically synthesizing more derivatives before studying their structure–efficacy relationship so to obtain better candidate drugs with high efficiency and low toxicity, and finally moving towards larger clinical trials for upfront therapy, similarly to those reported as evidenced-based TCM developed for treating other musculoskeletal disorders [[5], [6], [7], [8]]. Moreover, interdisciplinary new technologies will facilitate substantial advancements in treating this aggressive cancer, such as novel drug delivery systems, which have the promising potential on triggering intracellular drug delivery to improve the efficacy of OS therapy [9]. | true | true | true |
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PMC9630827 | 35999654 | James Sinnett-Smith,Tarique Anwar,Elaine F. Reed,Yaroslav Teper,Guido Eibl,Enrique Rozengurt | Opposite Effects of Src Family Kinases on YAP and ERK Activation in Pancreatic Cancer Cells: Implications for Targeted Therapy | 23-08-2022 | Abstract Pancreatic ductal adenocarcinoma (PDAC) remains an aggressive disease that is expected to become the second cause of cancer fatalities during the next decade. As therapeutic options are limited, novel targets, and agents for therapeutic intervention are urgently needed. Previously, we identified potent positive crosstalk between insulin/IGF-1 receptors and G protein–coupled (GPCR) signaling systems leading to mitogenic signaling in PDAC cells. Here, we show that a combination of insulin and the GPCR agonist neurotensin induced rapid activation of Src family of tyrosine kinases (SFK) within PANC-1 cells, as shown by FAK phosphorylation at Tyr576/577 and Tyr861, sensitive biomarkers of SFK activity within intact cells and Src416 autophosphorylation. Crucially, SFKs promoted YAP nuclear localization and phosphorylation at Tyr357, as shown by using the SFK inhibitors dasatinib, saracatinib, the preferential YES1 inhibitor CH6953755, siRNA-mediated knockdown of YES1, and transfection of epitogue-tagged YAP mutants in PANC-1 and Mia PaCa-2 cancer cells, models of the aggressive squamous subtype of PDAC. Surprisingly, our results also demonstrate that exposure to SFK inhibitors, including dasatinib or knockdown of YES and Src induces ERK overactivation in PDAC cells. Dasatinib-induced ERK activation was completely abolished by exposure to the FDA-approved MEK inhibitor trametinib. A combination of dasatinib and trametinib potently and synergistically inhibited colony formation by PDAC cells and suppressed the growth of Mia PaCa-2 cells xenografted into the flank of nude mice. The results provide rationale for considering a combination(s) of FDA-approved SFK (dasatinib) and MEK (e.g., trametinib) inhibitors in prospective clinical trials for the treatment of PDAC. | Opposite Effects of Src Family Kinases on YAP and ERK Activation in Pancreatic Cancer Cells: Implications for Targeted Therapy
Pancreatic ductal adenocarcinoma (PDAC) remains an aggressive disease that is expected to become the second cause of cancer fatalities during the next decade. As therapeutic options are limited, novel targets, and agents for therapeutic intervention are urgently needed. Previously, we identified potent positive crosstalk between insulin/IGF-1 receptors and G protein–coupled (GPCR) signaling systems leading to mitogenic signaling in PDAC cells. Here, we show that a combination of insulin and the GPCR agonist neurotensin induced rapid activation of Src family of tyrosine kinases (SFK) within PANC-1 cells, as shown by FAK phosphorylation at Tyr576/577 and Tyr861, sensitive biomarkers of SFK activity within intact cells and Src416 autophosphorylation. Crucially, SFKs promoted YAP nuclear localization and phosphorylation at Tyr357, as shown by using the SFK inhibitors dasatinib, saracatinib, the preferential YES1 inhibitor CH6953755, siRNA-mediated knockdown of YES1, and transfection of epitogue-tagged YAP mutants in PANC-1 and Mia PaCa-2 cancer cells, models of the aggressive squamous subtype of PDAC. Surprisingly, our results also demonstrate that exposure to SFK inhibitors, including dasatinib or knockdown of YES and Src induces ERK overactivation in PDAC cells. Dasatinib-induced ERK activation was completely abolished by exposure to the FDA-approved MEK inhibitor trametinib. A combination of dasatinib and trametinib potently and synergistically inhibited colony formation by PDAC cells and suppressed the growth of Mia PaCa-2 cells xenografted into the flank of nude mice. The results provide rationale for considering a combination(s) of FDA-approved SFK (dasatinib) and MEK (e.g., trametinib) inhibitors in prospective clinical trials for the treatment of PDAC.
One of the deadliest types of cancer has been and still is pancreatic ductal adenocarcinoma (PDAC). An estimated 48,220 patients will succumb to this disease, putting PDAC as the third leading cause of cancer mortality in the United States (1). Furthermore, PDAC is projected to become the second leading cause of cancer-related deaths before 2030 (2). Considering the failure to date to treat PDAC, there is an urgent need to identify novel targets and therapeutic agents to treat this devastating disease. There is consensus about the importance of mutated KRAS (encoding KRAS) in the initiation of PDAC, occurring in >90% of human PDAC (3), a notion supported by preclinical models of the disease (4). Although a critical role of KRAS mutations in PDAC development is generally accepted, recent studies demonstrated that KRAS is dispensable for the survival of the most aggressive subtype of PDAC, that is, the squamous subtype tumors (5). Therefore, it is important to identify additional oncogenic drivers that might represent targetable vulnerabilities of the disease. The structurally related nonreceptor Src family of tyrosine kinases (SFK) have been implicated in the regulation of cellular cytoskeletal organization, migration, and proliferation, and involved in invasion and metastasis in multiple solid tumors, including PDAC (6–8). The SFK comprises 12 members two of which, Src and YES1, are expressed prominently in human PDAC cell lines (9). An early study demonstrated that administration of the SFK inhibitor dasatinib (10) prevented metastatic dissemination in preclinical models of PDAC but did not interfere with the growth of the primary tumor (11). Subsequent clinical trials in PDAC patients using dasatinib in combination with gemcitabine (12, 13) or 5-fluorouracil and oxaliplatin (14) failed to demonstrate significant clinical benefit in patients with PDAC. The mechanism(s) leading to dasatinib resistance in PDAC are poorly understood. Intriguingly, SFKs phosphorylate wild type and oncogenic (G12D) KRAS on tyrosine residues (Tyr32 and Tyr64), thereby inducing conformational changes that inhibit KRAS stimulation of RAF/MEK/ERK (15, 16). Conversely, the pro-oncogenic tyrosine phosphatase SHP2 (17, 18), which dephosphorylates KRAS (19), promoted RAF/MEK/ERK signaling. Consequently, SFK-mediated tyrosine phosphorylation of KRAS in PDAC could function in a tumor-suppressive capacity via KRAS inactivation (15, 19) and thus, SFK inhibitors could induce KRAS hyper-activation. Given the apparent inhibitory effects of SFK on KRAS-dependent signaling, we hypothesized that SFKs promote progression of PDAC acting through a downstream target(s) that circumvents the requirement of KRAS-stimulated proliferation. A plausible candidate is the transcriptional co-activator YES1-Associated Protein (YAP), a major effector of the Hippo, growth factor, G protein–coupled receptor (GPCR) and integrin signaling pathways and a key regulator of development, organ-size, tissue regeneration, and tumorigenesis (20–22). When localized in the nucleus, YAP binds and activates predominantly the TEA-domain DNA-binding transcription factors (TEAD 1–4) thereby stimulating the expression of multiple genes. Recent evidence indicates that YAP acts as a potent oncogene in PDAC (23–25). Accordingly, YAP is overactive in PDAC tumor samples (26–28) and higher expression of YAP is correlated with poorer survival in patients with PDAC (25, 29). Importantly, YAP is highly activated in the squamous subtype of PDAC (30), which exhibits reduced dependency on KRAS for survival (31, 32). Accordingly, amplification and overexpression of Yap can substitute for mutant Kras expression in promoting PDAC in preclinical models (26). Several studies in other cell types indicated that SFKs lead to YAP activation (33–35), but the mechanisms are cell-context dependent and the role of SFKs in YAP regulation in pancreatic cancer cells has not been examined before. In previous studies, we identified potent positive crosstalk between insulin/IGF-1 receptors and GPCR signaling systems leading to mitogenic signaling in PDAC cells (36). Subsequently, we reported that stimulation of these cells with insulin and the GPCR agonist neurotensin promoted YAP nuclear localization and activity (37). Here, we demonstrate that the SFKs play a major role in mediating YAP nuclear localization and colony growth of pancreatic PANC-1 and Mia PaCa-2 cancer cells, models of the aggressive squamous subtype of PDAC (32). Mechanistically, the SFK members YES and Src stimulate YAP phosphorylation on Tyr357 and the phosphorylation of this residue plays a critical role in the regulation of YAP nuclear localization. Finally, we demonstrate that exposure of PDAC cells to SFK inhibitors, including dasatinib (10), induces ERK activation and that a combination of SFK inhibitor and MEK inhibitor potently inhibited colony formation by these cells and suppressed the growth of Mia PaCa-2 cells xenografted into the flank of nude mice. The results provide rationale for considering a combination(s) of FDA-approved SFK (e.g., dasatinib) and MEK (e.g., trametinib) inhibitors in prospective trials for the treatment of PDAC.
PANC-1 and Mia PaCa-2 were maintained in DMEM supplemented with 10% FBS. Capan-2 were maintained in McCoy's 5A medium supplemented with 10% FBS. Human pancreatic cancer cell lines were obtained from ATCC on the following dates: PANC-1 (February 2021, April 2021, April 2022), Mia Paca-2 (February 2021, April 2021, April 2022), and Capan 2 (August 2021). These cell lines were tested for mycoplasma and authenticated by the ATCC using short-tandem repeat analysis. All cell lines were used within 15 passages after recovery from frozen stocks. No authentication or mycoplasma testing was done by the authors.
The cultures were directly lysed in 2 × SDS-PAGE sample buffer [200 mmol/L Tris-HCl (pH 6.8), 2 mmol/L EDTA, 0.1 M Na3VO4, 6% SDS, 10% glycerol, and 4% 2-mercaptoethanol], followed by SDS-PAGE and transfer to Immobilon-P membranes (Millipore). Membranes were blocked with 5% nonfat dried milk in PBS and then incubated overnight with the desired antibodies diluted in PBS containing 0.1% Tween. Primary antibodies bound to immunoreactive bands were visualized by enhanced chemiluminescence (ECL) detection with horseradish peroxidase-conjugated anti-mouse or anti-rabbit antibody and a FUJI LAS-4,000 mini luminescent image analyzer. Quantification of the bands was performed using the FUJI Multi Gauge V3.0 analysis program.
Immunofluorescence of PDAC cells was performed by fixing the cultures with 4% paraformaldehyde followed by permeabilization with 0.4% Triton X-100. Cultures were then incubated for 2 hours at 25°C in blocking buffer (BB), consisting of PBS supplemented with 5% BSA, then incubated at 4°C overnight with a YAP mouse mAb (1:200) diluted in BB. Bound primary antibody was detected using Alexa Fluor 488-conjugated goat-anti mouse (1:1,000) for 1 hour at 25°C. Nuclei were stained using a Hoechst 33342 stain (1:10,000). Images were captured as uncompressed 24-bit TIFF files captured with an epifluorescence Zeiss Axioskop and a Zeiss (Achroplan 40/0.75W objective) and a cooled (−12°C) single CCD color digital camera (Pursuit, Diagnostic Instruments) driven by SPOT version 4.7 software. Alexa Fluor 488 signals were observed with a HI Q filter set 41001 and TRITC images with a HI Q filter set 41002c (Chroma Technology).
For YAP localization the average fluorescence intensity in the nucleus and just outside the nucleus (cytoplasm) was measured to determine the nuclear/cytoplasmic ratios. All Image analysis was performed using Zeiss analysis imaging software. The selected cells displayed in the appropriate figures were representative of 80% of the population.
All siRNA transfection experiments were performed using Lipofectamine RNAiMAX (Life Technologies) following manufacturer's instructions. The siRNA concentration used was 10 nmol/L. PANC-1 and Mia PaCa-2 cells were transfected with the plasmid containing a cDNA encoding FLAG-tagged YAP wild type and mutants from Addgene by using Lipofectamine 3000 (Invitrogen) as suggested by the manufacturer. Analysis of the cells transiently transfected was performed 24 hours after transfection.
For cell colony formation, 500 PANC-1, Mia PaCa-2, or Capan-2 cells were plated into 35 mm tissue culture dishes in DMEM containing 10% FBS. After 24 hours of incubation at 37°C, cultures were incubated with DMEM or McCoy's medium containing 3% FBS either in the absence or presence of tramenitib, dasatinib, or their combination. Colonies, consisting of at least 50 cells, were stained with Giemsa. Colony numbers from at least three dishes per condition were determined after 8 to 10 days of incubation and repeated in three to five independent experiments. The combination index (CI) was calculated using the equation CI = TCx/Tx + DCx/Dx, where Tx and Dx represented concentrations of trametinib (T) or dasatinib (D) added singly to produce x% inhibition, whereas TCx and DCx were the concentrations of trametinib and dasatinib in combination to elicit the same effect. CI < 1 indicated synergism.
RNA was extracted to measure gene expression. Following cDNA synthesis, real-time qPCR was performed using CTGF- or CYR61-specific primers. CTGF or CYR61 mRNA expression levels were normalized to 18S mRNA levels. Data are presented as the mean of three replicates with error bars representing SEM. All reactions were performed using the Applied Biosystems StepOne system and TaqMan Fast Advanced Master Mix. The following primers were used CTGF (Assay ID: Hs01026927_g1), CYR61 (Assay ID: Hs99999901_s1), and 18S (Assay ID: Hs99999901_s1) all were from Life Technologies.
Early passage Mia PaCa-2 cells were harvested, and 4 × 106 cells were implanted into the right flanks of 5-week-old male nu/nu mice (weight 20.5 g ± 0.8, mean ± SEM, n = 32). The male nu/nu mice were maintained in a specific pathogen-free facility at the University of California at Los Angeles. The animals were randomized into control and treated groups (8 mice per group). Treatment was initiated when the tumors reached a mean diameter of 3 to 4 mm, and the first day of treatment in both cases was designated as day 0. Mice were treated three times weekly with either control vehicle or dasatinib (10 mg/kg) or trametinib (0.5 mg/kg) or a combination of dasatinib and trametinib. Compounds were administered by oral gavage in 80 mmol/L citrate solution with 0.5% hydroxypropylmethyl cellulose and 0.2% Tween-80. Tumor volume (V) was measured with an external caliper and it was calculated as V = 0.52 (length × width2). All animal experiments in this study were approved by the UCLA Chancellor's Animal Research Committee (protocol no.: 2011-118).
DMEM, McCoys's medium, FBS, goat anti-mouse IgG secondary antibody conjugated to Alexa Fluor 488 (Thermo Fisher Scientific, Catalog No. A-11029; RRID:AB_2534088). Primary antibodies used were as follows: phospho-FAK Tyr861 (Thermo Fisher Scientific, Catalog No. 44-626-G; RRID:AB_2533703 final dilution 1:1,000); YAP (63.1, Santa Cruz Biotechnology, Catalog No. sc-101199; RRID:AB_1131430, final dilution 1:200), GAPDH (Santa Cruz Biotechnology, Catalog No. sc-365062; RRID:AB_10847862, final dilution 1:500), phospho-YAP Tyr357 (Abcam, Catalog No. ab62751; RRID:AB_956486, final dilution 1:1000); Flag, DYKDDDDK Tag (9A3) Mouse mAb (Cell Signaling Technology, Catalog No. 8146; RRID:AB_10950495, final dilution 1:500), YAP (Cell Signaling Technology, Catalog No. 14074; RRID:AB_2650491 final dilution 1:1,000), phospho ERK1/2 (Thr202/Tyr204, Cell Signaling Technology, Catalog No. 9106; RRID:AB_331768, final dilution 1:1,000), ERK1/2 (Cell Signaling Technology, Catalog No. 4696), phospho-FAK (Tyr576/577, Cell Signaling Technology, Catalog No. 3281; RRID:AB_331079, final dilution 1:1,000), FAK (Cell Signaling Technology, Catalog No. 3285; RRID:AB_2269034, final dilution 1:1000), YES (Cell Signaling Technology, Catalog No. 3201, RRID:AB_11178531, final dilution 1:1,000), Src (Cell Signaling Technology, Catalog No. 2123; RRID:AB_2106047, final dilution 1:1,000) and Src Y416, (Cell Signaling Technology, Catalog No. 6943; RRID:AB_10013641, final dilution 1:1,000). Dasatinib (S1021), Saracatinib (AZD0530, #S1006), PP2 (#S7008), Trametinib (#S1021), and KPT-330 (# S7252) were all from Selleckchem. CH6953755 (HY135299) was from Medchemexpress LLC. All RT-qPCR reagents were obtained from Thermo Fisher Scientific. pcDNA Flag Yap1 (Addgene plasmid; RRID:Addgene_18881) and pcDNA Flag Yap1 Y357F (Addgene plasmid; RRID:Addgene_18882) were gifts from Yosef Shaul; pCMV-flag S127A YAP was a gift from Kunliang Guan (Addgene plasmid; RRID:Addgene_27370). siRNAs were from Santa Cruz Biotechnolgy (Src no. sc-29228 and YES1 no. sc-29860). All other reagents were of the highest grade available.
Each experiment was repeated three times independently. Unless otherwise noted, data are presented as mean ± SEM. Differences in YAP nuclear/cytoplasmic ratios, protein phosphorylation, and colony formation were determined using Student t test and were considered significant if P < 0.05. The growth of Mia PaCa-2 cells xenografted in the flank of nude mice was analyzed by ANOVA.
Data were generated by the authors and included in the article. The data generated in this study are available within the article and its supplementary data files.
As a first step to define the role of SFK members in patients with PDAC, we used an interactive open-access database (www.proteinatlas.org/pathology). We found that higher expression of YES1 is significantly associated with unfavorable prognosis (survival) in PDAC (Fig. 1A). Only 7% of the patients with higher levels of YES1 expression survived for 5 years whereas 51% of the subset with the lower levels of YES1 mRNA survived for 5 years or more (P < 0.001). Higher expression of Src is also associated with unfavorable prognosis (P < 0.015) in PDAC (Supplementary Fig. S1). These findings not only are inconsistent with a tumor suppressive role of SFKs in PDAC but imply that SFKs, especially YES1, are pro-oncogenic. However, the mechanism(s) involved remain poorly understood. Current evidence indicates that the transcriptional co-activator YAP plays a major role in PDAC development (25, 30, 38, 39). Using the web-based Gene Expression Profiling Analysis (GEPIA) tool (40), we found a strong correlation between YES1 and YAP mRNA expression in PDAC (R = 0.7; P = 1.6e−27; Fig. 1B). We also found a statistically significant correlation between Src and YAP mRNA expression in PDAC (Supplementary Fig. S1). The associations of YES and Src with patient survival and YAP expression prompted us to examine the mechanism(s) by which SFKs regulate YAP activity in human PDAC cells.
Previously, we demonstrated potent positive crosstalk between insulin/IGF-1 receptors and GPCR signaling systems leading to PDAC cell proliferation (36, 37, 41) but the effect of crosstalk on SFK activity was not determined. To assess SFK activity in intact PDAC cells, we determined the phosphorylation of focal adhesion kinase (FAK) at the activation loop Tyr576/577, a sensitive biomarker of SFK activity within intact cells (42, 43). Stimulation of PANC-1 cells with 5 nmol/L neurotensin and 10 ng/mL insulin induced a marked increase in FAK phosphorylation at Tyr576/577, which was prominent at the earliest time-point examined (1 min) and persisted for at least 120 minutes (Fig. 1C). Treatment with the SFK inhibitor dasatinib (10) abolished basal and stimulated FAK phosphorylation at Tyr576/577 (Fig. 1D). Saracatinib, a different anilinoquinazoline inhibitor of SFK activity, also inhibited FAK phosphorylation at Tyr576/577 in these cells (Fig. 1D; Structure of inhibitors in Supplementary Fig. S2). SFK activation in response to agonists was also evident in cells transferred to serum-free medium for 24 hours or not subjected to prior serum starvation (Fig. 1E). Stimulation with neurotensin and insulin also induced FAK phosphorylation at Tyr576/577 in Mia PaCa-2 cells, an effect prevented by dasatinib or saracatinib treatment (Fig. 1F). Stimulation with neurotensin and insulin also increased FAK phosphorylation at Tyr861, another site phosphorylated by SFKs and of Src on Tyr416, a key autophosphorylation site in the members of the family (44). Exposure to dasatinib or saracatinib prevented the increase in the phosphorylation of FAK at Tyr861 and SFK on Tyr416 (Fig. 1G). Collectively, the representative Western blots shown in Fig. 1 indicate that stimulation with neurotensin and insulin elicits rapid SFK activation in PDAC cells.
Next, we determined whether SFK inhibition prevents YAP nuclear import in PDAC cells. Previously, we showed that cell density regulates YAP localization in PANC-1 and Mia PaCa-2. At low cell density, YAP was predominantly localized in the nuclei of these cells, whereas YAP was prominently in the cytoplasm (therefore inactive) of confluent PDAC cells (37, 41). In agreement with previous results (37, 41), stimulation with neurotensin and insulin induced robust YAP nuclear localization in confluent PANC-1 cells. Exposure to dasatinib, saracatinib or PP2, a different SFK inhibitor, blocked the nuclear import of YAP (Fig. 2A). Accordingly, SFK inhibitors markedly decreased the expression of the YAP/TEAD-regulated genes CTGF and Cyr61 induced by neurotensin and insulin in PANC-1 cells (Supplementary Fig. S3A). Similarly, SFK inhibitors prevented YAP nuclear import in Mia PaCa-2 (Fig. 2B) and Capan 2 cells (Supplementary Fig. S3B). The quantification of the nuclear/cytoplasmic immunofluorescence ratio of YAP in multiple cells is shown in Fig. 2C (PANC-1), Fig. 2D (Mia PaCa-2), and Supplementary Fig. S3C (Capan 2). These results indicate that the SFKs play a major role in promoting YAP nuclear localization in PDAC cells. To identify the mechanism(s) by which SFK regulate YAP localization in pancreatic cancer cells, we determined the effect of neurotensin and insulin on YAP tyrosine phosphorylation in PDAC cells. As shown in Fig. 3A, stimulation with these agonists induced a marked increase in the phosphorylation of YAP at Tyr357 in PANC-1 cells, which was prominent within 10 minutes and persisted for at least 120 minutes. Dasatinib or saracatinib treatment markedly attenuated the increase in YAP phosphorylation at Tyr357 (Fig. 3B; quantification in Fig. 3C). Neurotensin and insulin also induced a time-dependent increase in YAP Tyr357 phosphorylation in Mia PaCa-2 cells (Supplementary Fig. S4A). Dasatinib or saracatinib treatment also decreased YAP Tyr357 phosphorylation in these cells (Supplementary Fig. S4B). Similar results were obtained with Capan-2 cells (Supplementary Figs. S4C and S4D). As indicated above, YES1 is the SFK member strongly correlated with unfavorable PDAC prognosis and YAP expression. Recently, the aminopyrazole derivative CH6953755 has been identified as a preferential YES1 tyrosine kinase inhibitor (45). We determined whether this inhibitor also prevents YAP phosphorylation at Tyr357 in PDAC cells. Exposure of PANC-1 cells to CH6953755 prior to stimulation with neurotensin and insulin prevented YAP phosphorylation at Tyr357 (Fig. 3D) and nuclear translocation (Supplementary Fig. S5). To substantiate the results obtained with chemical inhibitors, we determined whether siRNA-mediated knockdown of YES1 averts YAP phosphorylation at Tyr357. Transient transfection of PANC-1 cells with siRNAs targeting YES1 decreased the level of YES1 protein and prevented the increase in YAP phosphorylation at Tyr357 induced by neurotensin and insulin (Fig. 3E; quantification in Fig. 3F). Collectively, the results show, for the first time, that YES1 plays a major role in mediating YAP phosphorylation on Tyr357 and nuclear localization in response to crosstalk between GPCR and insulin receptor signaling pathways in PDAC cells.
As mentioned above, cell density regulates YAP localization in PANC-1 and Mia PaCa-2. At low cell density, YAP was localized in the nuclei of these cells, whereas YAP was prominently in the cytoplasm of confluent cultures of PDAC cells (37, 41). In agreement with these findings, YAP was localized prominently in the nucleus of PANC-1 and Mia PaCa-2 cells growing at low cell densities (Fig. 4A and B). Dasatinib or saracatinib treatment induced a striking relocalization of YAP from the nucleus to the cytoplasm (Fig. 4A and B). These results indicate that SFK controls the localization of YAP in low-density, rapidly growing human PDAC cells. To determine the role of YAP phosphorylation in the regulation of YAP localization in PDAC cells, we used wild type and mutant FLAG-tagged YAP expressed in low-density cultures of PANC-1 cells. As shown with the endogenous YAP, the wild-type FLAG-YAP localized predominantly in the nucleus of low-density PANC-1 cells and redistributed to the cytoplasm in response to dasatinib treatment (Fig. 4C). Phosphorylation of Ser127 by the LATS1/2 of the Hippo pathway is a recognized mechanism by which YAP is retained in the cytoplasm. Accordingly, expression of FLAG-YAP with Ser127 mutated to Ala (FLAG-S127A-YAP) localized in the nucleus. Interestingly, dasatinib treatment induced cytoplasmic localization of FLAG-S127A-YAP, implying that tyrosine phosphorylation overrides regulation through the Hippo pathway (Fig. 4C). Crucially, a FLAG-YAP with Tyr357 mutated to Phe (FLAG-Y357F-YAP) was excluded from the nucleus of low-density PANC-1 cells, indicating that YAP phosphorylation on Tyr357 plays a critical role in controlling YAP localization. Interestingly, FLAG-Y357F-YAP accumulated in the nucleus of cells treated with KPT-330, a potent inhibitor of exportin 1 (XPO-1), the major nuclear-cytoplasmic exportin (Fig. 4C). Similar results were obtained when the FLAG-YAP plasmids were transfected to low-density cultures of Mia PaCa-2 cells (Fig. 4D). The results indicate that FLAG-Y357F-YAP is not permanently sequestered in the cytoplasm but enters into the nucleus from where it is excluded via XPO-1 and imply that YAP phosphorylation on Tyr357 interferes with its nuclear export. Having established that either SFK inhibitors or mutation of Tyr357 to Phe promotes cytoplasmic YAP localization in PANC-1 cells, we next determined whether the SFK inhibitors also inhibit the proliferation of these cells. As shown in Fig. 4E, dasatinib inhibited colony formation by PANC-1 cells in a dose-dependent manner.
Although the preceding results suggest that dasatinib could be useful in the treatment of PDAC through inhibition of the SFK/YAP axis, clinical trials in patients with PDAC using dasatinib with gemcitabine (12) or 5-fluorouracil and oxaliplatin (14) were unsuccessful. We conjectured that SFK inhibitors not only hinder YAP activation but also activate a compensatory pathway(s) that mediates resistance to these drugs (46). In line with this possibility, we found that treatment with dasatinib induced a marked increase in ERK activity in unstimulated PANC-1 cells or in cells exposed to neurotensin and insulin, as shown by ERK dual phosphorylation on Thr202 and Tyr204 (Fig. 5A; quantification in Supplementary Fig. S6A). Dasatinib-induced ERK activation was completely abolished by exposure to trametinib, a potent MEK inhibitor. Trametinib prevented dasatinib-induced ERK overactivation in PANC-1 cells at a concentration as low as 30 nmol/L (Fig. 5B). Saracatinib or PP2 treatment also enhanced ERK activation, and this effect was abrogated by trametinib (Fig. 5C). Furthermore, siRNA-mediated knockdown of YES1, SRC, or both decreased their protein levels and promoted ERK overactivation (Fig. 5D; quantification in Supplementary Figs. S6B and S6C). Dasatinib or saracatinib treatment markedly increased basal ERK activity in Mia PaCa-2 cells to a level comparable with that produced by agonist stimulation (Supplementary Fig. S6D).
Because trametinib prevented dasatinib-induced ERK activation in PDAC cells, we next determined whether trametinib potentiates the inhibitory effect of dasatinib on colony formation by these cells. As illustrated in Fig. 6A and B dasatinib, at a concentration that has a modest inhibitory effect on colony formation (0.03 μmol/L), markedly potentiated the inhibitory effect of trametinib (0.03 μmol/L). Colony formation by Mia PaCa-2 or Capan 2 cells was drastically inhibited at even lower concentrations. A combination of dasatinib and trametinib each at a concentration as low as 0.003 μmol/L abolished colony formation in either Mia PaCa-2 (Fig. 6C and D) or Capan 2 cells (Supplementary Fig. S7). The combination index (CI) of dasatinib and trametinib was <1 (i.e., 0.35, 0.4, and 0.38 for PANC-1, Mia Paca-2, and Capan 2, respectively), indicating synergistic interaction between these drugs in inhibiting colony growth in PDAC cell lines. These results show that a combination of dasatinib and trametinib inhibits colony formation of human PDAC cells at clinically relevant concentrations. Furthermore, administration of a combination of dasatinib (10 mg/kg) and trametinib (0.5 mg/kg) by oral gavage suppressed the growth of Mia PaCa-2 cells xenografted in the flank of nude mice (Fig. 6E).
PDAC is a highly aggressive disease that is expected to become the second cause of cancer fatalities during the next decade (2). As therapeutic options are limited, novel targets and agents for combinatory therapeutic intervention are urgently required (47). In this context, the highly conserved transcriptional co-activator YAP is attracting intense interest. YAP is a context-specific oncogene (20), which is overactive in PDAC patient tumor samples (26) and associated with poor PDAC survival (25). YAP is highly activated in the squamous subtype of PDAC (30), which exhibits reduced dependency on KRAS for survival (31, 32). Accordingly, overexpression of Yap can substitute for mutant Kras expression in promoting PDAC in preclinical models (26) and depletion of KRAS protein has minimal effects on the survival of PANC-1 cells (48), a model of squamous subtype of PDAC. Because either YAP or its upstream pathways is rarely mutated in PDAC, the signaling mechanism(s) leading to YAP activation is of critical importance. Several studies implicated SFK in YAP activation, acting through Hippo-dependent (33, 34) and/or Hippo-independent pathways (35) but the role of SFKs in YAP activation in PDAC cells remained unknown. Here, we show that insulin and the GPCR agonist neurotensin, a potent mitogenic combination, induced rapid SFK activation within intact PANC-1 cells, as judged by FAK phosphorylation at Tyr576/576 and Tyr861, sensitive biomarkers of SFK activity within intact cells (42) and Src416 autophosphorylation, a critical event in the activation of all members of the SFK (44). Treatment with either dasatinib or saracatinib suppressed SFK activity within PDAC cells. Crucially, these SFK inhibitors blocked YAP nuclear localization stimulated by neurotensin and insulin, implying that SFKs promote YAP nuclear accumulation in human PDAC cells. SFK have been implicated in the regulation of YAP localization but the mechanisms are cell-context dependent and the role of SFKs in YAP regulation in PDAC cells was not examined before. Here, we show that stimulation of PDAC cells with neurotensin and insulin induced a marked and persistent increase in YAP phosphorylation at Tyr357. Exposure to dasatinib or saracatinib completely prevented the increase in YAP phosphorylation on Tyr357. Furthermore, CH6953755, a preferential YES1 inhibitor (45), also abrogated YAP phosphorylation on Tyr357, suggesting that YES1 plays a major role in mediating YAP phosphorylation in response to agonist stimulation. Accordingly, siRNA-mediated knockdown of YES1 severely impaired YAP phosphorylation on Tyr357. Collectively, these results show, for the first time, that YES1 plays a critical role in promoting YAP tyrosine phosphorylation in PDAC cells. The importance of these mechanistic findings is emphasized by the Kaplan–Meier curves displayed in Fig. 1, showing that high YES1 expression is associated with shorter survival of patients with pancreatic cancer. As mentioned, YAP is localized prominently in the nucleus of PDAC cells growing at low cell densities (37, 41). Dasatinib or saracatinib treatment induced striking relocalization of YAP from the nucleus to the cytoplasm in sparse cultures of PDAC cells. These results suggested that SFK-mediated tyrosine phosphorylation controls the localization of YAP in low-density, rapidly growing human PDAC cells. In line with this notion, a FLAG-YAP with Tyr357 mutated to Phe (FLAG-Y357F-YAP) was excluded from the nucleus of PDAC cells. These results substantiate the conclusion that YAP phosphorylation at Tyr357 plays a major role in promoting YAP nuclear localization in these cells. Interestingly, FLAG-Y357F-YAP accumulated in the nucleus of cells treated with KPT-330 (49), a potent inhibitor of exportin 1 (XPO-1), the major nuclear-cytoplasmic exportin. The results suggest that FLAG-Y357F-YAP is not irreversibly trapped in the cytoplasm but enters into the nucleus from where it is efficiently excluded via XPO-1. We conclude that YAP phosphorylation at Tyr357 reduces the rate of its nuclear export thereby leading to YAP nuclear accumulation. The preceding results suggest that dasatinib could be useful in the treatment of PDAC through inhibition of the SFK/YAP axis. However, clinical trials in patients with PDAC using dasatinib in combination with gemcitabine (12) or 5-fluorouracil and oxaliplatin (14) were unsuccessful. We considered that SFK inhibitors not only hinder YAP activation but concomitantly trigger activation of a compensatory pathway(s) that mediates resistance to these drugs (46). Accordingly, we found that treatment of PDAC cells with SFK inhibitors increased basal ERK activity and further enhanced ERK activation by mitogenic agonists. Previous studies showed that dasatinib (50), like RAF inhibitors (51), induced BRAF/CRAF dimerization leading to MEK/ERK activation in a Ras-dependent manner. Dasatinib was thought to act as a weak inhibitor of RAF isoforms leading to paradoxical activation of the pathway (50). Our results do not support this mechanism in PDAC cells because we also observed ERK activation in response to saracatinib, a structurally different SFK inhibitor which does not inhibit BRAF or CRAF, even at high concentrations (52). Importantly, we also show that siRNA-mediated knockdown of Src/YES also induce ERK activation. We therefore conclude that pharmacologic or genetic suppression of SFK activity leads to ERK activation through a mechanism that differs from that used by RAF inhibitors. Recent studies showed that SFK directly phosphorylates KRAS on Tyr32 and Tyr64 and inhibits its ability to activate RAF/MEK/ERK (15, 53). Consequently, it is plausible that exposure to SFK inhibitors induces RAS hyper-activation, thereby leading to enhanced RAF/MEK/ERK signaling in PDAC cells by blocking SFK-mediated inhibitory phosphorylation of RAS, a proposition that warrants further experimental work. Our results also demonstrate that SFK-induced ERK activation was completely abolished by cell exposure to the FDA-approved MEK inhibitor trametinib. Importantly, the combination of dasatinib and trametinib, each inhibitor at clinically relevant concentrations, completely blocked YAP phosphorylation on Tyr357 and ERK activation and prevented colony growth by PDAC cells in a synergistic manner. Indeed, colony formation by PANC-1, Mia PaCa-2, or Capan 2 was only abolished when both pathways (Src/YAP and MEK/ERK) were inhibited. The dasatinib/trametinib combination also inhibited the growth of Mia PaCa-2 cells xenografted in nude mice. Thus, these results provide a mechanistic rationale to consider that a combination of the FDA-approved inhibitors dasatinib with trametinib should be considered for clinical trials in PDAC. Our study is in agreement with other recent reports that identified empirically the combination of dasatinib and trametinib as a plausible therapeutic intervention for cancers with KRAS-activating mutation (54, 55) but differs from these studies because the results presented here provide a mechanistic rationale for the interaction between the inhibitors, namely, dasatinib inhibits YAP tyrosine phosphorylation and nuclear localization and trametinib prevents the compensatory ERK activation induced by SFK inhibition. Our results also support the development of novel SFK/panRAF inhibitors, including CCT3833, which inhibits SFK but does not activate ERK and is being evaluated in an early-phase clinical trial (56). In summary, the findings presented here identify an important interplay between SFKs, YAP and MEK/ERK in PDAC sensitivity to drug combinations and support clinical testing of a combination of FDA-approved SFK and MEK inhibitors in PDAC treatment and the development of novel dual SFK/RAF inhibitors.
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PMC9630829 | Jenna C. Carlson,Mohanraj Krishnan,Samantha L. Rosenthal,Emily M. Russell,Jerry Z. Zhang,Nicola L. Hawley,Jaye Moors,Hong Cheng,Nicola Dalbeth,Janak R. de Zoysa,Huti Watson,Muhammad Qasim,Rinki Murphy,Take Naseri,Muagututi’a Sefuiva Reupena,Satupa‘itea Viali,Lisa K. Stamp,John Tuitele,Erin E. Kershaw,Ranjan Deka,Stephen T. McGarvey,Tony R. Merriman,Daniel E. Weeks,Ryan L. Minster | A stop-gain variant in BTNL9 is associated with atherogenic lipid profiles | 12-10-2022 | identification of disease genes,Polynesia,cardiovascular disease risk factors,genetics of complex traits,isolated population | Summary Current understanding of lipid genetics has come mainly from studies in European-ancestry populations; limited effort has focused on Polynesian populations, whose unique population history and high prevalence of dyslipidemia may provide insight into the biological foundations of variation in lipid levels. Here, we performed an association study to fine map a suggestive association on 5q35 with high-density lipoprotein cholesterol (HDL-C) seen in Micronesian and Polynesian populations. Fine-mapping analyses in a cohort of 2,851 Samoan adults highlighted an association between a stop-gain variant (rs200884524; c.652C>T, p.R218∗; posterior probability = 0.9987) in BTNL9 and both lower HDL-C and greater triglycerides (TGs). Meta-analysis across this and several other cohorts of Polynesian ancestry from Samoa, American Samoa, and Aotearoa New Zealand confirmed the presence of this association (βHDL-C = −1.60 mg/dL, pHDL-C = 7.63 × 10−10; βTG = 12.00 mg/dL, pTG = 3.82 × 10−7). While this variant appears to be Polynesian specific, there is also evidence of association from other multiancestry analyses in this region. This work provides evidence of a previously unexplored contributor to the genetic architecture of lipid levels and underscores the importance of genetic analyses in understudied populations. | A stop-gain variant in BTNL9 is associated with atherogenic lipid profiles
Current understanding of lipid genetics has come mainly from studies in European-ancestry populations; limited effort has focused on Polynesian populations, whose unique population history and high prevalence of dyslipidemia may provide insight into the biological foundations of variation in lipid levels. Here, we performed an association study to fine map a suggestive association on 5q35 with high-density lipoprotein cholesterol (HDL-C) seen in Micronesian and Polynesian populations. Fine-mapping analyses in a cohort of 2,851 Samoan adults highlighted an association between a stop-gain variant (rs200884524; c.652C>T, p.R218∗; posterior probability = 0.9987) in BTNL9 and both lower HDL-C and greater triglycerides (TGs). Meta-analysis across this and several other cohorts of Polynesian ancestry from Samoa, American Samoa, and Aotearoa New Zealand confirmed the presence of this association (βHDL-C = −1.60 mg/dL, pHDL-C = 7.63 × 10−10; βTG = 12.00 mg/dL, pTG = 3.82 × 10−7). While this variant appears to be Polynesian specific, there is also evidence of association from other multiancestry analyses in this region. This work provides evidence of a previously unexplored contributor to the genetic architecture of lipid levels and underscores the importance of genetic analyses in understudied populations.
Atherogenic lipid profiles—increased total cholesterol (TC), low-density lipoprotein cholesterol (LDL-C), and triglycerides (TGs) as well as decreased high-density lipoprotein cholesterol (HDL-C)—are well-documented and heritable risk factors for cardiovascular disease (CVD) worldwide. While behavioral modifications and medication have been successful in improving lipid profiles, CVD is still the leading cause of death worldwide, particularly among people of Polynesian and Pacific Island ancestry.1, 2, 3, 4, 5 The examination of the genetic underpinnings of lipid variation through genome-wide association studies (GWASs) has identified numerous genetic associations. These discoveries have furthered the understanding of CVD and potential therapeutic targets; however, this research has primarily come from studies in European-ancestry populations. Recent efforts to diversify research on this topic have highlighted the importance of including diverse populations for gene discovery, yielding novel associations, improved fine mapping, and better polygenic risk scores. Despite this, limited effort has focused on Polynesian populations, whose unique population history including genetic drift from founder effects, small population sizes, and population bottlenecks may provide insight into the biological foundations of variation in lipid levels, which would not only benefit Polynesian individuals but also those from other populations.7, 8, 9 One region of interest is 5q35, which has previously been associated with HDL-C levels in Micronesian and Samoan populations., The causal variant at this locus, and the biological mechanism underlying this association with an atherogenic lipid profile, is unknown. The aim of this study was to fine map this association signal in a cohort of 2,851 Samoan adults and replicate this association in several independent Polynesian cohorts from Samoa, American Samoa, and Aotearoa New Zealand. We identified a strong candidate causal variant at this locus, rs200884524—a stop-gain variant in BTNL9—that is associated with lower HDL-C and higher TG concentrations.
An overview of the analytical methods is presented in Figure S1.
We performed association testing between variants on 5q35 and lipid levels in a discovery cohort comprising 2,851 Samoan adults (at least 18 years old) drawn from a population-based sample recruited from Samoa in 2010 (Table 1), data for which are available from dbGaP (accession number: phs000914.v1.p1). The sample selection, data collection methods, and phenotyping, including the laboratory assays for serum lipid and lipoprotein levels, have been previously described., Briefly, serum lipid levels (TC, HDL-C, LDL-C, and TG) were derived from fasting whole-blood samples collected after a minimum 10-h overnight fast. LDL-C levels were estimated using the Friedewald equation, which does not accurately estimate LDL-C in individuals with TG ≥400 mg/dL, and, therefore, those individuals are missing LDL-C values. Analyses were performed without consideration of hypolipidemic medication, as it was not measured. However, individuals who reported taking medication for heart disease were excluded from analyses, as previous sensitivity analyses identified an association between self-reported use of heart disease medication and TC and LDL-C. This study was approved by the institutional review board of Brown University and the Health Research Committee of the Samoa Ministry of Health. All participants gave written informed consent via consent forms in the Samoan language. Genotyping was performed using Genome-Wide Human SNP 6.0 arrays (Affymetrix), and extensive quality control was conducted via a pipeline developed by Laurie et al. Additional details for sample genotyping and genotype quality control are described in Minster et al. A Samoan-specific genotype reference panel for imputation was created using the whole-genome sequence (WGS) data from the Trans-Omics for Precision Medicine (TOPMed) program from the National Institutes of Health’s Heart, Lung, and Blood Institute (NHLBI). The reference panel is comprised of high-quality (i.e., passing all quality control [QC] filters and with a minimum depth of 10), bi-allelic markers from the freeze 8 call subset of the 1,284 individuals from the Samoan Adiposity Study. The reference panel was phased using Eagle v.2.4.1. The Samoan-specific reference panel was then used to impute genotype data for the remaining Samoan participants in the discovery cohort who were not in the reference panel. Briefly, genotyped variant coordinates were converted to the hg38 genome build using LiftOver (https://genome.ucsc.edu/cgi-bin/hgLiftOver). Then, data were aligned to the reference panel using Genotype Harmonizer, using the mafAlign option to align to the minor allele when LD alignment failed and the minor allele frequency (MAF) was ≤30% in both the input and reference sets. The resulting variants were then phased with Eagle v.2.4.1, and genome-wide imputation was performed using Minimac4. Imputed genotype dosages from variants passing QC (r2 > 0.3) were combined with genotyped variants for association testing. We performed preliminary association testing between variants in a 1-Mb region around the sentinel SNP from the preliminary GWAS (hg38 chr5:180414895-181414895) and the four lipid levels (TC, HDL-C, LDL-C, and TG) in the discovery cohort using linear mixed modeling with inverse-normally transformed traits, marginally rescaled variance (to restore it to the original variance before the transformation), and additive genotype coding as implemented in the GENESIS R package, adjusting for the following fixed effects: three principal components of ancestry (generated through PC-AiR, which together accounted for >98% variation), age, age2, sex, age × sex interaction, and age2 × sex interaction. Age was mean centered for all analyses to avoid multicollinearity issues. Relatedness was measured through an empirical kinship matrix generated with GENESIS and was modeled through a random effect. Then, to estimate the effect size of rs200884524 for use in the meta-analysis across cohorts, we used a linear mixed-modeling strategy on the original lipid levels (i.e., not transformed), adjusting for the same fixed and random effects as implemented in the lmekin function in the coxme R package. These models were also repeated for several cardiometabolic traits as described in Minster et al. to examine the potential pleiotropic effects of rs200884524. As a sensitivity analysis to check for robustness against deviations from normality, the association tests with rs200884524 were repeated using lipid-level residuals that were adjusted for three principal components of ancestry, age, age2, sex, and age × sex and age2 × sex interactions and were then inverse-normally transformed using RankNorm function in the RNOmni R package. To preserve the interpretation of the effect estimates, the results of the untransformed trait analyses are presented. The results of the sensitivity analysis were similar to the models in which lipid levels were not transformed (Table S1). To detect a potential secondary signal, conditional analyses were run in the 5q35 region in the discovery cohort. rs200884524, the lead SNP in the region, was modeled as a fixed-effect covariate; the rest of the model parameters stayed the same. Bayesian fine mapping of the discovery cohort results was performed using PAINTOR v.2.1. Annotations of variant impact (high, moderate, low/modifier) as indicated by VEP v.91.1 were used to inform the fine mapping. Evidence of selection at this locus was assessed using nSL (number of segregation sites by length) analysis in 419 unrelated Samoans from the discovery cohort with no evidence of non-Oceanic admixture. WGS was completed by TOPMed, and a MAF of >0.01 was used. Haplotype phasing was completed using Eagle v.2.4.1. nSL was calculated with selscan in R.
Two cohorts consisting of a total of 1,466 Samoan and American Samoan adults (at least 18 years old) were used for replication. The first was a longitudinal study of adiposity and CVD risk factors among adults from American Samoa and Samoa recruited between 1994 and 1995, during which lipid levels were measured from fasting serum samples. LDL-C levels were estimated using the Friedewald equation, which does not accurately estimate LDL-C in individuals with TG ≥400 mg/dL, and, therefore, those individuals are missing LDL-C values. Detailed descriptions of the sampling, recruitment, and phenotyping have been reported previously.29, 30, 31 The second study included adults from American Samoa and Samoa, recruited in 2002–2003, and was drawn from an extended family-based genetic linkage study of cardiometabolic traits.32, 33, 34 Probands and relatives were unselected for obesity or related phenotypes, and all individuals self-reported Samoan ancestry. The recruitment process, criteria used for inclusion in this study, and phenotyping have been described in detail previously., To account for temporal differences, each of the two studies was modeled separately—Samoa/American Samoa 1994–1995 (n = 557) and Samoa/American Samoa 2002–2003 (n = 909). Participants in these two cohorts were genotyped using the Infinium Global Screening Array-24 v.3.0 BeadChip (Illumina, CA, USA) with custom content that included rs200884524. These studies were approved by the institutional review boards of Brown University and the American Samoa Department of Health, as well as the Health Research Committee of the Samoa Ministry of Health. All participants gave written informed consent.
A total of 1,810 adults (at least 16 years old) of Polynesian (NZ Maori and Pacific) ancestry were recruited from Aotearoa New Zealand as part of a study of risk factors for gout, type 2 diabetes, and kidney disease. Lipid parameters (VLDL and TG) have previously been associated with gout in an Aotearoa NZ Polynesian cohort. Participants were divided into three replication cohorts based on the self-reported ancestry of their grandparents (East Polynesia [Aotearoa New Zealand Māori and Cook Island Māori] n = 1,109; West Polynesia [Samoa, Tonga, Pukapuka, and Niue], n = 603; and Mixed East/West Polynesia, n = 98). A separate Māori sample set from the rohe (area) of the Ngāti Porou iwi (tribe) of the Tairāwhiti (East Coast of the North Island of New Zealand) region was also included in the East Polynesia group. This sample set was recruited in collaboration with the Ngāti Porou Hauora (Health Service) Charitable Trust. The details of sampling, recruitment, phenotyping, and creation of principal components of ancestry have been previously reported., Briefly, lipid levels were measured from serum samples at Southern Community Laboratories (Dunedin, NZ). LDL-C levels were estimated using the Friedewald equation, which does not accurately estimate LDL-C in individuals with TG ≥400 mg/dL, and, therefore, those individuals are missing LDL-C values. Genotyping of rs200884524 was carried out using the TaqMan SNP genotyping assay technology (Applied Biosystems, Foster City, CA, USA) using a LightCycler 480 Real-Time Polymerase Chain Reaction (PCR) system (Roche, Indianapolis, IN, USA). This study was approved by the New Zealand Multi-Region Ethics Committee and the Northern Y Region Health Research Ethics Committee. All participants gave written informed consent.
Association testing for rs200884524, the sentinel SNP in the 5q35 region as identified in the discovery cohort, and the lipid levels (TC, HDL-C, LDL-C, and TG) was performed in each of the five replication cohorts using linear mixed modeling, adjusting for the following fixed effects: principal components (PCs) of ancestry (four PCs generated through PC-AiR were used for the Samoan/American Samoan replication cohorts, three PCs for the Aotearoa New Zealand replication cohorts), polity (an indicator of Samoa/American Samoa in the two respective cohorts), age, age2, sex, age × sex interaction, and age2 × sex interaction. Age was mean centered within each cohort for all analyses to avoid multicollinearity issues. Relatedness was measured through empirical kinship matrices and modeled as a random effect using the lmekin function in the coxme R package. For the Samoan/American Samoan replication cohorts, the empirical kinship matrix was generated with GENESIS;, for the Aotearoa New Zealand replication cohorts, the empirical kinship matrix was calculated in PLINK v.1.9, as described previously.
The effects of rs200884524 on the four lipid traits from the discovery and 5 replication cohort analyses were combined using an inverse-variance fixed-effect meta-analysis as implemented in the rmeta R package. Additionally, meta-analyses were conducted separately across the two Samoan/American Samoan replication cohorts and the three Aotearoa New Zealand replication cohorts. Heterogeneity was assessed with Cochran’s Q statistic.
Descriptions of age, TC, HDL-C, LDL-C, and TG levels in the discovery and replication cohorts are given in Table 1. Generally, the cohorts were very similar (with the exception of TG, which varies more widely across cohorts). The association analyses in the discovery cohort in the 1-Mb region on 5q35 (Figures S2–S5) highlighted an association between a stop-gain variant (rs200884524; c.652C>T, p.R218∗) in the butyrophilin-like 9 (BTNL9) gene with HDL-C (p = 4.05 × 10−8; Figure S2). Conditional analyses on rs200884524 showed some evidence of a secondary signal approximately 0.449 Mb upstream from the lead variant in the region (rs71680280; CA>C, conditional p = 6.52 × 10−6, linkage disequilibrium [LD] with rs200884524 r2 = 0.035, Figure S2). Bayesian fine mapping pointed to rs200884524 with high posterior probability (PP) of causality (PP = 0.9987); all other variants in the region had PP <0.10. rs200884524 did not show evidence of association with any other cardiometabolic phenotype measured (Table S1). The estimated effects of rs200884524 on the four lipid levels for each cohort are given in Table 2. rs200884524 was significantly associated with lower average HDL-C levels overall (meta-analysis β = −1.60 mg/dL, p = 7.63 × 10−10; Figure 1), with no evidence of heterogeneity of effect across cohorts (p = 0.65). Similarly, rs200884524 was significantly associated with higher average TG levels overall (meta-analysis β = 12.00 mg/dL, p = 3.82 × 10−7, heterogeneity p = 0.75). Sensitivity analyses using inverse-normal-transformed phenotypes showed similar results (Table S2). rs200884524 accounts for 0.94% and 0.68% of the variation in HDL-C and TG, respectively, in the discovery cohort. rs200884524 has MAF <0.0001 in gnomAD v.3.1.1 (accessed February 16, 2022); of the 14 alleles seen in gnomAD, 7 are from East Asians, with no observed homozygotes. However, we observed a MAF of 0.223 in the Samoan discovery cohort with 163 observed homozygotes. The MAFs of rs200884524 ranged from 0.049 to 0.233 in the Samoan/American Samoan and Aotearoa New Zealand replication cohorts, with higher frequencies observed among the Western Polynesian participants (Table 2). Consistent with these disparate allele frequencies, rs200884524 also showed evidence of positive selection in the discovery cohort (nSL score of 1.79, 98.9 percentile in the Samoan genome).
We found strong evidence of association between a stop-gain variant in BTNL9, rs200884524, and atherogenic serum lipid profiles (lower HDL-C, higher TG) across several independent cohorts of Polynesian individuals. Interestingly, this variant appears to be Polynesian specific—common in those of Western Polynesian ancestry (which includes Samoa), rarer in those of Eastern Polynesian ancestry, and absent from other non-Polynesian populations. When compared with effect sizes of other index variants from GWASs of HDL-C, the effect size of rs200884524 was moderate to large, which is similar to other findings in this population.,, Moreover, the identification of rs200884524 was achieved using a population-specific genotype imputation panel, highlighting the importance of such panels when working with isolated populations. Previous work (using the discovery cohort from these results) observed a suggestive association between this region and HDL-C but was limited to SNP array data only rather than the dense data from imputed genotypes. The sparse genotypes in this region did not facilitate accurate fine mapping. In fact, we previously nominated MGAT1 as the causal gene for this locus as the stop-gain variant in BTNL9 was not genotyped. Once the dense information from imputed genotypes was available, it was easy to identify the stop-gain variant rs200884524 as a variant of interest, which led us to include it with the custom content selected for genotyping in the Samoan/American Samoan replication cohorts. These results highlight the necessity of population-specific imputation reference panels and the fruitfulness of gene discovery in isolated populations. There is evidence that rs200884524 plays a role in nonsense-mediated decay (NMD) efficacy; the variant is located in exon 4 of 11 in BTNL9 and has strong mRNA degradation predicted (NMDetective score 0.6,). While BTNL9 is not constrained/predicted to be loss-of-function intolerant (predicted LoF observed/expected = 0.94, gnomAD v.2.1.1), the role of the BTNL9 protein in human disease is still unknown. Despite the T allele of rs200884524 having a very low frequency in other populations, the association between variants in this region and lipid levels does not appear to be Polynesian specific. Results from studies of the UKBiobank and the Global Lipids Genetics Consortium demonstrate evidence of association in this region as well. Specifically, an intron variant in nearby BTNL3 (∼61 kb away from rs200884524) was significantly associated with HDL-C in a GWAS of ∼459,000 individuals of European descent (rs138354839, pHDL-C = 9.70 × 10−13). Additionally, variants in and upstream of BTNL8 (∼128–169 kb away from rs200884524) were associated with total and HDL-C in a large, multiancestry meta-analysis (rs138692142, pHDL-C = 8.09 × 10−17; rs188238483 pTC = 3.39 × 10−9). Each of these variants is rare in the Samoan discovery cohort presented here (MAF = 0.009, 0.0003, and 0.00003, respectively). The positions of these variants in relationship to the stop gain are depicted in Figure 2. Furthermore, there were significant associations between pLoF variants in BTNL9 and both apolipoprotein A and HDL-C observed in gene-based studies of European-ancestry individuals also from the UKBiobank.48, 49, 50 Both associations are driven by a single variant (rs367635312, pApoA = 1.93 × 10−9, pHDL-C = 1.48 × 10−7, MAF = 0.0125 in non-Finnish Europeans). This variant is also rare in the Samoan discovery cohort (MAF = 0.00016). Interestingly, the region also harbors a ∼55-kb common deletion with consequence in BTNL8 and BTNL3 (DEL_5_65831 gnomAD SVs v.2.1). This deletion is common in those of Asian, American, and European descent but rare in African and Oceanic (Papuan and Melanesian) populations. The frequency of this deletion in Polynesians is unknown as Polynesians have differing genetic ancestries from Papuans and Melanesians. Functional analysis has shown that this deletion down-regulates BTNL9 in lymphoblastoid cell lines. This deletion is in high LD (r2 > 0.80) with 26 SNVs in the UKBiobank., The SNV in very high LD with the deletion (rs72494581, LD with deletion 0.97) showed minimal evidence of association with HDL-C in the Pan-UKBiobank (p = 0.056 in meta-analysis across all populations, p = 0.037 in individuals of European descent)., Because of the low allele frequency of rs200884524 in non-Polynesian populations, it is unknown if the stop-gain variant is in LD with this deletion. Further work is needed to explore the interplay of this deletion with rs200884524 to examine the causal mechanism and resulting biological impact on lipids. Little is known about the function of BTNL9 and the mechanism by which it may impact serum lipids; however, it has been previously associated with cardiomyopathy,54, 55, 56 pre-eclampsia, and various cancers., BTNL9 has a cell- and tissue-specific expression pattern—it is most highly expressed in human B cells of adipose tissue, lung, thymus, spleen, and heart. Moreover, BTNL9 is localized to the plasma membrane and binds to immune cell surfaces. BTNL9 belongs to the butyrophilin (BTN) family of membrane proteins, which is part of a superfamily of immunoglobulin (Ig) receptors. Several studies suggest a role of BTN proteins in inflammatory and immunological functions,,63, 64, 65, 66, 67, 68, 69, 70, 71, 72 supported by the shared extracellular characteristics and homology of the BTN proteins with the B7 family (which includes ligands and receptors for T cell activity). BTNL9 is member of a BTN subfamily along with BTNL3 and BTNL8, which all diverged from a common ancestor. While BTNL3 and BTNL8 are primate specific, BTNL9 is conserved across several mammalian species; however, the protein structure differs across species. Human BTNL9 has two substitution mutations that result in the loss of the IgC domain that is present in other mammals. In mice, the receptor to Btnl9 was shown to be expressed in immune cells, supporting the hypothesized immune function., Future functional work is needed to identify the biological role of BTNL9 in lipid and lipoprotein metabolism, immune function, and, ultimately, cardiovascular (and potentially cancer) risk. Additional work is necessary to characterize the relationship between variation in BTNL9 and lipid levels, especially analyzing the impact of the nearby deletion in BTNL8/BTNL3, as well as any potential interaction between immune cells and lipid transport pathways. However, these findings fine map the suggestive association in 5q35 to a stop-gain variant in BTNL9, providing evidence of a new contributor to the genetic architecture of lipids. This work highlights the importance of measuring association in non-European populations as it can provide insight not only into population-specific findings and benefits but also into cross-population associations, which may lead to therapeutic targets able to benefit multiple population groups.
The discovery cohort data used for this study are available through dbGaP (accession numbers: phs000914.v1.p1 and phs000972.v5.p1). The Samoan/American Samoan and Aotearoa New Zealand replication cohorts’ data have not been deposited in a public repository because participants did not give consent for data sharing. Code used for data analysis will be made available upon request. | true | true | true |
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PMC9630969 | Xiang Peng,Chen Feng,Yan-Tao Wang,Xiang Zhang,Yan-Yan Wang,Yue-Ting Sun,Yu-Qin Xiao,Ze-Feng Zhai,Xin Zhou,Bing-Yang Du,Chao Wang,Yang Liu,Tian-Hong Li | miR164g-MsNAC022 acts as a novel module mediating drought response by transcriptional regulation of reactive oxygen species scavenging systems in apple | 30-08-2022 | Abstract Under drought stress, reactive oxygen species (ROS) overaccumulate as a secondary stress that impairs plant performance and thus severely reduces crop yields. The mitigation of ROS levels under drought stress is therefore crucial for drought tolerance. MicroRNAs (miRNAs) are critical regulators of plant development and stress responses. However, the complex molecular regulatory mechanism by which they function during drought stress, especially in drought-triggered ROS scavenging, is not fully understood. Here, we report a newly identified drought-responsive miRNA, miR164g, in the wild apple species Malus sieversii and elucidate its role in apple drought tolerance. Our results showed that expression of miR164g is significantly inhibited under drought stress and it can specifically cleave transcripts of the transcription factor MsNAC022 in M. sieversii. The heterologous accumulation of miR164g in Arabidopsis thaliana results in enhanced sensitivity to drought stress, while overexpression of MsNAC022 in Arabidopsis and the cultivated apple line ‘GL-3’ (Malus domestica Borkh.) lead to enhanced tolerance to drought stress by raising the ROS scavenging enzymes activity and related genes expression levels, particularly PEROXIDASE (MsPOD). Furthermore, we showed that expression of MsPOD is activated by MsNAC022 in transient assays. Interestingly, Part1 (P1) region is the key region for the positive regulation of MsPOD promoter by MsNAC022, and the different POD expression patterns in M. sieversii and M. domestica is attributed to the specific fragments inserted in P1 region of M. sieversii. Our findings reveal the function of the miR164g-MsNAC022 module in mediating the drought response of M. sieversii and lay a foundation for breeding drought-tolerant apple cultivars. | miR164g-MsNAC022 acts as a novel module mediating drought response by transcriptional regulation of reactive oxygen species scavenging systems in apple
Under drought stress, reactive oxygen species (ROS) overaccumulate as a secondary stress that impairs plant performance and thus severely reduces crop yields. The mitigation of ROS levels under drought stress is therefore crucial for drought tolerance. MicroRNAs (miRNAs) are critical regulators of plant development and stress responses. However, the complex molecular regulatory mechanism by which they function during drought stress, especially in drought-triggered ROS scavenging, is not fully understood. Here, we report a newly identified drought-responsive miRNA, miR164g, in the wild apple species Malus sieversii and elucidate its role in apple drought tolerance. Our results showed that expression of miR164g is significantly inhibited under drought stress and it can specifically cleave transcripts of the transcription factor MsNAC022 in M. sieversii. The heterologous accumulation of miR164g in Arabidopsis thaliana results in enhanced sensitivity to drought stress, while overexpression of MsNAC022 in Arabidopsis and the cultivated apple line ‘GL-3’ (Malus domestica Borkh.) lead to enhanced tolerance to drought stress by raising the ROS scavenging enzymes activity and related genes expression levels, particularly PEROXIDASE (MsPOD). Furthermore, we showed that expression of MsPOD is activated by MsNAC022 in transient assays. Interestingly, Part1 (P1) region is the key region for the positive regulation of MsPOD promoter by MsNAC022, and the different POD expression patterns in M. sieversii and M. domestica is attributed to the specific fragments inserted in P1 region of M. sieversii. Our findings reveal the function of the miR164g-MsNAC022 module in mediating the drought response of M. sieversii and lay a foundation for breeding drought-tolerant apple cultivars.
Various abiotic stresses such as drought and high soil salinity inevitably accompany plant growth and development [1–4]. Among them, drought stress severely reduces crop yields by at least 40% worldwide, a proportion that continues to rise due to climate change [5, 6]. Apple (Malus sp.) is one of the most economically important fruit trees and is widely cultivated globally, but apple fruit quality and yield have dramatically declined with the more common occurrence of drought stress [3, 4]. Characterization of the molecular components and signaling pathways related to drought stress represents the first step toward improving drought resistance and developing efficient strategies for breeding drought-tolerant apple cultivars. Malus sieversii Roem., an ancestral species of modern cultivated apples, has become a valuable resource for exploring drought response mechanisms and is the commonly used apple rootstock in some arid and semi-arid regions with exceptional drought stress tolerance [7–9]. However, little is currently known regarding how M. sieversii exhibits such prominent drought tolerance, which severely hampers our ability to breed drought-tolerant apple cultivars. Drought responses in plants are characterized by reduced leaf water potential and lower turgor pressure, which result in stomatal closure and eventually influence cell growth and elongation [2, 10, 11]. When drought extends beyond a given duration, plant cells start to excessively accumulate reactive oxygen species (ROS), which leads to oxidative damage, including membrane peroxidation, protein denaturation, nucleic acid damage, and ultimately cell death [12, 13]. To counteract the oxidative burst caused by ROS accumulation, plants employ tightly controlled ROS scavenging systems composed of enzymatic and non-enzymatic antioxidants. The enzymatic pathways include superoxide dismutase (SOD), peroxidase (POD), catalase (CAT), ascorbate peroxidase (APX) and glutathione peroxidase (GPX), while the non-enzymatic system mainly comprises antioxidants, such as ascorbic acid (AsA), carotenoids, α-tocopherol, and glutathione. Enzyme activity and the contents of these reducing substances are positively correlated with plant resistance to abiotic stress such as drought and salt stress [14, 15]. Therefore, plants with higher drought tolerance are thought to be better equipped at engaging their ROS scavenging systems. For instance, M. sieversii exhibits higher POD and SOD activities under drought stress, suggesting that these two enzymes and the transcript levels of their encoding genes are differentially regulated in M. sieversii compared to other apple cultivars [14]. In model plants, NAM, ATAF1/2, and CUC2 (NAC) transcription factors confer resistance to abiotic stress by regulating ROS scavenging systems [16–18]. For example, the Arabidopsis thaliana (Arabidopsis) NAC transcription factor JUNGBRUNNEN1 (JUB1), the expression of whose encoding gene is induced by hydrogen peroxide (H2O2), dampens intracellular H2O2 levels and enhances tolerance to various abiotic stresses by inducing its direct target DEHYDRATION-RESPONSIVE ELEMENT BINDING PROTEIN 2A (DREB2A) [17]. In rice (Oryza sativa), the stress-responsive NAC transcription factor SNAC3 confers drought tolerance through modulation of ROS [18]. Nevertheless, the role of NAC transcription factors in regulating ROS scavenging systems under drought stress remains to be explored in apple. MicroRNAs (miRNAs) are endogenous small single-stranded non-coding RNAs that play vital regulatory roles in plant drought tolerance. Multiple miRNAs have been reported to cleave transcripts of several drought-responsive genes and hence prevent the accumulation of their encoded proteins [19–23]. For instance, drought stress substantially downregulates the expression of miR169a and miR169c, while the abundance of their target transcript from NUCLEAR FACTOR Y A5 (NF-YA5) accumulates upon drought stress. Transgenic Arabidopsis plants overexpressing NF-YA5 display enhanced drought resistance [24]. The abundance of miR165/166 and their target transcript β-1,3-GLUCANASE 1 (BG1) also respond to drought stress. Lower miR165/166 expression leads to a greater accumulation of BG1 transcripts and thus abscisic acid (ABA) contents, ultimately enhancing drought tolerance in Arabidopsis [25]. The miR164 family was first identified as a class of miRNAs that function in plant growth and development in Arabidopsis, various miR164 loci participate in the development of lateral root and shoot apical meristems, the establishment of the cotyledon boundary and floral organs, fruit ripening, and pathogen-induced and age-dependent cell death [26–34]. Roles for miR164 in abiotic stress, especially drought stress, have emerged in recent years. miRNA transcriptome analysis in maize (Zea mays) identified multiple drought-responsive miR164 isoforms [35]. In other plant species such as wheat (Triticum aestivum), populus (Populus trichocarpa) and Medicago (Medicago truncatula), drought stress downregulates the transcription of miR164 genes [36–39]. Such regulation in different species for miR164 family members suggested that they might play crucial roles in drought responses. However, the functional role of miR164 in drought stress is poorly studied. In the present study, we identified and characterized miR164g and its target transcript MsNAC022 in M. sieversii from our previous high-throughput small-RNA and degradome sequencing [40]. A new member of the apple miR164 family, miR164g plays a crucial role in the drought response of M. sieversii miR164s (msi-miR164s). We show here that overexpression of msi-miR164g in Arabidopsis produces drought-sensitive seedlings, while overexpression of its target MsNAC022 in Arabidopsis and the cultivated apple line ‘GL-3’ enhances drought resistance. Phenotypic analysis revealed that the activity of ROS scavenging enzymes and the expression of their encoding genes are prominently higher in MsNAC022 overexpression plants. Furthermore, two insertions in the MsPOD promoter resulted in its greater transcriptional activation by MsNAC022, suggesting that improved ROS scavenging activity may contribute to the differences of drought tolerance between M. sieversii and Malus domestica.
M. sieversii is an ancestral species of modern apple cultivars that shows exceptional drought tolerance. To determine the candidate genes responsible for the drought resistance, we previously performed high-throughput small RNA sequencing and identified various small non-coding RNAs (snRNAs) and their targets in drought-exposed M. sieversii [40]. Here, we selected the novel miRNA namely miRNc11, which was differentially expressed under short-term drought stress treatments, for investigation of its regulatory role in the drought response. We conducted specific stem–loop reverse transcription PCR to validate the new predicted miRNc11 in M. sieversii and M. domestica. The identified free mature miRNA only exists in M. sieversii and aligned with miRNc11 exactly (Fig. 1A; Fig. S1, see online supplementary material). Sequence analysis also revealed that the miRNc11 mature sequence displays high sequence similarity to the msi-miR164 family, with only one nucleotide difference from msi-miR164a at nucleotide 21 in the 5′ end and two nucleotide differences from msi-miR164b/c/d/e/f at positions 17 and 21 in the 5′ end (Fig. S2, see online supplementary material). The difference between miRNc11 and msi-miR164a at position 21 in the 5′ end resulted in better alignment with the recognition sites in the target gene (Fig. 1B). We thus named this novel miRNc11 msi-miR164g, as the third mature sequence of msi-miR164 family. We also constructed a phylogenetic tree using the sequences of 126 miR164 precursors from 36 plant species (Fig. S3, see online supplementary material). We determined that miR164s are conserved throughout the plant kingdom and that each plant species has 1–11 miR164 family members (Fig. S3B, see online supplementary material). Quantitative real-time PCR of msi-miR164a/b/g revealed their high expression levels in roots relative to leaves under normal growth conditions. In addition, msi-miR164g expression was two to three times higher than that of msi-miR164a and msi-miR164b, indicating that msi-miR164g largely contributes to the function of the miR164 family in M. sieversii (Fig. 1C). Promoter analysis of the msi-miR164g locus identified multiple cis-acting elements related to drought, salinity, ABA, and low temperature signaling, indicating that msi-miR164g may accumulate in response to multiple environmental stimuli (Table S1, see online supplementary material). To test whether msi-miR164 responded to drought stress, we mimicked drought conditions using 20% (w/v) PEG 6000 treatment and examined the expression patterns of msi-miR164a/b/g in M. sieversii. The results showed that msi-miR164g expression is much more induced over the course of the treatment than that of msi-miR164a/b expression. In particular, msi-miR164g expression in roots and leaves were rapidly down-regulated at 2 h of drought treatment, indicating that msi-miR164g respond to drought stress (Fig. 1D, E).
The miR164 targets identified in other plant species belong to the NAC gene family [27, 30, 32, 39, 41]. To determine which candidate NAC gene msi-miR164g might target in apple, we performed an alignment search using the mature msi-miR164g sequence and the apple transcript database. The transcript for gene MD10G1198400 showed the best alignment with msi-miR164g, making it an excellent candidate target for msi-miR164g; we named this gene MsNAC022 based on the amino acid sequence similarity (Fig. S4, see online supplementary material). Using the msi-miR164g sequence as a query against the Arabidopsis transcript database retrieved the same candidate NAC genes as ath-miR164 (Fig. S5, see online supplementary material), indicating that the miR164g-NAC module is conserved among plant species. To ascertain the effect of msi-miR164g on the abundance of MsNAC022 transcripts, we performed a dual luciferase-based miRNA sensor assay to qualitatively and quantitatively evaluate the cleavage of MsNAC022 transcripts by msi-miR164g. The putative cleavage sites of msi-miR164g were located in the open reading frame of MsNAC022; we thus constructed a sensor vector LUC-MsNAC022 that expresses the firefly luciferase (LUC) reporter gene cloned in-frame with the MsNAC022 cleavage sites under the control of the cauliflower mosaic virus 35S promoter. We also generated a second sensor, LUC-mMsNAC022, with synonymous mutations in the cleavage sites. The transient infiltration of either sensor construct alone in Nicotiana benthamiana leaves resulted in strong relative LUC activity; by contrast, co-infiltration of the LUC sensors with a construct overexpressing pre-msi-miR164g dramatically reduced LUC activity from LUC-MsNAC022, but not from LUC-mMsNAC022 harboring a mutation in the presumed msi-miR164g target site (Fig. 2A, B). These results were consistent with the notion that msi-miR164g targets the MsNAC022 transcript. To confirm the cleavage sites, we conducted sequencing of a degradome library with high-throughput 5′ rapid amplification of cDNA ends (RACE). We established that MsNAC022 transcripts are cleaved at the site complementary to msi-miR164g between nucleotides 10 and 11 from the 5′ end of the miRNA (Fig. 2C). Consistently, MsNAC022 transcript levels exhibited a pattern opposite to that of msi-miR164g in response to drought. This feature of MsNAC022 expression was more apparent in leaves, as evidenced by induction after 2 h, followed by repression after 4 h and second rising wave until 24 h (Fig. 2D). Collectively, these findings support the notion that msi-miR164g directly cleaves MsNAC022 transcripts to decrease their abundance in response to drought. Bioinformatics analysis indicated that MsNAC022 carries a NO APICAL MERISTEM (NAM) domain, a canonical domain of the NAC family of transcription factors (Fig. 2E). We determined the transcriptional activity of MsNAC022 using a yeast expression system by separately fusing MsNAC022 and MsDREB6.2 (as positive control) to the DNA binding domain of LexA [42]. Both LexA-MsDREB6.2 and LexA-MsNAC022 activated the transcription of the LacZ reporter gene, indicating that MsNAC022 is a transcriptional activator (Fig 2F). As might be expected for a transcription factor, we colocalized MsNAC022-GFP fusion protein with the nuclear localization transcription factor MsDREB6.2-RFP fusion protein to the nucleus of N. benthamiana leaf epidermal cells (Fig. S6, see online supplementary material).
To investigate the potential function of the msi-miR164g-MsNAC022 module, we individually overexpressed msi-miR164g and MsNAC022 in Arabidopsis. We isolated three independent homozygous T3 transgenic lines for each construct with high expression levels for analysis, which msi-miR164g transcripts were increased by 27–30 and 14–16 times in transgenic lines leaves and roots, respectively. As for MsNAC022 transcripts, the expression levels were increased by 29–32 and 17–19 times in transgenic lines leaves and roots, respectively (Fig. S7A–E, see online supplementary material). Consistent with the published phenotypes associated with overexpression of its Arabidopsis counterparts as positive regulators of root development, MsNAC022 overexpression (OE) resulted in longer and more elaborate root systems (Fig. S8C, D, see online supplementary material). By contrast, msi-miR164g OE plants exhibited shorter primary roots compared to wild type (Fig. S8A, B, see online supplementary material). To further investigate the drought response of msi-miR164g-OE and MsNAC022-OE plants, we conducted drought survival assays. After long-term water deprivation, msi-miR164g-OE plants exhibited significantly lower survival rates than the wild type (P < 0.05; 56% in wild type; 27%,40%, and 33% in the three msi-miR164g-OE lines), while MsNAC022-OE plants fared better under the same conditions (P < 0.05; 45% in wild type; 65%, 64%, and 61% in the three MsNAC022-OE lines) (Fig. S8E–H, see online supplementary material). In addition, transcript abundance for four miR164-targeted AtNACs, especially Arabidopsis NAC022, was much lower in the three msi-miR164g-OE lines compared to the nontransgenic wild-type control, confirming the targeting of NAC022 transcripts by miR164g (Fig. S8I–L, see online supplementary material). Moreover, as drought and salinity both trigger osmotic stress, we examined the phenotypes of msi-miR164g-OE and MsNAC022-OE lines in response to mannitol osmotic and salt treatment. Msi-miR164g-OE lines were more sensitive to osmotic and salt stress, while MsNAC022-OE plants showed greater tolerance against these stresses (Fig. S9A, B, see online supplementary material). Collectively, these results suggest that plant resistance to drought, osmotic and salt stresses are negatively and positively modulated by msi-miR164g and MsNAC022, respectively.
To substantiate the function of the msi-miR164g-MsNAC022 module in M. sieversii, we used Agrobacterium (Agrobacterium tumefaciens)-mediated transformation of the apple cultivar ‘GL-3’ to generate transgenic plants overexpressing miR164g-resistant forms of MsNAC022. We selected three independent transgenic lines (MsNAC022-OE-1, OE-2, OE-4) with high MsNAC022 transcript levels for functional studies (Fig. S7F, G, see online supplementary material). The MsNAC022 transcripts were increased by 5.62–6.35 and 2.36–2.94 times in transgenic lines leaves and roots, respectively (Fig. 3A–C). Phenotypic analysis showed that as in the Arabidopsis assays (Fig. S8C, D, see online supplementary material), overexpression of MsNAC022 also resulted in an enhanced roots system in apple, with both longer and more numerous adventitious roots (AR) compared to the non-transgenic ‘GL-3’ apple plants (Fig. 3D, E). To investigate the role of MsNAC022 on drought stress, we performed a 14-d drought treatment on soil-grown nontransgenic ‘GL-3’ and MsNAC022-OE plants. The decrease in soil moisture caused more leaf curling and wilting in ‘GL-3’ plants than in MsNAC022-OE lines after 7 d and 14 d of drought treatment (Fig. 3F; Fig. S10A, see online supplementary material). In addition, MsNAC022-OE lines recovered from severe drought stress more completely than ‘GL-3’ plants after 7 d rewatering (Fig. 3F). Moreover, MsNAC022-OE lines formed vigorous roots systems and exhibited higher relative water content in their leaves relative to nontransgenic control plants (Fig. 3G–J). MsNAC022-OE plants also showed greater tolerance to salinity and mannitol osmotic treatment (Fig. S11A, see online supplementary material), which was in line with the phenotypes observed upon MsNAC022 overexpression in Arabidopsis. Many studies have reported that drought inhibits photosynthesis [43, 44]. To determine whether MsNAC022 might mitigate such adverse effects, we monitored the photosynthetic capacity of MsNAC022-OE plants during drought stress. Photosynthetic rate (Pn), stomatal conductance (Gs) and transpiration rate (Tr) all decreased as water deprivation was imposed for up to 14 d, but increased upon rewatering. Pn in MsNAC022-OE apple plants was higher than that in ‘GL-3’ across the entire experiment, while Gs and Tr in MsNAC022-OE apple plants were higher than those in ‘GL-3’ at the beginning of the experiment (0 d, before drought), reached the same lower levels as ‘GL-3’ plants after 14 d of drought stress, but then also recovered faster than the wild-type plants upon rewatering (Fig. 4A–C). In addition, instantaneous water-use efficiency (WUEI) which refers to the relationship between plant productivity and water use, was also higher in MsNAC022-OE lines compared to ‘GL-3’ under drought stress (Fig. 4D). These results indicate that MsNAC022 enhances drought tolerance by raising both photosynthesis rate and WUEI. Drought and salinity stress are accompanied by excessive ROS that cause oxidative damage [2, 13, 16, 45]. To address the exceptional performance of MsNAC022-OE plants in the face of drought stress, we examined the accumulation of H2O2 using 3,3′-diaminobenzidine (DAB) staining and that of superoxide (O2•–) with nitro blue tetrazolium (NBT) staining. MsNAC022-OE transgenic plants displayed a lighter staining pattern for both DAB and NBT compared to ‘GL-3’, indicating that the activity of ROS scavenging systems is higher in MsNAC022-OE plants (Fig. 4E, F). We then turned to a quantification of the underlying enzymes required for ROS scavenging. SOD, POD, and CAT activity levels were all elevated in the leaves of MsNAC022 overexpression lines compared to nontransgenic ‘GL-3’ plants (Fig. 4G–I). The leaves of MsNAC022 plants accumulated more proline but exhibited lower levels of malondialdehyde (MDA), a marker of cellular oxidative stress (Fig. S11B, C, see online supplementary material). Together, these results suggest that the increased fitness of MsNAC022-OE plants upon drought stress is linked to the higher activity of their ROS scavenging systems.
We wished to test whether the transcript levels of the drought stress-responsive genes and ROS scavengers also changed in MsNAC022-OE plants. Expression levels of seven typical drought stress-responsive genes and two DREB transcription factor were higher in MsNAC022-OE plants under normal conditions and drought stress. Likewise, the expression levels of six ROS scavenging genes, MdSOD, MdPOD, MdCAT, MdGST (GLUTATHIONE S-TRANSFERASE), MdGPX, and MdAPX, were both significantly increased in MsNAC022-OE apple plants under normal conditions and drought stress (Fig. 5A–L; Fig. S12A-R, see online supplementary material). Of the six ROS scavenger genes tested here, the expression of MdPOD and MdSOD displayed the strongest increase in MsNAC022-OE apple plants, which prompted us to investigate whether MsNAC022 directly activates their transcription (Fig. 5I–L). We performed a transient expression assay using a dual-luciferase system in N. benthamiana leaf cells, in which we placed the LUC reporter gene under the control of the MsPOD or MsSOD promoters, yielding the reporter constructs MsPODpro:LUC and MsSODpro:LUC. LUC activity derived from the MsPODpro:LUC and MsSODpro:LUC reporters increased substantially when co-infiltrated in N. benthamiana leaves with the effector construct 35S:MsNAC022, compared to the control vector (62-SK) (Fig. 5M–O). In addition, we identified several NAC binding sites among the cis-elements in the MsPOD and MsSOD promoters. However, we failed to detect direct binding between MsNAC022 and the MsPOD or MsSOD promoters in a yeast one-hybrid assay (Fig. S13, see online supplementary material), indicating that other transcription factors might be recruited to connect MsNAC022 to the MsPOD and MsSOD promoters and activate their transcription.
To further dissect the function of MsNAC022, we selected the MsPOD promoter for detailed analysis. We divided the 2.7-kb MsPOD promoter into three fragments based on the distribution of predicted NAC binding motifs to drive the transcription of the β-GLUCURONIDASE (GUS) reporter gene. We then co-infiltrated N. benthamiana leaves with the 35S:MsNAC022 effector and each MsPOD:GUS construct (Fig. 6A). Staining and relative GUS activity analysis showed that MsNAC022 activates transcription from the P1 promoter fragment, but not from P2 or P3 (Fig. 6B–D). We confirmed these results in a dual-luciferase assay with each MsPOD promoter fragment driving the transcription of LUC (Fig. 6E–H). The P1 region of the MsPOD promoter was 910 bp in length and spanned the region from −1819 bp to −2729 bp relative to the ATG start codon. A comparison of the P1 regions from M. sieversii and M. domestica identified insertion polymorphisms within this region. The MsPOD promoter harbored two fragments (−2143 bp to −2178 bp and − 2213 bp to −2592 bp) of 35 bp and 379 bp, respectively, that are absent from the MdPOD promoter (Fig. 7A; Table S2, see online supplementary material). Both insertions were located within the P1 fragment. We thus characterized the MdPOD promoter by dividing the 2.3-kb promoter into three fragments to drive LUC or GUS transcription. MsNAC022 activated the transcription of both reporter genes from the P1 region only, as with the MsPOD promoter (Fig. 7B–D). We also compared the transcriptional output of the MsPOD and MdPOD promoters when driving GUS or LUC. The MsPOD:GUS construct resulted in a higher GUS activity than did MdPOD:GUS when co-infiltrated with the 35S:MsNAC022 effector, suggesting that the transcriptional activation of MsPOD by MsNAC022 is greater than that of MdPOD. We noticed that the P1 region from M. sieversii (Ms-P1:GUS) resulted in transcription levels about two-fold higher than the P1 region from M. domestica (Md-P1:GUS), which explaining the difference in promoter strength between the two apple varieties (Fig. 7E–G). We obtained similar results in a dual-luciferase assay (Fig. 7H–J). Based on these results, the specific P1 region within the MsPOD promoter was attributed to enhanced transcriptional activation of MsPOD by MsNAC022, leading to higher accumulation of ROS scavenging systems that partially contribute to the higher drought tolerance observed in M. sieversii. In light of the different promoter strengths seen for the M. sieversii and M. domestica POD promoters in transient assays, we measured the transcript levels of NAC022 and POD in the two varieties, under drought stress and recovery conditions. When grown under normal conditions, NAC022 and POD expression was higher in roots relative to leaves, similar to miR164g (Fig. 8A, B; Fig. 1C). NAC022 and POD expression levels were higher in M. sieversii compared to M. domestica. Upon drought stress, NAC022 and POD transcript levels increased rapidly in M. sieversii and M. domestica, with M. sieversii displaying a more pronounced rise. Moreover, NAC022 and POD expression declined to a greater extent in M. sieversii after 6 h recovery than in M. domestica, indicating a more flexible and plastic response to changes in moisture conditions (Fig. 8C–F). Collectively, these results indicate that the NAC022-POD module may be partiallly responsible for the different drought response of M. sieversii and M. domestica.
Apples are one of the most valuable fruit crops whose productivity and growth is severely restricted by adverse environmental conditions, of which drought is one of the most severe encountered by apples in arid and semi-arid growing areas [3, 4, 46]. Therefore, it is of great scientific and breeding value to identify drought-related components and their underlying molecular mechanisms in apples. miRNAs and their respective targets have roles in responding to various stresses [24, 25, 47–50]. Among these, miR164 is a versatile miRNA with a vital role in plant growth and development. miR164 mediates responses to various abiotic stresses such as salinity, osmosis and drought [26–31, 36, 37, 51–53]. However, the molecular mechanisms by which miR164 and its targets regulate abiotic stress, particularly drought response, are still poorly understood in apples. Our study characterized the novel miRNA locus msi-miR164g, which was differentially expressed under short-term drought stress treatments in M. sieversii (Fig. 1D and E). Complementary sequence analysis and cleavage assays determined that msi-miR164g directly regulates MsNAC022 transcript levels in M. sieversii (Fig. 2A–C). Overexpression of msi-miR164g or MsNAC022 in Arabidopsis altered tolerance to drought stress. In contrast to the negative regulation of drought stress by msi-miR164g, overexpression of MsNAC022 conferred enhanced drought tolerance in transgenic Arabidopsis and apple (Fig. S8E and F, see online supplementary material; Fig. 3F). Several antioxidant enzymes required for ROS scavenging exhibited high activity levels in MsNAC022-OE apple lines, and their encoding genes were highly expressed (Fig. 4G–I; Fig. 5I–L; Fig. S12K–L, see online supplementary material). In particular, we showed here that MsNAC022 activates MsPOD transcription (Fig. 5N and O). POD promoter analysis in M. sieversii and M. domestica revealed the presence of specific insertions in M. sieversii that contribute to the greater activation of POD transcription by MsNAC022 in this variety (Fig. 7). Together, these results suggest that, in response to drought stress, elevated MsNAC022 transcript levels lead to an activation of MsPOD transcription to detoxify the accumulated ROS and thus enhance drought tolerance. We observed that msi-miR164g among all msi-miR164s most prominently responds to osmotic treatment. Furthermore, expression analysis of all msi-miR164s revealed that msi-miR164g has a higher basal expression level than other msi-miR164 family members under either normal conditions or stress treatment (Fig. 1C–E). We also noted that mature msi-miR164g is highly similar to the recognition sites of its target gene (Fig. 1B). Based on these observations, we speculate that miR164g may be the main contributing member of the M. sieversii miR164 family. In our drought treatment assay, msi-miR164g in root and leaf exhibited the declined expression trend at 2 h, indicating that msi-miR164g respond to drought rapidly (Fig. 1D and E). In contrast to its low level in leaf, msi-miR164g mainly expressed in roots (Fig. 1C). Considering that root is the first place to perceive drought stress, thus the effect of drought treatment on msi-miR164g expression is more pronounced in root at 2 h (Fig. 1D). miRNAs cleave the transcripts of their target gene. Previous studies have proved that miR164 directly target the type of NAC transcription factors [18, 27, 30, 32, 34, 39]. The NAC transcription factors directly regulate an array of stress-related genes expression to control drought response [16, 18, 52]. In this study, we established that MsNAC022 transcripts are targeted by msi-miR164g for cleavage, through dual-luciferase-based miRNA sensor assays and degradome sequencing (Fig. 2A–C). In support of this notion, the transcript levels of MsNAC022 and msi-miR164g showed an opposite relationship under drought stress conditions (Fig. 2D). Furthermore, overexpression of msi-miR164g and MsNAC022 resulted in drought-sensitive and drought-tolerant transgenic plants, respectively (Fig. S8E and F, see online supplementary material; Fig. 4F). Thus, we conclude that msi-miR164g negatively mediates drought tolerance by downregulating MsNAC022 transcript levels. Similarly, NAC transcription factors JUB1 and SNAC3 were also reported to improve the drought resistance by enhancing the ROS scavenging systems in tomato and rice, respectively [17, 18]. In addition, the positive regulation on drought tolerance was also identified in maize NAC transcription factors ZmNAC11, which improves water-use efficiency and upregulates the expression of drought-responsive genes [41]. However, the miR164-targeted NAC genes in rice act negatively on drought tolerance, implying that the function of this pathway in abiotic stress is divergent in different plant species [52]. Plant water uptake is ultimately determined by the size, properties, and distribution of the root system [54, 55]. Thus, water deficit induces changes in root architecture to adapt to the adverse environment. Previous studies have shown that overexpression of miR164-targeted NAC transcription factors induces lateral root development in transgenic Arabidopsis [30]. Similar results were reported in maize and soybean (Glycine max), as overexpression of ZmNAC1 or GmNAC020 resulted in more lateral roots and greater root density [32, 56]. In our study, transgenic Arabidopsis and apple plants overexpressing MsNAC022 also showed enhanced root system, especially in transgenic apple plants, the number and length of adventitious roots were significantly increased (Fig. S8C and D, see online supplementary material; Fig. 3A, D, and E). After 14 days of drought stress, the root difference of transgenic apple was more significant than that of the control plant, indicating that MsNAC022 significantly affected the root development of the transgenic plant during drought stress, thus conferring the transgenic apple plant excellent adaptability to drought stress (Fig. 3F–J). Similar to our study, miR167a expression was significantly reduced in Arabidopsis under high osmotic stress, which subsequently increased the expression level of target genes and promoted lateral root development, while root structure optimization enhanced the tolerance of transgenic plants under osmotic stress [50]. Our results suggest that the msi-miR164g-MsNAC022 module also affects apple root architecture, thereby conferring apple plants’ response to drought, salt, and osmotic stresses. However, the regulatory mechanisms by which msi-miR164g-NAC022-mediated root traits enhance stress resistance is unknown. Thus, more detailed molecular and biochemical studies are required to substantiate the regulation of root development by MsNAC022 in future studies.
Although ROS production is critical for growth, signaling, and development, their reactivity is a double-edged sword. Low ROS levels constitute a stress-signaling component that is beneficial for acclimation in response to stress [12, 57]. However, an overaccumulation of ROS becomes extremely deleterious, initiating oxidative stress that results in cellular damage and ultimately cell death. Under drought stress, photosynthetic rates drop, leading to enhanced electron leakage and photorespiration, both producing excess ROS [13]. Thus, steady-state ROS levels must be tightly regulated. To mitigate the damage caused by ROS overproduction, drought stress upregulates ROS scavenging systems, in the form of both enzymatic and non-enzymatic antioxidants [15]. The level of induction of the antioxidant systems is tightly correlated with the degree of drought tolerance exhibited by plants. Modulation of ROS scavenging systems affects plant survival rates under drought stress. For instance, overexpression of AUTOPHAGY-RELATED 18a (MdATG18a) increases CAT and POD activities, thus enhancing the drought tolerance of transgenic apples [58]. Likewise, in our previous work, overexpression of the miR171i target gene SCARECROW-LIKE PROTEINS 26.1 (MsSCL26.1) in apples markedly induced MdAPX and MONODEHYDROASCORBATE REDUCTASE (MsMDHAR) expression, thereby increasing AsA levels and improving drought tolerance in apples [40]. In the current study, we showed that overexpression of MsNAC022 promotes SOD, POD and CAT activities in transgenic apple lines compared to nontransgenic ‘GL-3’ plants under drought stress (Fig. 4G–I). In agreement with this observation, H2O2 accumulation was much lower in MsNAC022-OE apple leaves compared to the ‘GL-3’ control under abiotic stress, indicating that greater ROS mitigation may contribute to higher drought tolerance (Fig. 4E and F). Furthermore, the genes required for ROS scavenging systems were induced in MsNAC022-OE apple plants (Fig. 5I–L; Fig. S12K–R, see online supplementary material). MsNAC022 activated transcription from the MsSOD and MsPOD promoters (Fig. 5M–O). These results collectively demonstrate that the mi164g-MsNAC022 module may mediate the drought response of apples by regulating the expression of genes encoding ROS scavenging enzymes. In addition, our failure to observe direct binding of MsNAC022 on the MsSOD and MsPOD promoters in a yeast one-hybrid assay suggests that MsNAC022 might not act alone and may recruit other transcription factors or proteins to activate transcription. In our research, seven drought stress-related response and two DREBs gene were significantly increased in MsNAC022-OE plants relative to nontransgenic apple plants. Upon drought stress, the response levels of seven drought stress-related genes and two DREB genes were more significant imposed compared to normal conditions (Fig. 5E–H; Fig. S12A–J, see online supplementary material). Furthermore, previous studies have reported that NAC transcription factors can form homodimers or heterodimers or interact with DREBs or C-REPEAT/DRE BINDING FACTORs (CBFs) to enhance plant tolerance to drought and cold, respectively [59–63]. Based on the important regulatory function of DREB transcription factors in plant drought stress, we speculate that the DREB transcription factors may be a potential co-factor of MsNAC022 [42]. Hence, elucidating the concrete action of MsNAC022 in regulating the downstream ROS pathway is our next research interest and will be focused on in the near future.
M. sieversii is the likely progenitor of modern cultivated apple(M. domestica Borkh.) [64]. Among all cultivated rootstocks, M. sieversii is one of the most drought-resistant species in China [7–9]. However, some excellent drought response genes and regulatory mechanisms of M. sieversii may have become weaker or even eliminated in long-term domestication, resulting in the lowerobserved drought tolerance of M. domestica. Understanding the molecular genetic mechanisms governing drought responses in M. sieversii will help breeding programs enhance the survival of important commercial apple cultivars during periods of drought. Upon osmotic treatment, MITOGEN-ACTIVATED PROTEIN KINASE (MAPK) expression is rapidly upregulated, and M. sieversii displays the highest MAPK expression levels compared to other apple species [65]. Moreover, our previous study reported that MsDREB6.2 regulates cytokinin metabolism and participates in stomatal regulation, root development and aquaporin gene expression, thereby enhancing drought tolerance [42]. Based onthese results, the drought response machinery in M. sieversii is thought to be more flexible and plastic than that in M. domestica. Despite much progress, the molecular mechanisms behind thediscrepancy of drought tolerance between M. sieversii and M. domestica are not well understood. In our study, we showed that upon drought stress, ROS overaccumulation slows down in MsNAC022-OE plants overexpressing a miR164g-resistant form of the gene (Fig. 4E and F). As one of the main ROS scavengers, POD transcription was activated by MsNAC022 specifically through the P1 region (Figs 5N and O, and 6). Furthermore, sequence comparison of the P1 region highlighted two insertions present only in M. sieversii (Fig. 7A; Table S2, see online supplementary material). We further established that MsNAC022 activates transcription from the Ms-P1 promoter fragment to greater levels than with Md-P1, which lacks these twounique insertions (Fig. 7E–J). In addition, NAC022 and POD expression was more strongly induced in response to changes in moisture conditions in M. sieversii than in M. domestica (Fig. 8C–F). Based on these findings, we propose a putative model for the transcriptional regulation of ROS scavenger systems that is mediated by the miR164g-MsNAC022 module, which may contribute to the enhanced drought tolerance of M. sieversii (Fig. 8G). Under drought stress, rapidly declining miR164g levels alleviate the cleavage of MsNAC022 transcripts, whose encoding transcription factor in turn activates the transcription of its downstream gene MsPOD and other ROS scavenger genes. As MsNAC022 differentially activates MsPOD and MdPOD, the enhanced ROS scavenging systems might partially contribute to the higher drought resistance of M. sieversii. Drought stress is becoming one of the most critical determinants that limit apple production. It is highly desirable to breed new apple varieties with high water uptake efficiency or drought tolerance through biotechnology or molecular marker-assisted breeding, but such efforts have met only limited success thus far. M. sieversii was widely used because of its excellent drought resistance [7–9]. Dissecting the unique molecularmechanism of drought responses in M. sieversii will provide valuable genetic resources for breeding new drought-resistant rootstocks. Our work identified a novel regulatory module acting in M. sieversii drought response, which may have applications in apple breeding to increase plant fitness during drought stress through modulating the miR164g-MsNAC022 circuitry, ultimately mitigating ROS damage.
Tissue-cultured plants of M. sieversii and M. domestica were grown at 23°C and 40% relative humidity under a 16 h/8 h (day/night) photoperiod as described [40]. The apple plant ‘GL-3’ (M. domestica Borkh.) from the laboratory of Dr Zhihong Zhang (Liaoning, China) was also cultured under the conditions described above, which will be used for genetic transformation and abiotic stress treatments as previously described [66]. Rooted M. sieversii and M. domestica plants that were hydroponically precultured in half-strength Hoagland nutrient were subjected to drought stress. The plants were treated as described in previous studies with a solution of 20% (w/v) PEG 6000 or 30% PEG 6000 (Xilong Scientific, Shantou, China) for various periods of time [40, 42]. Samples were then separately harvested at 0, 2, 4, 12, and 24 h after drought stress and 6 and 24 h after rewatering, and were quickly frozen in liquid nitrogen for RNA isolation and expression analysis. The wild-type and transgenic Arabidopsis used in this study were in the Columbia-0 ecotype background. After sterilizing, seeds were planted on half-strength MS medium and then stratification at 4°C for 3 d. At 14 d later, seeding was transplanted to soil and grown in the greenhouse at 22°C with 16 h light/8 h dark cycle.
The msi-miR164g and the cleaving to miR164g-targeted gene MsNAC022 was screened via analyses of miRNAs databases of high-throughput small-RNA sequencing and degradome sequencing of M. sieversii which were obtained in our previous study [40]. To validate the new predicted miRNA, stem-loop reverse transcription PCR were used to obtain the mature of miR164g from M. sieversii, then were cloned into the pTOPO-blunt sample vector (Aidlab, Beijing, China) and clones were confirmed through sequencing. The coding sequence of MsNAC022 was amplified by reverse transcription PCR (RT-PCR) of M. sieversii RNA, and confirmed through sequencing. Primer sequences were listed in Table S3, see online supplementary material.
The M. sieversii genomic DNA was extracted using a Plant Genomic DNA Kit (Tiangen, Beijing, China). We checked the msi-miR164g promoter reference sequences by blast the GDR apple genomic database (https://www.rosaceae.org/) with the msi-miR164g precursor sequences obtained from our miRNAs databases, then we designed a specific primer and cloned the miR164g promoter sequences from M. sieversii. The msi-miR164g promoter sequences were submitted to PLANTCARE (http://bioinformatics.psb.ugent.be/webtools/plantcare/html/) to perform cis-acting elements analysis.
The Arabidopsis total RNA was extracted with TRIzol Reagent (CWBIO, Beijing, China). Small RNA was extracted using an EASYspin Plant microRNA Extract kit (RN40, Aidlab, Beijing, China). The DNA was removed by treating with RNase-free DNase I (RN34, Aidlab, China). After the detection of RNA samples quality by agarose electrophoresis, the same amount of RNA samples (1 μg) was used to generate cDNA with oligo dT primers and specific stem-loop reverse transcription primers of miRNA164 family members, respectively. The qRT-PCR was performed with SYBR Green Mix (CW0659, CWBIO, China). NCBI primer-blast online tools (https://www.ncbi.nlm.nih.gov/tools/primer-blast/index.cgi?LINK_LOC=BlastHome) and qPrimerDB (https://biodb.swu.edu.cn/qprimerdb/) online database tools were used to aid in the design of specific primers. The reactions were incubated in a Rotor-Gene Q Machine (Qiagen, Hilden, Germany), AtActin2 and Histone H3 were used as internal controls for Arabidopsis and apple, respectively. Melting curve assay were performed to detection the specificity of qRT-PCR reactions. Gene amplification efficiencies and relative expression levels were analysed using the previous reported method [67]. All primers were listed in Table S3, see online supplementary material.
A total of 126 miR164 precursors from 36 plant species and the NAC transcription factors amino acid sequences from Arabidopsis and apple were used as queries in BLAST searches of the miRBase 22.1, TAIR, and GDR databases, respectively. The phylogenetic analysis was conducted using MEGA X and the neighbor-joining method with 1000 bootstrap replicates [68]. The phylogenetic trees were modified using the Evolview v3 tool [69].
The msi-miR164g potential cleavage sites of MsNAC022 were introduced into the AvrII/AgeI sites of the pGreen-dual-luc-ORF-sensor vector as previously described, also its synonymous mutation sequences which were used for a negative control [70]. The primary of msi-miR164g with 482 bp sequence from M. sieversii was driven by the cauliflower mosaic virus 35S (CaMV35S) promoter in the pGreenII 0029 62-SK vector. The pGreen-dual-luc-ORF-sensor vector also carries the REN gene and was used as a positive control. Transformation and infiltration were performed as described [71].
To investigate the transcriptional activation activity of MsNAC022, the full-length MsNAC022 sequence lacking the termination codon was introduced into pEG202 vector. Sequencing-confirmed plasmid was transformed into yeast strain EGY48, and MsDREB6.2 was used for positive control [42]. Transcriptional activity assay was performed as described [72]. For the colocalization of MsNAC022, the full-length MsNAC022 sequence without the termination codon was fusion expressed with GFP by inserting into the pCAMBIA1302 vector (https://cambia.org/welcome-to-cambialabs/cambialabs-projects/cambialabs-materials-and-methods-developed-in-cambialabs/). The nuclear localization transcription factor MsDREB6.2[ was fusion expression with RFP by inserted into the pCAMBIA1300 vector modified by us. Vectors were transformed into A. tumefaciens strain GV3101. After Agrobacterium microbial concentration reached OD600 = 1.0, they were equally mixed and then were transiently transformed in N. benthamiana leaves and confocal fluorescence observation was performed as described [73].
To generate the msi-miR164g overexpression vector, the 482 bp sequence from M. sieversii containing the miR164g stem-loop structure was cloned and inserted into the downstream of the CaMV35S promoter in plant transformation vector pCAMBIA1301. Similarly, the coding sequence of MsNAC022 with synonymous mutations in the miR164g cleavage sites was amplified using overlapping PCR and fused into pCAMBIA1301 vector to construct the MsNAC022 overexpression vector. A. tumefaciens strain EHA105 transformed with these vectors was used for genetic transformation in Arabidopsis and apple ‘GL-3’ plants as previously described [74, 75]. After Hygromycin B selection, we performed PCR analysis to identify the presence of the transgenes and the Hygromycin coding sequences in putative transgenic lines; total RNA was also isolated to check the overexpression of msi-miR164g and MsNAC022 by qRT-PCR. In addition, apple leaves GUS staining assay was performed to identification the reporter gene of MsNAC022-OE transgenic ‘GL-3’ apple lines by a modified method [40, 76].
For drought stress treatment, the wild type plants and the overexpression T3 Arabidopsis lines were transplanted to the same pot (48 cm × 20 cm × 13 cm) and grew for 2.5 weeks, then were grown under drought stress for 2 weeks or 3 weeks (water was withheld); 5-month-old nontransgenic ‘GL-3’ apple plants and transgenic plants were also transplanted to the same pot and performed 2 weeks drought stress treatment (water was withheld). For salinity and osmotic treatment, 150 mM NaCl and 250–300 mM mannitol were added into the half-strength MS medium to treat the 14-d wild-type plants and the overexpression T3 Arabidopsis lines as described [40, 77]; 200 mM NaCl and 300 mM mannitol were also added into the MS subculture medium to treat nontransgenic ‘GL-3’ apple plants and transgenic plants as described [40, 66].
During drought stress, the photosynthetic capacity of MsNAC022-OE plant was monitored on sunny days between 9 and 11 a.m. with a photosynthetic apparatus (Li-6400; LICOR, Huntington Beach, CA, USA). Five measurements were performed for each group as previously described [58]. The relative water content of leaves was determined as described [3].
The measurement of ROS physiological indicators, SOD, POD, and CAT activity, Proline and MDA levels in leaves was performed as previously described [3, 78]. DAB and NBT staining using a modified method as described [79].
Expression levels of typical stress-responsive genes RD22, RD29A, RD29B, RD26, ERD1, ERD10, and LEA7 were examined in transgenic apple plants by qRT-PCR, as well as the various ROS scavenging systems related genes which were identified in the previous research, such as MdPOD, MdSOD, MdCAT, MdGST, MdGPX, and MdAPX [40, 80–83]. We also selected two DREB transcription factors MdDERB2A and MdDERB6.2 based on their essential roles in drought stress response [42, 84]. The genomic DNA was extracted from M. sieversii and M. domestica using a Plant Genomic DNA Kit (TIANGEN, China). Then promoters of MsPOD, MdPOD, and MsSOD were cloned and fused to pGreenII 0800-LUC vector, respectively. The full-length CDS of MsNAC022 was driven by the CaMV35S promoter in the pGreenII 0029 62-SK vector, then corresponding vectors were transformed into A. tumefaciens strain GV3101 harboring the pSoup plasmids. LUC promoter activity analysis was examined as described [85], as well as the LUC activities of the full-length and different fragments of MsPOD and MdPOD promoters.
The promoter of MsPOD and MdPOD was divided into three fragments according to the distribution of NAC binding element, the full-length promoters and different fragments were inserted into the pCAMBIA1301 vector, which contains the GUS reporter gene vector of 35S:LUC that was used for internal control. Vectors were transformed into A. tumefaciens strain GV3101. After Agrobacterium concentration was shaken and adjusted to OD600 = 1.0, various Agrobacterium that harbour effector, reporter, and internal controls were equally mixed and left to stand in the dark for about 1 h before infiltration. Transient transformation in N. benthamiana leaves was performed as described [70]. The histochemical staining of GUS and GUS activity analysis were performed as described [76].
All statistical analyses were performed via the one-way ANOVA followed by Duncan’s multiple range test, using the SPSS22.0 for Windows (SPSS Inc., Chicago, IL, USA). Three independent biological replicates were performed for each determination. Data are shown as mean ± standard deviation (SD), differences asterisks between these results were considered as statistically significant (*P < 0.05, **P < 0.01).
Sequence data from this article can be found in the TAIR databse (https://www.arabidopsis.org) and the Genome Database for Rosaceae website (https://www.rosaceae.org/): MsNAC022 (MD10G1198400); MsDREB6.2 (MD15G1365500); MdRD22 (MD15G1098800); MdRD29A (MD01G1201000); MdRD29B (MD07G1268800); MdRD26 (MD03G1222700); MdERD1 (MD06G1128400); MdERD10 (MD15G1003900); MdLEA7 (MD03G101800); MdDREB2A (MD01G1158600); MdDREB6.2 (MD15G1365500); MdPOD (MD00G1112500); MdSOD (MD00G1051500); MdCAT (MD06G1008600); MdGPX (MD06G1081300); MdGST (MD04G1111600); MdAPX (MD08G1150400); AtNAC022 (AT1G56010); AtNAC1L (AT3G12977); AtNAC080 (AT5G07680); AtNAC100 (AT5G61430).
We thank Prof. Zhihong Zhang from Shenyang Agricultural University for providing the ‘GL-3’ apple plants. This work was supported by the National Key R&D Program of China (Grant No. 2019YFD1000102–02), the National Natural Science Foundation of China (Grant No. 31972390), the Construction of Beijing Science and Technology Innovation and Service Capacity in Top Subjects (Grant No.CEFF–PXM2019_014207_000032), and the 2115 Talent Development Program of China Agricultural University.
X.P., Y.L., and T.-H.L. conceptualized this project and designed all experiments. Y.-T.W., X.P., and C.F. treated plant materials and performed the RNA extraction and gene expression analysis. X.P., C.F., and X.Zha performed the dual luciferase and GUS reporter assay. Y.-Y.W. and X.Zha contributed to the phylogenetic analysis of miR164 and NAC family members. Y.-Y.W. and Y.-T.S. contributed to the transcriptional activity analysis and subcellular localization of MsNAC022. Y.-Q.X., Z.-F.Z., and X.Zho. measured the physiological indicators. C.F. monitored the photosynthetic characteristics of MsNAC022-OE plants during drought stress. B.-Y.D. and C.W. contributed to the plasmid construction and Arabidopsis and apple genetic transformation. X.P., Y.L. and T.-H.L. wrote and revised the manuscript. All authors have read and agreed to the published version of the manuscript.
The data that support the findings of this study are available from the corresponding author upon reasonable request.
The authors declare no competing interests.
Supplementary data is available at Horticulture Research online.
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PMC9631435 | Siqi Cheng,Weihong Chen,Mingmin Zhao,Xing Xing,Lei Zhao,Bowen Ren,Na Li | Case report: A late-onset cobalamin C defect first presenting as a depression in a teenager 10.3389/fgene.2022.1012558 | 20-10-2022 | CblC defect,MMACHC,depression,genotype–phenotype correlation,case report | Background: The cobalamin C (cblC) defect, a common inborn disorder of cobalamin metabolism due to a genetic mutation in MMACHC, can cause combined methylmalonic acid and homocysteine accumulation in blood, urine, or both. In this article, a late-onset case was reported, and the patient first presented with depression identified with the MMACHC gene. We summarized the clinical features of the cblC defect, the relationship between genotype and phenotype, and the clinical experience concerning the diagnosis and treatment of the cblC defect. Case presentation: Initially presented with depression, the 16-year-old female patient showed progressive abnormal gait and bilateral lower limb weakness after 3 months. Blood routine examination suggested severe hyperhomocysteinemia, and screening for urine organic acids found elevated methylmalonic acid. Family gene sequencing showed mutations detected in MMACHC. She had a compound heterozygous mutation, while the c.271dupA (p.R91Kfs∗14) was only detected in her father and the c.482 G>A (p.R161Q) was only detected in her mother. Hence, she was diagnosed with a cblC defect and treated with B vitamin supplements. The muscle strength of both lower limbs improved notably. Conclusion: This case indicated that depression could be a presenting sign of cblC-type methylmalonic aciduria and homocysteinemia, and enhanced the genotype–phenotype relationship of the cblC defect, which will contribute to further understanding of this emerging disease. | Case report: A late-onset cobalamin C defect first presenting as a depression in a teenager 10.3389/fgene.2022.1012558
Background: The cobalamin C (cblC) defect, a common inborn disorder of cobalamin metabolism due to a genetic mutation in MMACHC, can cause combined methylmalonic acid and homocysteine accumulation in blood, urine, or both. In this article, a late-onset case was reported, and the patient first presented with depression identified with the MMACHC gene. We summarized the clinical features of the cblC defect, the relationship between genotype and phenotype, and the clinical experience concerning the diagnosis and treatment of the cblC defect. Case presentation: Initially presented with depression, the 16-year-old female patient showed progressive abnormal gait and bilateral lower limb weakness after 3 months. Blood routine examination suggested severe hyperhomocysteinemia, and screening for urine organic acids found elevated methylmalonic acid. Family gene sequencing showed mutations detected in MMACHC. She had a compound heterozygous mutation, while the c.271dupA (p.R91Kfs∗14) was only detected in her father and the c.482 G>A (p.R161Q) was only detected in her mother. Hence, she was diagnosed with a cblC defect and treated with B vitamin supplements. The muscle strength of both lower limbs improved notably. Conclusion: This case indicated that depression could be a presenting sign of cblC-type methylmalonic aciduria and homocysteinemia, and enhanced the genotype–phenotype relationship of the cblC defect, which will contribute to further understanding of this emerging disease.
Cobalamin C defect (cblC, OMIM 277400), a kind of complementation group disease of defective cblC metabolism described by Rosenberg in 1975 (Gravel et al., 1975), can result in both methylmalonic acid (MMA) and homocysteine (Hcy) accumulation in vivo, which was first reported by professor Mudd in 1969 (Mudd et al., 1969). It is an inherited metabolic disease caused by a mutation in MMACHC (OMIM 609831) (Lerner-Ellis et al., 2006). A CblC defect is the most common genetically inherited reason for combined methylmalonic aciduria and homocysteinemia, constituting the main biochemical phenotype of China and accounting for 60%∼80% of the domestic methylmalonic aciduria (Liu et al., 2012; Zhang et al., 2007). The clinical features of the cblC defect vary by age of disease onset. Differing from the early-onset type (<1 year old), the late-onset cases (>4 years old) usually have milder clinical manifestations and a better prognosis after early intervention (Martinelli et al., 2011). Sometimes there is a failure to detect cblC defects early due to their heterogeneous phenotypic spectrum of presentations. Normally, the cblC defect produces a series of multisystem neurological symptoms, such as psychological and behavioral abnormalities, leukoencephalopathy, encephalopathy, subacute combined degeneration of the spinal cord, lower extremity weakness, gait disorder, and seizures (Martinelli et al., 2011; Carrillo-Carrasco et al., 2012). However, there are few case reports focusing on cblC patients initially manifesting as depression. Here, we reported one novel late-onset cblC case, where the patient first presented with depression, carrying heterozygous variants in MMACHC, and also explored the pathophysiological mechanism. To date, only a small number of studies have found a potential association between the cblC genotype and its phenotype, which can help predict the age of disease onset and severity (Matos et al., 2013; Yu et al., 2015). Therefore, more comprehensive studies on the clinical and genetic features of cblC patients are warranted.
The 16-year-old girl was born at full term with a spontaneous delivery from a non-consanguineous Chinese Han family. Her parents were healthy and denied a family history of neurologic illness. The girl first suffered from low mood, low self-esteem, self-blame, and visual hallucinations. Additionally, the neuropsychological scale at admission, particularly self-reporting inventory-90 and patient health questionnaire-9, pointed toward the occurrence of depression. A routine blood examination suggested moderate iron-deficiency anemia (hemoglobin of 86 g/L) and moderate homocysteinemia (Hcy of 62 μmol/L, reference range: 5–15 μmol/L). Brain MRI showed patchy and symmetric white matter hyperintensities in the posterior horn of the bilateral lateral ventricles. She was diagnosed with depression by her psychiatrist and was orally administered antidepressants, iron, and methylcobalamin (Mecbl). The depression and hallucination symptoms were alleviated after pharmacological therapy for only 2 weeks. After 3 months, she showed gradually progressive abnormal gait and bilateral lower limb weakness without depression relapse, and she could hardly even stand without assistance. Physical examination also revealed hyperactive reflexes, bilateral ankle clonus, a positive Babinski sign, and suspicious deep sensory impairment. Iron-deficiency anemia and homocysteinemia were still detected with hyperuricemia. A review of the cerebral MRI found that the previous lesions displayed no change and were not enhanced on the spoiled gradient recalled (SPGR) sequence. The results of the special blood test were negative for pepsinogen I and II, pro-gastrin-releasing peptide, immune, and rheumatic factors. Moreover, neither serum nor cerebrospinal fluid discovered positive results of myelin oligodendrocyte glycoprotein antibody, aquaporin 4 antibody, glial fibrillary acidic protein antibody, and autoimmune encephalitis-related auto-antibodies. After excluding other diseases leading to multisystem involvement, inborn metabolic diseases were considered. The detailed information of this patient is shown in Table 1. Screening for urine organic acids was performed after 3 months of cobalamin supplement and still, elevated methylmalonic acid of 21.2 mmol/mol creatinine (reference range: 0.0–4.0 mmol/mol creatinine) was found. Amino acids and acylcarnitines analysis in blood were normal. Notably, with family whole exome sequencing and Sanger sequencing performed, it was detected that a compound heterozygous mutation existed in the MMACHC gene. One frameshift variant was c.271dupA (p.R91Kfs∗14), inherited from her mother, and the other variant, belonging to the missense type, was c.482 G>A (p.R161Q) from her father. According to the American College of Medical Genetics and Genomics (ACMG), both variants are pathogenic (Lerner-Ellis et al., 2006; Lerner-Ellis et al., 2009; Richards et al., 2015) (Figure 1). From the second onset, the patient got an intramuscular injection of vitamin B1 (50 mg/d) and cyanocobalamin (0.5 mg/d), intravenous injection of Mecbl (1 mg/d), and oral medication of vitamin B6 and folic acid tablets. The patient was not treated with betaine, for betaine is not available in our hospital. After 1 month of treatment, she could stand and walk for a short distance by herself; thus, she was allowed to be discharged from the hospital. The long-term oral administration of vitamin B and intermittent injections were executed after discharge. In the following 2 months, the muscles of both lower limbs gradually strengthened, so that she could walk indoors. The concentrations of MMA and Hcy were lowered.
As expected, the cblC defect is characterized by a heterogeneous clinical picture involving multipart nervous system symptoms, such as psychological and behavioral abnormalities, leukoencephalopathy, encephalopathy, and subacute combined degeneration of the spinal cord, extremity weakness, ataxia, and seizures. Notably, some studies have indicated that psychological and behavioral abnormalities are a presenting sign of late-onset cblC defect, among which schizophrenia and auditory hallucinations were common, rather than depression (Martinelli et al., 2011; Carrillo-Carrasco et al., 2012; Wei et al., 2019). Our case report demonstrates that depression is also one of the initial manifestations of the late-onset cblC, which usually misleads psychologists or psychiatrists to diagnose it as a plain mood disorder. However, the mechanism of depression in cblC patients remains unclear. Recently, some studies have pointed out that the underlying pathogenesis of depression might involve neurotransmitters and the related amino acid metabolism disorder (Wang et al., 2021), oxidative stress and inflammation (Behl et al., 2022), mitochondrial dysfunction, and energy metabolism disturbance (Weger et al., 2020). Some biochemical alterations in cblC patients may have harmful effects on the development of depression in multiple ways (Figure 2). First, Hcy elevation and vitamin B12 deficiency of cblC defect can result in depression via inhibiting the S-adenosyl-methionine-dependent synthesis of variable neurotransmitters and their biological activities, such as catecholamines, namely, dopamine, norepinephrine, epinephrine, and noncatecholamines, namely, serotonin, due to impairment in the methylation pathway (Bhatia and Singh, 2015). Additionally, rising Hcy and cysteine sulfinic acid can produce N-methyl-D-aspartate receptor agonists and reactive oxygen species, both of which have neurotoxic effects on dopaminergic neurons, thus inducing depression (Bhatia and Singh, 2015). Second, the dysfunction of mitochondrial energy metabolism and tricarboxylic acid cycle, caused by the elevated blood MMA level, can reduce neuroplasticity and impair hippocampal neurogenesis (Proctor et al., 2020; Forny et al., 2021), which is widely acknowledged to play a vital role in depression. Third, Hcy, together with MMA, also exacerbates oxidative stress and inflammation that are involved in key depressive pathogenic pathways. Thus, we deemed that the elevated Hcy and MMA levels in cblC patients, especially the former one (Moradi et al., 2021), are linked to depression. This connection is consistent with the fact that depression occurs in late-onset methylmalonic aciduria and homocysteinemia patients more frequently than in early-onset methylmalonic aciduria patients. Understanding the association between depression and cblC disease and its influence on the pathophysiologic mechanism of depression will be helpful in the early diagnosis of cblC disease and the etiology-based treatment of uncommon depression. Cobalamin, essential for human growth and development, is converted to two active forms: adenosylcobalamin is the coenzyme of mitochondrial methylmalonyl-CoA mutase, converting methylmalonyl-CoA to succinyl-coenzyme A, whose inborn error can cause MMA accumulation due to methylmalonyl-CoA increase; and Mecbl serves as the coenzyme of cytoplasmic methionine synthase, associated with the synthesis of methionine from Hcy, whose congenital disorder can lead to Hcy accumulation and methionine reduction. Details of the metabolic pathways of the cblC defect are shown in Figure 2. To date, inherited cobalamin deficiencies mainly contain more than 10 types, such as methylmalonyl-CoA deficiency, haptocorrin deficiency, cblA-G, cblJ, cblX, and cblK. Except for cblX, which is X-linked recessive, all the other recognized types are autosomal recessive inheritance. Some types generate a block in the synthesis pathway of both adenosylcobalamin and Mecbl, such as cblC, cblD, cblF, and cblJ, leading to combined methylmalonic aciduria and homocysteinemia (Watkins and Rosenblatt, 2022). Among these types, cblC is the most common inborn disorder. The MMACHC mutation was first identified in 2006 (Lerner-Ellis et al., 2006) and consists of nearly 100 variants recorded in the Human Gene Mutation Database (HGMD) up to now, which are responsible for the cblC defect. Many variants differ in RNA stability or residual function of the protein, which may produce, at least, a certain discrepancy in the phenotype and severity of the disease (Maquat, 2004). Previous published evidence indicated that the mutation spectrum of the MMACHC gene seemed to vary in different regions across the world. A study by Wang et al. suggested that c.609G>A (p.W203X), c.658_660delAAG (p.K220del), c.80A>G {p.Q27R [r.(spl?)]}, and c.482 G>A (p.R161Q) mutations were the most common mutations in the Chinese population, while the most frequent mutations in the Caucasian population were c.271dupA (p.R91Kfs∗14), c.394C>T (p.R132X), and c.331C>T (p.R111X) (Wang et al., 2019). However, little difference in the genotype–phenotype correlations of the different regions was observed. Present studies conformably believed that c.271dupA (p.R91Kfs∗14), c.609G>A (p.W203X), and c.331C>T (p.R111X) were mainly related to an early-onset and severe presentation, whilst c.482 G>A (p.R161Q) mainly caused the late onset and mild phenotype in any region (Carrillo-Carrasco et al., 2012; Morel et al., 2006; Wang et al., 2019; Yu et al., 2015). The mutation in our case was a compound heterozygous mutation in the MMACHC gene, that is, c.271dupA (p.R91Kfs∗14) and c.482 G>A (p.R161Q), causing a late onset with mild symptoms, which does not prove the aforementioned correlation. The reason for that may be two points. On one hand, it seems that the compound heterozygotes could result in quite a moderate disease, between the severe early-onset form associated with homozygosity for c.271dupA (p.R91Kfs∗14) and the slight late-onset phenotype related to homozygosity for c.482 G>A (p.R161Q); on the other hand, the late-onset form of the disease usually occurs in patients who possess compound heterozygotes for the c.271dupA (p.R91Kfs∗14) mutation and a missense mutation (Morel et al., 2006). Moreover, the fact that the diverse genetic constitutions in the cblC patients make it difficult to establish the entire connection between genotype and phenotype. Despite this, with the rising awareness of newborn screening and prenatal diagnosis, this genotype–phenotype relationship is still expected to forecast the onset age and disease severity and provide a guide for treatment strategy proposal. Furthermore, case series need to focus on their connection, even on specific clinical manifestations, such as depression, encephalopathy, extremity weakness, gait abnormalities, and seizures, which help to predict the delayed presentation and prognosis. CblC diagnosis typically depends on either biochemical examination or genetic analysis. Homocysteine in blood or urine, blood amino acids, free carnitine, acylcarnitines, and urine organic acids are vital biochemical assays. Importantly, mass spectrometry and gas chromatography-mass spectrometry are recommended to, respectively, detect blood levels of acylcarnitines, including propionylcarnitine (C3) and acetylcarnitine (C2), and urinary organic acids, including MMA and methylcitric acid. Particularly, using combined mass spectrometry and gas chromatography-mass spectrometry, newborn screening has effective identification ability, boosts early diagnosis, and allows better prognosis (Jin et al., 2021). At the same time, blood vitamin B12, folic acid, and antibody to intrinsic factor need to be detected to exclude the diagnosis of cobalamin disturbance due to secondary etiologies. Finally, genetic sequencing could facilitate the achievement of a definitive diagnosis and detailed genotyping, such as cblC disease based on the MMACHC gene mutation. With the improvement of detection means, cblC can be rapidly diagnosed if the physician is conscious of it. However, in the past reported cases, the time to unequivocal diagnosis needs more than 2 months, even 2 years (Wei et al., 2019). Unfortunately, it took us 3 months to make a clear diagnosis, although some points in this immature patient, for example, abnormal homocysteinemia and multiple neuropsychiatric symptoms, have provided a clue of the inherited metabolic disease for us. The major reason for the prolonged time to diagnosis is misdiagnosis or missed diagnosis during the initial visit. Thereby, physicians need to pay more attention to the understanding of cblC symptoms and heterogeneous clinical spectrum and enhance the awareness of disease screening. Currently, depression is a very common disorder characterized by paroxysm and easy recurrence, and is closely related to living difficulties, abnormal personality development, and suicide in adolescents, which also, inevitably, increase family and social burdens. Although the possibility of a coincidence cannot be completely excluded in this case, the clinical features of this patient’s depression were not typical, for only one attack occurred and the condition improved quickly. Thence, we considered that depression could be a presenting sign of cblC-type methylmalonic aciduria and homocysteinemia, which also indicated that cblC disease might be a cause of depression. Psychiatrists should extend biochemical or genetic analysis looking for this rare disease to those patients with atypical depression, for example, patients with markedly elevated Hcy levels, anemia, intelligence, or fitness decline, which provide crucial clues for genetic metabolic disease. Moreover, extremely elevated serum Hcy levels, anemia, and intelligence or fitness decline provide crucial clues for genetic metabolic disease. In this case, the acylcarnitine profile was normal, which is, indeed, totally beyond our expectations, as the test was conducted after medication treatment for several weeks. It suggested that we conduct the blood and urinary biochemical examinations as early as possible, for an improper test time could lead to completely confusing results. White matter hyperintensity shown in brain MRI is also one of the common image presentations. Fortunately, in this case, when homocysteinemia was detected, the patient was immediately administered a cyanocobalamin and Mecbl supplement, so that she could have a better prognosis. Thence, once cblC is considered, treatment with parenteral cobalamin and betaine is suggested as soon as possible after the biochemical examination, which helps to correct the metabolic disorder. Most notably, hydroxocobalamin is the only form of cobalamin proven to benefit patients with cblC disease, and cyanocobalamin/Mecbl is not recommended as a regular course of treatment (Carrillo-Carrasco et al., 2012; Baumgartner et al., 2014). The treatment of cyanocobalamin/Mecbl can only be applied to the patient with the presence of a partially functional MMACHC protein in heterozygosity (Anna et al., 2022) Previous studies and our case study revealed that although the late-onset cblC patient could show improved clinical manifestation and long-life span through active treatments, urine MMA and blood Hcy levels of the patients could not be reduced to normal (Matos et al., 2013; Wei et al., 2019), which indicated that MMA metabolic disturbance would last for a relatively long period, and the delayed complication could follow as well. These complications include encephalopathy, hydrocephalus (Zhang et al., 2019), optic neuropathy and retinopathy (Alowain et al., 2019), pulmonary hypertension, late-onset diffuse lung disease, cardiorespiratory disease (Liu et al., 2017; Zhang et al., 2020), proteinuria, renal thrombotic microangiopathy, and kidney function decline (Chen et al., 2020; Liu et al., 2017). Thereby, the late-onset cblC patients should be treated perpetually with hydroxocobalamin supplement and routine metabolic monitoring. During the follow-up, the physician needs to pay attention to the signs of these complications and conduct relevant imaging examinations if necessary. Except for the regular therapy, a lot of novel gene therapies, including adenovirus gene addition, genome editing, lentiviral gene therapy, and systemic mRNA therapy, would be effective for permanent long-term correction of metabolic disorders caused by methylmalonyl-CoA mutase gene mutation (Chandler and Venditti, 2019; Schneller et al., 2021). However, it should be noted that there have been no gene therapies available for cblC at present, and more attempts can be conducted to seize the appropriate genomic therapies.
Collectively, our case report has expanded the cblC clinical symptom spectrum, indicating that the consideration of depression as one kind of the initial sign of cblC would be helpful in the early diagnosis. Literature review about genotype–phenotype correlation and clinical experience could also remarkably improve physicians’ perspectives and patient prognosis for early diagnosis and treatment. | true | true | true |
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PMC9631443 | Hannah G. Daniels,Breanna G. Knicely,Anna Kristin Miller,Ana Thompson,Rina Plattner,Eva M. Goellner | Inhibition of ABL1 by tyrosine kinase inhibitors leads to a downregulation of MLH1 by Hsp70-mediated lysosomal protein degradation 10.3389/fgene.2022.940073 | 20-10-2022 | mismatch repair,nilotinib dasatinib,DNA repair,Hsp70,lysosome | The DNA mismatch repair (MMR) pathway and its regulation are critical for genomic stability. Mismatch repair (MMR) follows replication and repairs misincorporated bases and small insertions or deletions that are not recognized and removed by the proofreading polymerase. Cells deficient in MMR exhibit an increased overall mutation rate and increased expansion and contraction of short repeat sequences in the genome termed microsatellite instability (MSI). MSI is often a clinical measure of genome stability in tumors and is used to determine the course of treatment. MMR is also critical for inducing apoptosis after alkylation damage from environmental agents or DNA-damaging chemotherapy. MLH1 is essential for MMR, and loss or mutation of MLH1 leads to defective MMR, increased mutation frequency, and MSI. In this study, we report that tyrosine kinase inhibitors, imatinib and nilotinib, lead to decreased MLH1 protein expression but not decreased MLH1 mRNA levels. Of the seven cellular targets of Imatinib and nilotinib, we show that silencing of ABL1 also reduces MLH1 protein expression. Treatment with tyrosine kinase inhibitors or silencing of ABL1 results in decreased apoptosis after treatment with alkylating agents, suggesting the level of MLH1 reduction is sufficient to disrupt MMR function. We also report MLH1 is tyrosine phosphorylated by ABL1. We demonstrate that MLH1 downregulation by ABL1 knockdown or inhibition requires chaperone protein Hsp70 and that MLH1 degradation can be abolished with the lysosomal inhibitor bafilomycin. Taken together, we propose that ABL1 prevents MLH1 from being targeted for degradation by the chaperone Hsp70 and that in the absence of ABL1 activity at least a portion of MLH1 is degraded through the lysosome. This study represents an advance in understanding MMR pathway regulation and has important clinical implications as MMR status is used in the clinic to inform patient treatment, including the use of immunotherapy. | Inhibition of ABL1 by tyrosine kinase inhibitors leads to a downregulation of MLH1 by Hsp70-mediated lysosomal protein degradation 10.3389/fgene.2022.940073
The DNA mismatch repair (MMR) pathway and its regulation are critical for genomic stability. Mismatch repair (MMR) follows replication and repairs misincorporated bases and small insertions or deletions that are not recognized and removed by the proofreading polymerase. Cells deficient in MMR exhibit an increased overall mutation rate and increased expansion and contraction of short repeat sequences in the genome termed microsatellite instability (MSI). MSI is often a clinical measure of genome stability in tumors and is used to determine the course of treatment. MMR is also critical for inducing apoptosis after alkylation damage from environmental agents or DNA-damaging chemotherapy. MLH1 is essential for MMR, and loss or mutation of MLH1 leads to defective MMR, increased mutation frequency, and MSI. In this study, we report that tyrosine kinase inhibitors, imatinib and nilotinib, lead to decreased MLH1 protein expression but not decreased MLH1 mRNA levels. Of the seven cellular targets of Imatinib and nilotinib, we show that silencing of ABL1 also reduces MLH1 protein expression. Treatment with tyrosine kinase inhibitors or silencing of ABL1 results in decreased apoptosis after treatment with alkylating agents, suggesting the level of MLH1 reduction is sufficient to disrupt MMR function. We also report MLH1 is tyrosine phosphorylated by ABL1. We demonstrate that MLH1 downregulation by ABL1 knockdown or inhibition requires chaperone protein Hsp70 and that MLH1 degradation can be abolished with the lysosomal inhibitor bafilomycin. Taken together, we propose that ABL1 prevents MLH1 from being targeted for degradation by the chaperone Hsp70 and that in the absence of ABL1 activity at least a portion of MLH1 is degraded through the lysosome. This study represents an advance in understanding MMR pathway regulation and has important clinical implications as MMR status is used in the clinic to inform patient treatment, including the use of immunotherapy.
DNA mismatch repair (MMR) is the repair process that repairs small insertions and deletions, and base/base mispairs generated and not corrected by the replicative DNA polymerase during DNA replication (Li, 2008; Fishel, 2015). This correction of replication errors by MMR is necessary for genomic stability, and MMR deficiency leads to increased mutation rates and genomic instability (Kolodner, 1995; Gupta and Heinen, 2019). MMR defects and the resulting genomic instability promote cancer development, specifically colorectal and endometrial cancers (Borresen et al., 1995; de la Chapelle, 2004; Boland and Goel, 2010; Kastrinos and Stoffel, 2013; Lynch et al., 2015). Proteins in the MMR pathway also play various roles outside of repairing replication errors. These include preventing recombination between divergent sequences (Spies and Fishel, 2015; Tham et al., 2016) and promoting apoptosis in the presence of exogenously generated mispairs that are recognized by but not repaired by MMR, such as those formed after damage by alkylating agents or platinum agents (Jiricny, 2006; Topping et al., 2009; Fu et al., 2012; Gupta and Heinen, 2019). Loss of mismatch repair results in resistance to these classes of chemotherapeutics (Modrich and Lahue, 1996). MMR defects have recently been positively correlated with increased response to immunotherapy, presumably due to their increased mutational burden and neoantigen production (Kubecek et al., 2016; Zhao et al., 2019). Canonical eukaryotic MMR involves the recognition of the mispair by MutSα, a heteroduplex consisting of MSH2 and MSH6, followed by DNA nicking and excision licensing of the mispair after recruitment of MutLα, a heteroduplex consisting of endonuclease MLH1 and PMS2. Exonuclease 1 (EXO1) is then recruited to excise the mispair, or this step is carried out by one of the more recently elucidated EXO1-independent subpathways (Goellner et al., 2015; Calil et al., 2021). Finally, the replicative DNA polymerases, PCNA, and RFC carry out DNA synthesis to fill in the gap, and ligase completes repair (Bowen et al., 2013; Goellner et al., 2015; Bowen and Kolodner, 2017). EXO1-independent MMR can proceed through either multiple rounds of MLH1-PMS2 nicking (Amin et al., 2001; Smith et al., 2013; Goellner et al., 2014) or FEN1 activity can compensate during the excision step (Calil et al., 2021). Exogenously induced mispairs can occur when damaged bases, such as O6 methylguanine generated by SN1 DNA alkylating agents, become preferentially paired with an incorrect base by the replicative polymerases (Li et al., 2016). Repair of O6-methylguanine lesion is usually performed by direct reversal by methylguanine methyltransferase (MGMT); however, the absence of or deficiencies in MGMT lead to unrepaired lesions, which upon DNA replication can generate O6meG/T mispairs (Kaina et al., 2007). This mispair is then a substrate for MMR recognition. While these damage induced mispairs are recognized by the MMR machinery, they are ultimately unable to be repaired as the damage is on the template strand and therefore result in signaling the cell for apoptosis (Meikrantz et al., 1998; Stojic et al., 2004; Li et al., 2016). Studies have shown that the components of MutSα and MutLα heteroduplexes are required for this DNA damage response (Dosch et al., 1998; Cejka et al., 2003). Loss of either of these critical MMR components leads to resistance to alkylation-induced apoptosis. Due to the importance of MMR in the maintenance of genomic stability, the regulation of the MMR response pathway is of particular interest. Regulation of the MutSα complex has been shown in several studies to have cell cycle-dependent transcriptional regulation of MSH2 and MSH6 (Edelbrock et al., 2013; O'Brien and Brown, 2006; Seifert and Reichrath, 2006). Studies have shown that transcriptional regulation involving hypermethylation of the MLH1 promoter relates directly to MMR deficiency in tumors (Esteller et al., 1998; Niv, 2007). Additionally, mechanisms such as phosphorylation and protein degradation have been suggested to elicit posttranslational regulation of MLH1 and PMS2 (Jia et al., 2016; Weßbecher and Brieger, 2018). Phosphorylation of the MutLα heteroduplex has primarily been shown to occur on serine/threonine residues by various kinases (Jia et al., 2016; Hinrichsen et al., 2017b; Weßbecher and Brieger, 2018; Weßbecher et al., 2018). Degradation of the MutLα proteins often occurs due to complex instability caused by mutations in one of the heteroduplex partners, as MLH1 and PMS2 are thought to be obligate heterodimers, and heteroduplex formation is required for activity (Mohd et al., 2006; Abildgaard et al., 2019). Additionally, PCNA phosphorylation has been shown to control MMR activity (Ortega et al., 2015; Tong et al., 2015). In this study, we show that MLH1 levels are decreased by tyrosine kinase inhibitor, Dasatinib, and the more specific and clinically relevant tyrosine kinase inhibitors, imatinib and nilotinib. This effect was observed in both non-cancerous HEK293 cells and in cells derived from colorectal human tumors. Further, we show that of the shared Dasatinib and imatinib/nilotinib targets, knockdown of the ABL1 kinase by siRNA also decreases MLH1 protein expression. This decrease in expression is sufficient to disrupt MMR function, as cells treated with tyrosine kinase inhibitors or Abl siRNA became partially resistant to alkylation-induced apoptosis. This regulation of MLH1 by Abl kinase and tyrosine kinase inhibitors is posttranslational as mRNA levels remain steady after treatment, and we observe phosphorylation of MLH1 that is lost with ABL1 inhibition. We propose that Abl prevents MLH1 from being targeted for degradation by the chaperone HSP70 and that in the absence of ABL1 activity, at least a portion of MLH1 is degraded through the lysosome. Together this study elucidates a new mechanism of MMR regulation. Furthermore, as many tyrosine kinase inhibitors targeting ABL1 are approved for clinical use, this study has interesting implications on the potential side effects on MMR in cancers typically treated with tyrosine kinase inhibitors. Additionally, this study opens possibilities of clinically modulating MMR for enhanced response to immunotherapy.
Imatinib mesylate (imatinib) was obtained from Tocris Biosciences; 10 mM stock was prepared in dimethylsulfoxide (DMSO) (Fisher Scientific) and stored at −20°C. Nilotinib was obtained from Cell Signalling Inc.; 10 mM stock was prepared in DMSO and stored at −20°C. Dasatinib was obtained from Cell Signalling Inc.; 10 mM stock was prepared in DMSO and stored at -20°C. 6-Thioguanine 98% (6 TG) was obtained from TCI America (T0212-1G); 40 mM stock was prepared in NaOH and stored at −20°C. Methylnitronitrosoguanidine (MNNG) was obtained from Sigma-Aldrich (Cat #129941); 10 mM stock was prepared in DMSO and stored at −20°C. Antibodies used in this study include MLH1 (#3515, #4256), PCNA (#2586, #13110), ABL1 (#2862), and Phospho-Tyrosine MultiMab (#8954) (Cell Signalling Inc.), IRDye 800CW Donkey anti-Rabbit IgG Secondary Antibody and IRDye 800CW Goat anti-Mouse IgG Secondary Antibody (Li-Cor Biosciences).
Human embryonic kidney 293 (HEK293) cells and SW480 cells from American Type Culture Association (ATCC) were cultured in DMEM (Sigma-Aldrich) containing 10% Fetal Bovine Serum (Gibco) and 1% Penicillin-Streptomycin solution (Gibco) at 37°C in 5% CO2/95% air.
Knockdown of various tyrosine kinase targets was achieved using Trilencer-27 Oligo Duplex siRNA (OriGene). Duplexes were resuspended following the manufacturer’s protocol. Transfections with the siRNA were performed using Invitrogen Lipofectamine RNAiMAX Transfection Reagent (Invitrogen) following the manufacturer’s protocol. Initially, the percent protein knockdown of each duplex was determined by Western Blot analysis. After determining the duplex with the most efficient protein knockdown, future experiments were performed using only the most effective duplex.
For imatinib and nilotinib treatments, HEK293 cells were seeded into a 12-well plate at 110,000 cells per well. After allowing to incubate for 24 h at 37°C, the cells were treated at approximately 40% confluency with indicated doses of imatinib or nilotinib as well as a control dose of DMSO. After another 24 h incubation period, the media on the wells was removed and replaced with the following 6 TG doses, one dose per row: NaOH (Control), 5 µM, 10 µM. Cells were then allowed to incubate at 37°C for 48h before being trypsinized and counted via cell counter. For siRNA cytotoxicity assays, HEK293 cells were first seeded into a 6-well plate with 600,000 cells per well. After 24 h of incubation at 37°C, cells were transfected with scrambled control or siRNA using the RNAiMax transfection protocol. Cells were placed back in the incubator for another 24 h, then trypsinized, seeded into 12-well plates, treated with 6 TG, and counted following the steps outlined above. Live cell counts were recorded, and percentages were plotted for graphical analysis.
Cells were lysed by first removing all media from the plates and washing twice with ice-cold PBS. Cells were lysed using 200–500 µl RIPA lysis buffer (Thermo Scientific) containing either complete protease inhibitor cocktail (Roche) or a combination protease/phosphatase inhibitor cocktail (Cell Signalling). Cellular extracts were collected and stored at −20°C.
HEK293 cells were seeded into a 6-well plate with 600,000 cells per well and allowed to incubate for 24 h at 37°C. Cells were then either treated with imatinib or nilotinib or transfected with siRNA. After another 24 h incubation (excluding treatment timecourse experiments), RNA extraction of the cells was performed using the Qiagen RNA Extraction Kit. RNA concentrations were determined using a Nanodrop, then RNA was converted to cDNA using the Superscript cDNA Conversion Kit (Thermofisher). Following the RT-PCR SYBR Green protocol, RT-PCR was set up in a 96-well plate with MLH1 and PCNA primers, with PCNA primers acting as the control. The experiment was run and analyzed using the QuantStudio application and software.
Cells were washed with PBS and resuspended in cytoplasm extract buffer (20 mM HEPES, 10 mM KCl, 0.1 mM EDTA, 1 mM DTT, and protease inhibitor) and then chilled on ice for 10 min 0.75% Nonidet P-40 (NP-40) lysis buffer was added, and the solution was pipetted to mix and vortexed for 10 s. The cells were centrifuged at 800 x g for 3 min at 4°C to separate nuclei from the cytoplasm (supernatant). The cytoplasm extract was placed in a separate tube, and the nuclei pellet was resuspended in 25% sucrose/cytoplasm extraction buffer and pipetted to disperse. The cells in 25% sucrose/cytoplasm extraction buffer were underlaid with half the volume of 50% sucrose/cytoplasm extraction buffer and centrifuged at 10,000 x g for 15 min at 4°C. The supernatant was removed, and the nuclei pellet was lysed in PBE150Na [50 mM Tris-HCl at pH 7.5, 1 mM ethylenediaminetetraacetic acid (EDTA) at pH 8.0, 150 mM NaCl, 0.5% sodium deoxycholate and 1% NP-40, containing 1x Complete protease inhibitor cocktail (Roche Diagnostics GmbH, Germany)]. The pellet was then sonicated and centrifuged at 10,000x g for 15 min at 4°C. The supernatant was collected as the nuclear extract.
Co-immunoprecipitations of endogenous proteins were performed using magnetic protein A/protein G beads (Thermo Scientific), followed by a conjugation step to either the IgG control or antibody of interest. Beads were blocked with BSA for 1 h, followed by washes. Conjugated beads were incubated with samples at 4°C for 3 h rotating, followed by increasing salt washes. Beads were boiled with 6x loading buffer, and samples were run on SDS-PAGE gels followed by Western blot.
Kinase assays were performed using recombinant ABL1 (ABL1) (25ng, Invitrogen P3049) and MLH1 (100ng, Origene TP301607) with the addition of kinase buffer (20 mM Tris pH 7.4, 10mM MgCl2, 1 mM DTT) and 1 mM cold ATP. Each reaction was assembled in separate 1.5 ml microcentrifuge tubes and placed in a heat block at 37°C. The reaction was quenched by adding 4x Laemeli buffer and heating at 95°C for 5 min. The samples were then analyzed by Western blot. Initially, we performed a timecourse and quenched the reaction at the following time points after the addition of ATP: 0, 15, 30, and 60 min. We determined that 15 min after ATP addition was sufficient enough to observe tyrosine phosphorylation (Data not shown).
Immunoblots were imaged using LiCor infrared secondary antibodies and a LiCor Odyssey Infrared Western Blot Imaging system. Band intensity was calculated using the LiCor software and normalized to the band intensity of the loading control. Calculations of the mean, standard error, statistical analysis, and comparison of each set of experimental means were performed with Graphpad Prism 9.0 (Graphpad Software Inc., La Jolla, CA, United States). Comparison between conditions was performed with either an unpaired t-test or 1-way Anova.
There is interest in investigating SRC-kinase inhibitors in advanced colorectal cancer patients in combination with other chemotherapy (Parseghian et al., 2017; Scott et al., 2017). During investigations into the relationship between MMR status and response to tyrosine kinase inhibitors, we unexpectedly observed that Dasatinib-treated cells had reduced levels of MLH1 protein. In MMR proficient SW480 colorectal cells treated with increasing doses of Dasatinib, MLH1 expression was decreased in a dose-dependent manner, with a statistically significant effect seen at 5 μM Dasatinib, resulting in roughly 50% MLH1 expression remaining. (Figure 1A). Dasatinib has upwards of 70 known targets (Hantschel et al., 2008). With the understanding that Dasatinib is a third-generation SRC-kinase inhibitor with a wide range of targets, we decided to use earlier generation drugs more commonly used in the clinic, imatinib and nilotinib, to narrow down the kinase target responsible for the decrease in MLH1 protein expression. Imatinib and nilotinib have the same seven known targets (Hantschel et al., 2008). We treated SW480 cells with either imatinib or nilotinib and confirmed that MLH1 protein expression was decreased in a similar manner to Dasatinib treatment (Figure 1B). When comparing the targets between Dasatinib and imatinib/nilotinib, there are six common target kinases: Kit, PDGFR, ABL1, DDR1, ARG (ABL2), and the fusion kinase present only in certain leukemias, BCR-Abl (Hantschel et al., 2008) (Figure 1C). Based on our observations, we believed the kinase target responsible for the decrease in MLH1 protein expression was common between all three tyrosine kinase inhibitors.
To determine if this was a common phenomenon or specific to cancer cells, we used HEK293 cells going forward due to their proficient MMR mechanism and lack of cancer-specific alterations. To confirm that imatinib and nilotinib have a similar effect on MLH1 protein expression, HEK293 cells were treated with a varying dose range of each drug, respectively. Results show that imatinib and nilotinib treatment decrease MLH1 protein expression in HEK293 cells, similarly to what was observed in SW480 cells. (Figure 2A). We looked at other MMR proteins, including MSH2, MSH6, and Exo1, after nilotinib treatment in HEK293 cells and determined the effect of the drug to primarily be on MLH1 protein expression (Supplementary Figure 1A). We also observed changes to the MLH1 obligate binding partner, PMS2, after nilotinib treatment (Supplementary Figure 1B). To determine if the approximately 50% reduction in MLH1 levels was sufficient to affect the efficiency of the MMR pathway, cell viability after imatinib or nilotinib treatment in combination with 6 thioguanine (6TG) was examined. 6TG is a purine antimetabolite that is incorporated into the DNA during replication, ultimately leading to an O6 methylguanine mismatch recognized by MMR. The mispair cannot be repaired, resulting in apoptosis when MMR is functional and resistance to 6TG when MMR is defective (Meyers et al., 2004; O'Brien and Brown, 2006). Loss of MLH1 protein results in resistance to 6TG due to loss of MMR-induced apoptotic signaling in response to the O6-T mispair (WE et al., 1998; Niv, 2007; Zhao et al., 2019). We determined that imatinib or nilotinib treatment alone did not change cell viability (Figure 2B). However, in a 72-h survival assay, a 24-h pre-treatment with imatinib or nilotinib decreased cell death after 6 TG treatment compared to cells pretreated with the vehicle control (Figure 2C). Based on these data, we suggest that treatment with tyrosine kinase inhibitors leads to an impaired MMR damage response by the downregulation of MLH1 in a variety of non-cancerous and cancerous cell lines.
After determining that imatinib and nilotinib treatment impairs MMR-mediated apoptosis, we questioned which of the known kinase targets were responsible. There are seven known targets of imatinib and nilotinib: BCR/ABL, c-Kit, DDR1, PDGFRα, ABL1 (ABL1), ARG (ABL2), and NQO1 (Hantschel et al., 2008). BCR/ABL is a fusion kinase only present in certain leukemia cell lines, not HEK293 cells, leaving six targets of interest. Of these six targets, ABL1 specifically has been suggested to physically interact with MLH1 (Wagner et al., 2008; Fukuhara et al., 2014; Li et al., 2018). Based on this, we obtained siRNA duplexes for ABL1 and a target kinase not implicated in MMR, discoidin domain receptor tyrosine kinase 1, DDR1. HEK293 cells were transfected with each siRNA duplex separately, and Western blot analysis was performed to determine knockdown efficiency and changes to MLH1 protein expression. Knockdown of ABL1 resulted in a corresponding decrease in MLH1 protein expression levels similar to that seen with imatinib or nilotinib treatment (Figure 3A, Supplementary Figure 2A). In contrast, knockdown of DDR1 resulted in no significant change to MLH1 protein expression (Figure 3B; Supplementary Figure 2B). To confirm that decreased MLH1 protein expression by ABL1 knockdown also affected MMR-mediated apoptosis, we performed the short-term cytotoxicity assay with 6TG treatment in HEK293 cells with either ABL1 knockdown or scrambled siRNA control. ABL1 knockdown increased resistance to MMR-mediated apoptosis after 6TG treatment to a similar level as that seen with nilotinib or imatinib (Figure 3C). To determine the level of ABL1 activity in the SW480 and HEK293 cells and to confirm that imatinib/nilotinib treatment effectively inhibited ABL1 activity in these cells, we tested phosphorylation of Crk-like protein (CrkL). CrkL is phosphorylated by ABL1 and can be considered a surrogate marker of overall ABL1 activity (Ganguly et al., 2012; Jain et al., 2017; Tripathi et al., 2020). We determined that the SW480 cell line has increased ABL1 activity compared to HEK293 cells and that imatinib treatment does decrease pCrkL expression, however, only partially in the HEK293 cells (Supplementary Figure 3A). On the other hand, nilotinib treatment in HEK293 cells resulted in almost complete abolishment of ABL1 activity, indicated by the lack of pCrkL protein expression (Supplementary Figure 3B). We conclude that the ABL1 kinase is likely the imatinib/nilotinib target kinase responsible for decreased MLH1 protein expression and the subsequent impairment of MMR.
The MLH1 protein is highly stable (>24 h) (Supplementary Figure 3C), and while it is well known that MLH1 expression can be controlled by promotor methylation, there are few studies looking into its degradation (Kane et al., 1997; Hinrichsen et al., 2017a; Abildgaard et al., 2019). To determine the mechanism of MLH1 downregulation after ABL1 inhibition or loss, we first investigated any changes to mRNA levels by RT-PCR. We observed no significant mRNA change between control or treated samples in ABL1 knockdown or inhibitor conditions at the 24-h timepoint at which protein loss is observed (Figure 4A). No mRNA change is observed after nilotinib treatment at any prior timepoint either (Supplementary Figure 4A). Based on these results, we shifted our focus to mechanisms of posttranslational downregulation, starting with the potential tyrosine phosphorylation of MLH1 by ABL1. The ABL1 kinase phosphorylates various proteins on tyrosine residues for protein activation, protein degradation, or protein stability (Wang, 1993; Yuan et al., 1996; Shaul, 2000). To determine if MLH1 is phosphorylated by ABL1, ABL1, and MLH1 were overexpressed in HEK293 cells in the presence or absence of nilotinib, followed by immunoprecipitation of MLH1 and immunoblot using a pan-phosphotyrosine antibody. Western blot analysis showed a phosphotyrosine band corresponding to the size of MLH1 in the ABL1 and MLH1 overexpressed sample that was no longer detectable after ABL1 inhibition by nilotinib (Figure 4B). These results indicate that ABL1 tyrosine phosphorylates MLH1, and this phosphorylation is blocked after ABL1 inhibition. Thus, ABL1-mediated phosphorylation of MLH1 may prevent MLH1 degradation. To determine if this was a direct phosphorylation event by ABL1, recombinant ABL1 (ABL1) and MLH1 proteins were incubated with ATP in an in vitro kinase reaction. The reaction was run on an SDS-PAGE gel and probed with an anti-phosphotyrosine antibody. A phosphorylated band was observed at the 85 KDa molecular weight corresponding to MLH1 only when incubated with ABL1 protein (Figure 4C). Together, we conclude that ABL1 directly phosphorylates MLH1 on a tyrosine residue (Figure 4C). Next, we determined whether the observed loss of MLH1 after ABL1 inhibition occurs via lysosomal or proteasomal degradation pathways. HEK293 cells were co-treated with nilotinib and either Bafilomycin or MG-132 to inhibit lysosomal or proteasomal degradation, respectively. We observed that when cells were co-treated with Bafilomycin and nilotinib for 24 h, there was a rescue of MLH1 protein expression levels compared to cells treated with nilotinib alone (Figure 5). In contrast, the proteasomal inhibition by MG-132 did not prevent MLH1 downregulation after nilotinib treatment (Supplementary Figure 4B). We also observed no ubiquitination of MLH1 when MLH1 and HA-tagged ubiquitin were co-expressed, and MLH1 was immunoprecipitated and then probed with the anti-HA antibody (Supplementary Figure 4C).
A recent study showed that MLH1 mutant variants predicted by computational modeling to be unstable were more rapidly degraded by the proteasome than WT MLH1, presumable due to cellular unfolded protein response. The authors also showed that these unstable variants of MLH1 had increased interaction with the Hsp70 chaperone protein (Abildgaard et al., 2019). We hypothesized that Hsp70 may be a universal chaperone for MLH1 and tested whether Hsp70 was required for MLH1 degradation in the absence of ABL1 activity. We first examined whether Hsp70 interaction with MLH1 is dependent on ABL1. Using subcellular fractionation followed by immunoprecipitation of endogenous MLH1 and immunoblotting for Hsp70, we found that MLH1 and Hsp70 interact in the cytoplasm, and this interaction was significantly increased after silencing of ABL1 (Figure 6A). We also overexpressed MYC-FLAG-tagged MLH1 and treated cells with nilotinib, followed by immunoprecipitation of MLH1 and immunoblot for Hsp70. Like ABL1 knockdown, treatment with nilotinib increased Hsp70 interaction with MLH1 (Figure 6B). To determine whether binding to Hsp70 had an impact on MLH1 degradation, we co-treated cells with nilotinib and an Hsp70 inhibitor, YM-01. YM-01 treatment rescued the reduction in MLH1 protein expression induced by nilotinib treatment (Figure 6C). Taken together, we propose that ABL1 is important for maintaining MLH1 protein stability in at least a subset of cellular MLH1 pools, potentially by phosphorylation of MLH1. In the absence of ABL1, MLH1 is targeted by Hsp70 for lysosomal degradation.
This study identifies a novel mechanism of MLH1 regulation through the involvement of the ABL1 kinase, Hsp70, and lysosomal degradation. We first observed changes in MLH1 protein expression levels after treatment with the FDA-approved tyrosine-kinase inhibitor, Dasatinib. Since this inhibitor targets a large number of kinases, we systematically narrowed down the target kinase responsible by using tyrosine-kinase inhibitors with a narrower range of targets, imatinib and nilotinib, and siRNA to knock down specific kinases. ABL1 has previously been reported to physically interact with MLH1 (Kim et al., 2007), so we chose to focus on that target kinase first. We show here that ABL1 kinase knockdown by siRNA recapitulates the MLH1 down-regulation, MMR impairment, and Hsp70 binding that we observe with the nilotinib or Dasatinib treatment. We also tested DDR1 siRNA as a comparison for a protein not known to impact MMR, and we did not observe any MLH1 protein change. HEK293 cells do not have appreciable levels of PDGFR or c-Kit (Zaslavsky et al., 2005; Nakagomi and Hirota, 2007), so it is unlikely these contribute to the MLH1 degradation observed. While the degree of MLH1 protein reduction was roughly equivalent between pharmacological inhibition and siRNA knockdown of ABL1 in HEK293 cells, we cannot rule out the possibility that either c-Kit or PDGFR may influence MMR in specific tumor backgrounds where they are highly expressed or mutated. We did not evaluate the role of NQO1 as it is a quinone reductase instead of a non-receptor tyrosine kinase (Hantschel et al., 2008). The fusion protein BCR-ABL is produced by the Philadelphia chromosome translocation found in leukemia. This fusion produces a dysregulated constitutively active form of ABL1. Given our findings here that normal ABL1 activity is important for MLH1 protein stability in at least a portion of cellular MLH1, it raises interesting questions as to what MLH1 dynamics are in leukemia cells harboring BCR-ABL. It also raises questions about MMR function in solid tumors with hyperactive ABL1, such as subsets of melanoma or triple-negative breast cancer (Srinivasan and Plattner, 2006; Srinivasan et al., 2008; Ganguly et al., 2012; Ganguly and Plattner, 2012). Skorski et al. report that the Leukemia cells with the BCR-ABL fusion also have dysfunctional MMR, including increased mutation frequencies and decreased sensitivity to alkylating agents (Stoklosa et al., 2008). This report, together with our findings, suggests a balance of ABL1 activity is required for MMR, with both too little or too much activation resulting in MMR deficiency, although potentially through different mechanisms. Here, we identified one MMR protein whose stability is controlled by ABL1 activity. However, there are likely other MMR proteins whose activity or stability is controlled by kinase activity. Li et al. published a series of papers in which they showed that PCNA is phosphorylated on tyrosine residue 211 by EGFR (Ortega et al., 2015). This phosphorylation can be induced by arsenic exposure (Tong et al., 2015). Phosphorylation of PCNA prevented MMR activity by altering the interaction of PCNA with critical MMR proteins (Ortega et al., 2015). Bardelli et al. showed that targeting EGFR/BRAF downregulates MMR activity (Russo et al., 2019). However, treatment with either EGFR inhibitor alone or EGFR inhibitor with BRAF inhibitor decreased numerous repair pathway proteins, including those involved in homologous recombination and base excision repair. MMR was downregulated across most of the critical protein components, including MLH1, PMS2, MSH2, and MSH6. In this case, the regulation appears to be at the mRNA level and corresponds with mRNA upregulation of error tolerance pathways such as translesion polymerases (Russo et al., 2019). We observe protein downregulation but not mRNA downregulation after ABL1 inhibition by either siRNA or tyrosine kinase inhibitors. In our study, we also observe a concurrent loss of PMS2 protein levels but not a loss of MSH2 and MSH6. PMS2 is an obligate dimer with MLH1, and loss of MLH1 protein leads to loss of PMS2 protein (Wu et al., 2003; Kosinski et al., 2010). We focused on MLH1 in this study as MLH1 is the common partner for the MutL heterodimers. MLH1 dimerizes with MLH2, MLH3, and PMS2, although MLH1-PMS2 plays the most prominent role in MMR (Wang et al., 1999; Campbell et al., 2014). We observe phosphorylation of MLH1 after immunoprecipitation and immunoblot with a pan-phosphotyrosine antibody. We also observed phosphorylation of MLH1 by ABL1 in an in vitro kinase assay with purified recombinant ABL1 and MLH1, confirming there is a direct phosphorylation between ABL1 and the MLH1 component of the MLH1-PMS2 heterodimer. While the effect on MLH1 was consistent with ABL1 inhibition or knockdown, it consistently only lowered MLH1 levels by 25%–50%, even when ABL1 was knocked down to about 30% expression. Phosphorylation by overexpressed ABL1 in HEK293 cells also appears only to affect a moderate portion of the pulled-down MLH1 (Figure 4). The cellular conditions or the specific cellular pool of MLH1 in which ABL1 controls MLH1 expression are still unknown. MLH1 is primarily nuclear but has some minor cytoplasmic expression and is imported by a nuclear localization sequence (Wu et al., 2003). ABL1 has expression in both the nucleus and the cytoplasm. ABL2, also known as ARG, shares many roles with ABL1 but has some distinct roles and is only cytoplasmic (Ganguly et al., 2012). ABL2 is commonly also targeted by imatinib/nilotinib; thus, we cannot rule out ABL2 as also influencing MLH1. The potential impact of ABL2 in addition to ABL1 is currently under investigation. Utilizing drugs Bafilomycin and MG-132 to inhibit lysosomal or proteasomal degradation, respectively, we found that by inhibiting lysosomal degradation by Bafilomycin, MLH1 protein expression loss was prevented in the presence of ABL1 inhibition. Findings from a recent computational study led us to investigate the potential involvement of chaperone protein, Hsp70 (Abildgaard et al., 2019). Though Hsp70 is mainly reported to be involved in protein refolding, it has also been shown to be involved in presenting proteins for degradation by autophagy (Fernandez-Fernandez et al., 2017). Immunoprecipitation results showed increased interaction between a subset of the MLH1 cellular pool and Hsp70 after ABL1 inhibition. After inhibition of Hsp70 by pharmacological inhibitor YM-01, MLH1 protein expression loss was completely prevented in cells with ABL1 inhibition. In combination with previous studies, these data suggest that Hsp70 may act as a chaperone for MLH1 degradation in general. Overall, this study demonstrates a previously uncharacterized regulatory pathway of MLH1 by ABL1 and suggests that phosphorylation of MLH1 is required for its protein stability. ABL1 inhibition causes a loss of phosphorylation in at least a subset of cellular MLH1, leading to Hsp70 chaperone binding and lysosomal degradation. These data have interesting and important clinical implications given the rise of immunotherapy use in MSI-high/MMR deficient tumors (Kubecek et al., 2016). These data may present a strategy for sensitizing MSI-low/MMR proficient tumors to immunotherapy, especially as tyrosine kinase inhibitors are FDA-approved drugs with a known safety profile. | true | true | true |
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PMC9631484 | Qian Wang,Ang Dong,Jinshuai Zhao,Chen Wang,Christipher Griffin,Claudia Gragnoli,Fengxia Xue,Rongling Wu | Vaginal microbiota networks as a mechanistic predictor of aerobic vaginitis | 20-10-2022 | microbial interaction network,evolutionary game theory,aerobic vaginitis,quasidynamic ordinary differential equations,microbiota | Aerobic vaginitis (AV) is a complex vaginal dysbiosis that is thought to be caused by the micro-ecological change of the vaginal microbiota. While most studies have focused on how changes in the abundance of individual microbes are associated with the emergence of AV, we still do not have a complete mechanistic atlas of the microbe-AV link. Network modeling is central to understanding the structure and function of any microbial community assembly. By encapsulating the abundance of microbes as nodes and ecological interactions among microbes as edges, microbial networks can reveal how each microbe functions and how one microbe cooperate or compete with other microbes to mediate the dynamics of microbial communities. However, existing approaches can only estimate either the strength of microbe-microbe link or the direction of this link, failing to capture full topological characteristics of a network, especially from high-dimensional microbial data. We combine allometry scaling law and evolutionary game theory to derive a functional graph theory that can characterize bidirectional, signed, and weighted interaction networks from any data domain. We apply our theory to characterize the causal interdependence between microbial interactions and AV. From functional networks arising from different functional modules, we find that, as the only favorable genus from Firmicutes among all identified genera, the role of Lactobacillus in maintaining vaginal microbial symbiosis is enabled by upregulation from other microbes, rather than through any intrinsic capacity. Among Lactobacillus species, the proportion of L. crispatus to L. iners is positively associated with more healthy acid vaginal ecosystems. In a less healthy alkaline ecosystem, L. crispatus establishes a contradictory relationship with other microbes, leading to population decrease relative to L. iners. We identify topological changes of vaginal microbiota networks when the menstrual cycle of women changes from the follicular to luteal phases. Our network tool provides a mechanistic approach to disentangle the internal workings of the microbiota assembly and predict its causal relationships with human diseases including AV. | Vaginal microbiota networks as a mechanistic predictor of aerobic vaginitis
Aerobic vaginitis (AV) is a complex vaginal dysbiosis that is thought to be caused by the micro-ecological change of the vaginal microbiota. While most studies have focused on how changes in the abundance of individual microbes are associated with the emergence of AV, we still do not have a complete mechanistic atlas of the microbe-AV link. Network modeling is central to understanding the structure and function of any microbial community assembly. By encapsulating the abundance of microbes as nodes and ecological interactions among microbes as edges, microbial networks can reveal how each microbe functions and how one microbe cooperate or compete with other microbes to mediate the dynamics of microbial communities. However, existing approaches can only estimate either the strength of microbe-microbe link or the direction of this link, failing to capture full topological characteristics of a network, especially from high-dimensional microbial data. We combine allometry scaling law and evolutionary game theory to derive a functional graph theory that can characterize bidirectional, signed, and weighted interaction networks from any data domain. We apply our theory to characterize the causal interdependence between microbial interactions and AV. From functional networks arising from different functional modules, we find that, as the only favorable genus from Firmicutes among all identified genera, the role of Lactobacillus in maintaining vaginal microbial symbiosis is enabled by upregulation from other microbes, rather than through any intrinsic capacity. Among Lactobacillus species, the proportion of L. crispatus to L. iners is positively associated with more healthy acid vaginal ecosystems. In a less healthy alkaline ecosystem, L. crispatus establishes a contradictory relationship with other microbes, leading to population decrease relative to L. iners. We identify topological changes of vaginal microbiota networks when the menstrual cycle of women changes from the follicular to luteal phases. Our network tool provides a mechanistic approach to disentangle the internal workings of the microbiota assembly and predict its causal relationships with human diseases including AV.
Aerobic vaginitis (AV) is an inflammatory vaginal dysbiosis that affects many aspects of health and reproduction for both pregnant and non-pregnant women worldwide (Marconi et al., 2013; Han et al., 2019; Wang et al., 2020). It is known that the occurrence of this disease is accompanied by the relative change of microbial population sizes among operational taxonomic units (OTUs). For example, the transition of Firmicutes (mainly Lactobacillus crispatus and L. iners)-dominated microflora to Actinobacteria and Bacteroidetes-boosting microflora leads to vaginal dysbiosis and inflammation symptoms (Donders et al., 2002; Wang et al., 2020). However, results from cultivation studies show that the most common AV-related bacteria may also be represented by other types of microbes, such as Streptococcus agalactiae, Staphylococcus aureus, Enterococcus faecalis, coagulase-negative staphylococci (e.g., S. epidermidis), and Escherichia coli (Donders et al., 2002, 2017; Fan et al., 2013; Tang et al., 2020). In clinics, some patients met the diagnostic criteria of AV, but no underlying pathogens were identified by cultivation (Donders et al., 2002), making therapeutic intervention less effective (Donders et al., 2017). This suggest that AV is not merely related to a few aerobic microbes, but rather involves multiple bacteria that interact with each other to form intricate but well-orchestrated networks. There is a wealth of literature on the methodological development of microbial interaction networks (Proulx et al., 2005; Faust and Raes, 2012; Vidanaarachchi et al., 2020; Matchado et al., 2021). Correlation-based networks can characterize the strength of interactions, but fail to identify the causality of interactions (Steuer et al., 2002). Bayesian networks are directed graph models, with the power to detect the causality of interactions but cannot determine the sign of the causality (Friedman et al., 2000). Most of these approaches can only reconstruct an overall network from a large number of samples, failing to characterize sample-sample heterogeneities (Kuijjer et al., 2019). Dynamic networks can capture the full information of network structure and function (Gardner et al., 2003; Sontag et al., 2004; Bansal et al., 2006; Srividhya et al., 2007; Wu et al., 2014; Chen et al., 2017), but their use in practice is impaired by the unavailability of temporal or perturbed data. Wu and team have developed a series of statistical models for inferring informative, dynamic, omnidirectional, and personalized networks (idopNetworks) from static abundance data (Chen et al., 2019, 2022; Griffin et al., 2020; Wu and Jiang, 2021). Chen et al. (2019) examined the statistical behavior of idopNetworks and their application condition. More recently, idopNetworks have been applied to predict neuroblastoma risk from a complete set of genes (Sun et al., 2020) and characterize cell crosstalk across fetal germs and their microenvironment (Wang et al., 2022), overcoming the limitation of individual genes as predictors. These networks can chart how genes are co-expressed differentially across tissues to affect human health (Wu and Jiang, 2021). Taken together, idopNetworks have emerged as a generic tool to characterize detailed topological changes in networks that describe complex systems. In this article, we modified and implemented our network tool to reconstruct microbial interaction networks for the vaginal microbiota and reveal how microbial interactions are causally related to AV. We analyze a data set collected from a well-designed AV case-control study (Wang et al., 2020). The study monitored microbial abundance profiles from the vaginal microbiota of AV patients and healthy individuals by 16S rRNA gene sequencing. We characterized topological factors that distinguish AV-related microbial networks from healthy networks and analyzed networks changes across pH gradients. Beyond the phenomenological investigation of microbe-AV relationships based on individual microbes, our networks provide a systematic, mechanistic dissection of these relationships.
A study was initiated to assess the vaginal microbial profiles of AV patients compared with healthy individuals (Wang et al., 2020). The study includes a total of 240 participants, i.e., 80 gynecological (AV) outpatients (as the cases) at Tianjin Medical University General Hospital, China, and 160 healthy women (as the controls) who received routine examinations at the same hospital during the same period. There were strict scientific and ethical criteria for selecting these participants, as detailed in Wang et al. (2020), where the participants’ sociodemographic factors and multifaceted life behaviors were also obtained. The majority of the participants had information about the phase of their menstrual cycle; the phase-identified participants from each category (cases and controls) were classified into two groups, one in the proliferative phase (45 cases and 82 controls) and the second in the secretory phase (34 cases and 72 controls). The pH level of the vagina is associated with its healthy state; a normal vaginal pH value is between 3.8 and 4.4, whereas a pH value beyond 4.4 is abnormal for the vagina. We classify all participants including the cases and controls into three groups, one with pH = 3.8 (133 subjects), the second with pH = 4.0–4.4 (41 subjects), and the third with pH 4.6–5.4 (66 subjects). The second group is intermediate between normality and abnormality. Wang et al. (2020) described a detailed procedure for assessing vaginal microbial profiles at operational taxonomic units (OTUs) for the participants by 16S rRNA gene sequencing. By a series of bioinformatics and statistical analysis, the microbiota in the vagina were identified at different taxonomical levels from phyla to classes to orders to families to genera to species. At each level of taxa, there exist some missing microbes whose abundance was zero. We exclude these microbes from network modeling.
Chen et al. (2019) proposed a computational model for recovering idopNetworks from gene expression data. We modify this model to learn microbial interaction networks from static abundance data of vaginal microbes. Suppose there are m microbes that are measured in the vagina of each participant, regardless of its category from the cases or controls. We assume that these microbes constitute a dynamic system in which microbe-microbe interactions change from one participant to the next. Let gji denote the abundance level of microbe j in participant i and define as the habitat index (HI) of this participant. It can be seen that gji and Ei establish an allometric part-whole relationship, which can be quantified by a power equation, expressed as where αj and βj are the proportionality coefficient and allometric exponent of the power equation for microbe j existing in participant i. Since gji is expressed as a function of Ei, we use gj(Ei) in place of gji. Parameters αj and βj determine how microbe j changes its abundance level across participants. We argue that the pattern of microbial interactions in a system can be interpreted through lens of evolutionary game theory. In the interactive system, a microbe attempts to maximize its abundance and fitness based on its intrinsic capacity and the strategy of other microbes that interact with it (Wu and Jiang, 2021). This attempt continues until a Nash equilibrium is reached. Allometry scaling theory in equation (Marconi et al., 2013) formulates a basis of expanding evolutionary game theory into its quasi-dynamic representation by which the pattern of how different microbes interact with each other across participants can be characterized. This representation can be expressed as a system of quasi-dynamic ordinary differential equations (qdODEs), i.e., with the time derivative replaced by the HI derivative, where describes the (independent) abundance level of microbe j when it is assumed to be in isolation and describes the (dependent) expression level of microbe j regulated by microbe j′. The HI-varying independent abundance level can be fitted by power equation (Marconi et al., 2013) with parameters ϕj, whereas the dependent abundance level is fitted by a non-parametric approach with parameters ϕj←j’. We code independent components of each microbe as nodes and dependent components of each pair of microbes as edges in a (mathematical) graph (network) so that a causal network can be reconstructed. Since dependent components can be positive or negative, the networks reconstructed from equation (Han et al., 2019) can reveal the causality of microbial interactions.
Given that living systems are usually not fully interconnected in order to buffer against environmental stochasticity (May, 1973; Gravel et al., 2016), the microbial networks to be reconstructed should be sparse (Goyal et al., 2022; Yonatan et al., 2022). There are two strategies that can be used to ensure network sparsity. The first is to implement variable selection into a regression model built on the basis of equation (Han et al., 2019), by which a small set of the most significant microbes that link with a given microbe are chosen. Through this variable selection, m summations of dependent components for microbe j, as shown in equation (Han et al., 2019), are reduced to dj summations, because the microbe j is found to link with only dj (dj < < m) other microbes. We then solve this reduced system of qdODEs. The second strategy is to cluster all microbes into distinct modules each with a smaller number of microbes that are more strongly linked with each other than with those from different modules. Such network decomposition is consistent with developmental modularity theory, widely recognized to explain a living system’s stability and robustness in response to environmental change. We implement the procedure of the first strategy to reconstruct sparse networks for each module. Meanwhile, by taking and using the mean abundance level of all microbes within each module, we can identify the networks among modules. By linking a module-module network and its descent microbe-microbe networks, we can reconstruct multilayer and multiplex microbial idopNetworks.
We formulate a likelihood of microbial abundance data to solve qdODEs for network reconstruction (Wu and Jiang, 2021). Let yj = (yj(E1),…,yj(En)) denote a vector of abundance data for vaginal microbe j (j = 1, …, m) measured for n different samples (participants). We assume that m microbes interact with each other across samples in a way described by qdODE-based evolutionary game theory. The likelihood of the microbial data measured from n samples is written as where fi(⋅) is an m-variate longitudinal normal probability function with mean vector and covariance matrix Σ. Explicitly, we write the mean vector as where μj (Ei) is fitted by a system of qdODEs in equation (Han et al., 2019). These equations represent a fully interconnected network model, stating that each microbe is linked with all other m – 1 microbe. However, as mentioned above, such a full network is thought to be vulnerable, whereas a sparsely interconnected network can better buffer against stochastic perturbations (May, 1973; Gravel et al., 2016; Goyal et al., 2022; Yonatan et al., 2022). Through variable selection, we choose a small set of the dj most significant microbes that are linked with a given microbe j as a node in the network. Thus, the full model of equation (Han et al., 2019) reduced to a reduced model in which a microbe j is only linked with a small number of microbes. We implement a non-parametric approach to model the independent and dependent components, specified by ODE parameters ϕj and ϕj←j′ (j = 1, …, m; j′ = 1, …., j – 1, j + 1, …, m) for the reduced qdODEs. The covariance matrix has a symmetrical structure as follows: where the covariance matrices for microbe j and between microbes j and j′ across samples are expressed as Since each sample represents an independent subject, it is reasonable to assume that measurement errors of the same microbes or different microbes are independent among different samples. Under this assumption, matrices in Equations 5A, 5B can be simplified as diagonal matrices in which the elements off the main diagonal are all zero. Meanwhile, we assume that residual variances for microbe j and residual covariances between microbe j and j′ are constant across samples. Thus, the covariance matrix of equation (Fan et al., 2013) only contains two types of parameters (j = 1, …, m) and σjj′ (j′ = 1, …., j − 1, j + 1, …, m). By maximizing the likelihood, we implement the fourth-order Kutta-Runge algorithm in the estimation of all qdODEs that explain microbe-dependent independent and dependent abundance components and microbe-dependent residual variances and covariances. Such networks inferred from maximum likelihood estimation are stable in network topology. The maximum likelihood estimates (MLEs) of dependent abundance levels of one microbe regulated by another microbe are encapsulated in the idopNetwork, filled with bidirectional, signed, and weighted interactions and characteristic of each participant.
The above procedure was used to reconstruct microbial idopNetworks in different contexts, e.g., healthy group vs. AV group, proliferative group vs. secretory group, and groups across pH gradient, etc., and allow context-known networks to be tested and compared. Consider C contexts of interest for network comparison. Let ycj = (ycj(Ec1),…,ycj(Ecnc)) denote a vector of the abundances of vaginal microbe j (j = 1, …, m) measured for cn different samples from context c (c = 1, …, C). Under the assumption of independence in measurement error among different contexts, we formulate a joint likelihood of the microbial data measured in all C contexts as where fci(⋅) is an m-variate longitudinal normal probability function with mean vector μc = (μc1,…,μcm) and covariance matrix Σc from context c. It is straightforward to solve the likelihood (Wang et al., 2020) by implementing the algorithmic procedure described in the previous section, including estimating the MLEs of qdODE parameters ϕcj and ϕcj←j′ (j = 1, …, m; j′ = 1, …., j – 1, j + 1, …, m; c = 1, …, C) that model independent and dependent abundance components in context c, respectively. Consider two different contexts, c1 and c2 (c1≠c2 = 1,…,C), under which microbial networks are reconstructed. To test whether the overall link structure of these microbial networks is context-dependent, we formulate the following hypotheses: simultaneously for all j = 1, …, m; j′ = 1, …., j – 1, j + 1, …, m. Under the null hypothesis, the strength and direction of links between the same pair of microbes j and j′ are identical between contexts c1 and c2. We calculate the log-likelihood ratio as the test statistic using likelihood values under the null and alternative hypotheses and compare it against the critical threshold determined from permutation tests. Likewise, we can test whether a specific link between microbes j and j′ is context-dependent by formulating a similar hypothesis procedure. We compare and test the differences in microbial network structure between the healthy and AV groups, between proliferative and secretory phases, and between different pH value levels. From these tests, we find key interaction links that determine context-dependent differences. These links can serve as a mechanistic predictor of AV risk.
The vaginal tract is viewed as an ecological habitat that is colonized by the microbiota. The sum of abundance of all microbes in a vagina, define as the habitat index (HI), may reflect the ecological carrying capacity of the vagina. We calculate the HI of each sampled vagina using Wang et al.’s (Wang et al., 2020) case-control microbial data involving AV patients (n = 80) and matched healthy subjects (H) (n = 160). We find that the HI value is slightly smaller in the AV group than in the H group (Figure 1A). Large pH values in vagina are thought to be associated with the degree of AV (Amabebe and Anumba, 2018; Lin et al., 2021). The HI decreases fairly remarkably from pH = 3.8 (healthy state) to pH = 4.6–5.4 (diseased state) (Figure 1B). At a middle range of pH (4.0–4.4) where both H and AV groups carry, the HI does not much differ between the two groups, but the AV group is considerably more variable than the H group. The HI at the proliferative phase is larger for the healthy group than AV group, but the HI of two groups tends to be convergent at the secretory phase (Figure 1C). In summary, total vagina microbes change from a healthy state to an AV state, but this change depends on the physiological states of vaginas. A more mechanistic understanding of this context-dependent change is sorely needed.
Taxonomic microbes: The abundance level of individual microbes establishes a part-whole relationship with HI across samples. This relationship obeys a physical principle that can be fitted by the allometric scaling power equation. As such, we can express the abundance values of individual microbes collected in discrete samples as a quasi-dynamic function of HI (Griffin et al., 2020). We choose the richest 17 identified phyla and a mix of unidentified phyla (denoted as others) for data modeling and analysis. Among all the phyla studied, only Firmicutes is more abundant and also increases its abundance with HI at a greater slope in the healthy group than in the AV group (Figure 2A). The abundance of Actinobacteria, Bacteroidetes, Fusobacteria, and Tenericutes is much richer in the AV than healthy group; the abundance of the first three phyla increases with HI in the AV group but decreases with HI in the healthy group, whereas the abundance of Tenericutes decreases its abundance with HI in both groups. We further plot the abundance of individual genera against HI, which is also found to obey the power equation (Figure 2B). Genus Lactobacillus from Firmicutes has greater abundance and also increases its abundance with HI in the healthy group than in the AV group. All other genera are either much more abundant over a full range of HI in the AV and healthy group, such as Streptococcus and Aerococcus from Firmicutes and Gardnerella, Atopobium, and Prevottella from the other phyla, or are consistent between the two groups. Overall, only Lactobacillus is a favorable genus, contributing to maintaining a healthy vaginal ecosystem. Functional microbes: We classify 104 identified genera and a mix of other unidentified genera into 12 modules M1–M12, each composed of functionally similar genera (Figure 3A and Supplementary Table 1). We find that M8 only contains Lactobacillus, confirming that this genus functions differently from other genera. In general, only three modules (25%), i.e., M2, M5, and M6, cannot be used to distinguish healthy and AV groups, whereas as many as 75% of modules can serve as predictors of AV risk, the majority of which are more abundant in the AV group than in the healthy group. From the plots of the abundance of the five richest species from Lactobacillus over HI, we find that only L. crispatus is favorable for a healthy vaginal ecosystem over a full range of HI, whereas L. iners, L. jensenii and L. johnsonii are favorable only at a high level of HI (Figure 3B). An imbalance in vaginal microbial ecosystems can cause the alternation of pH values, thus, varying pH values may be associated with AV (Lin et al., 2021). We classify the pH-AV association into three categories, pH = 3.8 (healthy, n = 133), 4.0–4.4 (sub-healthy, n = 44), and 4.6–5.4 (AV risk, n = 66). We implement three-variate functional clustering (Wang et al., 2012) to classify 105 genera into distinct functional modules based on the similarity of how genera change their abundance with HI jointly under three categories of pH levels. We identify 13 modules, each containing a different number of genera and with a different HI-varying pattern (Figure 3C and Supplementary Table 2). All modules, except for M9, regardless of their number of genera, are more abundant in the AV category than in healthy and sub-healthy categories. Module M9 is only composed of one genus Lactobacillus, further confirming that Lactobacillus plays an important role in improving vaginal microbial ecosystems.
Individual microbes can serve as a predictor of AV risk at different levels of taxa and in terms of their functional discrepancies. The mechanistic role of individual microbes as a predictor can be better understood through microbial interaction networks. We reconstruct idopNetworks at the taxonomical (phylum) and functional levels. Taxonomic networks: Figure 4 illustrates 18-node bacterial idopNetworks among phyla for healthy and AV groups. We find that the taxonomic networks display remarkable discrepancies in topological architecture between the two groups (Figure 4A). The healthy network appears to be denser than the AV network due to a higher number of relatively weak links, suggesting that a healthy vagina can maintain a better balance between system function and stability. The number of outgoing links exerted by each phylum and the number of incoming links received by each phylum differ dramatically between the two networks (Figure 4A). These differences are also expressed in the relative number of positive and negative outgoing regulation and the relative number of positive and negative incoming regulation interactions for each phylum. For example, although existing as a predominant phylum in both networks, Firmicutes more actively regulates other phyla, with the number of outgoing links being up to one time larger in the healthy group than in the AV group. In particular, Firmicutes inhibits Bacteroidetes in the healthy vagina but promotes Bacteroidetes in the AV vagina. Actinobacteria is inhibited by only one phylum in the AV group but by many phyla in the healthy group. Comparative analysis based on power fitting of Figure 2 shows that the transition of healthy to AV states is associated with increasing quantities of phyla, such as Actinobacteria, Bacteroidetes, Fusobacteria, and Tenericutes. It is interesting to note that these phyla each have a much higher level of independent abundance in the AV than in the healthy group (Figure 4B). Cyanobacteria and Acidobacteria are expressed, to a similar extent, in the healthy and AV groups, suggesting that they are neutral to health state. Yet, the independent abundance level of these two phyla reduces considerably from healthy to AV states. The level of independent abundance of a microbe is directly related to its intrinsic capacity, determining its fitness in a condition where it cannot derive any resources from its peers. Phyla Actinobacteria, Bacteroidetes, Fusobacteria, and Tenericutes are inhibited by far fewer phyla in the AV group than in the healthy group (Figure 4B), which increases the likelihood that these microbes cause AV risk. On the other hand, phyla Cyanobacteria and Acidobacteria are promoted by many more phyla in the healthy group than in the AV group, increasing their capacity to maintain favorable ecological homeostasis in a healthy vagina. Taken together, idopNetworks provide a mechanistic interpretation of how different microbes at the phylum level interact with each other to bring about changes in the vaginal ecosystem from an eubiosis state to dysbiosis and vice versa. Functional networks: Figure 5 shows 12-node idopNetworks with functional modules for the healthy and AV groups. Considerable differences are found in network topology between two groups (Figure 5A). Module M8, composed of only genus Lactobacillus [predominant lactic acid bacteria (Gustafsson et al., 2011; Valenti et al., 2018)], has a considerably higher intrinsic capacity (described by independent abundance) than all other modules in the H network, but its intrinsic capacity reduces dramatically in the AV network. Unlike module M8, the intrinsic capacity of many other modules, especially M4, M6, M7, and M11, displays a pronounced increase from a healthy state to an AV state. The H network has two leaders, M1 and M11, which exert many outgoing links to other modules, but such leaders do not exist in the AV network (Figure 5A). Figure 5B shows the decomposition of the net HI-varying change curve into its independent and dependent component curves for each module. M2, M5, and M6 have similar observed microbial abundance between different health states, but their underlying ecological mechanisms are different. M2 is promoted by M1 in the healthy group, but in the AV network although the former is still promoted, even to a larger extent, by the latter, the large independent component of M2 is counteracted by strikingly strong inhibition from M5. Similar interpretations can be made for M5 and M6. The decomposition curves of Figure 5B can also mechanistically explain the reason why eight modules each have increased abundance in the AV group when compared to the healthy group. For example, M1 is activated by AV disease, displaying a large independent component, thus although it is inhibited by M5 and L. crispatus, its net abundance is still quite remarkable in the AV group. The independent component of M4 is strikingly larger in the AV group than in the healthy group, but although promoted by M11 in the AV group and inhibited by L. crispatus in the AV group, the net abundance of M4 is still much larger in the latter than in the former. Although M12’s independent component is virtually very large in the healthy group, its net abundance is reduced because it is strongly inhibited by other modules. Yet, despite its smaller independent component in the AV group, M12 is promoted by M1 and inhibited by M5, ultimately making M12’s net abundance level larger in the AV group than in the healthy group. As an important module, M8, i.e., Lactobacillus, we identify its five most abundant species to characterize the detailed role of each species in mediating network change from a healthy state to an AV state. As seen from the power fitting (Figure 3B), L. crispatus is consistently much richer in the healthy group than in the AV group. The independent abundance level of all species, especially L. crispatus and L. iners, reduces from a healthy state to an AV state, showing that these species participate in shifting vaginal ecosystems from symbiosis to dysbiosis (France et al., 2016). In the H network, all five species are promoted or inhibited by other modules, and none of them exerts outgoing links, neither to each other nor to any other microbes from other modules (Figure 5A). Just as Lactobacillus is one of the most important genera (Wang et al., 2012; Valenti et al., 2018), L. crispatus is one of the most important species in this genus, which is favorable to maintain a heathy vaginal state (France et al., 2016). It is interesting to note that L. crispatus is only one abundant species that establishes a mutualistic cycle with module M11. Although M11, containing nine infrequent genera, Enterococcus, Gemella, Mageeibacillus, Megasphaera, Mycoplasma, Peptoniphilus, Phyllobacterium, Staphylococcus, and Veillonella, are unfavorable to vaginal health, it simultaneously serves as an inhibitor of other unfavorable microbes (such as M3, M6, M10, etc.) and as a promotor of the favorable L. crispatus in the H network (Figure 5B). In the AV network, none of Lactobacillus’ five species receives any incoming link from other modules, suggesting that their growth is purely dependent on their own capacity. L. crispatus is a primary leader, exerting numerous outgoing links, not only in the subnetwork composed of its species counterparts, but also in the entire functional network (Figure 5A). Because of these multifaceted roles in the AV network, L. crispatus’ capacity to exploit and digest resources for symbiotic maintenance is largely weakened. This may be one important cause or consequence of AV. In addition, L. crispatus, as a favorable species, unexpectedly promotes some unfavorable modules, such as M6 and M9 (Figure 5B) and also promotes its peers L. iners and L. gasseri. As seen from the power fitting (Figure 3B), these peers are not always favorable for healthy vagina ecosystems; in some cases, they are positively associated with AV. The independent abundance of L. iners is reduced in the AV group, but its observed abundance is augmented from promotion from L. crispatus (Figure 5B). L. jensenii, L. gasseri, and L. johnsonii each are promoted by M1, but inhibited to a larger extent by M11, in the H network, whereas each of these three species is only promoted by L. crispatus in the AV network (Figure 5B).
We reconstruct 13-node functional idopNetwork for different pH categories (Figure 6). Each module displays pH-dependent differences in HI-varying abundance curves (Figure 3C), and these differences can be mechanistically explained by the networks (Figure 6A). For example, M11-M13 are observed to be much more abundant in the AV category than in healthy and sub-healthy categories. From the decomposition curves of Figure 6C, we can see that some of these differences (such as M7, M10, M12 and M13) are due to increasing independent abundance when the vaginal environment becomes alkaline, whereas some, such as M2, M3, M5, M6, M8, and M11) result strong inhibition from other modules in healthy and sub-healthy vaginas. It is interesting to see that as a whole, Lactobacillus has a greater independent component in the AV category than in the healthy category (Figure 6C). However, Lactobacillus is more strongly promoted by M7 in the healthy vagina than M12 in the diseased vagina, making its overall abundance level higher in the former than in the latter. During the transition from healthy to sub-healthy state, the intrinsic reproductive capacity of Lactobacillus is strengthened, but because of new inhibition from M12, its overall abundance is reduced. Considering their unique role in transiting the vagina from a healthy state to AV by maintaining its subacidity, we choose the five most abundant species of Lactobacillus, including L. crispatus, L. iners, L. gasseri, L. jenseni, and L. johnsonii, to be added into pH-varying functional networks (Figure 6A). We analyzed the proportions of these five species (Figure 6B). L. crispatus and L. iners are two predominant species, together occupying 90% of genus Lactobacillus in the healthy vagina, but they are different in the isomers of lactic acid they produce as end products of fermentation. L. crispatus can produce L- and D-lactic acid, whereas L. iners can only produce L-lactic acid (Amabebe and Anumba, 2018). We find that from categories 1 to 3, L. crispatus decreases its population proportion in order: 63.3%—37.4%—25.0%, whereas L. iners increases its population proportion in order: 33.4%—55.4%—60.7%, suggesting that the relative abundance of these two species is a predictor of AV risk. An increasing proportion of L. crispatus to L. iners is favorably associated with the healthy state. It is possible that 61% is a threshold for reciprocal transition between health and AV; i.e., if L. crispatus reaches 61% or higher of the microbiotic population, the vaginal microbiota maintains a healthy environment, whereas if L. iners reaches 61% or higher, the vaginal microbiota are dysbiotic. Why does the relative proportion of L. crispatus vs. L. iners decrease from the healthy to the AV state? This can be explained from the structure of idopNetworks. L. crispatus exhibits a larger independent component in a more acid vagina than in a more alkaline vagina, whereas an inverse pattern is found for L. iners (Figure 6C). Although L. crispatus in both conditions is promoted by a module, M11 in the acid condition and M12 in the alkaline condition, this regulation is unidirectionally commensalistic in the former but bidirectionally altruistic/predatory in the latter. Thus, while M12 leads to the increasing abundance of L. crispatus, this increase quickly inhibits the existence of M12, reducing its capacity to promote L. crispatus. Although L. iners receives promotion from a module in both conditions, it is simultaneously inhibited by the other module in a healthy vagina. For this reason, the increase of L. iners’ abundance is limited when vaginal pH level is more acid.
A women’s menstrual cycle includes three cyclic stages, the follicular phase, the luteal phase, and the menstrual phase with different endometrial characteristics in each phase. The follicular and luteal phases are facilitated by follicle-stimulating hormone (FSH) and luteinizing hormone (LH), respectively. In the follicular phase, estrogen-dominant hormone mediates the regeneration of the functional layer of the endometrium, whereas progesterone drives the endometrium to undergo various changes in preparation for embryo implantation in the luteal phase. These phase-dependent hormonal changes in the endometrium are associated with alterations in the proportion of different types of immune cells (Figure 7). We find that microbiota flora in the vagina vary with the shift of physiological states in the upper reproductive tract (Figure 7). We reconstruct idopNetworks among the nine most abundant species of Lactobacillus with all remaining microbes treated as others in the vagina, separately for the follicular and the luteal phases (Figure 7). In the healthy group, the interconnection density of the network composed of Lactobacillus species reduces dramatically from follicular to luteal phases, whereas such a phase-dependent change does not occur for the AV group. At the follicular phase, the intrinsic capacity of L. crispatus to expand its abundance reduces from the healthy to the unhealthy state, but this capacity stays stable over health state in the luteal phase. L. iners reduces its intrinsic capacity for reproduction from the healthy to unhealthy states to a greater extent at the luteal than the follicular phases. For the healthy group, L. crispatus reduces its intrinsic growth capacity, accompanied by the increase of L. iners’ intrinsic capacity, from follicular to luteal phases, whereas the intrinsic growth capacity of both species does not markedly change between two phases. Taken together, the Lactobacillus network is more adaptive in its overall topological features, especially the relative intrinsic growth capacity of two key species, L. crispatus and L. iners, as a function of physiological change to the endometrium. Also, while L. crispatus is a key determinant of AV risk at the follicular phase, this determinant is replaced by L. iners at the luteal phase.
Most studies linking microbiota with natural and health processes focus on comparing the relative abundance of individual microbes between different regimes. By comparing the 10 major phyla identified in the vaginal ecosystem, Wang et al. (2020) found that Firmicutes (mainly Lactobacillus) predominates the vaginal microbiota in healthy women, whereas Actinobacteria and Bacteroidetes became much more abundant in women infected by AV. The role of Lactobacillus is speculated to include a reduction in the microenvironmental pH level, generating various bacteriostatic and bactericidal compounds, and competitively excluding other bacterial species (Wang et al., 2012; Valenti et al., 2018). However, without a mechanistic picture of how individual microbes, such as Firmicutes, Actinobacteria, and Bacteroidetes, mediate the occurrence of AV, the precise treatment of this disease by probiotic supplementary agents remains problematic. For example, L. crispatus is a key species of Lactobacillus to meliorate vaginal dysbiosis, but because it also plays a role in inhibiting the other microbes that promote it, probiotics containing this species requires a balance of multiple microbes to maximize its efficacy. In this article, we used a powerful network tool to dissect how each microbe interacts with all other microbes to determine AV, providing a unique way to predict AV risk by understanding the mechanisms underlying microbiota-host crosstalk. Our predictive model includes three hierarchical stages, the calculation of HI, allometric scaling fitting of individual microbe levels, and microbial interaction modeling by qdODEs. As compared to the healthy group, the HI of the AV group reduces only slightly (Figure 1), suggesting that the reduction of the total amount of microbes does not fully reflect AV risk. Yet, by plotting the abundance level of individual microbes at a specific level of taxon, a set of microbes that distinguish between healthy and AV groups can be identified. For example, Firmicutes are more abundant in healthy women than in non-healthy women, whereas Actinobacteria, Bacteroidetes, and Fusobacteria display an increasing abundance level in AV women (Figure 2A). The association between microbial abundance and AV can be more clearly dissected at the genus level; a striking decrease in Lactobacillus and a striking increase in multiple aerobes, such as genera Streptococcus, Aerococcus, etc., accompanies the onset of AV (Figure 2B). Taken together, the slope of allometric scaling curves for certain genera that change their abundance with HI can be used as a powerful predictor of AV risk. The network model provides a mechanistic understanding of predictive models for AV risk. In a highly dense bioenvironment, such as the vagina, the function of any single microbe is regulated by other microbes (Ravel et al., 2011; Chen et al., 2021). The transition of vaginal microflora from a healthy (symbiotic) to abnormal (dysbiotic) state is not only characterized by the change of abundance of individual key microbes, but also through the interaction networks of all microbes as a cohesive whole. We reconstruct taxonomic microbial networks using natural taxa of microbes as network nodes, and find that microbes are not fully interconnected at the phylum level in symbiotic vaginal microflora, but with a density being higher than that in dysbiotic vaginal microflora (Figure 3). There has been a long debate on the complexity-stability relationship in living systems (Valenti et al., 2018). One view suggests that complex communities enhance community stability (Gravel et al., 2016; Goyal et al., 2022; Yonatan et al., 2022). However, this view is challenged by May (1973) who used mathematical models to find the positive association of community destabilization and complexity. The two distinct views implies that community stability is not related to community complexity in a linear way, rather their relationship is non-linear. The non-linear hypothesis well explains our discovery; i.e., microbial links in the vaginal ecosystem are maintained at a threshold level, below or above which vaginal microflora becomes abnormal. More importantly, we find that the decrease of abundance in Firmicutes is due to both intrinsic and extrinsic factors, i.e., the reduction of its carrying capacity (described by the independent component) and the negative regulation it receives from Actinobateria (described by the dependent component) (Figure 3B). In the healthy vagina, Firmicutes is simultaneously upregulated and downregulated by multiple microbes and, ultimately, receives slight overall positive regulation after regulation in different signs is canceled out. This result suggests that, in order to improve the dysbiosis of vaginal microflora through Firmicutes, using probiotics that only contain this microbe is not sufficient, rather it should be mixed with positive regulators. Several phyla, such as Actinobacteria, Bacteroidetes, Proteobacteria, etc., are activated, exhibiting a striking increase in their independent abundance, in abnormal vaginal microflora. As such, specific medications can be designed to control these microbes so that the vaginal microenvironment can be improved. This strategy may be efficient for Actinobacteria because it is only downregulated by Firmicutes. Yet, for the other phyla that are regulated by many different regulators, special attention should be paid to the development of medications that can balance the co-occurrence of the regulatees and regulators. For example, to control Proteobacteria, its positive regulators, Cyanobacteria and Verrucomicrobia, should be controlled simultaneously as a whole. Our idopNetworks chart a detailed roadmap of how and how much each phylum regulates, or is regulated by, every other phylum across subjects (Figure 3B), from which an optimal strategy to control specific microbes can be designed and delivered. We have also reconstructed functional microbial networks with functional units as network nodes. Although microbes from different taxa are phylogenetically different, they may perform a similar function. This allows us to classify different microbes at different taxonomic levels into distinct functional modules. This classification has two advantages. First, functional networks based on these modules can better explain the mechanistic relationships among microbes and their impact on disease outcome. Second, it provides a way to reconstruct networks from high-dimensional data. Clinically more useful and informative microbial networks should be reconstructed with a fine-grained unit, such as genera, species, strains, or even genes. However, such networks will have too many nodes to be coded into a graph because of computational burden and instability. The classification produces different modules, each with a smaller number of entities that make it possible to reconstruct networks. We classify 104 genera identified in healthy and AV-infected women into multiple modules based on the similarity of HI-varying abundance change over different health states and vaginal pH gradients. Results from functional networks based on modules well support those from taxonomic networks at the phylum level, while providing an additional insight into microbial interactions and their impact on AV risk. In both functional clustering over health states (heathy vs. AV) and pH levels (low, middle, high), genus Lactobacillus is always attributed to a module that only is composed of this genus itself. This result reflects the unique role of Lactobacillus, one of the most often found inhabitants in the vagina, in maintaining the vaginal ecosystem of healthy women (Goyal et al., 2022). Analysis of functional networks shows that Lactobacillus is positively regulated by the other functional module containing many species in healthy vaginal microbiota, but this positive regulation is largely weakened when the vaginal ecosystem become dysbiotic (Figure 6). This is accompanied by an increasingly alkaline microenvironment. By further dissecting the role of Lactobacillus at its species level, this genus is predominated by two species, L. crispatus and L. iners, with its next three most abundant species together accounting for only 3–14% of the total population (Figure 6B). Compared to L. iners, L. crispatus has a greater capacity to produce lactic acids that break down carbohydrates for energy when oxygen levels are low. In healthy vaginas, L. crispatus is promoted by other microbes, but it turns to serve as a regulator to regulate other microbes, even including promoting unfavorable microbes, in less healthy vaginas. These multiple tasks would, with no doubt, affect its role in maintaining vaginal symbiosis. Also, when a vagina becomes alkaline, L. crispatus receives positive regulation from, but while exerting inhibition to, the same microbial module. This aggressive/altruistic relationship forms a paradox for L. crispatus’ role in maintaining vaginal symbiosis and alleviating vaginal dysbiosis. In the healthy vagina at a normal pH, L. crispatus and L. iners account for about 63% and 33% of the total genus mass, respectively. This relative proportion is in agreement with the golden dissection hypothesis of animal conflict (Wang et al., 2019; He et al., 2021; Wu et al., 2021), with which the more abundant L. crispatus (>61%) tends to cooperate with the less abundant L. iners (<38%). This relative proportion changes when pH values increase. In the abnormal vagina at a more alkaline pH, the proportions of L. crispatus and L. iners become 25 and 60.7%, respectively. This proportion suggests the surrender-resistance hypothesis of animal conflict (Wu et al., 2021), by which the less abundant L. crispatus would be sacrificed if it chooses to cooperate, but it may benefit from its choice of conflict with the more abundant L. iners. In either case, the two species tend to compete against each other, leading the vaginal ecosystem to be dysbiotic (Pacha-Herrera et al., 2022). We report a detailed application of idopNetworks as a mechanistic predictor of AV risk. It is not surprising that this application produces interpretable results about the interactive mechanisms underlying the microecological balance of vaginal microflora, given that the development of this model was derived from the integration of biologically meaningful evolutionary game theory and prey-predator equation (Chen et al., 2019, 2022; Griffin et al., 2020; Wu and Jiang, 2021). The results from this application could be potentially more useful for the design of effective medications to treat AV when a more sample size is used and when more powerful statistical solution for curve fitting and stochastic modeling is developed. At the same time, the modified model can be used to reveal other microbial community assembly, such as the gut microbiota, and their impacts on health and natural processes.
The original contributions presented in the study are included in the article/Supplementary material, further inquiries can be directed to the corresponding authors.
The studies involving human participants were reviewed and approved by Tianjin Medical University General Hospital. The patients/participants provided their written informed consent to participate in this study.
QW, FX, and RW conceived of the project. AD and JZ performed data analysis. CW provided the data. ChG and ClG critically reviewed the manuscript. QW and RW wrote the manuscript. All authors contributed to the article and approved the submitted version. | true | true | true |
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PMC9631634 | 35819450 | Maximilian Englert,Katja Aurbach,Isabelle C. Becker,Annika Gerber,Tobias Heib,Lou M. Wackerbarth,Charly Kusch,Kristina Mott,Gabriel H. M. Araujo,Ayesha A. Baig,Sebastian Dütting,Ulla G. Knaus,Christian Stigloher,Harald Schulze,Bernhard Nieswandt,Irina Pleines,Zoltan Nagy | Impaired microtubule dynamics contribute to microthrombocytopenia in RhoB-deficient mice | 06-09-2022 | Key Points RhoB-deficient mice display microthrombocytopenia. RhoB exerts nonredundant functions in the megakaryocyte lineage compared with RhoA and regulates microtubule dynamics. | Impaired microtubule dynamics contribute to microthrombocytopenia in RhoB-deficient mice
RhoB-deficient mice display microthrombocytopenia. RhoB exerts nonredundant functions in the megakaryocyte lineage compared with RhoA and regulates microtubule dynamics.
Blood platelets are small anucleated cells with central roles in hemostasis and thrombosis. Platelets derive from large precursor cells called megakaryocytes (MKs) that are found predominantly in the bone marrow (BM). MK development is characterized by endomitosis (DNA replication without cell division) and cytoplasmic maturation, which comprises the biosynthesis of platelet-specific granules and the formation of an internal demarcation membrane system, which serves as a membrane reservoir for platelet production. Platelet biogenesis is a complex cellular process, initiated by mature MKs in direct contact with the BM sinusoids, and involves the penetration of endothelial cells and the extension of large cytoplasmic protrusions into the sinusoidal lumen, which are shed and undergo maturation in the peripheral circulation to become bona fide platelets. The different steps of platelet biogenesis highly depend on rearrangements of both the microtubule (MT) and actin cytoskeletal network. Consistently, genetic defects impairing cytoskeletal dynamics can result in thrombocytopenia, as shown in several human disorders and corresponding mouse models. Rho GTPases act as molecular switches by cycling between an inactive guanosine diphosphate–bound and an active guanosine triphosphate–bound state. In the latter conformation, Rho GTPases interact with multiple effector proteins to regulate diverse processes including cytoskeletal dynamics, cell adhesion, cell migration, cell polarity, cell cycle, and vesicle trafficking. Multiple members of the Rho family have been established as critical regulators of platelet production and function. We have previously reported that cytoplasmic MK maturation and subsequent transendothelial platelet biogenesis requires balanced signaling of RhoA and Cdc42. Mice lacking RhoA in the MK lineage display macrothrombocytopenia (decreased platelet count and increased platelet size), accompanied by a pronounced intravascular mislocalization of whole MKs. Higher vertebrates express 3 Rho subfamily proteins, RhoA, RhoB, and RhoC, which are 87% homologous on the protein level. RhoB is the most divergent member, with a unique C-terminal region containing a variety of lipid modifications, which result in its distinct localization to the plasma membrane, Golgi, and endosomes, in contrast to the other 2 members of this subfamily. In immune cells, RhoB is involved in cytokine trafficking and cell survival and affects cell adhesion and migration through β2 and β3 integrins but is not required for podosome assembly in macrophages. Although the role of RhoB in MKs and platelets remains unknown, its messenger RNA (mRNA) expression has been identified in both cell types. Human MKs, human platelets, and mouse platelets express RhoB mRNA at lower levels than RhoA or RhoC, whereas in mouse MKs, RhoB shows the highest abundance, followed by RhoA, and comparably lower levels of RhoC. The protein levels of RhoB in human platelets were detected at low levels, whereas in mouse platelets, it was shown to be more abundant than RhoC. In this study, we investigated for the first time the role of RhoB in platelet biogenesis and function by using constitutive knockout mice. We show that, in contrast to RhoA deficiency, loss of RhoB results in microthrombocytopenia, which was accompanied by moderately impaired platelet activation and thrombus formation in vitro. Our results demonstrate that RhoA and RhoB have specific, nonredundant functions in the MK lineage and point to selective defects in MT dynamics in RhoB-deficient mice as a potential mechanism underlying the manifestation of microthrombocytopenia in vivo.
Constitutive RhoB-deficient mice (further referred to as RhoB−/−) were kindly provided by Ulla G. Knaus. Animal studies were approved by the district government of Lower Franconia (Bezirksregierung Unterfranken).
Data presented are mean plus or minus standard deviation (SD). Unpaired, 2-tailed t tests or Mann-Whitney U tests were used to determine statistical significance between 2 groups with normal or nonnormal distribution, respectively. Sample size (n) and statistical significance are reported in the figures or figure legends. Asterisks indicate statistically significant differences compared with wild-type (wt) (*P ≤ .05; **P ≤ .01; ***P ≤ .001). Statistical analysis was performed in GraphPad Prism 9 (GraphPad Software). Platelet count and size determination, life span, integrin activation, degranulation, glycoprotein expression, aggregation, adhesion under flow conditions, spreading, cold-induced MT disassembly, tail bleeding time, intravital thrombosis model, immunofluorescence microscopy, direct stochastic optical reconstruction microscopy (dSTORM), transmission electron microscopy (TEM) analysis, MK isolation, immunoblotting, and materials are described in detail in supplemental Methods.
The consequence of the loss of RhoB in platelets and MKs was studied in constitutive RhoB−/− mice and compared with wild-type (wt) littermate controls. The complete loss of RhoB on the protein level was confirmed by immunoblotting in platelets and MKs (Figure 1A; supplemental Figure 1A). The protein levels of other Rho GTPases, namely RhoA, RhoC, Rac1, and Cdc42, remained unaltered in the absence of RhoB, indicating no direct compensatory regulation among these proteins. Using capillary-based quantitative immunoblotting, we found that human platelets expressed lower levels of RhoB protein, which is in agreement with the published proteomics data (supplemental Figure 1B). Although RhoA-deficient mice displayed a characteristic macrothrombocytopenia, loss of RhoB led to microthrombocytopenia (Figure 1B-D). RhoB−/− mice also exhibited a minor decrease in red blood cell counts, suggesting that the protein may play a role in the homeostasis of megakaryocytic and erythroid lineages (supplemental Table 1). The decreased platelet size of RhoB−/− platelets was confirmed by TEM analysis (Figure 1E-F). Resting wt platelets contain an average of 8 to 12 MT coils, known as the marginal band, which are located in the periphery to maintain their spherical shape. Notably, the number of MT coils in RhoB−/− platelets was significantly reduced (wt: 9.48 ± 1.86 vs RhoB−/− 7.43 ± 2.05 MT coils per platelet) (Figure 1E,G). The amount of α- and dense (δ-) granules observed in platelets, on the other hand, was not affected by loss of RhoB (supplemental Figure 1C-D). These results demonstrate that RhoB deficiency affects platelet count and size in vivo.
We next determined the characteristics and function of RhoB−/− platelets in vitro by flow cytometry. Levels of surface glycoproteins (GPs) were not significantly different between RhoB−/− and wt platelets, except for a 13% reduction in GPV and an 18% reduction in GPVI receptor levels (supplemental Table 2), which is likely related to the decreased platelet size. Platelet activation induces a shape change from a resting discoid to a spherical form. RhoA-deficient platelets showed selective defects in platelet activation and shape change after stimulation of agonists that induce G-protein–coupled (Gα13, Gαq) signaling pathways, which resulted in impaired intravital thrombosis and a mild bleeding phenotype. To investigate whether the absence of RhoB had a similar effect, we analyzed platelet shape change and compared it to RhoA-deficient platelets by light transmission aggregometry upon stimulation with a low dose of the stable thromboxane A2 analog U46619 (Figure 2A). Under these conditions, shape change in RhoB−/− platelets was unaltered, whereas it was abrogated in RhoA-deficient platelets, demonstrating that RhoB is not essential for Gα13 signaling. However, the aggregation of RhoB−/− platelets in response to intermediate concentrations of thrombin, U46619, or convulxin (CVX) was moderately reduced, indicating an overall impairment of platelet function (Figure 2B). To investigate these findings further, we next assessed platelet activation by flow cytometry, which allows for a more detailed analysis of platelet activation. In contrast to the reported selective defect of RhoA-deficient platelets in response to thrombin and U46619, RhoB−/− platelets displayed moderately reduced activation when stimulated with most tested agonists. The most profound defect was observed upon stimulation of the collagen/GPVI signaling pathway with either collagen-related peptide (CRP) or CVX (Figure 2C). This was consistent for both integrin αIIbβ3 activation (as assessed by JON/A phycoerythrin [PE] binding) as well as degranulation (as assessed by binding of anti–P-selectin fluorescein isothiocyanate [FITC]). We next measured adenosine triphosphate (ATP) release in lysates of activated platelets using a CellTiter-Glo assay. Although, there was no significant difference between the genotypes; RhoB−/− platelets display a tendency toward reduced ATP release in response to thrombin (supplemental Figure 2A). It was previously demonstrated that RhoB−/− macrophages displayed reduced level of β2 and β3, but not β1, integrins. Because murine platelets do not express β2 integrins, and the surface expression of β3 and β1 integrins was unaltered in RhoB−/− platelets (supplemental Table S2), we investigated platelet β1 integrin activation (as assessed by anti-integrin β1-FITC binding) by flow cytometry and found similar activation defects as for αIIbβ3 integrin activation (supplemental Figure 2B). We next performed in vitro flow chamber assays, in which anticoagulated whole blood was perfused over a collagen-coated surface at intermediate (1000 s−1) and high shear rates (1700 s−1). RhoB−/− platelets were able to form thrombi under these conditions; however, the overall surface coverage and relative thrombus volume were significantly reduced at intermediate shear compared with wt platelets (Figure 2D; supplemental Figure 2C), indicating that loss of RhoB predominantly affected the GPVI pathway, in accordance with our platelet activation results. To better understand the role of RhoB in GPVI signaling, we performed platelet activation using CVX (1 μg/mL, 90 seconds) in the presence of inhibitors of second-wave mediators followed by immunoblotting using an anti–phospho-tyrosine antibody. Under these conditions, we did not observe major differences in the pattern or intensity of proteins phosphorylated on tyrosine residues (supplemental Figure 2D). Rho-associated protein kinases (ROCK1 and ROCK2), downstream targets of Rho subfamily members, regulate myosin light chain 2 phosphorylation. Myosin light chain 2 phosphorylation was unaltered in platelets in response to thrombin stimulation, indicating that RhoA/RhoC can compensate for the loss of RhoB (supplemental Figure 2E). The platelet function defects observed in vitro in aggregation, integrin αIIbβ3 activation, P-selectin expression, and thrombus formation studies did not translate into altered arterial thrombus formation in vivo, which was determined by mechanical injury of the abdominal aorta (Figure 2E). Furthermore, unaltered tail bleeding times indicated that hemostasis was not affected in RhoB−/− mice (Figure 2F). These findings stand in contrast to the defects observed in RhoA-deficient mice in vivo and emphasize that RhoA and RhoB are involved in distinct platelet signaling pathways.
Decreased circulating platelet counts may manifest as a result of impaired platelet generation, an increased platelet clearance rate, or a combination of both. We have previously reported that macrothrombocytopenia in RhoA-deficient mice is associated with increased platelet turnover. We therefore investigated the lifespan of circulating, anti–GPIX-labeled platelets over a period of 5 days by flow cytometry (Figure 3A). We observed only a minor reduction of platelet lifespan in RhoB−/− mice, indicating that the reduction in platelet counts was predominantly caused by a direct impairment in platelet production. RhoB−/− mice exhibited splenomegaly; however, the overall spleen morphology and splenic MK numbers were unaltered (Figure 3B-D). Platelet removal was reported to be modulated by desialylation of surface glycoproteins, which, together with macrophage galactose lectin, mediate the clearance of platelets by Kupffer cells. However, RhoB−/− platelets did not show alterations in Erythrina cristagalli lectin binding to exposed galactose (Figure 3E) and in terminal galactose levels (determined by the ratio of neuraminidase treated platelets vs untreated platelets) (Figure 3F). The amount of RNA-rich, thiazole orange–positive platelets, an indicator of young, reticulated platelets, was not altered in RhoB−/− platelets (Figure 3G). Splenomegaly might be a consequence of the loss of RhoB in other cell types, potentially involving the erythroid lineage, as red blood cell numbers were significantly decreased (supplemental Table 1). Taken together, these results indicate that the thrombocytopenia in RhoB−/− mice was not caused by increased platelet clearance and suggest a direct role for RhoB in either MK maturation or platelet generation.
To better understand its role in MKs, we investigated the localization of RhoB in in vitro–differentiated mouse MKs and compared it to RhoA using immunofluorescence microscopy. We found a very distinct plasma membrane–associated staining for RhoB and a more diffuse, cytoplasmic staining for RhoA (Figure 4A), which is in agreement with their subcellular localization in other cells. Next, we stained healthy human bone marrow in situ for RhoB and found a similar staining pattern (Figure 4B).
We next investigated the outcome of RhoB deficiency on MK maturation and function. MK numbers in the BM compartment were unaltered (Figure 5A). Investigation of the ultrastructure of BM MKs by TEM in situ revealed a normally developed demarcation membrane system and granules in RhoB−/− MKs (Figure 5B). In situ analysis of BM cryosections stained for CD105 (vessel marker) and GPIX (MK marker) revealed that RhoB had no influence on MK localization near the BM vasculature with sinusoidal contact (Figure 5C-D). Together, these results indicated that RhoB is dispensable for MK maturation and localization in vivo. This is in marked contrast to the intrasinusoidal MK mislocalization observed in RhoA-deficient mice, which again emphasizes the differential functions of RhoB and RhoA not only in platelets but also in MKs. We next analyzed the ability of RhoB−/− MKs to produce proplatelets in vitro. For this, BM-derived MKs were cultured for 3 days in the presence of thrombopoietin and hirudin to induce proplatelet formation, followed by staining for F-actin and α-tubulin (Figure 5E-H). We could not detect alterations in the kinetics of proplatelet formation between enriched wt and RhoB−/− MKs during the observation time of 48 hours (Figure 5E), indicating that the ability to fragment into proplatelets was still preserved in the absence of RhoB. However, we observed an overall decreased proplatelet tip size and simultaneous unaffected F-actin distribution (Figure 5F,H; supplemental Figure 3A). Strikingly, a high proportion (78.1%) of proplatelets formed by RhoB−/− MKs displayed elongated irregular shaped proplatelet tips with a less uniform tubulin staining and/or enlarged proplatelet shafts compared with the wt, indicating a disorganized MT network in these cells (Figure 5G-H; supplemental Figure 3A). To assess the functionality of F-actin dynamics in the absence of RhoB, we additionally analyzed the characteristic integrin- and actin-dependent formation of podosomes in in vitro–differentiated MKs upon adhesion on collagen-coated cover slips. Importantly, podosome formation along collagen fibers, as well as the overall signal intensity of F-actin, was similar in wt and RhoB−/− MKs (supplemental Figure 3B-D). Consistently, and in contrast to observations of MKs with impaired podosome formation (eg, upon Cdc42 or Profilin-1 deficiency), loss of RhoB did not affect the total MK spreading area (supplemental Figure 3E). The distribution of tubulin was also not altered between wt and RhoB−/− MKs under these adhesive conditions (supplemental Figure 3F), pointing to a specific requirement of RhoB in the regulation of MT dynamics during proplatelet formation. Collectively, these results demonstrate that RhoB is a critical modulator of MT, but not actin dynamics, in MKs during proplatelet formation, which might provide an explanation for the decreased size of circulating RhoB−/− platelets in vivo.
To investigate the mechanism underlying the tubulin organization defect during proplatelet formation upon loss of RhoB, we investigated cytoskeletal regulators in MKs and platelets. Total protein levels of α-tubulin, and β1-tubulin, which is exclusively expressed in the MK lineage, were unaltered in BM-derived in vitro–differentiated MKs and platelets (supplemental Figure 4A). Protein levels of downstream effectors of the Rho subfamily proteins, such as nonmuscle myosin IIA and IIB, that influence cell migration and contractility, or the formins mDia1 and mDia2, which orchestrate actin and microtubule remodeling during PPF, were also unchanged (supplemental Figure 4B-C). Levels of the podosome-associated proteins vinculin and Arp2 were not affected, in line with functional podosome formation in RhoB−/− MKs (supplemental Figure 4B). The levels of the MT plus-end tracking proteins end-binding protein 3 and adenomatous polyposis coli (APC), which was recently reported as a negative regulator of proplatelet formation in mice, were also unchanged in RhoB−/− MKs (supplemental Figure 4A,C). These findings indicated that RhoB may directly influence MT dynamics during platelet formation. Due to the heterogeneity of MK cultures, we decided to focus on the circulating platelets (ie, the terminal in vivo products of proplatelet formation). We analyzed MT and F-actin distribution in adherent, spread platelets in vitro. For this, platelets were incubated for 5, 15, and 30 minutes on fibrinogen-coated cover slips, which induces extensive spreading of wt platelets that is supposed to be driven by integrin outside-in signaling. Similar to the normal adhesion and spreading of MKs on collagen, we did not observe a difference in the spreading kinetics between wt and RhoB−/− platelets (Figure 6A-B), indicating that integrin outside-in signaling is functional in the absence of RhoB. To analyze the distribution of F-actin and MTs, spread platelets were stained for α-tubulin and F-actin. In line with results from MKs, the distribution of F-actin was not affected by RhoB deficiency. Consistently, F-actin content and assembly upon CRP or thrombin stimulation were unaltered in RhoB−/− platelets (supplemental Figure 5A), emphasizing that RhoB is dispensable for F-actin dynamics in MKs and platelets. In contrast, the α-tubulin network appeared markedly altered in RhoB−/− platelets with shorter MT filament length (Figure 6C). We therefore next visualized the MT network in spread platelets by superresolution microscopy (dSTORM) as described previously. Indeed, we observed a high proportion of RhoB−/− platelets with fewer peripheral MT coils and an overall less dense MT network compared with the wt (Figure 6D; supplemental Figure 5B). These results were in line with the decreased number of tubulin coils in resting RhoB−/− platelets (Figure 1E,G) and pointed to a critical role of RhoB in MT dynamics. MTs are highly dynamic structures that are constantly assembled and disassembled. MT disassembly into dimers can be artificially induced by exposure to cold (4°C), and reassembly into fibers occurs spontaneously at 37°C. Although both wt and RhoB−/− platelets displayed MT coils at their periphery at 37°C, which disassembled upon cold storage, only RhoB−/− platelets were not able to reassemble MT coils after disassembly (wt: 83 ± 2% vs RhoB−/−: 23 ± 5%) (Figure 6E-F; supplemental Figure 5C). MTs can be modified through PTMs, which serve the adaption to different cellular functions. In platelets, PTMs of α-tubulin have been reported to be involved in MT rearrangements, including acetylation of residue K40 and detyrosination/tyrosination (in C-terminal tyrosines, often also called glutaminylation). Acetylation and detyrosination are indicators for stable, longer-lived MTs, whereas more dynamic MTs are tyrosinated and deacetylated. We therefore investigated the acetylation status of the α-tubulin K40 residue by immunoblotting. RhoB−/− platelets and MKs showed pronouncedly decreased levels of acetylated α-tubulin (Figure 6G-I), which was by tendency also observed in MK lysates. In contrast, levels of detyrosinated and polyglutaminylated α-tubulin were unaltered in both platelets and MKs (Figure 6G-H). Thus, deficiency in RhoB results in short-lived MTs that are either per se more unstable or constantly remodeled. Reduced acetylation levels were not due to a change in levels of a major deacetylation enzyme, HDAC6 (supplemental Figure 4C). In summary, these results reveal that RhoB has a nonredundant role as an important regulator of MT turnover or stability and suggest that the impaired MT dynamics contribute to defective platelet biogenesis and microthrombocytopenia in RhoB−/− mice.
Here, we show that, in contrast to MK-specific deficiency of RhoA or Cdc42, loss of the small Rho GTPase RhoB results in microthrombocytopenia in mice, which might involve a profound defect in MT stability. The majority of platelet disorders associated with mutations in cytoskeletal genes are characterized by macrothrombocytopenia, which is reflected in transgenic mouse models of the respective diseases. Microthrombocytopenia, on the other hand, is a rare clinical condition. It is a characteristic of the Wiskott-Aldrich syndrome (WAS) and X-linked thrombocytopenia, both of which are caused by mutations in the WAS gene, as well as congenital autosomal-recessive small-platelet thrombocytopenia caused by mutations in adhesion and degranulation-promoting adaptor protein (ADAP). Both WAS protein (WASP) and ADAP are primarily known as regulators of the actin cytoskeleton: ADAP is a scaffolding protein involved in receptor-induced actin cytoskeletal dynamics in platelets. WASP is a downstream effector of Cdc42 and, via the Arp2/3 complex, leads to actin cytoskeletal rearrangements associated with filopodia formation. In line with this pathway, loss of Arp2 in MKs in mice, or mutations in the Arp2/3 complex component ARPC1B in humans, leads to microthrombocytopenia. Interestingly, deficiency of Profilin-1, another protein associated with actin turnover, in MKs results in a WAS-like phenotype in mice. Profilin-1 and Arp2 deficiency is linked to altered MT stability, which resembles our observations on the RhoB−/− mice. However, there are 2 striking differences: first, a major characteristic of mouse models with WAS- or ADAP-like phenotypes is the release of proplatelets into the BM compartment, referred to as “ectopic platelet release.” Second, platelets and MKs lacking ADAP, Arp2, or WASP display pronounced defects in F-actin dynamics, including aberrant podosome formation. In contrast, RhoB−/− MKs do not show signs of ectopic platelet release, and our results indicate largely normal F-actin organization, including podosome formation in vitro. Furthermore, protein levels of Profilin-1 and Arp2 are unaffected in the absence of RhoB. Together, our findings point to impaired MT, but not actin, dynamics as a potential mechanism underlying the manifestation of microthrombocytopenia in vivo. Our results suggest that the characteristic phenotype of ectopic platelet release is critically dependent on aberrant actin dynamics upon dysregulation of the WASP/Arp2/3 pathway. In this study, we provide evidence that in MKs and platelets, RhoB and RhoA have very distinct functions. RhoA-deficient mice display largely normal MK maturation; however, a transmigration of entire MKs into BM sinusoids contributes tomacrothrombocytopenia. The phenotypic differences might be explained by the unique subcellular localization of RhoB compared with RhoA. Although RhoB was specifically localized close to the plasma membrane, RhoA was more evenly distributedthroughout the cytoplasm of MKs. Although we only found low levels of RhoB in human platelets, studies using transcriptomic approaches identified RhoB mRNA in human MKs, which we were able to confirm on the protein level (Figure 4). In murine MKs, we identified RhoB mRNA as most abundantly expressed among the Rho subfamily members. In contrast to the selective Gα13/Gαq-signaling defect in RhoA-deficient platelets, RhoB−/− platelets showed an impaired platelet activation in vitro independent of the agonist used, but most pronounced upon stimulation of the collagen receptor GPVI, which may partially be explained by the 18% reduction in GPVI receptor levels. Upon inhibition of second-wave mediators, GPVI signaling was not altered, suggesting that reduced thrombus formation in vitro might be a consequence of reduced signaling downstream of G-protein-coupled receptors (activated by adenosine diphosphate or thromboxane A2). This is supported by the lower tendency of ATP secretion in RhoB−/− platelets and together could point to a reduction in the feedback activation as a potential reason underlying the reduced response of platelets to GPVI agonists. Despite the differences observed in platelet activation, aggregation, and thrombus formation in vitro, arterial thrombus formation and bleeding time were not altered in the absence of RhoB, highlighting that additional signaling pathways can fully compensate the reduced activation of platelets in response to GPVI stimulation, similar to what has been described in Grb2-deficient mice. Interestingly, RhoB activity was shown to regulate Rac1 translocation to endosomes in endothelial cells. Because Rac1 is a critical regulator of PLCγ2 activation downstream of GPVI in platelets, loss of RhoB may affect Rac1 localization and function in platelets, causing predominantly GPVI-related signaling defects. However, in contrast to Rac1-deficiency, lamellipodia formation was not affected in the absence of RhoB. RhoB has mostly been described as a regulator of actin dynamics in diverse cell types where it, similar to RhoA, regulates stress fiber formation and cell migration. Interestingly, the F-actin distribution of RhoB−/− platelets and MKs appeared unaltered, which may indicate that RhoA is able to compensate for the loss of RhoB in regulating actin dynamics in the MK lineage. In contrast, RhoB−/− platelets displayed a pronounced MT assembly defect, evident by the inability to reassemble MT coils after cold storage, altered α-tubulin distribution in spread platelets, and decreased numbers of MT coils in resting platelets. MT dynamics are critical for the formation of proplatelets during the later stages of thrombopoiesis in vitro and in vivo. Consistently, and in line with our observations in platelets, the MT organization of RhoB−/− proplatelets was profoundly impaired, resulting in aberrantly sized and overall smaller proplatelet tips. These results thus provide a potential explanation for the decreased size of circulating platelets in RhoB−/− mice. This stands in contrast to the phenotype of Tubb1−/− mice, in which the complete loss of the tubulin β1 results in a pronounced macrothrombocytopenia. These findings imply that altered MT regulation can have different consequences compared with tubulin deficiency. Mechanistically, our results indicate that the marked inability to acetylate α-tubulin, a posttranslational modification associated with stable, long-lived MTs, contributes to their decreased stability in RhoB−/− platelets and MKs. Previous studies on the platelet marginal band have shown that α-tubulin is heavily acetylated in platelets and contributes to the kinetics of platelet spreading. Furthermore, acetylation levels of K40 of α-tubulin are associated with increased tubulin longevity, suggesting that decreased acetylation levels might correspond to either direct MT instability or increased MT turnover. This may explain the shortened MT coils in spread RhoB−/− platelets. Moreover, the finding that RhoB−/− platelets are not able to rebuild MT coils after cold-induced disassembly suggests that the formed MT coils are more susceptible to stress. The signaling pathways that link RhoB to tubulin acetylation remain to be defined in future studies. The tubulin-lysine deacetlyase HDAC6 or the acetyltransferase ATAT1 are obvious candidates, which could potentially be involved in controlling acetylation levels downstream of RhoB, although HDAC6 levels were unchanged in platelets or MKs. Loss of the MT plus-end tracking protein APC in MKs and platelets resulted in decreased α-tubulin acetylation at K40. However, protein levels of APC were unaltered in RhoB−/− MKs. Additional target proteins linking RhoB to the MT cytoskeleton could be twinfilin 1 (twf1) and cofilin1, which were recently shown as modulators of the actin/tubulin crosstalk during platelet biogenesis. Other PTMs of tubulin with important roles in platelets and MKs, including detyrosination and polyglutaminylation, were also unaltered, indicating a specific role for RhoB in regulating tubulin acetylation. In summary, our results reveal that impaired MT dynamics, in combination with normal cytoplasmic MK maturation, might contribute to microthrombocytopenia in RhoB−/− mice. Although genetic RhoB deficiency in humans has not been described to date, mutations resulting in persistent RhoB activation were shown to result in systemic capillary leak syndrome and an increased risk of cerebral palsy. The analysis of platelets from these patients may provide new insights into the roles of RhoB in the MK lineage in humans.
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PMC9631689 | 35667091 | Katarzyna I. Jankowska,Douglas Meyer,David D. Holcomb,Jacob Kames,Nobuko Hamasaki-Katagiri,Upendra K. Katneni,Ryan C. Hunt,Juan C. Ibla,Chava Kimchi-Sarfaty | Synonymous ADAMTS13 variants impact molecular characteristics and contribute to variability in active protein abundance | 21-09-2022 | Key Points ADAMTS13 sSNVs affect mRNA thermodynamic stability and may disturb mRNA-splicing sites. Synonymous variations may affect ADAMTS13 function and contribute to large variability in protein expression levels in healthy individuals. | Synonymous ADAMTS13 variants impact molecular characteristics and contribute to variability in active protein abundance
ADAMTS13 sSNVs affect mRNA thermodynamic stability and may disturb mRNA-splicing sites. Synonymous variations may affect ADAMTS13 function and contribute to large variability in protein expression levels in healthy individuals.
Although commonly assumed to be silent, synonymous single nucleotide variants (sSNVs) can cause protein deficiency or dysfunction severe enough to lead to disease through various mechanisms. Synonymous variants may alter constitutive splice sites or activate cryptic splice sites, which can result in unstable messenger RNA (mRNA) or defective protein. Synonymous changes may affect thermodynamic stability and secondary structure of mRNA or codon usage frequency, resulting in altered translational kinetics and cotranslational folding of a protein. Recent studies suggest that intermittent ribosome stalling at key mRNA regulatory sites can affect protein abundance, folding, and even posttranslational modifications, and the placement of certain stable structural elements within the mRNA sequence is not random. Moreover, synonymous variants can disturb microRNA (miRNA)-binding sites in the coding sequence, which can lead to developmental defects and disease. Synonymous variants can affect cytosine-guanine dinucleotide (CpG) sites, guanine-cytosine (GC) content, and codon usage biases, which may change the rate of translation due to ribosomal pausing. Several studies found that ribosomal pauses relate to cotranslational folding of protein domains, which in turn determines the final protein conformation. Ultimately, by affecting gene regulatory signature, mRNA structure, and pre-mRNA processing, sSNVs can influence protein characteristics, including expression, function, and immunogenicity. ADAMTS13 (MIM:604134) controls the hemostatic function of von Willebrand factor (VWF; MIM: 613160) by splitting highly adhesive, ultra-large VWF multimers into smaller forms. VWF is critical to the initial stage of thrombosis by tethering platelets to the endothelium at sites of vascular injury. Regulation of VWF by ADAMTS13 prevents the spontaneous formation of platelet thrombi. Deficiency of ADAMTS13 increases VWF thrombogenic potential and may lead to microvascular thrombosis such as thrombotic thrombocytopenic purpura (TTP) or congenital TTP, also known as Upshaw-Schulman syndrome (USS). ADAMTS13 plays a crucial role in pediatric stroke pathogenesis, and lower levels of ADAMTS13 have been associated with increased risks of coronary heart diseases and myocardial infarction. Recently, clinical studies have shown the development of acquired TTP followed by COVID-19 infection and a strong association between low ADAMTS13 plasma levels and increased mortality in patients with COVID-19. The ADAMTS13 gene is located on chromosome 9 and is ∼37 kb long containing 29 exons. ADAMTS13 mRNA is ∼4 kb long and encodes 1427 amino acids. This multidomain protein comprises a signal peptide, propeptide, metalloprotease, disintegrin-like domain, first thrombospondin type 1 repeat (TSP1), Cysteine-rich, and spacer domains. The distal C-terminus includes 7 additional TSP repeats and two CUB (C1r/C1s, urinary epidermal growth factor, bone morphogenetic protein) domains, that are unique for ADAMTS13. The metalloprotease domain of ADAMTS13 modulates ADAMTS13 protein activity via cooperative binding to one Zn2+ ion and three Ca2+ ions. Among human ADAMTS13 variants listed in a database of single nucleotide polymorphisms (dbSNP), ∼200 disease-causative SNVs have been identified in patients with TTP, all of which were non-synonymous and detected as haplotypes. Other SNVs may result in reduced plasma ADAMTS13 activity and disrupted ADAMTS13–VWF interactions by changing untranslated regions (UTRs), splice regulatory regions, or the coding sequence. In addition, various truncated forms of ADAMTS13 are detectable in plasma, and several alternatively spliced mRNA variants have been characterized. Three genome-wide association studies (GWAS) revealed high heritability of ADAMTS13 levels (59.1%) and identified hundreds of non-synonymous and synonymous SNVs at the ADAMTS13 locus that collectively explained ∼20.0% of large ADAMTS13-level variation in healthy individuals. Of 376 sSNVs registered in the National Center for Biotechnology Information dbSNP database, 357 sSNVs have been identified in healthy individuals, and 19 sSNVs have been identified in patients with USS (supplemental Table 1). The impact of USS-associated sSNVs in the context of deleterious co-occurring mutations is currently unknown. Such sSNVs may have an additive negative impact, or they may help to enhance ADAMTS13 expression. For instance, the USS synonymous variant c.354G>A (rs28571612) yielded higher extracellular and intracellular ADAMTS13 expression levels and higher specific activity according to in vitro measurements. Due to limited data from GWAS and few experimental validations, the impact of specific sSNVs on ADAMTS13 function remains difficult to interpret. Nevertheless, prior studies show that variants in the ADAMTS13 gene resulting in deficient ADAMTS13 activity may disturb VWF activity and lead to coagulopathies such as USS. Understanding the factors that contribute to ADAMTS13 expression and disturb normal protein activity could help reduce diagnostic errors, prevent TTP development, and improve treatment. Considering the substantial number of observed synonymous variants, comprehensive experimental verification is difficult to perform. Use of computational predictors and modeling can reveal sSNVs that are most likely to affect protein function and disease. This approach can help focus experimental studies on a smaller subset of synonymous variants most likely to have a detectable impact on protein characteristics. To better understand the contributions sSNVs may make to protein biogenesis using ADAMTS13 as a model, we performed comprehensive in silico analysis of all known ADAMT13 sSNVs. Our results highlight sSNVs that may alter mRNA splicing, change mRNA-folding energy, disturb miRNA-binding sites, and affect synonymous codon usage. This in silico analysis provides a generalizable approach to characterize the effects of sSNVs influencing other genes and diseases.
The ADAMT13 variants were obtained from the National Center for Biotechnology Information dbSNP database (https://www.ncbi.nlm.nih.gov/SNP/snp_ref.cgi?locusId=11093; GRCh38.p7). All SNVs from ADAMTS13 were filtered to include only synonymous variants in the open reading frame. Of >1000 SNVs, all 376 sSNVs were selected for further evaluation and compared with the wild-type (WT) ADAMTS13 (NM_139025). All sSNVs obtained from the dbSNP (supplemental Excel Worksheet 1) include 357 neutral variants and 19 sSNVs that were identified in patients with USS (USS variants). Although the USS variants are labeled as “being-likely” to cause the disease, this has not yet been confirmed. To our knowledge, there are no confirmed disease-associated ADAMTS13 sSNVs.
Determination of mRNA-folding energy, stability, and structure were performed by using mFold, NUPACK, kineFold, remuRNA, and RNAFold. Splicing impact of ADAMTS13 sSNVs was evaluated by using MaxEntScan (MES), NNsplice, SpliceSiteFinder-like, and GeneSplicer. Evaluation of miRNA-binding sites within the coding region of ADAMTS13 sSNVs was performed by using miRDB, Paccmit-CDS, and TargetScan. Relative synonymous codon usage (RSCU) and relative synonyms bicodon usage (RSBCU) were calculated as previously described. Codon pair score without natural log (CPS), rare codon (RC) enrichment, and codon adaptation index (W) were computed as previously described. RC clustering for ADAMTS13 sSNVs was computed by using %MinMax. Protein folding energy was estimated by using a coarse-grained cotranslational folding energy model. Complete descriptions of the in silico methods are given in the supplemental Methods, and data produced are provided in Excel Worksheets 1 to 3.
A total of 376 naturally occurring sSNVs of ADAMTS13, including 357 from a healthy population and 19 from patients with USS, were identified in the dbSNP; they affect almost 9% of nucleotides in the mRNA of ADAMTS13 and >26% of codons. Most sSNVs have been identified in exon 25. Many sSNVs have been found within the metalloprotease domain and C-terminal region of ADAMTS13, in exons 24 to 29, which encode the TSP7-8 and CUB1-2 domains (Figure 2A). The distribution of base changes across the sSNVs of ADAMTS13 shows that the most frequently occurring base changes were C>T and G>A, agreeing with prior studies reporting synonymous base pair change frequencies. Of 19 USS variants, 11 were identified within the TSP2-8 and CUB1 domains. Accounting for exon length, sSNVs most frequently affected exons 1, 5, 8, and 25 (∼11% sSNV per exon length) that encoded signal peptide, metalloprotease, and T8 domains, respectively (Figure 2B). In contrast, sSNVs were rarely found in exons 2 to 4, 9, and 23 encoding the propeptide, metalloprotease, disintegrin-like, and T6 domains, respectively (∼6% sSNV per exon length). The most frequent types of ADAMTS13 sSNVs, C>T and G>A (Figure 2C), lower GC content. Most identified ADAMTS13 sSNVs are very rare, with a frequency <0.001. The 12 most frequently occurring sSNVs are listed in Table 1. Interestingly, 6 of these variants were identified in patients with USS. The frequencies of all 376 sSNVs are included in supplemental Excel Worksheet 1.
We next evaluated RNA-folding energy of ADAMTS13 sSNVs. Such changes may affect antigen and activity levels of the protein. Experimental measurement of mRNA structure remains a challenge, especially to a degree of sensitivity that could detect structural differences resulting from single nucleotide changes. However, algorithms such as mFold, kineFold, remuRNA, or NUPAC can evaluate the stability of mRNA fragments by calculating Gibbs free energy (ΔG) of possible secondary structures. Using these 4 algorithms, we calculated ΔΔG () for all ADAMTS13 sSNVs (Figure 3A; supplemental Figure 1A). According to at least one algorithm, 67 sSNVs caused significantly altered folding energy compared with WT (P < .05) (Figure 3B-C). (The supplemental Methods provides a description of P value computation, and supplemental Excel Worksheet 1 provides the P values). Although the sSNVs generally resulted in increased ΔG and were predicted to cause structural instability, we found some sSNVs, particularly in exons 1, 6, 14, and 19, which caused significant decreases in ΔG and thus more stable mRNA structures (Figure 3B). Others have proposed that thermodynamically stable mRNA secondary structures should have a selective advantage. The synonymous variant c.999G>A results in the highest positive ΔΔG by mFold and remuRNA and consistently positive ΔΔG by the other 2 tools. Variants c.1521G>A and c.2385G>A displayed significantly positive ΔΔG by mFold and kineFold. The lowest negative ΔΔG predicted by mFold was found in variant c.2067C>A. This variant had significantly negative ΔΔG predicted by kineFold and remuRNA and was the only sSNV with significantly negative ΔG calculated by 3 tools. Variant c.1462C>A displayed the lowest negative ΔΔG calculated by NUPAC and significantly negative ΔΔG calculated by kineFold. Finally, the variant c.4041C>T within exon 28 was predicted to significantly decrease stability according to mFold and kineFold (Figure 3C). None of the 19 USS variants (supplemental Figure 1B) and 2 of the 12 high-frequency variants (supplemental Figure 1C) yielded significant ΔΔG. We next predicted full-length mRNA secondary structure of highly frequent variants (Table 1) and from variants resulting in most positive (c.999G>A) and most negative (c.1462C>A) ΔΔG by RNAfold. ΔG and the optimal mRNA secondary structure of either sSNV differ from those of WT ADAMTS13 mRNA (Figure 3D; supplemental Figure 2; supplemental Table 2). We observed moderate correlations between ΔΔG for the 4 algorithms used, with the strongest correlation between mFold and remuRNA, whereas kineFold exhibited the weakest correlations among all 4 (Figure 3E). Although few variants were predicted to significantly affect ΔΔG by multiple algorithms, the direction of the impact (increase or decrease) was generally consistent across the different algorithms (Figure 3C). In summary, numerous sSNVs may affect ADAMTS13 mRNA thermodynamic stability, and these alterations in mRNA secondary structure may have strong effects on gene function and play an important role in disease onset and progression.
We next examined ADAMTS13 sSNVs for their impact on GC content and CpG sites. The majority of ADAMTS13 sSNVs reduce GC content (Figure 4A), especially in exons 4, 12, 16, 21, and 24. Of the 376 ADAMTS13 sSNVs, 144 affected CpG sites (Figure 4B). Only 55 sSNVs increased GC content, and 31 sSNVs resulted in a creation of new CpG sites (Figure 4C). The sSNV 33T>G may create a new CpG site in exon 1, potentially affecting the DNA methylation state. Previous studies have shown that CpG sites are frequently methylated and that methylation in the first exon suppresses gene expression, suggesting that this variant may influence ADAMTS13 expression. As indicators of structural stability, observations of moderate correlations between ΔG and GC content of both WT and variant sequences were not surprising. However, weak correlations were observed between ΔΔG and ΔGC content (Figure 4D).
We next examined the possible impact of sSNVs on splicing and miRNA regulation of ADAMTS13 (Figure 2A-B; supplemental Excel Worksheets 2-3), which are 2 common mechanisms by which sSNVs can affect protein structure. We identified 7 variants predicted to affect constitutive splice donor or acceptor sites (Figure 5A), which might result in exon skipping or intron retention. In addition, 36 variants were identified that may activate cryptic splice sites, and although these cryptic sites are not associated with previously characterized ADAMTS13 splice isoforms, they may still limit expression of constitutive ADAMTS13 transcript. Seven variants were predicted to affect special cryptic sites, which are associated with alternative splice products of ADAMTS13 (NM_139026 and NM_139027). Five of these variants weakened special cryptic sites, potentially resulting in decreased production of alternative transcripts. The c.909C>T variant was predicted to strengthen the special exon 8 acceptor site, potentially decreasing primary transcript expression in favor of the NM_139026 transcript. The c.918C>T variant was predicted to strengthen (NNsplice) and weaken (MES) the special exon 8 acceptor site, making it difficult to predict this variant’s impact. Variants predicted to affect splicing were found in 15 different exons and influence positions that encode several protein domains, including metalloprotease domain (10 sSNVs), disintegrin-like domain (15 sSNVs), Cysteine-rich domain (2 sSNVs), spacer domain (5 sSNVs), and TSP repeats (10 sSNVs) (Figure 5A; supplemental Table 3). To further validate predictions of splice impact, we used additional tools (SpliceSiteFinder-like and GeneSplicer) for variants predicted to affect splicing by MES or NNsplice. For variants predicted to affect constitutive splice sites, limited agreement was found between the tools used (Figure 5C). However, we found 5 variants predicted to affect cryptic splice sites with complete agreement between 4 splicing tools and 4 additional variants with agreement between 3 splicing tools (Figure 5D). Because ADAMTS13 sSNVs may affect miRNA-binding sites and cause translational suppression or protein degradation, we next examined variants for their likelihood to affect miRNA-ADAMTS13 binding based on Paccmit-CDS, TargetScan, and miRDB. We found 9 variants predicted to affect miRNA-binding sites by all 3 tools (Figure 5B). These 9 variants resulted in gain of binding sites for 10 different miRNA species and loss of binding sites for 4 miRNA species. Because TargetScan and miRDB are tools primarily used to assess miRNA binding in 3′UTR, we expected to see some disagreement between these tools and Paccmit-CDS, which was specifically developed to assess miRNA binding to mRNA-coding regions. We found substantially higher overlap between TargetScan and other algorithms when using extended seed (nucleotides 2-8 of the miRNA) rather than nucleotides 1 to 7 as seed sequences (Table 2; supplemental Table 4; supplemental Figure 3); thus, the TargetScan values resulted from using bp 2 to 8 (Figure 5E; Table 2). Paccmit-CDS predicted gain/loss of 3408 miRNA-binding sites resulting from sSNVs. Of these 3408 affected miRNA-binding sites, 14 were predicted by TargetScan and miRDB (Figure 5B; Table 2), and 186 were predicted by TargetScan (Figure 5E). From these predicted miRNAs, miR-221-5p and miR-1248 were shown to be expressed in liver cells, where ADAMTS13 is predominantly expressed. Further confirming its relevance to the liver, miR-221 displayed upregulation in liver fibrosis and promoted human hepatocellular carcinoma migration.
We next evaluated how sSNVs could change ADAMTS13 synonymous codon preference. Here, P values <.05 were considered significant. A total of 83 sSNVs exhibited significantly different codon usage or codon pair usage based on RSCU and RSBCU values, respectively (Figure 6A; supplemental Figure 4). From this group, 22 sSNVs displayed significantly different codon adaptation index (ΔW) (Figure 6B). ADAMTS13 sSNVs commonly inserted RCs (supplemental Table 5), which are expected to decrease translation rate. Among them, variants c.360G>A, c.451C>T, c.1125G>A, c.1557G>A, c.2754G>A, c.3501G>A, c.3810C>A, and c.4185G>A led to decreases in codon usage metrics. Only 7 synonymous variants significantly increased indices of codon usage (supplemental Figure 4). We also calculated changes in CPS for sSNVs of ADAMTS13 to measure the impact of codon pair usage while controlling for codon usage. Forty-one sSNVs exhibited significantly different CPS values (Figure 6C). Three sSNVs (c.138G>T, c.225 T>C, and c.1989G>A) exhibited the largest decrease in CPS values. Only two sSNVs (c.738A>G and c.2703A>G) resulted in increased CPS for both affected codon pairs, although the increase was only significant for the 5′ codon pair. As expected, ΔRSCU showed a strong linear correlation with Δ%MinMax (supplemental Figure 5A), and we found a stronger correlation between CPS and RSBCU for 3′ codon pair than 5′ codon pair (supplemental Figure 5B-C). Next, we computed rare and common codon clustering for ADAMTS13 sSNVs using %MinMax. Of these, 31 variants resulted in significantly decreased %MinMax, and 5 resulted in significantly increased %MinMax (Figure 6D). Variants that decrease %MinMax imply the insertion of RCs, which can reduce local translation rate; variants that increase %MinMax imply the loss of RCs, which may be necessary for proper cotranslational folding. In addition, we investigated RC enrichment at positions of ADAMTS13 sSNVs and identified several loci with significant RC enrichment (supplemental Figure 6). RCs may inhibit translational initiation or slow translation elongation, which in turn can promote mRNA degradation and even affect protein folding. The highest increase in RC values were observed at positions 1551, 2439, 2448, and 3684 in exons 13, 20, 20, and 26, respectively. Synonymous variations in these positions may affect ADAMTS13 translational rate and disturb protein folding. When comparing variants that significantly affect RSCU, RSBCU, W, and %MinMax, we identified 10 sSNVs located in multiple different exons (Figure 6E) that significantly affect all parameters. We found 3 of these variants also significantly affected RC but we found no sSNVs that also significantly affect CPS (supplemental Figure 7).
Next, we investigated the regions essential for ADAMTS13 functionality to assess their vulnerability to sSNVs. Detailed crystal structure evaluation of ADAMT13 proximal domain MDTCS compared with identified sSNV positions revealed 13 sSNVs affecting loci critical for metal binding and protein activity (Figure 7A-C; Table 3). C.564C>T affected a codon directly involved in Ca2+ binding (Figure 7B). Another variant, c.1980G>A, is localized within the loop 660-672 that is essential in interaction with VWF and/or CUB domains relevant to “closed” ADAMTS13 conformation (Figure 7C). Nevertheless, 4 of these 13 sSNVs were predicted to affect mRNA splicing: c.546C>T, c.552G>A, c.567C>T, and c.834G>T (Figure 5A). Two were predicted to affect miRNA-binding sites: c.1833C>T and c.1980G>A (Table 2). Two variants significantly affected mRNA folding energy (supplemental Figure 8A). CUB1 domain amino acids Cys1254 and Cys1275 are essential for proper secretion and proteolytic activity of ADAMTS13, suggesting relevance of c.3762C>T and c.3825C>T variants. Furthermore, 16 additional sSNVs were identified in codons encoding cysteine, whose disulfide bonds stabilize the overall structure of ADAMTS13, and 42 sSNVs were identified in codons encoding proline, which introduces rigid turns into the peptide chain and sets α‐helix and β-sheet borders. Because proline and cysteine substantially influence protein structure, we expect more severe consequences from variants affecting proline or cysteine codons than from variants affecting other codons. Variants affecting cysteine codons did not significantly influence mRNA stability or codon usage (supplemental Figure 8B). Interestingly, based on amino acid frequency in ADAMTS13, neutral sSNVs affecting cysteine codons were 15% less frequent than expected (supplemental Table 6). Proline codons, in addition to those encoding alanine and threonine, were among the most frequently affected codons by sSNVs of ADAMTS13 (Figure 7D; supplemental Table 6). Proline codons are often affected by sSNVs in signal peptide, propeptide, and disintegrin-like domains (supplemental Figure 9). Several variants affecting proline codons were predicted to influence other examined parameters (supplemental Figure 8C). Seven variants may affect splicing (Figure 6), and 2 may affect miRNA binding (Table 2). In addition, threonine, alanine, and proline codons were more commontly affected by ADAMTS13 sSNVs, whereas tyrosine codons were less commonly affected by sSNVs than was expected (supplemental Table 6; supplemental Figure 9). Synonymous mutation in leucine codons dominate in metalloprotease and CUB domains, whereas sSNVs in neutral alanine codons were seen most frequently in thrombospondin domains. USS variants mostly encode leucine valine and serine codons (Figure 7D; supplemental Figure 9H). Finally, because changes in translation kinetics may affect cotranslational folding, we calculated ADAMTS13 folding energy of the nascent protein chain. We identified three variants (c.3150G>A, c.3177G>A, and c.2338T>C) downstream from areas of large changes in cotranslational folding energy (Table 4) and which change RSCU or RSBCU. Together, this implies these variants have the greatest potential impact on protein secondary structure.
Although most identified ADAMTS13 sSNVs are very rare, common sSNVs mainly coexist as haplotypes, making it very challenging to find individuals with a single sSNV to evaluate its effect. Conversely, cost and complexity remain substantial obstacles for comprehensive characterization of sSNVs affecting protein properties and disease states in vitro. Our in silico analysis of biomolecular characteristics of ADAMTS13 reveals a powerful opportunity for high-throughput screening of sSNVs likely to affect protein properties, even for large complex genes such as ADAMTS13. By integrating findings from a variety of in silico tools assessing different characteristics, we can comprehensively assess multiple mechanisms by which sSNVs may exert a biological impact. Results in the current study focus on ADAMTS13 sSNVs, but our approach using in silico tools to assess several different biomolecular mechanisms is generalizable to any gene. The estimated heritability of ADAMTS13 antigen levels suggests that most of the population variance of plasma ADAMTS13 is the result of genetic factors, including sSNVs. Our findings suggest numerous synonymous variants that may affect ADAMTS13 properties through multiple mechanisms, including pre-mRNA splicing, miRNA silencing, and translation kinetics. Although there is no single ADAMTS13 sSNV known to be responsible for USS, there are few documented examples in the literature that associate sSNVs of ADAMTS13 to pathologic states. In addition to USS variants (supplemental Table 1), other sSNVs has been identified and clinically evaluated (supplemental Table 7). Most of the sSNVs identified in patients with TTP have been labeled as haplotype with other synonymous variants that are associated with the disease,- and thus it is difficult to evaluate the role of sSNVs in these individuals. The genotype analysis of ADAMTS13 in 14 neonates diagnosed with congenital heart disease (CHD) identified 4 patients with thrombosis and 10 patients without thrombosis (supplemental Table 8). One patient (patient 2) who developed thrombosis at the site of surgery has ADAMTS13 haplotype with 3 sSNVs (c.420T>C [rs3118667], 1716G>A [rs372789831], c.2280T>C [rs3124767]), one not-TTP variant (c.2699C>T, rs685523), and one TPP variant (c.1370C>T, rs36220240) and was asymptomatic despite the presence of deleterious mutation previously linked to congenital TTP (c.1370C>T, rs36220240). Moreover, 10 patients with CHD exhibited high to normal ADAMTS13 antigen and activity levels (∼50%, which is considered the lower normal range in neonates) and have not developed thrombosis, whereas missense mutations (c.1342C>G [rs2301612, p.G448E], c.1370C>T [rs36220240, p.P457L], and c.2699C>T [rs685523, p.A900V]) often identified in patients with TTP were accompanied by sSNV variants. One CHD patient (patient number 7 without thrombosis) had single sSNV: c.2280T>C (rs3124767) in the ADAMTS13 gene and high ADAMTS13 antigen (63.6%) and activity (43.6%) levels. In addition, in vivo evaluation of the ADAMTS13 haplotype seen in patient 2 showed that the deleterious effect seen for the TTP variant (c.1370C>T, rs36220240, p.P457L) can be rescued by synonymous variants (supplemental Table 7). These studies suggest that sSNVs identified in neonates may have synergistic protective effects on ADAMTS13 functions. GWAS of plasma ADAMTS13 concentrations from healthy donors showed that individuals with c.420T>C or c.4221C>A variants exhibited ∼6% increase in ADAMTS13, whereas in patients with variants c.1716G>A or c.2280 C>T, the ADAMTS13 level was ∼5% lower. These synonymous variants were shown to alter ADAMTS13 expression and activity levels in in vitro studies (supplemental Tables 7 and 9). Variant c.420T>C (rs3118667) also exhibited a 3% increase in ADAMTS13 activity by GWAS and has been significantly associated with pediatric stroke. In addition, c.4221C>A was significantly more frequent in populations with major adverse cardiac events, cerebrovascular events, and Thai malaria. Overall, although the effect from single sSNVs is not dramatic, coexistence of 2 or more variants may have a synergistic effect and cause significant changes in ADAMTS13 functions. Combined, this evidence suggests that ADAMTS13 sSNVs affect plasma ADAMTS13 antigen and activity levels, emphasizing their relevance to cardiovascular disease and coagulopathy. In our in silico studies, the sSNVs identified in patient samples (supplemental Table 7) were located within RC-enriched regions (supplemental Table 9). Significantly high RC enrichment would suggest a cluster of conserved RCs around the region, thus amplifying potential impact from changes to more common codons. In addition, validation of RNA minimum free energy of full-length ADAMTS13 variants that have been identified in human subjects displayed significant differences in their RNA secondary structure (compared with WT), which may explain district antigen and activity level in those variants (supplemental Figure 2; supplemental Table 2). To summarize, we have presented a comprehensive in silico overview of all reported ADAMST13 sSNVs that may be used as point of reference to understand the clinical consequences of ADAMTS13 sSNVs. We found numerous sSNVs that affect ADAMTS13 GC content and CpG sites. Several methods were used to evaluate codon and codon pair usage changes that may affect translation rate and cotranslational folding of ADAMTS13. Moreover, our results show that 67 sSNVs confer significantly different mRNA folding energy compared with WT. Next, our analysis revealed 46 sSNVs that may affect ADAMTS13 splicing, including two USS variants, c.936C>T (rs36219562) and c.1797C>T (rs36221216). Nine sSNVs were found that can affect the binding sites of 14 miRNAs. Although the effect of miRNA binding within coding region may not be as high as in 3′UTR, they may still contribute to observed variance in ADAMTS13 expression. Calculation of cotranslational folding in the nascent protein chain identified 3 variants that may substantially affect protein folding. In addition, we characterized potential effects of these sSNVs on protein structure, especially with respect to the ADAMTS13-active site. Our previous evaluation of some ADAMTS13 SNVs found that even the variants with moderate changes in ΔG or codon usage can affect protein properties. Although further experimental evaluation is needed to fully validate the role of synonymous variation in protein function, this study has identified several ADAMTS13 sSNVs that are most likely to affect ADAMTS13 protein properties. Deficiency or dysfunction of ADAMTS13 can lead to thrombotic pathologies, including TTP, USS, myocardial infarction, and ischemic stroke, and may contribute to COVID-19–associated coagulopathy. As we show here, rare synonymous ADAMTS13 variants may markedly contribute to the natural variation observed in the healthy population and might explain differential susceptibility to thrombosis. Most of these ADAMTS13 sSNVs have not been identified in prior GWAS, often being systematically excluded. Better understanding of these sSNVs and appreciating how they contribute to variability in ADAMTS13 abundance and specific activity highlight the broader importance of considering sSNVs when assessing potential causes for differential gene expression, protein abundance, and structure.
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PMC9631948 | Yunjie Duan,Yongxing Du,Zongting Gu,Xiaohao Zheng,Chengfeng Wang | Prognostic value, immune signature and molecular mechanisms of the APOBEC family members APOBEC1, APOBEC3A, APOBEC3G and APOBEC3H in pancreatic adenocarcinoma 10.3389/fmolb.2022.1036287 | 20-10-2022 | pancreatic adenocarcinoma,APOBEC family,biomarkers,prognosis,immune infiltration | Background: Increasing evidence supports that the APOBEC family is associated with development of a variety of cancers. However, the function of APOBEC1/3A/3G/3H in pancreatic adenocarcinoma (PAAD) is still unclear. Methods: Comprehensive bioinformatic analysis using R (version 3.6.3), TISIDB, Metascape etc. were performed to study the clinicopathological characteristics, prognostic value, immune features and functional mechanisms of the APOBEC1/3A/3G/3H in PAAD. Results: APOBEC1/3A/3G/3H showed significantly elevated expression in PAAD than para-cancerous or normal tissues. Their high expression or amplification were significantly correlated with worse clinicopathological characteristics and prognosis in PAAD patients. In addition, the role of APOBEC1/3A/3G/3H in the immune regulation is diverse and complex, the high expression of APOBEC1 may inhibit the infiltration level of many kinds of immunoreactive tumor-infiltrating cells, which may be an important factor leading to immune escape of PAAD cells. Mechanistically, APOBEC1/3A/3G/3H played an activating role in multiple oncogenic pathways, including the EMT, RAS/MAPK and TSC/mTOR pathways. Moreover, we found that the expression level of APOBEC3G was positively correlated with the sensitivity of gemcitabine and doxorubicin. Conclusion: APOBEC1/3A/3G/3H play an oncogenic role in the development of PAAD and might serve as new biomarkers or therapeutic targets. | Prognostic value, immune signature and molecular mechanisms of the APOBEC family members APOBEC1, APOBEC3A, APOBEC3G and APOBEC3H in pancreatic adenocarcinoma 10.3389/fmolb.2022.1036287
Background: Increasing evidence supports that the APOBEC family is associated with development of a variety of cancers. However, the function of APOBEC1/3A/3G/3H in pancreatic adenocarcinoma (PAAD) is still unclear. Methods: Comprehensive bioinformatic analysis using R (version 3.6.3), TISIDB, Metascape etc. were performed to study the clinicopathological characteristics, prognostic value, immune features and functional mechanisms of the APOBEC1/3A/3G/3H in PAAD. Results: APOBEC1/3A/3G/3H showed significantly elevated expression in PAAD than para-cancerous or normal tissues. Their high expression or amplification were significantly correlated with worse clinicopathological characteristics and prognosis in PAAD patients. In addition, the role of APOBEC1/3A/3G/3H in the immune regulation is diverse and complex, the high expression of APOBEC1 may inhibit the infiltration level of many kinds of immunoreactive tumor-infiltrating cells, which may be an important factor leading to immune escape of PAAD cells. Mechanistically, APOBEC1/3A/3G/3H played an activating role in multiple oncogenic pathways, including the EMT, RAS/MAPK and TSC/mTOR pathways. Moreover, we found that the expression level of APOBEC3G was positively correlated with the sensitivity of gemcitabine and doxorubicin. Conclusion: APOBEC1/3A/3G/3H play an oncogenic role in the development of PAAD and might serve as new biomarkers or therapeutic targets.
Pancreatic adenocarcinoma (PAAD) is one of the most invasive and lethal human cancers (Ioannou et al., 2012). It is highly malignant, prone to distant metastasis and has a poor prognosis. The 5-year survival rate of patients is only 7.2% (He et al., 2022). As per global reports in 2018, pancreatic adenocarcinoma remains the twelfth most common cancer in men and the eleventh in women. Cancer-related deaths have pancreatic adenocarcinoma as the seventh leading cause (He et al., 2022). Complete surgical resection is the only cure, but due to the lack of specific pancreatic adenocarcinoma screening methods, less than 20% of patients are diagnosed with local tumors, and most pancreatic adenocarcinoma patients cannot receive surgical treatment (Lin et al., 2019). Mucin-related markers such as CA19-9 are the most widely used tumor markers of pancreatic adenocarcinoma, but their value as screening markers is limited by their low specificity (Ringel and Löhr, 2003). Therefore, it is very important to explore new biomarkers and therapeutic targets to improve the prognosis of patients with pancreatic adenocarcinoma. The apolipoprotein B mRNA-editing catalytic polypeptide (APOBEC) family has 11 members, including APOBEC1, APOBEC3A, APOBEC 3G and APOBEC 3H (Wang et al., 2008), all of which have a zinc-dependent cytidine deaminase domain (ZDD) (Smith et al., 2012). The expressed product is a highly efficient cytidine deaminase that can convert cytosine to uracil. It can also be used as an inhibitor of retroviral replication and retrotransposon migration through deaminase-dependent and deaminase-independent mechanisms. APOBEC1 can edit apolipoprotein B mRNA to regulate cholesterol metabolism, and its expression products have a carcinogenic effect on abnormal deamination of genomic DNA (Wolfe et al., 2020). In addition, APOBEC1 is also a potential endogenous mutation factor (Niavarani et al., 2018). Changes related to APOBEC1 mutation activity may enhance its carcinogenic potential (Saraconi et al., 2014) and promote the occurrence and development of many cancers, including gastrointestinal tumors and esophageal cancer (Niavarani et al., 2018; Buisson et al., 2019). APOBEC3A is the only APOBEC enzyme that has a great preference for hairpin substrates (Buisson et al., 2019). It is the cause of genetic heterogeneity in tumors and has become the main driver of cancer cell mutagenesis. Its overexpression can lead to DNA damage (Biayna et al., 2021) and inducement of invasive breast cancer (Kim et al., 2020). APOBEC3G is closely related to increased DNA damage and DNA repair disorders. Misregulation of its expression products can lead to somatic mutations in many cancers and affect the stability of the genome (Iizuka et al., 2017). Related studies have found that high expression of APOBEC3G can promote the occurrence and development of a variety of malignant tumors, including hepatocellular carcinoma, multiple myeloma and esophageal squamous cell carcinoma (Wang et al., 2008; Ding et al., 2011; Talluri et al., 2021). The expression of APOBEC3H in head and neck squamous cell carcinoma is upregulated and may regulate the immune response through its demethylation activity, which has been identified as a potential target for immunotherapy of head and neck squamous cell carcinoma (Liu J et al., 2020). In addition, its strong retroviral restriction and hypermutation activity are also the main causes of breast cancer and lung cancer cell mutation (Starrett et al., 2016; Liu Q et al., 2020). However, the role of APOBEC1, APOBEC3A, APOBEC3G and APOBEC3H in pancreatic adenocarcinoma is not clear, and related studies are very scarce, which motivated us to carry out relevant bioinformatics analysis. In this study, we comprehensively analyzed the possible functions and mechanisms of APOBEC family members APOBEC1, APOBEC3A, APOBEC3G and APOBEC3H in the occurrence and development of PAAD by using public databases and a variety of bioinformatics analysis techniques. First, we analyzed the difference in APOBEC1/3A/3G/3H expression between pancreatic adenocarcinoma samples, matched adjacent samples and normal pancreatic tissues using The Cancer Genome Atlas (TCGA) and Genotype-Tissue Expression (GTEx) databases. Then, based on the R (version 3.6.3) and Kaplan‒Meier Plotter databases, we analyzed the relationship between their expression levels and clinicopathological features and overall survival of patients with PAAD. Finally, the potential mechanism of APOBEC1/3A/3G/3H involved in the occurrence and development of PAAD was explored by gene variation, immune infiltration, gene enrichment and protein-protein interaction (PPI) analysis. In summary, our research reveals that APOBEC1/3A/3G/3H plays a carcinogenic role in the occurrence and development of PAAD and is expected to become a new biomarker and therapeutic target for this type of malignant tumor.
This study was approved by the academic Committee of the Cancer Hospital of Chinese Academy of Medical Sciences and strictly followed the principles of the Helsinki Declaration. All the data in this study were retrieved from online databases, and no human or animal experiments were involved.
In this study, R (version 3.6.3) was used to analyze the expression level of APOBEC1/3A/3G/3H in cancerous and paracancerous tissues in the TCGA database and normal pancreatic tissues in the GTEx database (https://xenabrowser.net/datapages/). The statistical significance of APOBEC1/3A/3G/3H expression was evaluated by the Wilcoxon test and p < 0.05 was considered statistically significant. Then, the correlation between the expression levels of APOBEC1/3A/3G/3H and clinical variables was analyzed by R (version 3.6.3). The statistical significance of APOBEC1/3A/3G/3H expression was evaluated by Fisher’s test and p < 0.05 was considered statistically significant. In addition, we searched the immunohistochemical staining results of APOBEC3A/3G/3H in PAAD and normal pancreatic tissues using HPA database (www.proteinatlas.org). HPA database offering the possibility to explore the tissue-elevated proteomes in tissues and organs and to analyze tissue profiles for specific protein classes. Comprehensive lists of proteins expressed at elevated levels in the different tissues have been compiled to provide a spatial context with localization of the proteins in the subcompartments of each tissue and organ down to the single-cell level.
The Kaplan‒Meier Plotter database (http://www.kmplot.com/) is an online survival analysis tool capable of performing univariate and multivariate survival analysis using any custom-generated data which was used to analyze the correlation between the expression of APOBEC1/3A/3G/3H and OS and RFS in pancreatic adenocarcinoma and p < 0.05 was considered statistically significant (Lánczky and Győrffy, 2021). The “survival” R package (version 2.38) was utilized to calculate log-rank p values, and p values below 0.05 were considered statistically significant. The “pROC” R package (version 1.17.0.1) and “ggplot2″ R package (version 3.3.3) were used to analyze and draw ROC curves. The values of the area under the ROC curve (AUC) were between 0.5 and 1 (Nagy et al., 2021).
The cBioPortal database (http://www.cbioportal.org/) provides a Web resource for exploring, visualizing, and analyzing multidimensional cancer genomics data. The portal reduces molecular profiling data from cancer tissues and cell lines into readily understandable genetic, epigenetic, gene expression, and proteomic events (Gao et al., 2013). The cBioPortal database was used to analyze the gene variation of APOBEC1/3A/3G/3H in pancreatic adenocarcinoma, and the correlation between the variation and some clinicopathological features was further determined. The statistical significance of the difference was evaluated by the chi-squared test and p < 0.05 was considered statistically significant.
TISIDB database (http://cis.hku.hk/TISIDB/) integrated multiple types of data resources in oncoimmunology and we can cross-check a gene of interest about its role in tumor–immune interactions through literature mining and high-throughput data analysis, and generate testable hypotheses and high-quality figures for publication (Ru et al., 2019). The TISIDB database was used to analyze the correlation between the expression levels of APOBEC1/3A/3G/3H and the infiltration level of immune infiltrating cells and the expression level of immune molecules in pancreatic adenocarcinoma. The difference was evaluated by Spearman’s test and p < 0.05 was considered statistically significant. Then, the relationship between the expression levels of APOBEC1/3A/3G/3H and immune score and matrix score was analyzed by the SangerBox analysis tool (http://sangerbox.com/tool). The statistical significance of the difference was evaluated by Spearman’s test and p < 0.05 was considered statistically significant.
The LinkedOmics database (http://www.linkedomics.org/) contains multiomics data and clinical data for 32 cancer types and a total of 11158 patients from TCGA project. It is also the first multiomics database that integrates mass spectrometry based global proteomics data generated by the Clinical Proteomic Tumor Analysis Consortium on selected TCGA tumor samples (Vasaikar et al., 2018). The 400 genes most closely related to APOBEC1/3A/3G/3H coexpression were selected by using the LinkedOmics database, and the volcano map was drawn by R (version 3.6.3). The first 50 gene heatmaps that were positively correlated with APOBEC1/3A/3G/3H expression were drawn by R (version 3.6.3) and annotated by GeneCards (https://www.genecards.org/). Metascape (https://metascape.org) is a web-based portal designed to provide a comprehensive gene list annotation and analysis resource for experimental biologists. In terms of design features, Metascape combines functional enrichment, interactome analysis, gene annotation, and membership search to leverage over 40 independent knowledgebases within one integrated portal (Zhou et al., 2019). The Metascape database visualization of APOBEC1/3A/3G/3H and their coexpressed genes Biological Process (BP), Cellular Components (CC), Molecular Function (MF) and Kyoto Encyclopedia of Genes and Genomes (KEGG) were used. Wayne diagrams of 8 genes coexpressed with APOBEC3A, APOBEC3G and APOBEC3H were drawn by R (version 3.6.3), and the eight genes were analyzed by GO and KEGG enrichment analysis. TIMER database (http://timer.cistrome.org/) provides 6 major analytic modules that allow users to interactively explore the associations between immune infiltrates and a wide-spectrum of factors, including gene expression, clinical outcomes, somatic mutations, and somatic copy number alterations (Mermel et al., 2011; Li et al., 2017). The correlation between APOBEC1/3A/3G/3H expression levels was analyzed by the TIMER database. The statistical significance of the difference was evaluated by Spearman’s test and p < 0.05 was considered statistically significant. In addition, the pathway enrichment of APOBEC1/3A/3G/3H was completed by using the GSCALite database (http://bioinfo.life.hust.edu.cn/web/GSCALite/). GSCALite database is a user-friendly web server for dynamic analysis and visualization of gene set in cancer and drug sensitivity correlation, which will be of broad utilities to cancer researchers (Liu et al., 2018).
Functional links between proteins can often be inferred from genomic associations between the genes that encode them: groups of genes that are required for the same function tend to show similar species coverage, are often located in close proximity on the genome (in prokaryotes), and tend to be involved in gene-fusion events. The STRING database (https://string-db.org/) is a precomputed global resource for the exploration and analysis of these associations (Von Mering et al., 2003). The genes with the strongest interaction with APOBEC1/3A/3G/3H proteins were obtained by using the STRING database, and the functional protein interaction network of APOBEC1/3A/3G/3H was established. Additionally, the interaction intensity of different genes in the PPI network was scored by Cytoscape (version 3.9.1) software (Shannon et al., 2003; Doncheva et al., 2019).
The GSCALite database was used to analyze the correlation between the expression level of APOBEC3G and the sensitivity of many kinds of chemotherapy or targeted therapies. The difference was evaluated by Spearman’s test and p < 0.05 was considered statistically significant.
Based on the data from the TCGA database and GTEx database, we analyzed the transcription levels of APOBEC1/3A/APOBEC3G/3H in many kinds of cancer tissues, adjacent tissues and normal tissues. The results showed that the transcription levels of APOBEC1/3A/3G/3H in 28 kinds of cancer tissues, including PAAD, esophageal carcinoma (ESCA) and stomach adenocarcinoma (STAD), was higher than that in paracancerous tissues and normal tissues but lower (Figures 1A–D) in 13 other kinds of cancer tissues, including lymphoid neoplasm diffuse large B-cell lymphoma (DLBC), kidney chromophobe (KICH) and lung squamous cell carcinoma (LUSC). The above findings suggest that there are differences in the expression of APOBEC1/3A/3G/3H across cancers and may play different roles in different cancers. In addition, we searched the immunohistochemical staining results of APOBEC3A/3G/3H in PAAD (Supplementary Figures S2A,C,E) and normal pancreatic tissues (Supplementary Figures S2B,D,F) using the HPA database.
First, we analyzed the relationship between the expression levels of APOBEC1/3A/3G/3H and the clinicopathological characteristics of PAAD patients based on the data from the PAAD project in the TCGA database and R (version 3.6.3). As shown in Table 1, high expression of APOBEC1 was significantly correlated with higher pathologic stage (p < 0.020), and high expression of APOBEC3H was significantly correlated with higher N stage (p < 0.003). Next, we analyzed the correlation between the expression level of APOBEC1/3A/3G/3H and the prognosis of patients with PAAD through the Kaplan‒Meier Plotter database. The results showed that the high expression of APOBEC1/3A/3G/3H was significantly correlated with shorter overall survival (OS) (Figure 2A), and the high expression of APOBEC1 was also significantly correlated with shorter recurrence-free survival (RFS) (p < 0.05) (Figure 2B). Finally, we drew ROC curves based on data from the TCGA and GTEx databases to distinguish normal and pancreatic adenocarcinoma tissues, and APOBEC1/3A/3G/3H showed high accuracy in predicting normal and tumor outcomes. The ROC curve Figure 2C shows that APOBEC1 AUC is 0.961, APOBEC3A AUC is 0.748, APOBEC3A AUC is 0.696–0.799, AUC is 0.969, AUC is 0.87, and AUC is 0.838–0.916. In summary, these results show that APOBEC1/3A/3G/3H is generally upregulated in PAAD, and the high expression levels of APOBEC1/3A/3G/3H are related to the poor clinicopathological features and prognosis of PAAD patients. These results also indicate the potential role of APOBEC1/3A/3G/3H in the occurrence and progression of PAAD, suggesting that APOBEC1/3A/3G/3H can be used as a prognostic marker in PAAD patients.
To further explore the mechanism of the differential expression of APOBEC1/3A/3G/3H in pancreatic adenocarcinoma, we used the cBioPortal online tool to analyze the gene variation of APOBEC1/3A/3G/3H. APOBEC1/3A/3G/3H had genetic variation in 5 samples (3%) from patients with pancreatic adenocarcinoma, of which the gene with the highest frequency of mutation is APOBEC1 (2%), and the main type of variation is amplification (Figure 3A). Based on this, we analyzed the clinicopathological features of patients with APOBEC1/3A/3G/3H gene mutation and nonmutation PAAD. The results showed that there was a significant correlation between the amplification variation of APOBEC1/3A/3G/3H and the invasion of surrounding tissues of pancreatic adenocarcinoma (Figures 3B–E). There was also a significant correlation between the amplification variations of APOBEC3A/3G/3H and the higher N stage of PAAD patients (Figures 3F–H). The above results suggest that APOBEC1/3A/3G/3H will be amplified and mutated in PAAD tissues, which will lead to an increase in APOBEC1/3A/3G/3H expression and poor clinicopathological features of PAAD patients, which may be another important factor leading to worse prognosis in PAAD patients.
First, we used the data from the PAAD project in the TISIDB database to explore the relationship between APOBEC1/3A/3G/3H expression and the level of tumor infiltrating cells and multiple immunomodulators based on various immunological markers in PAAD. Our results show that the roles of APOBEC1/3A/3G/3H in tumor immune regulation are not consistent. First, APOBEC1 was negatively correlated with the infiltration level of many kinds of immunoreactive tumor-infiltrating cells, including Tem_CD4, Tem_CD8 and NK cells (Figure 4A), and negatively correlated with the expression level of most immune promoters (Figure 4B). Therefore, the high expression of APOBEC1 may inhibit the immune response to PAAD, which may be an important factor leading to immune escape of PAAD cells. In contrast, APOBEC3A/3G/3H were positively correlated with the infiltration level of many kinds of immunoreactive tumor-infiltrating cells, including Tem_CD4, Tem_CD8 and NK cells (Supplementary Figure S1), and positively correlated with the expression level of most immunomodulators (immunoenhancers, MHC molecules, chemokines and chemokine receptors) in PAAD (Figures 4A,C–F), indicating that APOBEC3A/3G/3H may play immune-promoting roles in the PAAD tumor microenvironment. Then, based on the data from the PAAD project in the TCGA database, we used the SangerBox analysis tool to further explore the relationship between APOBEC1/3A/3G/3H expression levels and immune scores and matrix scores. The results showed that APOBEC1 was negatively correlated with the immune score and matrix score of PAAD, while APOBEC3A/3G/3H showed the opposite correlation (Figures 4G,H). These results suggest that the role of the APOBEC family in the immune regulation of the PAAD tumor microenvironment is complex and diverse. Special attention should be given to the immune escape of PAAD cells caused by high expression of APOBEC1, which may be a potential target for the treatment of PAAD.
To further explore the mechanisms of APOBEC1/3A/3G/3H in the occurrence and development of PAAD, we first obtained 400 coexpressed genes (Supplementary Material S1) of APOBEC1/3A/3G/3H in pancreatic adenocarcinoma from the LinkedOmics database and generated the volcano map (Figure 5A). We displayed the first 50 genes that were positively related to the APOBEC1/3A/3G/3H table in the heatmaps (Figure 5B) and then annotated them with GeneCards. The results showed that many oncogenes, including MCU, MAP3K8, GZMK and TNFAIP8L2, were positively correlated with the expression of APOBEC1/3A/3G/3H. Then, we carried out GO and KEGG enrichment analyses of APOBEC1/3A/3G/3H and their 400 coexpressed genes, which were based on the data from the PAAD project in the Metascape database. GO analysis showed that APOBEC1/3A/3G/3H and their 400 coexpressed genes mainly acted on BP terms (Figure 5C), such as “inflammatory response”, “positive regulation of immune response” and “leukocyte activation”; CC terms, such as “side of membrane”, “immunological synapse” and “secretory granule membrane” (Figure 5D); and MF terms, such as “immune receptor activity”, “chemokine activity” and “deoxycytidine deaminase activity” (Figure 5E). KEGG analysis showed that APOBEC1/3A/3G/3H and their 400 coexpressed genes may play important roles in many pathways related to immune activation, such as the “T-cell receptor signaling pathway”, “TNF signaling pathway”, and the “Tolllike receptor signaling pathway” (Figure 5F). Then, we obtained eight genes (ICOS, GBP5, TIGIT, GMFG, CCL5, CYTIP, SP140 and PRF1) coexpressed with APOBEC3A, APOBEC3G and APOBEC3H by drawing a Wayne diagram (Figure 5G) and then carried out GO and KEGG enrichment analyses of these eight genes (Figure 5H). The results showed that the target genes mainly acted on “regulation of leukocyte cell‒cell adhesion”, “regulation of T-cell activation”, “leukocyte cell‒cell adhesion” and “NOD-like receptor signaling pathway”. In addition, we also found that the expression level of APOBEC1 was negatively correlated with APOBEC3G and APOBEC3H (cor = -0.121/cor = -0.061), while the coexpression correlation between APOBEC3G and APOBEC3H was higher (cor = 0.665) (Figure 5J). Finally, we further analyzed the mechanisms of APOBEC1/3A/3G/3H in many carcinogenic pathways by using PAAD project data from the GSCA database. As shown in Figure 5J, APOBEC3A/3G/3H play an activating role in the “EMT signaling pathway”, and APOBEC3A is also an activating factor of the “RTK signaling pathway” and “TSC/mTOR signaling pathway”. APOBEC1 plays an active role in the “RAS/MAPK signaling pathway”, “RTK signaling pathway” and “TSC/mTOR signaling pathway". In summary, these results further indicate that APOBEC1/3A/3G/3H play important roles in the immune regulation of the tumor microenvironment in PAAD. In addition, the coexpression of APOBEC1/3A/3G/3H and a variety of tumor-promoting genes and their activation of a variety of tumor-promoting pathways may be a potential mechanism for promoting the occurrence and development of PAAD.
To construct and analyze the PPI network of APOBEC1/3A/3G/3H in patients with pancreatic adenocarcinoma, we used the STRING database to identify 23 genes (Figure 6A) (Supplementary Material S2) with the strongest PPI with APOBEC1/3A/3G/3H and then drew the related PPI network with Cytoscape software, in which the larger the circle and darker the color, the greater the number of PPIs associated with the gene. The results showed that CUL5 had a higher score (Figure 6B) (Supplementary Material S3) in the APOBEC1/3A/3G/3H PPI network, which indicates that CUL5 plays an important role in this PPI network, which is closely related to APOBEC1/3A/3G/3H. Another interesting finding is that SMARCA4 may be the key gene (Figure 6A) that induces the occurrence of PPI between the SWI/SNF family and APOBEC family.
Finally, we used the GSCALite online database to analyze the expression level of APOBEC3G and the sensitivity of a variety of chemotherapy and targeted drugs. As shown in Figure 6C, the expression level of APOBEC3G was positively correlated with the sensitivity to many targeted or chemotherapeutic drugs, including gemcitabine and doxorubicin. Therefore, gemcitabine and doxorubicin may have a better therapeutic effect in PAAD patients with high APOBEC3G expression.
APOBEC proteins are a “double-edged sword”; they act as antiviral factors, and their overexpression or sustained expression may induce carcinogenicity and lead to a higher mutation load in a variety of human tumors, including bladder cancer, lung cancer, head and neck cancer and breast cancer (Iizuka et al., 2017). Although some studies have revealed the carcinogenic role of APOBEC family members APOBEC1, APOBEC3A, APOBEC3G and APOBEC3H in esophageal cancer, breast cancer, liver cancer and head and neck squamous cell carcinoma (Yamanaka et al., 1995; Saraconi et al., 2014; Kim et al., 2020; Liu Q et al., 2020), their function in PAAD is still unclear; additionally, there are very few related studies and specific bioinformatics analysis has not been carried out. This study is the first bioinformatics analysis of the function of APOBEC1/3A/3G/3H in PAAD. It reveals the function and possible mechanisms of APOBEC1/3A/3G/3H in the occurrence and development of PAAD from the aspects of gene expression, gene variation, immune infiltration, gene enrichment, protein interaction and drug sensitivity. APOBEC1 can deaminate single-stranded DNA or RNA, and its deamination activity is related to cancer (Wolfe et al., 2020). In addition, overexpression of APOBEC1 leads to the editing of additional cytidine sites in the substrate, which is considered to be the main cause of abnormal mutation in cancer cells (Fujino et al., 1998). It has been reported that high expression of APOBEC1 is associated with poor prognosis of adrenocortical carcinoma, endometrial carcinoma, mesothelioma and thyroid carcinoma (Niavarani et al., 2018). In this study, we found that APOBEC1 was also highly expressed in PAAD tissues and was significantly associated with higher PAAD tumor grade and shorter OS and RFS. Therefore, APOBEC1 may lead to a worse prognosis of PAAD patients through the same mechanism. In addition, APOBEC1 can not only enhance genomic variation by introducing intraframe genomic insertions and deletions into normal pluripotent cells, leading to cancer (Niavarani et al., 2018), but our study also found that the APOBEC1 gene itself can have amplification mutations within the PAAD tissue; this will lead to increased expression of APOBEC1, resulting in pancreatic adenocarcinoma being more invasive and may be another important factor leading to poor prognosis in PAAD patients. Another interesting finding is that APOBEC1 plays an active role in the “RAS/MAPK signaling pathway”, “RTK signaling pathway” and “TSC/mTOR signaling pathway”. The “RAS/MAPK signaling pathway” and “RTK signaling pathway” can promote the growth and proliferation of tumor cells (Ledda and Paratcha, 2007; Rezatabar et al., 2019), while the “TSC/mTOR signaling pathway” can promote tumor angiogenesis (Jham et al., 2011), which may be another mechanism by which APOBEC1 plays a carcinogenic role. APOBEC3A, APOBEC3G and APOBEC3H have similar functions; their catalytic activity is necessary for DNA damage activation (Landry et al., 2011) and has strong antiviral activity (Wang et al., 2008). However, some studies have found that this antiviral ability may be at the expense of an increased risk of host genome changes, and uncontrolled genomic deamination is potentially harmful and may be the cause of genomic instability and cancer (Landry et al., 2011). Related studies have shown that the high expression of APOBEC3A/3G/3H is related to the occurrence and development of colon cancer, multiple myeloma and head and neck squamous cell carcinoma (Wang et al., 2008; Ding et al., 2011; Liu J et al., 2020). Our study found that APOBEC3A/3G/3H are also highly expressed in PAAD tissues, and the APOBEC3A/3G/3H gene itself can be amplified and mutated, which leads to an increase in the expression levels of APOBEC3A/3G/3H, resulting in uncontrolled deamination of the cellular genome. This potential mechanism may be an important factor leading to more aggressive PAAD and worse prognosis of patients. The “EMT signaling pathway” is a classical cancer-promoting pathway that leads to the formation of secondary metastatic lesions by activating the motor and invasive abilities of tumor cells. It plays a cancer-promoting role in many kinds of tumors, including pancreatic adenocarcinoma, prostate cancer and breast cancer (Kong et al., 2021; Nowak and Bednarek, 2021; Zhao et al., 2021; Peng et al., 2022). The activation of APOBEC3A/3G/3H in the “EMT signaling pathway” may be another mechanism to promote the occurrence and development of PAAD. Although cancer immunotherapy has been shown to improve the survival rate of many kinds of cancer patients, the remission rate of PAAD patients is still very low (Ribas and Wolchok, 2018). Therefore, it is important to find new immunotherapy targets and develop new immunotherapy strategies. We analyzed the immune characteristics of APOBEC1/3A/3G/3H in PAAD. The results showed that APOBEC1 was negatively correlated with the infiltration level of many kinds of immunoreactive tumor infiltrating cells, the expression level of most immune promoters, immune score and matrix score, indicating that the high expression of APOBEC1 may inhibit the immune response to PAAD, while the immunomodulatory effects of APOBEC3A/3G/3H are the opposite. Previous studies have found that APOBEC3A/3G/3H play important immune-promoting roles in the innate immune system and have antiviral activity against a variety of retroviruses (Wang et al., 2008; Landry et al., 2011; Garg et al., 2015), which is consistent with our GO and KEGG analysis results. However, the antiviral activity of APOBEC3A/3G/3H is achieved by targeted deamination of cytidine residues of single-stranded DNA produced during viral genomic RNA reverse transcription. Overexpression of APOBEC3A/3G/3H may lead to DNA fragmentation and increase genomic instability, which may lead to cancer risk (Wang et al., 2008). Therefore, the immunosuppressive effect of APOBEC1 in the PAAD tumor microenvironment and the genomic instability caused by the high expression of APOBEC3A/3G/3H in PAAD tissues may be important factors in the occurrence and development of PAAD. These results suggest that APOBEC1/3A/3G/3H can be used as potential targets for PAAD immunotherapy and as a molecular index for predicting the efficacy of immunotherapy. Another interesting finding is the strong PPI between CUL5 and SMARCA4 and APOBEC1/3A/3G/3H. The CUL5 protein is involved in the formation of the E3-specific ligase complex and is responsible for ubiquitin protein transport to its target substrate for ubiquitin-dependent degradation (Li et al., 2021). The traditional view is that CUL5 is a potential tumor suppressor that can inhibit the proliferation, migration and invasion of renal cell carcinoma, endometrial carcinoma, prostate cancer, gastric cancer and lung cancer cells (Zhu et al., 2019; Wang et al., 2020) and maintains genomic stability (Yu et al., 2003). Recent studies have found that the complex composed of CUL5 can induce ubiquitination and degradation of APOBEC3G (Yu et al., 2003), which may be the mechanism for maintaining genomic stability. Our analysis shows that there is also a strong PPI between CUL5 and APOBEC1/3A/3H, suggesting that CUL5 may also avoid excessive damage to APOBEC1/3A/3H by inducing ubiquitin and degradation of DNA; this needs to be verified by further research. SMARCA4, a member of the SWI/SNF protein family, has helicase and ATP enzyme activities and can regulate the transcription of some genes by changing the structure of the surrounding chromatin (Jubierre et al., 2016). Some studies have found that SMARCA4 is highly expressed in pancreatic adenocarcinoma and participates in many processes, such as cancer cell growth and proliferation (Jubierre et al., 2016). The strong PPI between SMARCA4 and APOBEC1/3A/3G/3H may be the synergistic effect that promotes the occurrence and development of PAAD, which also needs be verified through further research. In terms of clinical transformation, our study found that the expression level of APOBEC3G was positively correlated with the sensitivity to a variety of targeted or chemotherapeutic drugs, including gemcitabine and doxorubicin. Therefore, gemcitabine or doxorubicin may have a better therapeutic effect in PAAD patients with high APOBEC3G expression. This study is the first bioinformatics analysis of the function of APOBEC family members APOBEC1, APOBEC3A, APOBEC3G and APOBEC3H in PAAD. We found that there are differences in the expression of APOBEC1/3A/3G/3H across cancers, and may play different roles in different cancers; however, the high expression and amplification variation of APOBEC1/3A/3G/3H are significantly related to worse clinicopathological features and prognosis of PAAD patients. In addition, APOBEC1/3A/3G/3H may promote the occurrence and development of PAAD by activating a variety of carcinogenic pathways and regulating PAAD tumor immunity. Another important finding is the possible synergy between SMARCA4 and APOBEC1/3A/3G/3H in promoting the occurrence and development of PAAD. In terms of clinical transformation, our study found that gemcitabine or doxorubicin may have a better therapeutic effect in PAAD patients with high expression of APOBEC3G. However, this study has some limitations. For example, the number of databases included in this study is somewhat insufficient. In addition, this study is only a bioinformatics analysis of the function and mechanism of APOBEC1/3A/3G/3H in the occurrence and development of PAAD. Future experimental studies can further confirm the tumor-promoting role of APOBEC1/3A/3G/3H in PAAD.
In short, our results show that the APOBEC family members APOBEC1, APOBEC3A, APOBEC3G and APOBEC3H may play a carcinogenic role in the occurrence and development of PAAD and are expected to become new biomarkers and therapeutic targets of PAAD. However, further studies are needed to verify our findings and to promote the clinical application of APOBEC1, APOBEC3A, APOBEC3G and APOBEC3H in PAAD. | true | true | true |
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PMC9632196 | Jing Zhang,Congmin Yi,Jiaojiao Han,Tinghong Ming,Jun Zhou,Chenyang Lu,Ye Li,Xiurong Su | Gut microbiome and metabolome analyses reveal the protective effect of special high‐docosahexaenoic acid tuna oil on d ‐galactose‐induced aging in mice | 15-07-2022 | aging,gut microbiome,metabolome,protective effect,special high‐DHA tuna oil | Abstract Aging is closely related to altered gut function and its microbiome composition. To elucidate the mechanisms involved in the preventive effect of special high‐docosahexaenoic acid tuna oil (HDTO) on senescence, the effects of different doses of HDTO on the gut microbiome and metabolome of d ‐galactose‐induced aging mice were studied. Deferribacteres and Tenericutes and uridine might be used as indicator bacteria and characteristic metabolites to identify aging, respectively. HDTO markedly improved the impaired memory and antioxidant abilities induced by d ‐galactose. At the phylum level, the abundance of Firmicutes and Tenericutes was significantly increased upon d ‐galactose induction, while that of Bacteroidetes, Proteobacteria, and Deferribacteres was significantly decreased. At the genus level, the variation mainly presented as an increase in the abundance of the Firmicutes genera Ligilactobacillus, Lactobacillus, and Erysipelothrix, the decrease in the abundance of the Bacteroidetes genera Bacteroides and Alistipes, the Firmicutes genus Dielma, and the Deferribacteres genus Mucispirillum. HDTO supplementation reversed the alterations in the intestinal flora by promoting the proliferation of beneficial flora during the aging process; the metabolic pathways, such as glycine–serine–threonine metabolism, valine–leucine–isoleucine biosynthesis, and some metabolic pathways involved in uridine, were also partially restored. Furthermore, the correlation analysis illustrated an obvious correlation between gut microbiota, its metabolites, and aging‐related indices. Moreover, it is worth noting that the metabolic regulation by dietary intervention varied with different HDTO doses and did not present a simple additive effect; indeed, each dose showed a unique modulation mechanism. | Gut microbiome and metabolome analyses reveal the protective effect of special high‐docosahexaenoic acid tuna oil on d ‐galactose‐induced aging in mice
Aging is closely related to altered gut function and its microbiome composition. To elucidate the mechanisms involved in the preventive effect of special high‐docosahexaenoic acid tuna oil (HDTO) on senescence, the effects of different doses of HDTO on the gut microbiome and metabolome of d‐galactose‐induced aging mice were studied. Deferribacteres and Tenericutes and uridine might be used as indicator bacteria and characteristic metabolites to identify aging, respectively. HDTO markedly improved the impaired memory and antioxidant abilities induced by d‐galactose. At the phylum level, the abundance of Firmicutes and Tenericutes was significantly increased upon d‐galactose induction, while that of Bacteroidetes, Proteobacteria, and Deferribacteres was significantly decreased. At the genus level, the variation mainly presented as an increase in the abundance of the Firmicutes genera Ligilactobacillus, Lactobacillus, and Erysipelothrix, the decrease in the abundance of the Bacteroidetes genera Bacteroides and Alistipes, the Firmicutes genus Dielma, and the Deferribacteres genus Mucispirillum. HDTO supplementation reversed the alterations in the intestinal flora by promoting the proliferation of beneficial flora during the aging process; the metabolic pathways, such as glycine–serine–threonine metabolism, valine–leucine–isoleucine biosynthesis, and some metabolic pathways involved in uridine, were also partially restored. Furthermore, the correlation analysis illustrated an obvious correlation between gut microbiota, its metabolites, and aging‐related indices. Moreover, it is worth noting that the metabolic regulation by dietary intervention varied with different HDTO doses and did not present a simple additive effect; indeed, each dose showed a unique modulation mechanism.
Aging is an irreversible reality that cannot be relieved by most people. Aging is a natural process that cannot be prevented. The body's ability to adapt to the environment is progressively weakened, and its antioxidant capacity also decreases with age. The generation and removal of free radicals is out of balance, and health status is worsening, especially the decline in immune function (Campisi et al., 2019; Finkel & Holbrook, 2000). In addition, some studies have suggested that the composition and number of human gut microbiota are prominently altered during the aging process (Yamauchi et al., 2020). As a result, the risk of several diseases significantly increases, such as cognitive impairment, cardiovascular diseases, diabetes, and cancer (Ahima, 2009; Drew, 2018; Wang & Bennett, 2012). However, the current aging has indeed become the most important trend in the change of the world's population age structure; according to statistics, the elderly population tripled from 4% to 13% in the last century and is expected to grow sharply to reach 20% of the population by 2025 and 33% by 2050 (Azman & Zakaria, 2019; Sander et al., 2015), which will bring tremendous pressure and unprecedented challenges to the world economy. Thus, carrying out research on delaying aging and reducing age‐related diseases, continuously deepening the understanding of the underlying mechanisms of the aging process, and finding effective antiaging treatments and interventions are vital to the development of human health (Clark & Walker, 2018; Ragonnaud & Biragyn, 2021). Healthy aging constitutes a real economic challenge for nations in the 21st century. Currently a reasonable diet and balanced nutrition are the most effective ways to suppress aging (Juárez‐Fernández et al., 2020). Studies have shown that adherence to a healthier diet may contribute to better physical, cognitive, and mental health during old age (Nijholt et al., 2016). Furthermore, increasing dietary intake of blue fish and long‐chain (LC) n‐3 PUFAs may help delay the accumulation of age‐related deficits (García‐Esquinas et al., 2019). Fish oil diet may be one of the best options for delaying aging and preventing various aging‐related diseases. Thus, the use of fish oil has increased in recent years. Fish oil is a rich source of n‐3 polyunsaturated fatty acids (PUFAs), particularly docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA), which are dietary fats with an array of health benefits (Jamshidi et al., 2020; Su et al., 2008). Among them, DHA is a key component of all cell membranes, and is found in abundance in the brain and retina. It can improve the fluidity of neuronal cell membranes, thereby affecting signal transduction of neuronal cells. By preventing macular degeneration, Alzheimer's disease, Parkinson's disease, and other brain diseases, while enhancing memory and neuroprotection, DHA can play an important role in ensuring healthy aging (Cardoso et al., 2016). Furthermore, there is evidence that habitual consumption of fish may reduce the risk of cognitive decline, dementia, and Alzheimer's disease (Cunnane et al., 2009). Tuna oil is a typical DHA‐rich fish oil, and the tuna oil employed in this study is a further concentrated tuna oil with a higher DHA content. The potential mechanism of dietary intervention with high DHA tuna oil to suppress aging was preliminarily explored from the changes in the gut microbiome and metabolome of mice with mimetic aging induced by d‐galactose.
High‐docosahexaenoic acid tuna oil (HDTO) utilized in the study was obtained from Ningbo Today Food Co., Ltd. Its fatty acid profile was determined by gas chromatography–mass spectrometry (GC‐MS; Agilent 7890/M7‐80EI system with a DB‐WAX column [60 m × 0.25 mm × 0.25 μm]) (Zhang et al., 2020).
Seventy‐two ICR mice (4‐ to 5‐week‐old males) with an average weight of 23.2 ± 2.1 g were purchased from the Laboratory Animal Center of Zhejiang Province (SCXK [Zhejiang] 2014‐0001). All experiments were performed in accordance with the Guide for the Care and Use of Laboratory Animals prepared by the Ningbo University Laboratory Animal Center (affiliated with the Zhejiang Laboratory Animal Common Service Platform) and experimental procedures and animal care were performed. All animal protocols were approved by the Ningbo University Laboratory Animal Center under permit number SYXK (ZHE 2008‐0110). After 1 week of acclimatization, 72 male ICR mice were randomly assigned to six groups: control group (Control), aging model group (Model), positive drug group (d‐GALDON), low‐dose HDTO treatment group (d‐GALLTO), medium‐dose HDTO treatment group (d‐GALMTO), and high‐dose HDTO treatment group (d‐GALHTO). Each group was housed at 23 ± 1°C, under a 12:12‐h light/dark cycle and 60 ± 5% relative humidity. The modeling experiment lasted for 6 weeks. The control group was intraperitoneally injected with saline (1000 mg/kg/day), and the model and each diet intervention group were intraperitoneally injected with d‐galactose (800 mg/kg/day). During the modeling process, the mice could obtain food and water normally. Both the control group and the model group were given saline (800 mg/kg/day) by gavage; the positive drug group was given donepezil (1.2 mg/kg/day) by gavage; in the low‐, medium‐, and high‐dose HDTO groups, 65, 260, and 820 mg/kg/day HDTO were administered intragastrically, respectively. In the last week of the experiment (the sixth week), nine mice were randomly selected to conduct the Morris water maze experiment to assess the alterations in spatial memory and working memory of the mice. At the same time, the feces of each group were collected and stored at −80°C for subsequent experiments.
Four marked points were set at equal distances on the upper edge of the barrel as the water entry point for the mice. The projection points of the four water entry points on the water surface and the bottom of the bucket divided the water surface and the bucket equally into four quadrants (I, II, III, and IV), and a circular platform with a diameter of 9 cm was hidden in quadrant I, 2 cm below the water surface. In each test, the mouse head was placed toward the wall and randomly placed into the water from one of the other three quadrants outside the platform. The time required for the mouse to find the submerged platform and stand on it was recorded. If the mouse found the platform and stayed on it for 10 s, its escape latency was recorded; if not, the escape latency was recorded as 90 s. Each mouse was put into the pool from the four water entry points as one training session, and the interval between the two training sessions was 15–20 s, which lasted for 5 days.
On the sixth day, the platform was removed and the mice were placed in the water maze in the quadrant opposite to the previous location of the platform. The time and distance that the mice spent in the target quadrant and the number of times the mice traversed the former location of the platform were recorded.
Blood was collected from the orbital plexus and the serum was further isolated by centrifugation at 1500 g at 4°C for 15 min and then stored at −80°C. Serum levels of high‐density lipoprotein cholesterol (HDL‐C), low‐density lipoprotein cholesterol (LDL‐C), total cholesterol (TC), and malondialdehyde (MDA), as well as the activities of catalase (CAT) and superoxide dismutase (SOD) were measured using commercially available kits (Nanjing Jiancheng Bioengineering Institute) according to the manufacturer's instructions.
Fecal samples were sent to LC‐Bio for 16S rRNA gene sequencing. The V3–V4 region of the 16S rRNA gene was amplified using primers 338F (5′‐ACTCCTACGGGAGGCAGCAG‐3′) and 806R (5′‐GGACTACHVG GGTWTCTAAT‐3′). The samples were sequenced using an Illumina MiSeq platform. Sequences with ≥97% similarity were assigned to the same operational taxonomic unit (OTU). Alpha and beta diversity analyses were conducted using QIIME (version 1.8.0). Linear discriminant analysis (LDA) scores derived from the LDA effect size (LEfSe, https://huttenhower.sph.harvard.edu/galaxy/root?tool_id=lefse_upload) were used to identify the specific bacteria (p < .05, LDA score > 3.6) (Segata et al., 2011). Correlation analysis was conducted using Spearman's correlation in R software (version 3.6.3).
Fecal samples were dried with a freeze dryer (Labconco) and then smashed. Fifty milligrams of smashed stool was placed in 2‐ml microcentrifuge tubes, followed by the addition of 400 μl phosphate‐buffered saline (PBS; 1.9 mM Na2HPO4, 8.1 mM NaH2PO4, 150 mM NaCl, pH 7.4). After 2 min of vortex mixing, the mixture was homogenized for 10 min using an ultrasonic homogenizer. The fecal slurry was centrifuged for 20 min at 4°C (26,000×g), and 200 μl of the supernatant was transferred to a dry EP tube. Then, 400 μl of ice methanol was added to the precipitate, which was vortexed for 10 min. After centrifugation at 26,000×g for 10 min, 200 μl of supernatant was pipetted and combined with the first 200 μl of supernatant, mixed well, and centrifuged. Finally, 300 μl of the mixture was transferred into a glass vial, and 2 μl of tetracosane standard solution was added, and the solvent was evaporated under a stream of nitrogen, followed by trimethylsilyl derivatization before GC‐MS analysis (Pechlivanis et al., 2014).
Analyses were conducted using an Agilent 6890/7000C Triple Quadrupole (Agilent) equipped with an HP‐5MS fused‐silica capillary column (30 m × 250 μm i.d.; Agilent J&W Scientific). The injection volume was 2 μl in split mode (split ratio 1:10), and the solvent delay time was set to 3.5 min. The initial oven temperature was maintained at 60°C for 3 min, increased to 200°C at a rate of 5°C/min, maintained for 1 min, then increased at 5°C/min to 300°C and maintained for 10 min. The temperatures of the injector, transfer line, and electron impact (EI) ion source were set to 280°C, 250°C, and 280°C, respectively. The electron energy was 70 eV, and mass data were collected in full scan mode (m/z 50–800). Pretreatment of the resulting MS data. The metabolites were identified using the NIST database. After removing any known pseudo‐positive peaks from the datasets, such as peaks caused by noise, column bleed, and N‐methyl‐N‐(trimethylsilyl)‐trifluoroacetamide (MSTFA) derivatization procedure, a CSV file was obtained that listed m/z and retention time with corresponding intensities for each metabolite from every sample in the positive dataset. An internal standard (tetracosane‐n‐heptane) calibration was used to reduce the deviation between individual samples. Finally, the normalized dataset was imported into SIMCA‐P software (v14.1, Umetric) for multivariate statistical analysis and plotting of fecal metabolite profiles, where supervised partial least squares discrimination analysis (PLS‐DA/OPLS‐DA) was applied to identify metabolites that significantly contributed to the classification. Metabolic features with variable importance in projection (VIP) values of >1.0, and p < .05 in the OPLS‐DA model were considered to be significantly different metabolites in the paired comparison. The metabolites selected above were subjected to enrichment analysis and pathway topology analysis using MetaboAnalyst (http://www.metaboanalyst.ca) to determine biologically meaningful metabolic patterns and the most impacted pathways.
Data are shown as means ± standard error of the mean (SEM) unless otherwise noted, and the data were analyzed using SPSS 23.0, GraphPad Prism version 8.0, and OriginPro software. Student's t test was used to identify differences between the two groups. For data whose distribution did not conform to the Gaussian model of heterogeneity, nonparametric Kruskal–Wallis analysis was employed. Differences were considered statistically significant at p < .05.
Aging is a complex process that is accompanied by the occurrence of multiple chronic diseases. Although it is inevitable, researchers are still trying to understand the underlying mechanism of this process and determine possible ways to regulate aging to delay its consequences (Kolovou et al., 2014). DHA is a type of n‐3 polyunsaturated fatty acid that has drawn much attention and has a significant effect on counteracting aging and improving cognition. Decreased DHA levels are associated with cognitive decline during aging (Cardoso et al., 2016). Loss of DHA from nerve cell membranes can result in central nervous system dysfunction, anxiety, irritability, dyslexia, impairment of memory and cognitive functions, and prolonged reaction time (Gow & Hibbeln, 2014; Hellhammer et al., 2012; Ross, 2009; Stonehouse et al., 2013). However, DHA cannot be synthesized by the human body, and therefore must be obtained from the diet. Fish oil diet is a recommended way to replenish DHA in modern diets. Tuna oil is a typical DHA‐rich fish oil. Nevertheless, the causal relationship between fish oil supplementation and cognitive function improvement is still unclear and further research is needed (Daiello et al., 2015). Therefore, we explored the effects of HDTO diet intervention on the changes in intestinal flora and metabolome of d‐galactose‐induced aging mice by supplementation with HDTO.
The Morris water maze test was conventionally employed to determine the spatial learning and memory abilities of mice. As indicated in Figure 1a, the 5‐day continuous testing revealed that the escape latency of d‐galactose‐induced aging mice to the platform was markedly longer than that of control mice (p < .05), while the platform area crossing times were obviously reduced after the platform was removed (p < .05). These results suggest that administration of d‐galactose significantly impaired the spatial memory and learning ability of mice. Compared with the model group, the escape latency of the mice with HDTO diet intervention was correspondingly shortened. Nonetheless, with the increase in fish oil supplement dose, the shortening of escape latency did not reflect a dose‐dependent relationship. Mice administered with a positive drug showed a reduction in escape latency only on the first day. Meanwhile, the results in Figure 1b indicate that when the aging mice were treated with HDTO and a positive drug, their platform area crossing times were obviously increased, and the time was increased from 3 to more than 4. However, there was no significant difference between the intervention and treatment effects, and the three doses of fish oil supplementation did not show a dose‐dependent reversal effect. The above results suggested that the aging model was well constructed, and HDTO diet intervention improved the spatial learning and memory ability of d‐galactose‐induced aging mice.
Serum lipid and lipoprotein levels increase with age, and accumulating studies have shown that oxidative stress is closely related to biological aging (Liu et al., 2020; Zhao et al., 2018). Given that, the activities of SOD and CAT, as well as TC, HDL‐C, LDL‐C, and MDA levels in d‐galactose‐induced mice were determined. Compared with the control group, d‐galactose injection led to a significant increase in the levels of LDL‐C and TC in the serum of mice (p < .05), and a decrease in the level of HDL‐C. Then, in comparison to the aging group, HDTO dietary supplementation significantly reduced the LDL‐C level (p < .05), and the TC level also decreased, but only when the HDTO dose reached the highest level had a remarkable effect (p < .05) and was restored to a level similar to that of the control group. Positive drug treatment also caused a significant increase and decrease in HDL‐C and LDL‐C levels (p < .05), but the TC level increased insignificantly. The results of the oxidation biomarkers in Figure 2b showed that in comparison with the control group, the MDA content of the model group mice was significantly elevated, and the activity of CAT and SOD were significantly reduced, and the activity of CAT was reduced by nearly three times. HDTO supplementation reversed the levels of these serum indicators in d‐galactose‐induced aging mice. All three doses of HDTO restored SOD activity to the level of the control group, but medium‐dose HDTO had no significant effect. With the increase in HDTO dose, CAT activity gradually restored to the level of the control group (p < .05), and the MDA content was reduced by 6.67%, 8.89%, and 3.33%, respectively, under the three HDTO doses, which did not comply with the dose effect. Positive drug treatment had a significant reversal effect on CAT activity (p < .05), and SOD activity increased marginally, but the increase in MDA content induced by d‐galactose could not be suppressed. These results indicate that HDTO exhibits significant hypolipidemic and antioxidant effects in d‐galactose‐induced aging mice, but these effects did not conform to the dose effect.
d‐galactose injection led to a natural aging status but did not cause a decline in the abundance of the intestinal flora of mice. The observed species and Chao1 index increased significantly (Figure 3a,b). Donepezil administration further increased the observed species and Chao1 index. In the three HDTO diet intervention groups, with the increase in HDTO dose, the observed species and Chao1 index showed a gradual decreasing trend. In comparison to the control group, the Shannon index of the intestinal bacteria in the model group at the OTU level increased slightly, while the Simpson index decreased; however, compared with the model group, the positive drug treatment and the three HDTO diet intervention groups all further increased the Shannon and Simpson indices; however, with the increase in HDTO dose, the increasing extent of Shannon index gradually decreased, but statistical significance was not observed (Figure 3c,d). As shown in Figure 4, the flora distribution in the control group, the model group, the positive drug treatment, and the three diet intervention groups were remarkably separated, suggesting that the bacterial composition of each group was different. Furthermore, the flora distribution of d‐galactose‐induced aging mice with low, medium, and high doses of HDTO diet intervention gradually approached that of the control group with the increase in the intervention dose. Therefore, the species composition and structure of the gut microbiota in the aging mice supplemented with high‐dose HDTO were more similar to those of the control group. From the perspective of spatial distribution, the flora distribution of the donepezil treatment group was closer to that of the model group. It can be seen from the above results that d‐galactose injection caused an increase in the abundance and diversity of gut microbiota in mice, but the 6‐week HDTO diet intervention failed to significantly restore these changes to that of the control. However, the structural composition of the intestinal bacteria of the mice in each group was significantly different, and the structural composition of the intestinal bacteria of the mice in the HDTO diet intervention groups was more similar to that of the control group. The alteration in the intestinal flora of each group due to differences in diet was further analyzed at the species level (Figure 5). At phylum level (Figure 5a), the dominant bacterial community in the control, model, positive‐drug treatment, and three HDTO dietary intervention groups included Bacteroidetes (the relative abundances were 44.33%, 34.55%, 69.14% and 76.15%, 70.98%, 80.46%, respectively), Firmicutes (the relative abundances were 39.73%, 54.52%, 19.46% and 15.36%, 16.07%, 16.40%, respectively), Proteobacteria (the relative abundances were 12.23%, 8.56%, 7.80% and 8.30%, 10.22%, 2.77%, respectively), Deferribacteres (the relative abundances were 1.41%, 0.77%, 1.52% and 0.15%, 2.55%, 0.28%, respectively), and Tenericutes (the relative abundances were 0.62%, 1.60%, 2.08% and 0.03%, 0.17%, 0.06%, respectively). d‐galactose injection significantly decreased the abundances of Bacteroidetes, Proteobacteria, and Deferribacteres, while that of Firmicutes and Tenericutes significantly increased. Compared with the model group, HDTO diet intervention remarkably reversed the changes in Bacteroidetes, Firmicutes, and Tenericutes in the mice flora induced by d‐galactose, and even the extent of increase and decrease was more obvious, while the changes in the abundance of Proteobacteria and Deferribacteres reversed only at medium doses of HDTO. The positive drug treatment notably reversed the abundance changes of Bacteroidetes, Firmicutes, Deferribacteres, and Tenericutes. At the genus level, d‐galactose injection prominently elevated the abundance of Ligilactobacillus, Lactobacillus, and Erysipelothrix, and decreased that of Bacteroides, Alistipes, Dielma, and Mucispirillum in the gut microbiota of mice. Among them, only the abundance alterations in Ligilactobacillus, Bacteroides, Alistipes, and Erysipelothrix were reversed by HDTO diet intervention (Figure 5b). The effects of the positive drugs were similar. LDA effect size (LEfSe) analysis was further employed to identify specific phenotypes (with LDA scores >3.6) that significantly differed in response to d‐galactose or HDTO (Figure 6). Compared with the control group, the abundance of Pseudescherichia in the model group mice increased significantly, and Dielma decreased significantly; compared with the three HDTO diet intervention groups, the biomarkers with significant difference were Pseudescherichia, Erysipelothrix, Spiroplasma, Falsiporphyromonas, Duncaniella, Desulfovibrio, Bacteroides, Breznakia, and Desulfitobacterium. The positive drug administration caused a prominent alteration in the abundance of Pseudescherichia, Spiroplasma, Odoribacter, Roseburia, Erysipelatoclostridium, Duncaniella, Desulfovibrio, Bacteroides, and Millionella in the gut microbiota of d‐GALDON mice. These results indicated that HDTO supplementation significantly ameliorated the structure and composition of the gut microbiota in d‐galactose‐induced mice.
GC‐MS/MS was used to determine the metabolites in fecal samples. To confirm the effect of HDTO diet intervention on the metabolic pattern of d‐galactose‐induced aging mice, and to identify the metabolites that significantly changed, partial least squares discriminant analysis (PLS‐DA) and orthogonal partial least squares discriminant analysis (OPLS‐DA) was used to analyze the data measured by GC‐MS/MS. As shown in Figure 7, all the groups achieved separation from each other within the 95% confidence interval, and on the t2 axis, it could be discerned that HDTO diet intervention restored the metabolic disorders of aging mice. The metabolic characteristics of the d‐GALDON group were similar to those of the model group. OPLS‐DA analysis was further performed to ascertain the metabolites that had the greatest effect on clustering, and those metabolites with variable importance in the projection (VIP) value ≥1.5, and Student's t test p value <.05 were screened as the discriminating metabolites (Figure S1). Control, d‐GALDON, d‐GALLTO, d‐GALMTO, and d‐GALHTO were compared with the model group, and 49 metabolites were identified. They are mainly carbohydrates and amino acids. In comparison with the model group, HDTO diet intervention at dose of 65 mg/kg/day significantly elevated the concentration of d‐arabinose, d‐erythopentose, l‐5‐oxoproline, l‐serine, tyrosine, l‐valine, l‐threonine, l‐methionine, l‐leucine, inositol, glycerol, uridine, and uracil, and markedly decreased the content of l‐fucose, cellobiose, and glycine; HDTO diet intervention at dose of 260 mg/kg/day significantly elevated the concentration of d‐galactose, d‐xylose, d‐glucose, 2‐α‐mannobiose, l‐serine, l‐proline, l‐valine, l‐threonine, glycerol, uridine, and pyroglutamine acid, and markedly decreased the content of d‐arabinose, l‐fucose, cellobiose, l‐5‐oxoproline, l‐aspartic acid, glycine, and amphetamine; at intervention dose of 820 mg/kg/day, the significantly increased metabolites were d‐galactose, d‐xylose, d‐arabinose, d‐glucose, d‐rhamnose, l‐threonine, l‐methionine, uridine, uracil, pyrimidine, lactic acid, and succinic acid, while l‐5‐oxoproline, l‐aspartic acid, and glycine were those metabolites that were remarkably decreased. Metabolic pathway analysis (MetPA) was performed using metaboanalyst 5.0 (p < .05). The occurrence of disorders of eight metabolic pathways might be associated with d‐galactose supplementation (Figure S2), namely aminoacyl–tRNA biosynthesis, valine–leucine–isoleucine biosynthesis, glycine–serine–threonine metabolism, pantothenic acid and CoA biosynthesis, glycolysis/gluconeogenesis, glutathione metabolism, alanine–aspartic acid–glutamic acid nucleotide metabolism, and acetaldehyde acid–dicarboxylic acid sugar metabolism. The metabolic pathways changed by positive drug treatment included aminoacyl–tRNA biosynthesis, valine–leucine–isoleucine biosynthesis, valine–leucine–isoleucine degradation, glycine–serine–threonine metabolism, pantothenic acid and CoA biosynthesis, galactose metabolism, glutathione metabolism, glyoxylic acid–dicarboxylate metabolism, and cysteine–methionine metabolism. The effects of three doses of HDTO diet on the metabolic alterations in d‐galactose‐induced aging mice were compared. The metabolic pathways changed by all three doses were: aminoacyl–tRNA biosynthesis, pantothenic acid–CoA biosynthesis, glutathione metabolism, and glycine–serine–threonine metabolism. The metabolic pathways changed by low‐ and medium‐dose HDTO diet interventions included phenylalanine–tyrosine–tryptophan biosynthesis, glyoxylic acid–dicarboxylic acid esters metabolism; the metabolic pathways that are altered by medium‐ and high‐dose HDTO diet interventions were β‐alanine metabolism, alanine–aspartic acid–glutamate metabolism, and valine–leucine–isoleucine biosynthesis was a metabolic pathway that was altered by both low‐ and high‐dose HDTO. The metabolic pathways individually regulated by low‐dose HDTO diet intervention were galactose metabolism, cysteine–methionine metabolism, and valine–leucine–isoleucine degradation, and the pathways of amino sugar–nucleotide sugar metabolism and glycolysis/gluconeogenesis were regulated by medium‐ and high‐dose HDTO diet interventions, respectively. It can be seen from the above results that the metabolic regulation by HDTO diet intervention would vary with different doses, but by no means a simple additive effect. Each dose had its own regulatory mode. Moreover, the changes in metabolic pathways caused by positive drug treatment were all observed in d‐galactose‐induced aging mice with HDTO diet intervention.
The relationships between six biochemical indices and significantly different genera of gut microbiota were analyzed by establishing a correlation matrix to calculate Spearman's correlation coefficient. As indicated in Figure 8, for the serum lipid metabolism indices, LDL‐C was negatively correlated with Bacteroides, Desulfovibrio, and Duncaniella (|r| ≥ .6), while positively correlated with Spiroplasma (r = .82, p < .05); HDL‐C was positively correlated with Bacteroides, Desulfovibrio, and Duncaniella (r ≥ .6), and negatively correlated with Spiroplasma (r > .9, p < .01); TC was positively related to Erysipelothrix (r = .6). For the oxidative stress parameters, SOD was positively and negatively correlated with Falsiporphyromonas and Spiroplasma (|r| ≥ .6); CAT was negatively correlated with Breznakia and Erysipelothrix (|r| ≥ .6), while MDA was positively related to Erysipelothrix (r = .6).
The correlation between 10 biomarkers of gut microbiota screened from the comparison between the control group, three doses of HDTO group, and the model group and 28 differential metabolites was analyzed. As shown in Figure 9, Spiroplasma was negatively correlated with l‐methionine and uracil; Falsiporphyromonas was positively correlated with l‐valine and uridine; Erysipelothrix was negatively correlated with 2‐α‐mannobiose and d‐galactose; Duncaniella was positively correlated with l‐threonine, l‐methionine, and uracil; Dielma and Desulfitobacterium were negatively correlated with l‐proline and l‐fucose, respectively; Desulfovibrio was positively correlated with d‐xylose and l‐proline, and negatively correlated with d‐cellobiose; Breznakia was negatively correlated with 2‐α‐mannose and d‐galactose, and positively correlated with l‐5‐oxoproline; Bacteroides was positively correlated with l‐methionine and l‐fucose.
The results of this study suggest that HDTO has a protective effect on lipid metabolism disorders, oxidative stress, and cognitive impairment in d‐galactose‐induced mimetic aging mice. In addition, the gut microbiota plays a vital role in the body and regulates health conditions and disease progression. As reported in the literature, the composition, diversity, and functional features of the intestinal microbiota of aging‐associated gut microbiota are altered (Vaiserman et al., 2017). Therefore, establishing nutritional strategies aimed at counterbalancing the specific alterations occurring in the microbiota has been proposed as a promising treatment for age‐related metabolic and neurodegenerative diseases (Bana & Cabreiro, 2019). Dietary interventions targeting the flora can regulate host health and aging by enhancing antioxidant activity, improving immune homeostasis, inhibiting chronic inflammation, regulating fat deposition and metabolism, and preventing insulin resistance (Clements et al., 2018; Cӑtoi et al., 2020). It was found in our study that d‐galactose caused an increase in the abundance of mouse gut microbiota, while its diversity was not markedly altered, which was not entirely consistent with reports in the literature (Vaiserman et al., 2017). However, the changes in the observed species, Chao1, Shannon, and Simpson indices still reflected the trend of recovery to the control group status with the increase in HDTO intervention doses. From the perspective of species level, as reported in literature, 80% of the microbiota detected in the aging model was divided into three dominant species: Firmicutes, Bacteroidetes, and Actinobacteria (Lay et al., 2005); the ratio of Firmicutes/Bacteroidetes in adults determined by q‐PCR was significantly lower than that of the elderly (Mariat et al., 2009). In our study, Bacteroidetes and Firmicutes were the dominant flora, accounting for more than 80% of the total population. However, their distribution was remarkably altered by injection of d‐galactose in mice. The abundance of Bacteroidetes decreased from 44.33% to 34.55%, while the abundance of Firmicutes increased from 39.73% to 54.52%; thus, the ratio of Firmicutes/Bacteroidetes increased significantly, while HDTO diet intervention restored all these abundance changes. The decrease in the abundance of Proteobacteria was inconsistent with the description in the literature on the change of this phylum abundance in the aging state, and when the HDTO supplement reached the medium dose, the abundance level of these bacteria were restored to that of the control. Shin et al. (2015) reported that Proteobacteria was a potential diagnostic marker of dysbiosis, and according to some current research, the response to dysbiosis was an increase in its abundance. However, it remains controversial whether in related dysbiosis, Proteobacteria abundance is increased or decreased, and its individual change can be used as a marker, which still needs to be further explored (Ma et al., 2020). Parabacteroides has also been reported to exclude potential pathogens from colonizing the gut, which is beneficial for the host (Yang et al., 2018). Deferribacteres have been proposed to be age related (Godoy‐Vitorino et al., 2010). HDTO supplementation at medium‐dose levels restored its abundance level to that of the control group. The abundance of Tenericutes increased significantly in the aging model, and this change was consistent with the results of the inflammatory aging model (Kim et al., 2016). Yang et al. (2020) postulated that the bacteria Tenericutes phylum might be one of the key players in gut microbiota alteration associated with cognitive function upon diet intervention. HDTO supplementation significantly prevented this increase. An in‐depth study at the genus level can better understand which genera contribute to the alteration in the overall level of the phylum. The gut microbiota in the d‐galactose‐induced mice significantly increased the abundance of Ligilactobacillus, Lactobacillus, and Erysipelothrix of Firmicutes, and decreased the abundance of Bacteroides and Alistipes of Bacteroidetes, Dielma of Firmicutes and Mucispirillum of Deferribacteres. The metabolites that were markedly altered in the mimetic aging mice mainly included amino acids (l‐leucine, l‐methionine, l‐5‐oxoproline, l‐aspartic acid, glycine, proline, l‐valine, l‐threonine, tyrosine, l‐methionine, and l‐serine), sugars (d‐rhamnose, d‐erythritol, d‐xylose, 2‐α‐mannobiose, d‐arabinose, glucose, d‐galactose, l‐fucose, and d‐cellobiose), nucleic acids (uridine, pyrimidine, and uracil), organic acids (pyroglutamic acid, succinic acid, and lactic acid), alcohols (inositol and glycerol), and amphetamines. Amino acids are important building blocks required for protein synthesis and are essential for the synthesis of many bioactive molecules that participate in signal transduction, hormone production, reproduction, and muscle development (Dai et al., 2015). The levels of l‐serine and l‐threonine in the fecal metabolites of aging mice were significantly reduced, and glycine content increased. The inconsistent alterations in the levels of the three amino acids might reflect the disorder of the glycine–serine–threonine metabolic pathway, while glycine–serine–threonine metabolism is a pivotal metabolic hub related to life span (Aon et al., 2020). Branched chain amino acids (leucine, valine, isoleucine) play a key physiological role in regulating protein synthesis, metabolism, food intake, and aging (Le Couteur et al., 2020). l‐valine in the metabolites of d‐galactose‐induced aging mouse model was notably downregulated, and the level of l‐threonine was also significantly reduced. The biosynthetic pathway of valine–leucine–isoleucine may be disturbed. Uridine is a precursor of phospholipid synthesis, and its content is significantly reduced in the metabolites of model mice. Studies on elderly animals and neurodegenerative disease models have shown that combined supplementation of dietary phospholipid precursors (such as uridine, omega‐3 fatty acids, and choline) can increase cephalin, neurite outgrowth, synaptic proteins, dendritic spine formation, and nerve transfer (Perez‐Pardo et al., 2018). It has also been documented in the literature that phospholipids are associated with lower cholesterol levels (Ipsen et al., 1987; Ledreux et al., 2016). In our study, the levels of TC and LDL‐C were remarkably increased, the HDL‐C levels were notably decreased, and the mice showed abnormal serum lipid metabolism. This may be because d‐galactose disrupts uridine‐involved metabolism, thereby reducing the level of phospholipids and ultimately leading to an increase in cholesterol levels. Zhou et al. (2015) found that cholesterol levels in the liver of d‐galactose‐induced mice increased significantly. Compared with the model group, HDTO supplementation inordinately increased uridine levels; accordingly, serum cholesterol levels returned to the level of the control group. The microbial biomarkers Bacteroides, Desulfovibrio, and Duncaniella were negatively correlated with LDL‐C, Spiroplasma was significantly positively correlated with LDL‐C, Bacteroides, Desulfovibrio, and Duncaniella were positively correlated with HDL‐C, Spiroplasma was negatively correlated with HDL‐C, and Erysipelothrix was positively correlated with TC levels. 5‐Oxyproline is a product of glutathione metabolism, and is formed by the reduction of glutathione by glutamyl transpeptidase and glutamyl cyclotransferase. Glutathione is an antioxidant that removes reactive oxygen species produced by oxidative stress. The level of l‐5‐oxoproline in d‐galactose‐induced aging mice was significantly increased, owing to the possibility that d‐galactose induced a sudden increase in oxidative stress in mice and promoted the metabolism of glutathione. The content of l‐5‐oxoproline increased. However, when the supplemental dose of HDTO reached 260 mg/kg/day, its content in the fecal metabolites of d‐galactose‐induced aging mice returned to the level of the control group. The significant decrease in d‐arabinose in d‐galactose‐induced aging mice was related to a significant decrease in the abundance of Bacteroidetes in the intestine (Hor et al., 2019).
The modulatory effects of HDTO administration on aging mice were investigated. Behavioral experiments and serum biochemical indicators indicated that d‐galactose successfully induced senescence in mice, decreased memory and learning ability, increased blood lipid levels, and decreased antioxidant capacity. HDTO diet intervention significantly improved various aging manifestations. The abundance and diversity of gut microbiota in mice were elevated upon d‐galactose senescence induction. Specifically, at the phylum level, the abundance of Firmicutes and Tenericutes was significantly increased, while that of Bacteroidetes, Proteobacteria, and Deferribacteres was significantly decreased. At the genus level, the variation mainly presented as an increase in the abundance of the Firmicutes genera Ligilactobacillus, Lactobacillus, and Erysipelothrix, the decrease in the abundance of Bacteroidetes genera Bacteroides and Alistipes, Firmicutes genus Dielma, and Deferribacteres genus Mucispirillum. The significantly differential gut microbiota showed an obvious correlation with its metabolites and aging‐related indices. Meanwhile, d‐galactose resulted in disorders of eight metabolic pathways, and the metabolites with obvious changes mainly included amino acids, sugars, and uridine. HDTO diet intervention partially reversed the above alterations, and the reversal effect was different with the HDTO dose.
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PMC9632290 | Ying-Ying Zhao,Qian-Ming Xiang,Jia-Li Chen,Li Zhang,Wei-Long Zheng,Di Ke,Rong-Shu Shi,Kong-Wu Yang | SLC25A25-AS1 over-expression could be predicted the dismal prognosis and was related to the immune microenvironment in prostate cancer | 20-10-2022 | prostate cancer,SLC25A25-AS1,prognosis,biomarker,immune microenvironment | It has been established that long-chain coding RNA (lncRNA) SLC25A25-AS1 is associated with cancer progression. However, the roles and mechanisms of SLC25A25-AS1 in prostate cancer (PC) have not been reported in the literature. The present study explored the relationship between SLC25A25-AS1 expression and PC progression via comprehensive analysis. The pan-cancer expression of SLC25A25-AS1 was identified using data from The Cancer Genome Atlas (TCGA) database and tissue specimens from our hospital. The expression levels of SLC25A25-AS1 in various subgroups based on the clinical features were identified. The prognostic value of SLC25A25-AS1 and SLC25A25-AS1 co-expressed lncRNAs in PC patients was assessed by survival analysis and ROC analysis, and prognosis-related risk models of SLC25A25-AS1 were constructed. The relationship between SLC25A25-AS1 and the PC immune microenvironment was investigated using correlation analysis. SLC25A25-AS1 expression in PC was significantly increased and correlated with the T stage, clinical stage, Gleason score (GS), and dismal prognosis. SLC25A25-AS1 overexpression exhibited good performance in evaluating the prognosis of PC patients. The area under the curves (AUCs) of the 1-, 3-, and 5-year overall survival (OS) for SLC25A25-AS1 was 1, 0.876, and 0.749. Moreover, the AUCs for the 1-, 3-, and 5-year progress free interval (PFI) for SLC25A25-AS1 were 0.731, 0.701, and 0.718. SLC25A25-AS1 overexpression correlated with the infiltration of CD8 T cells, interstitial dendritic cells (IDC), macrophages and other cells. AC020558.2, ZNF32-AS2, AP4B1-AS1, AL355488.1, AC109460.3, SNHG1, C3orf35, LMNTD2-AS1, and AL365330.1 were significantly associated with SLC25A25-AS1 expression, and short OS and PFI in PC patients. The risk models of the SLC25A25-AS1-related lncRNAs were associated with a dismal prognosis in PC. Overall, SLC25A25-AS1 expression was increased in PC and related to the prognosis and PC immune microenvironment. The risk model of SLC25A25-AS1 have huge prospect for application as prognostic tools in PC. | SLC25A25-AS1 over-expression could be predicted the dismal prognosis and was related to the immune microenvironment in prostate cancer
It has been established that long-chain coding RNA (lncRNA) SLC25A25-AS1 is associated with cancer progression. However, the roles and mechanisms of SLC25A25-AS1 in prostate cancer (PC) have not been reported in the literature. The present study explored the relationship between SLC25A25-AS1 expression and PC progression via comprehensive analysis. The pan-cancer expression of SLC25A25-AS1 was identified using data from The Cancer Genome Atlas (TCGA) database and tissue specimens from our hospital. The expression levels of SLC25A25-AS1 in various subgroups based on the clinical features were identified. The prognostic value of SLC25A25-AS1 and SLC25A25-AS1 co-expressed lncRNAs in PC patients was assessed by survival analysis and ROC analysis, and prognosis-related risk models of SLC25A25-AS1 were constructed. The relationship between SLC25A25-AS1 and the PC immune microenvironment was investigated using correlation analysis. SLC25A25-AS1 expression in PC was significantly increased and correlated with the T stage, clinical stage, Gleason score (GS), and dismal prognosis. SLC25A25-AS1 overexpression exhibited good performance in evaluating the prognosis of PC patients. The area under the curves (AUCs) of the 1-, 3-, and 5-year overall survival (OS) for SLC25A25-AS1 was 1, 0.876, and 0.749. Moreover, the AUCs for the 1-, 3-, and 5-year progress free interval (PFI) for SLC25A25-AS1 were 0.731, 0.701, and 0.718. SLC25A25-AS1 overexpression correlated with the infiltration of CD8 T cells, interstitial dendritic cells (IDC), macrophages and other cells. AC020558.2, ZNF32-AS2, AP4B1-AS1, AL355488.1, AC109460.3, SNHG1, C3orf35, LMNTD2-AS1, and AL365330.1 were significantly associated with SLC25A25-AS1 expression, and short OS and PFI in PC patients. The risk models of the SLC25A25-AS1-related lncRNAs were associated with a dismal prognosis in PC. Overall, SLC25A25-AS1 expression was increased in PC and related to the prognosis and PC immune microenvironment. The risk model of SLC25A25-AS1 have huge prospect for application as prognostic tools in PC.
Long non-coding RNAs (lncRNAs) are a class of RNAs longer than 200 nucleotides that can participate in the regulation of epigenetics, cell cycle, cell differentiation, and other functions (1–7). For instance, the lncRNA SNHG1 is significantly upregulated in oral cancer, and its inhibition can suppress oral cancer cell proliferation. Current evidence suggests that SNHG1 can regulate oral cancer growth by regulating the miR-421/HMGB2 signaling pathway (1). Moreover, overexpression of lncARSR could increase the resistance of colorectal cancer (CRC) cells to oxaliplatin in vitro and in vivo. Inhibition of lncARSR expression could reduce cell viability and promote cell apoptosis, and its upregulation could induce the tumorigenesis of CRC cells (3). LncRNA AP003469.4 was significantly increased in hepatocellular carcinoma (HCC) tissues. AP003469.4 overexpression was an influencing factor for dismal prognosis in HCC patients and was related to short overall survival (OS) and disease-free survival (DFS). Downregulation of AP003469.4 expression could delay the cell proliferation, cycle transition, invasion, and migration and promote cell apoptosis (6). This finding indicated that inhibiting or promoting the expression of lncRNAs could delay cancer progression. Prostate cancer (PC) patients reportedly have a dismal prognosis and high mortality (8, 9). It was estimated that there will be more than 1,400,000 new cancer cases and more than 370,000 deaths in 2020 (9). The mainstay of treatment for PC patients is a comprehensive approach based on surgery. However, PC patients often have a dismal prognosis due to drug resistance and metastasis. An increasing body of evidence suggests that targeted therapy could improve the prognosis of cancer patients (10–13) and has gradually become a research hotspot. In recent years, lncRNA SLC25A25-AS1 has been associated with cancer progression (14, 15). The expression of SLC25A25-AS1 was significantly increased in the tissues and cells of non-small cell lung cancer (NSCLC). SLC25A25-AS1 overexpression was associated with shorter OS in patients with NSCLC. Importantly, SLC25A25-AS1 silencing could hinder cell proliferation, enhance the apoptosis rate of cells, and limit cell migration, invasion and tumor growth in NSCLC. Overall, SLC25A25-AS1 could exert a tumor-promoting effect by mediating the miR-195-5p/ITGA2 signaling pathway (14). Moreover, SLC25A25-AS1 has been reported to be significantly decreased in CRC tissues, and SLC25A25-AS1 overexpression could inhibit cell growth in CRC (15). No studies have hitherto reported the roles of SLC25A25-AS1 in PC progression. Therefore, this study sought to investigate the SLC25A25-AS1 expression in PC using a comprehensive analysis and the relationship between the overexpression level of SLC25A25-AS1 and the clinicopathological characteristics, diagnosis and prognosis of PC patients. The prognosis-related risk models of SLC25A25-AS1 were subsequently constructed. Finally, the relationship between SLC25A25-AS1 and the PC immune microenvironment was analyzed through correlation analysis to provide candidate targets for treating PC patients ( Figure S1 ).
The expression data of TPM and FPKM types in pan-cancer tissues (730 cases of normal tissues and 10,363 cases of tumor tissues) were obtained from The Cancer Genome Atlas (TCGA) (https://portal.gdc.cancer.gov) database, and the expression data of TPM type in pan-cancer tissues (727 cases of normal tissues, and 9,807 cases of tumor tissues) were downloaded from the XENA-TCGA (http://xena.ucsc.edu/) database in March 2022. The mean expression levels of SLC25A25-AS1 in normal and cancer tissues were compared.
Cancer and normal tissues from patients (N=8) with a pathological diagnosis of PC treated in our hospital were analyzed. These PC patients provided informed consent for using their data. Our study was approved by the Ethics Committee of the Affiliated Hospital of Zunyi Medical University. SLC25A25-AS1 expression in PC patient tissues was detected by Reverse Transcription-Polymerase Chain Reaction (RT-PCR) technique. Total RNAs from PC patient tissues were collected according to standard specifications, and the reverse transcription process was performed after quantification. The PCR procedure was performed after primer addition, and the relative SLC25A25-AS1 expression level in PC tissues was calculated.
The clinicopathological characteristics and prognostic data of PC patients were retrieved from TCGA database. The PC patients were grouped according to the clinicopathological characteristics, and different groups were compared to identify statistically significant differences. The expression data of SLC25A25-AS1 were analyzed, and PC patients were arranged into two groups according to the SLC25A25-AS expression value, and their prognostic values were compared by Kaplan-Meier (K-M) survival analysis. The relationship between the SLC25A25-AS1 expression and prognostic values in each subgroup based on the clinicopathological characteristics of PC patients were compared using K-M survival analysis.
Receiver operating characteristic (ROC) analysis is a widely acknowledged method used to assess the diagnostic accuracy of a particular test (16, 17). Over the years, ROC analysis has been used to assess the diagnostic value of molecules in cancer. AUC value’s closer to 1 were associated with higher diagnostic values. In the present study, the diagnostic values of SLC25A25-AS1 and SLC25A25-AS1 related lncRNAs expression levels in PC patients with the OS and progress free interval (PFI) at 1, 3 and 5 years were evaluated using ROC analysis.
Immune cell scores of PC tissues were analyzed by the ssGSEA algorithm (18). The correlation analysis explored the relationship between the SLC25A25-AS1 expression and the PC immune infiltrating cells. The immune cell expression values were arranged from low to high, and the immune cells were divided into two (high- and low-expression) groups by the median values of the immune cells, and the SLC25A25-AS1 expression level in the high- and low-immune cell expression groups were explored.
The lncRNAs of OS and PFI in PC were analyzed by COX regression, and the significant OS- and PFI-related lncRNAs were screened using the criteria: P < 0.05 and P < 0.001. SLC25A25-AS1 co-expressed lncRNAs in PC tissues were acquired based on the screening criteria correlation coefficient r > 0.6 or r < -0.6 and P < 0.001. The intersected lncRNAs between the OS-related lncRNAs, PFI-related lncRNAs, and SLC25A25-AS1 co-expressed lncRNAs were visualized by a Venn plot. The intersected lncRNAs were defined as SLC25A25-AS1-related prognostic lncRNAs.
The expression data of SLC25A25-AS1 related lncRNAs were retrieved from TCGA and matched and merged with the PC patient prognosis data. The expression data of SLC25A25-AS1 related lncRNAs were arranged, and the PC patients were grouped by the median expression value. K-M survival analysis was used to compare the relationship between SLC25A25-AS1-related lncRNAs and the OS and PFI of patients. patients. The value of SLC25A25-AS1-related lncRNAs AC020558.2, ZNF32-AS2, AP4B1-AS1, AL355488.1, AC109460.3, SNHG1, C3orf35, LMNTD2-AS1, and AL365330.1 for evaluating the OS and PFI at 1, 3, and 5 years was assessed by ROC analysis.
The relationship between SLC25A25-AS1, AC020558.2, ZNF32-AS2, AP4B1-AS1, AL355488.1, AC109460.3, SNHG1, C3orf35, LMNTD2-AS1 and AL365330.1 expression with OS and PFI in PC patients was analyzed by LASSO algorithm. The risk models of SLC25A25-AS1-related lncRNAs were constructed. K-M survival analysis identified the prognosis roles of PC patients in high- and low-risk groups (19).
The expression levels of SLC25A25-AS1 in the tissues and clinicopathological features of PC patients were explored using the Student’s t-test. K-M survival and ROC analysis were used to investigate the prognostic values of SLC25A25-AS1 and the co-expressed lncRNAs of SLC25A25-AS1. Correlation analysis was applied to understand the relationship between SLC25A25-AS1 expression and the PC immune microenvironment. A P-value < 0.05 was statistically significant.
We found that SLC25A25-AS1 was significantly overexpressed in CHOL, KICH, LIHC, LUAD, LUSC, PCPG, PC, STAD and UCEC tissues and downregulated in COAD, KIRC, KIRP and THCA tissues using the TPM type data from TCGA and XENA-TCGA database ( Figures 1A, 1B ). It was found that SLC25A25-AS1 was significantly overexpressed in CHOL, KICH, LIHC, LUAD, LUSC, PCPG, PC, STAD, and UCEC tissues and downregulated in COAD, KIRC, KIRP and THCA tissues based on FPKM data from TCGA database ( Figure 1C ).
Analysis of our clinical samples consistently showed that SLC25A25-AS1 expression was significantly up-regulated in most PC patients ( Figure S2 ). SLC25A25-AS1 expression was significantly elevated in T3 stage PC patients than in T2 stage patients ( Figure 2A ), and was significantly increased in N1 stage PC patients compared with N0 stage PC patients ( Figure 2B ). SLC25A25-AS1 expression was significantly increased in PC patients with positive surgical margins (R1) compared with PC patients negative for residual tumor (R0) ( Figure 2C ). SLC25A25-AS1 expression was significantly elevated in partial remission (PR) patients compared with complete remission (CR) patients ( Figure 2D ). Moreover, SLC25A25-AS1 expression was significantly elevated in PC patients with stable disease compared with the PC patients in CR ( Figure 2E ), and SLC25A25-AS1 was significantly increased in PC patients with Gleason Scores (GS) of 8 and 9 compared to PC patients with GS of 7 ( Figures 2F, G ). In terms of OS and PFI, higher SLC25A25-AS1 expression correlated with a poorer prognosis ( Figures 2H, I ). K-M survival analysis showed a significant correlation between SLC25A25-AS1 overexpression and a short OS and PFI ( Figure 3 ).
ROC analysis found that SLC25A25-AS1 overexpression has significant value in evaluating the prognosis of PC patients. The AUC of SLC25A25-AS1 for predicting OS at 1, 2 and 3 years was 1 ( Figure 4A ), 0.876 ( Figure 4B ) and 0.749 ( Figure 4C ), respectively. The AUC of SLC25A25-AS1 for PFI at 1, 2 and 3 years was 0.731 ( Figure 4D ), 0.701 ( Figure 4E ) and 0.718 ( Figure 4F ), respectively.
COX regression analysis revealed that SLC25A25-AS1 overexpression was an independent factor for short PFI in PC patients ( Tables 1 , 2 ). In addition, SLC25A25-AS1 overexpression was significantly realted to the dismal prognosis in subgroup PC patients using K-M survival. In this respect, T2-3 stage, T3 stage, T3-4 stage, N0 stage, M0 stage, CR, white people, age <=60 years, age >60 years, R0, R1, PSA (<4 ng/ml), and GS, SLC25A25-AS1 overexpression were independent predictors of the PFI in PC patients ( Figure 5 and Figure S3 ).
SLC25A25-AS1 overexpression was significantly correlated with the levels of neutrophils, CD8 T cells, macrophages, Tem, pDCs, iDCs, T helper cells, Tcm, TFH, Th17 cells, mast cells, Th2 cells, DCs, eosinophils and Th1 cells using the correlation analysis ( Figure 6 and Figure S4 ). SLC25A25-AS1 expression levels in CD8 T cells, macrophages, NK CD56bright cells, cytotoxic cells, mast cells, pDC, T helper cells, Th17 cells, neutrophils, Tem, TFH, and Th2 cells were found by grouping by the median expression value of immune cells which showed significant differences ( Figure 7 ).
Based on the predefined screening criteria, 143 lncRNAs were co-expressed with SLC25A25-AS1 ( Table S1 ), and 193 lncRNAs were significantly associated with OS in PC (P < 0.05), and 437 lncRNAs were associated with PFI (P < 0.001). The Venn plot showed 9 overlapping lncRNAs for the SLC25A25-AS1 co-expressed lncRNAs and was related to OS and PFI of PC patients, including AC020558.2, ZNF32-AS2, AP4B1-AS1, AL355488.1, AC109460.3, SNHG1, C3orf35, AL365330.1, and LMNTD2-AS1. Figure 8 shows that SLC25A25-AS1 was correlated with AC020558.2, ZNF32-AS2, AP4B1-AS1, AL355488.1, AC109460.3, SNHG1, C3orf35, LMNTD2-AS1, and AL365330.1 expression.
K-M survival analysis showed that SLC25A25-AS1-related AC020558.2, ZNF32-AS2, AP4B1-AS1, AL355488.1, AC109460.3, SNHG1, C3orf35, LMNTD2-AS1, and AL365330.1 overexpression were significantly correlated with a short OS and PFI in PC patients ( Figure S5 and Figure 9 ). ROC analysis found that overexpression of AC020558.2, ZNF32-AS2, AP4B1-AS1, AL355488.1, AC109460.3, SNHG1, C3orf35, LMNTD2-AS1, and AL365330.1 had significant value for the evaluation of the 1-, 3- and 5-year OS and PFI in PC patients ( Figure 10 and Figure S6 ).
The AC020558.2, ZNF32-AS2, AP4B1-AS1, AL355488.1, AC109460.3, SNHG1, C3orf35, LMNTD2-AS1, AL365330.1 and SLC25A25-AS1 expression data were merged with the prognosis data of PC patients. The risk factors for OS as AC020558.2 and SLC25A25-AS1 were screened by LASSO analysis. In the risk model of AC020558.2 and SLC25A25-AS1, the survival time of high-risk PC patients was significantly lower than low-risk patients ( Figure 11A ). LASSO analysis revealed that the risk factors for PFI were AC020558.2, AL355488.1, SNHG1, LMNTD2-AS1, and SLC25A25-AS1. In the risk model of AC020558.2, AL355488.1, SNHG1, LMNTD2-AS1, and SLC25A25-AS1, the PFI of high-risk PC patients was significantly lower than low-risk patients ( Figure 11B ).
There is a rich literature available suggesting that lncRNAs play important biological roles in PC progression (20–23). LncRNA TMPO-AS1 is increased in PC, and is relevant to a dismal prognosis. TMPO-AS1 overexpression could reduce cancer cell apoptosis and promote cell proliferation, cell cycle and migration (20). Interfering with LINC01679 expression could promote cell proliferation, metastasis, and tumor growth and inhibit cell apoptosis. Moreover, LINC01679 regulated PC cell growth and metastasis by regulating the miR-3150a-3p/SLC17A9 signaling pathway. LINC01679 and SLC17A9 expression levels were closely relevant to the dismal prognosis of PC patients (21). LOC440040 expression level was significantly upregulated in PC tissues and cells. LOC440040 overexpression in PC patients was associated with a short OS. LOC440040 overexpression was an independent risk lncRNA for dismal prognosis in PC patients. Inhibition of LOC440040 expression could inhibit cancer cell proliferation, migration and invasion (22). This finding indicated that inhibiting or promoting the expression of lncRNAs could delay cancer progression and improve the prognosis of cancer patients. At present, overwhelming evidence substantiates that SLC25A25-AS1 plays an important role in the occurrence and development of cancer (14, 15). SLC25A25-AS1 overexpression was relevant to a dismal prognosis in patients with NSCLC. Inhibition of SLC25A25-AS1 expression could reportedly slow cancer cell growth and migration (14), while overexpression of SLC25A25-AS1 could inhibit the growth of CRC cells (15). In our study, bioinformatics and PCR analyses substantiated that SLC25A25-AS1 was overexpressed in PC tissues. TCGA database analysis showed that SLC25A25-AS1 overexpression was significantly relevant to the T stage, pathological stage, and GS in PC patients. Moreover, SLC25A25-AS1 overexpression was associated with a short OS and PFI and could predict a dismal prognosis in PC patients. SLC25A25-AS1 overexpression was significantly relevant to the OS and PFI in PC patients with T2-3 stage, T3 stage, T3-4 stage, N0 stage, M0 stage, CR, R0, R1, PSA (<4 ng/ml), and GS. Our preliminary findings suggest that SLC25A25-AS1 functions as an oncogene in PC progression and is expected to be a future biomarker for this patient population. The risk models have been used to assess the dismal prognosis of cancer patients (24–28). The expression levels of AC012085.2, UBXN10-AS1 and LINC00261 were significantly downregulated, while the AP004608.1, AC104667.2, and AC008610.1 expression levels were upregulated in PC tissues. The risk model based on AP004608.1, LINC00261, AC012085.2, AC104667.2, UBXN10-AS1, and AC008610.1 was applied to evaluate the dismal prognosis of PC patients. In addition, a lncRNA-miRNA-mRNA regulatory network was established based on 6 autophagy-related AC008610.1, LINC00261, AC012085.2, AP004608.1, UBXN10-AS1, and AC104667.2, 17 miRNAs, and 12 autophagy genes to understand the signaling mechanisms underlying PC progression (25). NOL12 expression is reportedly upregulated in hepatocellular carcinoma (HCC) tissues and cells and correlates with a short OS. Inhibition of NOL12 expression levels could inhibit HCC cell growth and metastasis. The risk model based on the NOL12-related genes was an independent prognostic factor in patients with HCC. The survival of HCC patients in the low-risk group was significantly better than in the high-risk group (28). We found SLC25A25-AS1-related AC020558.2, ZNF32-AS2, AP4B1-AS1, AL355488.1, AC109460.3, SNHG1, C3orf35, LMNTD2-AS1, and AL365330.1 overexpression levels were associated with a short OS, and PFI, and exhibited significant value in diagnosing 1-, 3-, and 5-year suvival time in PC patients. In the risk model constructed, the OS and PFI of high-risk PC patients were significantly lower than low-risk patients, which indicated that our constructed risk model has huge prospects for application in evaluating the prognosis of PC patients. Immunotherapy has attracted significant interest in recent years for cancer treatment (12, 29–32). Programmed cell death 1/programmed cell death ligand 1 (PD-1/PDL-1) and immune checkpoint inhibitors targeting PD-1/PD-L1 play an important role in the treatment of pan-cancer patients. For example, PD-L1 overexpression was associated with dismal clinical prognosis in PC patients. The downregulation of PD-L1 expression levels in PC yields an anti-tumor effect (31). We found that SLC25A25-AS1 overexpression was significantly relevant to the PC immune microenvironment. Moreover, SLC25A25-AS1 overexpression was significantly relevant to the CD8 T cells, macrophages, Tcm, and other immune cell levels. Overall, we preliminarily demonstrated that the SLC25A25-AS1 expression level was relevant to the PC immune microenvironment. In this study, the roles of SLC25A25-AS1 in PC progression were comprehensively analyzed, indicating that SLC25A25-AS1 has huge prospects as a prognostic biomarker for PC patients. However, the clinical values of SLC25A25-AS1 and the risk model warrant further validation in clinical samples and analysis of the roles and signaling mechanisms of SLC25A25-AS1 in PC cell growth, migration, and tumorigenicity should be conducted in xenograft models. Future studies should focus on inhibition of the expression of SLC25A25-AS1 in PC cells and establishing xenograft models. In addition, the roles of SLC25A25-AS1 expression in PC immune cell infiltration should be investigated in vitro and in vivo. Taken together, our findings corroborate that the expression of SLC25A25-AS1 is significantly increased in PC tissues and associated with clinicopathological features and dismal prognosis. SLC25A25-AS1 overexpression was significantly relevant to the infiltration of CD8 T cells, iDCs, macrophages, etc. Besides, AC020558.2, ZNF32-AS2, AP4B1-AS1, AL355488.1, AC109460.3, SNHG1, C3orf35, LMNTD2-AS1, and AL365330.1 were significantly associated with the OS and PFI in PC patients. The risk models based on SLC25A25-AS1-related lncRNAs were relevant to a dismal prognosis and are expected to become prognostic tools for this patient population.
The original contributions presented in the study are included in the article/ Supplementary Material . Further inquiries can be directed to the corresponding authors.
The studies involving human participants were reviewed and approved by The Ethics Committee of the Affiliated Hospital of Zunyi Medical University. The patients/participants provided their written informed consent to participate in this study.
K-WY and R-SS established the central thrust of our study. Y-YZ and Q-MX drafted the manuscript, which was revised by DK, and J-LC. LZ and W-LZ analyzed the research data. All authors contributed to the article and approved the submitted version.
Our study was funded by the Zunyi City Joint Fund (Zun Shi Ke He HZ Word (2021) No. 73, and Zun Shi Ke He HZ Word (2022) No. 244).
We are extremely grateful for the support and help from ourdepartment at the Affiliated Hospital of Zunyi Medical University.
The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher. | true | true | true |
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PMC9632624 | Zi-Rui Huang,Qi-Zhen Huang,Ke-Wen Chen,Zi-Feng Huang,Yun Liu,Rui-Bo Jia,Bin Liu | Sanghuangporus vaninii fruit body polysaccharide alleviates hyperglycemia and hyperlipidemia via modulating intestinal microflora in type 2 diabetic mice | 17-10-2022 | Sanghuangporus vaninii,polysaccharide,hypoglycemic and hypolipidemic activity,inflammation,intestinal microflora | The disease of type 2 diabetes mellitus (T2DM) is principally induced by insufficient insulin secretion and insulin resistance. In the current study, Sanghuangporus vaninii fruit body polysaccharide (SVP) was prepared and structurally characterized. It was shown that the yield of SVP was 1.91%, and SVP mainly contains small molecular weight polysaccharides. Afterward, the hypoglycemic and hypolipidemic effects and the potential mechanism of SVP in T2DM mice were investigated. The results exhibited oral SVP could reverse the body weight loss, high levels of blood glucose, insulin resistance, hyperlipidemia, and inflammation in T2DM mice. Oral SVP increased fecal short-chain fatty acids (SCFAs) concentrations of T2DM mice. Additionally, 16S rRNA sequencing analysis illustrated that SVP can modulate the structure and function of intestinal microflora in T2DM mice, indicating as decreasing the levels of Firmicutes/Bacteroidetes, Flavonifractor, Odoribacter, and increasing the levels of Weissella, Alloprevotella, and Dubosiella. Additionally, the levels of predicted metabolic functions of Citrate cycle, GABAergic synapse, Insulin signaling pathway were increased, and those of Purine metabolism, Taurine and hypotaurine metabolism, and Starch and sucrose metabolism were decreased in intestinal microflora after SVP treatment. These findings demonstrate that SVP could potentially play hypoglycemic and hypolipidemic effects by regulating gut microflora and be a promising nutraceutical for ameliorating T2DM. | Sanghuangporus vaninii fruit body polysaccharide alleviates hyperglycemia and hyperlipidemia via modulating intestinal microflora in type 2 diabetic mice
The disease of type 2 diabetes mellitus (T2DM) is principally induced by insufficient insulin secretion and insulin resistance. In the current study, Sanghuangporus vaninii fruit body polysaccharide (SVP) was prepared and structurally characterized. It was shown that the yield of SVP was 1.91%, and SVP mainly contains small molecular weight polysaccharides. Afterward, the hypoglycemic and hypolipidemic effects and the potential mechanism of SVP in T2DM mice were investigated. The results exhibited oral SVP could reverse the body weight loss, high levels of blood glucose, insulin resistance, hyperlipidemia, and inflammation in T2DM mice. Oral SVP increased fecal short-chain fatty acids (SCFAs) concentrations of T2DM mice. Additionally, 16S rRNA sequencing analysis illustrated that SVP can modulate the structure and function of intestinal microflora in T2DM mice, indicating as decreasing the levels of Firmicutes/Bacteroidetes, Flavonifractor, Odoribacter, and increasing the levels of Weissella, Alloprevotella, and Dubosiella. Additionally, the levels of predicted metabolic functions of Citrate cycle, GABAergic synapse, Insulin signaling pathway were increased, and those of Purine metabolism, Taurine and hypotaurine metabolism, and Starch and sucrose metabolism were decreased in intestinal microflora after SVP treatment. These findings demonstrate that SVP could potentially play hypoglycemic and hypolipidemic effects by regulating gut microflora and be a promising nutraceutical for ameliorating T2DM.
Diabetes mellitus is a metabolism disorder disease mainly characterized by high levels of blood glucose (BG) (1). According to the Diabetes Alliance survey, the adult diabetes rate in the world was as high as 8.3% in 2019, and this proportion will increase to 9.6% by 2045. Furthermore, type 2 diabetes mellitus (T2DM) has multifactorial and complex pathogenesis (2). The main hazard of T2DM is the long-term high BG, which leads to the occurrence of some chronic diseases and complications, including hyperlipidemia, inflammation, damage, and lesions of tissues and organs such as kidneys, eyeballs, hearts, and blood vessels (3). The existing strategy to alleviate diabetes mainly involves oral anti-hyperglycemic agents (biguanides, sulfonylureas, glucagon-like peptide-1 (GLP-1) receptor agonists, insulin sensitizers, α-glucosidase inhibitors, and thiazolidinedione) (4), which may be accompanied by many side effects (5). Therefore, it needs to be committed to developing novel antidiabetic drugs from natural sources with outstanding biological activity and low toxicity. Growing evidences suggest that 16S rRNA sequencing has provided an important and effective platform to provide a standard method for studying intestinal microflora (6, 7). The interactions between the host and intestinal microflora powerfully affect the wellness and illness of the host. Hence, Dysbiosis or imbalance of the intestinal microflora is a hallmark of metabolic disease (8). Recently, fungal-derived polysaccharides have been shown to ameliorate metabolic syndrome by regulating intestinal microflora, such as Ganoderma lucidum, Hericium erinaceus, and Grifola frondosa (9). The treatment of diabetes by polysaccharides is associated with their fermentation by intestinal microflora to short-chain fatty acids (SCFAs), which play a key role in alleviating T2DM (10, 11). “Sanghuang” is a popular medicinal fungus available, with a reputation of being “forest gold” in China and “Meshimakobu” in Japan (12). There are various confusing names for Sanghuangporus, Phellinus linteus, Phellinus baumii, Inonotus sanghuang, and “Sanghuang,” and others (13, 14). However, the three species of Sanghuangporus sanghuang, Sanghuangporus baumii, and Sanghuangporus vaninii were well-recognized in China (15). Recently, some modern pharmacological studies have illustrated “Sanghuang” has anti-tumor, hypoglycemic, antioxidant, and hepatoprotective effects (16). It was reported that P. linteus polysaccharide improves insulin signaling by modulating the ratio of phosphatidylcholine to phosphatidylethanolamine and the ratio of S-adenosylmethionine to S-adenosylhomocysteine in mouse plasma to lower BG concentrations, thereby increasing glucose tolerance ability (17). In addition, P. linteus water extract could decrease serum lipid by suppressing the activity of 3-hydroxy-3-methylglutaryl coenzyme-A coenzyme, which is a reductase in the liver (18). P. linteus polysaccharide extract could reduce lipopolysaccharide content, reduce inflammatory factors and reverse insulin resistance by regulating the abundance of intestinal microbiota that produces SCFAs (19). However, there is no research investigating the antidiabetic effects of S. vaninii fruit body polysaccharide (SVP). Hence, this work was designed to prepare and characterize the SVP, and explore the underlying mechanism of oral SVP against T2DM in mice from the perspective of intestinal microflora. Moreover, the correlation between intestinal microflora, biochemical indexes, and SCFAs were investigated, providing a scientific reference for exploiting innovative active foods for people with T2DM-induced hyperglycemia and hyperlipidemia.
Firstly, the dried powders of S. vaninii fruit body were treated with 30 times the volume of 70% (v/v) ethanol by ultrasound at 60°C for 1 h twice, to remove alcohol-soluble substances. Secondly, the residue was ultrasound (45 kHz, 300 W) with 50 times the volume of distilled water for 1 h at 60°C, and subsequently extracted by water for 2 h twice. Then, the combined supernatant was concentrated at 60°C and then added to four volumes of 95% (v/v) ethanol and kept at 4°C overnight. After centrifugation, the crude polysaccharide was collected and washed with ethanol, acetone, and ether. Next, the crude polysaccharide was further treated by the Sevag method to remove protein. Finally, the deproteinized supernatant was lyophilized to yield SVP after 48 h dialysis membranes (3.5 kDa cut-off), and the extraction yield of SVP was 1.91 ± 0.25%.
The total sugar content was measured using the phenol-sulfuric acid method, and the protein content was determined by Bradford’s method. The results showed that the total sugar and protein contents were 60.06 ± 1.67 and 3.41 ± 0.64%, respectively. Moreover, Monosaccharide composition analysis was analyzed using the 1-phenyl-3-methyl-5-pyrazolone (PMP) derivatization method according to the previous protocol (20). The average molecular weight of SVP was analyzed according to a previous study (21). For analysis of fourier transform-infrared (FT-IR) spectrometry, the dried SVP (0.5 mg) was mixed with KBr powder and pressed into a 1 mm pellet, then the characteristic organic functional groups (scan range: 4,000 to 370 cm–1) were analyzed by a Bruker VERTEX 33 spectrometer (Bruker Corporation, Germany) (22).
Forty male ICR mice (SPF, 4 weeks age) were sourced from Wu Shi’s Experimental Center Laboratory (Fuzhou, China). All mice were kept in clean conditions with a 12 h light/dark cycle and extensive ventilation, the temperature was set at 22–26°C, and the relative humidity was controlled at 50–60%. All mice were treated with a normal-fat diet (NFD) and water ad libitum in the first week. After that, the mice were randomly divided into two groups: (1) NC group: mice were fed a NFD for 4 weeks (n = 8); (2) HSHCD group: mice were fed a high-sucrose and high-cholesterol diet (15% lard, 15% sucrose, 1% cholesterol, 10% yolk, 0.2% sodium deoxycholate, and 58.8% NFD) for 4 weeks (n = 32). After 4 weeks of intervention, the HSHCD group mice were injected intraperitoneally with streptozotocin (STZ) solution at a dose of 45 mg/kg three times during 1 week. And the mice in the NC group were intraperitoneally injected with an equal volume of citric acid buffer solution. The fasting blood glucose (FBG) levels were detected by the BG meter, and the successfully modeled T2DM mice (FBG value >11.1 mmol/L) were selected for next experiment. Finally, the T2DM mice from HFD group were also randomly divided into four groups (n = 8): DC (diabetic control), MET (oral 100 mg/kg/day of metformin), SVP-L (oral 100 mg/kg/day of SVP), and SVP-H (oral 300 mg/kg/day of SVP). The body weight (BW) of each mouse was measured every 2 weeks. After the experiment, the feces of each mouse were collected into a special centrifuge tube, snap-frozen in liquid nitrogen and stored at −80°C. The blood of mice was collected by eyeball, and then centrifuged at 3,000 × g for 10 min to obtain serum and stored at −80°C.
All animals fasted overnight, and the values of BG were detected and marked as BG0 h. Then, all the mice were immediately treated with oral administration of glucose solution (2 g/kg B.W.). Next, the BG concentrations of mice after 0.5, 1, and 2 h were also detected and marked as BG0.5 h, BG1 h, and BG2 h, respectively. Finally, the area under the curve (AUC) were calculated by formula: AUC = 0.5 × (0.5 × BG0 h + BG0.5 h + 1.5 × BG1 h + BG2 h).
The levels of total cholesterol (TC), triglyceride (TG), high-density lipoprotein-cholesterol (HDL-c), low-density lipoprotein-cholesterol (LDL-c), free fatty acid (FFA), total bile acid (TBA), and glycated serum protein (GSP) were determined by assay kits (Jiancheng, Nanjing, China). In addition, fasting insulin (FINS), GLP-1, tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), and interleukin-10 (IL-10) levels were determined by ELISA kits (Chundu, Wuhan, China). Besides, the levels of insulin-related indexes were also calculated by formulas: HOMA-IRI = FBG (mmol/L) × FINS (mIU/L)/22.5; HOMA-ISI = 1/[FBG (mmol/L) × FINS (mIU/L)].
The fecal sample of each mouse (250 mg) was put into a 2 ml centrifuge tube with 1 ml ultra-pure water. The mixture was centrifuged (4°C, 135,399 × g) for 5 min, and then the supernatant was added to 0.1 ml 50% concentrated sulfuric acid (v/v) and 1 ml anhydrous ether. The mixture was centrifuged (4°C, 135,399 × g) for 10 min. The sample was prepared by taking the ether layer with a 0.22 μm filter membrane. Different concentrations of SCFAs standard products were prepared. Then the GC-MS program was conducted according to a previous study (23).
The DNA genome was extracted from feces, and the V3–V4 region of the bacterial 16S rRNA was amplified with universal primers 338F and 806R. The 16S rRNA gene sequencing libraries of bacteria were produced by using a TruSeq DNA PCR-Free Sample Preparation Kit (Illumina, San Diego, CA, USA). The raw reads were obtained from Illumina NovaSeq PE250 platform (24), and the high-throughput sequencing was conducted by Novogene Co., Ltd. (Beijing, China).
Principal component analysis (PCA) in different groups was performed using SIMCA-14.1 (UMETRICS, Sweden). The differential abundances of intestinal microflora between paired groups using STAMP 2.1.3. The correlation data were analyzed and visualized as heatmap by R 4.1.0 with packages of “psych” and “pheatmap.” The correlation network was visualized by Cytoscape 3.9.1.
The results of this work were presented as mean ± standard deviation (SD), and the GraphPad Prism 8.4 was used for the statistical significance of one-way ANOVA analysis with Turkey’s test, and the significance level was set at p < 0.05 and marked with different letters.
The bioactivities of polysaccharides are hugely influenced by their chemical composition and structure characteristics (25). The results showed SVP is mainly composed of mannose, rhamnose, glucuronic acid, galacturonic acid, galactose, and arabinose in a molar ratio of 35.23, 6.45, 2.33, 9.64, 16.13, and 28.94%, respectively (Supplementary Table 1). The results of average molecular weight showed that SVP were mainly composed of 4 peaks, and the mass fractions of peak 1, 2, 3, and 4 were 4.7% (Mw 1051.4 kDa), 65.8% (Mw 31.2 kDa), 12.5% (Mw 12.5 kDa), and 4.9% (Mw 12.3 kDa), respectively (Supplementary Figure 1 and Supplementary Table 2). The above results exhibited that the main components of SVP are small molecular weight polysaccharides. According to several studies in recent years, low molecular weight polysaccharides usually have higher antioxidant (26) and antitumors (27), hypoglycemic activities (28), and potential as prebiotics (29). FT-IR spectroscopy is a powerful technique for the identification of characteristic organic groups in the bioactive polysaccharide and proteins. As shown in Figure 1, the strong absorption peak at 3,427 cm–1 was attributed to the tensile vibration of O-H. The small absorption peak at 2,932 cm–1 represents the stretching and bending vibrations of C-H (22). The two absorption peaks at 1,651 and 1,555 cm–1 are related to the stretching vibrations of C=O. The absorption peaks at 1,427 and 1,366 cm–1 accorded with the stretching and bending vibrations of C-H. The absorption peaks at 1,286 and 1,247 cm–1 were associated with the asymmetric O=S=O. The strong absorption peak at 1,080 cm–1 indicates the stretching vibration of C-O-C (21). The stretching vibration at 917–877 cm–1 were related to both α-glycosidic and β-glycosidic bonds between glucosamine units.
Body weight (BW) loss is one of the typical symptoms of STZ-injected diabetic mice (30). Compared with NC group, a significant (p < 0.01) weight loss of mice in DC group was observed from 0 to 4 weeks of the experiment (Figure 2A). After 2 weeks of intervention, SVP-L supplementation strongly (p < 0.01) reversed the weight loss in T2DM mice. However, BW of MET and SVP-H group mice did not show statistical differences from DC group. Compared with DC group, BW of MET, SVP-L, and SVP-H group mice were significantly (p < 0.01) increased at fourth week of experiment. These results suggested that long-term administration of SVP could improve impaired energy metabolism, carbohydrate utilization, and the symptom of weight loss in T2DM mice. It was illustrated FBG values of T2DM mice were markedly (p < 0.01) higher than that of NC group mice at initial stage of experiment (Figure 2B). At second week of intervention, except for MET group, the SVP intervention significantly (p < 0.05) reduced the FBG values in contrast to DC group mice. In addition, oral administration of metformin, SVP-L, and SVP-H for 4 weeks significantly (p < 0.01) decreased the FBG levels of T2DM mice by 18.33, 37.30, and 43.15%, respectively. It was demonstrated that SVP could regulate BG with a dose-dependent effect.
Oral glucose tolerance test (OGTT) is often used to assess the ability of glucose toleration in human or experimental animals. Generally, the BG concentration will be maintained in a relatively stable range, indicating that the body has a strong tolerance (31). The BG levels in each group increased and peaked at 0.5 h, then gradually decreased (Figure 3A). In comparison with NC group, the AUC values were strongly (p < 0.001) elevated by 246.06% in DC group (Figure 3B), suggesting glucose tolerance of HFD rats was impaired. In comparison with DC group, the AUC values of MET, SVP-L and SVP-H group mice were all significantly decreased (p < 0.001), and the reduction ratios were 29.74, 27.93, and 36.14%, respectively. The experiment showed that SVP intervention decreased the levels of AUC of OGTT in a dose-addicted manner, and could partly restore the glucose tolerance in T2DM mice. GSP is a delicate indicator of diabetes, which is not affected by the fluctuation of interim BG concentration and could provide feedback on the brief-term remedial effect of the drug (32). GSP level in DC group was significantly (p < 0.001) increased compared with NC group (Figure 3C). By contrast, MET, SVP-L, and SVP-H intervention for 4 weeks significantly (p < 0.05) decreased the GSP levels by 12.55, 11.42, and 16.57%, respectively. GLP-1 is not only a gastrointestinal hormone but also a neurotransmitter, and it was used to combat T2DM as a new therapeutic (33). As shown in Figure 3D, a significant loss of GLP-1 concentration was found in DC group compared with NC group. In contrast to DC group, MET, SVP-L, and SVP-H intervention for 4 weeks all significantly elevated the GLP-1 concentrations in T2DM mice. In short, it can be speculated that SVP has a similar hypoglycemic ability with metformin. The HOMA of β-cell function (HOMA-β) was developed to quantize basic insulin secretion. And the increased HOMA-IRI level and decreased HOMA-ISI level are the primary characteristics of T2DM (22). Compared with NC group, the HOMA-IRI level in DC group was significantly (p < 0.001) increased whereas the HOMA-ISI level in DC group was significantly (p < 0.001) decreased (Figures 3E,F), suggesting that DC group mice had severe insulin resistance and lower insulin sensitivity. Moreover, compared with DC group, levels of HOMA-IRI in SVP-L and SVP-H groups were obviously (p < 0.001) decreased by 48.82 and 52.69%, respectively. At the same time, SVP-L and SVP-H significantly increased (p < 0.001) the values of HOMA-ISI by 103.84 and 117.29%, respectively, which showed a dose-dependence. These results suggest that SVP treatment could partly improve the insulin resistance and restore the insulin sensitivity in T2DM mice. The long-term hyperglycemia in diabetic patients will inevitably cause damage to various organs, and further lead to a series of chronic complications, especially the hyperlipidemia (2, 21). The concentrations of TC, TG, LDL-c, FFA, and TBA in serum were all significantly (p < 0.001) higher in DC group mice than those in NC group mice (Figures 4A–F). After 4 weeks of experiment, MET and SVP intervention significantly decreased the above lipid biochemical indexes. Especially, SVP-H intervention reduced TC, TG, LDL-c, FFA, and TBA levels by 56.49% (p < 0.001), 22.96% (p < 0.01), 48.68% (p < 0.001), 59.42% (p < 0.001), and 63.69% (p < 0.001), respectively. Interestingly, in contrast to DC group, there were no statistical differences of serum HDL-c levels in NC, MET, SVP-L, and SVP-H groups, which is consistent with a previous study (34). In short, it was indicated that SVP could prevent T2DM-induced hyperlipidemia, thereby reducing the risk of diabetic complications (35).
Chronic low-grade inflammation often occurs in patients with islet impairment and T2DM (36). Abnormally increased or decreased inflammatory cytokines could lead to islet dysfunction in diabetic patients (37). Compared with NC group, the serum concentrations of both TNF-α and IL-6 were obviously (p < 0.001) increased (Figures 4G,H) in DC group, which is consistent with a previous report (38). However, the serum IL-10 concentration was significantly (p < 0.05) decreased (Figure 4I) in DC group. In contrast to DC group, decreased TNF-α levels were observed in MET (4.70%, p < 0.05), SVP-L (1.91%), and SVP-H (6.98%, p < 0.05) groups, and the reduced IL-6 levels were found in MET (9.40%, p < 0.01), SVP-L (9.48%, p < 0.01), and SVP-H (11.38%, p < 0.001) groups. On the contrary, serum IL-10 level was significantly increased by 20.12% (p < 0.01), 15.80% (p < 0.05), and 27.53% (p < 0.001) in MET, SVP-L, and SVP-H groups, respectively. The results demonstrated that SVP intervention could ameliorated the inflammation status in T2DM mice.
The advantage of polysaccharides in improving diabetes is that they are fermented into SCFAs by intestinal microflora, the lack of which may lead to further development of T2DM (39). Compared to NC group mice, the concentrations of acetic acid, propionic acid, isobutyric acid, n-butyric acid, isovaleric acid, and n-valeric acid in DC group were strongly reduced by 50.76% (p < 0.001), 26.47% (p < 0.05), 70.60% (p < 0.001), 82.73% (p < 0.001), 87.89% (p < 0.001), and 54.21% (p < 0.001) (Figure 5). After oral administrated by SVP-H for 4 weeks, the SCFAs concentrations were significantly (p < 0.001) increased by 203.83, 220.56, 315.82, 723.30, 886.77, and 342.76%, respectively. The results demonstrated oral SVP can markedly enhance the SCFAs production by intestinal microflora in vivo.
Recently, intestinal microflora has received great attention, and its structure and stability are important for the maintenance of intestinal balance (40). Studies have shown that altering specific microorganisms in the intestine can affect the development of diabetes (41, 42). In this study, 16S rRNA based sequencing analysis was conducted to investigate the effects of SVP on the structure of intestinal microflora. It was illustrated that the phylum level of microflora was dominated by Firmicutes, Bacteroidetes, Verrucomicrobia, Actinobacteria, Proteobacteria, etc. (Figure 6A). Obviously, the Firmicutes population was increased from 55.92% in NC group to 80.52% in DC group. Simultaneously, the relative abundance of Bacteroidetes was reduced from 31.01 to 13.63% in DC group mice. However, the Firmicutes abundances of MET and SVP-H group were decreased to 30.71 and 42.55%, respectively, whereas the Bacteroidetes abundances of MET and SVP-H group were increased to 32.23 and 31.41%. Afterward, the Firmicutes/Bacteroidetes (F/B) ratio in DC group (5.91) was strongly higher than that of NC group (1.80). After treatment with metformin and SVP, the F/B values were decreased to 0.95 and 1.35, respectively. Usually, the F/B value is used as an essential indicator of the degree of intestinal microflora disturbance, and it is positively related hyperglycemia and hyperlipidemia in many studies (43–45). As shown in Figure 6B, the Lactobacillus, Akkermansia, Dubosiella, Bacteroides, unidentified_Enterobacteriaceae, Parabacteroides, and other genera dominated the fecal microorganisms. Compared with NC group, Lactobacillus population in DC group was strongly elevated, whereas the relative abundances of Akkermansia, Dubosiella, Alistipes, and others were decreased. In contrast to DC group, Lactobacillus population in the MET and SVP-H groups were obviously decreased. By contrast, Akkermansia, Dubosiella, Bacteroides, and other genera abundances were significantly increased. In short, these findings revealed that oral SVP could alter the T2DM-induced imbalanced structure of intestinal microflora at phylum and genus levels. The α-diversity analysis demonstrated that the SVP-H intervention could partly reverse the reduction of Shannon, Simpson, Chao1, and ACE indexes of intestinal microflora in T2DM mice (Supplementary Table 3). Furthermore, β-diversity is the difference in diversities across samples or environments, which indicated differences in intestinal microflora composition in this study (46). PCA is an effective indicator for evaluating β-diversity (47). It was illustrated that an obvious separation between NC and DC groups (Figure 6C), indicating T2DM could induce huge structural changes of intestinal microflora. In detail, the symbols of NC group are mainly distributed beside the positive x-axis whereas those of DC group are mainly enriched beside the negative y-axis. Interestingly, the symbols of MET and SVP-H group were mainly distributed at the first and second quadrant, which have great distances from NC group and DC group. It was indicated that SVP had the potential to alleviate T2DM by changing the composition of intestinal microflora to restore gut dysbiosis.
Furthermore, using PICRUSt2 to predict the functional potential of microbial communities through marker gene sequencing maps is a common strategy in amplicon sequencing (48). Compared to the previous version, PICRUSt2 provides a more credible algorithm to explore predicted metabolic function modules in intestinal microflora. Through the KEGG database’s comparison and analysis, the intestinal microbiota’s metabolic function in different groups was analyzed (49). Compared with NC group, there are 18 up-regulated functional modules in DC group (Figure 7A), such as Fatty acid degradation [PATH:ko00071], Quorum sensing [PATH:ko02024], D-Alanine metabolism [PATH:ko00480], Glycerolipid metabolism [PATH:ko00561], Glycolysis/Gluconeogenesis [PATH:ko00010], etc. By contrast, 22 functional modules of GABAergic synapse [PATH:ko04727], Longevity regulating pathway-worm [PATH:ko04212], Oxidative phosphorylation [PATH:ko00190], Histidine metabolism [PATH:ko00340], Thiamine metabolism [PATH:ko00730], etc. were reduced. Compared with DC group, there are 22 up-regulated functional modules of the top 40 modules in MET group, such as Arginine and proline metabolism [PATH:ko00330], GABAergic synapse [PATH:ko04727], Flote biosynthesis [PATH:ko00790], Citrate cycle (TCA cycle) [PATH:ko00020], etc. and the down-regulated functional modules contains Purine metabolism [PATH:ko00230], Quorum sensing [PATH:ko02024], Glycerolipid metabolism [PATH:ko00561], Starch and sucrose metabolism [PATH:ko00500], and so on (Supplementary Figure 2). As shown in Figure 7B, the 23 up-regulated and 17 down-regulated functional modules were illustrated in SVP-H group in contrast to DC group. For instance, the modules of Citrate cycle (TCA cycle) [PATH:ko00020], GABAergic synapse [PATH:ko04727], Histidine metabolism [PATH:ko00340], Longevity regulating pathway-multiple species [PATH:ko04213], Insulin signaling pathway [PATH:ko04910] were increased whereas Purine metabolism [PATH:ko00230], Glycolysis/Gluconeogenesis [PATH:ko00010], Fatty acid degradation [PATH:ko00071], Taurine and hypotaurine metabolism [PATH:ko00430], Starch and sucrose metabolism [PATH:ko00500] were decreased after SVP intervention in intestinal microflora of T2DM mice, which are similar to the effects of MET intervention in this study. These results provide a novel insight into the beneficial effects of SVP intervention on the T2DM-induced imbalance of microbial function.
Linear discriminant analysis (LDA) at the genus level was applied to further explore the effects of SVP intervention on the characteristic intestinal microbes in NC, DC, MET, and SVP-H groups. As shown in Figure 8, total of 25 characteristic microbes were analyzed with a threshold of LDA >3 and p < 0.05 among NC, DC, MET, and SVP-H groups. In detail, there were eight characteristic microbes enriched in NC group, namely Dubosiella, Alloprevotella, Marvinbryantia, Bifidobacterium, Enterorhabdus, Romboutsia, Turicibacter, and Faecalibaculum. In addition, three characteristic microbes were enriched in DC group, namely Lactobacillus, Flavonifractor, and Odoribacter. After metformin treatment, Akkermansia, Streptococcus, Candidatus_Stoquefichus, and Parasutterella populations were significantly increased in T2DM mice. Afterward, SVP-H group has 10 characteristic microbes, including Candidatus_Soleaferrea, Bacteroides, Parabacteroides, Anaerovorax, Cuneatibacter, Erysipelatoclostridium, Weissella, Holdemania, Klebsiella, and Enterococcus. Spearman correlation analysis was used to analyze the associations among characteristic microbes, biochemical indexes, and fecal SCFAs. It was illustrated (Figure 9A) that the increased relative abundances of characteristic microbes of Flavonifractor, Lactobacillus, Odoribacter in DC group were positively related to the levels of GLP-1, HOMA-ISI, BW, isovaleric acid, isobutyric acid, acetic acid, n-butyric acid, IL-10, n-valeric acid were negatively associated with the TNF-α, LDL-c, HOMA-IRI, TC, TBA, GSP, FBG, TG, FFA, and IL-6 concentrations. In contrast to these three genera, the relative abundances of Candidatus_Soleaferrea, Parabacteroides, Bacteroides, Weissella, and others which enriched in SVP-H group showed an opposite correlation with biochemical indexes and SCFAs. Moreover, the visualized network plot (Figure 9B) illustrated the strong correlation with a threshold of | r| > 0.6 and p < 0.01. On the one hand, Flavonifractor is one of the characteristic microbes in DC group, which was significantly positively related to the levels of TBA and TNF-α, and negatively correlated to BW level. It was reported that Flavonifractor belongs to Firmicutes and is positively associated with the level of ALT, AST, and TG in high-fat diet rats (50) and many circulating inflammatory markers (such as IL-1β, IL-6, and IL-21) in patients with end-stage kidney disease (51). The above results indicated that the increased abundances of Flavonifractor would regulate the levels of lipid metabolism-related indexes and inflammatory factors, and eventually lead to the occurrence of T2DM. On the other hand, Odoribacter, a genus belongs to Bacteroides phylum, also presents a higher abundance in DC group. And the relative abundance of Odoribacter was significantly negatively related to n-valeric acid and propionic acid levels, which is similar with a previous study (52). Besides, Odoribacter was reported to be enriched in hypercholesterolemic subjects (53) and highly abundant in db/db mice (54). By contrast, oral SVP increased the relative abundances of Weissella, Alloprevotella, Dubosiella, Erysipelatoclostridium, etc. in T2DM mice. Of which, Erysipelatoclostridium and Weissella are important SCFAs-producing microorganisms, and the relative abundance of Weissella in this study was significantly positively associated with n-valeric acid, n-butyric acid, isovaleric acid, isobutyric acid whereas significantly negatively related to FFA level, which is consistent with many previous studies (55, 56). In addition, the relative abundance of Alloprevotella in this study was significantly positively associated with HOMA-ISI and negatively related to levels of FBG, GSP, TBA, TG, TC, and HOMA-IRI. It was reported that Alloprevotella has shown anti-inflammatory activities (57) and is negatively related to obesity and relative dyslipidemia (58), which is similar with our findings. Moreover, it was revealed the relative abundance of Dubosiella was significantly positively related to GLP-1, HOMA-ISI, BW and significantly negatively related to TG, HOMA-IRI, FBG, TC, LDL-c, TBA, and GSP levels, which is consistent with a previous study that a significant reduction of Dubosiella was found in T2DM mice. And it was shown that the Dubosiella abundance showed a positive relationship with levels of HDL-c, GSH-Px, T-AOC, SOD, and a negative relationship with TC, LDL-c, MDA and FBG levels (59). Additionally, the relative abundance of Dubosiella was also found made great contribution to the perturbations of lipid metabolism in the disease process (60). It was suggested that Dubosiella may contribute to alleviation of insulin resistance and lipid metabolism. Interestingly, it was reported that the Erysipelatoclostridium is one of the dominant intestinal genera in obesity mice (61), which was considered to be harmful to human health (62). However, the relative abundance of Erysipelatoclostridium in this study was significantly positively associated with propionic acid, n-valeric acid, acetic acid, isovaleric acid, n-butyric acid, isobutyric acid and significantly negatively related to FFA level. A previous study also showed Erysipelatoclostridium abundance was positively associated with the production of acetic acid and butyric acid (63). These findings demonstrated SVP could modulate the structure and function of intestinal microflora that produce more SCFAs to alleviate T2DM and its complications in T2DM mice.
In the present research, characterization of SVP showed that it mainly contains small-molecular polysaccharides. And oral SVP strongly reduced the T2DM-induced abnormally elevated levels of FBG, GSP, OGTT, AUC of OGTT, HOMA-IRI, and markedly increased the levels of BW, GLP-1, HOMA-ISI in mice, indicating that SVP treatment could ameliorate the hyperglycemic state of T2DM. Oral administration of SVP in T2DM mice also could improve the diabetic complications of hyperlipidemia and inflammation response, indicating as lower concentrations of TC, TG, LDL-c, FFA, TBA, TNF-α, IL-6, and higher levels of IL-10 in serum. Especially, oral SVP strongly increased the concentrations of acetic acid, isobutyric acid, n-butyric acid, isovaleric acid, and n-valeric acid in feces of T2DM mice. Furthermore, 16S rRNA sequencing analysis demonstrated SVP treatment altered the structure of intestinal microflora and improve the gut imbalance, indicating as increased relative abundances of Weissella, Alloprevotella, Dubosiella, etc., and decreased relative abundances of Lactobacillus, Flavonifractor, Odoribacter, etc. The predicted metabolic functions of the intestinal microflora were also changed after SVP treatment in T2DM mice, such as higher levels of Citrate cycle, GABAergic synapse, Insulin signaling pathway, and lower levels of Purine metabolism, Taurine and hypotaurine metabolism, Starch and sucrose metabolism. These results illustrate that SVP has well potential to be a possible diabetes treatment.
The data presented in this study are deposited in the NCBI SRA BioProject repository, and the accession number is PRJNA867313.
The animal study was reviewed and approved by the Animal Ethics Committee of Fujian Agriculture and Forestry University.
Z-RH conceived and designed the experiment and drafted the manuscript. Q-ZH and K-WC performed the experiments and collected the data. Z-FH and YL assisted with the interpretation of the data and checked the statistical analyses. R-BJ and BL provided the resources and reviewed the manuscript. All authors contributed to the article and approved the submitted version. | true | true | true |
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PMC9632790 | Kevin Gozzi,Ngat T. Tran,Joshua W. Modell,Tung B. K. Le,Michael T. Laub | Prophage-like gene transfer agents promote Caulobacter crescentus survival and DNA repair during stationary phase | 03-11-2022 | Gene transfer agents (GTAs) are prophage-like entities found in many bacterial genomes that cannot propagate themselves and instead package approximately 5 to 15 kbp fragments of the host genome that can then be transferred to related recipient cells. Although suggested to facilitate horizontal gene transfer (HGT) in the wild, no clear physiological role for GTAs has been elucidated. Here, we demonstrate that the α-proteobacterium Caulobacter crescentus produces bona fide GTAs. The production of Caulobacter GTAs is tightly regulated by a newly identified transcription factor, RogA, that represses gafYZ, the direct activators of GTA synthesis. Cells lacking rogA or expressing gafYZ produce GTAs harboring approximately 8.3 kbp fragment of the genome that can, after cell lysis, be transferred into recipient cells. Notably, we find that GTAs promote the survival of Caulobacter in stationary phase and following DNA damage by providing recipient cells a template for homologous recombination-based repair. This function may be broadly conserved in other GTA-producing organisms and explain the prevalence of this unusual HGT mechanism. | Prophage-like gene transfer agents promote Caulobacter crescentus survival and DNA repair during stationary phase
Gene transfer agents (GTAs) are prophage-like entities found in many bacterial genomes that cannot propagate themselves and instead package approximately 5 to 15 kbp fragments of the host genome that can then be transferred to related recipient cells. Although suggested to facilitate horizontal gene transfer (HGT) in the wild, no clear physiological role for GTAs has been elucidated. Here, we demonstrate that the α-proteobacterium Caulobacter crescentus produces bona fide GTAs. The production of Caulobacter GTAs is tightly regulated by a newly identified transcription factor, RogA, that represses gafYZ, the direct activators of GTA synthesis. Cells lacking rogA or expressing gafYZ produce GTAs harboring approximately 8.3 kbp fragment of the genome that can, after cell lysis, be transferred into recipient cells. Notably, we find that GTAs promote the survival of Caulobacter in stationary phase and following DNA damage by providing recipient cells a template for homologous recombination-based repair. This function may be broadly conserved in other GTA-producing organisms and explain the prevalence of this unusual HGT mechanism.
Horizontal gene transfer (HGT) is a powerful and common process in bacteria, enabling the facile acquisition of new DNA that can rapidly and dramatically alter the physiology or survival of a cell. HGT impacts both the short-term adaptability of bacteria and their long-term patterns of evolution [1,2]. Bacterial HGT mechanisms traditionally fall into 3 categories: transformation or natural competence, bacteriophage-mediated transduction, and direct cell–cell conjugation. A fourth, less studied mechanism involves gene transfer agents (GTAs), which were first identified and characterized in the α-proteobacterium Rhodobacter capsulatus [3]. GTAs are thought to derive from defective, “domesticated” prophages that mediate gene transfer via phage-like protein capsids filled with double-stranded DNA [4]. In contrast to phages, which usually preferentially package their own DNA, GTA particles package genomic DNA fragments relatively nonspecifically [5,6]. In some cases, the packaged DNA may be depleted for the GTA-encoding locus due to high levels of local transcription that occlude the packaging machinery [5]. Notably, the length of the DNA fragment packaged into a GTA is not sufficient to contain the entire GTA locus, thus GTAs cannot transfer themselves horizontally [7]. Moreover, in R. capsulatus and likely other GTA-producing species, several genes necessary for GTA production are separated in the genome from the locus encoding the structural components of the GTA [8]. In short, GTAs are not infectious phage-like particles and instead are likely a means of disseminating DNA to other cells in a population. However, their properties and functions remain very poorly understood. Genome sequence analysis suggests that GTAs are widespread in the α-proteobacteria and were present in one of the common ancestors of this clade [9]. Given the numerical dominance of α-proteobacteria in marine environments, GTAs may be critical in such environments where HGT is extremely prevalent [10]. GTAs have been discovered in some other taxa, including the δ-proteobacterium Desulfovibrio desulfuricans, the spirochete Brachyspira hyodysenteriae, and the archaeal methanogen Methanococcus voltae, but they have not been as closely examined in those organisms [11–14]. The physiological benefit(s) of GTAs remain unclear. For R. capsulatus, GTAs are produced during starvation or other stress conditions, whereas for Bartonella grahamii, the fastest growing cells in a population are both the producers and receivers of GTAs [6,15,16]. However, in neither case have GTA-producing cells been shown to harbor an advantage over non-producers. GTAs are frequently postulated to spread beneficial alleles through a population [6,7], but evidence supporting this function is lacking. Moreover, mathematical modeling indicates that it is more favorable for a population if cells that acquire a beneficial allele simply grow and propagate the allele via vertical inheritance rather than distribute the allele via GTAs [17]. This work further suggested that in a mixed population of GTA producer and non-producers, the non-producers will eventually take over. Here, we identified and characterized the GTA produced by the α-proteobacterium Caulobacter crescentus, an organism traditionally studied to understand cell cycle regulation and cellular asymmetry in bacteria. We found that GTA production in Caulobacter is tightly regulated by the transcription factor RogA, which directly represses 2 genes, gafYZ, that are necessary and sufficient, if expressed together, to activate GTA transcription. Cells lacking rogA or overexpressing gafYZ transcribe the primary, 21-gene GTA locus and several auxiliary loci, which combine to produce a particle that packages genomic DNA fragments of approximately 8.3 kbp. The production of GTAs leads to cell lysis and the release of particles into the surrounding environment. These released GTAs can transfer genetic material into a recipient cell, which can recombine the GTA-delivered DNA onto its chromosome. Importantly, we find that GTA production allows C. crescentus to better survive in stationary phase and to tolerate DNA damage during stationary phase by using GTA-derived DNA as a template for homologous recombination-based repair. Collectively, our work provides the first experimental characterization of Caulobacter GTAs and now demonstrates a physiological benefit of a GTA, which may be shared by other GTA-producing organisms.
We previously identified a RecA-independent mechanism by which C. crescentus cells respond to DNA damage, particularly double-strand breaks, culminating in activation of the transcription factor DriD in an SOS-independent manner [18] (Fig 1A). To identify additional factors involved in this pathway, we performed transposon mutagenesis on a strain harboring a reporter of DriD activity in which the promoter of the target gene didA was fused to lacZ (Fig 1B). In the absence of DNA damage, wild-type colonies harboring the reporter appear white, indicating low basal didA promoter activity. A loss-of-function mutation in a negative regulator was predicted to result in high promoter activity and blue colonies. After screening >10,000 colonies, multiple independent insertions were identified in CCNA_00080, a previously unannotated gene predicted to encode a 213-amino acid protein. Bioinformatic analyses suggested that CCNA_00080, now named RogA (Repressor of GTA activators), encodes a putative LexA-like transcriptional repressor with an N-terminal helix-turn-helix DNA-binding domain and a C-terminal LexA/cI-like autopeptidase domain though key residues required for cleavage of LexA/cI are missing (Fig 1B). To verify that RogA negatively regulates didA expression, we generated a marked rogA deletion strain (ΔrogA::tetR) in which all but the first 5 and last 5 codons of rogA were replaced by the tetracycline resistance cassette. Using our PdidA-lacZ reporter, we found that this ΔrogA mutant had approximately 4-fold higher didA promoter activity in stationary phase with a more modest 2-fold increase in exponential phase (Fig 1C). These increases in didA expression were complemented by providing a wild-type copy of rogA on a low-copy plasmid. The elevated expression of didA in the ΔrogA mutant was abolished when driD was deleted (Fig 1C). Compared to wild-type, further DNA damage in the ΔrogA mutant during early stationary phase did not result in an increase of didA expression, suggesting rogA is upstream of DNA damage activation of this pathway (S1 Fig). Together, these findings suggest that RogA indirectly regulates didA by somehow modulating DriD’s expression or activity, possibly by causing an accumulation of double-stranded DNA breaks and ssDNA, which are known to activate DriD [18,19].
To identify other genes regulated by RogA, we used RNA-seq to examine transcriptional changes in a ΔrogA mutant compared to the wild-type strain. Notably, we observed strong (approximately 500 to 1,000 fold) up-regulation of a 21-gene cluster (locus 9 in Fig 1D and 1E) predicted to encode a putative GTA similar to that found in R. capsulatus (S2A Fig). The GTA of C. crescentus has never been characterized and bioinformatic studies have suggested that C. crescentus may have an incomplete GTA cluster [20]. In addition to the GTA cluster, we also observed approximately 40-fold up-regulation of an operon consisting of CCNA_01111 and CCNA_01112 (locus 5 in Figs 1D and 1E and S2B), which are homologous to the N- and C-terminal portions, respectively, of the GTA activator gafA in R. capsulatus [21]. CCNA_01111 was previously named hdaB as overexpressing this gene can impact chromosomal DNA replication [22]; however, given the homology to GafA and the results presented herein, we propose GafY and GafZ as names for CCNA_01111 and CCNA_01112 for consistency with R. capsulatus. To better understand the prevalence of GafY, GafZ, and RogA, we searched for homologs across 1,331 α-proteobacteria genomes. Homologs of RogA have not previously been implicated in GTA regulation in other species. However, RogA is highly conserved in α-proteobacteria, with at least 1 homolog in 819 of the 1,331 sequenced genomes. Additionally, RogA commonly co-occurs with core GTA genes (CCNA_02880–02861) and auxiliary gene homologs such as gafYZ and CCNA_02456 (S2C Fig). We identified 759 GafZ homologs with 494 containing an adjacent GafY homolog. GafY and GafZ homologs were also found more often as separate genes than fused into one, as in R. capsulatus. As noted above, DriD was identified as an activator of didA in response to double-stranded DNA breaks [18]. Given that maturation of bona fide GTA particles would involve the production of linear DNA fragments, we hypothesized that such fragments drive DriD-dependent activation of didA in the ΔrogA mutant (Fig 1C). To test if GTA-packaged DNA was accumulating in the ΔrogA mutant, total DNA was isolated from cells in stationary phase, which was when didA expression was highest in the ΔrogA mutant (Fig 1C), and examined by gel electrophoresis (Fig 2A). In addition to the bright, low-mobility band representing genomic DNA, we observed a second band approximately 8 to 10 kb in length for DNA extracted from the ΔrogA mutant, but not wild-type cells or ΔrogA cells complemented with wild-type rogA (Fig 2A). This putative GTA DNA in a ΔrogA mutant was not seen if the primary GTA locus was also deleted (Fig 2A). Similarly, the putative GTA band was not seen in a ΔrogA mutant also harboring a deletion of either of the putative GTA activators gafY and gafZ (Fig 2A). Given that gafY and gafZ were necessary for GTA DNA band production in a ΔrogA mutant, we tested whether these 2 genes were also sufficient to induce GTA production. The gafY and gafZ coding regions were cloned either individually or together on a high-copy plasmid, with expression driven by a xylose-inducible promoter. The GTA band strongly accumulated only when both gafY and gafZ were induced (Fig 2B). In this experiment, rogA was still present, indicating that gafY and gafZ act downstream of rogA, with RogA likely serving to repress gafYZ expression and GafYZ acting as an activator of the GTA gene cluster (Fig 2C). Overexpression of gafYZ, and gafY alone to a lesser degree, in stationary phase was also sufficient to activate expression of the didA promoter, further suggesting that GTA production leads to DNA strand breaks throughout the cell (S2D Fig). To better understand the role of GafYZ in activating the Caulobacter GTA system, we performed RNA-seq on cells harboring a xylose-inducible copy of gafYZ or an empty vector, each treated with xylose for 3 h in stationary phase. Cells overexpressing gafYZ exhibited a >1,000-fold induction of genes in the GTA cluster (Fig 2D). We also observed strong induction of several loci distal to the GTA-encoding cluster (Fig 2D, loci #1–8, 10). We observed robust up-regulation of ccna_02456, which has homology to the GTA tail fiber gene in R. capsulatus. Locus 4 contained the gene ccna_01082, which is predicted to encode a homeodomain-related protein with no known function. The remaining up-regulated genes in loci #1–3, 5–8, and 10 contained hypothetical genes with no known function. Consistent with the notion that GafY and GafZ function downstream of RogA, we observed a high correlation between the genes most up-regulated in cells overexpressing gafYZ or lacking rogA (r2 = 0.92; Fig 2E). In both cases, GTA activation led to a dramatic shift in bulk gene expression, with more than 8% of all mRNA reads for the ΔrogA mutant mapping to the GTA cluster and accessory genes, and approximately 49% for gafYZ overexpression, compared to <0.1% in wild-type cells (Fig 2F). Collectively, these results indicate that RogA represses GTA expression and that gafYZ are necessary and sufficient to induce GTA expression.
In R. capsulatus, the GTA activator GafA is a single polypeptide [21], whereas in C. crescentus, the GafA homolog is split into 2 proteins, GafY and GafZ (S2B Fig). To test whether GafY and GafZ form a complex, we performed a co-immunoprecipitation (IP) with a ΔrogA strain expressing an N-terminal FLAG-tagged version of gafZ from its native promoter. Anti-FLAG antibodies were used to IP FLAG-GafZ from cell lysate and the resulting eluate was then blotted with an anti-GafY polyclonal antibody. We observed significant enrichment of GafY in the IP compared to the pre-IP input control (Fig 2G). Another cytoplasmic protein, ParB, was not enriched in the IP sample, suggesting that the enrichment of GafY was specific. We hypothesized that RogA and GafY, which encode DNA-binding proteins, and potentially GafZ, act directly as transcription factors at target promoters. To determine their direct targets, we used chromatin immunoprecipitation paired with deep sequencing (ChIP-seq). For RogA and GafY, we used polyclonal antibodies raised against each protein and compared wild-type cells to the corresponding deletion strain. For GafZ, we used an anti-FLAG antibody and compared cells producing FLAG-GafZ to isogenic cells producing untagged GafZ. GafY was enriched immediately upstream of all but 1 of the 10 loci that were most up-regulated during GTA expression (ΔrogA and gafYZ overexpression), including the major GTA cluster and gafYZ itself (Fig 2D and 2H and S3A Fig). GafZ was found only within the promoter and coding regions of the major GTA cluster, with the highest occupancy within CCNA_02880, the first gene of the GTA cluster (Fig 2D and 2H and S3B Fig). RogA was enriched only at the promoters of gafYZ and CCNA_02002, a gene whose expression did not change during GTA induction (Fig 2D and S3C Fig). To validate our ChIP-seq analysis, we tested whether purified RogA (see Methods) can bind the gafYZ promoter in vitro. Using overlapping approximately 40-bp DNA fragments, we scanned the gafYZ promoter for RogA binding via surface plasmon resonance (SPR). We observed strong binding of RogA to 3 DNA fragments that span the predicted core promoter region for gafYZ, consistent with RogA being a repressor of this locus (S4A and S4B Fig). This region featured 2 inverted repeats of GGAA-N4-TTCC (S4C Fig), as did the promoter of CCNA_02002. We also observed strong interaction between purified GafYZ complex and the GTA promoter region (S4D Fig). We were unable to predict a binding motif for GafYZ in the promoters of target genes. Taken all together, our results demonstrate that RogA silences GTA production by binding directly to and repressing the promoter of gafYZ, which encode the direct activators of the GTA cluster and several auxiliary genes and operons.
To determine whether C. crescentus cells can produce functional GTA particles, we first sought to characterize the DNA fragments produced in cells lacking rogA. We isolated approximately 8 to 10-kbp DNA fragment produced by ΔrogA cells (Fig 2A) and then digested this DNA with SalI, a restriction endonuclease with thousands of restriction sites across the C. crescentus genome. The SalI-digested DNA ran as a smear with no distinct bands, suggesting that the GTA DNA is heterogeneous, as a less complex input would generate distinct bands (S5A Fig). We also used qPCR to show that various genomic regions could be amplified using the putative GTA DNA as template (S5B Fig), further indicating that the GTA DNA is heterogeneous. To more precisely assess the GTA DNA, we isolated it from gafYZ-overexpressing cells in stationary phase and then sequenced it using PacBio Long-Read DNA sequencing. The GTA DNA fragments were 8.3 kb on average and always comprised a single, continuous region of the C. crescentus genome. Notably, we detected all regions of the genome, though with approximately 10-fold range in relative abundance (Fig 3A). Two broad peaks were centered around 1.0 and 3.0 Mbp, the midpoints of the 2 arms of the chromosome, with sequencing depth minima near oriC and ter. There was no major decrease in packaging of the GTA locus itself (Fig 3A inset). GTA production in R. capsulatus causes lysis and death of the producing cells [23]. Similarly, we found that inducing gafYZ in C. crescentus led to approximately 100-fold decrease in viable cells after 60 min and approximately 1,000-fold decrease after 4 h (Fig 3B) with a concomitant increase in extracellular protein throughout the time course, indicative of cell lysis (Fig 3C). The lethality of overexpressing gafYZ was completely GTA-dependent (Fig 3B). To ensure that the lethality observed was not an artifact of overexpressing gafYZ, we also tracked cell lysis as ΔrogA and wild-type cells entered stationary phase. We observed a marked increase in cell lysis for the ΔrogA strain, as measured by protein release into the supernatant, as cells reached an OD600 of approximately 1.3 (Fig 3D), matching the stationary phase-dependence of our didA reporter (Fig 1C) in the ΔrogA mutant. We also observed a significant decrease in colony-forming units (CFUs) in the ΔrogA mutant compared to wild-type cells as they entered stationary phase, further suggesting GTA production correlates with cell lysis and lethality (S5C Fig). To test that cells were indeed lysing, we stained gafYZ overexpressing cells with propidium iodide to track cell death as GTA production was induced. We observed a similar appearance of dead cells after 2 h of induction (S5D Fig), in line with the increase in protein release observed (Fig 3C). No homologs of lysis-associated genes in R. capsulatus [5] were identified in the C. crescentus genome nor do any of the genes up-regulated during GTA expression have homology to holin/endolysin genes.
To determine if the cell lysis observed was associated with the release of GTA particles, we added SYBR-gold, a DNA-staining dye, to agarose pads and imaged cells harboring an inducible copy of gafYZ. In cells expressing gafYZ, there were many SYBR-gold positive, phase-contrasting particles smaller than an individual cell (Fig 4A). These particles could be precipitated with polyethylene glycol and separated from other cellular components after centrifugation through a sucrose gradient (S5E Fig) with the putative capsid proteins producing a banding pattern by SDS-PAGE reminiscent of that seen with R. capsulatus GTAs [24] (S5F Fig). Transmission electron microscopy (TEM) revealed many spherical particles in the supernatant of ΔrogA cells, but not wild-type cells (Fig 4B). These particles were approximately 40 nm in diameter with a spherical head and no visible tail, despite the presence of genes predicted to encode a tail tube, a hub/baseplate protein, and tail fibers (Fig 1D and S2A Fig). It is possible the tail is too short to resolve, which would differ from the long tails of RcGTA [25], or that sheer forces during the preparation for TEM led to loss of tails, in line with the observed sensitivity of GTAs to various purification methods [3]. Similar particles were also observed in PEG-precipitated supernatant from cells overexpressing gafYZ (S5G Fig). To test whether these particles could mediate genetic transfer, we generated “donor” strains harboring an inducible copy of gafYZ to drive GTA expression and a tetracycline resistance marker at chromosome position 1.0 Mb. The donor strain was then mixed with a “recipient” strain bearing a chloramphenicol resistance marker and inducer (0.3% xylose) was added. The co-incubated cells were plated after 4 h, selecting for doubly resistant colonies indicative of successful transfer. For donor or recipient cells incubated alone, no colonies were detected (Fig 4C). In contrast, when mixing donor and recipient cells, doubly resistant colonies emerged, with the highest number arising when GTA production was induced in stationary phase at OD600 = 1.4 (Fig 4C and 4D). The maximum rate of transfer of the tetR cassette from the highly packaged 1.0 Mb position in these conditions was approximately 1 × 10−6 per recipient cell. A key feature of GTA-mediated transfer is that transfer is DNase resistant [26]. Indeed, we observed no difference in transfer rates when the supernatant was treated with excess DNase to digest any exogenous DNA during co-incubation (compared to no DNase added), indicating that transfer does not occur through transformation by free DNA (Fig 4E). To test whether transfer required recipient cells to also be in stationary phase, we mixed stationary phase donor cells with either exponential or stationary phase recipient cells. There was no major difference in the transfer rate of tetR (Fig 4F). We also varied the donor:recipient cell ratio and found that the rate of transfer decreased once recipients outnumbered donors (Fig 4F). Finally, we tested transfer from a donor harboring a tetR cassette at 2.0 Mb, a genomic region that was packaged less frequently based on our long-read sequencing (Fig 3A). The efficiency of transfer was approximately 5- to 10-fold lower from this strain at 2 and 3 h post-induction of gafYZ, though comparable after 4 h (Fig 4F). Taken all together, our results demonstrate that Caulobacter cells can produce functional GTA particles capable of transferring DNA from one cell to another. Donor cells must be in stationary phase to produce functional GTA particles but can transfer to cells in either exponential or stationary phase.
We next sought to determine if there was any physiological benefit to GTAs in C. crescentus. Because GTA production and transfer increased upon entry to stationary phase, we compared wild-type and ΔrogA strains during stationary phase. For longer-term co-incubations, the ΔrogA mutant was used as opposed to gafYZ overexpression, as overexpression over long time periods would require inducer to be added several times because the inducer (xylose versus glucose) gets consumed. Over the course of 120 h, we observed approximately 10- to 30-fold increase in the survival of the GTA-producing ΔrogA mutant compared to wild-type cells (Fig 5A and 5B). Expressing rogA from its native promoter on a plasmid in the ΔrogA strain restored survival to wild-type levels. This long-term survival benefit of the ΔrogA mutant was lost if the GTA locus was also deleted, demonstrating that the phenotype observed was GTA dependent (Fig 5A and 5B). If the GTAs produced by ΔrogA cells promote survival, then this benefit should be cell-non-autonomous and therefore diffusible to other cells in the population. To test this idea, we mixed differentially marked wild-type and ΔrogA::tetR mutant cells. This co-incubation increased the viability of wild-type cells approximately 10-fold after 96 h in stationary phase relative to wild-type cells grown identically but alone (Fig 5C). There was also a corresponding decrease in survival of the ΔrogA cells when grown with wild-type cells, suggesting that the wild-type cells may act as “cheaters” and receive the benefit from GTA producers with no cost (Fig 5C). These findings support the notion that GTAs act as public goods and can benefit non-producing cells in the population. Given that we identified GTAs by studying DriD and the SOS-independent DNA damage response, we tested whether this benefit required DriD. We found that ΔrogA cells also bearing a deletion in driD still provided a benefit to co-incubated cells, suggesting the SOS-independent DNA damage response may not play a direct role in GTA production (S6A Fig). We considered 3 potential models to explain the beneficial effects of GTA production on the viability of C. crescentus in stationary phase: (1) GTAs allow cells to acquire and spread new (relative to the wild-type genome), beneficial alleles; (2) GTA production causes cell lysis, which causes subsequent release of beneficial nutrients and DNA from the cytoplasm into the supernatant; or (3) GTAs allow cells to acquire and use wild-type DNA to repair or revert deleterious mutations. To address the first model, we tested if the surviving wild-type cells following co-incubation with ΔrogA cells (Fig 5C) had acquired beneficial mutations from the ΔrogA strain and could better survive extended stationary phase. In many bacteria, mutations arise in stationary phase that provide a growth advantage; such mutations are heritable [27,28]. In principle, GTAs could be promoting the observed, approximately 10-fold increase in survival of wild-type cells co-incubated with the ΔrogA strain by driving distribution of such beneficial mutations. However, of 12 colonies tested here, all exhibited long-term survival comparable to naïve wild-type cells that had not been co-incubated with GTA producers (S6B Fig). This finding argued against model 1. Additionally, model 1 would have required any beneficial allele that arose in the GTA producer to somehow spread to most other cells in the population, which was unlikely given the timescale (72 to 96 h) of our experiments. To distinguish between models 2 and 3, we first asked whether the survival benefit conferred by GTA-producing cells was recombination dependent. If the benefit results from nutrient release by lysed cells or direct delivery of nutrients by GTAs, it should be recombination independent. However, if the benefit involves GTA-mediated transfer of genetic material, it would require recombination. We used a recombination-deficient strain, rec526, that grows comparably to the wild type but is recombination deficient [29]. Whole-genome sequencing of the rec526 strain revealed a truncation in recA due to a premature nonsense mutation (Q129*). When co-incubated for 96 h with either ΔrogA cells or wild-type cells, we observed no significant difference in the survival of the rec526 cells (Fig 5D). This result suggested that the diffusible benefit from GTA-producing cells requires recombination and that GTAs may provide a template for recombination-dependent DNA repair.
The role of DNA uptake via competence machinery for DNA repair has been studied across many species [30]. To further test whether GTAs provide templates for DNA repair to promote survival following DNA damage, we exposed stationary phase cultures of different strains to UV light. Intriguingly, we observed a 21 ± 4-fold increase in survival of the ΔrogA strain, compared to wild-type cells (Fig 5E). This benefit was completely ablated if the GTA-encoding locus was also deleted. The increased survival of ΔrogA cells was only observed for stationary-phase cells with wild-type, ΔrogA, and ΔrogA Δgta strains all exhibiting equivalent survival following UV treatment in exponential phase (S6C Fig). We also observed approximately 10-fold increase in the survival of UV-damaged wild-type cells exposed to GTA-rich supernatant from the ΔrogA strain (Fig 5F). Conversely, there was approximately 10-fold decrease in the survival of UV-damaged ΔrogA cells that recovered in GTA-poor supernatant from wild-type cells compared to recovering in ΔrogA supernatant. GTA-rich supernatant from ΔrogA cells provided no benefit to UV-damaged rec526 cells, further supporting the conclusion that the benefit of GTAs is recombination dependent (Fig 5F). We next tested whether DNA from GTAs can be used by cells to repair a specific DNA lesion, such as a double-strand break. For these experiments, we used a strain harboring both a vanillate-inducible, site-specific restriction enzyme (I-SceI) and a corresponding single restriction site on the chromosome [31]. Induction of this I-SceI system leads to a lethal double-strand break on the chromosome, resulting in sensitivity to vanillate. The strain also bore a chloramphenicol resistance marker to facilitate selection. This strain was grown to stationary phase and then co-incubated with an inducible GTA-producing “donor” strain (or a control strain) with the wild-type sequence in place of the I-SceI site (Fig 6A, top). Inducers were added to trigger the double-strand break in the recipient and GTA production in the donor. We then selected against the donor strain by plating on chloramphenicol while also either including vanillate, to further induce a double-strand break, or not. If GTAs mediate transfer of wild-type DNA that can be used to repair the break and thereby eliminate the I-SceI site, we would expect greater survival on vanillate for the recipient strain when incubated with a GTA-producing strain than the control strain. Only about 1 × 10−3 cells were vanillate resistant after 4 h of co-incubation with GTA-producing or control donor cells, indicating that most cells still had an intact I-SceI site and the double-strand break was lethal, as expected (Fig 6A, t = 4 h). However, the recipient strain grown with GTA-producing donors had approximately 100-fold more vanillate-resistant colonies after 48 to 72 h than when grown with the control strain (Fig 6A). Sequencing confirmed that most (12 of 14) vanillate-resistant colonies from the culture with a GTA-producing donor had indeed replaced the I-SceI site with wild-type DNA (Fig 6A). In contrast, none (of 13) vanillate-resistant colonies from the culture with a control donor strain had lost the I-SceI site. We conclude that recipient cells can use the DNA from GTAs to repair their chromosomes. The frequency of transfer and integration of wild-type DNA that replaces the I-SceI site is likely rare, but those rare recombinants can then grow unabated in the presence of vanillate. We also tested whether GTAs promote survival following treatment with the double-strand break agent zeocin (Fig 6B). We observed a substantial increase in survival of the ΔrogA strain compared to the wild type. This increased survival was GTA dependent as deleting any of 3 genes within the major GTA locus (CCNA_02880 –terminase; CCNA_02877 –capsid portal; CCNA_02872 –major capsid) in the ΔrogA strain restored zeocin survival to approximately wild-type levels. A full deletion of the GTA locus actually produced hypersensitivity to zeocin suggesting that a gene within the GTA locus promotes resistance to double-strand breaks. Indeed, deleting the gene CCNA_02873, which is on the antisense strand of the GTA locus and annotated as a putative bleomycin resistance gene, was responsible for the hypersensitivity of the ΔrogA Δgta strain. Complementation of rogA in the ΔrogA ΔCCNA_02873 strain did not restore sensitivity to wild-type levels, further suggesting that loss of CCNA_02873 alone was responsible for the increased zeocin sensitivity. Collectively, these results are consistent with the notion that GTAs may promote survival to double-strand breaks. To test a more general role of GTAs in mediating survival after DNA damage, we treated wild-type, ΔrogA, and ΔrogA Δgta strains with 3 other DNA-damaging agents (mitomycin C, MMC; hydroxyurea, HU; and ethyl methanesulfonate, EMS) and 2 non-DNA-damaging antibiotics (cephalexin and vancomycin) using disk diffusion assays. A small disk of filter paper impregnated with a given drug was placed on a hard agar plate with a top layer of soft agar containing cells taken from stationary phase cultures. The resulting inhibition zone radius is inversely proportional to the survival of the strain in the presence of the drug. The ΔrogA strain survived better than the wild-type or the ΔrogA Δgta strain in the presence of MMC and HU (Fig 6C and 6D), which can induce double-strand breaks directly or via replication fork collapse, respectively. For MMC, we found no difference between the different strains if the cells plated came from exponential phase cultures (Fig 6C and 6D). There was also a small increase in survival against EMS, which causes DNA damage (base alkylation) that is repaired by mismatch or base excision repair, but can cause double-strand breaks at high levels [32]. In contrast to MMC, HU, and EMS, no significant difference in survival was detected for ΔrogA cells in the presence of cephalexin or vancomycin, which inhibit cell growth independent of DNA damage (Fig 6C and 6D). Taken together, our results suggest that GTAs can improve a recipient cell’s ability to survive DNA damage.
GTAs are intriguing, but still poorly understood, vehicles for HGT, particularly in α-proteobacteria. Caulobacter crescentus was not previously known to produce GTAs and bioinformatic studies even suggested it may not produce functional particles [9]. However, our work indicates that Caulobacter can indeed produce GTAs. Moreover, these GTAs, each harboring approximately 8.3-kb fragment of the genome, can transfer genetic material to recipient cells. Enrichment of packaging was seen around the 1.0 and 3.0 Mbp positions, which result from chromosomal arm cohesion, localization of those regions to mid-cell, and proximity to the GTA cluster [33]. The DNA transferred can enable cells to acquire beneficial alleles or wild-type DNA that can be used for recombination-based repair (Fig 7). The physiological benefits of GTA production have long been a mystery in the field. GTAs can transfer beneficial alleles, such as antibiotic resistance genes, that aid survival during a subsequent selection imposed in the lab, but whether a similar scenario arises in nature is unknown. Here, we presented evidence that GTA production may provide a long-term survival benefit to C. crescentus. A GTA-producing population of ΔrogA cells survived long-term stationary phase approximately 10-fold better than wild-type cells in a GTA-dependent manner. This benefit appears to be a public good as co-incubating a GTA-producer with a non-producer increased survival of the latter. However, the benefit was not the transfer of a beneficial allele as the wild-type cells that survived better in the presence of ΔrogA cells behaved like naïve, monocultured wild-type cells in a second round of stationary phase challenge (S6 Fig). Importantly, the benefit of GTAs to recipient cells depends on homologous recombination. This result suggests that the DNA provided by GTAs is not used simply as a nutrient source and instead must be integrated into the genome. We found that GTA-producing cells better survived UV-induced DNA damage in stationary phase, again in a recombination-dependent manner. Thus, our results suggest that GTA production confers a benefit to C. crescentus during long-term growth conditions, potentially by acting as a reservoir of DNA for the repair of damaged chromosomal DNA. Consistent with this model, we found that GTA production was less advantageous when cells were challenged with other forms of DNA damage, such as alkylating agents, that can be repaired through pathways other than homologous recombination. UV damage, which typically leads to interstrand crosslinks, depends on homologous recombination to be repaired. Moreover, we found that GTAs were beneficial to a population of cells in which we introduced a single, site-specific double-strand break, a lesion that also requires homologous recombination for repair. C. crescentus does not encode a non-homologous end-joining system like some bacteria [34]. A hallmark of C. crescentus, and many α-proteobacteria, is that they often harbor only a single chromosome—particularly in stationary phase [35]—meaning they lack a sister chromosome template for homologous recombination-based repair. GTAs may represent a solution to this problem. Whether providing cells with templates for DNA repair is a bona fide function of GTAs in wild populations of cells remains to be shown. The role of DNA uptake during competence, or natural transformation, has been studied and reviewed at length [36], with several potential models suggested: The DNA is used as a nutrient source [37], for DNA repair [30,38], or for increasing genetic diversity [39]. Notably, our results suggest GTAs act to benefit cells by providing a source of DNA during DNA-damaging conditions, in line with competence studies suggesting a role in DNA repair. One caveat of our model is that the incoming DNA includes only approximately 8-kb region of the genome. However, given large population sizes and the potentially large “burst size” from GTA producers, repair could still occur at a high enough frequency to explain our observations that damaged wild-type cells benefit from co-incubation with GTA producers and can use GTAs to repair specific lesions (Fig 6A). Increasing recombination within a population via GTA production, especially in a species like C. crescentus that lacks competence machinery, may also help to avoid the accumulation of deleterious mutations, a phenomenon known as Mueller’s Ratchet [40]. Another evolutionary conundrum regarding GTA production is that the benefit of GTAs must outweigh the heavy cost of producing GTAs, which requires cell lysis and the altruistic sacrifice of some cells for others in the population. Unlike natural transformation, where the production of uptake machinery occurs in the receiving cell and thus any benefit of transformation could outweigh the cost of machinery production, the energetic burden of GTA production appears to be solely on the producer, which will then go on to lyse and guarantee cell death. This evolutionary constraint necessitates a population-wide benefit to outweigh the cost of programmed cell death in individuals, such as seen in division of labor models or social evolution theory [41,42]. There are some examples of direct altruism in bacteria, including phage abortive infection, in which infected cells in a population die to prevent phage propagation and thereby save uninfected neighbor cells [43], and biofilm development, where cell lysis releases structural components needed to make a robust biofilm matrix [44]. However, the benefit to neighboring cells is less clear for GTAs produced by bacteria like Caulobacter in aqueous, high-diffusion environments where particles may be easily washed away from clonal neighbors. To counter this, GTA production may be linked to high cell density to promote transfer, as seen in this work and other GTA systems [16,45,46]. It may also be necessary for cells to ensure that any GTAs released can only be received by a closely related cell, e.g., a dedicated cell receptor/tail-fiber pair could restrict the benefit only to other cells of the same species as is seen in R. capsulatus [16]. In this way, a recipient cell could be informed of the incoming DNA’s origin, similar to the role of uptake sequences in transformation [47,48]. Further studies investigating whether the GTAs of C. crescentus can transfer DNA to closely related species will shed light on species-specificity and potential GTA receptors. The lack of apparent tails of the GTA in C. crescentus despite encoding tail proteins is surprising, but may be a result of physical forces during purification having disrupted tail structures. Further work to purify GTAs with other methods or visualization of GTAs in vivo with cryo-electron tomography, as well as testing which genes in the GTA cluster are required for transfer may help resolve this issue. Finally, if the benefit of receiving GTAs requires recombination, and thus relies on high sequence similarity, the benefit would also be restricted primarily to closely related strains and species.
Not surprisingly, GTA biogenesis is a tightly regulated process as it ultimately leads to lysis of the producing cell. Recent work in R. capsulatus identified GafA as a direct transcriptional activator of GTA production [21], although the mechanism of activation is not known. In Caulobacter, and likely many other α-proteobacteria, GafY and GafZ appear to be a split, 2-gene homolog of GafA. As with GafA in R. capsulatus, GafY and GafZ are necessary and, when expressed together, sufficient for GTA production in Caulobacter. Although GafYZ form a heterocomplex, as evidenced by co-IP studies, our ChIP-seq analysis indicated that GafY alone binds the promoter regions of many operons, including the long, primary GTA locus, whereas GafZ was found only within the GTA locus. The binding pattern of GafZ suggests that it may travel with and possibly regulate RNA polymerase during the transcription of this 21-gene cluster. GafY (locus CCNA_01111 in C. crescentus) is homologous to the DNA-binding domain of DnaA (domain IV). A prior study found that this gene is induced in stationary phase and that overexpressing it increased the copy number of some genomic loci, inhibited cell division, and eventually led to cell death [22]. These observations are consistent with ours and each can be explained by the impact of GafY on GTA production documented here. Thus, we propose that GafY does not regulate oriC-based chromosome replication via DnaA and instead functions in GTA biogenesis. We also identified the transcription factor RogA as a direct repressor of GafYZ and ultimately GTA production. RogA homologs are found throughout the α-proteobacteria and are particularly enriched in the Rhizobiales, Rhodospirillales, Rhodobacterales, and Caulobacterales, where they may similarly control gafYZ or gafA homologs to control GTA production. RogA has a domain organization similar to LexA and λ cI, with an N-terminal helix-turn-helix domain and an S24 auto-peptidase-like domain, though lacks the key catalytic residues. Notably, we had identified rogA in a screen for regulators of didA, which encodes a DNA damage-inducible cell division inhibitor. The expression of didA is directly controlled by the transcription factor DriD, with strongest induction triggered by double-strand breaks [18]. Our work here indicates that RogA directly controls gafYZ, which then indirectly stimulate didA expression. In rogA mutants, gafYZ are induced, leading to induction of GTA synthesis. The assembly of GTA particles requires the packaging of 8.3-kb linear fragments of the genome, which then trigger DriD to activate didA and other DriD targets. It is not yet clear whether the induction of didA and its ability to inhibit cell division is somehow advantageous to GTA producer cells. Although RogA and GafYZ (or GafA) appear to be relatively common, conserved regulators of GTA synthesis, there may also be species- or clade-specific regulators. For example, the CckA-ChpT-CtrA phosphorelay and quorum-sensing proteins GtaI and GtaR regulate GTA synthesis in Rhodobacter [49–53], but not in Caulobacter [54]. The stringent response, mediated by ppGpp, also somehow promotes GTA synthesis in Rhodobacter [45]. In both Caulobacter and Rhodobacter, GTA synthesis occurs almost exclusively in stationary phase. Although cells can receive GTAs in either exponential or stationary phase, synthesis and subsequent release is restricted to stationary phase. However, entry to stationary phase is necessary, but not sufficient. To detect GTA synthesis, we had to delete rogA or overexpress gafYZ. There are likely external signals or cues that trigger GTA synthesis in wild-type cells, possibly by inhibiting RogA or by stimulating GafYZ activity. DNA-damaging agents are one prime candidate given our finding that GTAs can provide a template for homologous recombination-based DNA repair. Why GTA synthesis is restricted to stationary phase warrants further investigation. It may be that an additional signal is normally coincident with or also requires stationary phase. For example, in Rhodobacter, GTA production is under quorum-sensing control, possibly to ensure that released GTAs will efficiently find recipient cells.
The prevalence and conservation of GTAs, particularly in α-proteobacteria, has long suggested that they provide cells a benefit, but the nature of this benefit has remained enigmatic. Our work now points to a possible role for GTAs in promoting DNA repair by providing recipient cells with templates for homologous recombination. However, we cannot rule out a role in facilitating the acquisition of beneficial alleles in some conditions or specific scenarios. Whatever the case, our discovery that C. crescentus produces functional GTAs also now offers a new, highly tractable organism to further dissect the functions, biogenesis, and regulation of GTAs.
Plasmids and strains used in this study are listed in S1 and S2 Tables, respectively. DNA oligonucleotides used in strain construction and experiments are listed in S3 Table. For statistical tests, 2-tailed t tests were used with a significance cutoff of p-value <0.05.
C. crescentus CB15N genomic DNA was used as template for PCR amplifications unless otherwise noted. All PCR products were digested with noted restriction enzymes and ligated into the double-enzyme cut corresponding plasmid. Approximately 5 μL of ligation reactions were subsequently transformed into chemically competent E. coli DH5α cells. All resulting plasmids were verified by Sanger sequencing (Genewiz). To generate, pBXMCS-2::Pxyl-gafY, pBXMCS-2::Pxyl-gafZ, and pBXMCS-2::Pxyl-gafYZ, the coding sequence of gafY, gafZ, or the native operon of gafYZ were PCR-amplified with primers oKRG92 and oKRG93, oKRG400 and oKRG401, or oKRG92 and oKRG401, respectively. Amplicons were digested with NdeI and SacI and ligated into cut pBXMCS-2. To generate, pRVMCS-2::ProgA-rogA, the promoter and coding sequence of rogA was PCR-amplified with primers oKRG127 and oKRG128, digested with SacII and AflII and ligated into cut pRVMCS-2. To generate pNPTS138::tetR 1.0Mb, a tetR cassette was cloned with approximately 600-bp flanking sites near the 1.0 Mb position as follows: Approximately 600-bp region including part of the coding region of CCNA_00928 up to the intergenic region to CCNA_00929 was amplified by PCR using primers oKRG446 and oKRG447. Approximately 600-bp region consisting of the CCNA_00928-CCNA_00929 intergenic region and some of the CCNA_00929 coding region was amplified by PCR using primers oKRG448 and oKRG449. The tetracycline resistance cassette was amplified from the plasmid pMCS-5 [55] using primers oKRG440 and oKRG441. These 3 PCR products were fused using fusion PCR with primers oKRG446 and oKRG449, gel purified, digested with EcoRI and SalI and ligated into pNPTS138. To generate pNPTS138::tetR 2.0Mb, an identical approach was used as for pNPTS138::tetR 1.0 Mb, instead with the tetR cassette cloned with approximately 600-bp flanking sites near the 2.0 Mb position. The regions upstream and downstream of the CCNA_01861-CCNA_01862 intergenic region were amplified using primers oKRG458 with oKRG459 and oKRG460 with oKRG461, respectively. Fusion PCR with the tetR cassette and ligation into pNPTS138 was performed as described for pNPTS138::tetR 1.0Mb. To generate pNPTS138::ΔrogA, a 600-bp region upstream of rogA that includes the first 9 nucleotides of rogA was amplified by PCR using primers oKRG102 and oKRG103. A 600-bp downstream of rogA that includes the last 9 nucleotides of rogA was amplified by PCR using primers oKRG104 and oKRG105. These 2 PCR products were fused using fusion PCR with primers oKRG102 and oKRG105, gel purified, digested, and ligated into a SpeI-AflII-cut pNPTS138. To generate pNPTS138::ΔrogA::tetR, a 600-bp region upstream of rogA that includes the first 9 nucleotides of rogA was amplified by PCR using primers oKRG102 and oKRG106. A 600-bp downstream of rogA that includes the last 9 nucleotides of rogA was amplified by PCR using primers oKRG107 and oKRG105. The tetracycline resistance cassette was amplified by PCR using primers oKRG440 and oKRG441 with the previously described pMCS-5 [55] as template. These 3 PCR products were fused using fusion PCR with primers oKRG102 and oKRG105, gel purified, digested, and ligated into a SpeI-AflII-cut pNPTS138. To generate pNPTS138::Δgta, a 600-bp region including 300-bp upstream of CCNA_02880 and 300 bp into the coding region of CCNA_02880 was PCR amplified using primers oKRG111 and oKRG110. A 600-bp downstream of CCNA_02861 that includes the last 300 nucleotides of CCNA_02861 was amplified by PCR using primers oKRG109 and oKRG108. These 2 PCR products were fused using fusion PCR with primers oKRG108 and oKRG111, gel purified, digested, and ligated into a EcoRI-cut pNPTS138. To generate pNPTS138::hfaB::kanR, upstream half and downstream half of hfaB were PCR amplified using primers oKRG114/oKRG115 and oKRG112/oKRG113. The kanamycin resistance cassette was PCR amplified with primers oKRG116 and oKRG117 and pBXMCS-2 as template. These 3 PCR products were fused using fusion PCR with primers oKRG112 and oKRG115, gel purified, digested, and ligated into a SpeI-AflII-cut pNPTS138. To generate pNPTS138::ΔgafY, a 500-bp region upstream of gafY that includes the first 9 nucleotides of gafY was amplified by PCR using primers oKRG118 and oKRG119. A 500-bp downstream of gafY that includes the last 9 nucleotides of gafY was amplified by PCR using primers oKRG120 and oKRG121. These 2 PCR products were fused using fusion PCR with primers oKRG118 and oKRG121, gel purified, digested, and ligated into a EcoRI-cut pNPTS138. To generate pNPTS138::ΔgafZ, a 500-bp region upstream of gafZ that includes the first 9 nucleotides of gafY was amplified by PCR using primers oKRG122 and oKRG123. A 500-bp downstream of gafY that includes the last 9 nucleotides of gafY was amplified by PCR using primers oKRG124 and oKRG125. These 2 PCR products were fused using fusion PCR with primers oKRG122 and oKRG125, gel purified, digested, and ligated into a SpeI-AflII-cut pNPTS138. To generate pNPTS138::ΔgafYZ, a 500-bp region upstream of gafY that includes the first 9 nucleotides of gafY was amplified by PCR using primers oKRG118 and oKRG119. A 500-bp downstream of gafZ that includes the last 9 nucleotides of gafY was amplified by PCR using primers oKRG126 and oKRG125. These 2 PCR products were fused using fusion PCR with primers oKRG118 and oKRG125, gel purified, digested, and ligated into a SpeI-AflII-cut pNPTS138. To generate pNPTS138::ΔrogA::tetR 2 (maintaining a longer stretch of the starting nucleotides of the open reading frame), a 500-bp region upstream of rogA that includes the first 30 nucleotides of rogA was amplified by PCR using primers NTP2194 and NTP2305 and a 500-bp downstream of rogA that includes the last 9 nucleotides of rogA was amplified by PCR using primers NTP2306 and NTP2197. The tetracycline resistance cassette was amplified by PCR using primers NTP2303 and NTP2304, and as template the previously described pMCS-5 [55]. All 3 resulting PCR products were gel purified and assembled together into BamHI-HindIII-cut pNPTS138 using 2× Gibson mastermix. To generate pNPTS138::ΔCCNA_02880, pNPTS138::ΔCCNA_02877, pNPTS138::ΔCCNA0041_02872, pNPTS138::ΔCCNA_02873, approximately 500-bp upstream region of each gene including 3 to 5 codons into the coding region was amplified using the primer pairs NTP2230 and NTP2231, NTP2493 and NTP2494, NTP2222 and NTP2223, or NTP2323 and NTP2324, respectively. The downstream region of each gene was amplified by PCR using primer pairs NTP2232 and NTP2233, NTP2495 and NTP2496, NTP2224 and NTP2225, or NTP2325 and NTP2326, respectively. Each upstream and downstream PCR products were gel purified and assembled together into BamHI-HindIII-cut pNPTS138 using 2× Gibson mastermix. To generate pENTR::gafY, the coding sequence of GafY was PCR-amplified using primers NTP2568 and NTP2569. The resulting PCR product were gel purified and assembled with the pENTR plasmid backbone using 2× Gibson mastermix (NEB). To generate pML333::his6-mbp-gafY, DNA containing gafY was recombined into a Gateway-compatible destination vector pML333 via an LR recombination reaction (Invitrogen). For LR recombination reactions: 1 μL of purified pENTR::gafY was incubated with 1 μL of pML333, 1 μL of LR Clonase II mastermix (Invitrogen), and 2 μL of water in a total volume of 5 μL. The reaction was incubated for an hour at room temperature before being introduced into E. coli DH5α cells by heat-shock transformation. Cells were then plated out on LB agar + carbenicillin. Resulting colonies were restruck onto LB agar + carbenicillin and LB agar + kanamycin. Only colonies that formed on LB + carbenicillin plates were used for culturing and plasmid extraction. To generate pET21b::rogA-his6, the coding sequence of RogA was PCR amplified using primers NTP2291 and NTP2292. The resulting PCR product was gel purified and assembled into an NdeI-HindIII-cut pET21b using a 2× Gibson mastermix. To generate pCOLADuet-1:his6-gafZ, the coding sequence of GafZ was PCR amplified using primers NTP2693 and NTP2694. The resulting PCR product was gel purified and assembled into an EcoRI-HindIII-cut pCOLADuet-1 plasmid backbone using a 2× Gibson mastermix. To generate pCOLADuet-1:his6-gafZ gafY, the coding sequence of (tagless) GafY was PCR amplified using primers NTP2691 and NTP2692. The resulting PCR product was gel purified and assembled into an NdeI-KpnI-cut pCOLADuet-1:his6-gafZ plasmid backbone using a 2× Gibson mastermix. To generate pNPTS138::flag-gafZ, a 500-bp region upstream of the FLAG-tag insertion site was amplified by PCR using primers NTP2598 and NTP2599. A 500-bp downstream of the FLAG-tag insertion site was amplified by PCR using primers NTP2600 and NTP2601. The resulting PCR products were gel purified and assembled together into a BamHI-HindIII-cut pNPTS138 using a 2× Gibson mastermix. To generate pNPTS138::ΔgafY::8xTAG, a 500-bp region upstream of the 8xstop codon (TAG) insertion site was amplified by PCR using primers NTP2642 and NTP2643. A 500-bp downstream of the 8xTAG insertion site was amplified by PCR using primers NTP2644 and NTP2645. The resulting PCR products were gel purified and assembled together into a BamHI-HindIII-cut pNPTS138 using a 2× Gibson mastermix.
For strain construction, insertions and deletions were verified by PCR using primers outside the genetic alteration, as appropriate. Double-crossover gene knock-ins were performed with 2-step recombination as described previously [56]. Transcriptional reporters (PdidA-lacZ) and antibiotic cassette markers (kanR) were inserted at the hfaB locus, which encodes for holdfast protein and is a dispensable region in CB15N [57] commonly used for genomic insertions. To generate ML3660 and ML3661, the insert in the plasmid pNPTS138::ΔrogA::tetR was introduced by 2-step recombination to CB15N and ML2169, respectively. To generate ML3662, the insert in the plasmid pNPT-spec-ΔdriD was introduced by 2-step recombination to ML3660. To generate ML3663 and ML3664, the plasmid pRVMCS-2::ProgA-rogA was introduced by electroporation into ML3661 and ML3660, respectively. To generate ML3665, ML3666, ML3667, ML3668, and ML3669, the inserts in plasmids pNPT-spec-ΔdriD, pNPTS138::Δgta::kanR, pNPTS138::ΔgafY, pNPTS138::ΔgafZ, and pNPTS138::ΔgafYZ were introduced into ML3660 by 2-step recombination, respectively. To generate ML3670, ML3671, ML3672, and ML3673, the plasmids pBXMCS-2 (empty), pBXMCS-2::Pxyl-gafY, pBXMCS-2::Pxyl-gafZ, and pBXMCS-2::Pxyl-gafYZ were introduced into CB15N by electroporation. To generate ML3674 and ML3675, the inserts in plasmids pNPTS138::ΔrogA and pNPTS138::Δgta were introduced by 2-step recombination to CB15N, respectively. To generate ML3676, the plasmid pBXMCS-2::Pxyl-gafYZ was introduced by electroporation into ML3675. To generate ML3677 and ML3678, the plasmids pNPTS138::tetR 1.0Mb and pNPTS138::tetR 2.0 Mb were first introduced by 2-step recombination to CB15N, respectively. The plasmid pBXMCS-2::Pxyl-gafYZ was then subsequently introduced to each of these intermediate strains to generate ML3677 and ML3678. To generate ML3679, the insert in plasmid pNPTS138::hfaB::kanR was introduced by 2-step recombination to CB15N. Then, the plasmid pXGFPC-6 was integrated into this strain at the xylose promoter by 1-step recombination. To generate ML3680, an I-SceI restriction cut site located near the CCNA_00727 locus was introduced into ML2169 by ΦCr30-mediated transduction from the strain ML2466. The resulting strain was then used as the recipient strain for a second ΦCr30-mediated transduction of the i-SceI gene integrated at the vanillate locus and marked by a chloramphenicol resistance cassette from ML2466. To generate ML3681, the plasmid pXGFPC-2 (empty) was introduced into ML3680 by electroporation. To generate ML3682, ML3683, and ML3685, the plasmids pBXMCS-2 (empty), pBXMCS-2::Pxyl-gafY, and pBXMCS-2::Pxyl-gafYZ were introduced into ML2169 by electroporation. To generate NTS2275, which bears a deletion of rogA with the 30 nucleotides of the ORF remaining, the insert in plasmid pNPTS138::ΔrogA::tetR 2 was introduced into CB15N by 2-step recombination. To generate NTS2481, which contains a FLAG tag at the N-terminus of GafZ, the insert in the plasmid pNPTS138::flag-gafZ was introduced into CB15N by 2-step recombination. To generate NTS2501, which bears an 8xstop codon (TAG) downstream of the start codon of gafY to disrupt gafY without affecting expression of gafZ, the plasmid pNPTS138::ΔgafY::8xTAG was introduced into CB15N by 2-step recombination. To generate NTS2515 and NTS2489, the rogA::tetR allele from NTS2275 was introduced into NTS2501 and NTS2481 by ΦCr30-mediated transduction, respectively. To generate NTS2316, NTS2445, NTS2314, and NTS2403, the individual gene deletions from pNPTS138::ΔCCNA_02880, pNPTS138::ΔCCNA_02877, pNPTS138::ΔCCNA_02872, and pNPTS138::ΔCCNA_02873 were introduced by 2-step recombination to CB15N, respectively. To each of the resulting intermediate strains, the rogA::tetR allele from NTS2275 was introduced by ΦCr30-mediated transduction to generate the respective double mutants.
PCR was performed with Phusion HF DNA polymerase using 5X Phusion GC Reaction Buffer (NEB). Each reaction contained 10 μL buffer, 10 μL 3M Betaine monohydrate (Sigma), 4 μL dNTPs, 5 μL of a 10 μm forward and reverse primer mix, 50 ng template, 1 μL DMSO, 0.5 μL polymerase, and nuclease-free water to 50 μL. Two-step cycling was performed as follows: 98°C 30 s, 34× (98°C 10 s, 72°C 30 s/kb), 72°C 5 min. Fusion PCR was performed similarly with 50 ng of the largest template fragment and equimolar amounts of the smaller template fragments and an additional annealing step of 20 s at 60°C was added.
Caulobacter strains were grown at 30°C in rich medium (PYE) shaking in flasks. To induce or repress expression from the Pxyl promoter, liquid media was supplemented with 0.3% xylose or 0.2% glucose, respectively. To induce or repress expression from Pvan, liquid media was supplemented with or without 500 μM vanillic acid (Fluka). Mitomycin C (A&G) was added to liquid cultures at 1 μg/mL and to agarose pads and agar plates at 0.35 μg/mL unless otherwise indicated. To induce expression from Plac, plate media were supplemented with 75 or 100 μM IPTG (Sigma). Novobiocin (Fluka) and cephalexin (Sigma) were added to agar plates at 0.35 μg/mL and 7.5 μg/mL, respectively. Antibiotics were used at the following concentrations for strain construction and plasmid maintenance in Caulobacter cells in liquid:plates—kanamycin 5 μg/mL: 25 μg/mL; oxytetracycline 1 μg/mL: 2 μg/mL; chloramphenicol 2 μg/mL: 1 μg/mL; gentamycin 2.5 μg/mL: 5 μg/mL. Transformations and transductions were performed as previously described [58]. All cell survival and CFU enumeration assays were performed with 10-fold serial dilutions, unless otherwise noted.
Cells expressing lacZ driven by the didA promoter integrated at the chromosomal hfaB locus were mutagenized with the EZ-TN5 transposome kit (Epicentre) and were selected for transposon insertion on PYE plates containing kanamycin and 20 μg/mL X-gal. Dark blue colonies were isolated and transposon insertions were identified using rescue cloning with pir-116 electrocompetent E. coli cells (Epicentre, TSM08KR).
β-galactosidase activity was measured in mid-log (OD600 = 0.2–0.3) or stationary phase (OD600 = 1.2–1.3). Strains bearing the PdidA-didA reporter were permeabilized by adding 100 μL of chloroform to 800 μL of cells followed by vortexing. Cells were incubated at 30°C for 15 min prior to addition of ortho-nitrophenyl-β-galactoside. The assay and subsequent activity calculations were done as previously described [59].
RNA-seq was performed as previously described [60]. Briefly, RNA-seq libraries were sequenced by paired-end sequencing (75 nt) on an Illumina NextSeq500 sequencer at the MIT BioMicro Center. Custom scripts written in Python 2.7.6 were used to perform data analysis. The paired-end reads were mapped to Caulobacter NC011916.1 using bowtie2 with default parameters [61]. The read coverage was mapped by assigning each mapped base a value of 1/N where N equals the length of each paired read. mRNA abundance was calculated by first adding a pseudocount to all positions and then the number of reads mapped to a gene was divided by the gene’s length and normalized to yield the mean number of reads per kilobase per million sequencing reads (RPKM). Changes in gene expression were calculated by taking the log2 transformation of the ratio of the RPKM of each gene from the experimental condition (Pxyl-gafYZ in xylose or ΔrogA) to the respective control (Pxyl-empty in xylose or wild-type). RNA-seq data are available at GEO accession number GSE184480.
Total DNA extractions were performed by pelleting 1 mL of stationary phase cells. Pellets were resuspended in 600 μL Cell Lysis Solution (Qiagen) and lysed by incubation at 80°C for 5 min. A total of 50 μg of RNAse A was then added to the cell lysate and incubated at 37°C for 30 min to digest cellular RNAs. Proteins were precipitated from the lysate by adding 200 μL of Protein Precipitation Solution (Qiagen), vortexing and setting the sample on ice for 30 min, and pelleting for 10 min at 14,000 rpm. The supernatant was then mixed with 600 μL of isopropanol and inverted to precipitate the DNA. The DNA was pelleted via centrifugation at 14,000 rpm for 3 min, washed once with 70% ethanol, and resuspended in 100 μL of H2O.
To sequence the DNA packaged in GTA particles, cells bearing the Pxyl-gafYZ overexpression plasmid were induced with 0.3% xylose for 3 h after reaching an OD600 of 1.3. Total DNA was then extracted as above. The GTA DNA was separated from chromosomal genomic DNA via gel electrophoresis in a 1% agarose gel and subsequent gel extraction (Zymoclean Gel DNA extraction kit). The DNA was further purified by isopropanol precipitation and an ethanol wash and was resuspended in Qiagen elution buffer. The resulting purified GTA DNA was sequenced using PacBio SMRT sequencing technology at the MIT BioMicro Center. Briefly, the dsDNA library was prepared using the Express Template Prep Kit v2.0, consisting of end repair, DNA damage repair, adapter ligation, further purification, and quality control. The library was sequenced with a 10-h movie on a PacBio Sequel v3. Sequencing reads were analyzed using circular consensus sequencing. Full consensus reads were aligned to the Caulobacter NC011916.1 genome with Bowtie 2. PacBio sequencing data are available at GEO accession number GSE184478.
For induction of GTA expression, cells bearing Pxyl-gafYZ on a high-copy plasmid were grown up to stationary phase (OD600 = 1.2–1.3) in PYE with 0.2% (w/v) glucose, unless otherwise noted. Xylose was added to a final concentration of 0.3% (w/v) to induce expression. Cells were then used for enumerating survival with CFU assays, DNA extractions to isolate GTA-packaged DNA, or as donor cells for subsequent gene transfer assays. To quantify cell lysis, cultures at each time point were pelleted by centrifugation and cell-free supernatant was assayed for protein content using the Pierce Coomassie Plus Assay Kit (Thermo), following manufacturer’s protocol.
Cells bearing the Pxyl-gafYZ plasmid were induced with 0.3% xylose for 3 h after reaching OD600 = 1.3. Cells were pelleted, resuspended in PBS, and SYBR Gold (Invitrogen) was added at a 1:5,000 fold dilution from the stock. Cells were incubated at room temperature for 5 min. One microliter was spotted on 1.5% agarose in PBS pads and imaged with phase contrast and epifluorescence on a Zeiss Observer Z1 microscope using a 100×/1.4 oil immersion objective and an LED-based Colibri illumination system with MetaMorph software (Universal Imaging, PA). Dead cell staining with propidium iodide was performed similarly as SYBR Gold staining, with a final concentration of 1 μM propidium iodide.
Cultures of donor cells bearing the Pxyl-gafYZ plasmid and a tetR marker at the 1.0 Mbp locus, and recipient cells bearing the empty high-copy vector pBXMCS-2 and a chloramphenicol resistance cassette at the hfaB locus, were grown to OD600 = 1.3 in PYE containing 0.2% glucose to repress the xylose promoter and kanamycin to maintain plasmid selection. The cultures were mixed 1:1, or at noted ratios, in 250 mL flasks and induced with 0.3% xylose for the indicated amount of time. Cells were directly plated on PYE plates containing kanamycin (25 μg/mL) and either chloramphenicol (1 μg/mL), tetracycline (2 μg/mL), or both to select for transferants. To assay the benefit of GTA transfer during a single double-strand break, recipient cells bearing the inducible I-SceI double-strand break system and an empty pBXMCS-2 kanR-marked plasmid and donor cells bearing either the Pxyl-gafYZ or Pxyl-empty plasmid were grown up to stationary phase in the presence of 0.2% glucose to repress the xylose promoter and kanamycin to maintain plasmid selection. Cultures were mixed 1:1 in 250 mL flasks and induced with 0.3% xylose and 500 μM vanillate. At indicated time points, cells were enumerated for viability and loss of vanillate sensitivity on plates containing chloramphenicol to select for recipient cells with or without 500 μM vanillate to select for vanillate-resistant cells. Cells were subsequently picked, genomic DNA was extracted, and the region containing the I-SceI cut site was sequenced by PCR and Sanger sequencing using primers oKRG523 and oKRG524.
Sequence alignment of gafY and gafZ from C. crescentus to gafA in Rhodobacter capsulatus were performed using MUSCLE [62]. gafY and gafZ from C. crescentus NC011916.1 were concatenated into 1 gene and aligned against gafA (rcc_01865) from R. capsulatus NC014034. Sequences were visualized using Jalview [63]. Co-occurrence of gafY, gafZ, rogA, the GTA capsid (CCNA_02872), the GTA terminase (CCNA_02880), and a putative GTA tail fiber (CCNA_02456) across 1,336 representative bacterial taxa using String-DB [64].
Single colonies of the indicated strain were inoculated into 25 mL PYE in 250 mL flasks and grown at 30°C, shaking at 210 rpm. Cell survival was enumerated with 10-fold serial dilutions onto PYE plates. The marked ΔrogA::tetR, as opposed to a clean deletion, was used throughout for ease of quantifying donor and recipient cells where applicable. For co-incubation experiments, 25 mL of each indicated culture were grown overnight to stationary phase. Approximately 12.5 mL of the indicated cultures were mixed into fresh 250 mL flasks and cell survival was enumerated by serial dilutions on plates containing appropriate antibiotics. For experiments with filtered supernatant, stationary phase cultures of indicated strains were pelleted cells with centrifugation for 5 min at 8,000 rpm at 25°C and the supernatant was passed through a 0.20 μm SFCA filter (Corning) and used to resuspend pelleted recipient cells where indicated. Quantification of cells during co-culture with the rec- strain was performed with marked donors, either WT (hfaB::kanR) or ΔrogA::tetR cells, and markerless recipients, either WT or rec- strains. The co-incubation cultures were plated out to single colony and replica plated onto plates containing no antibiotic or the corresponding antibiotic of the donor and cells sensitive to antibiotic were enumerated.
UV treatment was performed with a UV Stratalinker 2400. Treatment with “high” UV damage was equal to 5 J/m2 (Fig 5F), while the rec- strain was exposed to 1 J/m2 (Fig 5F). Exposure of cells in exponential phase was 1 J/m2 (S6C Fig). For quantification of the rec- survival to UV when incubated with other strains, the rec- strain was enumerated using replica plating. Co-incubations were diluted out to single colonies before and after treatment on plates containing no antibiotic. Plates were then replica plated on plates containing antibiotic corresponding to the donor or plates without antibiotic, and colonies that only grew on antibiotic-free plates were enumerated, as these corresponded to the rec- strain. This was also performed with a markerless wild-type strain for comparison. Soft agar chemical sensitivity assays were done as described previously [65] with some modifications. Each strain was grown to deep stationary phase in PYE (48 h of growth). A total of 60 μL of cells were added to 3 mL of warm PYE with 0.3% agar and poured onto a PYE agar plate. A 6-mm sterile filter paper disk was impregnated with 20 μL of the tested compound, air dried, and placed on top of the solidified soft agar. Zones of inhibition were measured after 48 h of incubation at 30°C. The distance between the disk and the edge of bacterial lawn growth was quantified using Fiji [66].
Plasmid pML333::his6-mbp-gafY was introduced into E. coli Rosetta (DE3) competent cells (Merck) by heat-shock transformation. A 40-mL overnight culture was used to inoculate 4 L of LB medium + carbenicillin + chloramphenicol. Cells were grown at 37°C with shaking at 210 rpm to an OD600 of approximately 0.4. The culture was then left to cool down to 28°C before isopropyl-β-D-thiogalactopyranoside (IPTG) was added to a final concentration of 1.0 mM. The culture was left shaking for an additional 3 h at 28°C before cells were harvested by centrifugation. Pelleted cells were resuspended in a buffer containing 100 mM Tris-HCl (pH 8.0), 300 mM NaCl, 10 mM imidazole, 5% (v/v) glycerol, 1 μL of Benzonase nuclease (Merck), 1 mg of lysozyme (Merck), and an EDTA-free protease inhibitor tablet (Merck). The resuspended cells were then lysed by sonication (10 cycles of 15 s with 10 s resting on ice in between each cycle). The cell debris was removed by centrifugation at 28,000 g for 30 min and the supernatant was filtered through a 0.45 μm filter disk. The lysate was then loaded into a 1-mL HisTrap column (GE Healthcare) that had been pre-equilibrated with buffer A [100 mM Tris-HCl (pH 8.0), 300 mM NaCl, 10 mM imidazole, and 5% glycerol]. Protein was eluted from the column using an increasing imidazole gradient (10 mM to 500 mM) in the same buffer. Fractions containing His6-MBP-GafY were pooled and diluted to a conductivity of 16 mS/cm before being loaded onto a Heparin HP column (GE Healthcare) that had been pre-equilibrated with 100 mM Tris-HCl pH 8.0, 25 mM NaCl, and 5% glycerol. Protein was eluted from the Heparin column using an increasing salt gradient (25 mM to 1 M NaCl) in the same buffer. Fractions containing His6-MBP-GafY were pooled and analyzed for purity by SDS-PAGE. Glycerol was then added to His6-MBP-GafY to the final volume of 10%, and the protein was stored at -80 °C. Two mg of this protein was used to raise polyclonal antibody in rabbit (Cambridge Research Biochemicals, United Kingdom).
Plasmid pET21b::rogA-his6 was introduced into E. coli Rosetta (DE3) competent cells (Merck) by heat-shock transformation. A 40-mL overnight culture was used to inoculate 4 L LB medium + carbenicillin + chloramphenicol. Cells were grown at 37°C with shaking at 210 rpm to an OD600 of ~0.4. The culture was then left to cool down to 28°C before IPTG was added to a final concentration of 1.0 mM. The culture was left shaking for an additional 20 hours at 20°C before cells were harvested by centrifugation. Pelleted cells were resuspended in a buffer containing 100 mM Tris-HCl pH 8.0, 300 mM NaCl, 10 mM imidazole, 5% (v/v) glycerol, 1 μL of Benzonase nuclease (Merck), 1 mg of lysozyme (Merck), and an EDTA-free protease inhibitor tablet (Merck). The resuspended cells were then lyzed by sonification (10 cycles of 15 s with 10 s resting on ice in between each cycle). The cell debris was removed by centrifugation at 28,000 g for 30 min and the supernatant was filtered through a 0.45 μm filter disk. The lysate was then loaded into a 1-mL HisTrap column (GE Healthcare) that had been pre-equilibrated with buffer A [100 mM Tris-HCl pH 8.0, 300 mM NaCl, 10 mM imidazole, and 5% glycerol]. Protein was eluted from the column using an increasing imidazole gradient (10 mM to 500 mM) in the same buffer. RogA-His6 containing fractions were pooled and diluted to a conductivity of 16 mS/cm before being loaded onto a Heparin HP column (GE Healthcare) that had been pre-equilibrated with 100 mM Tris-HCl (pH 8.0), 25 mM NaCl, and 5% glycerol. Protein was eluted from the Heparin column using an increasing salt concentration (25 mM to 1 M NaCl) in the same buffer. RogA-His6 fractions were pooled together and analyzed for purity by SDS-PAGE. Glycerol was then added to RogA-His6 fractions to a final volume of 10%, and the protein was stored at −80 °C. Two mg of this protein was used to raise polyclonal antibody in rabbit (Cambridge Research Biochemicals, UK).
Plasmid pCOLADuet-1:his6-gafZ gafY was introduced into E. coli Rosetta (DE3) competent cells (Merck) by heat-shock transformation. A 40-mL overnight culture was used to inoculate 4 L LB medium + kanamycin + chloramphenicol. Cells were grown at 37°C with shaking at 210 rpm to an OD600 of approximately 0.4. The culture was then left to cool down to 28°C before IPTG was added to a final concentration of 1.0 mM. The culture was left shaking for an additional 3 h at 28°C before cells were harvested by centrifugation. Pelleted cells were resuspended in a buffer containing 100 mM Tris-HCl (pH 8.0), 300 mM NaCl, 10 mM imidazole, 5% (v/v) glycerol, 1 μL of Benzonase nuclease (Merck), 1 mg of lysozyme (Merck), and an EDTA-free protease inhibitor tablet (Merck). The pelleted cells were then lyzed by sonification (10 cycles of 15 s with 10 s resting on ice in between each cycle). The cell debris was removed by centrifugation at 28,000 g for 30 min and the supernatant was filtered through a 0.45 μm filter disk. The lysate was then loaded into a 1-mL HisTrap column (GE Healthcare) that had been pre-equilibrated with buffer A [100 mM Tris-HCl (pH 8.0), 300 mM NaCl, 10 mM imidazole, and 5% glycerol]. Protein was eluted from the column using an increasing imidazole gradient (10 mM to 500 mM) in the same buffer. Fractions containing the GafYZ complex fractions were checked by SDS-PAGE for purity. Proteins were subsequently used for SPR assay.
C. crescentus cells were grown in PYE at 28 °C with shaking at 220 rpm in biological duplicate. When the cultures (60 mL) reached either exponential phase (for IP of RogA) or stationary phase (for IP of GafY or FLAG-GafZ), formaldehyde was added to a final concentration of 1%. Cells were further incubated at room temperature for 30 min, then the fixation was quenched using 0.125 M glycine for 15 min at room temperature. Cells were washed 3 times with 1× PBS (pH 7.4) and were resuspended in 1.5 mL of buffer 1 [20 mM K-HEPES (pH 7.9), 50 mM KCl, 10% glycerol, and EDTA-free protease inhibitors]. Subsequently, the cell suspension was sonicated on ice using a probe-type sonicator (8 cycles of 15 s with 15 s resting on ice, amplitude setting 8) to lyse cells and to shear the chromatin to below 1 kb. The cell debris was cleared by centrifugation (20 min at 13,000 rpm at 4°C). The supernatant was then transferred to a new 2-mL tube and the buffer conditions were adjusted to 10 mM Tris-HCl (pH 8.0), 150 mM NaCl, and 0.1% NP-40. To immunoprecipitate FLAG-tagged GafZ, 100 μL of α-FLAG M2 agarose beads (Merck) was washed off their storage buffer by repeated centrifugation and resuspension in IPP150 buffer [10 mM Tris-HCl (pH 8.0), 150 mM NaCl, and 0.1% NP-40], beads were then introduced to the cleared supernatant and incubated with rotation at 4°C overnight. Protein A beads (Merck) with α-RogA and α-GafY polyclonal antibodies were employed to immunoprecipitate RogA and GafY, respectively. To immunoprecipitate RogA and GafY, 60 μL of α-RogA/α-GafY polyclonal antibodies were added to the cleared supernatant first, the mixture was then incubated overnight before protein A beads were added and further incubated for another 4 h. After the incubation, beads were washed 5 times at 4°C for 2 min each with 1 mL of IPP150 buffer, then twice at 4°C for 2 min each in 1× TE buffer [10 mM Tris-HCl (pH 8.0) and 1 mM EDTA]. Immunoprecipitated protein–DNA complexes were then eluted twice from the beads by incubating the beads first with 150 μL of the elution buffer [50 mM Tris-HCl (pH 8.0), 10 mM EDTA, and 1% SDS] at 65°C for 15 min, then with 100 μL of 1× TE buffer + 1% SDS for another 15 min at 65°C. The supernatant (i.e., the ChIP fraction) was then aspirated from the beads and was incubated at 65°C overnight to completely reverse the crosslinks. DNA from the ChIP fraction were then purified using a PCR purification kit (Qiagen) according to the manufacturer’s instruction and eluted out using 50 μL of EB buffer (Qiagen). Subsequently, the purified DNA was used to construct barcoded libraries suitable for Illumina sequencing using the NEXT Ultra II library preparation kit (NEB). Barcoded libraries were pooled together sequenced on the Illumina Hiseq 2500 at the Tufts university genomics facility. The generation and analysis of ChIP-seq profiles have been described previously [67]. Briefly, Hiseq 2500 Illumina short reads (50 bp) were mapped back to the C. crescentus NA1000 reference genome (NCBI Reference Sequence: NC011916.1) using Bowtie 1 [68]. Subsequently, the sequencing coverage at each nucleotide position was computed using BEDTools [69]. Finally, ChIP-seq profiles were plotted with the x-axis representing genomic positions and the y-axis is the number of reads per kilobase pair per million mapped reads (RPKM) using custom R scripts. ChIP-seq data are available at GEO accession number GSE184477.
C. crescentus cells (80 mL culture) were grown at 28 °C to stationary phase before cells were harvested by centrifugation. Cell pellets were washed with 1× PBS (pH 7.4), resuspended in 1.5 mL of lysis buffer [50 mM Tris-HCl (pH8), 150 mM NaCl, 1% Triton X-100, EDTA-free protease inhibitors, 10 mg/mL lysozyme, and 1 μL of Benzonase], and incubated at 37 °C for 20 min. Subsequently, the cell suspension was sonicated on ice using a probe-type sonicator (6 cycles of 15 s with 15 s resting on ice, amplitude setting 8). The lysate was cleared from the cell debris by centrifugation (13,000 rpm for 20 min at 4 °C). A total of 50 μL of this supernatant (i.e., the INPUT fraction) was kept for immunoblot analysis. The remaining supernatant was mixed with 50 μL α-FLAG magnetic bead as instructed by the μMACS Epitope Tag Protein Isolation Kit (Miltenyi Biotec). From here, all the subsequent steps were performed according to the instruction from the μMACS kit, except for the washing step. The magnetic columns were washed instead with 2 mL of IPP200 buffer [10 mM Tris-HCl (pH 8.0), 200 mM NaCl, and 0.1% NP-40], followed by 0.5 mL of 1× TE buffer [10 mM Tris-HCl (pH 8.0) and 1 mM EDTA]. The immunoprecipitated proteins (i.e., the IP fraction) were eluted using 50 μL of elution buffer [50 mM Tris-HCl (pH 6.8), 50 mM DTT, 1% SDS, and 1 mM EDTA]. For immunoblot analysis, 10 μg of the INPUT fraction and 5 μL of the IP fraction were loaded on a 4% to 20% Novex WedgeWell SDS-PAGE gels (Thermo Fisher Scientific). Resolved proteins were transferred to polyvinylidene fluoride (PVDF) membranes using the Trans-Blot Turbo Transfer System (BioRad), and the membrane was incubated with a 1:2,500 dilution of an α-FLAG antibody (Merck), or a 1:5,000 dilution of an α-ParB antibody (custom antibody, Cambridge Research Biochemicals, UK), or a 1:300 dilution of an α-GafY antibody (custom antibody, Cambridge Research Biochemicals, UK). Subsequently, the membranes were washed twice in a 1× TBS + 0.005% Tween-20 buffer before being incubated in a 1:10,000 dilution of an HRP-conjugated secondary antibody. Blots were imaged using an Amersham Imager 600 (GE Healthcare).
Overlapping single-stranded oligomers that span the promoter region of gafY were dissolved to 100 μM in water (gafY_1–11_F-R). They were annealed together with their complementary oligos in an annealing buffer [10 mM Tris-HCl (pH 8.0), 50 mM NaCl, and 1 mM EDTA] to form double-stranded DNA before being diluted to a working concentration of 1 μM in HPS-EP buffer [0.01 M HEPES (pH 7.4), 150 mM NaCl, 3 mM EDTA, and 0.005% surfactant P20] for SPR experiments. SPR measurements were recorded at 25°C using a Biacore 8K system (Cytiva). All experiments were performed using the Re-usable DNA Capture Technique (ReDCaT) as described previously [70]. Briefly, ReDCAT uses a sensor chip SA Cytiva that has streptavidin pre-immobilized to a carboxymethylated dextran matrix, to which a 20-base biotinylated ReDCaT linker is immobilized. This chip is then used to immobilize PgafY- dsDNA on the chip surface as each dsDNA also contain a single-stranded overhang complimentary to the ReDCaT linker on the surface. The DNA to be tested was flowed over the test flow cell on the chip at a flow rate of 10 μL/min and it annealed through the complementary DNA to the ReDCaT linker, thus was immobilized on the surface of the chip. Purified RogA-His6 protein, pre-diluted in HBS-EP buffer, was then flowed over the chip surface (both the surface with the immobilized DNA and a blank surface as a control), followed by HBS-EP buffer to observe RogA-His6 dissociation from the DNA. The chip was then regenerated using 1 M NaCl and 50 mM NaOH to remove any residual RogA-His6 protein and the test DNA. The cycle can then be repeated as many times as desired with new test DNA and protein concentration. The Biacore 8K has 8 channels and 2 flow cells so for each cycle, 8 different DNA samples can be tested (and each referenced against a reference flow cell). The SPR signal (response units) was monitored continuously throughout the process. All sensorgrams recorded during ReDCAT experiments were analyzed using Biacore Insight Evaluation software version 3.0.11.15423 (Cytiva). Data were then plotted using Microsoft Excel.
Overlapping single-stranded oligomers that spans the promoter region of CCNA_02880 (GTA terminase) were dissolved to 100 μm in water (02880_1–9_F-R). They were annealed together with their complementary oligos in an annealing buffer [10 mM Tris-HCl (pH 8.0), 50 mM NaCl, and 1 mM EDTA] to form double-stranded DNA before being diluted to a working concentration of 1 μM in HPS-EP buffer. A similar ReDCaT procedure was used to measure the interaction between the GafYZ complex and immobilized DNA, except that a custom binding buffer TSE-T buffer [10 mM Tris-HCl (pH 8.0), 200 mM NaCl, 1 mM EDTA, and 0.005% Tween 20] instead of HPS-EP buffer was used instead.
C. crescentus ΔrogA cells (in 200-mL culture) were grown in PYE at 28 °C to stationary phase. Cells were pelleted by centrifugation (8,000 rpm for 10 min at 4 °C). The supernatant that contained GTA particles was then transferred to a fresh 500-mL bottle and was filtered twice using a 0.22-μm filter funnel (Sartorius). The supernatant was concentrated using a 100-kDa MWCO Amicon concentrator (Merck) to approximately 25 mL. GTA particles in this concentrated supernatant were precipitated by incubating with a 5xPEG/NaCl solution [20% PEG-8000 and 2.5 M NaCl] on ice for 30 min. Precipitated GTA particles were collected by centrifugation (8,000 rpm for 20 min at 4°C) and were resuspended in 200 μL storage buffer [50 mM Tris pH 8.0, 150 mM NaCl, and 5% glycerol]. For TEM analysis, 3 μL of the purified GTA particles was pipetted on a 400-mesh copper grid (EM Resolutions) that had been glow discharged for 20 s at 10 mA in an Ace 200 (Leica Microsystems). After 60 s, excess solution was wicked away using Whatman No. 1 filter paper and leftover samples on the grid were stained using 2% (w/v) uranyl acetate solution. Grids were imaged using a Talos F200C transmission electron microscope (Thermo Fisher Scientific) operated at 200 kV, equipped with a 4 k OneView CMOS detector (Gatan, UK).
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PMC9633173 | Yue Ming,Zhihui Deng,Xianhua Tian,Yuerong Jia,Meng Ning,Shuhua Cheng | m6A Methyltransferase METTL3 Reduces Hippocampal Neuron Apoptosis in a Mouse Model of Autism Through the MALAT1/SFRP2/Wnt/β-catenin Axis | 21-10-2022 | Autism,Methyltransferase like-3,Long noncoding RNA MALAT1,DNA methyltransferase,Secreted Frizzled Related Protein 2,Wnt/β-catenin signaling,Hippocampal neurons | Objective Hippocampal neuron apoptosis contributes to autism, while METTL3 has been documented to possess great potentials in neuron apoptosis. Our study probed into the role of METTL3 in neuron apoptosis in autism and to determine the underlying mechanism. Methods Bioinformatics analysis was used to analyze expressed genes in autism samples. Institute of Cancer Research mice were treated with valproic acid to develop autism models. The function of METTL3 in autism-like symptoms in mice was analyzed with behavioral tests and histological examination of their hippocampal tissues. Primary mouse hippocampal neurons were extracted for in vitro studies. Downstream factors of METTL3 were explored and validated. Results METTL3, MALAT1, and Wnt/β-catenin signaling were downregulated, while SFRP2 was upregulated in the hippocampal tissues of a mouse model of autism. METTL3 stabilized MALAT1 expression by promoting m6A modification of MALAT1. MALAT1 promoted SFRP2 methylation and led to reduced SFRP2 expression by recruiting DNMT1, DNMT3A, and DNMT3B to the promoter region of SFRP2. Furthermore, SFRP2 facilitated activation of the Wnt/β-catenin signaling. By this mechanism, METTL3 suppressed autism-like symptoms and hippocampal neuron apoptosis. Conclusion This research suggests that METTL3 can reduce autism-like symptoms and hippocampal neuron apoptosis by regulating the MALAT1/SFRP2/Wnt/β-catenin axis. | m6A Methyltransferase METTL3 Reduces Hippocampal Neuron Apoptosis in a Mouse Model of Autism Through the MALAT1/SFRP2/Wnt/β-catenin Axis
Hippocampal neuron apoptosis contributes to autism, while METTL3 has been documented to possess great potentials in neuron apoptosis. Our study probed into the role of METTL3 in neuron apoptosis in autism and to determine the underlying mechanism.
Bioinformatics analysis was used to analyze expressed genes in autism samples. Institute of Cancer Research mice were treated with valproic acid to develop autism models. The function of METTL3 in autism-like symptoms in mice was analyzed with behavioral tests and histological examination of their hippocampal tissues. Primary mouse hippocampal neurons were extracted for in vitro studies. Downstream factors of METTL3 were explored and validated.
METTL3, MALAT1, and Wnt/β-catenin signaling were downregulated, while SFRP2 was upregulated in the hippocampal tissues of a mouse model of autism. METTL3 stabilized MALAT1 expression by promoting m6A modification of MALAT1. MALAT1 promoted SFRP2 methylation and led to reduced SFRP2 expression by recruiting DNMT1, DNMT3A, and DNMT3B to the promoter region of SFRP2. Furthermore, SFRP2 facilitated activation of the Wnt/β-catenin signaling. By this mechanism, METTL3 suppressed autism-like symptoms and hippocampal neuron apoptosis.
This research suggests that METTL3 can reduce autism-like symptoms and hippocampal neuron apoptosis by regulating the MALAT1/SFRP2/Wnt/β-catenin axis.
Autism is a severe neurobehavioral syndrome and often accompanied with deficient social interaction and communication capacities and repeated and stereotyped behaviors [1]. Autism affects males more than females which might due to the influence of fetal testosterone [2]. More importantly, autism is commonly diagnosed in early childhood [3]. Accumulated evidence reveals that autism is related to aberrant expression of genetic factors or environment factors, including alcohol abuse and cocaine during pregnancy [4]. To get insight to the molecular mechanism, hippocampal neuron apoptosis has been reported to contribute to the development of autism [5]. Recently, m6A modification has been reported to regulate gene expression by affecting mRNA stability, translation, and transcription elongation [6-8]. METTL3, a methyltransferase, governs predominant m6A modification in cells and engages in diverse biological processes, including cell apoptosis [9]. Importantly, a recent study reveals aberrant downregulation of METTL3 in autism hippocampal tissues while the molecular mechanism of how METTL3 regulates the development of autismlike behaviors remains exclusive [10]. METTL3 can mediate the expression of MALAT1 in adriamycin-resistant breast cancer through m6A [11]. Long noncoding RNAs (lncRNAs) are involved in the regulation of various critical biological processes by regulating gene expression, sponging microRNAs or functioning as a scaffold to recruit specific epigenetic factors to the certain sites [12,13]. Bioinformatics analysis predicted that DNA methyltransferase 1 (DNMT1), DNMT3A, and DNMT3B might interact with MALAT1. DNMT protein family has been reported to participate in regulation of biological process by promoting methylation on genes promoter [14]. DNMT1 is a maintenance methyltransferase, which is conducive to maintain the methylation pattern. DNMT3A and DNMT3B are the latest methylation enzymes, which participate in the establishment of tissue-specific DNA methylation patterns during development and in response to environmental factors [15]. Secreted frizzledrelated protein (SFRP) is glycoprotein containing a so-called frizzled-like cysteine-rich domain. This domain helps them bind to Wnt ligands or frizzled receptors, regulating Wnt signaling [16]. A previous study has revealed that SFRP2 promotes phosphorylation mediated degradation of β-catenin [17]. Meanwhile, Wnt/β-catenin signaling has been revealed to protect hippocampal neurons from apoptosis [18]. Therefore, this study aims to verify whether METTL3 inhibits autism-like behaviors and hippocampal neuron apoptosis by regulating the MALAT1/SFRP2/Wnt/β-catenin axis.
Animal experimental processes were approved by the Animal Care and Use Committee of Qiqihar Medical university. All experiments were in accordance with the Animal Research: Reporting of In Vivo Experiments guidelines on the Care and Use of Experimental Animals.
Differential analysis of the autism-related gene expression dataset GSE38322 retrieved from GEO database was conducted using R “limma” package to screen differentially expressed genes with p<0.05 as the threshold. GSE38322 dataset includes 36 samples, consisting of 18 normal samples and 18 autism samples. Venn diagram was then plotted and m6A-modified genes were screened. Interacting genes of significantly differential m6A-modified genes were predicted through STRING database and a protein-protein interaction (PPI) network was constructed. A confidence score of >0.7 was considered to be statistical significance [19]. Next, Cytoscape was used to plot and calculate the core degree, with genes with degree value >10 considered as hub genes. The differential m6A-modified gene with the highest core degree was selected [20]. Based on the previous reports, its downstream pathways were determined. MEM database was then applied for co-expression analysis to identify the correlation of the m6A-modified gene with downstream genes. The methylases possibly regulated by MALAT1 were predicted by RPISeq. Moreover, the downstream target, SFRP2 was determined based on previous studies and Methprimer database prediction results. STRING database was used to predict the interacting genes of SFRP2, which were then subjected to KEGG pathway enrichment analysis using KOBAS database to identify regulatory pathways.
Healthy adult ICR male (aged: 8 weeks old; weighing: 40– 70 g) and female mice (aged: 8 weeks old; weighing: 40–60 g) were obtained from Hunan SJA Laboratory Animal Co., Ltd. (Hunan, China). These mice were housed in the SPF laboratory at 25°C and 55% humidity under a 12-h light/dark cycle, with ad libitum access to food and water for acclimatization. The male and female mice were allowed to mate overnight at a ratio of 2:1. The next morning, the female mice were examined for pregnancy. Then, the pregnant mice were housed in separate cages and randomized into control and valproic acid (VPA)-treated groups. VPA (p4543, Sigma-Aldrich, St. Louis, MO, USA) was dissolved in 0.9% saline at a concentration of 250 mg/mL. Female mice were subjected to a single intraperitoneal injection of 600 mg/kg VPA on E12.5 day after conception, and control female mice were subjected to injection of the same amount of saline at the same time point. The new born mice from the model group were described as autism group; the mice from the control group were described as control group. The offspring were weaned on postnatal day [21]. The following experimentations were carried out only on the male offspring. After 35 days, behavior-related experiments were processed to validate the successful construction of the model.
Mice after modeling were deeply anesthetized by isoflurane and then injected with lentivirus (Genechem, Shanghai, China) to overexpress or silence METTL3, MALAT1, or SFRP2. Bilateral pores were drilled 1.7 mm posterior and 1.5 mm lateral to the anterior chimney of the mice and the viruses (0.6 μL, 2×106 transducing units/μL) were injected into the hippocampal region at a rate of 0.1 μL per min with the Nanoject II (Drummond Scientific Company, Broomall, PA, USA) system. The injection cannula was slowly retracted 5 min after lentivirus injection. Mice were randomized as follows (8 mice for each group): oe-negative control (NC), oe-MALAT1, oe-NC+sh-NC, oe-METTL3+sh-NC, oe-METTL3+sh-MALAT1, sh-NC, sh-SFRP2 (SFRP2 silencing), sh-SFRP2+dimethyl sulfoxide (DMSO), sh-SFRP2+nitazoxanide (NTZ; Wnt/β-catenin signaling inhibitor), oe-METTL3+DMSO, and oe-METTL3+ NTZ. Wnt/β-catenin signaling inhibitor NTZ was delivered to mice at a dose of 200 mg/kg daily. After 4 weeks, behavioral experiments were performed. Next, the hippocampal tissues of mice were removed and used for biochemical analysis.
The open field test was carried out for assessing the general locomotor activity and anxiety-like behavior utilizing a Plexiglas (100×100×40 cm) [21]. The three-chamber test was a commonly applied method to assay social approach behavior in mice with a 60 cm×40 cm white Plexiglas box. During the test phase, stranger 1 mice of the same sex and same age were randomly placed in metal cages in the left or right box. An unfamiliar object was placed on the metal cage on the other side of the box and the test mice were allowed to freely explore the three experimental compartments for 10 min. Video recordings were used to detect the residence time of the mice in each of the three experimental compartments, and to measure the time the mice spent in establishing social communication with stranger 1. The duration of direct contact between mice and stranger 1 or an empty metal cage was determined. With 3–5 cm around the metal cage defined as the contact range, the duration of mice entering each box was recorded. The head and four claws of mice entering a box was indicative of their existence in the box. Self-grooming test is performed to analysis repetitive performance of mice in restricted space utilizing a 500-mL glass beaker (7.5 cm diameter×10 cm tall) [21].
TUNEL staining kit (In Situ Cell Death Detection Kit; Roche, Basel, Switzerland) was applied for detection of neuron apoptosis in the hippocampal tissues of mice. Briefly, hippocampal tissue sections were incubated with anti-NeuN antibody (1:500, ab177487; Abcam, Cambridge, UK), and then stained with DIPA. For NC, after TUNEL staining, slides were incubated with the TUNEL labeling solution without terminal transferase. The data were expressed as the number of total TUNELand NeuN double-positive per 250 μm length of medial CA1. All measurements were implemented in a blinded manner. There sections were selected from each mouse and then observed under a fluorescence microscope (Olympus Optical Co., Ltd, Tokyo, Japan) in three fields of the CA1 region for cell counting. The number of TUNEL and NeuN double positive cells is the number of cells obtained by counting the field of view in the CA1 region of 250 μm in length [22].
The hippocampal tissues were fixed in 10% formaldehyde, paraffin-embedded and sliced into 4-μm-thick sections. The tissue sections were heated in a 60°C oven for 1 h, dewaxed, dehydrated and incubated with 3% H2O2 (Sigma-Aldrich) for 30 min. Afterwards, the sections were boiled in 0.01 M sodium citrate for 20 min at 95°C and blocked with normal goat serum (C-005; HaoranBio, Shanghai, China) at 37°C for 10 min. The sections were immunostained with anti-BAX antibody (Rabbit, 1:250, ab32503; Abcam) for 24 h at 4°C. The next day, the sections were incubated with biotin-labelled secondary antibody goat anti-rabbit (1: 500, ab6721; Abcam) for 10 min at room temperature. Afterwards, the sections were treated with horseradish peroxidase (HRP)-labeled streptavidin working solution (0343-10000U; Emmer Biotechnology Co., Ltd., Beijing, China) and developed with DAB (ST033; Guangzhou Weijia Technology Co., Ltd., Guangzhou, China). The sections were then counterstained with hematoxylin (PT001; Shanghai Bogoo Biotechnology Co., Ltd., Shanghai, China) for 1 min, dehydrated, cleared and sealed before observation under an optical microscope. BAX-positive expression is mainly exhibited by yellow-brown staining in the cytoplasm.
The neonatal mice within 24 h were sterilized with 75% medical alcohol, after which the brain of the mice was cut out and the hippocampal tissue was quickly separated, and cut into small pieces. The hippocampal tissue pieces were digested with 0.125% trypsin for 15 min at 37°C, ground and centrifuged. Cells were seeded in 10-mm dishes coated with polylysine (10 mmol/L) at a density of 1×106 cells/mL and maintained in Neural Basal Medium (Gibco, Carlsbad, CA, USA) with 2% B27 (Gibco) and 0.25% Glumax (Gibco). After 3 days, 2.5 μg/mL cytarabine (Sigma-Aldrich) was added to the medium for 24 h to suppress the proliferation of glial cells. Next, 50% of the medium was renewed every three days. Cells were cultured at 37°C with 5% CO2 for 14 days before experimentations [23]. Hippocampal neurons were then transduced with lentivirus (Genechem; multiplicity of infection=5) carrying sh-NC, sh-MALAT1-1, sh-MALAT1-2, sh-MALAT1-3, oe-NC, oe-MALAT1, sh-METTL3-1, sh-METTL3-2, sh-METTL3-3, and oe-METTL3. After 48 h, cells were harvested for subsequent experimentations.
On the 7th day of in vitro culture, primary mouse hippocampal neurons were fixed with 4% paraformaldehyde for 30 min, treated with 0.2% Triton X-100 at ambient temperature for 15 min, blocked with 3% bovine serum albumin (BSA) at 4°C for 30 min, and then incubated with mouse anti-NeuN (ab104224, 1:200; Abcam) in a wet box at 4°C overnight. Next, the cells were incubated with goat anti-mouse secondary antibody (ab150113, 1:200) at ambient temperature away from light for 2 h. 4’,6-diamidino-2-phenylindole (ab104139, 1:100; Abcam) was added for incubation at ambient temperature for 10 min. The results were observed under an inverted fluorescence microscope.
Total RNA was extracted from mouse hippocampal tissues and cells using a Trizol kit (Thermo Fisher Scientific Inc.). RNA was then reversely transcribed into cDNA employing Reverse Transcription kit (ABI, Applied Biosystems). Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) was processed utilizing an ABI7500 quantitative PCR instrument. GAPDH was used as a loading control and the 2-ΔΔCt method was employed to calculate the relative expression of target genes. Primers are provided by Sangon Biotech Co., Ltd. (Shanghai, China) and listed in Supplementary Table 1 (in the online-only Data Supplement).
The extracted protein sample was separated using freshlyprepared sodium dodecyl sulphate polyacrylamide gel electrophoresis, electrotransferred onto polyvinylidene fluoride membranes, blocked with 5% BSA for 2 h, and probed with primary antibodies overnight at 4°C. The membranes were then re-probed with HRP-conjugated secondary antibody goat anti-rabbit (ab6721, 1:2,000; Abcam) at room temperature for 1 h. Next, enhanced chemiluminescence reagent (EMD Millipore, Billerica, MA, USA) was used to visualize the results by X-ray film. Protein bands were assayed utilizing Image J software, with β-actin selected for normalization. Primary antibodies used: SFRP2 (12189-1-AP, 1:1,000; Proteintech, Chicago, IL, USA), β-catenin (ab16051, 1:2,000, Abcam), p-β-catenin (ab75777, 1:500; Abcam), BAX (ab32503, 1:1,000; Abcam), Bcl-2 (ab196495, 1:1,000; Abcam), and β-actin rabbit polyclonal antibodies (ab8227, 1:2,000; Abcam).
Genomic DNA from neurons were extracted with TIANamp Genomic DNA Kit (Tiangen Biotech, Beijing, China), converted by bisulfite, and stored at -80°C. MS-PCR was processed utilizing a methylation-specific kit (Tiangen Biotech) on purified DNA with methylated primers and unmethylated primers (Sangon) described in Supplementary Table 2 (in the online-only Data Supplement). PCR conditions consisted of 10 min at 95°C for initial denaturation, followed by 35 cycles of 95°C (45 s), annealing temperature (methylated [58°C] and unmethylated [57°C] of primers [30 s], and 72°C [45 s]) and a final elongation of 10 min at 72°C. The resultant products were visualized by agarose gel electrophoresis and analyzed by Image J.
The interaction between MALAT1 and indicated DNMTs were validated by RNA binding protein immunoprecipitation (RIP) kit (EMD Millipore). Cells were rinsed by pre-cold phosphate phosphate-buffered saline, lysed by equal volume lysis buffer for 5 min, and centrifuged at 8,000 g for 10 min. The supernatant was incubated with indicated beads pre-conjugated with indicated antibody overnight at 4°C. The samples were harvested in magnetic constellation and then digested by proteinase K and subjected to RNA extraction for following RT-qPCR. Used antibody: DNMT1 (10411-1-AP, 1:50; Proteintech), DNMT3A (20954-1-AP, 1:50; Proteintech), and DNMT3B (MA5-16165, 1:50; Invitrogen Inc., Carlsbad, CA, USA).
Chromatin immunoprecipitation (ChIP) assay was implemented employing ChIP Kit (EMD Millipore). Cells were fixed with 1% formaldehyde for 10 min to generate DNA-protein cross-linking and incubated with 0.125 M glycine for 5 min. Then, the cells were lysed and subjected to ultrasonic treatment at 120 w, 2 s on, and 5 s off for 15 cycles to produce appropriately sized fragments. The lysates were incubated antibodies against RNA polymerase II (positive control), IgG (negative control; ab205718, 1:100; Abcam), DNMT1 (10411-1-AP, 1:50; Proteintech), DNMT3A (20954-1-AP, 1:50; Proteintech), and DNMT3B (MA5-16165, 1:50; Invitrogen) at 4°C overnight. Protein agarose/sepharose was used to immunoprecipitate the endogenous DNA-protein complex, which was incubated at 65°C overnight to relieve cross-linking. Phenol/chloroform was then applied for extraction of the obtained DNA fragments. Finally, the DNA fragments were analyzed using qPCR.
Total RNA was isolated from neurons by TRIzol and mRNA was isolated using PolyATtract® mRNA Isolation Systems (AZ5300; Aidlab, Beijing, China). Anti-m6A antibody (Abcam, ab151230) or anti-IgG (ab109475, 1:100; Abcam) was prebound to protein A/G magnetic beads (Pierce) in IP buffer (20-mM Tris pH 7.5, 140-mM NaCl, 1% NP-40, and 2-mM EDTA) for 1 h. mRNA and magnetic beads complex was isolated and purified by IP buffer supplemented with ribonuclease inhibitors and protease inhibitors. RNA was eluted by wash buffer and purified by phenol-chloroform extraction. Primer sequences are described in Supplementary Table 1 (in the online-only Data Supplement). MALAT1 expression was assayed by RT-qPCR.
Neurons were incubated with 200 mM of 4-thiopyridine (4SU) (Sigma-Aldrich) for 14 h and crosslinked with 0.4 J/cm2 at 365 nm. The lysates were followed by immunoprecipitation with METTL 3 antibody at 4°C (ab240595, 1:200; Abcam), RNA was precipitated by [g-32-P]-ATP labeling and visualized by radioautocytography. Photoactivatable-ribonucleoside-enhanced-fragments were digested by protease K to remove proteins, and RNA was extracted for RT-qPCR to detect MALAT1 expression levels. The experiment was repeated three times.
Data are described as the mean±standard deviation from at least three independent experiments. Statistical comparison was assayed using unpaired t test when only two groups were compared or by Tukey’s test-corrected one-way analysis of variance (ANOVA) when more than two groups were compared. All statistical analyses were implemented with SPSS 20.0 software (IBM Co., Armonk, NY, USA; *p<0.05, **p<0.01, ***p< 0.001, ****p<0.0001).
Differential analysis of the GSE38322 dataset revealed 5,860 differential genes, consisted of 2,748 upregulated and 3,112 downregulated genes. A previous study has suggested that m6A modifications may be related to axonal regeneration in the nervous system [24]. We compared differential genes with m6A-modified genes and found that four m6A-modified genes were significantly differentially expressed in autism (Figure 1A). Next, we predicted five related genes of these four genes by STRING database and constructed a PPI network (Figure 1B). Based on this, we found the highest degree of METTL3 core in the network, which indicated METTL3 might be the key m6A-modified gene in autism. Meanwhile, analysis of the GSE38322 dataset found that METTL3 was significantly downregulated in autism samples (Figure 1C). As previously reported, METTL3 can stabilize MALAT1 expression by mediating m6A modification of it [25]. Besides, impaired spatial learning memory was found in METTL3 depleted mice [26]. Moreover, we determined that METTL3 was significantly co-expressed with MALAT1 by co-expression analysis using MEM (Figure 1D). The online website RPISeq predicted that MALAT1 could bind DNA methylation enzymes DNMT1, DNMT3A, and DNMT3B (Figure 1E), and the Methprimer database predicted the presence of large number of CPG islands in the SFRP2 promoter (Figure 1F). What’s more, previous study has documented that SFRP2 can undergo hypermethylation [27]. Thus, we speculated that MALAT1 could recruit DNA methylation enzymes (DNMT3B, DNMT3A, and DNMT1) to regulate the methylation status of SFRP2 in hippocampal neurons of the mouse model of autism. We used STRING database to predict the 10 interacting genes of SFRP2 (Figure 1G), and then the KEGG pathway enrichment of SFRP2 and its interacting genes by KOBAS obtained a total of 17 KEGG pathways, of which the most enriched signaling was Wnt signaling (Figure 1H). Published literature has shown that SFRP2 inhibits the Wnt/β-catenin signaling to promote neuron apoptosis [28], while activation of the Wnt/β-catenin signaling can inhibit neuron damage [29]. SFRP2 may regulate neuron apoptosis in autism via the Wnt/β-catenin signaling. Taken together, we speculated that METTL3 might affect SFRP2 methylation to govern the Wnt/β-catenin signaling by regulating MALAT1 expression via m6A modification, thus influencing neuron apoptosis in autism.
Then, we moved to determine the role of the aforementioned METTL3/MALAT1/SFRP2/Wnt/β-catenin axis in autism in vivo. We established a mouse model of autism and performed behavioral experiments when mice were 35 days old to validate the success of the model. In the open filed test, the autistic mice exhibited reduced duration and entries in the central area in comparison with control (Figure 2A). Meanwhile, the three chambers social test was carried to determine the social ability, and the results revealed that the autistic mice tended to stay longer time in non-social chamber than in social chamber in contrast to control mice (Figure 2B). Consistently, autistic mice spend more time in repetitive self-grooming than control mice (Figure 2C). Collectively, these data indicated the successful establishment of autism mouse models. The hippocampal tissues of mice were collected after behavioral experiments. Neurons were labeled with NeuN and TUNEL staining. The results revealed higher apoptosis rate of hippocampal neurons in the CA1 hippocampal tissues of autistic mice (Figure 2D). In addition, the results of RT-qPCR suggested that MALAT1 was under-expressed in hippocampal tissues of autistic mice (Figure 2E). Taken together, our data revealed decreased MALAT1 expression in hippocampal tissues of autistic mice.
The above findings of aberrant downregulation of MALAT1 in hippocampal tissues of a mouse model of autism allowed us to investigate the role of MALAT1 in autism. MALAT1 was overexpressed in hippocampal tissues of autistic mice and the overexpression efficiency was confirmed by RT-qPCR (Figure 3A). Open field test results revealed that MALAT1 overexpression significantly increased the duration and entries in central area of a mouse model of autism (Figure 3B). Moreover, three chambers test data demonstrated autistic mice treated with oe-MALAT1 stayed longer in social chamber than non-social chamber (Figure 3C). Consistently, autistic mice treated with oe-MALAT1 spent less time on self-grooming (Figure 3D). These results indicated that overexpression of MALAT1 alleviated the autism-like behaviors in mice. NeuN and TUNEL staining results indicated that ectopic expression of MALAT1 reduced neuron apoptosis rates in the CA1 hippocampal tissues of a mouse model of autism (Figure 3E). In addition, MALAT1 overexpression led to decreased BAX positive expression rates in hippocampal tissues of a mouse model of autism (Figure 3F). Meanwhile, western blot results demonstrated that overexpression of MALAT1 increased anti-apoptosis protein BCL2 expression while inhibiting BAX expression (Figure 3G). Collectively, overexpression of MALAT1 suppressed autism-like behaviors and hippocampal neuron apoptosis in a mouse model of autism.
A previous report revealed that METTL3 could stabilize MALAT1 by enhancing m6A modification [25]. Hence, we aimed to determine whether METTL3 can promote m6A modification of MALAT1 in autistic mice. RT-qPCR results showed that METTL3 was downregulated in the hippocampal tissues of autistic mice (Figure 4A). Moreover, Me-RIP assay data revealed that m6A level of MALAT1 was reduced in hippocampal tissues of a mouse model of autism (Figure 4B). Immunofluorescence for determination of the expression of neuron marker NeuN showed that the percentage of NeuN positive cells, that is, the neurons, was more than 95% (Figure 4C), which could be used in subsequent experiments. Furthermore, we silenced METTL3 gene in neurons by three independent shRNAs, and RT-qPCR was carried to validate the knockdown efficiency (Figure 4D). Among the three shRNAs, shMETTL3#2 showed superior silencing efficiency and was chosen for following experiments. We overexpressed and silenced METTL3 in primary mouse hippocampal neurons and found that oe-METTL3 significantly increased MALAT1 expression while sh-METTL3 reduced MALAT1 expression (Figure 4E). In addition, ectopic METTL3 expression elevated the m6A levels of MALAT1, while METTL3 silencing caused opposite results (Figure 4F). Importantly, photoactivatable-ribonucleoside-enhanced crosslinking and immunoprecipitation demonstrated that METTL3 overexpression facilitated its interaction with MALAT1, while precipitated MALAT1 was reduced when METTL3 was depleted (Figure 4G). Altogether, METTL3 promoted MALAT1 expression by enhancing the m6A level of MALAT1.
Considering the aforementioned results, we then sought to investigate whether METTL3 regulated autism-like behaviours and hippocampal neuron apoptosis by MALAT1. RT-qPCR results confirmed that MALAT1 and MALAT1 expression was significantly upregulated in hippocampal tissues of autistic mice treated with oe-METTL3 while further sh-MALAT1 led to decreased MALAT1 expression without affecting METTL3 expression (Figure 5A). Furthermore, we performed open field test, three chambers test, and self-grooming test to validate mice motor activity and social abilities (Figure 5B-D). The results showed that ectopic METTL3 expression increased the duration and entries in central area in autistic mice. Consistently, autistic mice treated with oe-METTL3 spent more time on social chamber and less time on self-grooming. However, the effect of METTL3 overexpression was reversed by MALAT1 silencing. Subsequently, NeuN and TUNEL staining results identified that METTL3 overexpression suppressed neuron apoptosis in the hippocampal tissues of autistic mice, while this effect was abolished by MALTA1 depletion (Figure 5E). In addition, METTL3 overexpression dramatically reduced BAX positive expression rates in the hippocampal tissues of a mouse model of autism, while increased BAX positive expression rates were noted due to further MALAT1 silencing (Figure 5F). Similarly, METTL3 overexpression suppressed BAX expression and promoted BCL-2 expression in hippocampal tissues of autistic mice but these results were rescued by depletion of MALAT1 (Figure 5G). Collectively, our data demonstrated METTL3 reduced autism-like behaviors and hippocampal neuron apoptosis by enhancing m6A level of MALAT1.
We then investigated the downstream factor of MALAT1 in autism. The results of western blot and RT-qPCR revealed that SFRP2 was upregulated in the hippocampal tissues of a mouse model of autism (Figure 6A and B). In addition, we silenced MALAT1 in hippocampal neurons by three independent shRNAs and RT-qPCR validated the knockdown efficiency (Figure 6C). Among those shRNAs, silencing efficiency of sh-MALAT1#1 was the best and it was chosen for following experiments. Furthermore, overexpression of MALAT1 reduced SFRP2 expression, while its depletion increased SFRP2 expression in neurons (Figure 6D and E). Moreover, MSP assay results revealed that overexpression of MALAT1 increased the methylation level of SFRP2, while the methylation level was downregulated after MALAT1 inhibition (Figure 6F). The results of RIP assay suggested that overexpression of MALAT1 increased DNMT1, DNMT3A, and DNMT3B-precipetated MALAT1, while it was reduced when MALAT1 silenced (Figure 6G), which indicated MALAT1 interacted with DNMT1, DNMT3A, and DNMT3B. Furthermore, ChIP assay revealed that overexpression of MALAT1 increased the enrichment of DNMT1, DNMT3A, and DNMT3B on the SFRP2 promoter, while this increase was reversed following MALAT1 silencing (Figure 6H). Collectively, MALAT1 promoted the methylation of SFRP2 and repressed SFRP2 expression by recruiting DNMT1, DNMT3A, and DNMT3B to the promoter region of SFRP2.
Previous literature has reported that activation of the Wnt/β-catenin signaling inhibits neuron damage[29] and SFRP2 functions as an antagonist of the Wnt/β-catenin signaling to promote neuron apoptosis [28]. Thus, in the next experiment, we proceeded to analyze whether SFRP2 participates in the apoptosis of hippocampal neurons in autistic mice by affecting the Wnt/β-catenin signaling. Western blot data showed that β-catenin phosphorylation level was increased in the hippocampal tissues of autistic mice, which led to degradation of β-catenin and a decrease in the protein expression of β-catenin (Figure 7A). Moreover, western blot and RT-qPCR results demonstrated that SFRP2 silencing inhibited β-catenin phosphorylation level and increased β-catenin protein expression, while further NTZ treatment elevated β-catenin phosphorylation level and decreased β-catenin protein expression without affecting SFRP2 mRNA and protein expression (Figure 7B and C). Furthermore, as shown in Figure 7D-F, SFRP2 silencing increased the duration and entries in central area in autistic mice. Consistently, autistic mice treated with sh-SFRP2 spent more time on social chamber and less time on self-grooming. However, the effect of SFRP2 silencing was reversed by NTZ treatment. Subsequent NeuN and TUNEL staining results showed that SFRP2 silencing suppressed hippocampal neuron apoptosis in autistic mice, while this effect was abolished by NTZ treatment (Figure 7G). Immunohistochemistry (IHC) data exhibited that SFRP2 silencing reduced BAX positive expression rates, which was reversed by NTZ treatment (Figure 7H). Similarly, SFRP2 silencing suppressed BAX expression and promoted BCL-2 expression in the hippocampal tissues from autistic mice. In contrast, opposite results were found in response to NTZ treatment (Figure 7I). Collectively, SFRP2 promoted activation of the Wnt/β-catenin signaling to enhance autism-like behaviors and hippocampal neuron apoptosis in a mouse model of autism.
Finally, we intended to elucidate the effect of METTL3 regulating the MALAT1/SFRP2/Wnt/β-catenin signaling on the hippocampal neuron apoptosis in vivo. The results of western blot and RT-qPCR revealed that overexpression of METTL3 upregulated MALAT1 and β-catenin expression accompanied with downregulation of SFRP2 expression and β-catenin phosphorylation level. Importantly, ectopic expression of SFRP2 or NTZ treatment in METTL3 overexpressed autistic mice reversed the upregulated β-catenin expression caused by METTL3 overexpression without affecting MALAT1 expression (Figure 8A and B). Furthermore, the results of open field test, three chambers test, and self-grooming test (Figure 8C-E) found that METTL3 overexpression increased the duration and entries in central area in a mouse model of autism. Consistently, oe-METTL3-treated autistic mice spent more time on social chamber and less time on self-grooming. However, the positive effect of METTL3 overexpression was reversed by ectopic SFRP2 expression or NTZ treatment. In addition, the results of NeuN and TUNEL staining suggested that METTL3 overexpression suppressed hippocampal neuron apoptosis in a mouse model of autism while this effect was abolished by SFRP2 overexpression and NTZ treatment (Figure 8F). Consistently, IHC data presented that METTL3 overexpressed reduced BAX positive expression rates, while this reduction was abrogated by SFRP2 overexpression and NTZ treatment (Figure 8G). Similarly, METTL3 overexpression repressed BAX expression and promoted BCL-2 expression in the hippocampal tissues of a mouse model of autism. Importantly, the upregulation of BCL-2 and downregulation of BAX were rescued by SFRP2 overexpression and NTZ treatment (Figure 8H). Taken together, METTL3/MALAT1/SFRP2 axis prevented autism-like behaviors and apoptosis of hippocampal neuron by regulating the Wnt/β-catenin signaling.
Although modification on RNA has been discovered for decades, its roles in the regulation of genes expression are wellstudied until now [6-8]. METTL3 forms complex with METTL14, which responsible for majority m6A modification in cells [30]. Accumulated evidence has demonstrated the vital role of METTL3 in biological processes including metastasis, chemoresistance, DNA repair, and apoptosis [31-33]. Interestingly, a recent study reveals an aberrant downregulation of METTL3 in individuals with autism [10]. However, the molecular mechanism of how METTL3 involved in the regulation of development of autism-like behaviors remains largely unknown. Here, our study firstly analyzed autism-related microarray dataset and found that METTL3 was downregulated in autism samples. Consistent with former studies and microarray data, we further validated significantly downregulated METTL3 expression in hippocampal tissues from a mouse model of autism. What’s more, restoration of METTL3 expression by lentivirus injection in autistic mice alleviated the impaired social communication capacities and motor activity of a mouse model of autism. Notably, our data revealed that ectopic METTL3 expression significantly prevented autism-like behaviors and hippocampal neuron apoptosis. Moreover, this study revealed that METTL3 could promote MALAT1 expression by facilitating its stability through m6A modification. In agreement with this result, a recent work has identified that METTL3 upregulates MALAT1 expression by promoting its stability via mA modification in the context of IDH-wildtype gliomas [34]. MALAT1 has been determined a significantly differentially expressed gene enriched in the nervous system, immune system and transcription and translationrelated pathways, which appears to be a promising candidate to track clinical improvement following an integrative treatment model in toddlers affected by autism spectrum disorder [35]. MALAT1 has been reported to regulate cell proliferation, metastasis, and chemoresistance [36-38]. Neuron apoptosis is associated with development of autism-like behaviors. Differential penetrance of autism-inducing genetic/epigenetic variants may reflect atypical developmental trajectories associated with Huntingtin functions, including apoptosis [39]. Additionally, neuron apoptosis may also offer an obvious link between autism and glutathione metabolism, which can affect and modulate DNA methylation and epigenetics [40]. MALAT1 has been validated to protect neurons from death [41]. But, its role in the development of autism-like behaviors and hippocampal neuron apoptosis as well as the associated mechanism remains largely unknown and calls for more in-depth investigation. Accumulating research has uncovered that Wnt/β-catenin signaling is essential for diverse biological processes, including cell cycle transition, cell proliferation, chemoresistance, and apoptosis [42-44]. Importantly, Wnt/β-catenin signaling is tightly involved in the modulation of autism-like behaviors [45]. How the β-catenin is controlled in living cells also has been well studied. For instance, phosphorylation of β-catenin can promote its proteasome dependent degradation [46]. SFRP2, a novel regulator of β-catenin, has been documented to regulate it expression in cardiac fibroblasts [47]. More importantly, our data revealed that SFRP2 was elevated in the hippocampal tissues of autistic mice and negatively correlated with β-catenin protein expression. Furthermore, this study revealed that SFRP2 promoted autism-like behaviors and hippocampal neuron apoptosis by blockage of the Wnt/β-catenin signaling. Notably, our study identified MALAT1 could interact with several DNMTs, DNMT1, DNMT3A and DNMT3B. More importantly, restoration of MALAT1 expression in a mouse model of autism significantly downregulated SFRP2 expression in hippocampal tissues. As far the molecular mechanism of how MALAT1 regulated SFRP2 expression, we revealed that MALAT1 interacted with indicated DNMTs and as a scaffold RNA to recruit the DNMT1, DNMT3A, and DNMT3B to the SFRP2 promoter which led to the hypermethylated CpG island in the SFRP2 promoter and transcription suppression. Taken together, our data clearly demonstrate METTL3 promotes MALAT1 expression by promoting m6A modification of MALAT1, increases SFRP2 methylation by recruiting DNMT1, DNMT3A, and DNMT3B to the promoter region of SFRP2 and activates the Wnt/β-catenin signaling. Thus, METTL3 represses autism-like symptoms and hippocampal neuron apoptosis (Figure 9). These findings highlight that METTL3 may serve as a novel biomarker for autism. However, further investigation is required based on the clinical specimens. | true | true | true |
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PMC9633303 | 35969377 | Xiaofan Duan,Xiaoxiao Xu,Yumei Zhang,Yuan Gao,Jiuli Zhou,Jin Li | DDR1 functions as an immune negative factor in colorectal cancer by regulating tumor‐infiltrating T cells through IL ‐18 | 24-08-2022 | colorectal cancer,DDR1,IL‐18,PD‐L1,tumor‐infiltrating T cell | Abstract Immunotherapies represented by programmed cell death protein 1/programmed cell death ligand 1 (PD‐1/PD‐L1) immune checkpoint inhibitors have made great progress in the field of anticancer treatment, but most colorectal cancer patients do not benefit from immunotherapy. Discoidin domain receptor 1 (DDR1), a tyrosine kinase receptor, is activated by collagen binding and overexpressed in various malignancies. However, the role of DDR1 in colorectal cancer and immunoregulation remains unclear. In this study, we found DDR1 is highly expressed in colorectal cancer tissues and negatively associated with patient survival. We demonstrated that DDR1 promotes colorectal tumor growth only in vivo. Mechanistically, DDR1 is a negative immunomodulator in colorectal cancer and is involved in low infiltration of CD4+ and CD8+ T cells by inhibiting IL‐18 synthesis. We also reported that DDR1 enhances the expression of PD‐L1 through activating the c‐Jun amino terminal kinase (JNK) signaling pathway. In conclusion, our findings elucidate the immunosuppressive role of DDR1 in colorectal cancer, which may represent a novel target to enhance the efficacy of immunotherapy in colorectal cancer. | DDR1 functions as an immune negative factor in colorectal cancer by regulating tumor‐infiltrating T cells through IL ‐18
Immunotherapies represented by programmed cell death protein 1/programmed cell death ligand 1 (PD‐1/PD‐L1) immune checkpoint inhibitors have made great progress in the field of anticancer treatment, but most colorectal cancer patients do not benefit from immunotherapy. Discoidin domain receptor 1 (DDR1), a tyrosine kinase receptor, is activated by collagen binding and overexpressed in various malignancies. However, the role of DDR1 in colorectal cancer and immunoregulation remains unclear. In this study, we found DDR1 is highly expressed in colorectal cancer tissues and negatively associated with patient survival. We demonstrated that DDR1 promotes colorectal tumor growth only in vivo. Mechanistically, DDR1 is a negative immunomodulator in colorectal cancer and is involved in low infiltration of CD4+ and CD8+ T cells by inhibiting IL‐18 synthesis. We also reported that DDR1 enhances the expression of PD‐L1 through activating the c‐Jun amino terminal kinase (JNK) signaling pathway. In conclusion, our findings elucidate the immunosuppressive role of DDR1 in colorectal cancer, which may represent a novel target to enhance the efficacy of immunotherapy in colorectal cancer.
Abbreviations CRC colorectal cancer CTLA‐4 cytotoxic T lymphocyte‐associated antigen‐4 DDR1 Discoidin domain receptor 1 IFN‐γ interferon‐γ IL‐18 Interleukin‐18 JNK c‐Jun amino terminal kinase PBMC peripheral blood mononuclear cell siRNA small interfering RNA TIL tumor‐infiltrating lymphocyte
Globally, CRC is the third most diagnosed malignancy and the second leading cause of cancer death. In the past decade, following the initial success of melanoma treatment, immunotherapy has rapidly become the mainstay of treatment for a variety of solid cancers, including a subset of colorectal cancer that are mismatch repair deficient (dMMR). , But this proportion of patients is small and most patients cannot benefit from immunotherapy. Moreover, studies have shown that tumor tissues that do not respond to immunotherapy often lack immune cell infiltration. , , Therefore, it is critical to understand the mechanisms responsible for “cold” immune tumors to boost antitumor immunity. Discoidin domain receptor 1 is a poorly characterized receptor tyrosine kinase (RTK) that binds to collagens, which are the major components of the extracellular matrix. DDR1 functions as a central extracellular matrix sensor to regulate cell adhesion. DDR1 can cross‐talk with several transmembrane receptors, including Notch and TGF‐β receptors, and influence their signaling upon collagen stimulation. DDR1 also promotes cell proliferation, motility, and invasion, depending on the tumor type and the nature of the microenvironment. , In colorectal cancer, DDR1 promotes cell survival through the Ras/Raf/MAPK pathways under genotoxic stress and supports the metastatic process through Wnt/β‐catenin‐dependent, as well as BCR‐dependent and PEAK1‐dependent, mechanisms. , It has also been found that DDR1 overexpression can induce colorectal cancer cell invasion through the upregulation of MMP‐2. Nevertheless, there has been no relevant report on the immune regulation of DDR1 in CRC. IL‐18, a member of the IL‐1 cytokine family, is similar to IL‐1β for being processed by caspase 1 to an 18 kDa‐biologically active mature form and mediates inflammation downstream of the NLRP3 and NLRP1 inflammasomes. , In the body, IL‐18 is constitutively expressed by several cell types, including macrophages and intestinal epithelial cells. IL‐18 promotes the enhancement of CD4 and CD8 cells proliferation and secretion of various cytokines. However, a connection between DDR1 and the infiltration of immune cells in colorectal cancer has not been established. Here, we report that DDR1 promotes colorectal cancer progression through the enhancement of the immunosuppressive microenvironment, and its mechanism of inhibiting immune cell infiltration is partially mediated by IL‐18 and PD‐L1. Furthermore, we also show that DDR1 regulates PD‐L1 expression through the JNK/c‐Jun pathway in CRC.
Human CRC cell lines (HCT116, HCT8, SW480, LoVo, DLD‐1 and RKO) were purchased from the Chinese Academy of Sciences in Shanghai. Mouse CRC cell line MC38 was obtained from Southern Medical University in Guangzhou, China. CRC cells were cultured in DMEM (Gibco) that was supplemented with 10% FBS (Gibco), 100 U/ml penicillin and 100 μg/ml streptomycin, and were maintained in the 5% CO2 incubator at 37°C. Collagen I (Sigma) was coated on culture plates to stimulate DDR1 (8 μg/cm2) as previously described.
Cells at 80% density were transfected with siRNAs (Transheep) using RFect reagent (BIOG Nucleic Acid Quick Swab Kit, China) and plasmid (Transheep) using Lipofectamine 3000 (Invitrogen). The target sequences for DDR1 siRNA and IL‐18 siRNA sequences are listed in Table S1. After 24 h or 48 h incubation, the cells were collected for subsequent experiments. Lentivirus CMV (pTSB011104/Puro) containing DDR1 overexpression plasmids and lentivirus CMV (pTSB201131/Puro) containing IL‐18 overexpression plasmids were purchased from the Transheep company. For the infection of lentivirus‐based constructs, cells at 80% confluency were incubated for 24 h in medium containing concentrated viral particles and polybrene (Sigma‐Aldrich). The transfected cells were allowed to grow for another 2 days and then selected with puromycin (Sigma‐Aldrich) for 1 week. The transfection efficiency was validated by qRT‐PCR or western blot.
To knockout DDR1 in SW480 cells, gene editing was performed using the CRISPR/Cas9 system. Oligonucleotides with BsmB1 restriction sites for guide RNAs were synthesized and cloned into LentiCRISPRv2 together with the puromycin selection marker by Transheep. The sequences of the cloned plasmids that were extracted from numerous selected colonies were confirmed by Transheep. SW480 cells were transfected with LentiCRISPRv2‐single guide RNA (sgRNA) DDR1 using Lipofectamine 3000, according to the manufacturer's protocol. Two sgRNAs, DDR1‐6‐156/rev and DDR1‐6‐183/fw were simultaneously transfected into cells to knock out DDR1 from two different sites. The two sgRNA sequences of DDR1 were: DDR1‐6‐156/rev:5′‐GTAACGCAGCCGGTAGCTCC‐3′; DDR1‐6‐183/fw: 5′‐CTACCGGCTGCGTTACTCCC‐3′. The cells were cultured with increasing concentrations of puromycin for 2 weeks, starting at 3 days post‐transfection. After routine digestion, the cells were separated using flow cytometry and inoculated into 96‐well plates. Single cell clones were expanded and evaluated for gene‐specific knockout through immunoblot analysis. DNA sequencing technology was used to confirm the purity of a single clone of DDR1‐KO SW480 cells, shown in Figure S1.
Cell proliferation assay was tested by 5‐ethynyl‐2′‐deoxyuridine (EdU) Cell Proliferation Assay Kit (BBI Life Sciences). Cells seeded into a 24‐well plate in triplicates were manipulated according to the manufacturer's instructions and then photographed using a fluorescence microscope. Red fluorescence indicated that the cells were in a proliferative state, and the number was counted and analyzed.
Total protein was extracted after lysing the cells with RIPA lysis buffer containing protease inhibitor cocktail. Proteins were separated using SDS‐PAGE and transferred to PVDF membranes. Membranes were then blocked with 5% BSA and incubated with primary antibodies. After that, membranes were washed and incubated with HRP‐conjugated secondary antibodies. Finally, the protein bands were visualized using an ECL detection reagent (Millipore). The primary antibodies used are listed in Table S2. Primary antibodies were detected using goat polyclonal rabbit (WELLBIO).
Total RNA was extracted using TRIzol (Invitrogen). The cDNA was reverse transcribed according to the manufacturer's instructions. Quantitative real‐time PCR (qRT‐PCR) was then performed using SYBR Green, and GAPDH was used as an internal control. Relative expression levels were determined using the 2−ΔΔCt method. Primers used are listed in Table S3.
All mice were housed and treated in accordance with protocols approved by the Animal Care and Use Committee of laboratory animal research center, Tongji University (TJBB04521101). MC38 cells (1.8 × 106 cells for each injection, with 200 ul phosphate‐buffered saline resuspension) were injected subcutaneously into 6‐week‐old female C57bL/6 mice. The length (L) and width (W) of the tumors were measured using an external caliper and the volume (V) of each tumor was calculated according to the equation [V = (L × W 2) × 0.5]. The mice were euthanized using carbon dioxide asphyxiation. Tumors were excised, weighed, and subjected to immunohistochemistry (IHC), WB, or immune cell profile analysis.
Paraffin‐embedded slides were deparaffinized, rehydrated and treated with 1× EDTA or citrate at 98°C for 10 min for antigen retrieval. The slides were further incubated in 3% H2O2 solution to block endogenous peroxidase. After blocking with 3% BSA, tissues were incubated with primary antibodies against PD‐L1 (Proteintech, #14‐5983‐82), PD‐1 (CST, #84651), DDR1 (CST, #5583), CD4 (Abcam, #ab183685) and CD8α (CST, #989941) overnight at 4°C. After rinsing with PBS, the slides were incubated with biotin‐conjugated secondary antibody, washed, and incubated with HRP‐conjugated streptavidin. The slides were counterstained with hematoxylin. Stained areas were calculated using ImageJ software and statistically analyzed.
Blood samples were collected from healthy donors. PBMCs were isolated with Ficoll–Hypaque (GE Life Sciences) by density gradient centrifugation within 2 h of sample collection. Total CD8+ T cells were purified from PBMCs by negative selection (BioLegend). For the mouse cell isolation, after careful removal of the mouse tumor tissue, portions were minced into 2 mm pieces. Using the Mouse Tumor Dissociation Kit (Miltenyi Biotec Inc.), these tissues were further dissociated into single cells by combining mechanical dissociation and enzymatic degradation of the extracellular matrix. Immune cells were enriched using a discontinuous Percoll (GE Life Sciences) gradient.
CD8+ T cells were sorted and co‐cultured with SW480 cells in 6‐well plates at a ratio of 10:1. Cells were stimulated with αCD3/CD28 (IBA Lifesciences). After 48 h, T cells were collected to determine the cytokine production. Before that, Leukocyte Activation Cocktail (BD) was added and sustained for 8–10 h to block IFN‐γ release. To verify the effect of IL‐18, rIL‐18 (Absin), control IgG (R&D) and IL‐18 neutralizing antibody (R&D) were added in a co‐culture system for 24 h.
For multicolor flow cytometry immunophenotypic analysis, cells were stained with the indicated antibodies and analyzed on a CytoFLEX (Beckman Coulter). The flow cytometric profiles were analyzed using counting 20,000–50,000 events using the CytoFLEX software. Information on antibodies is presented in Table S4 and Section 2 and gating strategies for immunophenotyping shown in Figure S2.
Cells were conditioned for 48 h in serum‐free medium. The medium was collected and centrifuged at 300 x g for 5 min to remove particles. IL‐18 production in the supernatant of SW480 and HCT116 were assessed by enzyme‐linked immunosorbent assay (ELISA) using a commercially available ELISA kit (Abcam) according to the manufacturer's recommendations.
Each experiment was done at least three times, and data are presented as the mean ± SD. All data were analyzed using GraphPad Prism software (version 8.0.1). The comparison between the two groups of values was performed by t‐test and one‐way analysis of variance (ANOVA) followed by Tukey's multiple comparison test, which was used for more than two groups. A value of p < 0.005 was considered as a significant difference. In figures, *p < 0.05, **p < 0.01, ***p < 0.001 and ****p < 0.0001.
To evaluate DDR1 expression on CRC, we examined the RNA‐seq data from multiple malignancies in The Cancer Genome Atlas (TCGA). We found that DDR1 is highly expressed in CRC tissues compared with the adjacent normal tissues (Figure 1A). To further determine the effects and the clinical significance of DDR1, we analyzed the relationship between the expression level of DDR1 and disease‐free survival (DFS) in patients with CRC using the PrognoScan database. Notably, we found that higher DDR1 expression positively correlated with poorer DFS (Figure 1B). These results indicated that a high level of DDR1 is a potential risk factor to poor prognosis in patients with CRC. To directly determine the role of DDR1 in CRC, we first examined the expression of DDR1 in various CRC cell lines. DDR1 was highly expressed in SW480, LoVo, and HCT116 cells, but expressed at relatively low levels in RKO and HCT8 cells (Figure 1C). Then we constructed four independent siRNAs and verified their knockdown efficacy in SW480, HCT116, and MC38 cells (Figure 1D). We selected siRNA‐1 for validation at the mRNA level in SW480, HCT116, and MC38 cells (Figure 1E). We also constructed cell lines with DDR1 overexpression using a lentiviral‐mediated method (Figure 1F,G). Next, we detected the effect of DDR1 on the proliferative capability in SW480, HCT116, and MC38 cells. However, after overexpression of DDR1, the proportion of cells in the proliferative phase was slightly increased only in HCT116 cells. No significant difference was observed in other cell lines (Figure 1H–K), indicating that DDR1 may not be a direct regulator of CRC cell growth.
We sought to test the role of DDR1 on tumor progression in fully immunized mice. We used DDR1‐OE MC38 cells to establish a subcutaneous CRC xenograft model in C57bL/6 mice (n = 7/group). The tumor volume was monitored. Cells overexpressing DDR1 formed much bigger tumors compared with the control group (Figure 2A–C). We considered whether immunity was involved in this difference. To determine whether DDR1 modulates the tumor immune environment, we analyzed the immune cell profile regulated by DDR1 from xenografts. Flow cytometry analysis showed that CD4+ T cells (CD45+CD3+CD4+) and CD8+ T cells (CD45+CD3+CD8a+) that infiltrated the tumors were significantly lower in the DDR1‐OE group than in the scramble controls (Figure 2D–G). To further validate the above results, we collected mouse tumor tissues for IHC staining. First, we stained DDR1 and confirmed its high expression (Figure 2H,I). Then, we stained CD4 and CD8a (Figure 2J–M) and reached a conclusion consistent with the flow cytometry results. It was further confirmed that DDR1 overexpression inhibited the infiltration of CD4+ T cells and CD8+ T cells in the tumor microenvironment.
The activated CD8+ T cells utilize two main pathways for killing their target cells: granule exocytosis and Fas ligand‐mediated apoptosis induction. CD8+ T cells also release IFN‐γ and tumor necrosis factor α (TNF‐α) to induce cytotoxicity in the cancer cells. Therefore, we compared the functional differences in IFN‐γ secretion by CD8+ T cells after co‐culture with SW480 cells that expressed different levels of DDR1. A DDR1‐KO cell line was established using CRISPR/Cas9 technology (Figure 3A). DDR1‐OE cell lines were constructed as described previously (Figure 3D). The flow cytometry (FCM) results indicated that DDR1‐KO could increase IFN‐γ synthesis in CD8+ T cells (Figure 3B,C), whereas DDR1‐OE suppressed its synthesis (Figure 3E,F). This suggests that DDR1 not only inhibits the infiltration of T cells, but also inhibits the function of CD8+ T cells.
Interleukins and related cytokines serve as a means of communication between adaptive immune cells and nonimmune cells and tissues. ILs can nurture an environment favoring cancer growth and are essential to generate tumor‐specific immune responses. , To further elucidate the underlying mechanisms in which DDR1 affects immunity, we screened the expression of several ILs and cytokines that may be relevant for immune cell infiltration (Figure S3). Among these cytokines, IL‐18 was attractive as it functions as an immunomodulatory factor, inducing IFN‐𝛾 from natural killer (NK) cells and T cells in tumor foci. , , , , Essentially, to convince the regulation of IL‐18 by DDR1, we examined the IL‐18 levels both in vivo and in vitro. We first confirmed that IL‐18 levels were reduced in DDR1‐OE tumor tissues by western blot and qRT‐PCR (Figure 4A,B). Then we examined the level of IL‐18 in DDR1‐KO SW480 cells using western blot, qRT‐PCR and ELISA, respectively, and showed that DDR1‐KO promoted the synthesis and secretion of IL‐18 (Figure 4C–E). Consistently, DDR1 knockdown profoundly provoked the synthesis and release of IL‐18 (Figure 4F–H), whereas DDR1 overexpression induced a decreased IL‐18 level in SW480 and HCT116 cells (Figure 4I–K). Overall, these results revealed that DDR1 regulated IL‐18 synthesis and secretion in CRC, which may contribute to the sequestration of immune cells and the release of other crucial immune factors.
To identify whether DDR1 regulates the activation of T cells through IL‐18, we first overexpressed IL‐18 in DDR1‐OE SW480 cells (Figure 5A). Consistent with our hypothesis, the FCM showed that the IFN‐γ level was elevated in the co‐culture system, when IL‐18 was added back in DDR1‐overexpressed cells (Figure 5B,C). We then knocked down IL‐18 with siRNA in DDR1‐KO SW480 cells (Figure 5D). As expected, IL‐18 knockdown in DDR1‐depleted cells further decreased IFN‐γ secretion from CD8+ T cells in the co‐culture system (Figure 5E,F). Furthermore, we exogenously changed the concentration of IL‐18 in the co‐culture system. rIL‐18 (100 ng/ml) stimulated CD8+ T cells to secret IFN‐γ robustly when co‐cultured with DDR1‐OE SW480 cells (Figure 5G,H). By contrast, IL‐18 neutralizing antibody (20 μg/ml) inhibited CD8+ T cells to secret IFN‐γ when co‐cultured with DDR1‐KO SW480 cells (Figure 5I,J). Therefore, IL‐18 is one of the major downstream components through which DDR1 inhibits the activation of CD8+ T cells in the co‐culture system in vitro. To explore the functional interaction between DDR1 and IL‐18 in vivo, we overexpressed IL‐18 in DDR1‐OE MC38 cells (Figure 6A). MC38 cells bearing DDR1‐OE + IL‐18‐OE formed much smaller tumors compared with DDR1‐OE cells (Figure 6B–D). FCM analysis revealed a significant increase in CD4+ and CD8+ T cells in IL‐18‐OE + DDR1‐OE tumors compared with DDR1‐OE tumors (Figure 6E–H). Together, these findings suggested that IL‐18‐OE attenuates the immunosuppression and tumor growth of DDR1‐OE and enhances the antitumor immune response in CRC.
Binding between PD‐L1 on cancer cells and PD‐1 on TILs results in the suppression of the T cell receptor (TCR) pathway and the inhibition of T cell activity. , , PD‐L1 expression plays an important role in tumor immune escape. Therefore, we analyzed whether PD‐L1 was regulated by DDR1 in our mouse model. PD‐1 and PD‐L1 staining were much higher in the DDR1‐OE tumors than in the scramble controls (Figure 7A–D). Then, we examined PD‐L1 expression using a western blot assay and found that PD‐L1 was markedly elevated in DDR1‐OE tumor tissues (Figure 7E). Accordingly, PD‐L1 protein levels were decreased in DDR1‐KD and DDR1‐KO cells (Figure 7F). Consistently, DDR1 promoted PD‐L1 expression in DDR1‐OE cells (Figure 7G,H). Many studies have discussed the mechanisms regulating PD‐L1 expression. Based on the available studies, we explored several pathways (JAK/STAT pathway, JNK/SAPK pathway, PI3K/AKT/mTOR pathway, and epidermal growth factor receptor [EGFR] pathway) in cells (Figure S4) that are possibly responsible for PD‐L1 regulation. The phosphorylated protein levels of JNK signaling including p‐JNK (Thr183/Tyr185) and p‐c‐Jun (Ser73) were increased in DDR1‐OE mouse tumor tissues (Figure 7E). Consistent results were obtained in cells (Figure 7F,G). Previous studies showed that JNK/c‐Jun signaling activation could significantly increase PD‐L1 expression, and that the activation of c‐Jun rendered promoters and enhancers of PD‐L1 accessible. , , To further clarify the mechanism, we used JNK inhibitor, SP600125 (10 μM), to suppress the phosphorylation of JNK for 24 h (Figure 7K). We observed the reduction of PD‐L1 in both SW480 and HCT116 cells (Figure 7K). Therefore, DDR1 may upregulate PD‐L1 expression at least partially through the JNK/c‐Jun pathway. We also explored the role of IL‐18 in the regulation of PD‐L1, VEGFA, and KRAS expression. These did not vary significantly under various IL‐18 expression levels (Figure S5). Recently, accumulating evidence has shown that vascular endothelial growth factor A (VEGFA) significantly upregulated the expression of inhibitory immune checkpoints that mediated the exhaustion of intratumoral CD8+ T cells. , We found that DDR1 was positively correlated with the expression of VEGFA in TCGA database (Figure S6A). Then we checked the expression of VEGFA in tumor tissues and cells (Figure 7E–G,I) and revealed that DDR1 enhanced VEGFA expression. In conclusion, we found that DDR1 upregulated PD‐L1 expression through the JNK/c‐Jun axis. In addition, DDR1 also regulated VEGFA expression, which may promoted CD8+ T cell exhaustion. Interestingly, we found a positive correlation between DDR1 and KRAS in TCGA database (Figure S6B) and verified that DDR1 promotes KRAS expression at both the protein and mRNA levels (Figure 7E–G,J). The specific mechanism remains to be further explored.
It is known that the accumulation of tumor‐specific CD4 + and CD8 + T cells is critical for an effective antitumor response. Here, we provide evidence that DDR1 is a major regulator controlling the infiltration of CD4+ and CD8+ T cells in CRC (Figure S7). Our study revealed that the infiltration of immune killer cells was decreased in DDR1‐OE tumors, suggesting that DDR1 could promote the formation of immunosuppressive microenvironments in CRC. CD4+ T cells promote the recruitment and effector function of tumor‐specific CD8+ T cells and activate innate killer cells in the tumor. , Furthermore, IFN‐γ exerts direct antiproliferative and proapoptotic antitumor effects. , We found that DDR1 is also responsible for the activity of CD8+ cells through inhibiting the synthesis of IFN‐γ in CRC. For the mechanism of DDR1 in regulating immunity, we found that IL‐18 was changed significantly upon DDR1 inhibition or overexpression. Early studies have shown that IL‐18 can induce the production of IFN‐γ and stressed its role as an inducer of Th1 responses. Recently, it was demonstrated that IL‐18 promoted the expansion and survival of effector cells including NK and CD8+ T cells that expressed IL‐18 receptor α/β chains and receptors containing an immunoreceptor tyrosine‐based activation motif (ITAM). , IL‐18 performs its biological functions by ligation of IL‐18 receptors (IL‐18R) α and β, and activates CD4+, CD8+ T, and NK cells through NF‐κB activation, leading to IFN‐γ production in target cells. , Therapeutically, immune checkpoint inhibitors and IL‐18 synergistically inhibited the growth of tumor cells without significant adverse events in animal models. A phase II study of recombinant IL‐18 (rIL‐18) was conducted in an untreated American Joint Committee on Cancer (AJCC) stage IV melanoma and rIL‐18, tested in this trial, was well tolerated. These results highlighted the potential of the IL‐18 pathway for immunotherapeutic intervention. The effect of DDR1 on T cell exhaustion should also be concerned. Exhausted CD8+ T cells in cancer frequently express high level of inhibitory receptors, including PD‐1, CTLA‐4, LAG‐3, and so forth. , , Our study found that the DDR1‐OE tumors had stronger PD‐1/PD‐L1 staining, which may result in an immunosuppressive microenvironment. VEGFA is not only an important driver of tumor angiogenesis, but also an important suppressive factor of antitumor immunity. , , Our study highlighted that DDR1 can regulate VEGFA expression, but also raised complex questions. For example, how does DDR1 regulate VEGFA? And what role does VEGFA play in T cell immersion upon DDR1 inhibition? Many studies have reported the regulatory mechanisms of PD‐L1 expression, but the role of DDR1 in this has not been examined. We found that DDR1 regulates the activity of JNK/c‐Jun pathway, which is essential for the expression level of PD‐L1 (Figure S7). Knockdown of JNK or treatment with the JNK inhibitor, SP600125, led to reduced PD‐L1 expression. , Mechanistically, DDR1 phosphorylation induced the phosphorylation and the nuclear localization of c‐Jun by activating JNK, and then promoted the transcription of PD‐L1. , , , Therefore, whether the combination of anti‐PD‐1/PD‐L1 antibodies, antiangiogenic therapy, and DDR1 inhibitor will have synergetic effects in CRC needs to be further explored, and may contribute a way in which to solve the limited application of immunotherapy. We also found that DDR1 was positively correlated with the expression of KRAS. Several studies have confirmed that DDR1 can improve the efficacy of KRAS‐mutant tumors. , , For example, combining DDR1 inhibition with chemotherapy prompted a synergistic therapeutic effect and enhanced the cell death of KRAS‐mutant tumors in vivo. It was also reported that the combined inhibition of DDR1 and Notch signaling could be an effective targeted therapy for patients with KRAS‐mutant lung adenocarcinoma. However, how DDR1 regulates KRAS expression and whether DDR1 improves the efficacy of immunotherapy in CRC patients with KRAS mutations require further investigation. Recently, Sun et al. reported the role of DDR1 in immunity in triple‐negative breast cancer (TNBC), which is consistent with our results. They also showed that DDR1 instigates immune exclusion by promoting collagen fiber alignment, which is an inspiring progress. In our study, we demonstrated that DDR1 plays a vital role in tumor growth in vivo through regulating immune cell infiltration in CRC. The immune‐regulatory function of DDR1 is at least partially mediated by IL‐18, VEGFA, and PD‐L1 expression. Therefore, targeting DDR1 may represent a new strategy to enhance the efficacy of immunotherapy, which provides a direction to convert current challenges into opportunities.
Xiaofan Duan and Xiaoxiao Xu designed the research and performed experiments; Yumei Zhang contributed to animal experiments of article revision. Yuan Gao contributed to data visualization. Xiaofan Duan and Xiaoxiao Xu wrote the original draft; Jin Li and Jiuli Zhou designed the research, supervised the study, and revised the manuscript. The work reported in this paper has been performed by the authors, unless clearly specified in the text.
This work was supported by the Top‐level Clinical Discipline Project of Shanghai Pudong (PWYgf2021‐07), National Natural Science Foundation of China (81772561) and the Shanghai Sailing Program (20YF1453300).
All authors declare that they have no conflict of interest. Jin Li, the corresponding author for this study, is the Associate Editor of Cancer Science.
Approval of the research protocol by an Institutional Reviewer Board: The research protocol was approved by the Ethics Committee of Shanghai East Hospital (EC.D [BG]0.016.02.1) and it conformed to the provisions of the Declaration of Helsinki. Informed consent: Informed consent was obtained from healthy donors. Registry and the registration no. of the study/trial: N/A. Animal studies: All animal experiments were approved by the Animal Care and Use Committee of laboratory animal research center, Tongji University (TJBB04521101).
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PMC9633311 | 35912545 | Haibin Ou,Lili Wang,Ziyao Xi,Hui Shen,Yaofei Jiang,Fuxiang Zhou,Yu Liu,Yunfeng Zhou | MYO10 contributes to the malignant phenotypes of colorectal cancer via RACK1 by activating integrin/Src/ FAK signaling | 26-08-2022 | colorectal cancer,integrin/Src/FAK signaling,MYO10,RACK1,tumorigenesis | Abstract Liver metastases still remain a major cause of colorectal cancer (CRC) patient death. MYO10 is upregulated in several tumor types; however, its significance and the underlying mechanism in CRC are not entirely clear. Here, we found that MYO10 was highly expressed in CRC tumor tissues, especially in liver metastasis tissues. MYO10 knockout reduced CRC cell proliferation, invasion, and migration in vitro and CRC metastasis in vivo. We identified RACK1 by LC‐MS/MS and demonstrated that MYO10 interacts with and stabilizes RACK1. Mechanistically, MYO10 promotes CRC cell progression and metastasis via ubiquitination‐mediated RACK1 degradation and integrin/Src/FAK signaling activation. Therefore, the MYO10/RACK1/integrin/Src/FAK axis may play an important role in CRC progression and metastasis. | MYO10 contributes to the malignant phenotypes of colorectal cancer via RACK1 by activating integrin/Src/ FAK signaling
Liver metastases still remain a major cause of colorectal cancer (CRC) patient death. MYO10 is upregulated in several tumor types; however, its significance and the underlying mechanism in CRC are not entirely clear. Here, we found that MYO10 was highly expressed in CRC tumor tissues, especially in liver metastasis tissues. MYO10 knockout reduced CRC cell proliferation, invasion, and migration in vitro and CRC metastasis in vivo. We identified RACK1 by LC‐MS/MS and demonstrated that MYO10 interacts with and stabilizes RACK1. Mechanistically, MYO10 promotes CRC cell progression and metastasis via ubiquitination‐mediated RACK1 degradation and integrin/Src/FAK signaling activation. Therefore, the MYO10/RACK1/integrin/Src/FAK axis may play an important role in CRC progression and metastasis.
Abbreviations CCK‐8 cell‐counting kit‐8 CRC colorectal cancer CRISPR clustered regularly interspaced short palindromic repeats DFS disease‐free survival IHC immunohistochemical staining KEGG Kyoto Encyclopedia of Genes and Genomes LC‐MS/MS liquid chromatograph mass spectrometer/mass spectrometer NSCLC non‐small cell lung cancer qRT‐PCR real‐time quantitative polymerase chain reaction siRNA small interfering RNA TCGA The Cancer Genome Atlas
Colorectal cancer (CRC) is the third most common diagnosed cancer, and about 50% of CRC patients develop metastatic disease. CRC metastasis is the leading cause of cancer mortality. Therefore, understanding the biological mechanism of metastasis is important for development of new treatment strategies and markers predictive of CRC metastasis. MYO10 (also known as Myosin‐X or Myo X) belongs to the family of unconventional myosins. MYO10 is broadly distributed in many tissues and involved in numerous essential biological process, for example, orientation of the mitotic spindle, endothelial cell migration and angiogenesis, filopodia formation, and cell motility. In addition, numerous studies have found that MYO10 is highly expressed in melanoma, breast cancer, , non–small cell lung cancer, prostate cancer, and cervical cancer, compared with normal tissues, and promotes proliferation, invasion, and migration of tumor cells. However, its functional role in metastasis, especially in liver metastasis, has yet to be fully characterized. Receptor of activated protein C kinase 1 (RACK1) is a member of the Trp‐Asp (WD) repeats family, which functions as a scaffold protein to transduce signals. RACK1 regulates numerous cellular processes, including growth, differentiation, invasion, and migration. , , , , The dysregulation of RACK1 is implicated in the development of numerous tumors types. Studies have shown that RACK1 promotes the progression and chemoresistance in liver cancer, while RACK1 inhibits tumor metastases of gastric cancer. Thus, RACK1 may exert variable or even opposing roles in different types of cells or tissues. In this study, we examined the role of MYO10 in progression and metastasis of CRC. Bioinformatic analyses found that MYO10 is highly expressed in CRC. Further experiments showed that MYO10 knockout inhibits tumorigenesis and metastasis in vitro and in vivo through the integrin/Src/FAK signaling pathway. In addition, MYO10 interacts with RACK1 and regulates its ubiquitination and proteasomal degradation in CRC. Therefore, MYO10 may play an important role in CRC tumorigenesis and hepatic metastasis.
Human CRC cell lines HCT116, SW620, SW480, DLD1, HCT8, HT29; human colonic epithelial cells NCM460; human embryonic kidney cells 293 T; and mouse colon cancer CT26 cells were purchased from the Cell Library of the Chinese Academy of Sciences. These cell lines were authenticated by short tandem repeat analysis and cultured in a 37°C, 5% CO2 incubator with culture media according to manufacturer's instructions.
For generation of the MYO10 knockout cell lines, the lentiCRISPR method was used. The guide RNAs (sgRNA) targeting MYO10 (Table S1) were designed at http://crispor.tefor.net/crispor.py. Lentivirus was produced according to the manufacturer's instructions. Cells were infected with lentiviral media, and then monoclonal cells were screened by a limiting dilution assay and confirmed by immunoblotting.
The siRNAs targeting RACK1 mRNA were designed and synthesized by Tsingke. Sequences were as follows: si‐RACK1: 5′‐CUCUGGAUCUCGAGAUAAATT‐3′, si‐Control: 5′‐UUC UCC GAA CGU GUC ACG UTT‐3′. The siRNAs and plasmids were transfected into cells using Lipofectamine® 3000 transfection reagent (Invitrogen) according to the instruction. The human MYO10 cDNA was subcloned into pEGFPC1 and pCDH‐CMV‐MCS‐EF1‐GFP vector. The human RACK1 and ITGB1 cDNA was subcloned into a pEnMCV‐3×FLAG‐SV40‐Neo vector (MiaoLingPlasmid). Lentiviral packaging was carried out according to the instruction. The CT26‐KO1 cells were subsequently infected with human‐MYO10‐OE lentivirus, and GFP‐positive cells were selected with FACS AriaIII flow cytometer (BD Biosciences).
Total RNA was extracted by the TRIZOL method and reverse‐transcribed using a reverse transcription reagents (Vazyme). Each reaction of 20 μl containing 2 μl of primers (Table S2), 500 ng cDNA, and 10 μl AceQ® SYBR Green Master Mix (Vazyme) and qRT‐PCR were performed in a CFX‐96 instrument (BIORAD).
RNA‐sequencing RNA samples were obtained from control and MYO10 knockout CT26 cells described above. Each sample was prepared in triplicate, and the nine samples were sent to BGI for high‐through sequencing with DNBseq platform. RNA‐sequencing data are available at NCBI under SRA accession number SRP355594.
Cells were collected and lysed in RIPA buffer and incubated on ice for 30 minutes and centrifuged for 15 minutes (13,000 × g, 4°C). The proteins were transferred to the PVDF membrane after electrophoresis. Then, the membranes were blocked in 5% skim milk for 2 hours, incubated overnight at 4°C with the appropriate diluted primary antibodies, washed 5 × 10 minutes in TBST (TBS with 0.1% Tween20), and incubated with secondary antibody diluted in TBST for 1 hour. After washing five times for 10 minutes with TBST, bands were visualized with an ECL Western blot detection system (Tianneng). Information on the antibodies used is listed in Table S3.
Cells were seeded in confocal dishes for 24 hours and fixed in 4% paraformaldehyde (PFA) (Aspen) for 30 minutes after 24 hours of incubation. The cells were treated with 0.1% TritonX‐100 for 15 minutes and blocked with 5% normal donkey serum for 1 hour at room temperature. Subsequently primary antibody incubation was performed overnight at 4°C. After rinsing five times with PBS, the cells were incubated with appropriate fluorescent secondary antibody (Antgene) or Rhodamine Phalloidin (Invitrogen) for 1 hour at room temperature shielded from light. Nuclei were counterstained with DAPI (VectorLabs). Images were captured using a confocal laser‐scanning microscope (SP8, Leica).
Cells were collected and lysed in IP buffer, and supernatant was collected and incubated with primary antibody overnight at 4°C. The protein‐antibody complex was incubated with protein A/G magnetic beads (MCE) for 6 hours at 4°C. Protein‐antibody‐bead complexes were washed five times in PBST (PBS with 0.5% TritonX‐100), and proteins were eluted by boiling in 1 × SDS‐loading buffer for 5 minutes at 100°C for analysis by Western blot or LC‐MS/MS mass spectrometry (BGI).
293 T cells were transfected with various combinations of plasmids, along with or without Myc‐Ub. Thirty‐six hours after transfection, cells were treated with MG132 for 6 hours before cell collection. Then cell lysates were immunoprecipitated with anti‐FLAG antibody and analyzed by immunoblot using anti‐Myc antibody to detect ubiquitinated proteins.
Cells were plated in 96‐well plates (1000 cells/well) in quadruplicate. Following 24, 48, 72, 96, and 120 hours of cell incubation, 10 μl of cell‐counting kit‐8 (CCK‐8) working solution (Vazyme) was added to the wells. Finally, absorbance at 450 nm was measured using a microplate reader (SpectraMax CMAX Plus, Molecular Devices).
For colony formation assay, the cells were plated in six‐well plates (500 cells/well) and cultured for 12‐14 days. After fixation in 4% PFA, colonies were stained with 0.1% crystal violet and counted. For soft‐agar assay, 1 × 104 cells were plated in six‐well plates in growth medium containing 0.6% agar on a layer of solidified media containing 1.2% agar. After 12‐14 days of growth, the colonies were counted.
Cells were seeded in six‐well plates (5 × 105 cells/well) and grown to 100% confluence. The scratch wound was generated using a 10‐μl pipette tip and then the medium was replaced by serum‐free medium. Scratch wounds were photographed at indicated time points.
Cells (5 × 104 cells/well) were plated in the upper chamber (Corning) with (Transwell invasion assay) or without (Transwell migration assay) matrigel (BD Biosciences), and growth media with 20% FBS were added to the lower chamber. Twenty‐four hours later, cells were fixed with 4% PFA, stained with 0.1% crystal violet, and counted under a microscope.
Four‐to‐five‐week‐old female BALB/c mice and BALB/c nude mice were purchased from Vital River Laboratories, housed in specific pathogen‐free facilities in the Laboratory Animal Facility of Zhongnan Hospital. The HCT8 cells (NC, KO1, and KO2 cells, 5 × 106 cells per mouse) and CT26 cells (NC, KO1, KO2, KO1‐Ctrl, and KO1‐homo‐MYO10 cells, 5 × 105 cells per mouse) were subcutaneously inoculated under the right armpits of mice or the dorsal flank area of the mice. Tumor size was measured every 2 days with Vernier calipers, and tumor volume was calculated using the standard formula: volume = length × (width)2/2. For the liver metastasis model, the spleen was exposed through incision and tumor cells (CT26‐NC, CT26‐KO1, HCT8‐NC, HCT8‐KO1) were injected. Twenty to twenty‐five days later, the mice were anesthetized using isoflurane, and MRI was conducted to scan tumors at the Wuhan Institute of Physics and Mathematics, Chinese Academy of Sciences, using a 7 T small animal MRI scanner (Biospec70/20USR). Then, all mice were sacrificed, and liver metastasis was evaluated. All animals were treated in accordance with guidelines of the Wuhan University Institutional Animal Care and Use Committee (ethics approval number: 2018042).
Immunohistochemical detection of Ki‐67 in paraffin embedded mouse subcutaneous tumors and liver tissues was carried out using an antibody against Ki‐67. DAB was used as a chromogen (reacted for 10 minutes), and hematoxylin was used as a counterstain. The histological sections were photographed under a light microscope (Olympus BX53).
Transcriptomic data and clinical data for CRC patients were downloaded from The Cancer Genome Atlas (TCGA) database (https://portal.gdc.cancer.gov/). The data of normal tissue samples were obtained from GTEx V8 release version (https://gtexportal.org/home/datasets). The dataset GSE41258 used was downloaded from the Gene Expression Omnibus (GEO) database (https://www.ncbi.nlm.nih.gov/geo/). Data analysis was performed in R software (version 4.1.2).
Paraffin‐embedded CRC tissue sections were obtained from Zhongnan Hospital at Wuhan University in China. The study was approved by the ethics committee of Zhongnan Hospital, Wuhan University, China (ethic approval number: 2013020).
Statistical analyses were performed using SPSS 16.0. Mean ± SD was used to express normally distributed data, and Student's t test was used to perform comparison. Categorical data were analyzed with either Fisher exact test or chi‐square test. Spearman rank correlation test was used in the correlation analysis. For disease‐free survival (DFS), Kaplan‐Meier and log‐rank tests were used to perform analysis. Statistical significance was considered as a P value <0.05.
Analysis of TCGA and GTEx databases indicates that MYO10 mRNA is upregulated in tumor tissues in CRC patients (Figure 1A). In addition, we found that elevated MYO10 expression is associated with liver metastasis (Figure 1B) and the higher MYO10 expression correlates with reduced DFS (Figure 1C). Then, we examined the level of MYO10 in CRC cells and observed that MYO10 was highly expressed in CRC cells compared with normal colon cell line NCM460 by qRT‐PCR and immunoblotting (Figure 1D,E). To further explore the biological role of MYO10, CRISPR/Cas9 technology was used to generate two independent knockout cell lines (KO1 and KO2) and control cells (NC) (Figure 1F). After MYO10 knockout, the proliferation, colony‐formation abilities, and 3D colony‐formation abilities of CRC cell lines were significantly decreased (Figure 1G–K). Taken together, these results suggested that MYO10 might be a vital regulator of proliferation in CRC cell lines.
Researchers found that MYO10 expression facilitates filopodia formation and invasion. To gauge the relationship between MYO10 and cell metastatic ability, immunofluorescence staining showed that the number and length of filopodia were significantly reduced after MYO10 knockout in CT26 and SW480 cells (Figure 2A–C). Moreover, the wound‐healing rates, migration, and invasion of CT26 and HCT8 cells were significantly attenuated by knockout of MYO10, compared with control cells (Figure 2D–H). Similar phenomenon was observed in SW480 cells (Figure S1A–E). These findings highlight that MYO10 acts as an important contributor to filopodia formation and elongation and metastasis in CRC cells.
To further evaluate the oncogenic role of MYO10 on CRC cells in vivo, murine subcutaneous models were established. We found that knockout of MYO10 significantly reduced the tumorgenicity of HCT8 cells in subcutaneous xenografts in BALB/c nude mice (Figure 3A,B). IHC staining showed that the percentage of Ki67 positive cells was greatly decreased following MYO10 knockout in xenograft tumors (Figure 3C). An unexpected result was that formation of subcutaneous tumors was not observed in the KO group (mice injected with MYO10 knockout CT26 cells) (Figure 3D,E). In summary, these data indicate that MYO10 knockout inhibits tumorigenesis of CRC cells in vivo. Next, we tested how deletion of MYO10 affected in vivo hepatic tumor seeding following intrasplenic injection of CT26 and HCT8 cells in BALB/c or BALB/c nude mice (Figure 3F). Twenty days later, we examined the abdomen of these BALB/c mice with a 7 T MRI scanner (Figure 3G). When the livers were dissected out, we found that transferred liver tumors were formed in 80% (4/5) of mice injected with CT26 control cells, but not in those injected with MYO10 knockout cells (Figure 3H,I). Additionally, we found that the liver metastasis tumors were significantly lower in the MYO10 knockout group (HCT8‐KO1) than in the control group (HCT8‐NC) in BALB/c nude mice (Figure 3J,K). HE and IHC staining of the liver tissue indicated the metastasis nodules in the control group but not in the CT26‐MYO10‐KO group, and in the BALB/c nude mice of the HCT8‐NC group, the diameter and number of liver metastasis foci were larger and higher than those in the MYO10 knockout group (Figure 3L). These results demonstrate that the knockout of MYO10 significantly inhibits CRC liver metastasis in vivo.
To obtain insight into the mechanism of action of MYO10, we sought to identify potential proteins targeting the MYO10 interaction. 293 T cells were transfected with GFP‐MYO10 plasmids and CT26 cells as endogenous sample. Whole cell lysates were collected for immunoprecipitation with anti‐GFP antibody or anti‐MYO10 antibody separately, and the binding partners were identified by LC‐MS/MS (Figures 4A, S2). We identified 543 human proteins and 611 mouse proteins, respectively (Table [Link], [Link]). Among them, RACK1 is associated with the organization of the actin cytoskeleton; we therefore chose this protein for subsequent studies. A correlation was found between MYO10 expression and the expression of RACK1 in the GSE41258 dataset (Figure 4B). Immunofluorescence staining indicated that MYO10 colocalized with RACK1 in HCT8 and CT26 cells (Figure 4C,D). Immunoprecipitation assay showed the interaction between both exogenous and endogenous MYO10 with RACK1 (Figure 4E–H). These results suggested that MYO10 interacts and colocalizes with RACK1.
To prove the underlying mechanism by which MYO10 regulates RACK1, we performed qRT‐PCR and immunoblotting and found a concomitant decrease in RACK1 protein levels but not in mRNA levels with MYO10 knockout (Figure 5A,B). Therefore, we postulated that RACK1 might be regulated by post‐translational modifications (PTMs) that affect its stability. Cycloheximide (CHX) chase experiments showed a shorter half‐life of RACK1 in MYO10 knockout HCT8 cells compared with the control cells, while opposite phenomena were observed in HCT116 cells overexpressing MYO10 (Figure 5C–F). It has been documented that RACK1 can be degraded by the ubiquitin‐proteasome pathway. , Ubiquitination assays showed that overexpression of MYO10 significantly decreased the level of polyubiquitination of RACK1 in 293 T cells (Figure 5G). In addition, we observed that the protein level of RACK1 elevated as the expression level of MYO10 increased, and the degradation of RACK1 was partly inhibited by the proteasome inhibitor MG132 (Figure 5H). Taken together, MYO10 maintains the stability of RACK1 via the ubiquitination‐proteasome pathway.
To delineate the molecular mechanisms through which MYO10 regulates proliferation and metastasis of CRC cells, high‐throughput mRNA sequencing (RNA‐seq) were conducted in two MYO10 knockout cells of CT26 (KO1 and KO2) and controls. The KO1 and KO2 groups exhibited a distinct gene expression profile, with 1327 and 256 differentially expressed genes (DEGs) when compared with control cells, respectively (fold change >1.5, p < 0.05) (Figure 6A). Moreover, KEGG enrichment analysis of 140 common DEGs revealed that “ECM‐receptor interaction” was the most enriched pathway (Figure 6B). Recently, it was reported that MYO10 regulates integrin β1 activity at filopodia tips. As shown in Figure 6C,D, GFP‐MYO10 was successfully coimmunoprecipitated by ITGB1‐FLAG. Additionally, endogenous interaction between MYO10 and ITGB1 was revealed in CT26 cells (Figure 6E). Fibronectin (FN) is the major ligand for integrin α5β1 and αvβ1. Knockout of MYO10 reduced FN‐dependent activation of FAK signaling (Figures 6F–G, S3). It was reported that RACK1 interacts with Src in osteoclasts. Likewise, we confirmed this interaction between RACK1 and Src in CT26 cells (Figure 6H–I). Moreover, we found that the protein level of RACK1 and the phosphorylated Src (Tyr416) and phosphorylated FAK (Tyr397) levels were decreased without alteration of the total protein levels after MYO10 knockout (Figures 6J, S4). Collectively, these results suggested that MYO10 might partly participate in the promotion of progression and metastasis through the integrin/Src/FAK pathway.
To further verify whether MYO10 promotes CRC progression and metastasis by the regulation of RACK1, the siRACK1 and the MYO10‐overexpressing HCT116 cells were prepared. Colony‐formation assay showed that the suppression of RACK1 partly inhibits the colony number increased by MYO10 overexpression (Figure 7A,B). Moreover, knocking down RACK1 reversed the MYO10‐induced cell migration of HCT116 cells (Figure 7C,D). Immunoblotting confirmed that overexpression of RACK1 could rescue the downregulation of p‐Src (Tyr416) and p‐FAK (Tyr397) protein level caused by MYO10 knockout (Figures 7E, S5). To confirm whether the poor tumorigenicity of CT26‐KO cells in vivo was caused by knockout of MYO10, we used a lentiviral vector to partly restore the protein level of MYO10 in CT26‐KO1 cell lines (Figure 7F). The engraftment potential of CT26‐KO1 cells was partly rescued in vivo by overexpression of human MYO10 gene, while the volume of the tumor relatively small as compared to the CT26‐NC group (Figure 7G–I). Immunofluorescence staining of human colorectal tissue microarrays revealed that FAK protein and FAK tyrosine phosphorylation (Tyr397) levels are elevated in CRC tumors compared with adjacent normal tissue; the same trend was observed for Src and Src tyrosine phosphorylation (Tyr416). Moreover, FAK and Src phosphorylation levels were higher in MYO10‐high tumor tissues as compared with MYO10‐low tumor tissues (Figure 7J,K). Together, these results demonstrate that MYO10 may act as an oncogene by regulating RACK1's functions in CRC.
Despite significant advances in the treatment of metastatic disease to the liver, hepatic metastases still remain a major cause of death in CRC patients. To date, few studies have been carried out to elucidate the relationship between MYO10 and CRC progression. In our study, the expression of MYO10 was upregulated in CRC patients and was correlated with a metastatic phenotype. Data from in vitro and in vivo experiments confirmed that MYO10 enhanced the progression and metastasis ability of CRC cells by interacting with RACK1. MYO10 reduced the proteasomal degradation of RACK1 and enhanced protein stability, which further induced the activation of the integrin/Src/FAK pathway. Together, our findings suggested that MYO10 promotes CRC progression and metastasis via RACK1 by modulating Src /FAK signaling activation (Figure 8). It has been reported that MYO10 is associated with filopodia formation and elongation. Studies showed that filopodia promote neurite outgrowth, , and knockdown of MYO10 impair neuronal adhesion. MYO10 is highly expressed in several types of cancer, and increased MYO10 promotes breast cancer invasion and metastasis. , Moreover, elevated MYO10 predicts poor prognosis and promotes cell proliferation and migration in cervical cancer. Some researchers found that MYO10‐driven filopodia are crucial for leader driven NSCLC collective invasion. Recently, it has been demonstrated that MYO10 drives genomic instability and inflammation in cancer. Here, we first reported the correlation between high expression of MYO10 and CRC development and further clarified the function and mechanism underlying this process. In the LC‐MS/MS results, RACK1 was found to be a potential MYO10‐interacting protein, and we further confirmed the interaction between MYO10 and RACK1. Researchers have found that RACK1 has different properties in different neoplasms, with some suppressing tumorigenesis while others promoting tumor progression. , , , , , It was found that RACK1 acts as a tumor‐promoting factor in CRC. Moreover, RACK1 promotes tumorigenicity of CRC by inducing cell autophagy. Our data showed that the protein level of RACK1 is downregulated in MYO10 knockout CRC cells, but there was no decrease in the mRNA level. The half‐life of the RACK1 protein was markedly decreased in MYO10 knockout cells, while MYO10 overexpression increased the half‐life of RACK1. In MYO10‐overexpressing cells, proteasome inhibitor MG132 inhibited the degradation of RACK1, thus indicating that MYO10 and RACK1 interaction may cause a structural change and result in the reduction of RACK1 ubiquitination and degradation. Recent studies suggested that MYO10 and talin regulate integrin activity at filopodia tips. Integrins α5β1 and αvβ1 interact with FN and other ECM proteins to facilitate cancer progression, and FN is the major ligand for integrin α5β1 and αvβ1, triggering the integrin/FAK signal pathway after binding. Studies revealed that MYO10 is not directly interacting with FN36. LC‐MS/MS and immunoprecipitation data revealed that MYO10 interacts with integrin β1. Although integrin β1 protein expression showed no significant change, knocking out MYO10 significantly reduced FN‐dependent activation of FAK signaling. The scaffold protein RACK1 links the integrin effector focal adhesion kinase (FAK) to other proteins. A study found that high RACK1 expression increased phosphorylation of Src at Tyr416 in neuroblastoma. Active Src (p‐Src Tyr416) localizes to filopodia and plays a critical role in filopodia induction. In our research, we confirmed the interaction between RACK1 and Src in CRC cells, and the phosphorylation levels of Src (Tyr416) and FAK (Tyr397) were significantly inhibited in MYO10 knockout cells. Thus, MYO10 interacts with RACK1 and regulates integrin/Src/FAK signaling in CRC cells. In conclusion, our studies demonstrated that MYO10 can promote the progression and metastasis of CRC. We found that MYO10 interacts with RACK1 and regulates the activation of integrin β1 and integrin/Src/FAK signaling in CRC. Our work provided new insights into the pathogenesis of CRC and may suggest an avenue for the treatment of liver metastasis.
National Natural Science Foundation of China (Grant/Award Number: ‘81370070’, ‘81472799’).
There are no competing interests any of the authors.
Approval of the research protocol by an Institutional Reviewer Board. Paraffin‐embedded colorectal cancer tissue sections were obtained from Zhongnan Hospital at Wuhan University in China. The study was approved by the ethics committee of Zhongnan Hospital, Wuhan University, China (ethics approval number: 2013020).
All clinical samples were obtained with informed consent by the patients.
All animals were treated in accordance with guidelines of the Wuhan University Institutional Animal Care and Use Committee (ethics approval number: 2018042).
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PMC9633315 | 35848898 | Rui Mao,Zhengyun Ren,Fan Yang,Peng Yang,Tongtong Zhang | Clinical significance and immune landscape of KIR2DL4 and the senescence‐based signature in cutaneous melanoma | 30-08-2022 | cutaneous melanoma,immunotherapy,KIR2DL4,prognosis,senescence‐related genes | Abstract Senescence is an effective barrier to tumor progression. Mutations that inhibit senescence and promote cell division are mandatory for the development of cancer. Therefore, it is particularly important to explore the differences between cutaneous melanoma (CM) patients with severe and mild degrees of senescence. We clustered all the patients with CM in the Cancer Genome Atlas (TCGA) database based on all the genes of the senescence pathway in the CellAge and MSigDB database. The prognosis, immunotherapy effect, tumor microenvironment score, NRAS mutation rate, expression of CD274, CTLA4, and PDCD1, and abundance of CD8+ T and natural killer (NK) cell infiltration in the younger group of patients (YG) were higher than those in the older group (OG). Compared with the American Joint Committee on Cancer (AJCC) stage, the risk scoring system stratified the risk of CM patients and guided immunotherapy more accurately. The nomogram model, which combined the AJCC stage and risk score, greatly improved the ability and accuracy of prognosis prediction. As KIR2DL4 is the core molecule in the risk scoring system (RSS), knocking down the KIR2DL4 of human NK cells in vitro can inhibit the cytotoxicity of NK cells and can also inhibit the secretion of tumor necrosis factor‐α and interferon‐γ by NK cells. In contrast, upregulation of KIR2DL4 can activate the MEK/ERK signaling pathway, which is the activation pathway of NK cells. Our RSS and nomogram model can accurately stratify the risk of CM patients and effectively predict the effect of immunotherapy and prognosis in CM patients. | Clinical significance and immune landscape of KIR2DL4 and the senescence‐based signature in cutaneous melanoma
Senescence is an effective barrier to tumor progression. Mutations that inhibit senescence and promote cell division are mandatory for the development of cancer. Therefore, it is particularly important to explore the differences between cutaneous melanoma (CM) patients with severe and mild degrees of senescence. We clustered all the patients with CM in the Cancer Genome Atlas (TCGA) database based on all the genes of the senescence pathway in the CellAge and MSigDB database. The prognosis, immunotherapy effect, tumor microenvironment score, NRAS mutation rate, expression of CD274, CTLA4, and PDCD1, and abundance of CD8+ T and natural killer (NK) cell infiltration in the younger group of patients (YG) were higher than those in the older group (OG). Compared with the American Joint Committee on Cancer (AJCC) stage, the risk scoring system stratified the risk of CM patients and guided immunotherapy more accurately. The nomogram model, which combined the AJCC stage and risk score, greatly improved the ability and accuracy of prognosis prediction. As KIR2DL4 is the core molecule in the risk scoring system (RSS), knocking down the KIR2DL4 of human NK cells in vitro can inhibit the cytotoxicity of NK cells and can also inhibit the secretion of tumor necrosis factor‐α and interferon‐γ by NK cells. In contrast, upregulation of KIR2DL4 can activate the MEK/ERK signaling pathway, which is the activation pathway of NK cells. Our RSS and nomogram model can accurately stratify the risk of CM patients and effectively predict the effect of immunotherapy and prognosis in CM patients.
Abbreviations AJCC American Joint Committee on Cancer CM cutaneous melanoma DMFS distant metastasis free survival DSS disease special survival KIR Killer cell immunoglobulin‐like receptor OG older group of patients OS overall survival RSS risk scoring system SASP senescence‐associated secretory phenotype TCGA the Cancer Genome Atlas TME tumor microenvironment YG younger group of patients
Cutaneous melanoma is a fatal skin cancer that originates from melanocytes, which produce melanin in the skin. Over the past few decades, its incidence among White people has increased dramatically, with 230,000 new cases per year worldwide (World Health Organization). While early localized malignant melanoma of the skin can be cured by surgery, the prognosis of metastatic melanoma is very poor. Although some patients with metastatic melanoma may benefit a lot from immunotherapy, other patients either develop drug resistance or do not experience any treatment effect. These clinical challenges drive us to discover new drug targets and drugs that can benefit patients who are inherently resistant to targeted therapy and immunotherapy. Aging plays an important role in the regulation of cancer cells. The carcinogenic transformation of normal cells can lead to aging and initially prevent their growth. However, malignant cells typically bypass this process through gene mutation or the epigenetic downregulation of tumor inhibition‐related pathways, such as the p53–p21 and p16ink4a–RB pathways. The age‐related accumulation of SASP cells can promote cancer progression by reprogramming the primary and metastatic microenvironment (including the premetastatic niche) over time to a state more prone to malignant cell growth. Senescent cells are produced throughout life and play a beneficial role in various physiological and pathological processes. In addition, with increasing age, the continuous accumulation of aging cells also brings adverse consequences. These nonproliferating cells occupy a key cell niche and synthesize proinflammatory cytokines, leading to diseases and incidence rates related to aging. CM is a complex tumor, and a variety of environmental and genetic factors are needed to guide the acquisition of malignant characteristics. Recently, the immunotherapy of CM has made exciting progress and ushered in a new era of CM treatment. Immunotherapy can cause an unprecedented sustained response in patients with advanced cancer compared with response to conventional chemotherapy. However, this response occurs only in a relatively small number of patients. The positive response of immunotherapy typically depends on the interaction between tumor cells and immune regulation in the tumor microenvironment (TME). Under these interactions, the TME plays an important role in inhibiting or enhancing the immune response. Understanding the interaction between immunotherapy and the TME is not only the key to analyzing the mechanism, but also to providing new methods to improve the efficacy of immunotherapy. Exploring different senescence patterns among CM patients and their inherent TME differences, mutation landscape, immunotherapy effect, and prognosis can provide new insights into the mechanism of occurrence and development of CM and clinical treatment. KIR is a transmembrane glycoprotein expressed by NK cells and T‐cell subsets. KIR protein is considered to play an important role in the regulation of the immune response. KIR2DL4, with two Ig domains and a long cytoplasmic tail 4, is a type of KIR, but its association with CM has not been studied. Previous studies have suggested that HLA‐G is recognized by KIR2DL4 receptors on NK cells, and this leads to immune tolerance and immune escape. However, in the absence of HLA‐G, KIR2DL4 interacts with IFN‐γ to promote the secretion of more proinflammatory and angiogenic factors in NK cells. , Therefore, it is speculated that there is some type of factor in melanoma that interacts with KIR2DL4 to promote NK cells to release cytotoxic factors to kill tumor cells. Here, using the SKCM data from TCGA, we systematically studied the different aging modes of CM patients, as well as the differences in somatic mutation, TME, immunotherapy, and prognosis between the two modes. In addition, to guide the clinical practice, we constructed an RSS and a prognostic nomogram to better predict the effect and prognosis of immunotherapy. Finally, using functional experiments in vitro, we explore the mechanism of the senescence‐related molecule KIR2DL4 in inhibiting the development of CM.
In this study, TCGA (https://portal.gdc.cancer.gov/) database provided a TCGA‐SKCM data set as a training cohort, containing the gene expression matrix of 459 CM samples and their corresponding clinical follow‐up data. The GSE22153, GSE15605, GSE22154, GSE43955, GSE46517, GSE54467, and GSE65904 data sets were downloaded from the GEO (https://www.ncbi.nlm.nih.gov/gds/) database. As for GSE54467 and GSE15605, we downloaded log2‐transformed and quantile normalization matrix data, whereas GSE22153, GSE46517, GSE22154, GSE46517, and GSE65904 data sets were converted and normalized by log2 after download. Usually, we used the median of the expression value as the expression of genes with multiple probes. The downloaded matrix was an mRNA expression profile in fragments per kilobase of transcript per million format. The samples used in this study had to meet the following criteria : samples with nonzero probe expression accounted for 80% of all samples ; patients had accurate survival status and follow‐up time. The GSE65904 data set, containing information about DSS (210 patients) and DMFS (150 patients), was used as a test cohort. In addition, the GSE22153, GSE22154, GSE46517, and GSE54467 data sets, containing only the OS data (236 patients), were integrated into the validation cohort. While integrating the validation data set, we used the combat function of R package “sva” to remove the batch effect. Table S1 shows the basic data of the data sets included in this study. CellAge (https://genomics.senescence.info/cells/), which contains 279 aging‐related genes, is a database of genes associated with cell senescence. The Molecular Signatures Database (MSigDB) , is a collection of annotated gene sets for use with GSEA software. The three aging gene sets (GOBP_AGING, GOBP_CELL_AGING, and GOBP_CELLULAR_SENESCENCE) were downloaded from the MSigDB database (http://www.gsea‐msigdb.org/gsea/msigdb/index.jsp). These three aging‐related gene sets contained nearly all the genes that play an important role in aging involved in the existing aging research (n = 304). We combined two aging‐related gene data sets (CellAge and MSigDB) to obtain 313 aging‐related genes. We intersected these 313 genes with TCGA‐SKCM dataset and all genes in the GEO data set involved in this study using an upset analysis, and we finally obtained 221 aging‐related genes. The “Upset” analysis was performed by executing the “Upset” function in the “UpSetR” package, and the VENN circle was drawn using the “ggvenn” function in the “yyplot” package.
The immune score and stromal score of each CM patient in TCGA‐SKCM cohort were calculated using the ESTIMATE and Xcell (https://xcell.ucsf.edu/) website, and the differences in the immune score and stromal score among the different groups were further analyzed using the Wilcoxon test. The estimate score is the combination of the stromal score and the immune score. It is an index to evaluate the purity of a tumor. Based on the matrix of TCGA‐SKCM cohort, we calculated the abundance of immune cells by using CIBERSORT, MCP Counter, TIMER (http://timer.comp‐genomics.org/), EPIC, and Xcell software packages and excluded the samples with p > 0.05. Finally, the Mann–Whitney U‐test was used to analyze the differences in the immune cell subtypes among the different groups.
The tumor mutation load (TMB) has attracted much attention in immunotherapy. TMB and PD‐L1 are two important biomarkers to predict the response to PD‐1 antibody therapy. We used the waterfall function of the “maftools” package to show mutations in patients with high senescence and low‐risk scores in TCGA‐SKCM cohort. Missense, nonsense, uninterrupted, silence, and frameshift/in‐frame insertions and deletions were counted, while synonymous mutations were excluded. The total number of somatic mutations was used to calculate the TMB score. According to the median of the TMB score, all CM samples with somatic mutations in TCGA data set were divided into a high TMB score group and a low TMB score group.
By using the “Survival” package of R software, we used a univariate Cox analysis to calculate the prognostic value of each SRG and selected an SRG as the seed SRG for the Cox‐lasso regression analysis. Then, a multivariate Cox regression analysis was performed to evaluate the prognostic characteristics of each SRG using the R packages “Survminer”, “Glment”, and “Survival”. The risk score of each patient in the training group was calculated using the Cox proportional hazards model (PH model): h^it=h^0texpxi′β^ (where exp is the prognostic gene expression level; β is the multivariate Cox regression model regression coefficient; and h0t is the baseline hazard function. This is a function that describes the “instantaneous mortality” that specifically refers to the instantaneous mortality of the observed object at the time of survival to t). All of the CM samples were randomly divided into a high‐risk score group and a low‐risk score group according to the median of the risk score. The Kaplan–Meier method was used for the survival analysis, and the log‐rank test was used to compare survival between the groups. The R software package, “SurvivalROC”, was used to draw the receiver operating characteristic curve (ROC) curve and calculate the corresponding area under the curve (AUC).
Figure S1 shows the entire analytical process of the study. First, we screened the coexisting SRGs in TCGA, GSE22153, GSE22154, GSE46517, GSE54467, and GSE65904 databases using UPSET and identified a total of 221 SRGs (Figure S2A). Then, we merged four data sets, GSE22153, GSE22154, GSE46517, and GSE54467, and eliminated the batch effect (Figure S2B). The results of the principal component analysis (PCA) before and after merging the data sets showed that the batch effect was well eliminated (Figure S2C,D). The determination of the K value is very important for the consistency of a cluster analysis. When the cumulative distribution function (CDF) reaches the approximate maximum value, the cluster analysis result is the most reliable. Typically, a K value with small decline slope of the CDF is used. Based on unsupervised clustering, TCGA‐SKCM was divided into two clusters (Figure 1A,B). According to this method, it is really the best when k = 2. According to the results of prognostic analysis between the two groups, cluster 1 showed great advantages in survival and nonrecurrence (Figure 1C,D). Figure 1E shows a multivariate heat map that contains data on the expression of 221 SRGs and clinicopathological data. There was a significant difference in the multiple SRGs between the two clusters (Figure 1F). The age of patients in cluster 2 was significantly higher than that of patients in cluster 1 (Figure S3A). Therefore, we labeled the clusters 1 and 2 as the younger group (YG) and older group (OG), respectively.
We analyzed the differential expression of 58,385 probes between the YG and OG and found that 356 protein‐coding genes were upregulated in the OG, and 1048 protein‐coding genes were upregulated in the YG (Figure S3B). The upregulated genes in the OG and YG were analyzed using a functional enrichment analysis of the Gene Ontology Biological Processes (GO‐BP) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways, respectively. The results of the KEGG and GO‐BP analyses showed that the upregulated genes in the YG were primarily concentrated in the T‐cell‐receptor signaling pathway, the B‐cell‐receptor signaling pathway, the positive regulation of lymphocyte activation, the positive regulation of biological process, PD‐L1 expression, and the PD‐1 checkpoint pathway in cancer, and Th17 cell differentiation (Figure 2A,B). According to the KEGG and GO‐BP analysis, the upregulated genes in the OG were primarily concentrated in the skin epidermis development, the regulation of epidermis development, the regulation of epidermal cell differentiation, the IL‐17 signaling pathway, the extracellular matrix (ECM)–receptor interaction, skin development, and the adaptive immune response (Figure 2C,D). The upregulated genes in the YG were primarily enriched in the immune response and immunotherapy pathways, whereas those in the OG were primarily enriched in the aging‐related pathways, such as cell and epidermal development and differentiation. Therefore, we speculated that the immune landscape and response to immunotherapy between the YG and the OG may be different.
Based on the results of the TIDE analysis, we further found that the response of patients in the YG to the immune checkpoint blockade was significantly higher than that in the OG (Figure 3A,B). In addition, the Kaplan–Meier (KM) analysis in patients who received immunotherapy and chemotherapy showed that the response of patients in the YG to immunotherapy and chemotherapy was significantly better than that of patients in the OG (Figure 3C,D). In addition, we compared the expression of multiple immune checkpoints in the B7 family and the CD28 family between the YG and the OG. The results showed that the expression levels of CD274, CTLA4, PDCD1, CD80, CD86, CD28, ICOS, and ICOSLG in the YG were significantly higher than that in the OG (Figure 3E). To explore the mechanism between the immunotherapy response and the tumor immune microenvironment, we further evaluated the differences in the tumor immune microenvironment between the two groups. The TME contains immune, interstitial, and endothelial cells, as well as inflammatory mediators and ECM molecules. The results showed that the immune, stromal, and estimated scores of patients in the YG were significantly higher than those of patients in the OG (Figure S4A). The results of Xcell validated that the tumor immune microenvironment score of the YG was higher than that of the OG (Figure S5C). Through a KM analysis, we found that the prognoses of CM patients with higher stromal scores, immune scores, and ESTIMATE scores were better than that of CM patients with lower scores (Figure S4B–D). Based on the fact that the prognosis of patients with different immune scores was quite different, we further explored the landscape of immune infiltration between the two groups of patients. Through a comparative analysis of the results of CIBERSORT and MCPcounter, we found that the infiltration abundances of CD8+ T cells, NK cells, monocyte lines, macrophage cell line, and B‐cell lines in patients in the YG were significantly higher than that in patients in the OG, while the abundances of neutrophils in the YG was significantly lower than that in patients in the OG (Figure S6A,B,D). Then, by regarding immune cells as prognostic factors and combining them with prognostic data, a univariate Cox regression analysis was conducted for each type of immune cells. We found that the CD8+ T cells, NK cell lines, B‐cell lines, monocyte lines, and macrophage cell line were associated with a better prognosis in patients with CM (Figure S6C,E). In addition, we compared the results of TIMER, EPIC, and Xcell and found that the CD8+ T cells, NK cells, monocyte lines, and B‐cell lines in patients in the YG were still significantly more abundant than those in patients in the OG (Figure S5A–C). Moreover, the abundance of melanocyte infiltration in patients in the OG was higher than that in patients in the YG (Figure S5C). The univariate Cox analysis also revealed that the abundances of B cells, NK cells, CD8+ T cells, B‐cell lines, and dendritic cells were associated with a better prognosis in patients with CM (Figure S7A–C). The abundance of melanocytes was associated with a poor prognosis in patients with CM (Figure S7A).
Figure S8 shows a summary of mutations in the CM patients in TCGA database provided by “maftool”. The mutation waterfall map (Figure S9) of all the CM patients indicates that the mutation rate of BRAF in CM patients was 47%. The mutation rate of recorder NRAS was 25%. Through the somatic mutation analyses of the YG and the OG, we showed that the total mutation rate and the mutation rate of common mutation genes in the OG were lower than those in the YG (Figure S10A,B). By calculating the tumor mutation load scores of the two groups, we found that the TMB of the YG was significantly higher than that of the OG (Figure S10D). The KM analysis showed that the prognosis of the CM patients with high TMB scores was significantly better than that of the CM patients with low TMB scores (Figure S10C). In addition, the NRAS mutation rate (21%) in the YG was significantly lower than that in the OG (38%) (Figure S10E).
In total, 94 SRGs (p < 0.05) selected using the univariate Cox regression analysis were included in the LASSO regression analysis (Figure S11A). According to the standard of lambda.1se (lambda value = 4) (Figure S11B,C), four molecules, namely, IL15, B2M, FOXM1, and KIR2DL4, were selected for the multivariate Cox regression analysis (Figure S11D). We calculated the risk score of the RSS according to the formula and according to the median risk score, and the CM patients in the training cohort and validation cohort were divided into a high‐risk score group and a low‐risk score group (Figure S12A,B). We found a significant difference in the expression of the four SRGs between the high‐risk score group and the low‐risk score group in both the training cohort and the validation cohort (Figure S12C,D). It was obvious that the expression of KIR2DL4 in the low‐risk score group was significantly higher than that in the high‐risk score group. In contrast, the expression of FOXM1 in the low‐risk score group was significantly lower than that in the high‐risk score group. To validate the prognostic ability of RSS, we performed ROC and KM analyses. In the training and validation cohorts, the OS and recurrence free survival (RFS) of the low‐risk score group were significantly better than those of the high‐risk score group (log‐rank test p‐value < 0.0001, Figure S13A,C,D). The results of the ROC analysis showed that the AUC of 5‐year OS in the training and validation cohorts was 0.711 and 0.830, respectively (Figure S13B,E). To further understand whether RSS has the same predictive effect on DSS and DMFS in patients with CM, we performed a similar analysis in the test cohort (n = 210). Similarly, the DSS and DMFS of CM patients with high‐risk scores were significantly lower than those of CM patients with low‐risk scores (log‐rank test p‐value < 0.001; Figure S13F,H). In addition, the ROC analysis showed that the RSS predicted that the AUC of 3‐year DSS and DMFS in patients with CM was 0.661 and 0.698, respectively (Figure S13G,I). These results suggest that RSS can accurately predict the prognosis of patients with CM. Based on the results of the TIDE analysis, we further found that the response to the immune checkpoint blockade among patients in the high‐risk score group was significantly lower than that in the low‐risk score group (Figure 4B,C). In addition, the KM analysis of the two groups of patients who received immunotherapy and chemotherapy showed that the response of the low‐risk score group to immunotherapy and chemotherapy was significantly better than that of the high‐risk score group (Figure 4A,D). In addition, we compared the expression differences of multiple immune checkpoints in the B7 family and CD28 family between the low‐risk score and high‐risk score groups. We found that the expression levels of CD274, CTLA4, PDCD1, CD80, CD86, CD28, ICOS, and ICOSLG in the low‐risk score group were significantly higher than those in the high‐risk score group (Figure 4E).
Based on the results of GEPIA2, the expression levels of B2M, KIR2DL4, and FOXM1 in CM patients were significantly higher than those in normal skin tissues, but there was no significant difference in the expression of IL15 between the two groups (Figure 5A). Next, we verified the expression of these four genes in two CM cohorts containing normal skin tissue samples. The results showed that the expression levels of B2M, FOXM1, and KIR2DL4 were still high in CM patients. Surprisingly, in the GSE46517 cohort, the expression of IL15 in the tumor group was significantly higher than that in the normal skin tissue (Figure 5B,C).
Clinically, the pathological stage is an important factor that affects the survival of patients with CM, whereas other key factors include sex and age. Therefore, we validated the ability of RSS to predict the prognosis of CM patients in different stages, age, and sex, both in the training and the validation cohorts. RSS accurately stratified the risk in all four stages, both sexes, and both age categories (age ≥ 65 years and age < 65 years) (Figure S14).
We obtained 13 frozen samples of skin melanoma patients who received nivolumab (NIVO) (PD‐1 inhibitors) and ipilimumab (IPI) (CTLA‐4 inhibitors) treatment from March 2018 to March 2022 from the Department of Pathology of the third people's Hospital of Chengdu. The methodology of immunohistochemistry and immunofluorescence had been provided in the supplementary document of the manuscript. Immunohistochemical and immunofluorescence staining were performed on it (Figure S15A,B). Obviously, the results of immunofluorescence suggest that KIR2DL4 is indeed co‐located with NK cells. The results from immunohistochemistry showed that, among the 13 specimens, 7 had high expression of KIR2DL4 and 6 had low expression. The OS time of CM patients with high expression of KIR2DL4 was significantly longer than that of patients with low expression of KIR2DL4 in CM patients (Figure S15C). In addition, the results of Kaplan–Meier analysis also suggested that OS in patients with high expression of KIR2DL4 was better than that of patients with low expression of KIR2DL4 (Figure S15D). We isolated NK cells from melanoma tissue using flow cytometry (Figure S15A) (sorting–culture–re‐sorting–re‐nourishment). We first explored whether KIR2DL4 directly regulated the cytolytic activity of NK cells. We knocked down KIR2DL4 from the isolated NK cells with siRNA, and then we washed and co‐cultured with melanoma cells to evaluate the cell lytic activity using a lactate dehydrogenase (LDH) assay. As shown in Figure S15B, the cytolytic activity of the NK cells decreased significantly compared with the control group, and the maximum killing rate was 1/4 of that of the control group. Next, we used enzyme‐linked immunosorbent assay (ELISA) to evaluate the effect of KIR2DL4 on the cytokine production in the NK cells. As shown in Figure S15C,D, the KIR2DL4 knockdown significantly reduced the production of IFN‐γ and TNF‐α by the NK cells. To explore how KIR2DL4 activates NK cells, we constructed an overexpression plasmid of KIR2DL4 and transfected it into the NK cells. The results showed that the classical NK cell activation pathway, namely, the MEK/ERK signal pathway, was significantly activated, and the phosphorylated MEK/ERK protein was significantly upregulated after overexpression of KIR2DL4 (Figure S15E).
To further stratify the risk in CM patients and validate the prognostic predictive ability of RSS, we constructed a prognostic nomogram. We included four variables, including age, sex, stage, and the risk score, into univariate and multivariate Cox regression analyses (Figure 6A,B). The risk score was independently associated with poor prognosis of patients with CM (HR = 2.613, p < 0.05). Next, we used these four features to construct a prognostic nomogram, and each subtype of the clinical features in this model corresponded to a specific score (Figure 6C). The vertical red line in the figure indicates the score of a CM patient (TCGA‐DA‐A960‐01) included in the model. She was female, 73 years old, the AJCC grade was grade II, and the risk score was 1.711751, belonging to the high‐risk score group. After calculation, her total risk score was 180, and the probability that her survival time was less than 3 years, 5 years, and 7 years was 0.517, 0.694, and 0.832 respectively. In fact, her survival time was 804 days, less than 3 years. The above results verified that our prognosis prediction model was accurate and effective. Finally, the total score of all clinical factors corresponded to a specific 3‐, 5‐, and 7‐year survival probability. The C index of the nomograms of the training cohort and the validation cohort were 0.697 (95% CI, 0.695–0.699) and 0.762 (95% CI, 0.746–0.778), respectively. The calibration curve of the training cohort showed that the predicted values were consistent with the observed values of the 3‐, 5‐, and 7‐year OS (Figure 6E). We calculated the total risk score of each patient according to each predictor in the nomogram model and took the median total risk score as the cut‐off value to divide all CM patients into high‐risk patients and low‐risk patients. The KM analysis showed that the survival of high‐risk patients was significantly poorer than that of low‐risk patients (Figure 6D). The ROC analysis showed that the AUC values of 3‐year, 5‐year, and 7‐year prognosis of CM patients by the total risk score reached 0.767, 0.758, and 0.781, respectively (Figure 6G). In addition, the AUC value of the total risk score was always higher than that of the AJCC stage. Finally, the clinical decision curve analysis showed that the patient benefit rate of the model excluding the risk score was significantly lower than that of the model excluding the AJCC stage (Figure 6F). Unsurprisingly, the model that included both the risk score and the AJCC stage showed the highest clinical benefit rate. In addition, The validation cohort can accurately validated the model (Figure S17).
The results of GSEA showed that the cell cycle pathway was enriched in the high‐risk score group (Figure S18). However, the pathways that were enriched in the low‐risk score group were mainly the immune response‐related pathways, such as the T‐cell‐receptor signaling pathway, B‐cell‐receptor signaling pathway, NK cell‐mediated cytotoxicity, primary immunodeficiency, cell adhesion molecules (CAM), cytokine–cytokine receptor interaction, NOD‐like receptor signaling pathway, PD‐L1 expression, and the PD‐1 checkpoint pathway in cancer, JAK/STAT signaling pathway, and Toll‐like receptor signaling pathway. This suggested that the patients in our low‐risk score group were in a state of immune activation, and the effect of immunotherapy was better.
Aging skin promotes the metastasis of CM because when fibroblasts are aging, ECM components are more single, and collagen cross‐linking and ECM contractility are reduced, and this inhibits the migration and recruitment of immune cells. The aggregation of CD4+ T cells and CD8+ T cells increases the immune ability and antitumor activity in CM. , NK cells are cytotoxic innate lymphocytes, which can produce effective responses to a variety of tumor cells. NK cells contribute to the editing of cancer immunity and are often defective or dysfunctional in cancer patients. The decrease in the number of NK cells and CD8+ T cells is associated with adverse outcomes. Tumor‐induced neutrophils acquire the ability to inhibit cytotoxic T lymphocytes carrying CD8 antigen, which limits the metastasis of CD8 T cells, affecting immunotherapy and this is not always conducive to the prognosis. In this study, we used the senescence gene set to divide CM patients into the YG and the OG. The immune score, stromal score, and TME score of the YG were higher than those of the OG. In addition, the results from five types of software for estimating the abundance of immune cell infiltration in tumor tissues showed that the abundance of B‐cell line, CD4+ T cells, CD8+ T cells, NK cells, monocytes, and macrophages in patients in the YG was higher than that in patients in the OG. However, the neutrophils in the YG were significantly lower than those in the OG. Univariate Cox regression analysis showed that CD8+ T cells, CD4+ T cells, and NK cells were protective factors for the prognosis of CM patients. Therefore, we speculated that the prognosis and response to immunotherapy in the YG were better than those in the OG. The subsequent TIDE and KM analyses confirmed our assumption. The possible mechanism is that the abundance of targeted cells such as CD8+ T cells and NK cells in the tumor tissues of patients in the YG is higher than that in the OG and that the expression of immune checkpoint molecules such as PD‐L1 and CTLA4 is higher than that in the OG, which can improve the effect of immunotherapy and prognosis. TMB has attracted much attention in immunotherapy. TMB and PD‐L1 are two important biomarkers to predict the therapeutic effect of PD‐1 antibody. According to Hodi et al., in patients who received an anti‐PD‐1 inhibitor NIVO or NIVO combined with anti‐CTLA‐4 inhibitor IPI or IPI alone, high (>median) TMB was associated with longer survival than low (≤median) TMB. In our study, the TMB score of the YG was significantly higher than that of the OG, and the prognosis of CM patients with a high TMB score was significantly better than that of patients with a low TMB score. The NRAS mutation is one of the mutation subtypes in patients with CM, which occurs in ~25% of patients. NRAS‐mutant melanoma is more invasive, resulting in a lower median OS time and lagging treatment options. , , Our results showed that the NRAS mutation rate of the OG was higher than that of the YG, but the specific mechanism needs to be further explored. In addition, the upregulated genes in the YG were mainly enriched in the immune response and immunotherapy pathways, while those in the OG were mainly enriched in aging‐related pathways such as cell and epidermal development and differentiation. Therefore, we speculate that patients in the YG have higher immune activity and better response to immunotherapy, while patients in the OG age more severely. Although the recent use of targeted therapy (BRAF, MEK) and immunotherapy (anti‐CTLA‐4, anti‐PD‐1) has significantly prolonged the median OS of patients with metastatic melanoma, the treatment is far from perfect because the clinical response is either short‐lived or restricted to a limited subgroup of melanoma patients. Therefore, developing biomarkers that can predict the effect of immunotherapy would provide a more accurate treatment plan for patients with CM. We found that the survival time, status, and recurrence survival rate of CM patients in the YG were significantly higher than those of patients in the OG. The upregulated genes in the YG were mainly enriched in immune response and immunotherapy pathways, whereas those in the OG were mainly enriched in senescence‐related pathways such as cell and epidermal development and differentiation. In addition, we also found that the effect of chemotherapy and immunotherapy and the response rate to immunotherapy in the YG were higher than those in the OG. Therefore, we speculate that senescence may affect the survival of patients with CM and the effect of immunotherapy. To further develop more practical and convenient senescence‐related biomarkers for predicting prognosis and immunotherapy effect, we screened out four SRGs that independently affected the prognosis of patients with CM and constructed a set of risk scores based on them. The KM and ROC analyses suggested that the RSS was able to accurately stratify the risk of CM patients and effectively predict the OS, RFS, DSS, and DMFS of CM patients. Surprisingly, the effect of chemotherapy and immunotherapy and the overall prognosis of patients with high aging score were significantly poorer than those of CM patients with low aging score. The expression of CD274, CTLA4, PDCD1, CD80, CD86, CD28, ICOS, and ICOSLG in the low‐risk score group was significantly higher than that in the high‐risk score group. Therefore, RSS can accurately predict the prognosis and immunotherapy effect of patients with CM. In addition, we showed that RSS was able to accurately stratify the risk of CM patients with different stages (stage I, stage II, stage III, and stage IV), sex (men and women), and age (age ≥ 65 years and age < 65 years). Finally, to analyze the effects of age, sex, AJCC stage, and RSS on the prognosis of CM patients and to quantify the contribution of each index to the prognosis, we constructed the prognostic nomogram for CM patients. The results of the training and the validation cohorts showed that the prediction ability of this model was better than that of RSS or AJCC stage alone. The results of GSEA showed that the pathways related to tumorigenesis were enriched in the high‐risk score group. However, the pathways that were enriched in the low‐risk score group were mainly the immune response‐related pathways. This further implies that the immune activity of the low‐risk score group is strong, and the immunotherapeutic effect is good, while the high‐risk score group has low immune activity, frequent carcinogenic mutations, and poor therapeutic effect. Our study found that KIR2DL4 was highly expressed in CM compared with normal skin tissues, and multivariate Cox regression analysis suggested that it was an independent protective factor for the prognosis of CM patients (HR = 0.640, p < 0.05). In addition, the expression of KIR2DL4 was significantly decreased in the OG and the high‐risk score group. Therefore, we speculated that the high expression of total KIR2DL4 in the YG and the low‐risk score group increased the activity of immune cells such as NK cells and T cells, thereby enhancing the immune activity and improving the prognosis and immunotherapy effect of these CM patients. The results of immunofluorescence showed that KIR2DL4 was co‐located with NK cells, and the overall prognosis and immunotherapy effect of CM patients with high expression of KIR2DL4 were better than those with low expression. In addition, the mechanism study showed that knocking down the KIR2DL4 in human NK cells in vitro can inhibit the cytotoxicity of NK cells, and can also inhibit the secretion of tumor necrosis factor‐α and interferon‐γ by NK cells. In contrast, upregulation of KIR2DL4 can activate the MEK/ERK signaling pathway, which is the activation pathway of NK cells. The proproliferative transcription factor FOXM1 is the main regulator of cell cycle, which is necessary to enter the S phase and mitosis on time. , Phosphorylation of FOXM1 greatly enhances its transcriptional activity, therefore inhibiting the senescence of melanocytes. In our study, as an independent risk factor for patients with CM (HR = 1.303, p < 0.05), FOXM1 was significantly upregulated in CM tissues compared with normal skin tissues. FOXM1 is likely to promote CM metastasis by inhibiting senescence, thereby resulting in a poor prognosis. Inactivation or loss of β2 microglobulin (B2M) is considered to be a determinant of melanoma resistance to immune checkpoint inhibitors. , In this study, B2M was highly expressed in tumor tissues, and it was more highly expressed in the YG compared with the OG. In addition, compared with the high‐risk score group, B2M was more highly expressed in the low‐risk score group. The immunotherapeutic effects of OG and high‐risk score group were poorer than those of YG and low‐risk score group, which is probably due to the loss and inactivation of B2M. Both CD8+ T cells and NK cells rely on cytokine IL‐15 to maintain balance in vivo. , Exogenous administration of IL‐15 can also promote the activation of CD8+ T cells and NK cells, which has been used as an adjuvant for cancer immunotherapy. , In the present study, IL15 was more highly expressed in the YG than in the OG. In addition, compared with the high‐risk score group, IL‐15 was more highly expressed in the low‐risk score group. Moreover, its adjuvant effect on immunosuppressive therapy may lead to better prognosis and immunotherapy effect in the YG and the low‐risk score group. In general, we developed a new RSS, which can predict the prognosis of patients with CM and the effect of immunotherapy. As the core molecule in the RSS, KIR2DL4 activated the NK cells by activating the MEK/ERK pathway, therefore promoting the release of cytotoxic factors from the NK cells and increasing the cytotoxic activity of the NK cells. Moreover, we constructed the prognostic nomogram model to better stratify the risk of CM patients and efficiently guide clinical decisions.
The authors would like to thank the staff of the National Center for Biotechnology Information and the National Cancer Institute.
RM: Conceptualization, Experiment, Methodology, Software, Investigation, Visualization, Writing original draft. ZR: Experiment. PY: Experiment. FY: Experiment, Software, Investigation. TZ: Conceptualization, Writing – original draft, Funding acquisition, Supervision.
The authors declare that they have no conflict of interest.
This study was approved by the Institutional Ethics Review Board of the Third People's Hospital of Chengdu (record #:2018S75; Chengdu, Sichuan, China), and was conducted in accordance with the Chinese ethical guidelines for human genome/gene research.
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PMC9633810 | Bratati Mukherjee,Ankit Tiwari,Ananya Palo,Niharika Pattnaik,Subrat Samantara,Manjusha Dixit | Reduced expression of FRG1 facilitates breast cancer progression via GM-CSF/MEK-ERK axis by abating FRG1 mediated transcriptional repression of GM-CSF | 03-11-2022 | Breast cancer,Metastasis | Multiple molecular subtypes and distinct clinical outcomes in breast cancer, necessitate specific therapy. Moreover, despite the improvements in breast cancer therapy, it remains the fifth cause of cancer-related deaths, indicating the involvement of unknown genes. To identify novel contributors and molecular subtype independent therapeutic options, we report reduced expression of FRG1 in breast cancer patients, which regulates GM-CSF expression via direct binding to its promoter. Reduction in FRG1 expression enhanced EMT and increased cell proliferation, migration, and invasion, in breast cancer cell lines. Loss of FRG1 increased GM-CSF levels which activated MEK/ERK axis and prevented apoptosis by inhibiting p53 in an ERK-dependent manner. FRG1 depletion in the mouse model increased tumor volume, phospho-ERK, and EMT marker levels. The therapeutic potential of anti-GM-CSF therapy was evident by reduced tumor size, when tumors with decreased FRG1 were treated with anti-GM-CSF mAb. We found an inverse expression pattern of FRG1 and phospho-ERK levels in breast cancer patient tissues, corroborating the in vitro and mouse model-based findings. Our findings first time elucidate the role of FRG1 as a metastatic suppressor of breast cancer by regulating the GM-CSF/MEK-ERK axis. | Reduced expression of FRG1 facilitates breast cancer progression via GM-CSF/MEK-ERK axis by abating FRG1 mediated transcriptional repression of GM-CSF
Multiple molecular subtypes and distinct clinical outcomes in breast cancer, necessitate specific therapy. Moreover, despite the improvements in breast cancer therapy, it remains the fifth cause of cancer-related deaths, indicating the involvement of unknown genes. To identify novel contributors and molecular subtype independent therapeutic options, we report reduced expression of FRG1 in breast cancer patients, which regulates GM-CSF expression via direct binding to its promoter. Reduction in FRG1 expression enhanced EMT and increased cell proliferation, migration, and invasion, in breast cancer cell lines. Loss of FRG1 increased GM-CSF levels which activated MEK/ERK axis and prevented apoptosis by inhibiting p53 in an ERK-dependent manner. FRG1 depletion in the mouse model increased tumor volume, phospho-ERK, and EMT marker levels. The therapeutic potential of anti-GM-CSF therapy was evident by reduced tumor size, when tumors with decreased FRG1 were treated with anti-GM-CSF mAb. We found an inverse expression pattern of FRG1 and phospho-ERK levels in breast cancer patient tissues, corroborating the in vitro and mouse model-based findings. Our findings first time elucidate the role of FRG1 as a metastatic suppressor of breast cancer by regulating the GM-CSF/MEK-ERK axis.
Breast cancer remains at the top of cancer-related deaths among women, with a mortality rate of 6.9% [1]. Epithelial to mesenchymal (EMT) transition, which comprises the detachment of cancer cells from their primary origin, intravasation, and formation of metastatic growth at distant sites, accounts for majority of the deaths associated with breast cancer [2]. Even after the significant progress in primary breast cancer therapy, metastasis and recurrence are the major cause of reduced survival [3]. Finding out the role and mechanism of additional genes which affect EMT can provide additional opportunities to improve the survival rate. FSHD region gene 1 (FRG1), which is mainly known for being the candidate gene for facioscapulohumeral muscular dystrophy (FSHD), has recently shown its potential as a tumor suppressor gene. Our earlier study first time reported the correlation of FRG1 downregulation with oral cavity, colorectal and gastric carcinoma [4]. Depletion of FRG1 levels increased cancerous properties of prostate cancer cell lines via activation of the p38-MAPK [5]. Mechanistically, reports have suggested FRG1’s involvement in pre-mRNA processing [6], F-actin-bundling [7], and angiogenesis [8]. The role of FRG1 in breast cancer is mostly unexplored. Expression profiling of the genes associated with triple-negative breast cancer (TNBC) using the MDA-MB-231 cell line showed FRG1 as one of the significantly downregulated genes which were connected with the migratory potential of breast cancer cells [9]. However, the exact biological function of FRG1 in breast cancer and its mechanism are entirely unknown. So far, many cytokines such as IL6/8/10, TNFα, IFNγ, growth factors TGFβ, bFGF, and VEGF have been reported to involve with EMT in breast cancer [10]. Granulocyte-macrophage colony-stimulation factor (GM-CSF), a potent hematopoietic growth factor mainly known for its immunomodulatory function in the tumor niche, has recently been reported to play a role in EMT in colon cancer [11]. Elevated expression of GM-CSF is clinically correlated with advanced histological grade, metastasis, and poor prognosis in patients with prostate cancer, breast cancer [12], and pancreatic ductal carcinoma [13]. Nevertheless, very little is known about the upstream regulation and downstream signaling mechanism coordinating GM-CSF-mediated metastatic colonization. Previously we found enhanced GM-CSF expression in FRG1-depleted prostate cancer cells [5]. In the current study, we first report the FRG1-mediated regulation of GM-CSF in breast carcinoma. Prior to this, no information was available on the detailed mechanism of how GM-CSF promotes EMT in any cancer. Here we have shown that FRG1 binds to the GM-CSF promoter and inhibits its expression. The loss of FRG1 resulted in increased cell proliferation, migration, and invasion triggered by increased levels of GM-CSF and the activation of the MEK/ERK pathway, in both the cell lines of luminal (ER+; MCF7) and basal (TNBC; MDA-MB-231) origin. We have validated our in vitro findings in breast cancer patient tissues and mouse models. We have also shown the therapeutic potential of anti-GM-CSF antibody in the mouse model. Overall, here we report the role and molecular mechanism of a new gene, FRG1, in breast cancer, which has the potential to be explored as a therapeutic target irrespective of molecular subtypes.
We prepared stable lines with FRG1 expression perturbation in estrogen receptor-positive (ER+) cells MCF7 which has moderate endogenous expression of FRG1 (Supplementary Fig. S1A). The reduction of FRG1 expression in MCF7 increased the proliferation rate in MTS and colony formation assays compared to the control group (Fig. 1A, B). Correspondingly, increased FRG1 expression decreased the rate of proliferation in both assays (Fig. 1E, F). To explore the metastatic potential, we checked the effect of FRG1 expression on cell migration. The wound-healing assay showed enhanced cell migration due to reduced FRG1 expression (Fig. 1C). Matrigel invasion assay corroborated these findings (Fig. 1D). We observed the opposite effect on wound healing and invasion of MCF7 cells with ectopic expression of FRG1 (Fig. 1G, H). Besides, we generated FRG1 knockout MCF7 cells (FRG1_KO) and checked its effect on the tumorigenic properties (Supplementary Fig. S1B, C). In accordance with our findings in the FRG1 knockdown group, we also observed increased cell proliferation (Supplementary Fig. S1B) and migration (Supplementary Fig. S1C) due to FRG1 knockout. To determine if the effect of FRG1 is molecular subtype-specific, we ectopically expressed FRG1 in TNBC cell line MDA-MB-231, which has low endogenous FRG1 expression (Supplementary Fig. S1A). In agreement with our observation in MCF7_FRG1_Ex cells, we observed a reduction in cell proliferation (Fig. 1I, J), migration (Fig. 1K), and invasion (Fig. 1L). Together these data suggest FRG1 expression can modulate the tumorigenic properties of breast cancer cell lines.
Intrigued by the effect of FRG1 expression level on the tumorigenic properties of breast cancer cells, we explored its effect on ERK and AKT, two frequently altered signaling pathways in cancers [14]. We observed that the reduced FRG1 level led to the activation of ERK and its upstream molecule MEK in MCF7 cells without the alteration in total-ERK or total-MEK levels (Fig. 2A). A similar trend was observed in FRG1 knockout MCF7 cells, where knockout of FRG1 led to increased expression of phospho-ERK (Supplementary Fig. S1D). On the other hand, ectopic expression of FRG1 decreased the activation of ERK and MEK in MCF7 (Fig. 2B) and MDA-MB-231 cells (Fig. 2C). As further corroboration of our findings, ectopic expression of FRG1 in MCF7_FRG1_KD cells nullified the change in phospho-ERK level (Fig. 2D). Unexpectedly, we found that FRG1 depletion reduced AKT activation at the 308 position, and there was no effect at the 473 position in MCF7 cells (Supplementary Fig. S2A, B). Increased FRG1 expression also affected the activation of AKT only at the 308 position, in the opposite manner (Supplementary Fig. S2C, D). Previous studies have reported that activation in ERK signaling can suppress the AKT pathway [15]. We inhibited the ERK pathway in MCF7_FRG1_KD cells and found the reversal of the phospho-AKT 308 suppression (Supplementary Fig. S2E), which confirmed that inhibition of AKT activation was ERK-mediated. As expected, no effect of ERK inhibition was found on the level of phospho-AKT 473 (Supplementary Fig. S2E). This data suggests that reduced FRG1 expression might be involved in breast cancer progression by the activation of the ERK pathway.
Loss of apoptotic control is a hallmark of cancer which permits cancer cells to survive longer and gives more time to accumulate the mutations [16]. Reduction in FRG1 level in MCF7 significantly decreased the downstream effector caspase 3/7 level (Fig. 3A). We made a coherent observation in a flow cytometry-based analysis of Annexin V/ propidium-iodide where FRG1 depletion reduced apoptosis in MCF7 cells (Fig. 3B). Mechanistically, we found that depletion of FRG1 reduced phospho-p53 level (Fig. 3C). No change in phospho-p38 level indicates reduced apoptosis in FRG1_KD cells may be independent of phospho-p38. (Fig. 3C). Several studies have reported ERK-mediated inhibition of p53 [17, 18]. To test this, we inhibited ERK and checked the levels of phospho-p53 in FRG1-depleted MCF7 cells. We observed restoration of phospho-p53 level in MCF7_FRG1_KD group treated with ERK inhibitor (Fig. 3D). Overall, our findings suggest that a reduced level of FRG1 leads to an ERK-mediated decrease of phospho-p53, which reduces apoptosis.
It is well established that EMT increases cell migration, leading to metastasis in cancer [19]. As reduced FRG1 level increased the migration in cell-based assays, we checked its effect on EMT markers snail, slug, and twist, which have long been reported to trigger EMT through ERK signaling [20–22]. As hypothesized, we detected significant upregulation of snail, slug, and twist in MCF7_FRG1_KD cells (Fig. 4A). To validate, we inactivated the ERK pathway in MCF7_FRG1_KD cells and found abrogation of the ERK-mediated upregulation of EMT marker snail (Fig. 4B). Scratch wound-healing assay also revealed a similar effect (Fig. 4C). As expected, ectopic expression of FRG1 in MDA-MB-231 cells reduced the expression of EMT markers (Fig. 4D). To further substantiate our findings, we treated MDA-MB-231_FRG1_Ex cells with ERK activator Ceramide, which restored the reduced level of phospho-ERK caused by ectopic expression of FRG1 (Fig. 4E). The same experimental setup also rescued snail expression levels (Fig. 4E) and cell migration (Fig. 4F). These findings also suggest that the effect of FRG1 on EMT is consistent in both the breast cancer cell lines. Our previous work [5] found that FRG1 perturbation changed the levels of inflammatory cytokines CXCL1, CXCL8, GM-CSF, PDGFα, and PDGFβ, which have been reported to affect EMT [11, 23, 24]. We found a significant increase in CXCL1, CXCL8, GM-CSF, PDGFα, and PDGFβ transcript levels due to FRG1 depletion (Fig. 4G) in MCF7 cells, the opposite effect was observed with the ectopic expression of FRG1 in MDA-MB-231 cells (Fig. 4I). Further analysis of the effect of altered FRG1 levels on GM-CSF concentration in the conditioned media harvested from MCF7 and MDA-MB-231 cells, confirmed our findings at protein levels (Fig. 4H, J). To ascertain the specificity of the observation, we inhibited the ERK pathway in MCF7_FRG1_KD cells, which counterbalanced the increase in CXCL1, CXCL8, PDGFα, and PDGFβ transcript levels caused by FRG1 depletion, suggesting that the effect was downstream of ERK (Supplementary Fig. S2F). To elucidate if CXCL1 and CXCL8 were responsible for ERK activation and downstream change in EMT markers [25] we inhibited their receptor CXCR2 by using CXCR2 antagonist Cpd 19 [26]. We found no effect in phospho-ERK and snail levels (Fig. 4K), which suggests ERK activation is not downstream of CXCR2-CXCL1/8. Interestingly, we found that ERK pathway inhibition in MCF7_FRG1_KD could not nullify the increase in GM-CSF level caused by FRG1 reduction (Supplementary Fig. S2F), which implies that the effect on GM-CSF is upstream of ERK. We propose that reduced FRG1 leads to ERK-mediated upregulation of cytokines except for GM-CSF, and FRG1 could act upstream of GM-CSF.
So far, the role of GM-CSF in breast cancer has not been fully understood. We checked the effect of GM-CSF levels in MCF7 cells and found that GM-CSF enhanced the tumorigenic properties of MCF7 cells by upregulating cell proliferation and migration (Supplementary Fig. S3A, B). Also, ectopic administration of GM-CSF upregulated the expression of phospho-ERK and snail (Supplementary Fig. S3C). In order to confirm that GM-CSF is downstream of FRG1 and upstream of ERK, we perturbed GM-CSF levels in the cells with altered expression of FRG1. GM-CSF inhibition resulted in the downregulation of phospho-ERK and snail in MCF7_FRG1_KD cells (Fig. 5A), which eventually reduced cell migration in scratch wound-healing assay (Fig. 5B). Correspondingly, the opposite effect was observed in MCF7_FRG1_Ex cells upon treatment with exogenous human recombinant GM-CSF (Fig. 5C, D). These results suggest that the effect of FRG1 on the ERK pathway might be GM-CSF mediated.
Based on the observations above, we hypothesized that FRG1 might be a direct transcriptional regulator of GM-CSF. Our previous work has shown “CTGGG” as a binding site for FRG1. We found six “CTGGG” sequences in the GM-CSF promoter region within 907 bp upstream of the transcription start site (Fig. 5E). Luciferase reporter assay using the promoter of GM-CSF (907 bp upstream of transcription start site in pGL4.23) revealed increased luciferase activity in HEK 293 T cells compared to the empty vector control (Fig. 5F). On the contrary, no change in luciferase activity was observed in HEK 293 T with FRG1 depletion, between the GM-CSF promoter and the control groups (Fig. 5F). When MDA-MB-231 cells with increased FRG1 expression levels were transfected with the same constructs, we found a substantial decrease in luciferase activity in cells containing GM-CSF promoter than control, while no change was observed in relative luciferase intensity in MDA-MB-231 (Control_Ev) cells with basal FRG1 expression between the GM-CSF promoter and the control groups (Fig. 5G). This observation strengthened that FRG1 more likely possessed an inhibitory effect on GM-CSF expression, which agrees with our previous observations. Additionally, to confirm the binding of FRG1 on the GM-CSF promoter, a ChIP assay was performed in HEK 293 T cells. As shown in Fig. 5H, enrichment of FRG1 protein on GM-CSF promoter fragment was found after immunoprecipitation with anti FRG1 antibody but not by the negative control IgG. This approves our hypothesis that FRG1 binds to the GM-CSF promoter. To further validate, a competitive EMSA on labeled oligos was carried out with an increased amount of unlabeled oligos. The result showed that oligos were sufficient to compete for the binding. Thereby drastic reduction in the intensity of the shift was observed. Furthermore, when the binding complex was subjected to FRG1 specific antibody, a shift was noted, indicating the binding of FRG1 to the oligos (Fig. 5I). These results confirm in vitro binding of FRG1 on the CTGGG site of GM-CSF promoter. Therefore, our data strongly support the notion that FRG1 hinders EMT progression in breast cancer by inhibiting GM-CSF-mediated ERK activation.
GEPIA-based analysis revealed lower expression of FRG1 transcripts in cancer patients (n = 1085) compared to normal (n = 291) (Fig. 6A) [27]. GEPIA also depicted that the patient group with a high level of FRG1 had a higher probability of disease-free survival (Fig. 6B). Kaplan–Meier plotter-based survival analysis also showed that breast cancer patients (containing wild-type p53) with a high level of FRG1 had a higher probability of recurrence-free survival (Supplementary Fig. S4A) [28]. To further authenticate TCGA dataset-based findings, we collected 194 breast cancer tissue samples with 56 normal adjacent tissues and performed IHC for FRG1 expression. We observed a significant downregulation of FRG1 levels in cancer patient samples compared to the normal tissue (Fig. 6C). Out of 194 cancer tissues, 57 indicated (29%) high FRG1 expression (AS: 7–8), 106 indicated (55%) moderate expression (AS: 3–6), and only 31 showed (16%) low level of FRG1 (AS: 1–2). In contrast, out of 56 normal tissues, 40 showed (71%) high FRG1 level (AS: 7–8), and 16 showed (29%) moderate FRG1 expression (AS: 3–6). No normal counterparts showed low FRG1 expression (AS: 1–2) (Fig. 6D). We observed an inverse relationship between FRG1 and phospho-ERK expression levels (n = 10) (Fig. 6E), strengthening our in vitro findings. Estrogen signaling is crucial for breast carcinogenesis as it profoundly contributes to the proliferation of breast cancer cells. So, we checked whether the loss of FRG1 activated the ER signaling. To find out the correlation, we segregated breast cancer patient tissue samples according to their ER/PR/HER2 status and checked the expression of FRG1. We did not see any significant difference in FRG1 expression levels between ER + breast cancer tissues and TNBC patients (median FRG1 AS: 6 versus 5) (Fig. 6F). In MCF7 cells, we found upregulation in phospho-ER due to FRG1 depletion (Supplementary Fig. S4B), and precisely the opposite in the case of ectopic expression of FRG1 in MCF7 cells (Supplementary Fig. S4C). To examine whether ER activation was concomitant to ERK activation, we treated FRG1_KD MCF7 cells with ERK inhibitor FR180204 and found a significant reduction in phospho-ER levels. This suggests an ERK-mediated upregulation of ER in FRG1-depleted MCF7 cells (Supplementary Fig. S4D). Taken together, our findings imply an inverse relation between FRG1 and ERK activation in breast cancer patients. As a downstream effect, FRG1 can affect ER activation in ERK-dependent manner.
To demonstrate the impact of FRG1 expression levels in vivo, we developed an orthotopic mice model by implanting 4T1 cells with depleted and elevated levels of FRG1, in the mammary fat pad of BALB/c mice. FRG1 knockdown significantly increased tumor volume and weight (Fig. 7A and Supplementary Fig. S5A). Correspondingly, ectopic expression of FRG1 significantly reduced tumor volume and weight (Fig. 7B and Supplementary Fig. S5B). Parallel to our cell line-based data, we found a reduced expression of phospho-ERK and snail in the FRG1 over-expression group (Fig. 7C). To assess its metastasis potential, we injected FRG1-depleted 4T1 cells in the tail vein of BALB/c mice. We observed more metastatic nodules in the lungs of the FRG1_KD group than in control (Fig. 7D). An opposite trend was identified in the set with FRG1 over-expression (Fig. 7E). These results corroborate our in vitro observations in the mouse model.
To investigate the therapeutic potential of anti-GM-CSF therapy in breast cancer patients with reduced FRG1 expression, we did a mouse model-based study. We injected 4T1_FRG1_KD and Control_Sc cells subcutaneously into mice (n = 4). After the tumor reached a palpable size (7 days), one set of 4T1_FRG1_KD mice (n = 4) was intraperitoneally administrated with anti-GM-CSF neutralizing monoclonal antibody (mAb) (10 mg/kg body weight), and the other set of 4T1_FRG1_KD mice (n = 4) was injected with control IgG antibody (10 mg/kg body weight) alternative days till day 21. We found a significant reduction in tumor size in the set treated with GM-CSF mAb compared to IgG (Fig. 7F and Supplementary Fig. S5C). In support of our in vitro data, we also observed a reduction in phospho-ERK and snail in GM-CSF treated group (Fig. 7G). Henceforth our in vivo findings establish the role of FRG1-mediated regulation of GM-CSF. It also indicates that loss of FRG1, disrupts the suppression of GM-CSF, which might promote the proliferation and EMT by activating ERK and its downstream targets.
Metastatic dissemination in breast cancer accounts for 90% of cancer-related deaths [29]. Therefore, elucidating the molecular mechanism underlying the metastatic process has attracted considerable attention from researchers. Loss of function of tumor suppressor genes is often associated with increased metastasis and poor patient survival. In the case of breast cancer, different molecular subtypes represent discrete clinical outcomes. Hence, the search for novel targets that can act independently of molecular subtypes, will contribute to the development of more effective therapy. Since the discovery of the FRG1 in 1996, most of the reports have highlighted its involvement in FSHD pathophysiology, muscle development, actin-bundling, and angiogenesis. Very few reports have shown an indirect association of FRG1 with cancer. Whole-exome sequencing identified some deleterious mutations in the FRG1 gene in calcifying fibrous tumor of the pleura [30]. Another whole-exome sequencing study, done in six follicular thyroid cell lines, reported a mutation in the FRG1 gene in cancer cell lines [31]. Our group showed FRG1-mediated activation of the p38-MAPK pathway in prostate cancer [5]. In the current study, our findings first-ever report the inverse association of FRG1 expression levels with breast cancer and unravel the underlying molecular mechanism using multiple model systems. Activation of ERK plays a pivotal role in cell proliferation, angiogenesis, and malignant transformation [32]. It supports tumor growth and cancer progression by upregulating various EMT-inducing factors [33]. TNBC, which is considered more aggressive and therapeutically challenging [34], is often associated with shorter patient survival with high expression of ERK [35]. The cross-talk between ERK and ERα signaling in luminal carcinoma may lead the cells toward chemoresistance [36]. In our case, we have found that reduced FRG1 expression led to the activation of ERK in both the breast cancer cell lines. We have further observed ERK-mediated elevated expression of the EMT markers snail, slug, and twist, which is consistent both in vitro and in vivo. Our findings may create enormous scope for research to develop novel therapeutics that can target upstream regulators of ERK and act irrespective of molecular subtypes of breast cancer. The cross-talk between ERK and AKT molecules is an important determinant of the cell fate towards survival or apoptosis [18]. Surprisingly, although there was no change in AKT 473 activation, the depletion of FRG1 reduced the activation of AKT 308. Previous studies have shown ERK-mediated suppression of AKT activation [37], as was the case in our study, where the suppression of AKT 308 activation was rescued upon ERK inhibition in MCF7 cells. This observation supports the previous finding that MEK downregulation decreases AKT activation in EGFR and HER2-driven breast cancer [15]. Also, the activation of AKT was found to protect HeLa cells from apoptosis [38]. In this context, several reports suggest multiple mechanisms for MEK/ERK-mediated downregulation of AKT activation [15, 38, 39]. ERK has been shown to negatively regulate Gab1-PI3K binding, reducing the downstream AKT signaling [39], [15]. Also, inhibition of ERK, reduces EGFR phosphorylation, resulting in augmented AKT phosphorylation [40]. In addition to AKT-P38-P53 mediated activation of apoptosis, several studies have reported ERK-mediated regulation of p53 and apoptosis [41]. Inactivation of ERK promoted translocation of apoptosis-inducing factor in the nucleus, thus, causing apoptosis [18]. Another report suggests ERK promotes increased MDM2 expression and thus promotes the degradation of p53 [17]. We found reduced expression of phospho-p53 in FRG1-depleted MCF7 cells, which was rescued by the inhibition of ERK. This is in parallel to the literature that supports the MEK/ERK pathway can downregulate the activation of p53 and promote cell survival [18]. We put forward our hypothesis that FRG1 depletion activates the ERK pathway, which attenuates AKT and p53 phosphorylation that results in ERK-dependent inhibition of the apoptotic pathway. Previously it was reported that GM-CSF promotes breast cancer pathogenesis by recruiting CCL-18+ macrophages into the tumor microenvironment [12]. Increased GM-CSF level in breast cancer is correlated with increased metastasis and poor patient survival [42]. Higher expression of GM-CSF receptors on nonhematopoietic cells in multiple tumor types has suggested its potential pro-tumorigenic factor, which is yet to be validated [43]. Expression of GM-CSF in skin carcinoma cells enhanced the metastatic growth and proliferation of cancer cells [44]. Also, in the head and neck [45], glioma [46], and osteosarcoma [47], autocrine stimulation of GM-CSF is reported to promote tumor growth. Although several reports have shown altered GM-CSF levels in multiple cancers, the underlying molecular mechanism in GM-CSF-mediated metastasis needs to be further addressed. The effect of the GM-CSF on the MAPK/ERK/ZEB1 pathway has been reported in colon cancer [11], but the detailed mechanism remains unexplored. The present study first time, discovered the in-depth role of GM-CSF in breast cancer and found that FRG1 acts as its repressor. When the expression of FRG1 is less in the cells, it leads to the expression of more GM-CSF, which in turn activates ERK-mediated EMT. Our findings have provided many missing links from FRG1 to GM-CSF to ERK to EMT. Besides, we have observed treatment with anti-GM-CSF mAb reduced tumor volume and EMT marker in the mouse model. Collectively, our study highlights the therapeutic potential of anti-GM-CSF therapy in cancer samples with low FRG1 expression. To validate our findings, we performed a retrospective study in clinical patient samples and found reduced FRG1 expression in 71% of breast cancer patients' tissues. Reports suggest expression of ER in breast cancer is associated with favorable clinical outcomes [48]. We observed that in ER+ and TNBC samples, there was no difference in FRG1 expression levels. It emphasizes that the role of FRG1 in breast cancer is not subtype-specific. Yet, our in vitro findings suggest activation of ER in FRG1-depleted MCF7 cell line in an ERK-dependent manner showing cross-talk between FRG1-mediated regulation of ERK and ER signaling. Taken together the in vitro and in vivo data, the therapeutic potential of FRG1-mediated signaling can be explored in all molecular subtypes of breast cancer. Furthermore, survival analysis in GEPIA and Kaplan–Meier plotter designates the association of high FRG1 level with a higher recurrence-free survival rate in breast cancer patients, indicating a favorable role of FRG1 towards prognosis determination. Observation in large and stage-specific cohorts can confirm the authenticity of this observation. In conclusion, reduced expression of FRG1 in breast cancer leads to transcriptional activation of GM-CSF, which promotes activation of ERK and EMT (Fig. 8). Identification of GM-CSF as an activator of ERK, and the cross-talk between AKT and ERK, can help in the development of a more efficient therapeutic strategy.
Our study included 194 breast cancer tissue samples with 56 adjacent normal tissues. From SRL diagnostics-Bhubaneswar, Apollo Hospitals-Bhubaneswar, and AHRCC-Cuttack, between 2014 to 2019, we collected tissues from 104 breast cancer patients, out of which 46 had the adjacent normal tissues. The patients who did not undergo any prior treatment before the surgery with known ER/PR/HER2 status, stage, and grade information as per the TNM system, were only included in the study. This study was approved by the Institutional Ethics Committee of NISER (NISER/IEC/2021-02) and AHRCC. For prospective sample collection, written consent was taken; for retrospective tissue FFPE blocks, the ethics committee waived the requirement of written consent. Additionally, a tissue microarray containing 90 tumors and 10 normal tissue samples was purchased (Biomax, MD, USA, #BC081120c).
From FFPE blocks, 5 µm thin sections were cut and deparaffinized by heating at 80 °C dry bath for an hour, then submerging the slides into xylene. Sections were rehydrated by a gradient of ethanol-100, 90, 70, 50%, and water. Heat-induced antigen retrieval was done in EnVision FLEX target retrieval solution (pH 9) high pH citrate buffer (DAKO, MN, USA) and blocked with EnVision FLEX Peroxidase blocking buffer (DAKO). Sections were incubated with primary and EnVision FLEX HRP secondary antibody (DAKO) for 2 hours and 30 minutes. We stained slides with EnVision FLEX DAB + Chromogen (DAKO) and counterstained them with Haematoxylin (Himedia, Mumbai, India). FRG1 expression was calculated using the Allred score (AS), as described previously [4]. Briefly, the Allred score was measured by combining the staining intensity of FRG1 protein in the cytoplasm and the percentage of cells stained positive for FRG1. Measurements of ‘staining intensity’ were categorized as weak “0–2”, moderate “3–6”, and strong “7–8”. FRG1 positive tumor tissue’s staining percentage were scored as “0” if 0%, “1” if 1%, “2” if 2–10%, “3” if 11–33%, “4” if 34–66% and “5” if ≥67%. Each sample was compared to the adjacent uninvolved tissue (if found) as a control.
Detailed methodology is provided in the supplementary information.
According to the manufacturer’s protocol, total RNA was extracted from the cells using an RNeasy mini kit (Qiagen, Hilden, Germany). Reverse transcription was performed with 1 µg of RNA using the verso cDNA synthesis kit (Thermo Scientific, Lithuania). Each qPCR reaction was carried out using 10 ng of cDNA, 2x SYBR Green PCR Master Mix (Thermo Fisher, CA, USA), and respective primers (Supplementary Table 1) in Applied Biosystem 7500 system (Thermo Fisher, CA, USA). All reactions were done in triplicate. GAPDH was used as an internal control. The relative expression of each transcript was calculated using the ΔΔCt method.
ELISA was performed to quantify the level of GM-CSF present in MCF7 and MDA-MB-231 cells with altered FRG1 expression. In total, 1 × 106 cells were plated in a 100 mm culture dish in their respective culture media. After 12 h, the media was replaced by the media containing 2% FBS. After 3 days of incubation, the media was collected and centrifuged at 4000 rpm (4 °C) for 10 min to get rid of the cellular debris. The supernatant was aliquoted and stored at −80 °C till further use. 100 μl of this supernatant was used to carry out the ELISA using Human GM-CSF Quantikine ELISA Kit (R&D Systems, MN, USA) according to the manufacturer’s protocol. OD values were taken at 450 nm and 540 nm in the Varioscan multimode microplate reader (Thermo). During the final analysis, values obtained at 450 nm was subtracted from the values at 540 nm.
Cell lysates were prepared in ice-cold RIPA buffer (Thermo Scientific, IL, USA), supplemented with protease-phosphatase inhibitor (Thermo Scientific, IL, USA). Protein was quantified by the BCA protein estimation kit (Thermo Scientific, IL, USA). About 30–40 µg of protein samples were resolved on 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto a PVDF membrane (Millipore, Bangalore, India). The membrane was blocked using 5% BSA (MP Biomedicals, OH, USA) for an hour and incubated overnight with the primary antibodies (Supplementary Table 2). Blots were washed in 1X TBST buffer and detected with respective horseradish peroxidase (HRP) conjugated secondary antibodies (Abgenex, Bhubaneswar, India). The chemiluminescence signal was developed by SuperSignalTM West Femto maximum sensitivity substrate (Thermo Scientific, IL, USA) and detected in Chemidoc XRS + (Bio-Rad, CA, USA). Images were analyzed in ImageJ (NIH, MD, USA) software. All the experiments were performed in triplicate.
Details are given in the supplementary information.
Caspase 3/7 activity was measured using the caspase-Glo 3/7 assay kit (Promega, WI, USA) as per the manufacturer’s instructions. Briefly, 1 × 103 cells were plated in a 96-well plate. After 24 h, 100 µl of caspase-Glo 3/7 reagent was added to each well and incubated for 20 min. Luminescence readings were measured in a Varioscan multimode microplate reader (Thermo Scientific, USA).
The cellular apoptosis was measured in FRG1-depleted MCF7 cells using Annexin FITC and PI staining kit (BD Pharmingen™, NJ, USA) following the manufacturer’s protocol, in FACS caliber (BD Biosciences, CA, USA). Flow cytometric data were analyzed by CellQuest Pro software (BD Biosciences).
GM-CSF promoter (accession no. P04141) was cloned into the pGL4.23 [luc2/minP] (Promega, WI, USA) vector. Appropriate cells were plated and transfected with 990 ng of pGL4.23_GM-CSF promoter construct and 10 ng internal control pGL4.74 [hRluc/TK] (Promega, WI, USA) using Lipofectamine 3000 (Invitrogen). After 48 h, cells were harvested using the lysis buffer provided with the Dual-Glo Luciferase assay kit (Promega, WI, USA). Firefly and renilla luminescence signal was quantified using Varioscan multimode microplate reader (Thermo Scientific). For each sample, firefly luciferase activity was normalized to renilla luciferase activity.
Nuclear extract was prepared from HEK 293 T cells expressing FRG1 using the NE-PER kit (Thermo Scientific, IL, USA). Oligonucleotides were commercially procured (IDT, IA, USA). The binding of FRG1 on the CTGGG site of the GM-CSF promoter was investigated using 3 fmol of biotinylated double-stranded oligonucleotides. Competitive EMSA was carried out using 3 pmol of unlabeled oligonucleotides. For the supershift assay, 1.5 µg of FRG1 antibody (Abcam, Cambridge, UK) was used following standard reaction conditions per the manufacturer’s protocol. Protein-DNA complexes were separated on 10% native polyacrylamide gels in 0.5X TBE, transferred on nylon membrane (Thermo Scientific, IL, USA), and crosslinked upon UV exposure for 5 min. Signal development was done using Chemiluminescent Nucleic Acid Detection Module Kit (Thermo Scientific) and detected in the Chemidoc XRS + system (Bio-Rad).
ChIP assay was carried out with the chromatin (1 × 106 cells) harvested from HEK 293 T cell line. Afterward, cells were processed using the ChIP kit (Abcam, USA) according to the manufacturer’s protocol. Briefly, first cells were washed using 1x PBS followed by cross-linking with 1.1% formaldehyde and Buffer A for 10 min at room temperature. Next, quenching of the cross-linking process was done using 1.5 M glycine for 5 min provided with the kit. The cell pellet was collected after centrifugation for 5 min at 4 °c. The cell pellet was lysed and then subjected to sonication to attain 200–600 bp fragments using a sonicator (Cole-Parmer, IL, USA) at maximum speed (30 times) for 35 s. Sheared chromatin was availed after centrifugation at 14,000 × g for 5 min. Immunoprecipitation was done by incubating the chromatin with the antibodies against FRG1 and negative control IgG at 4 °c overnight. After de-cross-linking and proteinase K treatment, immunoprecipitated chromatin was isolated and subjected to qRT PCR using the primers given in supplementary table 1.
GEPIA webserver (http://gepia.cancer-pku.cn/about.html) was used to ascertain the differential FRG1 transcript level (cancer patients vs. TCGA normal and GTEx, rest of the default parameters- p value cutoff 0.01, log2FC value cutoff 1, log scale, and jitter size 0.4) and disease-free survival (DFS) (group cutoff quartile) in BRCA dataset (accessed on 24 December 2021). We did relapse-free survival (RFS) analysis in Kaplan–Meier plotter (https://kmplot.com/analysis/, accessed on 16 Feb 2022) using mRNA gene chip data and auto-select the best cutoff in breast cancer patients with wild-type p53. Hazard ratio (HR) with a 95% of confidence interval (CI), and statistical significance were determined using log-rank test.
All animal experiments were performed following the protocol approved by the Institutional Animal Ethics Committee, NISER (NISER/SBS/IAEC/AH-109). Details of tumor development, metastasis, and in vivo GM-CSF inhibition experiments are given in the supplementary information.
Statistical analysis was performed using GraphPad Prism 6.0 version (GraphPad Software Inc., CA, USA) and Microsoft Excel (Microsoft, WA, USA). To determine the statistical significance of the difference in mean values, a two-tailed unpaired Student’s t-test was applied. Mann–Whitney U-test was used to measure the correlation between Allred scores for FRG1 expression in cancer and normal samples. Data were presented as mean ± SD. P value ≤0.05 was considered to be significant.
Consent for authorship changes Author Contribution Statement Supplementary figures Supplementary Materials and methods Supplementary Table 1 Supplementary Table 2 Original Data File | true | true | true |
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PMC9633872 | Zhidan Qi,Shen Wang,Ang Xuan,Xinyi Gu,Jin Deng,Chen Huang,Lei Zhang,Xiaofeng Yin | MiR-142a-3p: A novel ACh receptor transcriptional regulator in association with peripheral nerve injury | 15-10-2022 | MT: Non-coding RNAs,miR-142a-3p,Pgc-1α,acetylcholine receptor,neuromuscular junction,peripheral nerve injury | Long-term denervation leads to the disintegration of nicotinic acetylcholine receptor (nAChR) located at the endplate structure, which translates to deficits in functional activation despite nerve repair. Because of a lack of effective measures to protect AChR expression, we explored the effect of alterations in muscular miR-142a-3p on nAChR. In this study, we constructed a model of miR-142a-3p knockdown by transfecting a miR-142a-3p inhibitor short hairpin RNA (shRNA) into C2C12 myotubes, and we injected this miR-142a-3p inhibitor shRNA into the tibialis anterior (TA) muscle in uninjured mice and in denervated mice by transecting the sciatic nerve. Our results showed that miR-142a-3p knockdown led to an increased number and area of AChR clusters in myotubes in vitro and larger neuromuscular endplates in adult mice. Furthermore, miR-142a-3p knockdown delayed the disintegration of motor endplates after denervation. Last, upon miR-142a-3p knockdown in uninjured and denervated mice, we observed an increase in the mRNA levels of five AChR subunits as well as mRNAs of genes implicated in AChR transcription and AChR clustering. Together, these results suggest that miR-142a-3p may be a potential target for therapeutic intervention to prevent motor endplate degradation following peripheral nerve injury. | MiR-142a-3p: A novel ACh receptor transcriptional regulator in association with peripheral nerve injury
Long-term denervation leads to the disintegration of nicotinic acetylcholine receptor (nAChR) located at the endplate structure, which translates to deficits in functional activation despite nerve repair. Because of a lack of effective measures to protect AChR expression, we explored the effect of alterations in muscular miR-142a-3p on nAChR. In this study, we constructed a model of miR-142a-3p knockdown by transfecting a miR-142a-3p inhibitor short hairpin RNA (shRNA) into C2C12 myotubes, and we injected this miR-142a-3p inhibitor shRNA into the tibialis anterior (TA) muscle in uninjured mice and in denervated mice by transecting the sciatic nerve. Our results showed that miR-142a-3p knockdown led to an increased number and area of AChR clusters in myotubes in vitro and larger neuromuscular endplates in adult mice. Furthermore, miR-142a-3p knockdown delayed the disintegration of motor endplates after denervation. Last, upon miR-142a-3p knockdown in uninjured and denervated mice, we observed an increase in the mRNA levels of five AChR subunits as well as mRNAs of genes implicated in AChR transcription and AChR clustering. Together, these results suggest that miR-142a-3p may be a potential target for therapeutic intervention to prevent motor endplate degradation following peripheral nerve injury.
Motor endplates (MEPs) are the anatomical and functional link that mediates the cross-talk between motor neurons and skeletal muscle fibers. As neuromuscular junctions mature, MEPs become perforated and complex, resembling pretzels with arrays or branches that are innervated by one axon per neuromuscular junction. Structural and functional MEP defects have been implicated in a number of neuromuscular diseases, of which the most frequent is peripheral nerve injury. Peripheral nerve injury is a growing topic of interest, as it represents a common disability around the world. Our previous study showed that denervation led to a marked decrease in the volume of MEP fragments available for reinnervation and that MEP size was gradually reduced with a longer delay in the repair time. Despite tremendous progress in surgery to improve clinical outcomes, nerve repair results in satisfactory motor recovery in only 51.6% of patients. This incomplete restoration is due to the failure of regenerating nerves to establish a proper muscle-nerve interface because of MEP disintegration. These changes in MEPs may be due to acetylcholine receptor (AChR) dispersion after skeletal muscle denervation, which results in the decline of mature pretzel-shaped MEPs. A previous study showed that AChR aggregation induced by matrix metalloproteinase 3 (MMP3) deletion contributed to preventing MEP degradation and improving subsequent reinnervation. As the core protein at the MEP, AChR was analyzed in previous studies mainly on the basis of the general principles of AChR clustering, maturation, and stability. Although the regulation of acetylcholine receptor transcription is only beginning to be understood, it is a promising intervention for preventing MEP degradation and thereby contributing to dying-back motor neuronopathy, similar to the fused in sarcoma (Fus) and peroxisome proliferator-activated receptor gamma coactivator 1-α (Pgc-1α) transcriptional regulation.11, 12, 13 In our previous study, we constructed mouse models of sciatic nerve injury and analyzed the whole transcriptome involved in denervated gastrocnemius muscle. We performed a careful bioinformatics analysis of sequencing datasets and found that miR-142a-3p was consistently highly upregulated within 2 months after denervation. To date, extensive studies have demonstrated that miR-142 is a major regulator of cell fate decisions in the hematopoietic system and plays pleiotropic roles in embryonic development, cancer,17, 18, 19 viral infection, inflammation and immune tolerance.,Recent evidence has shown that miR-142a-3p can influence mitochondrial morphology and reduce lipid use, which indicates that miR-142a-3p might play important roles in skeletal muscle. This evidence suggests that miR-142a-3p may have a major effect on nicotinic AChR (nAChR) defects following peripheral nerve injury. Here, we constructed a model of miR-142a-3p knockdown by transfecting a miR-142a-3p inhibitor short hairpin RNA (shRNA) into C2C12 myotubes and injected this miR-142a-3p inhibitor shRNA into the tibialis anterior (TA) muscle to investigate the effect of muscular miR-142a-3p on AChR expression and AChR cluster formation in vitro and in vivo, and we explored whether miR-142a-3p knockdown could delay MEP degradation following denervation.
We performed a careful bioinformatics analysis of microRNA (miRNA) sequencing datasets of denervated gastrocnemius muscles in mice to look for differentially expressed miRNAs, which were obtained from our published literature. Gastrocnemius samples of denervation injury were obtained at 0 (control group), 1, 2, 4, and 8 weeks after injury. Differential miRNA expression between the control groups and denervated groups was present, with a log2 fold change (FC) > 2 after injury at 0 (control group), 1, 2, 4, and 8 weeks (Figure S1). After peripheral nerve injury, compared with 0 weeks, there was more than a 3-fold increase in miRNA expression levels from 1 to 8 weeks in the denervation injury group. In addition, Pgc-1α and Sorbs2 were significantly downregulated in denervated muscle (Figure S2). Both genes were predicted as target genes of miR-142a-3p on the basis of the target gene prediction software miRanda (http://cbio.mskcc.org/miRNA2003/miranda.html) and were also proven to regulate the transcription of a broad neuromuscular junction (NMJ) gene program and regulate AChR cluster formation.,
To explore the effect of miR-142a-3p knockdown on AChR clustering in C2C12 myotubes, C2C12 cells were transfected with either miR-142a-3p inhibitor shRNA or negative control (NC) shRNA using a lentiviral vector (Figure 1A). The knockdown efficiency is presented in Figure 1B. After differentiation for 7 days, NC-transfected myotubes showed only sporadic clustering of AChR on the cell membrane. However, miR-142a-3p inhibitor-transfected myotubes showed great increases in the number and area of AChR clusters (Figures 1C and 1D).
To construct a model of miR-142a-3p knockdown, healthy male Thy1-YFP-16 mice (weighing 22–25 g and aged 6–8 weeks) were randomly assigned to two experimental groups as follows: one group was injected with miR-142a-3p inhibitor short hairpin RNA into the tibialis anterior muscle, and the other group was injected with negative control inhibitor short hairpin RNA into the tibialis anterior muscle. For the uninjured groups, TA muscle tissue samples were harvested on day 7 after injection. We assessed whether miR-142a-3p knockdown in skeletal muscle induced morphological alterations in MEPs in vivo. Three-dimensional (3D) reconstruction showed that MEPs in the TA muscle were distributed in their original positions, as they appeared in the control group (shown in Figure 2A). MiR-142a-3p expression is shown in Figure 2B. Although the number of MEPs was not significantly changed, the volume of a single AChR-concentrated MEP was larger in the miR-142a-3p-knockdown mice than in the control mice (see Figures 2C and 2D). Confocal microscopy was used to visualize NMJs, and a representative confocal stack image is shown in Figure 2E. Consistent with the results above, in the TA, the endplate area was increased in miR-142a-3p-knockdown muscles by ∼21.5% compared with that in NC-treated muscles (Figure 2F). However, endplate innervation with clear colocalization did not differ between the miR-142a-3p-knockdown group and the control group (Figure S3A). MEPs always present a certain percentage of abnormalities at the postsynaptic levels. To quantify this change, we used a previously described scheme to characterize endplate morphology. Endplates were categorized as pretzel (mature with weblike pattern including multiple perforations) and fragmentation (immature and smaller size lacking perforations)., A representation of the morphology of AChRs encountered in muscle is shown in Figure S3B. The percentage of fragmentation was reduced in the miR-142a-3p-knockdown mice (see Figure 2G). In addition, we assessed the size of muscle fibers by measuring muscle mass and cross-sectional area, and no difference was found in these parameters between control and miR-142-3p-knockdown mice (see Figure 2H; Figures S3C and S3D). We next investigated whether miR-142a-3p expression in muscle could affect synaptic folds at the ultrastructural level using transmission electron microscopy (TEM), which are located at the interface between the motor neuron and the muscle and to a large extent governed the area of MEPs. Interestingly, the average length of these synaptic folds was increased in miR-142a-3p-knockdown muscles compared with NC-treated muscles (1014 ± 19.2 vs. 768.5 ± 19.3 nm, p < 0.001) (see Figure 2I; Figure S3E). To further explore the functional relationship in uninjured mice, rectangular pulses (0.9 mA, wave width 0.1 ms, and frequency 50 Hz) were delivered to induce muscular contraction, which was used to judge the responses of the TA to electrical stimulation of the sciatic nerve. However, we did not observe a significant difference (p < 0.05) in muscle strength between the miR-142-3p-knockdown animals and control animals (Figures S3F and S3G).
We assessed whether MEPs were maintained in vivo in the skeletal muscle of miR-142a-3p-knockdown mice following sciatic nerve transection (Figure 3A). MiR-142a-3p expression is shown in Figure 3B. Three-dimensional reconstruction of MEPs in the TA muscle revealed a greater number and larger volume of MEPs in the knockdown group than in the control group (volume: 6,501 ± 41.7 vs. 8,761.4 ± 93.1 μm3 [2 weeks], 4,364.9 ± 71.7 vs. 6,787.8 ± 323.6 μm3 [4 weeks], 3,255.5 ± 32.1 vs. 7,730.6 ± 578.3 μm3 [8 weeks]; number: 3,208.3 ± 73.9 vs. 3,018.3 ± 59 [2 weeks], 2,522.3 ± 56.1 vs. 2,762.3 ± 74.2 [4 weeks], 1,886 ± 42.5 vs. 2,542 ± 18.6 [8 weeks]) (Figures 3C and 3D). Endplates from control animals at several time points following denervation underwent progressive decreases in area and the percentage of pretzel-shaped MEPs (shown in Figure 3E). However, the decline in area was delayed significantly in miR-142a-3p-knockdown mice across the same time interval (Figure 3F). (area: 82.0% ± 2.5% vs. 107% ± 3.6% [2 weeks], 38% ± 0.7% vs. 93.9% ± 9.3% [4 weeks], 30.9% ± 3.5% vs. 99.8% ± 4.4% [8 weeks]). Following denervation, pretzel-shaped MEPs were also maintained in miR-142a-3p-knockdown animals (Figure 3G). A post hoc Bonferroni correction confirmed that the differences were significant at 3 time points. Measurements of muscle mass and cross-sectional area revealed that atrophy occurred at slower rates following denervation in miR-142a-3p-knockdown mice than in control mice at 2, 4, and 8 weeks postinjury (see Figure 3H; quantified in Figures 3I and 3J). Thus, miR-142a-3p knockdown delayed the rate of muscle atrophy.
The endplate size and number in miR-142a-3p-knockdown mice increased, which prompted us to investigate a possible role of miR-142a-3p in the regulation of Chrn gene expression. First, in uninjured mice, miR-142a-3p knockdown increased the mRNA and protein expression of each of the five Chrn genes in TA (Figures 4A–4C). In addition, some proteins are critical for concentrating AChRs to promote clustering, which anchors AChRs to the cortical cytoskeleton by interacting with cytoskeletal proteins or scaffold proteins, including Rapsyn, laminin α4 (Lama4), and laminin β2 (Lamb2). The mRNA expression of the three genes (Rapsyn, Lama4, and Lamb2) was also elevated compared with that in the control group (Figure 4D). To confirm the target gene of miR-142a-3p, luciferase assays were conducted, and a schematic diagram of the reporter containing either the wild-type 3′ UTR or the mutant 3′ UTR of Pgc-1α 3, Sorbs2, and mir-142a-3p is shown in Figure 4E. Treatment with the miR-142a-3p mimics significantly decreased the luciferase activity of the wild-type reporter of Pgc-1α in HEK-293 cells compared with treatment with the NC mimics (Figure 4F). Consistent with luciferase assays, in uninjured mice, miR-142a-3p knockdown upregulated the relative mRNA and protein expression of Pgc-1α in TA (Figures 4G–4I). However, Sorbs2, another important gene for regulating AChR clustering, was not directly targeted by miR-142a-3p, although the relative mRNA expression was upregulated in TA (Figure 4J). Importantly, the transcript levels of GA-binding protein A (Gabpa), which is the DNA-binding subunit and has been proven to bind to N-box elements to promote NMJ gene transcription by directly binding Pgc-1α, were also elevated (Figure 4K). Consistent with the results from uninjured mice, miR-142a-3p knockdown was still able to increase the mRNA expression of AChR-related genes in injured mice (Figures S4A–S4C).
The AChR is a transmembrane ligand-gated ion channel composed of five protein subunits whose clustering and maintenance at the postsynaptic endplate is a hallmark of the mammalian NMJ. The nAChR has been implicated as a potential molecular target for therapeutic intervention in a variety of neuromuscular diseases; for example, active AChR prevents the atrophy of denervated skeletal muscles and favors reinnervation. In this study, we found that miR-142a-3p knockdown could strongly upregulate the expression of five AChR subunits and simultaneously greatly relieve denervation-induced changes in skeletal muscle. MiR-142a-3p knockdown activated AChR clustering in C2C12 myotubes in vitro. We first transfected C2C12 cells with a miR-142a-3p inhibitor shRNA and found that the miR-142a-3p inhibitor shRNA and subsequent miR-142a-3p knockdown increased the number and average area of AChR clusters in myotubes in vitro. The receptors that were able to bind α-bungarotoxin (α-BTX) appeared as typical oval plaques, as previously reported, indicating proper morphology. The mature MEPs in vivo were pretzel shaped (mature with weblike patterns, including multiple perforations). However, the AChR clusters in myotubes in vitro lacked complex internal structures, which might be due to the absence of nerves, which is supported by the plaque-like shape of clusters that form in aneural muscles. Despite being immature, the number and area of these spots indicated the clustering ability of AChR. For example, a previous study showed that the average area of AChR clusters could increase up to 59% by applying electrical stimulation in vitro. In our study, compared with the control group, the average area of AChR clusters in the miR-142a-3p-knockdown group was doubled, which indicates a strong interaction to activate AChR clustering. Treatment with the miR-142a-3p inhibitor shRNA improved MEP morphology in uninjured mice in vivo. Treatment with the miR-142a-3p inhibitor shRNA increased the MEP area in the muscle of miR-142a-3p-knockdown uninjured mice by 21.5%, similar to the stimulus of resistance training, which has been reported to increase the endplate area by 16% without alterations in muscle fiber size. In addition, MEPs always present a certain percentage of abnormalities at the postsynaptic level in vivo. For example, the pretzel shape can be irregular or fragmented, and the percentage increases with age. Caloric restriction and exercise have been shown to decrease the percentage of fragmentation in aged mice to reverse these age-related synaptic changes. Thus, the improvement in MEP morphology induced by the miR-142a-3p inhibitor shRNA may indicate a better muscle condition. In addition, the number and distribution of MEPs were not significantly altered in vivo, which indicated that the newly synthesized AChR activated by miR-142a-3p knockdown entered the original AChR microclusters and then formed larger clusters. At the ultrastructural level, the increase in synaptic fold length indicates a greater structural postsynaptic surface, which can harbor more AChRs and show a larger size, triggered by muscle miR-142a-3p knockdown. However, there was no significant difference (p < 0.05) in muscle contractility between the miR-142a-3p-knockdown animals and control animals. Perhaps the intervention time was too short to promote the remodeling of the NMJ, including presynaptic modulation. Alternatively, miR-142a-3p in skeletal muscle does not influence the release of synaptic vesicles in presynaptic nerve terminals in vivo. Similarly, in a previous study, the volume of MEPs increased in Mmp3 null mutant mice, but electrophysiological recordings revealed no change in quantal content or miniature endplate potential (MEPP) frequency. We will perform further relevant experiments. MiR-142a-3p knockdown greatly relieved the degradation of denervated muscle in vivo, including endplate morphology and muscle mass. Our previous study showed that AChRs in MEPs stained with α-BTX were disintegrated and fragmented after denervation in vivo, especially with a significant decrease in the mean volume of a single MEP. The area and volume of MEPs in miR-142a-3p-knockdown mice remained broadly unchanged even after 8 weeks of denervation. Previous interventions that tried to delay the disintegration of denervated MEPs presented a positive effect on muscle function and health, such as the implantation of a flexible microelectrode array (MEA), a-calcitonin gene-related peptide (CGRP) treatment in vivo and Mmp3 deletion. In addition, we observed that treatment with the miR-142a-3p inhibitor shRNA led to strong maintenance of the percentage of pretzel-shaped MEPs for long-term denervation in vivo. Generally, following denervation, pretzel-shaped MEPs were diminished dramatically, which was indicative of endplate destabilization. The results above indicate that the MEPs of muscles injected with the miR-142a-3p inhibitor shRNA were more resistant to disassembly. Moreover, the rate of muscle atrophy was delayed following denervation in miR-142a-3p-knockdown mice. Given that neural repair following long-term denervation leads to improved functional endpoints when MEP stability is preserved, miR-142a-3p represents an important therapeutic target to relieve the degradation of denervated muscle. MiR-142a-3p knockdown enhanced the synthesis of five AChR subunits in uninjured and denervated mice in vivo. The genes encoding muscle nAChR include Chrna1, Chrnb1, Chrng, Chrnd, and Chrne, and arbitrary mutations in these genes have been shown to impair neurotransmission at the nerve endplate. Previous studies focused on the suppression of AChR degradation but not the stimulation of protein synthesis, such as the CGRP treatment described above. In a recent study, simultaneous reduced expression of the five Chrn genes in Fus-amyotrophic lateral sclerosis (ALS) model mice resulted in reduced endplate surface area and a loss of function. Interestingly, in our study above, the five Chrn genes were upregulated simultaneously, and MEP volume and area were increased. Thus, we speculate that simultaneous preservation of the five Chrn genes may result in an increase in MEP volume and area and better functional maintenance. To further determine how miR-142a-3p regulates AChR gene transcription, we constructed a luciferase reporter to demonstrate that Pgc-1α was directly targeted by miR-142a-3p in vitro. However, Sorbs2, another important gene for regulating AChR clustering, was not directly targeted by miR-142a-3p. Regarding the link between Pgc-1a and AChRs, a previous study found that when activated, Pgc-1α bound host cell factor (HCF) and the ets-related GA-binding protein transcription factor complex (Gabp α/β). PGC-1α enhanced the binding of Gabp to the N-box (the DNA-binding site for Gabp) by phosphorylation, an important sequence motif in the promoters of many NMJ synaptic genes. Thus, Pgc-1α elevated the expression of a broad neuromuscular junction gene program, including AChR α1, AChR δ, AChR ε and Rapsyn. In addition, Pgc-1α was also proven to protect skeletal muscle from atrophy. The expression of Gabpa, which has been demonstrated to mediate the transcriptional response of Chrn genes,39, 40, 41, 42 was also elevated by miR-142a-3p knockdown. Notably, a previous study showed that Pgc-1α can regulate only three subunits, AChR α1, AChR δ, and AChR ε, by binding to and coactivating Gabpa. However, miR-142a-3p knockdown enhanced the synthesis of five AChR subunits, AChR β1 and AChR γ, suggesting other mechanisms of AChR gene regulation induced by miR-142a-3p knockdown. The roles of Rapsyn and Laminins were reported as key genes in AChR concentration. Rapsyn binds tightly to AChR to the subsynaptic cytoskeleton to form a high-density network. In the laminin protein family, the loss of laminin α4 (Lama4) and laminin β2 (Lamb2) was shown to result in the disruption of the pretzel-shaped endplates., The knockdown of miR-142a-3p, however, affects the expression of Sorbs2, which is also required for AChR cluster formation. Upregulation of the expression of these genes above favors the maintenance of the pretzel shape. A schematic diagram of miR-142a-3p regulating AChR-related gene transcription is shown in Figure 5. There are several limitations in this study. We did not perform miR-142a-3p knockdown in the tibialis anterior muscle of Pgc-1α muscle-specific knockout mice, which demonstrated that AChR transcription is partly due to the regulation of Pgc-1α. In addition, some potential regulatory mechanism of miR-142a-3p should be further explored. For example, how miR-142a-3p regulates the gene expression of Sorbs2 is not clear. In addition, further studies may be required to explore the long-term effects of miR-142a-3p on NMJs in uninjured mice. Perhaps transgenic animals of miR-142a-3p knockdown could help elucidate the contribution of miR-142a-3p to adaptation of the NMJ. In summary, we found an important target that regulates a broad range of AChR transcription and thus led to an improvement in MEP morphology in many aspects in uninjured and denervated mice. To the best of our knowledge, this is the first time that a muscular microRNA has been directly implicated in AChR maintenance and stability. Furthermore, these findings also indicate a possible contribution of muscle miR-142a-3p to the treatment of other neuromuscular diseases with prominent NMJ pathology, including myasthenic syndromes, ALS and spinal muscular atrophy.
The data analysis presented refers to previously published data. Differentially expressed (DE) genes with corrected p values < 0.05 were considered statistically significant. Differentially expressed miRNAs whose log2 fold change was >2 after injury at each time point are displayed using Venn diagrams in Figure S1. Heatmaps were generated using MATLAB software. Then, the intersections of the colocalized or coexpressed target mRNAs with miR-142a-3p, on the basis of the target gene prediction software miRanda (http://cbio.mskcc.org/miRNA2003/miranda.html), were displayed in a Venn diagram (Figure S2). Seventy-one targets of miR-142a-3p were downregulated at all time points. A heatmap showing 14 downregulated mRNAs (log2 FC < −2) was generated using MATLAB software.
miRNA inhibitors and NC inhibitors were designed and synthesized by GeneChem (Shanghai, China) and were prepared as lentiviruses to transfect C2C12 cells and adenoviruses to transfect TA muscle cells. The C2C12 myoblast cell line was procured from American Type Culture Collection (ATCC). To obtain miR-142a-3p-knockdown myotubes, C2C12 myoblast cells were transfected with miRNA inhibitor and NC inhibitor plasmids in accordance with the manufacturer’s instructions. C2C12 myoblast cells were seeded into a 24-well plate containing the appropriate growth medium at 1 × 105 to 5 × 105 cells per well, and the cells were transfected when the cell density reached 30%–50%. The cells were cultured in a culture plate in a 5% CO2 incubator at 37°C for 72 h and observed with a fluorescence microscope. When the transfection rate was above 80%, the medium was discarded, and the cells were washed three times with PBS and then replaced with screening medium (containing a 3 μg/mL concentration of puromycin). After 4–10 h of screening, the transfection rate was detected, and the transfection rate was more than 80% for subsequent experiments. To induce C2C12 myoblasts to differentiate into myotube cells, when the cells were grown to 60%–70% confluence, the proliferation medium was discarded, and the cells were rinsed gently 3 times with Hank’s balanced salt solution (HBSS) and then cultivated with differentiation medium (DMEM high-glucose medium supplemented with 2% horse serum and 1% penicillin/streptomycin). The solution was changed every 2 days for 7 days, and the process of myotube formation was observed with an inverted microscope. During myotube differentiation, the same number of C2C12 myoblasts were converted to thick and spindle shapes, which was regarded as mature differentiation. AAV is a common family of viral vectors with hundreds of capsids that are designed for different gene therapy applications. Among these, AAV serotype 9 (AAV9) is particularly attractive for gene therapy because it can lead to robust and body-wide muscle transduction in animal models. For in vivo gene knockdown studies, 1.0 × 1011 IU/mL adenovirus particles 9 composed of miR-142a-3p inhibitor (AAV9-miR-142a-3p inhibitor) or NC inhibitor (AAV9-NC inhibitor) shRNA were injected into the mouse right TA muscle. The injection was performed by multiple-point injection at four different points in the upper and lower muscle with 5 μL per injection. In each animal, we injected 20 μL virus into the right TA muscle.
Healthy male Thy1-YFP-16 mice (weighing 22–25 g and aged 6–8 weeks) were obtained from The Jackson Laboratory (Bar Harbor, ME) and kept in the Laboratory Animal Centre of Peking University (Beijing, China). The mice were assigned under a randomized allocation to two experimental groups as follows: one group was injected with miR-142a-3p inhibitor short hairpin RNA into the tibialis anterior muscle, and the other group was injected with negative control inhibitor short hairpin RNA into the tibialis anterior muscle. For the uninjured groups, TA muscle tissue samples were harvested on day 7 after injection. The wet weight of the TA muscle was used to assess muscle mass. There were thirty mice for the uninjured groups and all the mice survived in this study. None of the mice showed toe self-biting or ulcers in the operated limbs. Six mice were used to obtain 3D images of MEPs in intact TA. Six mice were used for muscle strength measurement. To reduce the number of animals used in experiments, the TA muscles of eighteen mice were used for qPCR measurements and imaging studies at the same time. The detailed numbers used for qPCR measurements and imaging studies were specified in the legend. For the injured groups, there were twenty-four mice for each time point at 2, 4, and 8 weeks after denervation. Six mice were used to obtain 3D images of MEPs in intact TA. The remaining mice were used for qPCR measurements and imaging studies. This study was carried out in accordance with the principles of the Basel Declaration and recommendations of Chinese guidelines for the care and use of laboratory animals. All experiments complied with the ARRIVE (Animal Research: Reporting of In Vivo Experiments) guidelines and were carried out in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals (NIH publication 8023, revised 1978). The protocol was approved by the Ethics Committee of the Peking University People’s Hospital (permit 2020PHE089).
Male Thy1-YFP-16 mice were anesthetized with isoflurane, and then the sciatic nerve and its main branches in the right limbs were exposed. The sciatic nerve was transected 5 mm proximal to the bifurcation with a 3-mm-long nerve defect. The proximal and distal stumps were ligated with 5-0 nylon sutures separately and stitched to the adjacent muscles in reverse to prevent neural growth. For the injured groups, tissue samples were harvested at 2, 4, and 8 weeks after transection. The wet weight of the TA muscle was used to assess muscular atrophy.
AChR aggregates were visualized by incubating myotubes with a solution containing 1.0 μg/mL α-BTX-Alexa Fluor 647 in DMEM for 1 h at 37°C with 7.5% CO2. Cells were rinsed twice with warmed PBS and fixed immediately in a 4% paraformaldehyde (PFA)-PBS solution. After 30 min at room temperature in fixative, the cells were rinsed with PBS, and the cell nuclei were stained using DAPI for 10 min.
Alexa Fluor 647-conjugated α-BTX (α-BTX 647) (Invitrogen, Carlsbad, CA) was injected via the tail vein to label MEPs. After injecting fluorescent α-BTX (0.3 μg/g) via the tail vein with a 2 h conjugation time, the TA muscle was dissected. The TA muscle was removed and fixed in 4% PFA in PBS, stored at 4°C overnight, and washed in PBS 3 times the next day. Thereafter, the TA muscle was dehydrated at 4°C in 20% sucrose solution, dehydrated for 1 day in the dark and dehydrated in 30% sucrose solution for 2 days. The TA muscle tissues were embedded in OCT. For the quantification of endplate morphology and innervation status, tissues were sliced into 50 μm sections using a vibratome (VT1000 S; Leica, Wetzlar, Germany) and imaged at 25× magnification on a Zeiss LSM700 confocal microscope. Fiji software was used to obtain maximum intensity projections of a limited number of confocal sections to generate images of a select number of individual, nonoverlapping endplates. We analyzed more than 100 endplates per muscle. The surface area of each of these endplates was determined using the “freehand selection” and “analyze particles” tools in Fiji, and the average area of MEPs was calculated. The morphology of each of these endplates was determined and described as pretzel-shaped and fragmented. The pretzel was mature with a weblike pattern, including multiple perforations, and the fragments were immature and smaller in size, lacking perforations. To quantify innervation status, composite images of the red and green channels were generated. Endplates that displayed at least three green-positive dots were scored as innervated, and the percentage of innervated endplates per muscle was calculated and used for statistical analysis. For the quantification of endplate volume and the total number of endplates, the dissected and postfixed TA muscles were processed using a typical clearing method called 3DISCO, as described in our previous study. First, tetrahydrofuran (Sinopharm Chemical Reagent, Shanghai, China) and dibenzyl ether (Sigma-Aldrich, St. Louis, MO) were preprocessed with basic activated aluminum oxide (Sigma-Aldrich, St. Louis, MO) to remove the residual peroxides. Then, the fixed muscle samples were incubated with 50%, 70%, 80%, and 100% tetrahydrofuran for dehydration, each for 2–3 h (30 min for muscle slices) in glass vials while gently shaking. After that, the samples were placed into dibenzyl ether until they became completely transparent. Then, an UltraMicroscope I (LaVision BioTec, Bielefeld, Germany) equipped with a 2×/0.5 objective (dry, working distance 20 mm) was used to image the cleared intact muscles. This instrument could create a thin light sheet to illuminate the cleared biological samples while imaging perpendicular to the light sheet. For AChR labeled with α-BTX, 647 nm was applied as the excitation wavelength. The z step size was set to 5 μm. After setting the imaging parameters appropriately, images of the samples were acquired for subsequent processing and analysis. For both groups (uninjured mice and denervated mice), n = 3.
The TA muscles were dissected and postfixed in 2.5% glutaraldehyde and 2.5% PFA in PBS at 4°C overnight. Samples were embedded in Epon 812. Ultrathin sections were cut at 70 nm, contrasted with uranyl acetate and lead citrate and examined at 120 kV using an electron microscope (JEM 1400plus; JEOL). Synaptic fold lengths were determined for individual NMJs using ImageJ software (National Institutes of Health, Bethesda, MD), and lengths were measured cursively from the edge of the synapse to the end of each fold.
TA muscles from 0, 2, 4, and 8 weeks following denervation were fixed in 4% formalin and paraffin embedded. Muscles were sliced into transverse sections (20 μm) using a cryostat, and muscle sections were stained with H&E. One hundred fifty fibers per muscle were then analyzed for cross-sectional area using ImageJ software. For the NC inhibitor group, n = 3; for the miR-142a-3p inhibitor group at 2 weeks, n = 5; for both groups at 4 weeks, n = 4; and for both groups at 8 weeks, n = 5.
Plasmid transfections for luciferase assays in 293T cells were performed with 0.1 μg wild-type or mutant 3′ UTR luciferase reporter of Pgc-1α and Sorbs2 and then transfected with 0.8 μg miR-142a-3p mimics plasmid in a 24-well plate using Nanofectin transfection reagent (PAA, Cölbe, Germany) as described by the manufacturer. The 293T cells were divided into (1) WT-3′ UTR-PGC-1α + miR-142a-3p mimics, (2) MUT-3′ UTR-Pgc-1α + miR-142a-3p mimics, (3) WT-3′ UTR-Sorbs2 + miR-142a-3p mimics, and (4) MUT-3′ UTR-Sorbs2 + miR-142a-3p mimics. Luciferase activity was measured 48 h posttransfection using the Dual Luciferase Reporter Assay System (Promega) as recommended by the manufacturer.
Total RNA was extracted from C2C12 myotubes and TA muscles with TRIzol. Template DNA was removed by treatment with DnaseI. RNA purity was checked using a NanoPhotometer spectrophotometer (IMPLEN). Only when the OD260/OD280 of RNA was between 1.8 and 2.0 was the RNA used for subsequent reverse transcription (RT) using a 5X All-In-One RT MasterMix kit (G492; abm). Stem-loop RT primers were used for RT of miRNAs. All qRT-PCR data were analyzed using the Livak method, where ΔΔCt values were calculated and reported as relative quantification (RQ) values, which were calculated using the 2-ΔΔCt method. Primers for RT-PCR are listed in Table S1.
C2C12 myotubes and TA muscle tissues were homogenized in RIPA buffer containing 1 mM phenylmethylsulfonyl fluoride (PMSF) and Protease Inhibitor Cocktail (Roche). Lysates were centrifuged for 20 min at 12,000 × g (4°C). Supernatants were transferred to a separate tube, and the bicinchoninic acid (BCA) assay (Beyotime) was used for protein-level quantification. Proteins were separated using SDS-PAGE (Beyotime), transferred to polyvinylidene difluoride (PVDF) membranes (Millipore), and blocked for 1 h with 5% nonfat dry milk in Tris-buffered saline (TBS) at room temperature. Primary antibodies against the following targets were incubated overnight at 4°C: rabbit polyclonal to PGC1 alpha-N-terminal (1:1,000; Abcam), rabbit anti-CHRNA1 (α1) (1:1,000; Proteintech), rabbit anti-CHRNB1 (β1) (1:1,000; Proteintech), mouse anti-CHRND (δ) (1:5,000; Novus Biologicals), mouse anti-AChRε (1:1,000; Santa Cruz Biotechnology), and rabbit anti-CHRNG (γ) (1:1,000; MyBioSource). After 3 washes, the blots were incubated with the appropriate secondary antibodies (Abcam) at room temperature for 1 h. Enhanced chemiluminescence (ECL) detection reagent and X-ray film were used for protein detection.
Mice were deeply anesthetized with isoflurane for surgical procedures and then placed on the operating table in the right lateral position. After preoperative skin preparation and disinfection with 0.5% iodophor in the right lower limb, an L-shaped incision was made at the anterolateral site to expose the sciatic nerve and the tibialis anterior muscle. The PCLAB-UE biomedical signal acquisition and processing system (MicroStar Science and Technology Development, Beijing, China) was applied for muscle strength measurement with its supporting PZ-1 tension sensor (50 g measuring range). “Continuous contraction” and “0–50 g” were chosen as the measurement type and range. The image recording parameters were set as 25.00 mV/div and 2.000 s/div. First, a zero setting was performed with 50 g of balancing weight. With the immobilization of the tibial plateau, the tibialis anterior tendon was dissociated with knitting sutures and connected parallel to the tension sensor. Then, the MedlecSynergy electrophysiological device was used as the stimulation producer, and the stimulating signal was set as square waves with 0.9 mA, a wave width of 0.1 ms, and a frequency of 50 Hz. The sciatic nerve was given continuous stimuli to record the waveforms of muscular contraction. Finally, we analyzed every waveform of muscular contraction and measured its maximal muscle strength to calculate the difference between the two groups.
Data are presented as mean ± standard error of the mean (SEM). One-way analysis of variance with Bonferroni post hoc comparison was performed unless otherwise indicated. Statistical significance is reported as p < 0.05. | true | true | true |
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PMC9633926 | 36316110 | Pia Pužar Dominkuš,Aner Mesic,Petra Hudler | PLK2 Single Nucleotide Variant in Gastric Cancer Patients Affects miR-23b-5p Binding | 26-09-2022 | Gastric cancer,Genetic variation,Chromosomal instability,Cell cycle genes,MiR-23b-5p | Purpose Chromosomal instability is a hallmark of gastric cancer (GC). It can be driven by single nucleotide variants (SNVs) in cell cycle genes. We investigated the associations between SNVs in candidate genes, PLK2, PLK3, and ATM, and GC risk and clinicopathological features. Materials and Methods The genotyping study included 542 patients with GC and healthy controls. Generalized linear models were used for the risk and clinicopathological association analyses. Survival analysis was performed using the Kaplan-Meier method. The binding of candidate miRs was analyzed using a luciferase reporter assay. Results The PLK2 Crs15009-Crs963615 haplotype was under-represented in the GC group compared to that in the control group (Pcorr=0.050). Male patients with the PLK2 rs963615 CT genotype had a lower risk of GC, whereas female patients had a higher risk (P=0.023; P=0.026). The PLK2 rs963615 CT genotype was associated with the absence of vascular invasion (P=0.012). The PLK3 rs12404160 AA genotype was associated with a higher risk of GC in the male population (P=0.015). The ATM Trs228589-Ars189037-Grs4585 haplotype was associated with a higher risk of GC (P<0.001). The ATM rs228589, rs189037, and rs4585 genotypes TA+AA, AG+GG, and TG+GG were associated with the absence of perineural invasion (P=0.034). In vitro analysis showed that the cancer-associated miR-23b-5p mimic specifically bound to the PLK2 rs15009 G allele (P=0.0097). Moreover, low miR-23b expression predicted longer 10-year survival (P=0.0066) in patients with GC. Conclusions PLK2, PLK3, and ATM SNVs could potentially be helpful for the prediction of GC risk and clinicopathological features. PLK2 rs15009 affects the binding of miR-23b-5p. MiR-23b-5p expression status could serve as a prognostic marker for survival in patients with GC. | PLK2 Single Nucleotide Variant in Gastric Cancer Patients Affects miR-23b-5p Binding
Chromosomal instability is a hallmark of gastric cancer (GC). It can be driven by single nucleotide variants (SNVs) in cell cycle genes. We investigated the associations between SNVs in candidate genes, PLK2, PLK3, and ATM, and GC risk and clinicopathological features.
The genotyping study included 542 patients with GC and healthy controls. Generalized linear models were used for the risk and clinicopathological association analyses. Survival analysis was performed using the Kaplan-Meier method. The binding of candidate miRs was analyzed using a luciferase reporter assay.
The PLK2 Crs15009-Crs963615 haplotype was under-represented in the GC group compared to that in the control group (Pcorr=0.050). Male patients with the PLK2 rs963615 CT genotype had a lower risk of GC, whereas female patients had a higher risk (P=0.023; P=0.026). The PLK2 rs963615 CT genotype was associated with the absence of vascular invasion (P=0.012). The PLK3 rs12404160 AA genotype was associated with a higher risk of GC in the male population (P=0.015). The ATM Trs228589-Ars189037-Grs4585 haplotype was associated with a higher risk of GC (P<0.001). The ATM rs228589, rs189037, and rs4585 genotypes TA+AA, AG+GG, and TG+GG were associated with the absence of perineural invasion (P=0.034). In vitro analysis showed that the cancer-associated miR-23b-5p mimic specifically bound to the PLK2 rs15009 G allele (P=0.0097). Moreover, low miR-23b expression predicted longer 10-year survival (P=0.0066) in patients with GC.
PLK2, PLK3, and ATM SNVs could potentially be helpful for the prediction of GC risk and clinicopathological features. PLK2 rs15009 affects the binding of miR-23b-5p. MiR-23b-5p expression status could serve as a prognostic marker for survival in patients with GC.
Gastric cancer (GC) is the fourth most common cause of cancer-related death worldwide [1]. GC is a complex, multifactorial disease influenced by intricate interactions between environmental and genetic factors [2]. In a large study conducted by The Cancer Genome Atlas (TCGA) involving 295 patients with gastric adenocarcinoma, approximately 50% of tumors were characterized by chromosomal instability [3]. It is believed that this form of genomic instability leads to long-term accumulation of genomic changes, which eventually results in the transformation of normal cells into malignant cells [4]. Recent studies have suggested that chromosomal instability can be driven by low-penetrating changes, such as single nucleotide variants (SNVs) in cell cycle and DNA repair genes [56], as these are extremely polymorphic [7]. Cyclins and cyclin-dependent kinases, Aurora kinases, checkpoint kinases, and other kinases, such as polo-like kinases (PLK) and ataxia telangiectasia mutated protein (ATM), are essential for cell cycle progression and response to stress. PLK2 is involved in centriole duplication and the G1/S phase transition, whereas PLK3 is required for entry into the S phase and cytokinesis [89]. Both kinases appear to act as tumor suppressors. PLK2 is transcriptionally silenced through promoter methylation in hematologic B-cell malignancies [10], acute myeloid leukemia [11], and hepatocellular carcinoma [12]. PLK3 has been shown to be downregulated in B cells during Helicobacter pylori infection, a well-known GC risk factor [13]. PLK3 mRNA is undetectable or significantly downregulated compared to paired normal tissue in lung carcinomas [14], head and neck carcinoma [15], and carcinogen-induced rat colon tumors [16]. ATM is involved in cellular response to DNA damage, cell cycle regulation, chromatin remodelling, and apoptosis [1718]. Germline mutations in ATM result in ataxia telangiectasia syndrome, which manifests as a lifetime increased cancer risk [19]. A genome-wide association study performed on a European population demonstrated the association between loss-of-function SNVs in ATM and GC and showed that cancer occurs at a significantly earlier age in those carrying these variants than in non-carriers [20]. Low penetrating SNVs in these genes in combination with environmental factors could be crucial biomarkers to aid in disease prevention and early intervention strategies. Our aim was to assess the association of candidate SNVs in cell cycle genes, PLK2, PLK3, and ATM, with GC risk and clinicopathological features of the patients in a case-control study. Relevant associations were further evaluated using in silico analysis and in vitro luciferase reporter assay.
A total of 221 patients with gastric adenocarcinoma and 321 healthy individuals were enrolled in this retrospective, case-control study. The diagnosis was confirmed by histological examination of the tissues removed during surgery. The patients underwent surgery at the Department of Abdominal Surgery and the Department of Thoracic Surgery at the University Medical Center Ljubljana, Jesenice Hospital, Hospital Dr. Petra Držaja, and the Ljubljana Institute of Oncology. The controls were matched by ethnicity, free from any personal history of GC or other malignant neoplasms, and unrelated to the patients and to each other. Tissue and blood samples were frozen at −80°C until use.
All procedures followed were in accordance with the ethical standards of the responsible committee on human experimentation (institutional and national) and the Helsinki Declaration of 1964 and later versions. This study was approved by the Republic of Slovenia National Medical Ethics Committee (No. 0120-59/2019/3). This was a retrospective study.
Genomic DNA was isolated from adjacent non-tumor gastric and tumor tissue samples from patients with GC and peripheral blood samples of controls using the Wizard Genomic DNA Purification Kit (Promega Corporation, Madison, WI, USA) following the manufacturer’s instructions. The concentration and purity of isolated DNA were determined spectrophotometrically using a Synergy H4 Hybrid Microplate Reader (BioTek Instruments, Inc., Winooski, VT, USA).
Seven SNVs in cell cycle genes were selected for genotyping: rs963615 and rs15009 in PLK2; rs17883304 and rs1204160 in PLK3; and rs228589, rs189037, and rs4585 in ATM. The SNVs were selected according to the following criteria: a) previous reports of gene associations with GC, b) minor allele frequency (MAF) of ≥10% in Utah residents with Northern and Western European ancestry (CEU) population according to the 1000 Genomes Project Phase 3 project, and c) in silico analysis of functional annotations in the selected candidate regions using the publicly available single nucleotide polymorphism selection tool, SNPinfo (http://www.niehs.nih.gov/snpinfo) [21]. The SNVs were genotyped using the following TaqMan allelic discrimination assays, which were supplied as predesigned SNV genotyping assays: C_8962866_10 (rs963615), C_2839927_1_ (rs15009), C_63720915_10 (rs17883304), C_159308_10 (rs12404160), C_2283144_1_ (rs228589), C_2283145_10 (rs189037), C_1039793_20 (rs4585) (Applied Biosystems, Foster City, CA, USA). Genotyping was performed in 5-µL reaction mixtures containing 20x SNV genotyping assay, 2x Taq-Man Genotyping Master Mix (Applied Biosystems), and 10 ng of genomic DNA. The polymerase chain reaction (PCR) was performed according to the manufacturer’s protocol using ViiATM 7 Real-Time PCR System and QuantStudioTM Real-Time PCR Software (Applied Biosystems) with the following cycling conditions: 60°C for 30 seconds, 95°C for 10 minutes followed by 45 cycles at 95°C for 15 seconds, 60°C for 1 minute, and 60°C for 30 seconds. Random samples were selected and re-genotyped to confirm the consistency of obtained genotypes.
For SNVs in ATM, PLK2, and PLK3, raw genotyping data for genetic variants, rs228589, rs189037, and rs4585 in ATM; rs15009 and rs963615 in PLK2; and rs17883304 and rs12404160 in PLK3, were extracted. to perform haplotype analysis and generate the haplotype block structure, which entailed corrections for multiple comparisons by 10000 permutations, Haploview software version 4.2. [22] and SNP tools V1.80 (MS Windows, Microsoft Excel) [23] were used. In this regard, the solid spine of the linkage disequilibrium (LD) algorithm with a minimum Lewontin D′ value of 0.8 was chosen. To correct the occurrence of type I errors (false positive results), a permutation procedure was performed using Haploview (10000 permutations). This approach enables correction for multiple testing, but also considers the correlation between markers. Hence, permutation correction is less conservative than Bonferroni correction; however, it is suitable for independent tests with multiple markers [24].
The functional effects of intron and UTR SNVs were evaluated using the following publicly available bioinformatics tools: a) PROMO software within the ALGGEN (Algorithmics and Genetics Group) web server [2526] for the search for putative transcription factor motifs. We built the search using the following parameters: human species, all motifs, and all factors; b) MirSNP database for the prediction of miR-binding sites affected by SNVs [27]. The miRANDA algorithm used in the MirSNP database uses criteria where the seed region contains at least seven nt long. However, we additionally excluded miRs that could potentially bind to one allele with a 6 nt seed region, as this possibility has been observed and described previously in the literature [28]. For PLK2, which lies on the reverse strand, all alleles are reported in the forward orientation. Gene orientation was considered for in silico functional analysis. Sequences for in silico analysis of the studied SNVs in FASTA format were extracted from Ensembl Release 99 (http://www.ensembl.org) [29].
The PLK2 3’ UTR region with the wild-type (Wt) or polymorphic allele (Var) was cloned into the dual luciferase reporter vector pmirGLO (Promega Corporation). GC cells, MKN45, obtained from the Japanese Collection of Research Bio Resources Cell Bank (JCRB, Osaka, Japan) were transfected with miR-23a-5p, miR-23b-5p, or negative control mimics (Dharmacon, Lafayette, CO, USA) and pmirGLO-PLK2-3’UTR Wt, pmirGLO-PLK2-3’UTR Var, or pmirGLO as negative controls, using GenMute siRNA Transfection Reagent (SignaGen Laboratories, MD, USA) according to the manufacturer’s protocol. Luciferase activity was measured using the Dual Luciferase Reporter Assay (Promega Corporation) on the Synergy H4 Hybrid Microplate Reader (BioTek) luminometer, 48 hours after transfection. Experiments were performed in triplicate and replicated four times. Data are represented as the mean ± standard deviation and were compared using GraphPad Prism version 8.0.2 for Windows. Statistical significance was set at P<0.05.
The agreement of genotype frequencies with Hardy-Weinberg equilibrium (HWE) and the differences in genotype distribution between cases and controls were calculated using the chi-squared test. Minor genotype and allele frequencies for candidate SNVs were compared to the frequencies in populations from the 1000 Genome Project Phase 3 release. A generalized linear model was used to calculate odds ratios (ORs) and 95% confidence intervals (CIs) to examine the association of selected SNVs with the risk of GC and clinicopathological features. Dominant, recessive, and co-dominant genetic models were used for the analysis, depending on the genotype frequencies. All associations were calculated using R studio version 3.5.3 [30] and the SNPAssoc package for R [31]. When more associations were significant for one SNV, the genetic model with the lowest Akaike information criterion value was selected [32]. The associations between SNVs genotypes and overall patient survival were estimated using the Kaplan–Meier (KM) method and log-rank test using GraphPad Prism version 8.0.2 for Windows (GraphPad Software, San Diego, CA, USA, www.graphpad.com). Statistical significance was set at P<0.05. KM miR survival curves were generated using the KM Plotter online tool (https://kmplot.com) [33] and TCGA datasets (n=436 patients with GC). The TCGA dataset is available using the following link: https://portal.gdc.cancer.gov/. Differential expression of hsa-miR-23b-5p in tumors vs. normal or high vs. low-grade GC samples was extracted from the dbDEMC 3.0 database (https://www.biosino.org/dbDEMC/index) [34]. Expression datasets in dbDEMC are based on microarray or miRNA-seq platforms obtained from public repositories, including Gene Expression Omnibus (GEO), Sequence Read Archive, ArrayExpress, and TCGA.
A total of 221 patients with GC were enrolled in this study. Clinical and histological data were extracted from registered medical records and are presented in Table 1. Male patients accounted for 62.25%, and female patients accounted for 37.75% of the study cohort. Their mean age at diagnosis was 67.3 years. Data on histological type, according to Lauren's classification; location; grade of differentiation; and vascular, perineural, and lymphatic invasion were included in the study. SNVs from the three cell cycle genes, PLK2, PLK3, and ATM, are presented in Table 2. All selected SNVs were located in the non-coding regions of the genes. Genotype and allele frequencies in patients with GC and controls were comparable to those in populations from public databases (Supplementary Fig. 1). The distribution of genotype frequencies and HWE in patients with GC and controls for each studied SNV were analyzed using the chi-square test (Supplementary Table 1). Genotype frequencies did not deviate from the HWE (P>0.05).
We performed analyses based on generalized linear models to evaluate the association between candidate SNV genotypes and GC risk. Genotyping was performed on DNA isolated from non-tumor gastric tissues (Table 3). There were no significant associations between the cell cycle genes SNVs, PLK2 rs963615, rs15009, PLK3 rs17883304, and rs12404160, or ATM rs228589, rs189037, and rs4585 and GC risk. In the stratified analysis, there were significant associations between the PLK2 rs963615 CT genotype and lower GC risk in the male population according to the overdominant genetic model [(CC+TT):CT] (OR=0.59, 95% CI=0.37–0.93, P=0.023) and higher GC risk in the female population in the same genetic model [(CC+TT):CT] (OR = 2.03, 95% CI=1.09–3.80, P=0.026). A significant association was also found between the PLK3 rs12404160 AA genotype and a higher GC risk in the male population, according to the recessive genetic model [(GG+GA):AA] (OR=3.55, 95% CI=1.26–10.04, P=0.015).
For haplotype analysis, a single haplotype block was created that included either PLK2, PLK3, or ATM SNVs with an average Lewontin D' > 0.8 (D'=1.0 value shows the strongest LD between the two polymorphisms) (Fig. 1). Haplotype frequencies of the cases and controls are presented in Table 4. The frequency of the PLK2 C rs15009-C rs963615 haplotype was lower in the GC group than that in the control group, with borderline significance (Pcorr=0.050). This association was marginal; therefore, our results should be interpreted with caution and further validated in a larger cohort. The frequency of the PLK3 A rs17883304-T rs12404160 haplotype was significantly lower in the GC group than that in the control group (Pcorr=0.001). Additionally, the frequency of the ATM A rs228589-G rs189037-T rs4585 haplotype was significantly lower, and that of the T rs228589-A rs189037-G rs4585 haplotype was significantly higher in the GC group than in the control group (Pcorr<0.001).
Association analysis between SNVs genotypes and clinicopathological tumor features was performed on genotyping results from gastric tumor tissue DNA, given that variations in tumor tissue define the behavior and characteristics of the tumor. The categories with the most significant results are presented in Table 5. The non-significant results are shown in Supplementary Table 2. Genotype CT in PLK2 rs963615 was significantly associated with the absence of vascular invasion according to the overdominant genetic model [(CC + TT):CT)] (OR=0.38, 95% CI=0.18–0.81, P=0.012). Genotypes TA+AA, AG+GG, and TG+GG in ATM rs228589, rs189037, and rs4585, respectively, were significantly associated with the absence of perineural invasion (OR=0.39, 95% CI=0.16–0.95, P=0.034) according to the dominant genetic models [TT:(TA+AA)], [AA:(AG+GG)], and [TT:(TG+GG)], respectively.
The survival interval in our GC patient cohort ranged from 1.6–133.5 months (mean, 40.0 months). Survival analysis was performed to examine the effect of candidate SNVs on the prognosis of patients with GC (Fig. 2). Analysis was performed on SNVs where each genotype group comprised a minimum of five participants. No significant associations were found between candidate SNV genotypes and overall survival in patients with GC. However, a trend was observed for patients with the PLK2 rs15009 GG genotype, who had a 10-year survival rate of 63.5% in comparison to the patients with the CG or CC genotype (survival rate, 15.7% and 20%, respectively) (P=0.337, Fig. 2b).
Next, we performed in silico functional analysis of the candidate SNVs with statistically significant associations, including SNVs with observed trends in survival analysis. The 5’-UTR regions and introns typically contain enhancer and silencer regions and bind transcription factors to enhance or repress gene transcription [3536]. These motifs may also be present in the 3’ UTR [37]. Additionally, variations in the 3’ UTR may affect the binding of miRs and, therefore, the mRNA expression levels. The results of the disruption of transcription factor binding sites due to polymorphic sites and identification of miRs that differentially bind to these polymorphic sites are summarized in Table 6. In PLK2 rs963615, a TATA-binding protein (TBP) binding motif was created if the T allele was present. In PLK2 rs15009 Enkephalin transcription factor 1 (ENKTF-1) appeared to bind in the presence of the C allele, whereas Retinoid X receptor α:Retinoic acid receptor β (RXR-α:RAR-β), Multiprotein bridging factor-1 and vitamin D receptor binding motifs were created when the G allele was present. Moreover, putative binding sites for miR-23a-5p and miR-23b-5p were formed when the G allele of PLK2 rs15009 was present, in contrast with the C allele. In PLK3 rs12404160, a binding motif for Purine Rich Box-1 (PU.1) was created in the presence of the G allele. In ATM rs228589, differential binding motifs for transcription factors Yin yang 1 transcription factor (YY1) and polyoma enhancer activator 3 (PEA3) were formed in the presence of the A allele, whereas in the case of the T allele, the binding motifs for transcription factors glucocorticoid receptor α (GRα), signal transducer and activator of transcription-4, c-erythroblast transformation-specific transcription factor-1 (c-Ets-1), and erythroblast transformation-specific Like-1 protein (Elk-1) were generated. In ATM rs189037, a binding motif for retinoblastoma-associated protein-1 (E2f-1) is created in the presence of the G allele. Finally, in the presence of the T allele in ATM rs4585, binding sites for X-box binding protein-1 (XBP-1) and general transcription factor IID, as well as the binding site for miR-2964a-5p, were created.
Haplotype analysis results suggested that the PLK2 C rs15009-C rs963615 haplotype may have a marginal protective role in GC risk (Pcorr=0.050). We performed luciferase reporter assays to evaluate how the presence of different alleles (C/G) in the PLK2 3’-UTR region affects the binding of miR-23a-5p and miR-23b-5p, as both have been previously associated with GC [3839]. The relative luciferase activity of the pmirGLO-PLK2-3’UTR Var (G allele) was decreased by 12% when the cells were co-transfected with the miR-23a-5p mimic (P=0.085) and by 41% when they were co-transfected with the miR-23b-5p mimic (P=0.0097) compared to miR co-transfection in the negative control (Fig. 3B). The relative luciferase activity of the pmirGLO-PLK2-3’UTR Wt (C allele) was unaffected by co-transfection with both mimics (Fig. 3B). These results suggest that miR-23b-5p directly targets PLK2 when the G allele is present in PLK2 rs15009. We used the KM plotter online tool to analyze whether miR-23b-5p could have prognostic value in patients with GC (Fig. 3C). The analysis showed that low miR-23b-5p expression predicted longer 10-year survival in patients with GC (hazard ratio=1.52, P=0.0066). There was no significant correlation between miR-23a-5p expression and overall survival in GC patients using the KM plotter tool (P=0.304, data not shown). We also analyzed differential miR-23b-5p expression in the TCGA_STAD (The Cancer Genome Atlas Stomach Adenocarcinoma) dataset as well as available GEO datasets to evaluate miR-23b-5p expression in tumor vs. normal or high-grade vs. low-grade samples: the results are shown in Fig. 3D and E. MiR-23b-5p was downregulated (log FC=-1.26) in tumor sample compared to normal samples (TCGA_STAD dataset, Padj = 4.88×10-3) (Fig. 3D). Notably, in the GSE33743 dataset, miR-23b-5p was upregulated in tumors compared to normal samples (log FC=0.76, Padj=3.21×10-2). MiR-23b-5p expression was downregulated in high grade vs. low grade tumour samples from the TCGA_STAD dataset (Fig. 3E): in grade 2 vs. grade 1, log FC was −1.93 (Padj=2.98×10-16); in grade 3 vs. grade 1, log FC was −2.54 (Padj=2.52×10-24); in grade 4 vs. grade 1, log FC was −0.64 (Padj = 2.67 x10-2); in grade 4 vs. grade 2, log FC was −0.61 (Padj=4.02×10-3); and in grade 4 vs. grade 3, log FC was −1.90 (Padj=4.89×10-23). Mir-23b-5p expression was upregulated when comparing grade 3 vs. grade 2 TCGA_STAD datasets, where log FC was 1.29 (Padj=2.26×10-13) (Fig. 3E).
In this study, we investigated the associations between SNVs in cell cycle genes, PLK2, PLK3, and ATM, and GC risk and the clinicopathological features of patients with GC. Candidate SNVs with significant associations were further functionally analyzed to determine their potential regulatory effects on transcription factors and miRNA-binding sites. We also confirmed the putative PLK2 binding site for miR-23b-5p in vitro. GC is approximately twice as likely to be diagnosed in men than in women [1]. Incidence variation by sex has been attributed to sex-specific lifestyles and potential underlying hormonal mechanisms. The PLK2 rs963615 CT genotype could be used for selection of female persons at higher GC risk and as a determinant for lower GC risk in the male population. In the presence of the T allele, a binding motif for the transcription factor, TBP, was formed according to in silico functional analysis. The expression level of TBP has been confirmed to affect cell proliferation and transformation potential [40]. Additional sex-specific cellular factors may also contribute to TBP levels. Next, the PLK3 rs12404160 AA genotype could be used as a biomarker for the selection of male individuals at higher GC risk. In silico functional analysis implied a binding motif for the transcription factor, PU.1, in the presence of the G allele, suggesting that PU.1 may increase PLK3 transcriptional activity, which may result in tumor suppression. PU.1 has been previously identified as a tumor suppressor or oncogene in different types of leukemias, breast cancers [41], and gliomas [42]. PU.1 inhibition by small-molecule inhibitors or RNA interference decreases the tumor burden and increased the survival of patients with acute myeloid leukemia [43]. Further studies are necessary to confirm how PU.1 affects PLK3 transcription, how PU.1-associated actions differ in male and female patients, and whether it would be beneficial to inhibit or promote its activity in patients with GC. Haplotype analysis results suggest that the PLK2 C rs15009-C rs963615 haplotype may have a protective role against GC, although the significance was borderline. To our knowledge, PLK2 rs15009, which lies in the 3’ UTR region of the gene, has been studied only in association with the Reelin signaling pathway in Alzheimer’s disease, where the authors showed that CC and GG genotypes had a protective effect [44]. In silico analysis results for PLK2 rs15009 suggested that binding of RXR-α and RAR-β proteins in the presence of the G allele and binding of ELK-1 and ENKTF-1 in the presence of the C allele may alter the level of PLK2 expression. RXR-α and RAR-β are retinoic acid nuclear receptors that regulate apoptosis, cell cycle, and differentiation [45]. Lower RXR-α expression has been reported in patients with GC and is significantly associated with advanced disease stages [46]. RAR-β hypermethylation is predominantly associated with diffuse GC [44]. Notably, in this study, it was further established that RAR-β methylation status was statistically associated with invasion, differentiation, and location of the tumor in diffuse types, whereas these histopathological features were not associated with RAR-β methylation status in intestinal types of GC [47]. This indicates that different levels of DNA binding factors or their differential binding to polymorphic sites could profoundly affect their downstream pathways and, thus, influence the development of distinct tumor types. Similarly, it has been found that ELK-1 activity promotes cell migration and invasion and is involved in cancer development due to inflammation [4849]. Therefore, aberrant ELK-1 activity due to SNVs can affect PLK2 transcription and mRNA-associated processes. Using a luciferase reporter assay, we analyzed the binding of miR-23a-5p and miR-23b-5p to PLK2 rs15009, which was selected as the best potential binding candidate for in silico analysis. We confirmed that miR-23b-5p binds specifically to the PLK2 rs15009 G allele. It has been proposed that miR-23b plays a dichotomous role in cancer, either as a tumor suppressor or as an oncogene. MiR-23b expression is downregulated in human glioma, prostate, bladder, breast, and gastrointestinal cancers and has been shown to suppress tumor growth, invasion, angiogenesis, and metastasis and affect chemoresistance and tumor cell dormancy [505152535455]. In contrast, some studies have shown that the expression of miR-23b is upregulated, and that it may also function as an oncogene by promoting tumor growth, proliferation, and metastasis in prostate and breast cancer, as well as in GC [565758]. Analysis of paired tumor-normal samples from 160 gastric adenocarcinoma patients demonstrated that co-expression of miR-23a and miR-23b was significantly upregulated, particularly in specimens from patients at advanced stages (II-IV), and correlated with lymph node metastasis [38]. In addition, miR-23a/b enhanced tumor growth in a GC xenograft mouse model by inhibiting apoptosis of GC cells by directly targeting a tumor suppressor, programmed cell death 4 protein. The observed function of miR-23b as an oncomir is in line with our survival analysis results using the KM plotter on GC samples from the TCGA database. Lower miR-23b expression was associated with a higher 10-year survival rate. These results suggest that miR-23b-5p may serve as a prognostic factor for tumor progression and survival. Significantly shorter 5-year overall survival and disease-free survival were observed in patients with higher plasma miR-23b expression [59]. Notably, public dataset expression analysis showed that miR-23b-5p was downregulated in the TCGA_STAD cohort and upregulated in the GSE33743 cohort. It is worth noting that the first cohort included 436 tumor and 41 normal samples in the analysis, whereas the latter only analyzed 37 tumor and four normal samples. When comparing expression in high-vs. low-grade samples, miR-23b-5p was mostly downregulated, with the exception of the grade 3 vs. grade 2 comparison, where it was upregulated. These results suggest that miR-23b-5p may serve as a prognostic factor for tumor progression. Low miR-23b-5p expression appears to be associated with greater overall survival. Furthermore, comparison of its expression in high-and low-grade tumor tissues indicated that its expression could gradually decrease during tumor progression. This confirmed the dichotomous role of miR-23b, as discussed above. For better interpretation, analysis of samples grouped by sex, clinical and histological parameters, whether therapy is administered, and outcome, is necessary. Overall, the perplexing behavior of miRs confirms that in complex and heterogenic diseases such as GC, it is necessary to develop systems medicine approaches to decipher oncogenic mechanisms. Therefore, the protective role of the PLK2 C rs15009-C rs963615 haplotype in GC could be explained by PLK2 repression through miR-23b-5p binding to the G allele. To draw more definite conclusions from our study, we wanted to analyze a cohort in which the PLK2 rs15009 genotype, miR-23b-5p expression, and PLK2 mRNA expression would be known, possibly together with data on patient survival or response to therapy. Unfortunately, we were unable to obtain such datasets from publicly available databases. Nevertheless, we can theoretically consider two scenarios in which data on PLK2 rs15009 and miR-23b-5p expression levels would have clinical value. In the first scenario, individuals with the risk PLK2 rs15009 haplotype would also have overexpression of miR-23b-5p. This combination could result in the decreased expression and functional activity of PLK2 during the cell cycle. This would lead to tumor progression since PLK2 acts as a tumor suppressor [101112]. In the second scenario, individuals with the PLK2 risk haplotype, who have miR-23b-5p underexpression, would have PLK2 mRNA levels higher than usual. High PLK2 levels are associated with a protective role in cancer, but might also lead to chemoresistance in individuals receiving chemotherapy. Previous observations have shown that at higher expression levels, PLK2 significantly predicted a poorer outcome in patients with colorectal cancer by enhancing chemoresistance [60]. PLK2 has also been proposed to be an important determinant of chemotherapy sensitivity in ovarian cancer [61]. Its repression through miR-23b-5p in the presence of the rs15009 G allele could reduce chemoresistance. Therefore, depending on the disease grade and whether therapy is administered, PLK2 rs15009 could be useful as a biomarker of chemotherapy resistance prediction in GC in combination with miR-23b expression levels. This could be exploited in the future for the development of novel targeted PLK2 therapies using either miR or PLK2 inhibitors or miR mimics [62]. Interactions between other proteins and miRNAs should also be considered. The PLK2 rs963615 CT genotype was significantly associated with the absence of vascular invasion, whereas ATM rs228589, rs189037, and rs4585 were significantly associated with the absence of perineural invasion. These SNVs could be used in clinical settings to aid in prognosis and possibly in the determination of the most suitable treatment options for patients. The A allele of ATM rs228589 has been previously associated with a higher risk of chronic myeloid leukemia in the Indian population [63], whereas the T allele has been associated with a higher risk of breast cancer in the Jewish female population [64]. In a meta-analysis conducted by Zhao et al., the A allele in ATM rs189037 was significantly associated with breast, oral, and lung cancer risk in East Asian and Latino populations, but not in Caucasians [65]. Moreover, the AA genotype of rs189037 is significantly associated with a higher GC risk in the Chinese population [66]. It was also associated with higher TNM stage, overall tumor size, and survival prognosis. This is in concordance with our results, where ATM rs189037 AG + GG genotypes were associated with the absence of perineural invasion. ATM SNV rs4585 is associated with a lower risk of papillary thyroid carcinoma in the ATM haplotype, Crs373759-Grs664143-Trs4585 [67]. Notably, our results indicated that the ATM haplotype, Trs228589-Ars189037-Grs4585, was significantly more frequent, whereas Ars228589-Grs189037-Trs4585 was significantly reduced in patients with GC. The differences in associations between specific alleles and cancer risk may be due to the different ethnicities of the studied populations and different cancer types. In silico analysis resulted in several transcription factor candidates that may, through ATM activation or silencing, depending on specific allele binding, contribute to higher GC risk (haplotype analysis) or the absence of perineural invasion. Low ATM expression is generally associated with more aggressive tumors. In contrast, overexpression of ATM may lead to cisplatin resistance, resulting in a less favorable prognosis [68]. The A allele of ATM rs228589 forms a binding site for YY1 and PEA3; however, this binding motif is lost if an individual is a carrier of the T allele. YY1 expression is upregulated in GC cell lines and tissues, contributing to gastric carcinogenesis [69]. YY1 is also considered a potential therapeutic target [70]. PEA3 was upregulated in gastric adenocarcinoma samples, and together with the ERK signaling pathway, indicated poor survival prognosis [71]. In the presence of the G allele of ATM rs189037, a binding motif for E2f-1 is formed. E2f-1 has been previously associated with poor overall survival in GC [72]. However, E2f-1 overexpression in the MGC-803 GC cell line results in cell growth and proliferation inhibition, reduced invasion, and a higher apoptotic rate [73]. When the G allele is present, a binding site for STAT-4 and c-Ets-1 is formed. High STAT expression has been associated with better clinical outcomes in GC [74]. C-Ets-1 expression is correlated with H. pylori infection, a major risk factor for GC [75]. The T allele of ATM rs4585 participates in the formation of a binding motif for XBP-1. XBP-1 is involved in endoplasmic reticulum stress and unfolded protein response and it generally promotes cancer cell survival and tumor progression [76]. XBP-1 is a crucial factor that promotes tumor growth and invasion in GC while inhibiting apoptosis and autophagy [77]. Additionally, the hsa-miR-29641-5p binding site is formed in the presence of the T allele. Binding has been confirmed in vitro, and results in decreased ATM expression [78]. MiR-29641-5p has been characterized as an oncomiR in periampullary adenocarcinoma [79] and breast cancer [80]. Histological type, degree of differentiation, and molecular type of the tumor, as well as genetic background and cooperation between multiple miRs and miRNA-transcription factors may affect the final role of particular miR on gene expression. There are a few limitations to the power of SNV association studies. In complex diseases, the contribution of a particular SNV is usually modest. Study population size, allele frequencies, LD, and other parameters affect the power of the study [81]. We performed a candidate gene association study, which is a targeted approach compared to genome-wide association studies, where screening is untargeted and associations for marker SNVs are studied. Candidate gene association studies enhance the power of the study and are important when studying low-frequency SNVs or when the study population is small, as in our case. Our study included 221 patients with GC and 321 healthy controls. In small study populations, larger effects are easier to detect, whereas smaller effects might be missed, leading to false negatives. Another limiting factor is the minimum MAF, which is typically 0.05 [82]. To compensate for the small sample size, we selected SNPs with MAFs≥0.10. Enriching for more common alleles increases the power to detect associations. However, it is important to keep in mind that rarer SNVs may have larger effect sizes, and omitting them may be potentially counterproductive [83]. Moreover, we assessed the MAF in our study group and CEU to ensure that they were comparable. When performing the analyses of risk-associated haplotypes, we included adjustments for multiple comparisons to avoid false positives. In some cases, the sample sizes were small for association tests between SNVs and clinicopathological features. Unfortunately, clinical data were not available for all the patients. Additionally, some reported associations are marginal; therefore, our results should be interpreted with caution and further validated in a larger cohort. Our study indicates a possible role for PLK2, PLK3, and ATM SNVs in gastric tumorigenesis. We also show that PLK2 is targeted by miR-23b-5p in vitro and that low expression of miR-23b-5p in tumors is associated with better survival prognosis. Analyses of inter-individual genetic variability are of paramount importance in precision medicine, as they can significantly influence occurrence, course, and response to treatment. Low-penetrating variations in cell cycle genes may affect transcription factor and miR binding and can serve as biomarkers for tailored therapy selection, such as small-molecule or RNA inhibitors of transcription factors or oncomiR inhibitors. The current understanding of the molecular etiology and progression of GC is limited, and there is a critical need to explore novel genetic and molecular candidates that might contribute to the better management of this multifaceted disease. | true | true | true |
PMC9634078 | Marcella Martinelli,Caterina Mancarella,Luca Scapoli,Annalisa Palmieri,Paola De Sanctis,Cristina Ferrari,Michela Pasello,Cinzia Zucchini,Katia Scotlandi | Polymorphic variants of IGF2BP3 and SENCR have an impact on predisposition and/or progression of Ewing sarcoma | 21-10-2022 | Ewing sarcoma,polymorphisms,IGF2BP3,SENCR,cancer predisposition | Ewing sarcoma (EWS), the second most common malignant bone tumor in children and adolescents, occurs abruptly without clear evidence of tumor history or progression. Previous association studies have identified some inherited variants associated with the risk of developing EWS but a common picture of the germline susceptibility to this tumor remains largely unclear. Here, we examine the association between thirty single nucleotide polymorphisms (SNPs) of the IGF2BP3, a gene that codes for an oncofetal RNA-binding protein demonstrated to be important for EWS patient’s risk stratification, and five SNPs of SENCR, a long non-coding RNA shown to regulate IGF2BP3. An association between polymorphisms and EWS susceptibility was observed for three IGF2BP3 SNPs - rs112316332, rs13242065, rs12700421 - and for four SENCR SNPs - rs10893909, rs11221437, rs12420823, rs4526784 -. In addition, IGF2BP3 rs34033684 and SENCR rs10893909 variants increased the risk for female respect to male subgroup when carried together, while IGF2BP3 rs13242065 or rs76983703 variants reduced the probability of a disease later onset (> 14 years). Moreover, the absence of IGF2BP3 rs10488282 variant and the presence of rs199653 or rs35875486 variant were significantly associated with a worse survival in EWS patients with localized disease at diagnosis. Overall, our data provide the first evidence linking genetic variants of IGF2BP3 and its modulator SENCR to the risk of EWS development and to disease progression, thus supporting the concept that heritable factors can influence susceptibility to EWS and may help to predict patient prognosis. | Polymorphic variants of IGF2BP3 and SENCR have an impact on predisposition and/or progression of Ewing sarcoma
Ewing sarcoma (EWS), the second most common malignant bone tumor in children and adolescents, occurs abruptly without clear evidence of tumor history or progression. Previous association studies have identified some inherited variants associated with the risk of developing EWS but a common picture of the germline susceptibility to this tumor remains largely unclear. Here, we examine the association between thirty single nucleotide polymorphisms (SNPs) of the IGF2BP3, a gene that codes for an oncofetal RNA-binding protein demonstrated to be important for EWS patient’s risk stratification, and five SNPs of SENCR, a long non-coding RNA shown to regulate IGF2BP3. An association between polymorphisms and EWS susceptibility was observed for three IGF2BP3 SNPs - rs112316332, rs13242065, rs12700421 - and for four SENCR SNPs - rs10893909, rs11221437, rs12420823, rs4526784 -. In addition, IGF2BP3 rs34033684 and SENCR rs10893909 variants increased the risk for female respect to male subgroup when carried together, while IGF2BP3 rs13242065 or rs76983703 variants reduced the probability of a disease later onset (> 14 years). Moreover, the absence of IGF2BP3 rs10488282 variant and the presence of rs199653 or rs35875486 variant were significantly associated with a worse survival in EWS patients with localized disease at diagnosis. Overall, our data provide the first evidence linking genetic variants of IGF2BP3 and its modulator SENCR to the risk of EWS development and to disease progression, thus supporting the concept that heritable factors can influence susceptibility to EWS and may help to predict patient prognosis.
Ewing sarcoma (EWS), the second most common primary tumor of bone in the pediatric population, is a mesenchymal very aggressive cancer with high tendency to form distal metastasis and still unmet clinical needs (1). From a genetic point of view, EWS is characterized by a very low mutational burden (2–4) while its genetic landscape is thought to be driven by the aberrant transcript that derives from the fusion of EWSR1 gene with a member of the ETS family genes, in most of the cases represented by EWSR1-FLI chimera (5). EWS is not considered a heritable cancer but disparity in EWS epidemiological distribution, with higher incidence in European than in Asian and African population (6) together with some reports indicating EWS in siblings or cousins (7, 8) and reports of family aggregation of different malignant tumors between EWS patients and their relatives (9, 10) suggest that genetic susceptibility factors may exist for this tumor. Indeed, in the last decade several evidence of correlation between polymorphic variants and EWS risk has been reported (11–18). Through genome-wide association studies (GWAS), multiple genetic susceptibility loci (1p36.22, 6p25.1, 10q21, 15q15, 20p11.22 and 20p11.23) associated with EWS risk have been identified (16, 17). Most of these loci reside near GGAA repeat sequences and may condition the binding of the aberrant transcriptional factor EWS-FLI1. A noteworthy example is the locus 10q21, in which rs79965208 variant increases the number of consecutive GGAA motifs and the consequent EWS-FLI1-dependent enhancer activity, leading to EGR2 overexpression and favoring EWS susceptibility and aggressiveness (18). Deeply investigation of genes already known to be involved in the pathogenesis and progression of EWS has been performed by several groups as an alternative option to identify predisposing factors for this disease. In particular, analysis of single nucleotide polymorphisms (SNPs) in the EWSR1 gene revealed that the rs4820804 variant in homozygosis may increase the chance of chromosome breakage and occurrence of chromosomal translocation (11). The relationship between polymorphic variants in genes implicated in EWS pathogenesis and progression and their role in EWS susceptibility has been also studied for NROB1 and CAV1, two EWS-FLI1 target genes (19), and for CD99, another hallmark of EWS critically associated with EWS cell differentiation, migration and metastasis (20). Specifically, CD99 rs311059-T allele was found to be associated with early EWS onset, while rs312257-T variant was related with a reduced risk of relapse (12). In this study, we focused on the analysis of genetic polymorphisms of the Insulin-like growth factor 2 mRNA-binding protein 3 (IGF2BP3), a gene that encodes for an oncofetal RNA-binding protein that was found to be undetectable in most adult tissues but strongly expressed in embryos and in diverse tumor types (21). In EWS, high levels of IGF2BP3 were found to support cell migration and metastasis formation besides correlating with disease progression and poor patient’s prognosis (22–24). Thirty SNPs mapping on IGF2BP3 were genotyped in a cohort of 73 EWS Italian patients to evaluate the genetic influence of IGF2BP3 polymorphisms on EWS susceptibility and to establish whether a potential link between IGF2BP3 somatic variants and EWS progression exists. In addition, we analyzed five genetic polymorphisms of SENCR, a long non-coding RNA transcribed antisense from the 5’ end of the FLI1 gene, which was shown to regulate IGF2BP3 (25).
A cohort of 73 unrelated Italian patients with localized (58 cases) or disseminated (15 cases) EWS treated at the IRCCS Istituto Ortopedico Rizzoli (Bologna, Italy) was considered. Patients underwent local treatments (surgery; surgery plus radiotherapy; radiotherapy only, when the surgeon considered the lesion inoperable or due to patient refusal) and neo-adjuvant chemotherapy according to protocols that were previously reported in detail (26, 27). For radiotherapy administered in combination with surgery, the doses ranged between 45 Gy and 54 Gy, depending on the individual factors (age, site, size, surgical margins, response to chemotherapy); for radiotherapy administered alone, the doses ranged between 54-60 Gy. The timing of radiation therapy ranged between 4 to 6 weeks (26, 27). Clinical-pathological data are shown in Table 1 . Patients with localized EWS were followed-up for 120 months and clinical information updated. Ethical committee approval was obtained from the Comitato Etico di Area Vasta Emilia Centro (Codice CE AVEC 505/2019/Sper/IOR) and written informed consent was obtained. The study was conducted in accordance with the Declaration of Helsinki ethical guidelines. Control sample consisted of three populations among the 1000 Genome Project, i.e Toscani in Italia (TSI), Utah residents with Northern and Western European ancestry (CEU), and Iberian population in Spain (IBS). Genotypes for each polymorphism were obtained from the Ensembl.org genome browser (GRCh37/hg19).
Quality and concentration of DNA obtained from peripheral blood leukocytes or from muscle tissue using standard DNAzol procedure (Thermo Fisher Scientific, Foster City, CA, USA) were evaluated by Nanodrop (Thermo Fisher Scientific). Aliquots of 12 ng/μl DNA from each patient were plated for being processed by the Sequenom MALDI-TOF mass spectrometer MassArray system (as a service at Applied Biomedical Research Center, S. Orsola-Malpighi Polyclinic, Bologna, Italy). The SNPs were selected using the SNPclip tool [https://ldlink.nci.nih.gov/] among the Genome1000 Phase1 Vars (GRCh37/hg19). Caucasian, Italian and Iberian populations (CEU+TSI+IBS dataset) were used to explore the haplotype complexity of each locus considered, applying the thresholds R2 0.8 and MAF 0.07. To a selection of 30 SNPs distributed along the entire IGF2BP3 sequence with the minimal redundancy level (28) were added 5 SNPs of the SENCR gene. Assay design was performed using specific Sequenom software package (Sequenom, San Diego, California, USA). Primers were synthesized at Metabion (Martinsried, Germany) (sequences available upon request). Allele peaks were analyzed with the Sequenom Typer Analysis software.
The distribution of genotypes in patient and control groups was tested for deviations from the Hardy–Weinberg equilibrium using Pearson’s χ2 test. The PLINK software was used to test for allelic association within different sample subsets, defined by patient sex or age at disease onset within an alternate phenotype file (29). Odds ratios were calculated to estimate the level of association of the rare allele carriers, i.e heterozygotes versus non-carriers, as well as homozygotes versus non-carriers. A permutation procedure was used to generate empirical significance levels. Such procedure relaxed assumptions about normality of continuous phenotypes and Hardy-Weinberg equilibrium and faced with rare alleles and small sample sizes. Haplotype association analysis using a likelihood ratio approach was performed with the aid of UNPHASED software v3.1.7 for loci showing nominal evidence of association in allelic association analysis. The full model test was performed to obtain a global P for association. In addition, for the combinations of SNPs that showed a global P < 0.05 in the overall association test, a specific analysis was carried out to evaluate a difference in risk between one haplotype versus all the others pooled together. Association between IGF2BP3 or SENCR variants and overall survival (OVS) was also estimated. Survival curves for OVS were obtained using the Kaplan–Meier method, while the log-rank test was used to calculate univariate statistical significance. OVS was defined as the time from diagnosis to cancer-related death. Patients who were lost to follow-up were censored at the last contact date. The genetic variants significantly associated with OVS in univariate analysis were entered into a Cox proportional hazards model. Values of 95% confidence intervals (CIs) of hazard ratios (HRs) were provided (30). Analyses were performed with SPSS software, version 22.0. P value ≤ 0.05 were considered significant.
Genotyping was carried out on a cohort of 73 EWS patients whose clinical-pathological features are summarized in Table 1 . Among the 35 genotyped polymorphisms, three SNPs (rs17796758, rs62468200 and rs70954368) mapping on IGF2BP3 and one SNP (rs7930515) mapping on SENCR were excluded from any statistical analyses because of a low call rate. Pairwise association analysis was performed to test the impact of the remaining variants and results are shown in Table 2 . In details, the IGF2BP3 rs12700421 variant was found to be significantly less frequent in EWS patients than in the control group. Specifically, heterozygote genotype led to a reduced risk of developing EWS [ORhet = 0.47 (95% CI 0.24-0.91)]. A similar trend was observed for the IGF2BP3 rs13242065 variant [ORhet = 0.29 (95% CI 0.09-0.98)]. Instead, the adjacent rs112316332 variant showed association with increased risk of disease [ORhet = 1.94 (95% CI 1.08-3.49)]. The IGF2BP3 rs146075134 variant also showed a significant association level but it was excluded from further analyses because of a deviation from Hardy-Weinberg equilibrium observed in the control-group (P value < 0.01). The analysis of SENCR polymorphisms evidenced a protective effect of the variant allele at rs11221437, rs12420823, and rs4526784 [ORhet = 0.48 (95% CI 0.27-0.86); ORhet = 0.52 (95% CI 0.3-0.92); ORhet = 0.5 (95% CI 0.28-0.87) respectively]. An opposite role was observed for the SENCR rs10893909 (P = 0.0092), as the variant allele of this marker increased the risk of EWS more than three times when carried in homozygosis [ORhom = 3.33 (95% CI 1.35-8.19)]. To verify if the level of association varies in relation to patient sex or age at disease onset, stratified data were considered ( Tables 3 , 4 ). Females were found to be more prone than males to incur in EWS when carrying the IGF2BP3 rs34033684 variant [OR = 3.38 (95% CI 1.44-7.94)]. An increased risk for females [OR = 2.48 (95% CI 1.28-4.82)] was also observed for the SENCR rs10893909 variant allele ( Table 3 ). Of note, when females carry the variant at both rs34033684 and rs10893909, their risk to develop EWS is further increased [OR = 4.43 (95% CI 1.11-19.01)]. In patients with a later onset of EWS (> 14 years) a significantly lower frequency of the rare allele was observed both for IGF2BP3 rs13242065 and rs76983703 [OR = 0.19 (95% CI 0.04-0.78) and OR = 0.29 (95% CI 0.11-0.74), respectively] ( Table 4 ). The multipoint association analysis confirmed the role of the IGF2BP3 genetic region that includes rs112316332 and rs13242065 in influencing the risk of EWS development ( Supplementary Table 1 ). Significant P value levels were also obtained when the IGF2BP3 rs58201821, rs12533936, rs34033684, and rs6953027 SNPs, which map close to the rs112316332 and rs13242065 SNPs, were considered in the haplotype analysis. Both over- or under-represented haplotypes were found in EWS patients. Notably, haplotypes including four, five, or six SNPs had a higher level of association compared to the effect produced by single allele in pairwise analysis.
To search for the possibility that IGF2BP3 or SENCR SNPs impact on the probability for patients to have a different outcome, we stratified patients according to the presence (censored as POS) or absence (censored as NEG) of the variant allele at the 30 previously considered SNPs (26 in IGF2BP3 and 4 in SENCR, respectively). In order to limit possible drawbacks related to the presence of metastasis at diagnosis, an event known to be associated to a worse prognosis (31), we limited our analysis to 58 patients with primary, localized tumor homogeneously treated in a single Institution ( Table 1 , Dataset II). Kaplan-Meier curves and log-rank test performed on IGF2BP3 polymorphisms showed that the absence of the variant allele at rs10488282 and the presence at rs199653 or rs35875486 were significantly associated with a worse OVS at 120 months ( Figure 1 ). Multivariate analysis was performed for the three variables identified by univariate analysis and confirmed the prognostic value of the absence of the variant allele at IGF2BP3 rs10488282 ( Table 5 ) as an independent factor of worse outcome.
Susceptibility to the development of sporadic tumors is based on a complex interplay that includes various genetic and environmental factors whose degree of influence depends on the type of cancer. In pediatric cancer etiology, the genetic contribution is predominant over the environmental one. In EWS, the peak of incidence in the second decade of life draws attention to genetic predisposition rather than to environmental repercussion for the disease onset. The identification of EWS predisposing genetic factors can lead to clinical benefits for patients, highlighting new oncogenic pathways that may be useful either for the molecular diagnosis or for better therapy. Besides wide-scale approaches, an alternative option to identify germinal predisposing factors is to deeply investigate genes already known to be involved in the biology of cancer disease. Based on this approach, our study considers IGF2BP3 and SENCR as candidate genes for searching susceptibility genetic factors to EWS. To the best of our knowledge, this is the first study linking germline genetic variants of IGF2BP3 and of its putative modulator SENCR to the risk of EWS development, further supporting the concept that heritable factors can influence susceptibility to EWS (11–18). Nominal level of significance in pairwise association analysis was obtained with three IGF2BP3 SNPs (rs12700421, rs13242065 and rs112316332). The polymorphisms rs13242065 and rs112316332 were located in a gene region where multipoint association analysis provided evidence of association with different haplotypes. This region, bounded by rs58201821 and rs6953027, spans 29 Kbp across 5’-UTR and the second intron of the gene. According to ENCODE Registry of candidate cis-Regulatory Elements (cCREs) hosted in UCSC Genome Browser on Human GRCh38/hg38 Assembly, such region includes two cCREs showing a promoter-like signature proximal to a transcription start site (EH38E2540316 and EH38E2540292), and several predicted proximal and distal enhancers, suggesting for a potential regulatory function. The relevance of the region spanning across 5’-UTR and the second intron of IGF2BP3 for EWS predisposition was proved also when patients were stratified for sex (rs34033684) or age (rs13242065). Our finding corroborates the hypothesis that susceptibility factors act differently in females than in males and may influence the age of EWS occurrence. In addition to IGF2BP3 polymorphisms, our study also highlighted a potential value for SENCR genetic variants in EWS predisposition. All the four polymorphisms evaluated for SENCR were found to be significantly associated with a different risk to develop EWS and should be considered as inherited susceptibility factors of the disease. Although genetic variants in lncRNAs have been implicated as potential biomarkers in prediction of complex diseases (32), the genetic association between lncRNAs and EWS has yet to be explored. While the rs4526784 maps in the second exon of SENCR gene (http://genome.ucsc.edu/index.html) and may act by affecting the lncRNA sequence, the rs10893909, rs11221437 and rs12420823 map on the first intron of the gene and very likely influence EWS susceptibility in an indirect manner. For example, the rs10893909 and rs11221437 are located in regulatory regions annotated as proximal enhancer-like signature in ENCODE (EH38E1581272 and EH38E1581271, respectively) while according to the JASPAR database of transcription factor binding profiles (33) both the two variants disrupt transcription factor (TF) binding motifs. In particular, the rs10893909 variant was reported to disrupt a transcription factor binding motif with predicted affinity for several TFs, including NRF1 and KLF15 that were shown to cooperate with EWS-FLI1 (34). Likewise, the rs11221437 modifies a transcription factor binding motif with predicted affinity for CTCF, a TF involved in many cellular processes including the regulation of the transcriptional state-dependent 3D organization of the chromatin (35). In addition, we demonstrate for the first time that three allelic variants of IGF2BP3 may affect EWS patient’s outcome. Particularly the absence of the C allele at rs10488282 SNP was confirmed as an independent factor of prognosis at multivariate analysis, being associated with a poor survival for patients with localized EWS. Although mechanistic studies are needed to explain this observation, our findings support the hypothesis that genetic variants in the IGF2BP3 gene may significantly affect the progression of EWS. Considering the limits related to the low number of patients here considered and the rarity of the tumor, we offer this evidence to the scientific community for more extensive validation studies. Comprehensive genomic and epigenomic profiling has revealed that epigenetic factors likely play a critical role in EWS initiation and progression (5). RNA-binding proteins, along with microRNAs and lncRNAs, which dictate the entire RNA life cycle from alternative splicing to nuclear export, transcript storage, stabilization, subcellular localization and degradation [for a review, please consider (36)], may thus represent major regulators of tumor onset and progression. Over the past few years, studies have increasingly documented the contribution of IGF2BP3 to fundamental processes in cancer biology, such as cell growth, migration, and the response to drugs. Indeed, many tumor types upregulate IGF2BP3 compared to normal tissues but very limited information regarding the molecular regulatory mechanisms responsible for human IGF2BP3 expression is available [for a review see (21)]. Here we focused on SENCR, a gene coding for a lncRNA, recently found to play a critical role in the proliferation and migration of vascular smooth muscle cells (37), which may influence gene expression through multiple mechanisms, including interaction with RNA-binding proteins. The role and mechanism of action of the lncSENCR in malignant tumors remains largely unexplored. Our study supports deeper investigation on this lncRNA as a factor influencing cancer susceptibility.
The original contributions presented in the study are included in the article/ Supplementary Material . Further inquiries can be directed to the corresponding authors.
Ethical committee approval was obtained from the Comitato Etico di Area Vasta Emilia Centro (Codice CE AVEC 505/2019/Sper/IOR). The study was conducted in accordance with the Declaration of Helsinki ethical guidelines, and patient informed consent for research use of biobanking material was obtained.
MM, CZ, and KS: conception and design. MM, CM, LS, AP, PDS, CF, and MP: acquisition and analysis of data. MM, CZ, and KS: drafting the manuscript. MM and KS: study supervision. All authors contributed to the article and approved the submitted version.
The research leading to these results has received funding from AIRC under IG 2019 — ID. 22805 project — P.I. Scotlandi Katia, and from Ricerca Fondamentale Orientata (RFO, University of Bologna) to Zucchini Cinzia and Martinelli Marcella. The materials presented and views expressed herein are the responsibility of the authors only. The sponsor takes no responsibility for any use of the information presented herein. None of the funders played a role in study design; in the collection, analysis, and interpretation of data; in the writing of the manuscript; and in the decision to submit the paper for publication.
We thank the patients and their family for supporting this study. We thank Dr. Vilma Mantovani and Dr. Carlotta Cristalli for their technical support during experimental procedure of genotyping at CRBA (Applied Biomedical Research Center, S. Orsola-Malpighi Polyclinic, Bologna, Italy). We thank Dr. Marika Sciandra (Laboratory of Experimental Oncology, IRCCS Istituto Ortopedico Rizzoli, Bologna, Italy) for her technical support. The authors are grateful to Muscolo Skeletal Tumor Biobank-Biobanca dei Tumori Muscoloscheletrici (Biotum)—member of the CRB-IOR—which provided us the biological samples.
The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher. | true | true | true |
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PMC9634093 | 36335987 | Xiaoyan Xia,Zikui Li,Yaojun Li,Feng Ye,Xiaoming Zhou | LncRNA XIST promotes carboplatin resistance of ovarian cancer through activating autophagy via targeting miR-506-3p/FOXP1 axis | 17-10-2022 | Ovarian Cancer,Carboplatin | Objective Resistance to chemotherapy drugs makes ovarian cancer (OC) difficult to treat and ultimately kills patients. Long non-coding RNAs are closely related to carboplatin resistance in OC. In present study, we explored the role of lncRNA X-inactive specific transcript (XIST) on carboplatin resistance in OC. Methods Cell viability, proliferation, and apoptosis were assessed through 2,5-diphenyl-2H-tetrazolium bromide, colony formation, and flow cytometry assays, respectively. Microtubule-associated protein 1A/1B-light chain 3 expression was evaluated by immunofluorescence assay to analyze the cell autophagy. The interaction of XIST/miR-506-3p or miR-506-3p/forkhead box protein P1 (FOXP1) was analyzed using RNA immunoprecipitation (RIP) and dual-luciferases reporter assays. The function of XIST/miR-506-3p/FOXP1 axis in vivo was further confirmed by tumor xenograft study and immunohistochemistry. Results The expression of XIST and FOXP1 increased while miR-506-3p decreased in OC and carboplatin resistance cells. XIST silencing repressed the proliferative and autophagic capacities of carboplatin resistance cells while promoted the apoptosis. XIST overexpression led to the opposite results. XIST targeted miR-506-3p and downregulated its expression. MiR-506-3p inhibition facilitated the proliferative and autophagic capacities while suppressed the apoptosis of cells, XIST knockdown reversed these effects. MiR-506-3p bound to FOXP1. XIST knockdown or miR-506-3p overexpression reversed the increase of cell proliferative and autophagic abilities and the decrease of apoptosis rate induced by FOXP1 overexpression. XIST affected autophagy and carboplatin resistance in vivo via regulating the miR-506-3p/FOXP1 axis. Conclusion XIST knockdown inhibited autophagy and carboplatin resistance of OC through FOXP1/protein kinase B (AKT)/mammalian target of rapamycin pathway by targeting miR-506-3p. | LncRNA XIST promotes carboplatin resistance of ovarian cancer through activating autophagy via targeting miR-506-3p/FOXP1 axis
Resistance to chemotherapy drugs makes ovarian cancer (OC) difficult to treat and ultimately kills patients. Long non-coding RNAs are closely related to carboplatin resistance in OC. In present study, we explored the role of lncRNA X-inactive specific transcript (XIST) on carboplatin resistance in OC.
Cell viability, proliferation, and apoptosis were assessed through 2,5-diphenyl-2H-tetrazolium bromide, colony formation, and flow cytometry assays, respectively. Microtubule-associated protein 1A/1B-light chain 3 expression was evaluated by immunofluorescence assay to analyze the cell autophagy. The interaction of XIST/miR-506-3p or miR-506-3p/forkhead box protein P1 (FOXP1) was analyzed using RNA immunoprecipitation (RIP) and dual-luciferases reporter assays. The function of XIST/miR-506-3p/FOXP1 axis in vivo was further confirmed by tumor xenograft study and immunohistochemistry.
The expression of XIST and FOXP1 increased while miR-506-3p decreased in OC and carboplatin resistance cells. XIST silencing repressed the proliferative and autophagic capacities of carboplatin resistance cells while promoted the apoptosis. XIST overexpression led to the opposite results. XIST targeted miR-506-3p and downregulated its expression. MiR-506-3p inhibition facilitated the proliferative and autophagic capacities while suppressed the apoptosis of cells, XIST knockdown reversed these effects. MiR-506-3p bound to FOXP1. XIST knockdown or miR-506-3p overexpression reversed the increase of cell proliferative and autophagic abilities and the decrease of apoptosis rate induced by FOXP1 overexpression. XIST affected autophagy and carboplatin resistance in vivo via regulating the miR-506-3p/FOXP1 axis.
XIST knockdown inhibited autophagy and carboplatin resistance of OC through FOXP1/protein kinase B (AKT)/mammalian target of rapamycin pathway by targeting miR-506-3p.
Ovarian cancer (OC) is the main cause of gynecological cancer mortality in most developed countries. It does not only let women and family into physical and psychologic agony, but also cause serious social and economic load [12]. Currently, the most frequent treatment for OC is a combination of surgery and chemotherapy drugs. Although the development and popularization of chemotherapy drugs such as cisplatin and carboplatin have extended the survival time of OC patients to some extent [3], the long-term use of chemical drugs can cause cancer cells to develop drug resistance, so that many patients die from recurrent and progressive diseases [4]. Therefore, it is very important to explore and discover molecular targets for regulating chemotherapy resistance in OC. Long non-coding RNAs (lncRNAs), which play a role in the control of several tumor cell activities, such as proliferation, apoptosis, autophagy, epithelial-mesenchymal transition (EMT) and drug resistance, are possible markers for a range of human cancers [5]. In recent years, a growing body of evidence showed that lncRNA X-inactive specific transcript (XIST) was abnormally expressed in tumors and controlled the progression of multiple cancers [6], such as thyroid cancer [7]. Furthermore, the function of XIST in chemoresistance have gotten a lot of attention and have been widely explored. A previous study explored the mechanism by which carboplatin combined with XIST worked against retinoblastoma, and demonstrated that carboplatin could suppress cell proliferation and EMT in vitro by regulating the XIST/miR-200a-3p/Neuropilin 1 pathway [8]. It was reported that XIST was highly expressed in OC both in vivo and in vitro, which was linked to tumor grade and distant metastasis [9]. However, there are few studies on whether the combination of carboplatin and XIST can regulate the development of OC cells, which may be a potential therapeutic method. Thus, this study was to establish a theoretical foundation for the implementation of carboplatin and XIST targeted therapy from the perspective of molecular biology. MicroRNAs (miRNAs) were proved to regulate fundamental cellular processes and tissue specific functions through post-transcriptionally regulating gene expression by binding to 3’-untranslated region (3’-UTR) of mRNAs [1011]. Overexpression of miR-506-3p has been observed to suppress certain human tumors, including OC. For instance, miR-506-3p enhancement was suggested to act as a way to prevent the onset of OC [12]. Moreover, miR-506-3p was found to regulate autophagic and proliferative processes in post-burn skin fibroblasts by inhibiting Beclin-1 level [13]. MiR-506-3p enhanced cisplatin sensitivity in serous OC by regulating the Enhancer of zeste homolog 2/β-catenin pathway [14]. On the other hand, the role of miR-506-3p in OC carboplatin resistance needs to be further studied. Forkhead box protein P1 (FOXP1), a member of the FOXP subfamily of transcription factors, was found to be up-regulated in OC, and miR-29c-3p suppressed autophagy and drug resistance in OC cells by down-regulating FOXP1 [15]. Furthermore, protein kinase B (AKT)/mammalian target of the rapamycin (mTOR) pathway was extremely actived in OC, regulating a range of cellular functions and playing a key role in OC development [16]. It has also been reported that FOXP1 can stimulate AKT/mTOR pathway [17]. Therefore, the regulatory mechanism of the FOXP1/AKT/mTOR axis in OC is worth exploring. The function of the crosstalk of XIST, miR-506-3p and FOXP1 on OC cell growth, autophagy and carboplatin resistance was explored in this work. Our findings provided a feasible theoretical basis for increasing carboplatin sensitivity in OC therapy.
Human ovarian surface epithelial cells HOSE and OC cells SKOV3, A2780 and HO-8910 were offered by American Type Culture Collection (ATCC, Manassas, VA, USA), then maintained in Roswell Park Memorial Institute-1640 (Invitrogen, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum (FBS, Invitrogen), 100 U/mL penicillin and 100 μg/mL streptomycin (Invitrogen) at 37°C with 5% CO2. SKOV3 and A2780 cells were transfected with the following plasmids: short hairpin RNA XIST (sh-XIST), XIST overexpression (OE-XIST), miR-506-3p mimics, miR-506-3p inhibitor, and OE-FOXP1 alone or in combination: miR-506-3p inhibitor+sh-XIST, OE-FOXP1+miR-506-3p mimics, OE-FOXP1+sh-XIST and the corresponding negative controls using Lipofectamine 3000 (Invitrogen). All plasmids were purchased from RiboBio (Guangzhou, China).
Total RNA was isolated by TRIzol reagent (Invitrogen), then RNA quality was detected using NanoDro2000c (Thermo Scientific, Waltham, MA, USA). Next, TaqMan® miRNA reverse transcription kit was employed in miRNA qPCR assay, and for the other genes, random primers from the RT Master Mix kit were used to synthesize cDNAs, and qRT-PCR process was performed on an ABI 7900 system using SYBR Green Real-Time PCR master mixes (Thermo). The relative expressions were normalized to that of glyceraldehyde 3-phosphate dehydrogenase (GAPDH) mRNA or U6 using the 2-ΔΔCt method. Primers were showed in Table S1.
Total proteins were isolated, and the concentration was determined using BCA method. After proteins was separated by using 10%–12% SDS-PAGE, proteins were transferred to PVDF membranes (Millipore, Bedford, MA, USA). The 1% BSA in TBS buffer was used to block the membranes and cultivated at 4°C overnight with primary antibodies against FOXP1 (1:5,000, ab134055, Abcam, Cambridge, UK), microtubule-associated protein 1A/1B-light chain 3 (LC3) (1:3,000, ab51520), P62 (1:1,000, ab207305), B-cell lymphoma 2 (Bcl-2) (1:1,000, ab59348), Bcl-2 associated X (Bax) (1:5,000, ab32503), AKT (1:500, ab8805), phospho (p)-AKT (1:1,000, ab38449), mTOR (1:10,000, ab134903), GAPDH (1:1,000, ab8245), β-actin (1:1,000, ab8226). Then incubating with the corresponding secondary antibodies. The protein signaling was visualized by ECL reagent. GAPDH and β-actin as loading controls and all antibodies were purchased from Abcam.
Cells (5,000/well) were seeded into 96-well plates and incubated for 24 hours, then treated with carboplatin at different concentrations (0, 100, 150, 200, 250, 300 μM). After incubating for 48 hours, cells were cultured with MTT solution at 37°C and then dissolved in dimethyl sulfoxide. Cell viability was detected at 570 nm by a microplate reader (BioTek Instruments, Winooski, VT, USA).
Cells seeded into 6-well plates were cultured at 37°C until 100% confluence. The medium was changed every 2 days. After 14 days, the colonies were fixed with 4% paraformaldehyde and then stained with 0.1% crystal violet. A microscope was used to calculate the number of colonies.
Cells were washed with binding buffer and then centrifuged at 500 × g for 5 minutes at room temperature, then resuspended in cold PBS. Next, 10 µL Annexin V-fluorescein isothiocyanate and 10 µL propidium iodide were added and cultured for 15 min in the dark. Samples were analyzed through Becton-Dickinson flow cytometer.
After fixing with 4% formaldehyde, cells were cultured with 5% Tris buffered saline with Tween-20 (pH 8.3) diluted non-fat dry milk and incubated with the primary antibody LC3 (1:2,000, ab51520, Abcam) and corresponding secondary antibody. Next, 4’,6-diamidino-2-phenylindole (DAPI) was used to stain cell nuclear, and a confocal laser scanning microscope was used to analyze the immunofluorescence images.
RIP assay was conducted using a Magna RIPTM RNA-Binding Protein Immunoprecipitation Kit (Millipore). Cells were lysed with RIP lysis buffer and then immunoglobulin G (IgG) antibody and argonaute 2 (Ago2) antibody coated on magnetic beads overnight. Then the RNA complexes were purified. The co-precipitated RNA was extracted using TRIzolTM, and qRT-PCR was then used to analyze the purified RNA.
Briefly, the wild type (WT) putative miR-506-3p binding site of the XIST or FOXP1 3’-UTR was amplified and inserted into the pmirGLO vectors to establish recombinant luciferase reporter plasmids and named XIST-WT or FOXP1-WT. The matched mutant (MUT) miR-506-3p binding site was also cloned into the pmirGLO vectors to establish mutant recombinant luciferase reporter plasmids and named XIST-MUT or FOXP1-MUT. Then, cells were co-transfected with above plasmids and miR-506-3p mimics or mimics NC for 24 hours. Finally, luciferase intensity was tested by Dual-Luciferase Reporter Assay System (Promega, Madison, WI, USA).
BALB/c-nude mice (22–24 g, 6 weeks) were purchased from Animal Experiment Center of Chinese Academy of Sciences (Shanghai, China). SKOV3 cells stably expressed sh-NC, sh-XIST, sh-NC+CBP and sh-XIST+CBP were injected subcutaneously into the right flank of the mice (n=6) to perform tumorigenesis assay. Then we measured the tumor size using calipers every 5 days and calculated the volume. After 20 days, mice were sacrificed, and then xenograft tumor tissues were harvested. All experiments were performed according to protocols from the Frist Hospital of Hunan University of Chinese Medicine and approved by the Frist Hospital of Hunan University of Chinese Medicine and the Laboratory Animal Ethics Committee, which conforms to the relevant provisions of the National Laboratory Animal Welfare ethics.
Paraffin sections were deparaffinized and hydrated. After serial incubation with primary antibodies anti-LC3 (1:400, ab48394, Abcam), anti-cleaved caspase3 (1:50, #9661, Cell signaling, Danvers, MA, USA) and secondary antibody, then subjected to the liquid DAB substrate-chromogen system. Images were visualized using a Nikon ECLIPSE Ti microscope system and processed with Nikon software.
All data was presented as mean ± standard deviation. Data analysis was performed by Graphpad Prism (Version 7.0; San Diego, CA, USA) using Student’s t test or one-way analysis of variance. The value of p less than 0.05 was considered significant.
QRT-PCR was used to reconfirm the abnormal expression of XIST in OC cells. As shown in Fig. 1A, XIST was increased in SKOV3, A2780 and HO-8910 cells. Conversely, miR-506-3p showed down-regulation in OC cells (Fig. 1B). In addition, FOXP1 was observed to be overexpressed in OC cells (Fig. 1C and D). SKOV3 and A2780 cells with the most obvious differential expression were selected for subsequent experiments.
To study the biological function of XIST dysregulation in OC carboplatin resistant cells (SKOV3/CBP and A2780/CBP cells), we first measured XIST levels in SKOV3/CBP and A2780/CBP cells and observed that XIST was significantly over expressed (Fig. 2A). Subsequently, MTT results showed that as the concentration of carboplatin was increased, cell proliferative ability dropped progressively (Fig. 2B). To determine the impact of XIST knockdown or overexpression on carboplatin sensitivity, we first conducted the transfection efficiencies of sh-XIST and OE-XIST in SKOV3/CBP and A2780/CBP cells and observed that XIST significantly decreased or increased as expected (Fig. 2C). Then, the results indicated that knockdown of XIST suppressed the proliferation of carboplatin resistant OC cells, while enhancement of XIST promoted the proliferation (Fig. 2D). Furthermore, compared with the apoptosis rate in the control groups, XIST knockdown increased the cell apoptosis, while XIST overexpression reduced the apoptosis (Fig. 2E). Silencing of XIST promoted the expression of Bax (pro-apoptosis protein) and inhibited Bcl-2 (anti-apoptotic protein) levels, while overexpression of XIST led to the opposite results (Fig. 2F). An immunofluorescence technique was used to analyze the autophagy of SKOV3/CBP and A2780/CBP cells. We observed that knockdown of XIST inhibited cell autophagy, which was further confirmed by the decrease of LC3 II/I level and the increase of P62 level, Conversely, overexpression of XIST promoted cell autophagy (Fig. 2G-I). Taken together, XIST facilitated carboplatin resistance in OC cells, while depletion of XIST was able to improve the efficacy of carboplatin.
Since the downregulation of miR-506-3p in OC, we had reason to clarify the interaction of XIST/miR-506-3p. StarBase analysis demonstrated a binding sequence between XIST and miR-506-3p (Fig. 3A). Then, as shown in Fig. 3B, XIST and miR-506-3p were coimmunoprecipitated by Ago2 antibody instead of IgG antibody. Furthermore, co-transfection of XIST-WT and miR-506-3p mimics repressed luciferase activities, while the activities in the XIST-MUT reporter had no significant change (Fig. 3C). MiR-506-3p levels were increased after transfecting with sh-XIST or miR-506-3p mimics, while transfection of OE-XIST decreased miR-506-3p levels (Fig. 3D). Therefore, we concluded that miR-506-3p was directly bound to XIST. In addition, FOXP1 was found to be over expressed in carboplatin resistant OC cells (Fig. 3E). MiR-506-3p enhancement could suppress FOXP1 expression and the protein expression of mTOR and the phosphorylation of AKT (Fig. 3F and G). Subsequently, a binding site between miR-506-3p and FOXP1 was found by StarBase (Fig. 3H). As shown in Fig. 3I, FOXP1-WT co-transfected with miR-506-3p mimics inhibited luciferase activity, while there was no significant change after co-transfecting with FOXP1-MUT and miR-506-3p mimics, which further confirmed the targeted relationship between them. Taken together, XIST might positively regulate FOXP1 expression by targeting miR-506-3p.
Next, a rescue assay was designed and used in vitro to explore the function of the XIST/miR-506-3p pathway. Firstly, miR-506-3p was down-regulated in carboplatin resistant cells (Fig. 4A). Then, we silenced miR-506-3p levels in SKOV3/CBP and A2780/CBP cells (Fig. 4B). The proliferation of SKOV3/CBP and A2780/CBP cells was enhanced after knockdown of miR-506-3p, while simultaneous silencing of miR-506-3p and XIST blocked the promoting effects (Fig. 4C). In addition, the reduction in apoptosis rate caused by miR-506-3p knockdown was restored through silencing of XIST (Fig. 4D). Furthermore, the miR-506-3p inhibitor reduced the level of Bax and enhanced Bcl-2 expression, while knockdown of XIST reversed the effect (Fig. 4E). MiR-506-3p silencing increased autophagic cells, while the increase was arrested by co-transfection with sh-XIST (Fig. 4F). Knockdown of XIST eliminated the promoting effects of miR-506-3p silencing on LC3 II/I expression and the inhibiting effects on P62 levels (Fig. 4G and H). Taken together, the XIST/miR-506-3p pathway did influence carboplatin sensitivity in vitro.
To learn more about the impact of the XIST/miR-506-3p/FOXP1 pathway on OC cell sensitivity to carboplatin, we first measured the overexpression efficiency of OE-FOXP1 in SKOV3/CBP and A2780/CBP cells (Fig. S1A and B). As shown in Fig. S1C, the proliferation of SKOV3/CBP and A2780/CBP cells increased in the OE-FOXP1 group, while the promoting effect of FOXP1 overexpression was reversed through transfecting with miR-506-3p mimics or sh-XIST. Furthermore, miR-506-3p enhancement or XIST silencing abolished the inhibitory effects of FOXP1 enhancement on cell apoptosis (Fig. S1D). Then, we examined apoptosis related protein expression, the results showed that FOXP1 inhibited Bax expression while promoting Bcl-2 expression, however, knockdown of XIST or overexpression of miR-506-3p had the opposite result (Fig. S1E). We also found that autophagy was promoted in the OE-FOXP1 group, while this change rescued in OE-FOXP1+miR-506-3p mimics and OE-FOXP1+sh-XIST groups (Fig. S1F). In addition, overexpression of FOXP1 increased LC3 II/I levels and decreased P62 levels, whereas miR-506-3p enhancement or XIST silencing restored these alterations (Fig. S1G). Overexpression of FOXP1 enhanced FOXP1, mTOR and p-AKT levels, while co-transfection of miR-506-3p mimics or sh-XIST eliminated the effect of OE-FOXP1 (Fig. S1H). These findings demonstrated that XIST/miR-506-3p/FOXP1 axis enhanced the carboplatin resistance of OC cells by regulating autophagy.
To verify the role of the novel axis, XIST/miR-506-3p/FOXP1 in vivo, we constructed mice models. We observed that XIST knockdown or carboplatin treatment repressed the volume and weight of the tumor, and the tumor growth was further inhibited in the sh-XIST+CBP group (Fig. 5A-C). Subsequently, we observed that knockdown of XIST or treatment of carboplatin suppressed the expression of XIST and FOXP1 in vivo, while up-regulating miR-506-3p expression (Fig. 5D). Moreover, the results from immunohistochemistry indicated that LC3 was down-regulated while caspase3 was up-regulated in mice models injected with XIST silenced SKOV3 cells or treated with carboplatin (Fig. 5E). Thus, silencing of XIST enhanced the sensitivity to carboplatin through the miR-506-3p/FOXP1 axis in vivo.
Chemotherapy resistance has emerged as a significant stumbling block in the treatment of malignant tumor. The mechanism of drug resistance is complicated, which include tumor heterogeneity, reduced drug concentration to the target, alteration in drug target structure [18]. Carboplatin-based chemotherapy is the standard first-line treatment for OC patients, however, patients may relapse because of the developing carboplatin resistance [19]. Although the possible molecular mechanism of carboplatin resistance in OC had been elucidated by many publications [2021], clinical experiments based on these studies have not yet yielded satisfactory therapeutic effects [22]. In this study, we found that knockdown of XIST suppressed the resistance of OC cells to carboplatin, investigated for the first time the roles of the XIST/miR-506/FOXP1 axis on carboplatin resistance in OC. The dysregulation of lncRNAs was suggested to show vital effects in diverse processes of cancer development, including the initiation and progression [23]. In recent years, the publications of the functions of lncRNAs in drug resistance, such as the effect of lncRNA SNHG1 in sorafenib [24] and that of lncRNA PVT1 in gemcitabine [25], have also helped our understanding of tumor biology. The effect of XIST in drug resistance has also attracted increasing attention. For example, Schouten et al. suggested that XIST and 53BP1 could be used to identify patients with BRCA1-like breast cancer who have a high incidence and poor prognosis after high-dose chemotherapy [26]. Furthermore, XIST levels in OC were linked to the number of cancer stem cells (CSCs) and susceptibility to Taxol therapy [27]. We hypothesized that XIST affected carboplatin resistance in OC, and our results revealed that XIST promoted carboplatin resistance in vitro. Remarkably, XIST knockdown suppressed the autophagy. Autophagy was observed to be either tumor-suppressing or tumor-promoting in different cell context [2829]. Autophagy induction has been shown to help cells survive stress, hypoxia and starvation. Furthermore, autophagy is also activated as a defensive mechanism to mediate the drug resistance of cancer therapy [30]. We concluded that XIST might affect OC cell resistance to carboplatin through regulating autophagy, which is in line with a prior study that inhibition of autophagy lowered the survival of CSCs during anticancer treatment [31]. In addition, carboplatin and XIST targeted therapy inhibited tumor growth in mice more effectively. These findings suggested that inhibition of XIST was a valuable therapeutic approach to enhance carboplatin sensitivity. It is widely reported that lncRNA function as a ceRNA to bind specific miRNA, thereby regulating miRNA-mediated gene silencing [32]. To explore whether XIST could act as a ceRNA in OC carboplatin chemoresistance, we used bioinformatic analysis, RIP and dual-luciferase reporter assays, which observed XIST to engage in complementary binding with miR-506-3p. Thus, we hypothesized that XIST might modulate cell autophagy and OC cell resistance to carboplatin by serving as a miRNA sponge. More importantly, miR-506-3p was suggested to have important roles in cancer chemotherapy resistance regulation [1433]. For example, overexpression of miR-506-5p reversed erlotinib resistance in non-small-cell lung cancer, which was mediated by suppressing the Sonic Hedgehog pathway [34]. Meanwhile, in our study, miR-506-3p silencing promoted carboplatin resistance cell growth and autophagy. The rescue experiments demonstrated that XIST silencing abolished the function of miR-506-3p knockdown in vitro, further indicating that XIST enhanced the resistance to carboplatin of OC cells by down-regulating miR-506-3p. FOXP1 had been reported to positively regulate Bcl-2 levels, thereby affecting cell apoptosis [35] and acted as an oncogene in hepatocellular carcinoma [36], diffuse large B-cell lymphoma [37], and so on. In the study of OC, Li et al. [38] suggested that FOXP1 reversed the inhibition of miR-374b-5p on the proliferative, migration, and EMT abilities of OC cells, and the enhancement of miR-374b-5p on cell sensitivity to cisplatin. Furthermore, FOXP1 influenced autophagy and chemoresistance in OC through targeting miR-29c-3p [15]. Similarly, FOXP1 was targeted to miR-506-3p, and XIST knockdown or miR-506-3p enhancement abolished the promoting effects of FOXP1 overexpression on carboplatin resistance cell proliferation and autophagy in our work. In addition, we observed that FOXP1 overexpression upregulated AKT phosphorylation levels and mTOR expression, suggesting that FOXP1 contributed to the activation of the AKT/mTOR pathway, which was recognized as a key regulatory signal for autophagy [39]. Lee et al. [40] proved that carboplatin effectively suppressed the activation of the mTOR signaling cascade in OC cells. Therefore, we concluded that XIST knockdown inhibited autophagy and suppressed carboplatin resistance of OC cells through the FOXP1/AKT/mTOR pathway by targeting miR-506-3p. The biological functions of the XIST/miR-506-3p/FOXP1 pathway were also confirmed in vivo. Finally, these findings firstly demonstrated that XIST/miR-506-3p/FOXP1 axis was involved in OC carboplatin resistance and regulated autophagy, providing a theoretical basis for XIST to be a prognostic marker for OC chemosensitivity. | true | true | true |
PMC9634094 | 36245227 | Yan Zhu,Lijuan Yang,Jianqing Wang,Yan Li,Youguo Chen | SP1-induced lncRNA MCF2L-AS1 promotes cisplatin resistance in ovarian cancer by regulating IGF2BP1/IGF2/MEK/ERK axis | 23-08-2022 | Ovarian Cancer,Cisplatin,LncRNA MCF2L-AS1,SP1,IGF2BP1,IGF2/MEK/ERK Signaling Pathway | Objective Cisplatin resistance is a huge problem encountered in ovarian cancer treatment. Our study probed the roles and the underlying mechanisms of lncRNA MCF2L-AS1 in ovarian cancer cisplatin-resistance. Methods SKOV3 and IGROV-1 cells were subjected to gradually increasing concentrations of cisplatin to construct ovarian cancer cisplatin-resistance cells. Cell proliferation was evaluated by cell counting kit-8 and colony formation assays. Cell apoptosis was assessed using Annexin V and PI staining. The relationships between SP1, MCF2L-AS1 and insulin-like growth factor-2 mRNA binding protein 1 (IGF2BP1) were verified by RNA pull-down, RIP, ChIP and dual-luciferase reporter gene assay, respectively. Tumor xenograft experiment was employed to evaluate the effects of MCF2L-AS1 silencing on ovarian cancer cisplatin-resistance in vivo. TUNEL staining and immunohistochemistry were performed in tumor tissue. Results MCF2L-AS1 and IGF2BP1 were upregulated in cisplatin-resistant cells. MCF2L-AS1 silencing suppressed cell proliferation of cisplatin-resistant cells, while promoted the apoptosis, suggesting that MCF2L-AS1 knockdown suppressed ovarian cancer cells cisplatin-resistance. Meanwhile, MCF2L-AS1 silencing enhanced cisplatin sensitivity in ovarian cancer parental cells and IGF2BP1 overexpression impaired cisplatin sensitivity of parental cells. MCF2L-AS1 activated IGF2/MEK/ERK pathway through interacting with IGF2BP1. Transcription factor SP1 activated MCF2L-AS1 expression. MCF2L-AS1 knockdown inhibited ovarian cancer cisplatin-resistance in vivo. Conclusion SP1-induced MCF2L-AS1 promoted ovarian cancer cisplatin-resistance through activation of IGF2/MEK/ERK pathway via interacting with IGF2BP1. | SP1-induced lncRNA MCF2L-AS1 promotes cisplatin resistance in ovarian cancer by regulating IGF2BP1/IGF2/MEK/ERK axis
Cisplatin resistance is a huge problem encountered in ovarian cancer treatment. Our study probed the roles and the underlying mechanisms of lncRNA MCF2L-AS1 in ovarian cancer cisplatin-resistance.
SKOV3 and IGROV-1 cells were subjected to gradually increasing concentrations of cisplatin to construct ovarian cancer cisplatin-resistance cells. Cell proliferation was evaluated by cell counting kit-8 and colony formation assays. Cell apoptosis was assessed using Annexin V and PI staining. The relationships between SP1, MCF2L-AS1 and insulin-like growth factor-2 mRNA binding protein 1 (IGF2BP1) were verified by RNA pull-down, RIP, ChIP and dual-luciferase reporter gene assay, respectively. Tumor xenograft experiment was employed to evaluate the effects of MCF2L-AS1 silencing on ovarian cancer cisplatin-resistance in vivo. TUNEL staining and immunohistochemistry were performed in tumor tissue.
MCF2L-AS1 and IGF2BP1 were upregulated in cisplatin-resistant cells. MCF2L-AS1 silencing suppressed cell proliferation of cisplatin-resistant cells, while promoted the apoptosis, suggesting that MCF2L-AS1 knockdown suppressed ovarian cancer cells cisplatin-resistance. Meanwhile, MCF2L-AS1 silencing enhanced cisplatin sensitivity in ovarian cancer parental cells and IGF2BP1 overexpression impaired cisplatin sensitivity of parental cells. MCF2L-AS1 activated IGF2/MEK/ERK pathway through interacting with IGF2BP1. Transcription factor SP1 activated MCF2L-AS1 expression. MCF2L-AS1 knockdown inhibited ovarian cancer cisplatin-resistance in vivo.
SP1-induced MCF2L-AS1 promoted ovarian cancer cisplatin-resistance through activation of IGF2/MEK/ERK pathway via interacting with IGF2BP1.
Ovarian cancer is one of the lethal gynecological malignancies. Cisplatin is a well-known antitumor agent [1]. However, most patients with ovarian cancer will eventually die of recurrent and progressive disease because of resistance to cisplatin [2]. As reported, cisplatin resistance in ovarian cancer is a multifactorial process, which may result from a series of dysregulation of gene expression; however, the specific mechanism of cisplatin resistance in ovarian cancer is still poorly understood. Transcription factor SP is overexpressed in many cancers and is associated with poor prognosis, which both activates and suppresses the expression of a number of essential oncogenes and tumor suppressors [3]. As previously reported, SP1 was markedly upregulated in ovarian cancer, and SP1 knockdown could suppress cell migration, cell invasion and chemoresistance [45]. It is suggested that SP1 facilitates the malignant behavior of ovarian cancer cells, while its role in regulating ovarian cancer cell cisplatin resistance is unknown, which deserves further study. LncRNA refers to a single stranded RNA (ssRNA) with the transcripts more than 200 nts and function in many biological processes [6]. As widely reported, the development of ovarian cancer is accompanied with changes in expression pattern of large set of lncRNAs [78]. For instance, Xu et al. [9] demonstrated that lncRNA EBIC promoted ovarian cancer cisplatin-resistance. In addition, lncRNA CHRF was markedly upregulated in cisplatin resistant ovarian cancer cells and patients with resistant disease [10]. LncRNA MCF.2 cell line derived transforming sequence like-antisense RNA 1 (MCF2L-AS1) was confirmed as an oncogene in colorectal cancer and lung cancer [1112]. However, the expression and the role of lncRNA MCF2L-AS1 in ovarian cancer were never reported before. Herein, through the investigation of GEPIA database, it was found that lncRNA MCF2L-AS1 expression markedly increased in ovarian cancer, which caught our attention. Recent studies have revealed that SP1 could bind to the promoter of non-coding RNA to promote ovarian cancer progression [413]. In the current study, SP1 was predicted to have a binding site to lncRNA MCF2L-AS1 promoter by using JASPAR. However, whether SP1 could positively regulate lncRNA MCF2L-AS1 expression to promote ovarian cancer cisplatin-resistance needs further study. Insulin-like growth factor-2 mRNA binding protein 1 (IGF2BP1) is a member of the conserved RNA binding protein family [14]. Much evidence has confirmed that IGF2BP1 is involved in tumor cell proliferation, invasion, and chemo-resistance in a series of malignant tumors, including ovarian cancer [1516]. IGF2BP1 binds to targets such as insulin-like growth factor 2 (IGF2) in the cytoplasm [17]. Elevated IGF2 expression is associated with increased risk of developing various cancers, and IGF2 promotes the malignant behaviors of tumors by activating downstream pathways, such as MEK/ERK signaling pathway [1819]. As previously reported, IGF2BP1 promoted the proliferation of tongue squamous cell carcinoma cells by activating the IGF2/MEK/ERK signaling pathway [20]. RNA-protein interaction prediction (RPISeq) showed high affinity of MCF2L-AS1 to IGF2BP1, suggesting that lncRNA MCF2L-AS1 could interacted with IGF2BP1 to regulate IGF2/MEK/ERK signaling pathway. However, the regulatory relationship between lncRNA MCF2L-AS1, IGF2BP1 and IGF2/MEK/ERK in ovarian cancer and their roles in ovarian cancer remains unclear. Herein, we found that SP1-induced MCF2L-AS1 promoted ovarian cancer cisplatin-resistance through activation of the IGF2/MEK/ERK signaling pathway via interacting with IGF2BP1. Our findings provided new insights into the regulatory mechanisms of lncRNA MCF2L-AS1 in ovarian cancer cisplatin-resistance.
Human ovarian cancer cells (SKOV3 and IGROV-1 cells) were purchased from ATCC (VA, Manassas, USA). All cells were cultured in Dulbecco’s Modified Eagle Medium (Gibco, Gaithersburg, MD, USA) containing 10% fetal bovine serum (Gibco) with 5% CO2 at 37°C. SKOV3/DDP and IGROV-1/DDP cells were constructed as previously described [21]. Cells were treated with cisplatin for 2 days as a cycle. After completing 3 cycles of stimulation with the same concentration of cisplatin, the dose was increased. The initial concentration of cisplatin was 5 μM, and the final concentration of cisplatin was 70 μM. Only when cells remained the resistance to cisplatin after cultured in medium without cisplatin for at least 6 months, they were used for subsequent experiment. In subsequent experiments, for cisplatin treatment, SKOV3/DDP cells were subjected to 30 μM cisplatin, and IGROV-1/DDP cells were subjected to 36 μM cisplatin.
The overexpression plasmid of SP1 (Oe-SP1), the overexpression plasmid of IGF2BP1 (Oe-IGF2BP1), the short hairpin RNA of lncRNA MCF2L-AS1 (sh-MCF2L-AS1) and their negative controls were obtained from GenePharma (Shanghai, China). SKOV3, IGROV-1, SKOV3/DDP and IGROV-1/DDP cells were transfected with above plasmids using Lipofectamine™ 3000 (Invitrogen, Carlsbad, CA, USA).
Cells were seeded in 96-well plates with 5×103 cells per well and then incubated with CCK-8 reagent (10 μL, Sangon) for 2 hours. Absorbance was examined at 450 nm with the microplate spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA).
For colony formation analysis, 1×105/well cells were seeded on the 6-well plates plate and incubated for 1 week at 37°C. Then cells were stained with 0.1% crystal violet for 10 minutes (Solarbio, Beijing, China), and the colonies formed were counted manually.
Cells were collected and re-suspended in 500 μL of 1X Annexin-binding buffer (Beyotime, Shanghai, China). Then cells were incubated with 10 μL Annexin V-FITC and 5 μL PI stain (Beyotime) for 10 minutes. Samples were immediately analyzed using flow cytometry (BD Biosciences, New York, NY, USA).
LncRNA MCF2L-AS1 or NC was labeled with biotin (Roche, Basel, Switzerland) and transcribed with the Biotin RNA Labeling Mix (Roche) and T7 RNA polymerase (Roche). About 2×107 cells were dissolved in the soft lysis buffer plus 80 U/mL RNasin (Promega, Madison, WI, USA). Cell extract (2 μg) was incubated with biotinylated RNA (100 pmol) for 1 hour at 4°C. Washed streptavidin-coupled agarose beads (Invitrogen) were added to each binding reaction and further incubated for 1 hour to isolate the RNA-protein complex. The retrieved protein was assessed using western blot.
Cells were lysed with a complete RIP lysis buffer (Millipore, Burlington, MA, USA). Cell extract was incubated with immunoglobulin G (IgG) (1:50, ab172730; Abcam, Waltham, MA, USA) and IGF2BP1 (1:50, ab184305; Abcam) antibody at 4°C overnight. RNA samples were purified by phenol chloroform extraction, followed by quantitative real-time polymerase chain reaction (qRT-PCR) to determine MCF2L-AS1 transcripts enrichment.
ChIP assay was conducted by using ChIP kit (Beyotime). Briefly, cells were treated with 1% formaldehyde solution for 10 minutes and quenched with 125 mM glycine for 5 minutes. DNA was fragmented by sonication. Cell lysate was subsequently incubated with anti-SP1 (1:100, ab231778; Abcam) or anti-IgG (1:100, ab172730; Abcam) at 4°C overnight. Then, dynabeads protein G (Invitrogen) was added for 2 hours for DNA enrichment. Finally, immunoprecipitated DNAs were analyzed by qRT-PCR.
The SP1-binding site in the promoter of lncRNA MCF2L-AS1 was predicted using JASPAR database (http://jaspar.genereg.net/). Site-directed mutagenesis of the SP1 binding site in lncRNA MCF2L-AS1 promoter was performed using a site-directed mutagenesis kit (Stratagene, La Jolla, CA, USA). Wild-type (wt) and mutant-type (mut) reporter plasmids of lncRNA MCF2L-AS1 sequences were cloned into PGL3 vector (GenePharma). Then, cells were co-transfected with lncRNA MCF2L-AS1-wt or lncRNA MCF2L-AS1-mut plasmids and oe-SP1 or oe-NC by Lipofectamine™ 3000 (Invitrogen). The luciferase activity was examined using a dual-luciferase reporter assay system (Promega).
A total of 24 BALB/c nude mice were assigned to 3 groups: SKOV3/DDP group (injected with SKOV3/DDP cells), sh-NC group (injected with SKOV3/DDP cells with sh-NC transfection) and sh-MCF2L-AS1 group (injected with sh-MCF2L-AS1 cells with sh-MCF2L-AS1 transfection) (8 mice/group). 0.2 mL of above cell suspension containing 2×104 cells was injected into the back of each mouse. At 7 days after inoculation, all mice received 4 mg/kg cisplatin treatment every 3 day. The tumor volumes were determined by measuring their length (l) and width (w) and calculating the volume (V) as follows: V=lw2/2. At 21 days, the mice were euthanized and tumor tissues were weighted. The animal studies were approved by Animal Ethics Committee of The Fourth Affiliated Hospital of Nantong University.
The tumor tissues were fixed in 10% paraformaldehyde and tumor sections (4 μm in thickness) were prepared. After deparaffinization and antigen retrieval (Dako, Santa Clara, CA, USA), sections were then blocked with goat serum, avidin solution and biotin solution. Then sections were incubated with antibody against Ki67 (1:200, ab15580; Abcam) overnight followed by incubation with an appropriate secondary antibody (1:500, ab150077; Abcam) for 1 h. The sections were stained with diaminobenzidine and then counterstained with hematoxylin, dehydrated and mounted. Then, the sections were observed under an inverted microscope (Nikon, Tokyo, Japan). The number of positive cells was counted using ImageJ software version 1.8 (National Institute of Health, Bethesda, MD, USA).
The experimental operation was performed in accordance to the instructions of the TUNEL staining kit (Sigma-Aldrich, St. Louis, MO, USA). Sections were photographed using a microscope (Olympus, Tokyo, Japan).
Total RNA was isolated with TRIzol reagent (Thermo Fisher Scientific). cDNA was synthesized with HiFiScript cDNA synthesis kit (Life Technologies, Carlsbad, CA, USA). Then, the cDNA was used for qRT-PCR assay using SYBR (Thermo Fisher Scientific). The relative expressions of mRNAs were normalized by glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and calculated by 2−ΔΔCT method. The primers used in the study were listed as follows (5′-3′): SP1 (F): 5′-GACAGGACCCCCTTGAGCTT-3′, SP1 (R): 5′-GGCACCACCACCATTACCAT-3′; LncRNA MCF2L-AS1 (F): 5′-GATCAACGTTCAATCCACCG-3′, LncRNA MCF2L-AS1 (R): 5′-CGTCAAGATAGCGCAGCTTCC-3′; IGF2BP1 (F): 5′-CCAAGGAGGAAGTGAAGCTG-3′, IGF2BP1 (R): 5′-ATGTCTCGGATCTTCCGTTG-3′; GAPDH (F): 5′-CCAGGTGGTCTCCTCTGA-3′, GAPDH (R): 5′-GCTGTAGCCAAATCGTTGT-3′.
The proteins were isolated from cells by using RIPA buffer, and concentrations of protein were determined by a BCA Kit (Beyotime), which further transferred to a PVDF membrane (Millipore). Then, membranes incubated overnight with antibodies against Bax (1:1,000, ab32503; Abcam), B-cell lymphoma-2 (Bcl-2) (1:1,000, ab182858; Abcam), Cleaved Caspase-3 (1:1,000, ab2302; Abcam), IGF2BP1 (1:1,000, ab100999; Abcam), IGF2 (1:1,000, ab177467; Abcam), p-MEK (1:1,000, 2338; Cell Signaling Technology, Danvers, MA, USA), mitogen-activated protein kinase (MEK) (1:1,000, 4694; Cell Signaling Technology), p-ERK (1:1,000, 4370; Cell Signaling Technology), extracellular regulated protein kinase (ERK) (1:1,000, ab32537; Abcam) and GAPDH antibody (1:10,000, SAB2701826; Sigma-Aldrich). After washed with PBS-T, membranes were then incubated with the corresponding secondary antibody (1:10,000, ab7090; Abcam) for 60 minutes. The membranes were visualized and imaged by GEL imaging system (Bio-Rad, Hercules, CA, USA). The quantification of proteins was analyzed by the software Image J.
All data were obtained from at least 3 replicate experiments. Results were expressed as mean±standard deviation (SD). Statistical analysis was performed using SPSS 19.0 (IBM, Armonk, NY, USA). The comparisons of 2 different group parameters and multi-group were determined using Student’s t-test and one-way analysis of variance (ANOVA), respectively. The p-values less than 0.05 were considered significant.
Through GEPIA database, we found that MCF2L-AS1 was markedly upregulated in ovarian cancer tumor tissues compared to non-tumor tissues, indicating that MCF2L-AS1 was involved in ovarian cancer development. However, whether MCF2L-AS1 participates in ovarian cancer cisplatin-resistance remains unknown. To probe the roles of MCF2L-AS1 in regulating cisplatin resistance in ovarian cancer cells, cells were exposed to increasing concentrations of cisplatin as previously described [21] to construct SKOV3/DDP and IGROV-1/DDP cells. The results displayed that the half maximal inhibitory concentration (IC50) of cisplatin-resistant cells was greater than that of the parental cells (Fig. 1A). As revealed in Fig. 1B, MCF2L-AS1 expression was significantly elevated in resistant ones compared to parental cells. To further probe the relationship between MCF2L-AS1 expression and cisplatin resistance in ovarian cancer, we knocked down MCF2L-AS1 expression in SKOV3/DDP and IGROV-1/DDP cells (Fig. 1B). The results from CCK-8 assay and colony formation assay subsequently demonstrated that MCF2L-AS1 knockdown remarkably inhibited proliferation and colony formation of cisplatin-resistant cells (Fig. 1C-D). In addition, MCF2L-AS1 silencing promoted cell apoptosis of cisplatin-resistant cells (Fig. 1E). Similarly, MCF2L-AS1 knockdown significantly upregulated Bax and Cleaved Caspase-3 levels, while reduced Bcl-2 level (Fig. 1F). Moreover, we detected the effect of MCF2L-AS1 knockdown on cisplatin sensitivity in parental cells (SKOV3 and IGROV-1 cells). The expression of MCF2L-AS1 in SKOV3 and IGROV-1 cells was decreased after transfected with sh-MCF2L-AS1 (Fig. S1A). As shown in Fig. S1B, the IC50 of SKOV3 and IGROV-1 cells was significantly reduced after MCF2L-AS1 knockdown, suggesting that cisplatin sensitivity in parental cells was enhanced by MCF2L-AS1 silencing. Moreover, MCF2L-AS1 knockdown remarkably inhibited parental cell proliferation (Fig. S1C) and promoted cell apoptosis (Fig. S1D). Taken together, MCF2L-AS1 silencing remarkably inhibited cisplatin resistance in ovarian cancer cells.
We sought to identify the target of MCF2L-AS1 to explore the molecular mechanisms by which MCF2L-AS1 exerted its effects on ovarian cancer cisplatin-resistance. RPISeq prediction was first employed to identify MCF2L-AS1 interaction proteins and revealed that IGF2BP1 was a potential binding protein of MCF2L-AS1 (prediction using RF classifier: 0.7 score; prediction using SVM classifier: 0.77 score). RNA pull-down and RIP assay subsequently displayed that MCF2L-AS1 directly interacted with IGF2BP1 (Fig. 2A and B). As demonstrated in Fig. 2C and D, IGF2BP1 expression was significantly elevated in cisplatin-resistant cells. We observed that sh-MCF2L-AS1 transfection did not have any impact on the mRNA level of IGF2BP1 in cisplatin-resistant cells (Fig. 2E). However, the protein level of IGF2BP1 was markedly reduced by MCF2L-AS1 silencing, as well as the protein levels of IGF2/MEK/ERK signaling pathway-related proteins (IGF2, p-MEK and pERK) (Fig. 2F). All these results suggested that lncRNA MCF2L-AS1 activated the IGF2/MEK/ERK signaling pathway through interacting with IGF2BP1 protein.
Next, we aimed to explore the effects of IGF2BP1 on MCF2L-AS1-mediated biological functions in vitro. Thus, we overexpressed IGF2BP1 alone and co-transfected with sh-MCF2L-AS1 and oe-IGF2BP1 in SKOV3/DDP and IGROV-1/DDP cells. First, IGF2BP1 expression was markedly reduced following sh-MCF2L-AS1 transfection, while IGF2BP1 expression was significantly elevated in sh-MCF2L-AS1 + Oe-IGF2BP1 group compared to sh-MCF2L-AS1 + Oe-NC group (Fig. 3A). Overexpression IGF2BP1 remarkably promoted proliferation and colony formation of cisplatin-resistant cells, which could abolish the inhibitory effects of MCF2L-AS1 silencing on proliferation and colony formation of cisplatin-resistant cells (Fig. 3B and C). Besides, cell apoptosis of cisplatin-resistant cells was inhibited after IGF2BP1 overexpression, which reversed the promotion effect of MCF2L-AS1 silencing on cell apoptosis of cisplatin-resistant cells (Fig. 3D). Finally, IGF2BP1 overexpression significantly inhibited Bax and Cleaved Caspase-3 expressions, while promoted Bcl-2 expression, and the effects of MCF2L-AS1 silencing on the levels of these apoptosis-related protein were eliminated (Fig. 3E). Moreover, we detected the effect of IGF2BP1 overexpression on cisplatin sensitivity in parental cells (SKOV3 and IGROV-1 cells). It was observed that the IC50 of SKOV3 and IGROV-1 cells was significantly increased after IGF2BP1 overexpression (Fig. S2), suggesting that cisplatin sensitivity in parental cells was weakened by IGF2BP1 overexpression. In summary, IGF2BP1 overexpression could promoted cisplatin resistance, and reverse the effects of MCF2L-AS1 silencing on cisplatin resistance in ovarian cancer.
Much evidence has suggested that several transcription factors contribute to lncRNAs dysregulation in human malignancies [22]. The factors involved in MCF2L-AS1 dysregulation in ovarian cancer remained elusive. Through JASPAR database, we found that a classic transcription factor SP1 had potential binding sites to MCF2L-AS1 promoter region (Fig. 4A). ChIP assay subsequently revealed that SP1 could bind to P1 site of MCF2L-AS1 promoter (Fig. 4B). Subsequently, SP1 overexpression upregulated the luciferase activity of P1-WT, while the luciferase activity of P1-MUT was not affected (Fig. 4C), further suggesting that SP1 could bind to P1 site of MCF2L-AS1 promoter. Moreover, SP1 expression was significantly elevated in cisplatin-resistant cells (Fig. 4D and E). Subsequently, MCF2L-AS1 expression in cisplatin-resistant cells was markedly elevated after SP1 overexpression (Fig. 4F). In conclusion, SP1 activated MCF2L-AS1 expression in cisplatin-resistant ovarian cancer cells.
Next, we aimed to probe the effects of the SP1/lncRNA MCF2L-AS1 axis on cisplatin resistance in ovarian cancer. Thus, we co-transfected with oe-SP1 and sh-MCF2L-AS1 in cisplatin-resistant cells. First, SP1 and MCF2L-AS1 expressions significantly increased after SP1 overexpression; MCF2L-AS1 expression in oe-SP1 + sh-MCF2L-AS1 group was markedly reduced compared to oe-SP1 + sh-NC group, while SP1 expression was not significantly different (Fig. 5A). SP1 overexpression remarkably promoted proliferation and colony formation of SKOV3/DDP and IGROV-1/DDP cells, while this phenomenon was abolished by MCF2L-AS1 knockdown (Fig. 5B and C). Cell apoptosis were markedly suppressed by oe-SP1 transfection, while it was eliminated by sh-MCF2L-AS1 transfection in SKOV3/DDP and IGROV-1/DDP cells (Fig. 5D). After SP1 overexpression, Bax and Cleaved Caspase-3 levels in cisplatin-resistant cells were obviously reduced and Bcl-2 level was significantly elevated, while MCF2L-AS1 silencing attenuated this effect (Fig. 5E). In total, MCF2L-AS1 knockdown mitigated the promotion effects of SP1 overexpression on ovarian cancer cisplatin-resistance.
To probe the effect of MCF2L-AS1 on cisplatin resistance in vivo, SKOV3/DDP stably transfected with sh-NC or sh-MCF2L-AS1 were injected into the back of nude mice, and then all mice were received cisplatin treatment. As displayed in Fig. 6A-C, after cisplatin treatment, tumor injected with sh-MCF2L-AS1 grew slower than controls, suggesting that MCF2L-AS1 knockdown promoted the cell cytotoxicity induced by cisplatin treatment in vivo. In addition, IHC displayed that the protein level of Ki67 (proliferation marker) in tumor tissues was obviously decreased following MCF2L-AS1 knockdown (Fig. 6D). TUNEL positive cell in tumor tissues were increased by MCF2L-AS1 knockdown (Fig. 6E). As shown in Fig. 6F, MCF2L-AS1 expression in tumor tissues was significantly reduced after MCF2L-AS1 knockdown. Finally, we observed that Bax and Cleaved Caspase-3 levels increased, while the protein levels of IGF2/MEK/ERK signaling pathway-related proteins, IGF2BP1 and Bcl-2 in tumor tissues were significantly reduced after MCF2L-AS1 knockdown (Fig. 6G). In summary, MCF2L-AS1 silencing suppressed ovarian cancer cisplatin-resistance in vivo.
For researchers, the mechanism of ovarian cancer cisplatin-resistance is still elusive. Although many molecular medical studies have been performed to explain the mechanism of drug resistance in ovarian cancer, it is difficult to distinguish effective targets for regulating drug resistance. The epigenetic regulation of ovarian cancer may unravel this mystery. Herein, we found that SP1-induced MCF2L-AS1 promoted cisplatin resistance in ovarian cancer through targeting the IGF2BP1/IGF2/MEK/ERK signaling pathway. The important function of lncRNAs in regulating ovarian cancer cisplatin-resistance is obvious to all. For example, Miao et al. revealed that lncRNA ANRIL reduced the sensitivity of ovarian cancer to cisplatin by targeting miR-let-7a [23]. LncRNA MCF2L-AS1 was previously confirmed as an oncogene in several human malignancies, such as colorectal cancer [24] and lung cancer [12]. However, the role of MCF2L-AS1 in regulating cisplatin resistance in ovarian cancer remains unknown. Herein, our results revealed that MCF2L-AS1 was markedly upregulated in cisplatin-resistant cells. Besides, we found that MCF2L-AS1 silencing remarkably inhibited cell growth and promoted cell apoptosis of ovarian cancer parental cells and cisplatin-resistant cells at the first time. Additionally, our results firstly confirmed that MCF2L-AS1 silencing suppressed cisplatin resistance in ovarian cancer in vivo. It has been widely described that lncRNAs also function in regulating cisplatin sensitivity of ovarian cancer. For instance, Miao et al. revealed that lncRNA ANRIL silencing improved cisplatin-sensitivity of ovarian cancer cells [23]. LncRNA LINC01125 enhanced cisplatin sensitivity of ovarian cancer via acting on miR-1972 [25]. In the present study, our results revealed that the IC50 of SKOV3 and IGROV-1 cells was significantly reduced after MCF2L-AS1 knockdown, suggesting that cisplatin sensitivity in parental cells was enhanced by MCF2L-AS1 silencing. Moreover, MCF2L-AS1 knockdown remarkably inhibited parental cell proliferation and promoted cell apoptosis. Collectively, our results suggested that MCF2L-AS1 downregulation contributed to cisplatin sensitivity of parental cells and impaired cisplatin resistance of cisplatin-resistant cells. Platine-based chemotherapy agents are major drugs in oncology and are currently used in most solid malignancies. Of these, cisplatin and carboplatin have been the most widely used over past years. Many trials performed comparing the efficacy of carboplatin and cisplatin in ovarian cancer, either as single agent first-line therapy or in combination chemotherapy suggest equivalent results [26]. However, superior results in terms of either progression-free survival or overall survival have been obtained with cisplatin in 2 larger randomized studies of combination chemotherapy [26]. Our study aimed to explore the drug resistance mechanism of ovarian cancer, which layed a foundation for clinical research. Considering that the mechanism of action of these 2 drugs for ovarian cancer is similar, and the efficacy of cisplatin is better than that of carboplatin, it is more beneficial to explore the regulatory mechanism of cisplatin resistance in ovarian cancer. In the future, we will also conduct research on carboplatin resistance in ovarian cancer. Recent studies have suggested that SP1 is a key transcription factor regulating the abnormal expressions of lncRNAs in cancers [2728]. Meanwhile, it was widely reported that SP1 is a risk factor promoting cisplatin resistance in ovarian cancer [529]. Herein, SP1 was confirmed to bind to MCF2L-AS1 promoter by using ChIP and luciferase reporter assays, which was never reported before. In addition, as expected, MCF2L-AS1 knockdown could mitigate the promotion effects of SP1 overexpression on ovarian cancer cisplatin-resistance. For the first time, our study revealed that SP1 contributed to ovarian cancer cisplatin-resistance by activating the transcriptional activity of MCF2L-AS1. IGF2BP1 is a type of oncofetal RNA binding protein, which is upregulated in a variety of solid tumors, including ovarian cancer [16]. Meanwhile, the high expression of IGF2BP1 in ovarian cancer was also related to cisplatin resistance, specifically IGF2BP1 overexpression could enhance cisplatin resistance in ovarian cancer [30]. In the present study, IGF2BP1 expression was markedly elevated in SKOV3/DDP and IGROV-1/DDP cells. Moreover, this current report was the first one to indicate that MCF2L-AS1 could interact with IGF2BP1 protein. Our results revealed that MCF2L-AS1 knockdown significantly reduced IGF2BP1 protein level but not the mRNA level in cisplatin-resistant cells. It was suggested that MCF2L-AS1 might be involved in regulating the post-transcriptional level of IGF2BP1 through other regulatory mechanisms, such as acetylation, ubiquitination and other modifications. As reported, lncRNA NEAT1 could regulate the DDX5 protein to enhance its stability in colorectal cancer cells [31]. In addition, lncRNA HOTAIR regulated the ATXN1 protein but not the mRNA by ubiquitination [32]. However, the specific regulatory mechanism of MCF2L-AS1 on the post-transcriptional level of IGF2BP1 remains unknown and needs further study. By analyzing the functions in depth, it was evident that IGF2BP1 overexpression promoted cisplatin resistance in cisplatin-resistant cells and reversed the effects of MCF2L-AS1 silencing on cisplatin resistance. Moreover, our results revealed that IGF2BP1 overexpression resulted in increased IC50 of SKOV3 and IGROV-1 cells, suggesting that cisplatin sensitivity in parental cells was weakened by IGF2BP1 overexpression. According to reports, IGF2BP1 usually achieves its biological function in cancers by regulating the expression of target mRNA. IGF2, one of the targets of IGF2BP1, is associated with an acquired drug resistance in cancer [33]. Yang et al. [20] also revealed that IGF2BP1 could promote cell proliferation ongue squamous cell carcinoma cells through the activation of IGF2/MEK/ERK signaling pathway. Herein, we found that MCF2L-AS1 silencing markedly inhibited IGF2 level, as well as MEK/ERK signaling pathway-related proteins (p-MEK and pERK). Therefore, we speculated that lncRNA MCF2L-AS1 promoted cisplatin resistance in ovarian cancer through regulating IGF2BP1/IGF2/MEK/ERK axis. In summary, our study displayed that SP1-induced MCF2L-AS1 promoted ovarian cancer cisplatin-resistance through activation of IGF2/MEK/ERK pathway via interacting with IGF2BP1, which illustrated that MCF2L-AS1 had a potential therapeutic value for the treatment of cisplatin resistance in ovarian cancer patients. | true | true | true |
PMC9634122 | Qiufang Bao,Qiaomei Zheng,Shaoyu Wang,Wenlu Tang,Bin Zhang | LncRNA HOTAIR regulates cell invasion and migration in endometriosis through miR-519b-3p/PRRG4 pathway | 21-10-2022 | endometriosis,LncRNA HOTAIR,miR-519b-3p/PRRG4,invasion,migration | Endometriosis is a common benign disease in gynecology and has malignant biological behaviors, such as hyperplasia, invasion, metastasis, and recurrence. However, the pathogenesis of endometriosis remains unclear. The present study aimed to investigate whether LncRNA HOTAIR regulates cell invasion and migration in endometriosis by regulating the miR-519b-3p/PRRG4 pathway. The qRT-PCR results showed that the average relative expression of LncRNA HOTAIR was much higher in ectopic endometrial tissues than in eutopic endometrial tissues. Scratch and transwell assays showed that the cell migration and invasion ability of LncRNA HOTAIR overexpression group was significantly higher than those in the control group. Conversely, the LncRNA HOTAIR knockdown group showed the opposite results. Bioinformatics analysis suggested that the downstream target genes of LncRNA HOTAIR were miR-519b-3p and Prrg4. Knockdown of LncRNA HOTAIR can reduce the up-regulation of Prrg4 by miR-519b-3p and then inhibit the invasion and migration ability of endometrial stromal cells. In Conclusion, LncRNA HOTAIR can regulate the ability of invasion and migration of endometrial stromal cells, and its mechanism is proved by regulating the miR-519b-3p/PRRG4 pathway. | LncRNA HOTAIR regulates cell invasion and migration in endometriosis through miR-519b-3p/PRRG4 pathway
Endometriosis is a common benign disease in gynecology and has malignant biological behaviors, such as hyperplasia, invasion, metastasis, and recurrence. However, the pathogenesis of endometriosis remains unclear. The present study aimed to investigate whether LncRNA HOTAIR regulates cell invasion and migration in endometriosis by regulating the miR-519b-3p/PRRG4 pathway. The qRT-PCR results showed that the average relative expression of LncRNA HOTAIR was much higher in ectopic endometrial tissues than in eutopic endometrial tissues. Scratch and transwell assays showed that the cell migration and invasion ability of LncRNA HOTAIR overexpression group was significantly higher than those in the control group. Conversely, the LncRNA HOTAIR knockdown group showed the opposite results. Bioinformatics analysis suggested that the downstream target genes of LncRNA HOTAIR were miR-519b-3p and Prrg4. Knockdown of LncRNA HOTAIR can reduce the up-regulation of Prrg4 by miR-519b-3p and then inhibit the invasion and migration ability of endometrial stromal cells. In Conclusion, LncRNA HOTAIR can regulate the ability of invasion and migration of endometrial stromal cells, and its mechanism is proved by regulating the miR-519b-3p/PRRG4 pathway.
Endometriosis is a common benign disease in gynecology and has malignant biological behaviors, such as hyperplasia, invasion, metastasis, and recurrence (1–3). The main clinical features of endometriosis are dysmenorrhea, infertility, and menstrual irregularities (4). At present, the hypothesis of endometriosis includes the theory of retrograde menstruation, metaplasia of the coelom, vascular and lymphatic metastatic spread, epithelial mesenchymal transformation, altered inflammatory response, genetic susceptibility immunity, endometrial determination, and so on (4–6). However, the molecular pathogenesis of endometriosis is still not fully clarified. Long non-coding RNAs (LncRNAs) have a length >200 nucleotides and have no protein-coding function. Recently, LncRNAs have been proved to play important roles in cell proliferation, differentiation, apoptosis, and metastasis (7). Emerging evidence has demonstrated that LncRNAs may have the potential to influence the development and persistence of cancer and endometriosis by modulating inflammation, proliferation, giogenesis, and tissue remodeling (8). LncRNA HOTAIR (Homeobox transcript antisense RNA) is highly expressed in cervical cancer, endometrial cancer, and other cancers which is proved to be related to the occurrence and metastasis of tumor (9, 10). The overexpression of LncRNA HOTAIR is associated with tumor invasion, progression, metastasis, and poor prognosis (11). Recent research showed that the expression level of LncRNA HOTAIR was elevated in endometriosis. Zhang et al. (12) found that the expression level of LncRNA HOTAIR was elevated in ectopic lesions of endometriosis. Chang et al. (13) found that patients with aggressive endometriosis expressed higher levels of LncRNA HOTAIR. Genetic alterations in LncRNA HOTAIR may be one of the risk factors leading to endometriosis development. However, the specific mechanism of LncRNA HOTAIR in the pathogenesis of endometriosis is still unclear. A number of studies have found that LncRNAs can act as competing endogenous RNAs (ceRNAs) to bind microRNA (miRNA), regulate cell function, mediate the invasion and metastasis of cells (14). miRNAs are a class of 22 nucleotide non-coding small RNA molecules, which can bind to the 3′ untranslated regions of target gene mRNA mainly through base complementation. Moreover, they can regulate gene expression at the post-transcriptional level by degrading the target mRNA or inhibiting protein synthesis. Wang et al. (15) found that LncRNA HOTAIR can regulate the expression level of miR-326 negatively to regulate proliferation and migration in lung cancer. LncRNA HOTAIR can also serve as a sponge of miR-331-3p to regulate HER2 expression in gastric cancer (16). Zhang et al. (12) reported that exosomal LncRNA HOTAIR promotes the progression and angiogenesis of endometriosis via the miR-761/HDAC1 axis. Although there are a few reports on the regulation of endometriosis by LncRNA HOTAIR, its molecular mechanism has not been fully clarified. In this study, we found that LncRNA HOTAIR was much higher in ectopic endometrial tissues than in eutopic endometrial tissues, which could promote the invasion and migration ability of endometrial stromal cells. Bioinformatics analysis suggested that the downstream target genes of LncRNA HOTAIR were miR-519b-3p and Prrg4. Further studies indicated that LncRNA HOTAIR regulates cell invasion and migration in endometriosis by regulating the miR-519b-3p/PRRG4 pathway, which may prove to be potential markers and new targets for early diagnosis and treatment of endometriosis.
Paired ectopic and eutopic endometrial tissues were collected from 20 patients with ovarian endometriotic cysts, and normal endometrial tissues were collected from 10 patients without endometriosis who had surgery for other benign ovarian cysts between April 2020 and November 2020. Patients with endometriosis were pathologically confirmed. All patients between 20 and 40 years of age were examined in the Department of Gynecology and Obstetrics at the First Affiliated Hospital of Fujian Medical University in Fuzhou, People’s Republic of China. All patients in the present study had regular menstrual cycles, had not taking any combined hormonal contraception for at least six months prior to surgery, and had no other malignant, estrogen-dependent, immune, surgical, or inflammatory diseases. Patients were not classified according to menstrual cycle (menstrual cycle was divided into proliferative period, secretory period and menstrual period). We examined all cases and grouped them according to the time of menstruation: reproductive period and secretory period. There were 9 cases of reproductive period and 11 cases of secretory period in the experimental group, and 5 cases of reproductive period and secretory period in the control group. All samples for detecting LncRNA HOTAIR, miR-519b-3b and PRRG4 were from the same patient. All specimens were immediately frozen in liquid nitrogen and stored at -80°C. This study was approved by the Ethics Committee of the First Affiliated Hospital of Fujian Medical University, Fuzhou, Fujian, China (approval number:2020 [248]). All the patients signed an informed consent form.
We purified primary human endometrial stromal cells as described in previous study (12). In brief, ectopic endometrial samples from patients with ovarian endometriotic cysts (proliferative period) were cut into small pieces and digested by collagenase IV and deoxyribonuclease (Sangon Biotech, Shanghai, China) for 1 h. The tissue suspension was filtered through nylon cell strainers, and stromal cells were passed through the strainer in the filtrate. Then, the suspension was centrifuged at 1000 × g for 5 min at room temperature. This action was followed by culture in red blood cell lysis buffer for 10 min to remove erythrocytes. The separated cells were cultured in DMEM/F-12(Gibco, Grand Island, USA) supplemented with 10% fetal bovine serum (Gibco, Grand Island, USA) at 37 °C under humidified air containing 5% CO2. The media was replaced every two days. The purified cells were used for subsequent experiments. The plasmid of specific small interfering RNA targeting HOTAIR(si-HOTAIR) and negative control (si-NC), the plasmid of Prrg4 overexpression (PRRG4 OE) and the control (vector) were constructed and synthesized by Anti-HeLa Biological Technology Trade, Xiamen, Fujian, China. miR-519b-3p mimics, miR-519b-3p inhibitor, and NC inhibitor were purchased from RiboBio (Guangzhou, China).
The purified cells were cultured for 48 hours, then the plasmids of HOTAIR-OE, si-HOTAIR, and miR-519b-3p inhibitor were transfected into endometrial stromal cells by using Lipofectamine™2000 reagent according to the manufacturer’s instructions. After transfection for 48 h, qRT-PCR was used to detect the expression level of miR-519b-3p. qRT-PCR and WB analyses were conducted to detect the mRNA and protein expression levels of PRRG4. The endometrial stromal cells were divided into six groups for transfected si-NC, si-HOTAIR, si-HOTAIR+inhibitor NC, si-HOTAIR+miR-519b-3p inhibitor, si-HOTAIR+Vector, and si-HOTAIR+PRRG4 OE by using Lipofectamine™2000 reagent according to the manufacturer’s instructions, respectively. At 48 h after transfection, qRT-PCR and WB analyses were performed to detect the mRNA and protein expression levels of PRRG4.
In brief, 50 mg of tissues were obtained for ultrasonic homogenization. Total RNA was extracted using an RNA extraction kit (Novazan, Nanjing, China) following the manufacturer’s instructions. RNA concentration was measured using spectrophotometry, and purity was evaluated by the ratio of absorbance at 260 nm to 280 nm (A260/A280). qRT-PCR analysis was performed using an RT-PCR Kit (Novazan, Nanjing, China) according to the manufacturer’s instructions. Each sample was run in triplicate. The expression level of Prrg4 was normalized using 18s as the internal control and calculated using 2−ΔΔCt method. The sequences of the qRT-PCR primers were as follows: LncRNA HOTAIR, F:5′-AATAGACATAGGAGAACAC TT-3′, R:5′-AATCTTAATAGCAGGAGGAA-3′, miR-519b-3p F:5′-CGCGAAAGT GCATCCTTTTA-3′, R:5′-AGTGCAGGGTCCGAGGTAT-3′, 18s, F:5′-AGGCGC GCAAATTACCCAATCC-3′, R:5′-GCCCTCCAATTGTTCCTCGTTAAG-3′. PRRG4, F:5′-GGGAGAAGAAGTGTTTAC-3′, R:5′-CTGGCTTCCTCATAATTG-3′.
Transfected cells were harvested and rinsed using phosphate buffered saline (PBS). Total cell protein was extracted using protein lysate of RIPA, and concentration was detected with a BCA protein assay kit (Hyclone, Logan, USA). The extracted protein was collected for sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto the polyvinylidene fluoride membrane. The membranes were blocked with skim milk for 1 h, stored overnight with PRRG4 and GAPDH primary antibodies, cleaned with TBST, and probed with HRP-conjugated secondary antibody for 2 h at room temperature. Finally, ECL chemiluminescence solution was added to analyze the gray level of protein bands and calculate the protein level of PRRG4.
The PARIS™ Kit (Ambion, Austin, TX) was used for nucleus–cytoplasm separation. Approximately 5×106 endometrial stromal cells were placed in nucleoplasmic separation lysis fluid for resuspension and treated on ice for 10 min, which was inverted and mixed every 1 min. After centrifugation at 11,000 r/min for 10 min at 4°C, the supernatant was taken as the cytoplasm sample and precipitated into the nucleus. The content distribution of LncRNA HOTAIR in the nucleus and cytoplasm was detected by qRT-PCR. Then, the subcellular localization of LncRNA HOTAIR was determined. U6 and 18s were used as a positive control for the expression of nuclear RNA and cytoplasmic RNA, respectively.
We used TargetScan to predict the binding sites of miR-519b-3p with LncRNA HOTAIR or Prrg4. The gene fragments of LncRNA HOTAIR or Prrg4 were cloned and inserted into the luciferase reporter gene PmirGLO to construct wild-type plasmids of LncRNA HOTAIR or Prrg4 (HOTAIR-Wt, PRRG4-Wt). The mutant type plasmids of LncRNA HOTAIR or Prrg4 (HOTAIR-Mut, PRRG4-Mut) were constructed with the mutant sequences of LncRNA HOTAIR or Prrg4 gene fragments. The endometrial stromal cells were co-transfected with miR-519b-3p mimics and HOTAIR-Mut or HOTAIR-Wt by using Lipofectamine™2000 reagent (Thermo Fisher Scientific, San Jose, USA). The cells were then co-transfected with miR-519b-3p mimics and PRRG4-Mut or PRRG4-Wt with the same method. After 48 h in an incubator at 37°C and 5% CO2, the co-transfected cells were harvested. Luciferase activity was detected using the dual-luciferase assay reporter kit (Promega, Madison, WI, USA).
Cells from each group were transfected in a six-well plate and cultured until they reached 90% confluence. A wound was created with a pipette tip and cultured with serum-free DMEM/F12 medium (Gibco, Grand Island, USA). According to the images captured using an inverted microscope at 0 and 48 h, the migration ability of the cells was determined by calculating the distance to the edge of the cell scratches.
For cell invasion assays, 24-well transwell plates containing 8 μm pore size inserts was paved with 50 μL of 10 mg/mL Matrigel (BD Biosciences, San Jose, USA). Cells from each group were diluted in serum-free DMEM/F12 medium, and 200 μL of the solution containing 10000 cells was inoculated in the upper chamber. About 600 μL of 20% FBS medium was added to the lower chamber. After 48 h in the incubator, the cell culture medium was sucked up with a pipette, and cells in the upper layer were gently wiped off with a cotton swab. Cells in the lower layer were washed with PBS, fixed in 4% paraformaldehyde for 20 min, and finally stained with 1% crystal violet at room temperature for 10 min. Excess dye solution was then washed off, and the cells were photographed using a light microscope. Cells in five randomly selected fields were counted. Cell migration assays were performed using similar method but without matrigel.
GSE40186 data (https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE40186, miRNA expression profiles in endometriotic cyst stromal cells (ECSCs)), starBase (http://starbase.sysu.edu.cn/index.php), miRNet (http://mirdb.org/index.html), and LncBase (https://www.mirnet.ca/miRNet/faces/home.xhtml) were used to predict the downstream miRNA of LncRNA HOTAIR, and the intersection of the results was used to obtain the key miRNA. After obtaining the key miRNA, GSE86534 data (https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE86534,Genome-wide long non-coding RNAs and mRNAs analysis of the tissues related to ovarian endometriosis), MicroT-CDS (http://diana.imis.athena-innovation.gr/DianaTools/index.php?r=microT_CDS/index), miRDB (http://mirdb.org/index.html), mirDIP (http://ophid.utoronto.ca/mirDIP/index.jsp#r), miRTarBase (https://mirtarbase.cuhk.edu.cn/~miRTarBase/miRTarBase2019/php/index.php), and starBase were used to predict the target mRNA of miRNA, and the intersection of the results was used to obtain the key mRNA.
All statistical analyses were performed using SPSS Statistics version 21. Data are presented as means ± standard error of the mean (SEM) or median and range. Data from multiple groups were compared using one-way ANOVA, followed by Bonferroni’s post hoc test. Data were considered statistically significant at P<0.05.
To investigate the expression level of HOTAIR, ectopic and eutopic endometrial tissues from 20 ovarian endometriosis patients and normal endometrial tissues from 10 control patients were performed by qRT-PCR. The results suggested that the relative expression of LncRNA HOTAIR in ectopic endometrial tissues 8.45 (4.96 -11.23) was significantly higher than that in eutopic endometrial tissues 5.76(1.18-7.95) (P < 0.05). The relative expression of LncRNA HOTAIR in ectopic endometrial tissues was slightly higher than that in normal endometrial tissues 5.08 (3.24-6.55), but the difference was not significant (P =0.067) ( Figure 1A ).
To understand the role of LncRNA HOTAIR in regulating the migration and invasion of endometrial stromal cells, endometrial stromal cells were transfected with siRNAs designed against LncRNA HOTAIR (si-HOTAIR-1, si-HOTAIR-2), and negative control (si-NC), over expression LncRNA HOTAIR, and empty vector (NC) (used as control). The results showed that the overexpression of LncRNA HOTAIR transfected group in endometrial stromal cells exhibited significantly higher expression compared with control cells ( Figure 1B , P < 0.01). si-HOTAIR-1 and si-HOTAIR-2 transfected group exhibited significantly lower expression in endometrial stromal cells compared with control cells. si-HOTAIR-1 showed better effects, so si-HOTAIR-1 was used for subsequent experiments ( Figure 1C , P < 0.01). The results indicated that the transfection was successful and used for subsequent experiments. The cell scratch assays showed that the cell distance of endometrial cells in the overexpression LncRNA HOTAIR transfected group was significantly smaller than that in the control group (NC). The cell distance of endometrial cells in the si-HOTAIR transfected group was significantly larger than that in the control transfected group (si-NC) ( Figures 1D, E ). The transwell assays of invasion and migration showed that the number of transmembrane cells in the overexpression LncRNA HOTAIR transfected group was significantly higher than that in the control group (NC). The number of transmembrane cells in the si-HOTAIR transfected group was significantly lower than that in the control group (si-NC) (P < 0.01; Figures 1F–H ). These results suggest that regulating LncRNA HOTAIR expression may alter the migration and invasion ability of cells.
To examine the subcellular localization of LncRNA HOTAIR and whether it could act as a miRNA sponge in endometriosis, we determined the subcellular localization of LncRNA HOTAIR using nucleus–cytoplasm separation. The results of qRT-PCR showed that the expression of LncRNA HOTAIR in the cytoplasm was higher than that in the nucleus (P < 0.05) ( Figure 2A ). The expression of LncRNA HOTAIR was mostly located in the cytoplasm. 18s was mostly located in the cytoplasm, while U6 was mostly located in the nucleus. These finding suggests that LncRNA HOTAIR mainly located in the cytoplasm which may play a regulatory role in the pathogenesis of endometriosis by binding and interacting with miRNA. To predict the downstream miRNAs of LncRNA HOTAIR, we first screened out 271 miRNAs with low expression in endometriotic cyst stromal cells by using GSE40186 data (https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE40186, miRNA expression profiles in endometriotic cyst stromal cells (ECSCs)). Then, 45 miRNAs were predicted by starBase (http://starbase.sysu.edu.cn/index.php), 45 miRNAs were predicted by miRNet (http://mirdb.org/index.html), and 209 miRNAs were predicted by LncBase (https://www.mirnet.ca/miRNet/faces/home.xhtml). The four online prediction results were overlapped by Venn diagram analysis ( Figure 2B ). Two candidate miRNAs were screened out, namely, miR-519b-3p and miR-326. We continued to predict the downstream mRNAs of miRNA. First, we predicted the downstream targeted mRNAs for miR-519B-3p. We screened out 267 mRNAs with high expression in ovarian endometriosis by using GSE86534 data (https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE86534,Genome-wide long non-coding RNAs and mRNAs analysis of the tissues related to ovarian endometriosis). Then, a total of 1333 mRNAs were predicted by MicroT-CDS (http://diana.imis.athena-innovation.gr/DianaTools/index.php?r=microT_CDS/index), 988 mRNAs were predicted by miRDB (http://mirdb.org/index.html), 1235 mRNAs were predicted by mirDIP (http://ophid.utoronto.ca/mirDIP/index.jsp#r), 344 mRNAs were predicted by miRTarBase (https://mirtarbase.cuhk.edu.cn/~miRTarBase/miRTarBase2019/php/index.php), and 3174 mRNAs were predicted by starBase (http://starbase.sysu.edu.cn/index.php). These online prediction results were overlapped by Venn diagram analysis ( Figure 2C ). Finally, the downstream target mRNA (PRRG4) of miR-519b-3p was identified. However, the downstream target mRNA of miR-326 was not found.
To determine whether LncRNA HOTAIR can regulate endometrial stromal cells by sponging miR-519b-3p, we detected the expression level of miR-519b-3p in patients with endometriosis and control patients by qRT-PCR. The results suggested that the relative expression of miR-519b-3p in ectopic endometrial tissues 3.75(0.43-5.64) was significantly lower than that in eutopic endometrial tissues 6.06 (1.58-9.34) (P < 0.05). The relative expression of miR-519b-3p in ectopic endometrial tissues was lower than that in normal endometrial tissues 5.72(2.36-7.95). The differences were all statistically significant (P < 0.05) ( Figure 2D ). To investigate the expression level of PRRG4, we evaluated the ectopic and eutopic endometrial tissues from 20 patients with ovarian endometriosis and 10 control patients with normal endometrial tissues by qRT-PCR. The relative expression level of PRRG4 in ectopic endometrial tissues 11.06(7.13-13.16) was significantly higher than that in eutopic endometrial tissues 5.36(0.02-7.89). The relative expression level of PRRG4 in ectopic endometrial tissues was significantly higher than that in normal endometrial tissues 8.25(7.12-9.67). The differences were all statistically significant (P<0.01, Figure 2E ).
To further explore the underlying mechanism of LncRNA HOTAIR in endometriosis, we used TargetScan to predict whether miR-519b-3p might be the target gene of LncRNA HOTAIR ( Figure 3A ). Dual-luciferase reporter assay was used to validate the targeting relationship between LncRNA HOTAIR and miR-519b-3p. The transfection with miR-519b-3p mimics significantly repressed the luciferase activity of HOTAIR-wt in endometrial stromal cells (P<0.01) but had no effect on the luciferase activity of HOTAIR-mut ( Figure 3B ). Thus, HOTAIR may mediate the degradation of miR-519b-3p in endometriosis. Subsequently, to determine whether miR-519b-3p expression is affected by LncRNA HOTAIR, we transfected endometrial stromal cells with si-NC and si-HOTAIR and detected miR-519b-3p expression by qRT-PCR. The knocked down of LncRNA HOTAIR significantly increased the expression level of miR-519b-3p (P<0.01, Figure 3C ). These results suggested that miR-519b-3p was the target gene of LncRNA HOTAIR and negatively regulated by LncRNA HOTAIR.
To further explore the underlying mechanism of miR-519b-3p in endometriosis, we used TargetScan to predict whether Prrg4 might be the target gene of miR-519b-3p ( Figure 4A ). Dual-luciferase reporter assay was used to detect the targeting relationship between miR-519b-3p and PRRG4. The transfection with miR-519b-3p mimic significantly repressed the luciferase activity of PRRG4-wt in endometrial stromal cells (P<0.01) but had no effect on the luciferase activity of PRRG4-mut ( Figure 4B ). Thus, miR-519b-3p may mediate the elevation of PRRG4 in endometriosis. Subsequently, to determine whether PRRG4 expression is affected by miR-519b-3p, we transfected endometrial stromal cells with inhibitor NC and miR-519b-3p inhibitor and detected PRRG4 expression by qRT-PCR and WB. The result showed that miR-519b-3p significantly increased the mRNA and protein expression levels of PRRG4 (P<0.01, Figures 4C–E ). Collectively, these results suggested that Prrg4 was a target gene of miR-519b-3p and negatively regulated by miR-519b-3p.
To clarify the relationship among LncRNA HOTAIR, miR-519b-3p, and PRRG4 in endometriosis, we conducted a rescue experiment. The transfected endometrial stromal cells were divided into the following groups: cells in the si-NC group were transfected with the negative control plasmid (si-NC), cells in the si-HOTAIR group were transfected with si-HOTAIR plasmid, cells in the si-HOTAIR+inhibitor NC group were co-transfected with si-HOTAIR plasmid and inhibitor NC, cells in the si-HOTAIR+miR-519b-3p inhibitor group were co-transfected with si-HOTAIR plasmid and miR-519b-3p inhibitor, cells in the si-HOTAIR+Vector group were co-transfected with si-HOTAIR plasmid and empty plasmid for PRRG4, and cells in the si-HOTAIR+PRRG4 OE group were co-transfected with si-HOTAIR plasmid and overexpression PRRG4 plasmid. The mRNA and protein levels of PRRG4 were all reduced by si-HOTAIR in endometrial stromal cells, while down-regulating miR-519b-3p or up-regulating PRRG4 reversed the downregulation of PRRG4 levels (P<0.01, Figures 5A–C ). These finding suggested that LncRNA HOTAIR can competitively bind to miR-519b-3p to regulate the expression level of the target gene PRRG4, consistent with the ceRNA regulatory network predicted by bioinformatics analysis. The results of scratch assays ( Figures 5D, E ) and transwell assays ( Figures 5F–H ) showed that si-HOTAIR decreased the invasion and migration ability of endometrial stromal cells, whereas these effects were partially reversed by the down-regulation of miR-519b-3p or the up-regulation of PRRG4. Hence, miR-519b-3p inhibitor or overexpression of PRRG4 could partially reverse the downregulation effects of si-HOTAIR on the migration and invasion of endometrial stromal cells. Based on these results, LncRNA HOTAIR could combine with miR-519b-3p to regulate the expression level of PRRG4 and then regulate the migration and invasion behaviors of endometrial stromal cells.
Endometriosis is defined as presence of growing–functioning endometrial tissue outside the uterine cavity, and its specific pathogenesis is still unclear. Many hypotheses have been established for endometriosis, but none of them can fully explain its etiology and developmental mechanism (4, 5). Menstrual reflux/planting is the generally accepted hypothesis that the endometrial tissue of the menstrual period flows to the abdominal cavity through the fallopian tube, and then migrates and implants to form ectopic lesions. During this process, the reflux endometrium can form ectopic lesions through a series of processes such as adhesion, invasion, and migration and then develops into endometriosis. This disease has biological behaviors that similar to cancer such as proliferation, invasion, and metastasis of recurrent malignant tumors. Long non-coding RNAs (lncRNAs) are a class of RNAs longer than 200 nucleotides with no protein-coding function. Studies have shown that LncRNA plays an important role in the proliferation, differentiation, apoptosis, and metastasis of cancer cells, leading to tumor invasion, progression, metastasis, and poor prognosis. Scholars have focused on the role of LncRNA in endometriosis. Sun et al. (17) first performed microarray analysis of LncRNA expression in ovarian endometriosis and found that 948 LncRNA transcripts and 4088 mRNA transcripts were dysregulated in ectopic endometrial tissue compared with those in paired eutopic endometrial tissue. Wang et al. (14) discovered that LncRNAs are differentially expressed between ectopic and eutopic endometrial tissues, and some dysregulated LncRNAs can be used as non-invasive biomarkers for diagnosis of endometriosis. Ghazal et al. (18) proved that the expression level of H19 was significantly decreased in patients with endometriosis and may be involved in regulating the pathogenesis of infertility. HOX transcript antisense intergenic RNA (HOTAIR) is one of the most studied LncRNAs. It is transcribed from the antisense strand of the HOXC gene cluster and influences the expression of genes from the HOXD locus. LncRNA HOTAIR, with length of about 2200 nt, was located on chromosome 12q13.13 between HOXC11 and HOXC12 genes. Dysregulations of LncRNA HOTAIR were often involved in the occurrence and development of malignant tumors and were associated with tumor invasion, progression, and metastasis. LncRNA HOTAIR overexpression was found in patients with breast cancer, and its overexpression was associated with metastasis and poor prognosis (19). Qiu et al. (20) showed that LncRNA HOTAIR was highly expressed in ovarian cancer, which was positively correlated with the invasion ability of cells, and its overexpression was associated with invasive metastasis and poor prognosis. Zhang et al. (12) showed that the ectopic endometrium exhibited higher expression of HOTAIR compared with that in paired eutopic endometrium and normal endometrium. In this study, we found that the LncRNA HOTAIR expression increased in endometriosis, and the LncRNA HOTAIR level of ectopic endometrial tissues was significantly higher than that of eutopic endometrial tissues, thereby promoting the ability of migration and invasion in endometrial stromal cells and playing an important role in the pathogenesis of endometriosis. MicroRNAs (miRNAs), another class of endogenous non-coding RNAs with approximately 22 nucleotides in length, can bind to the 3′ untranslated regions (3′ UTR) of target genes to modulate gene expression mainly through base complementation. LncRNAs can act as competing endogenous RNAs (ceRNAs) to combine with miRNAs to regulate the downstream target mRNA and then play biological functions (21). Wang et al. (22) found that LINC00261 can act as ceRNA to regulate BCL2L11 expression by combining with miR-132-3p and participating in the invasion of endometriosis. Liu et al. (23)found that LncRNA-H19 could inhibit ectopic endometrial cell proliferation and invasion by modulating miR-124-3p and ITGB3. Many studies suggested that LncRNA HOTAIR can also be used as ceRNA to combine with target miRNAs and regulate cell functions. LncRNA HOTAIR can regulate cell migration and invasion through miR-152-3p6 in endometrial carcinoma (24). It can attenuate the inhibitory effect of miR-129-5p on cervical cancer cells and may be involved in the regulation of the progression of cervical cancer (25). Zhang et al. (12) reported that exosomal LncRNA HOTAIR promotes the progression and angiogenesis of endometriosis via the miR-761/HDAC1 axis. In the present study, we found that LncRNA HOTAIR could competitively bind to miR-519b-3p to control the expression level of miR-519b-3p. Decreasing the LncRNA HOTAIR expression can increase the expression level of miR-519b-3p and then regulate the invasion and migration ability of endometrial stromal cells. This result also suggests that LncRNA HOTAIR may act on multiple miRNAs to participate in the occurrence of diseases. Proline-rich γ-carboxyglutamic acid protein 4 (PRRG4) is one of the cell surface transmembrane proteins in the PRRG family that has γ-carboxyglutamate (Gla) residues at the extracellular site and WW binding motifs in the cytosole. Yamamoto et al. (26) showed that the gene deletion of Prrg4 may be related to WAGR syndrome, which is an autosomal hereditary disease. A recent study suggested that PRRG4 plays an important role in the metastasis and invasion of breast cancer cells (27). Zhang et al. (12) reported that Exosomal HOTAIR promoted the progression and angiogenesis of endometriosis by regulating the miR-761/HDAC1 axis and activating STAT3-mediated inflammation in vitro and in vivo. In the present work, we found that Prrg4 was the target gene of miR-519b-3p by bioinformatics analysis. The qRT-PCR analysis results showed that the expression level of PRRG4 increased in endometriosis, especially in ectopic endometrial tissues. Inhibiting the expression of miR-519b-3p can target up-regulate the expression level of PRRG4. Through the recovery experiment, we found that the low expression of LncRNA HOTAIR could down-regulate the expression level of PRRG4 and reduce the migration and invasion ability of endometrial stromal cells. Adding miR-519b-3p inhibitor or overexpressing PRRG4 could partially reverse the down-regulation of PRRG4 and the downregulation effects of si-HOTAIR on the migration and invasion of endometrial stromal cells. This result also suggests that LncRNA HOTAIR may affect multiple signal pathways to participate the molecular pathogenesis of endometriosis. In conclusion, LncRNA HOTAIR elevated the expression level of PRRG4 by sponging miR-519b-3p to promote the invasion and migration ability of endometrial stromal cells. The LncRNA HOTAIR/miR-519b-3p/PRRG4 pathway was involved in the pathogenesis of endometriosis and might be a potential marker and new target for early diagnosis and treatment of endometriosis.
The original contributions presented in the study are included in the article/supplementary material. Further inquiries can be directed to the corresponding author.
The studies involving human participants were reviewed and approved by Ethics Committee of the First Affiliated Hospital of Fujian Medical University. The patients/participants provided their written informed consent to participate in this study.
QB carried out most of the experiments and analyzed the data. BZ conceived and supervised the project, provided suggestion to the experiments, discussed the data and wrote the manuscript with contributions from QZ, WT, and SW. All authors contributed to editing the manuscript.
This work was supported by Scientific Research Project from the Education Department of Fujian Province (No. JAT190208) and Natural Science Fundation of Fujian Province (No.2022J01218).
The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
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PMC9634127 | Zhiyun Gu,Haojie Yin,Haiwei Zhang,Hui Zhang,Xiaoyu Liu,Xiaohua Zeng,Xiaodong Zheng | Optimization of a method for the clinical detection of serum exosomal miR-940 as a potential biomarker of breast cancer | 21-10-2022 | exosomes,reference genes,miR-940,RT-qPCR,breast cancer | Serum exosomal microRNAs (miRNAs) are potential biomarkers for tumor diagnosis. Clinically, reverse transcription-quantitative polymerase chain reaction (RT−qPCR) can be used to determine the expression of exosomal miRNAs in the serum of breast cancer patients. The prerequisites for obtaining meaningful serum exosomal miRNA data of breast cancer patients include a suitable extraction method for exosomes and RT−qPCR data standardized by internal reference genes. However, the appropriate methods for the extraction of exosomes and the applicability of reference genes for analyzing exosomal miRNAs in breast cancer patients remain to be studied. This study compared the effects of three exosome extraction methods as well as the expression of exosomal miRNA in different initial serum amounts and at different serum states to identify the selection of the best method for serum exosome extraction. Five candidate reference genes including miR-16, miR-484, miR-1228, miR-191 and miR-423 for standardizing serum exosomal miRNAs were screened using five algorithms and were used for the quantification of serum exosomal miR-940. Significant downregulation of serum exosomal miR-940 expression in breast cancer was detected using miR-191 and miR-1228, whereas no significant down or up regulation was observed with miR-484, miR-423 and miR-16. Previous studies have shown that the expression level of miR-940 is downregulated in breast cancer tissues. The absolute quantitative results showed that miR-940 was significantly downregulated in breast cancer serum exosomes, which was consistent with the results from the analysis using miR-191 or miR-1228 as reference genes. Therefore, miR-191 and miR-1228 could serve as reference genes for the relative quantification of serum exosomal miRNAs. This finding indicated the importance of rigorously evaluating the stability of reference genes and standardization for serum exosomal miRNA expression. Moreover, the level of serum exosomal miR-940 in breast cancer could reflect the presence of lymph node metastasis and the status of HER2/neu, which indicates its potential as a biomarker for breast cancer metastasis. In summary, an optimized protocol for the detection of serum exosomal miR-940 as a breast cancer marker was preliminarily established. | Optimization of a method for the clinical detection of serum exosomal miR-940 as a potential biomarker of breast cancer
Serum exosomal microRNAs (miRNAs) are potential biomarkers for tumor diagnosis. Clinically, reverse transcription-quantitative polymerase chain reaction (RT−qPCR) can be used to determine the expression of exosomal miRNAs in the serum of breast cancer patients. The prerequisites for obtaining meaningful serum exosomal miRNA data of breast cancer patients include a suitable extraction method for exosomes and RT−qPCR data standardized by internal reference genes. However, the appropriate methods for the extraction of exosomes and the applicability of reference genes for analyzing exosomal miRNAs in breast cancer patients remain to be studied. This study compared the effects of three exosome extraction methods as well as the expression of exosomal miRNA in different initial serum amounts and at different serum states to identify the selection of the best method for serum exosome extraction. Five candidate reference genes including miR-16, miR-484, miR-1228, miR-191 and miR-423 for standardizing serum exosomal miRNAs were screened using five algorithms and were used for the quantification of serum exosomal miR-940. Significant downregulation of serum exosomal miR-940 expression in breast cancer was detected using miR-191 and miR-1228, whereas no significant down or up regulation was observed with miR-484, miR-423 and miR-16. Previous studies have shown that the expression level of miR-940 is downregulated in breast cancer tissues. The absolute quantitative results showed that miR-940 was significantly downregulated in breast cancer serum exosomes, which was consistent with the results from the analysis using miR-191 or miR-1228 as reference genes. Therefore, miR-191 and miR-1228 could serve as reference genes for the relative quantification of serum exosomal miRNAs. This finding indicated the importance of rigorously evaluating the stability of reference genes and standardization for serum exosomal miRNA expression. Moreover, the level of serum exosomal miR-940 in breast cancer could reflect the presence of lymph node metastasis and the status of HER2/neu, which indicates its potential as a biomarker for breast cancer metastasis. In summary, an optimized protocol for the detection of serum exosomal miR-940 as a breast cancer marker was preliminarily established.
MicroRNAs (miRNAs) are short noncoding RNA molecules that can participate in the regulation of posttranscriptional gene expression (1). Such regulation is essential for embryonic development, tumor initiation, immune modulation, and other biological processes. Exosomes are 40-100-nm disc-shaped vesicles. Various types of cells can release exosomes under normal and pathological conditions (2). Exosomes are involved in cell–cell communication and are released from donor cells into the microenvironment to affect target cells. This biological process exerts important biological functions (1). To date, exosomes have been found in various body fluids, such as human blood, urine, tears, breast milk, and semen (3). An et al. verified that exosomes contain many proteins, cytokines, DNA, mRNAs, miRNAs, lncRNAs, and other nutritional elements (4). A previous study showed that exosomal miRNAs play an important role in communication between breast cancer cells (4, 5). A cohort study compared the serum levels of exosomal miRNAs between healthy women and breast cancer patients with different molecular subtypes and found that exosomal miRNAs can be regarded as blood-specific biomarkers for more aggressive tumors, such as triple-negative and hormone receptor-negative breast cancer (6). Another study noted that exosomes are potential biomarkers for ovarian cancer and breast cancer (7). Zhou et al. proved that the content level of miRNAs is significantly altered in serum exosomes from breast cancer patients, which indicates that exosomal miRNAs can be used as biomarkers for the identification of breast cancer (8). Additionally, Rodriguez et al. proved that the expression of exosomal miRNAs found at high levels in serum can be used as a biomarker of breast cancer (9). Several different histological subtypes of breast cancer have been identified, and each subtype has different levels of invasiveness, clinical presentations, treatment programs, and prognoses (10). The diagnosis of breast cancer must be made as soon as possible such that treatment can be started in time. The difference in the expression level of exosomal miRNAs between healthy people and breast cancer patients can be used as a biomarker for the prediction of breast cancer. Accurately measuring the level of miRNA in exosomes is the first step for its detection as a biomarker (11). RT–qPCR is a precise method for detecting and quantifying circulating miRNAs. To ensure that the measurements are comparable across different samples, the same volume of each body fluid is needed for RNA extraction. Despite using the same initial biofluid volume for each sample, the total RNA levels will not be consistent between samples due to different upstream procedures for RT–qPCR, such as sample preparation, exosome isolation, and miRNA extraction. Hemolysis may occur in clinically collected blood samples, and the effect on exosomal RNA is unknown (12). Therefore, standardized sample collection must be performed to address these problems. Notably, stable internal reference genes are essential for qPCR standardization and accurate miRNA quantification. Differences in expression results may not be caused by the disease itself but may instead be caused by differences in the processes used for sample acquisition, sample preservation, RNA extraction, and target gene quantification (13). Therefore, appropriate methods for exosome extraction and optimal reference genes with stable expression must be determined to accurately normalize the quantitative data of exosomal miRNA (14). To date, in the RT–qPCR analysis of breast cancer exosomal miRNA, a consensus has not been reached regarding appropriate exosome extraction methods and appropriate internal reference genes. MicroRNA-940 (miR-940) is a recently identified miRNA family member that is abnormally expressed in cardiovascular and neoplastic diseases. This miRNA has different expression levels in different diseases; in gastric cancer, the expression level of miR-940 is increased, whereas in cardiovascular disease, the expression level of miR-940 is decreased (15–17), which indicates its important regulatory role. Previous studies found that triple-negative breast cancer tissues express lower levels of miR-940 compared with normal tissues and that proliferation and migration in triple-negative breast cancer are affected by miR-940 (18). However, the clinicopathological relationship between serum exosomal miR-940 and breast cancer patients is unclear. To select a suitable methods for serum exosome extraction that can be used in clinical practice, we extracted exosomes from the same sample using three extraction methods, analyzed the expression differences of serum exosomal miRNAs among different methods, analyzed the effect of differences in the initial serum volume on the RT–qPCR results, and finally analyzed the changes in hemolytic blood sample exosomal miRNAs in blood samples and normal blood samples. Based on previous studies, various candidate reference genes were used to normalize the expression levels of exosomal miRNAs in breast cancer patients and healthy controls to screen the reference genes. A total of 5 candidate internal reference genes were selected based on previous reports of their suitability for RT–qPCR analyses of cancer using tissues or serum. Two of these genes were previously described as reference genes for exosome miRNA analysis: miR-16 and miR-484 (6). The remaining genes, miR-1228, miR-191, and miR-423, were obtained from other studies (19, 20). Five genes were initially screened as internal reference genes of serum exosomal miRNA, and the expression levels of the five candidate genes were then detected. The expression stability of these five genes was evaluated to determine suitable reference genes. The screened internal reference genes were used to evaluate the expression level of miR-940 and verified that the results of the screened internal reference genes were stable and reproducible. Moreover, the applicable clinical exosomes extraction method identified from our comparison of breast cancer serum and normal serum was used to explore the differential expression of exosomal miR-940 using the selected stable reference genes. The serum exosomal miR-940 copy number and clinical data for breast cancer patients were analyzed by absolute quantification, and the results confirmed that serum exosomal miR-940 could serve as a metastatic marker for breast cancer. This study provided the first identification of suitable reference genes for the clinical study of serum exosomal miRNA expression in breast cancer while screening pretreatment methods suitable for the clinical extraction of serum exosomes; moreover, this study demonstrated that serum exosomal miR-940 could serve as a potential metastatic marker for breast cancer.
This study was approved by the Ethics Committee of the Chongqing University Cancer Hospital. Blood samples were donated by the Chongqing University Cancer Hospital. The blood sample set was composed of 118 breast cancer patients and 40 healthy controls. The all blood samples collected from patients were collected before treatment (including radiotherapy, chemotherapy, and surgery). The blood samples from patients without any treatment were drawn on an empty stomach, whereas those from healthy controls were also drawn early in the morning. A total of 5 mL of whole blood was drawn from each donor. Peripheral blood was collected into a tube containing heparin sodium and centrifuged at 3000×g for 10 min. The upper part of the serum was collected into a brand new centrifuge tube and centrifuged at 12000×g for 10 min to remove a cellular component. All centrifugation operations were performed at 4 °C. The separated serum was stored at -80 °C. To standardize the treatment of different serum samples, exosomal RNA was extracted from 1000 µL of serum, and the samples were preliminarily standardized according to a uniform volume. We randomly selected 59 of them to verify the stability of the candidate genes. The remaining 59 cases were used to investigated the relationship between serum exosomal miR-940 expression level and patient physiological indicators, so we looked up the clinicopathological reports of these patients. The clinicopathological were provided by the Department of Pathology and specific clinical data were presented in Schedules 1 and 2 ( Supplementary Table 1 , Supplementary Table 2 ).
Exosomes were extracted by ultracentrifugation, membrane affinity, and precipitation. Equal portions of the collected serum were removed from the -80 °C refrigerator, thawed on ice, and centrifuged at 12000×g and 4 °C for 10 min, and 1 mL of serum was then collected. Ultracentrifugation was performed using a 10-mL sample consisting of 1 mL of serum sample and 9 mL of PBS, and the sample was centrifuged by ultracentrifuge (Optima L-100XP, Beckman, USA) at 4°C and 100000×g for 70 min. The supernatant was carefully removed from the ultracentrifugation tube, and the remaining 2 mL of liquid containing exosomes was mixed with 8 mL of PBS. The supernatant was centrifuged again at 100000×g and 4°C for 70 min. Exosomes were resuspended in 200 μL of PBS and stored at -80 °C. Precipitation was performed with ExoQuick exosome precipitation solution (SBIS, System Biosciences, USA). The reagents were added to 1 mL of serum, and the mixture was shaken thoroughly. The flocculent precipitate appeared and was allowed to rest overnight at 4°C. The supernatant was then centrifuged at 12000×g for 10 min, and the precipitate contained exosomes. The exosomes were resuspended in 200 μL of PBS and stored in a -80°C refrigerator. The membrane affinity method was performed by collecting exosomes using an exoEasy Maxi Kit (QIAGEN GmbH, Hilden, Germany). Briefly, buffer XBP and serum were mixed in equal volumes, 2 mL of the mixture of each sample was then added to the exoEasy spin column and centrifuged at 4200×g for 1 min, and the waste after centrifugation was discarded. Ten milliliters of buffer XWP was added to the spin column and centrifuged at 5000×g for 5 min to remove the buffer XWP. Next, 200 μL of Buffer XE was added to the spin column and incubated for 1 min. The XE buffer was collected after centrifugation at 5000×g for 5 min. Exosomes were resuspended in XE buffer and stored at -80 °C.
The extracted exosomes were characterized using different methods. The characteristics of the exosomes were determined by transmission electron microscopy (TEM) (Tecnai G2 F30S-TWIN, FEI, USA). The exosomal particle size was measured using a nanoparticle tracking analysis (NTA) instrument (ZetaView, Particle Metrix, Meerbusch, Germany) according to the experimental requirements. The characteristic proteins of exosomes, such as CD9, CD63, and TSG101, were analyzed by Western blotting. Monoclonal anti-CD63 (#98327) antibodies, anti-CD9 (#52090) antibodies, and anti-TSG101 antibodies (#72312) were obtained from Cell Signaling Technology (Denver, MA, USA).
The sequence of the target gene was obtained from the NCBI and miRBase databases, and the primers were designed according to the requirements of the SYBR Green method for RT–qPCR and were synthesized by Biotechnology (Biotechnology, Shanghai, China). The primer information of each gene designed is shown in Table 1 .
The exosomes extracted from each sample were lysed with 1 mL of QIAzol® (Qiagen GmbH, Hilden, Germany). The quality of the RNA was assessed by measuring the A260/A280 value of the extracted RNA using a NanoDrop™ One/OneC (Thermo Scientific, China). These cDNAs were obtained by reverse transcription of 1 ng of the extracted exosomal RNA using a miRcute Plus miRNA first-strand cDNA kit (TianGen, Beijing, China). The thermal cycling parameters for reverse transcription were 60 min at 42 °C and 3 min at 95 °C. The cDNA samples were diluted 10-fold in nuclease-free water and stored at −20 °C. The expression levels of candidate internal reference genes in the serum exosomes were analyzed by RT–qPCR, which was performed with 384-well reaction plates using a LightCycler 480 II (Roche, Germany). The qPCR conditions are shown in Table 2 . The data were analyzed using the software provided for the fluorescence quantitative PCR system. When the Ct value is greater than 35, the data have no meaning and are regarded as unexpressed, and this type of data is removed. Moreover, the arithmetic average of the Ct values of the three wells was used as the final Ct value of the miRNA PCR amplification.
The method used for copy number determination was described in the literature (21). Briefly, standard curves for miR-940 expression in serum exosomes were constructed by a serial dilution series of standard miR-940 ranging from 100 to 106 copies/μL. The plasmid copy number was calculated using the following equation: The corresponding logarithm template copy number was then plotted against the Ct values obtained by real-time qPCR. Statistical analyses were performed using Student’s t test (two-tailed) to analyze the differences between groups. All values are expressed as the means ± S.E.Ms. A value of P< 0.05 was regarded as statistically significant.
First, the standard deviation (SD) and coefficient of variation (CoV) of each sample were calculated. In addition, four commonly used methods were applied to more comprehensively assess the stability of the candidate genes: geNorm (22), NormFinder (23), BestKeeper (24), and the comparative ΔCt method (25). These four methods use online tools to evaluate reference gene expression (26). Moreover, statistical analyses were performed with the paired t test using SPSS Statistics 21 software, and a P value less than 0.05 indicated significant differences.
The first step in the evaluation of exosomal miRNAs is the successful isolation of exosomes from serum. Ultracentrifugation, membrane affinity, and precipitation were used to isolate exosomes from serum, and a physical examination by transmission electron microscopy (TEM) showed a spherical structure of 30~150 nm and a typical doughnut-like shape ( Figure 1A ). The characteristics of the exosomes were consistent with those previously reported (27). Nanoparticle tracking assessment (NTA) analysis showed that the average particle size of exosomes ranged from 110 nm to 140 nm ( Figure 1B ). A Western blot analysis of the characteristic proteins of serum exosomes (3), i.e., CD63, CD9, and TSG101, was performed ( Figure 1C ).
Twenty serum samples were randomly selected from our collected serum samples and divided into two groups to screen the best method for the clinical serum exosome extraction. We analyzed the effects of various exosome extraction methods and the starting amount of serum samples on the expression levels of three serum exosomal miRNAs, analyzed the expression levels of three serum exosomal miRNAs under hemolytic conditions, and established a pretreatment method suitable for the clinical detection of serum exosomal miRNAs. The results from the literature and our experiments confirmed that all three extraction methods could extract exosomes. Therefore, through the detection of miRNA expression levels in exosomes using three different extraction methods, the influence of different extraction methods on the serum exosomal miRNA levels was analyzed. miR-16, miR-1228 and miR-940 were selected: the first two represent internal reference genes, and the last one represents the target gene of this study. Among the three extraction methods, the exosomes extracted by ultrasonication had the lowest content of the three miRNAs tested. No significant differences in the contents of the three miRNAs were identified between exosomes extracted using the membrane affinity method and exosomes obtained using the precipitation method ( Figure 2A ). However, the coefficient of variation (CoV) of miRNA expression in exosomes extracted using precipitation was higher than the coefficient of variation (CoV) obtained for exosomes extracted using membrane affinity ( Table 3 ). Therefore, membrane affinity was selected for exosome extraction in all subsequent experiments. RT–qPCR was used to detect the expression levels of miR-16, miR-1228 and miR-940 in the exosomes extracted from the different initial volumes of serum from breast cancer patients. The initial serum levels were 250, 500 and 1000 μL. When the serum volume of breast cancer patients doubled, the Ct values of all miRNAs decreased over three cycles ( Figure 2B ). Therefore, for small blood samples, a serum volume lower than that detailed in the instructions could also be used for exosome extraction. In the process of preserving samples, hemolysis occurred in 10 samples, and we subsequently collected normal blood samples from these patients. Therefore, we studied the influence of hemolysis on the content of miRNA in the exosomes of the samples. Exosomal RNA was extracted from both hemolytic and normal blood samples, and the same method was used for the extraction of exosomal miRNA from hemolysis samples. The levels of miR-16, miR-1228 and miR-940 were detected. The RT–qPCR results showed that in hemolyzed blood, the expression levels of miR-16, miR-940 and miR-1228 in the extracted exosomes were significantly higher than those in serum exosomes without hemolysis ( Figure 2C ). This finding is likely because blood cells release exosomes during blood storage, which alters the amount of exosomes that will be collected through plasma/serum. In plasma or serum samples, the quantification of exosomal miRNAs may be impaired due to contamination with erythrocyte-derived miRNA caused by hemolysis. Therefore, in clinical serum exosome experiments, hemolysis may increase the expression level of target genes.
Because the primary requirement of internal reference genes is to present similar expression levels under diseased and healthy conditions, we compared the expression of candidate internal reference genes in serum exosomes under breast cancer and healthy conditions. We read out the Ct values of different candidate genes in each serum sample ( Figure 3A ). Among them, the serial numbers 1-20 correspond to samples from the controls, whereas the numbers 21-79 correspond to samples from breast cancer patients. The picture shows the Ct value of different candidate genes obtained for each serum sample. The Ct values ranged from 12.01 (miR-16) to 24.78 (miR-432). Subsequently, the expression levels of miR-16, miR-1228, miR-484, miR-191, and miR-423 in the serum exosomes of breast cancer patients and normal human serum exosomes were compared ( Figure 3B ). The results showed that the Ct value of each candidate gene was not significantly different between the serum exosomes from the breast cancer patients and those from the controls. We also showed the dispersion of the Ct values ( Table 4 ). The stability of the five candidate internal genes was analyzed and sorted using four algorithms, namely, BestKeeper, NormFinder, GeNorm, and ΔCT; however, the four methods used to analyze the stability of the internal genes were not the same ( Figure 3C ). BestKeeper compared the correlation coefficient (r), standard deviation (SD), and coefficient of variation (CV) generated by the pairing of each gene and ultimately determined a relatively stable internal reference gene. The principle was that a more stable internal reference gene would have a smaller standard deviation and coefficient of variation and a larger correlation coefficient. Gene stability was also judged according to the SD value. At SD>1, the expression of the internal reference gene was unstable. The results obtained with BestKeeper are shown in Figure 3C , which indicates high SD variation among miR-1228, miR-16, and miR-484. In this study, miR-423 was the most stable gene (std dev=1.012), followed by miR-16 (std dev=1.275). NormFinder software was designed by Claus et al. to screen out internal reference genes suitable for RT−qPCR. A linear scale was used for quantification of the raw data, analyze the stability of candidate genes and provide the stability value of each gene: a higher stability value indicates lower stability of the gene as an internal control and thus indicates that the gene is not suitable as an internal control in this experiment. Among the five candidate genes tested in this study, the results obtained using NormFinder showed that miR-1228 has the lowest stability value of 0.778, which indicates that this gene was the most stable internal reference gene in this experiment, followed by miR-423 (1.056) and miR-191 (1.139), and the least stable gene was miR-484. The standard for using GeNorm to evaluate the stability of the internal reference genes was to calculate the average coefficient of variation M value of the logarithmic conversion value of the ratio of the first gene to the remaining genes. The M value must be less than 1.5, and a smaller M value corresponds to greater stability of the gene as an internal reference. The final result obtained by calculation was two or more candidate combinations. The results obtained using GeNorm showed that the M values of miR-484, miR-423 and miR-16 in the sample were higher than 1.5, indicating that these candidate genes are unreliable and cannot be used as internal reference genes for the standardization of breast cancer serum exosomal RNA. The most stable gene combination was the combination of miR-1228 and miR-191, which had an expression stability M value of 1.391. The comparative ΔCt method was used to analyze the stability of the internal reference gene, and the result was a combination of two genes. The ΔCT method could eliminate the influence of coordinated regulation and evaluate the reference genes from various aspects. The results obtained using the ΔCt method indicated that miR-1228 and miR-423 constituted the most stable group. Because the four analysis methods use different algorithms, the results obtained were also different; thus, normalization and integration of the data were performed when necessary. RefFinder is a web tool that can synthesize the results from the four software programs to generate the final overall ranking of reference genes. According to the output, the most stable reference gene was miR-1228, and the lowest and most unstable reference gene was miR-484 ( Figure 3D ). These results indicated that miR-1228 may be used as the most stable reference gene in breast cancer research.
RT–qPCR analysis was applied to further evaluate the stability of each candidate reference gene in the sample. miR-940 exhibits low expression levels in the serum of breast cancer patients (28, 29). The expression level data of miR-940 were normalized ( Figure 4 ) using the RefFinder program recommended for miR-1228, miR-191, and miR-423 and the geNorm program recommended for miR-484. Although miR-16 was not recommended as a suitable reference gene by BestKeeper and geNorm, we used miR-16 to normalize the content of miR-940 because this gene has often been used for expression studies (30, 31). When using different internal reference genes, the fold change in serum exosomal miR-940 in each group was calculated ( Figure 4 ). The results from the normalization of miR-940 were used. miR-191 and miR-1228 showed that the serum exosomal miR-940 levels in breast cancer patients were significantly downregulated, whereas the results obtained by normalization using other candidate genes did not show the same result. This analysis showed that different normalization schemes may affect the quantitative expression of data. However, miR-940 should be assessed in a large sample study to confirm the reliability of the reference gene.
For the quantification of miR-940, a standard linear regression curve of the Ct values against the copy numbers was derived from serially diluted known amounts of miR-940 cDNA ( Figure 5A ). Based on the curve, the copy number of miR-940 transcripts per nanogram of exosomal RNA isolated from each cancer patient was determined. The copy number of miR-940 in serum exosomes of breast cancer patients was significantly lower than that of normal controls, which is similar to the results from the relative quantification of miR-940 using miR-191 and miR-1228 ( Figure 5B ), and this finding demonstrated that miR-191 and miR-1228 were appropriate reference genes. However, miR-16, miR-484 and miR-423 could not be used as reference genes for breast cancer exosomal miRNAs. These results indicated that the expression level of miR-940 in the serum exosomes of breast cancer patients was significantly downregulated. To investigate the potential physiological significance of circulatory exosomal miR-940, the correlation of the levels of exosomal miR-940 with a spectrum of pathophysiological parameters in cancer patients was tested ( Figure 5C ). The patients were grouped according to various pathological indicators, and the grouping results are shown in Table 5 . Among the 59 samples, we found that one of the breast cancer patients was male and the youngest, but we did not find any special pathological information, so we analyzed it in the same way. The patients were divided into two groups according to different parameters, and the corresponding miR-940 copy numbers were averaged within groups and compared with each other using a t test. The correlations between exosomal miR-940 and pathophysiological parameters in breast cancer patients are shown in Figure 5C , which showed that exosomal miR-940 levels were significantly lower in HER2/neu-positive patients than in HER2/neu-negative patients (median copy number: 9.43×1011 vs. 1.46×1012, P=0.017). The serum exosomal miR-940 levels were significantly higher in breast cancer patients without lymph node metastasis than in those with lymph node metastasis (median copy number: 8.7×1011 vs. 1.59×1012, P=1.110-11). However, the level of serum exosomal miR-940 was not related to the age, degree of differentiation, ER/PR status, Ki67 or TNM stage of the breast cancer patients (P>0.05). These results showed that the content of miR-940 in the serum exosomes of breast cancer patients was related to lymph node metastasis and HER2/neu expression status.
With the development of precise tumor therapy and the continuous improvement of liquid biopsy technology, extracellular vesicles that can be secreted by various cells, such as exosomes, have attracted increasing attention. Exosomal miRNAs are not as degraded as free miRNAs in human fluids, and this type of RNA may be more suitable for the detection of tumor markers (32). Breast cancer is the leading cause of cancer death among women. Differences in the expression levels of circulating exosomal miRNAs between healthy people and breast cancer patients could identify molecular markers for the diagnosis and prediction of breast cancer (33). However, the detection methods of serum exosomal miRNAs in breast cancer patients have not been unified in clinical practice. Based on previous experiments, we first analyzed the effects of various exosome extraction methods and the starting amount of serum sample on miR-940, miR-16, miR-16 and miR-1228, analyzed the expression levels of three serum exosome miRNAs under hemolysis, and established a preprocessing method suitable for the clinical detection of serum exosomal miRNAs. To clinically detect the expression level of exosomal miRNA in breast cancer, an appropriate volume of insoluble blood samples should be used for serum exosomal RNA extraction using the membrane affinity method. We then demonstrated that miR-1228 and miR-191 could be used as reference genes for breast cancer serum exosomal miRNA, whereas miR-16, miR-484 and miR-423 were not suitable as reference genes. We also found that the expression level of serum exosomal miR-940 could reflect the presence of lymph node metastasis in breast cancer patients and the expression level of serum exosomal HER2/neu in breast cancer patients, indicating its potential as a metastasis marker. As a novel diagnostic marker, exosomes have many advantages, such as their rapid, efficient and economic isolation and their important potential application value in clinical diagnosis and treatment (34). However, due to the complex formation environment and small diameter of exosomes, an ideal separation technology is key to limiting the research and application of exosomes. Among these, the preservation and pretreatment of samples is crucial. Ultracentrifugation was the first technique used for exosome isolation and remains the most common technique in articles (35). However, this method is time-consuming and complicated and requires a high amount of serum. Although precipitation can yield exosomes (36), the separated exosomes have very low purity because almost all soluble granules can settle, which is not conducive to downstream analysis. This study was used to extract exosomes using the membrane affinity column method with high efficiency and low loss obtained outside secreted RNA, which is suitable for clinical laboratory work and advantageous for downstream analysis (37). In addition, the storage state of serum samples is also very important in clinical testing, and hemolysis may occur. Studies have shown that the levels of various proteins, RNAs and DNAs in whole blood are greatly changed after hemolysis, whereas previous studies have shown that exosomal miRNAs are not as degraded as free miRNAs in body fluids. This study experimentally verified that hemolytic samples also have a great impact on the extraction of serum exosomal miRNA, which fills the gap in this aspect. Moreover, due to limited clinical serum resources, our study conducted a preliminary exploration of whether a sample size lower than that recommended by the protocol could achieve the same experimental effect. Among the many techniques for the analysis of miRNA transcription levels, RT–qPCR is considered the most accurate technique due to its high sensitivity and easy reproducibility (38). Although relative quantification is a common method, it needs a reference gene for the normalization of different samples to ensure high sensitivity because the expression differences in the data results may not be due to the disease itself but rather to differences in the processes used for sample collection, stabilization, RNA extraction and target quantification. Therefore, identification of the best reference genes suitable for study is necessary for the accurate standardization of exosomal miRNA data. However, suitable reference genes for serum exosomal miRNAs in clinical breast cancer patients have not been reported to date. Based on previous reports, five candidate internal reference genes were selected for RT−qPCR studies of breast cancer using tissue, serum or plasma. Among them, miR-484, miR-423 and miR-1228 were used in the study of serum miRNA, and miR-16 and miR-191 were used in the study of breast tissue. Due to differences in the environment of exosomes, tissues and body fluids, the effect of these genes in standardizing breast cancer serum exosomes has been questioned. Therefore, we selected five genes as candidate genes to study their applicability as internal references for serum exosomal miRNAs in breast cancer patients. The results demonstrate that both miR-1228 and miR-191 could be used as reference genes for breast cancer serum exosomal miRNA. Although the results from the software analysis showed that miR-191 was not suitable as a reference gene for breast cancer serum exosomes, the relative quantification of the target genes using miR-191 also yielded the same results as those obtained with miR-1228. Similarly, high differences were observed when different miRNAs were used under the differentiation conditions described by Roulex-Bonin and Coste, which suggested that miR-191-5p was the most stable reference gene (39). Therefore, we believe that miR-191 could also serve as a suitable reference gene. Previous studies have confirmed that the expression level of miR-940 in breast cancer tissues was significantly lower than that in adjacent tissues. Further studies found that miR-940 could inhibit the proliferation, invasion and migration of breast cancer cells by targeting and regulating CXC chemokine 2 (CXCR2) or ZNF24 (18, 40). Zhang et al. indicated that miR-940 induces malignant progression of breast cancer by regulating FOXO3 (41). In addition, the serum miR-940 levels in breast cancer patients predicted the efficacy of trastuzumab in patients with HER2-positive metastatic breast cancer (29). The downregulation of miR-940 levels in breast cancer tissues also led to the low content of free miR-940 in patient serum, and the stability of free miR-940 in serum was susceptible to environmental influences, which limited the ability of free miR-940 as a prognostic marker. To verify whether breast cancer serum exosomal miR-940 has potential as a tumor marker of breast cancer, the copy number of miR-940 in the serum exosomes of each patient was calculated by an absolute quantitative method, and the results demonstrated that the content of miR-940 in the serum exosomes of breast cancer patients was significantly lower than that in normal human serum exosomes. The relationship between the copy number of serum exosomal miR-940 and the clinical data of breast cancer patients was analyzed. The results showed that the expression level of miR-940 in the serum exosomes of breast cancer patients with lymph node metastasis was significantly downregulated. Therefore, the expression level of miR-940 in serum exosomes of patients can be used to judge whether a patient has lymph node metastasis. Similarly, we found that the expression level of miR-940 in the serum exosomes of HER2/neu-positive patients was significantly lower than that in those of HER2/neu-negative patients. However, as shown in Figure 5C , the differences were not as distinct due to the high dispersion of data points in both groups. Exosomal miR-940 alone may not be sufficiently accurate to judge the HER2/neu status of patients. We also demonstrated that serum exosomal miR-940 is a potential metastatic marker for breast cancer patients. Li et al. found that exosomal miR-940 was mainly secreted by tumor cells in vivo through an analysis of exosomes and exosome-free supernatant from primary breast cancer cells and peripheral immune cells and revealed that miR-940 expression is increased in trastuzumab-sensitive HER2-positive metastatic breast cancer patients and further increased in trastuzumab-resistant patients (29). Therefore, they speculated that serum exosomal miR-940 has the potential to be used as an indicator of trastuzumab sensitivity in HER2/neu-positive metastatic breast cancer. Wang et al. found that the expression level of exosomal lncRNA-HOTAIR is able to reflect the HER2/neu status (21). Therefore, we hypothesized that the expression levels of exosomal miR-940 and lncRNA-HOTAIR may be used to judge the HER2/neu status of breast cancer patients. Of course, further experiments are needed to verify our hypothesis. In conclusion, serum exosomal miR-940 can be used as a minimally invasive liquid biopsy for monitoring disease progression. In this study, we only selected some of the most frequently used reference genes in the literature and screened out the most stable reference genes. We did not sequence the RNA in the serum exosomes of breast cancer patients and normal people, and thus, more accurate reference genes may be obtained. When comparing clinical exosome extraction methods, the number of samples is small. If the sample size can be increased, the results may be more convincing. In addition, this paper describes one gene selected for miR-940, which shows that it can be used as a metastasis marker of breast cancer. We should test multiple miRNAs to increase its efficacy as a biomarker. Only one gene was selected. If multiple miRNAs are selected for combined detection with existing tumor markers, the reliability will be higher.
The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.
The studies involving human participants were reviewed and approved by The Ethics Committee of the Chongqing University Cancer Hospital. The patients/participants provided their written informed consent to participate in this study.
ZG and HY performed and analyzed the experiments and wrote the manuscript. HWZ, HZ, and XHZ helped to perform the experiments. XDZ designed and supervised the study. All authors have read and approved the final manuscript.
The present study was supported by a grant from the Fundamental Research Funds for the Central Universities (2019CDYGZD006).
The authors of the present study are grateful for the valuable comments from members of the department of Breast Cancer, Chongqing University Cancer Hospital. The authors of this study also thank JunLi Huang from Chongqing University for valuable comments.
The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher. | true | true | true |
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PMC9634614 | Apirat Chaikuad,Rezart Zhubi,Claudia Tredup,Stefan Knapp | Comparative structural analyses of the NHL domains from the human E3 ligase TRIM–NHL family | 27-09-2022 | TRIM NHLs,NHL domains,TRIM E3 ligases,protein interaction domains,β-propeller protein modules,genetic mutations | Family-wide structural analyses of human TRIM NHL domains reveal evolutionary divergence of their β-propeller architecture that might be essential for recruiting diverse interacting partners and for the roles of NHL domains as E3 ligases. | Comparative structural analyses of the NHL domains from the human E3 ligase TRIM–NHL family
Family-wide structural analyses of human TRIM NHL domains reveal evolutionary divergence of their β-propeller architecture that might be essential for recruiting diverse interacting partners and for the roles of NHL domains as E3 ligases.
Tripartite motif (TRIM) proteins form one of the largest subclasses of the RING-type E3 ubiquitin ligases, comprising more than 80 members. They commonly harbor a conserved architecture of three N-terminal motifs on their N termini, which include a RING domain, one or two B-box domains and a coiled-coil domain, which are important for E2 binding, protein–protein interaction and oligomerization, respectively (D’Amico et al., 2021 ▸). The presence of these three motifs is a hallmark of this subfamily, thus TRIMs are also referred to as RBCC proteins. However, a high degree of domain variation is observed for the C-terminal regions of TRIMs, which contain various protein modules that typically exert an intermolecular interaction function and thus probably play a significant role in substrate recruitment. Similar to other E3 ligases, TRIMs are key regulators of many biological processes, such as protein quality control and degradation through their involvement in ubiquitin–proteasome degradation, autophagy, apoptosis, DNA repair and tumor suppression (D’Amico et al., 2021 ▸; Hatakeyama, 2017 ▸), and, as recently shown, transcription regulation through their RNA-interaction activities (Williams et al., 2019 ▸; Schwamborn et al., 2009 ▸). In addition, emerging evidence has suggested other diverse roles of TRIMs from immune and cell stress responses to viral-entry restriction (Kato et al., 2021 ▸; Caddy et al., 2021 ▸; Stremlau et al., 2004 ▸). These roles overall highlight the importance of TRIMs in homeostasis, and indeed their dysregulation has been linked to diverse disease development (Hatakeyama, 2011 ▸; Meroni, 2020 ▸). Diversity of the C-terminal protein modules in combination with different numbers of B-box domains lead to the classification of TRIMs into 11 subtypes with an additional ‘unclassified’ group containing members that lack a RING-finger domain (Hatakeyama, 2017 ▸; Williams et al., 2019 ▸; D’Amico et al., 2021 ▸). The SPRY domain, known also as B30.2, is the most common domain, as it appears in more than 40 TRIMs. Other protein modules found at the C termini of TRIMs include, for example, the PHD–Bromodomain, NHL, COS, filamin and fibronectin type III (FN3), albeit less frequently. These diverse protein domains have different preferences for intermolecular interactions, hence they diversify various biological functions of TRIMs. For example, SPRY domains have been shown to function as a protein–protein interaction module comprising multiple and highly variable binding surfaces for diverse binding partners that have little similarity in topology, share no consensus-sequence motif and play different roles in diverse cellular processes (James et al., 2007 ▸; Kato et al., 2021 ▸). However, PHD–Bromodomains are more specific protein–protein interaction modules that specifically recognize the acetylated lysine, essentially that of histone, implicating thus the role of this class of TRIMs in epigenetics signaling (Tsai et al., 2010 ▸). The NHL domain, or so-called NHL repeats named after ncl-1, HT2A and lin-41, forms another protein module found in four TRIMs, including TRIM2, TRIM3, TRIM32 and TRIM71, that constitute the subclass VII. In addition, TRIM56 is another distantly related member due to the presence of an NHL-like domain (Kumari et al., 2018 ▸; Liu et al., 2016 ▸). This motif, which is present in many other proteins, folds into a conserved β-propeller structure; a scaffold that typically mediates interactions with diverse macromolecules including proteins and nucleic acids (Loedige et al., 2015 ▸; Couture et al., 2006 ▸). To date, little is known about human TRIM NHLs and their endogenous interaction partners. However, previous studies on TRIM orthologues established a role of NHL domains as a bona fide RNA-binding module (Loedige et al., 2015 ▸; Kumari et al., 2018 ▸; Williams et al., 2019 ▸). This RNA-binding function suggests a role of TRIM–NHL proteins as regulators of gene expression with a link to diverse aspects of RNA metabolisms. In addition, the RNA-binding activity may be important for a role of TRIM56 in suppression of influenza virus RNA synthesis (Liu et al., 2016 ▸). Although the NHL is evolutionarily conserved, diversity of intermolecular recognitions mediated by the central NHL binding cavities has been proposed, and this property could be linked to plasticity of this protein module that can thus in turn differentiate biological roles of TRIM–NHL proteins (Kumari et al., 2018 ▸). For example, a regulatory role of TRIM71 in controlling expression of genes promoting differentiation has been demonstrated, implicating its involvement in cellular plasticity and reprogramming of differentiated cells into pluripotent cells (Worringer et al., 2014 ▸). TRIM2 NHL has been shown to interact with the motor-protein myosin V, and the function of this E3 ligase has been linked to neuronal activity including neurons and axon growth (Balastik et al., 2008 ▸; Ohkawa et al., 2001 ▸). Diverse biological roles of TRIM32 have been documented ranging from neuronal differentiation, muscle homeostasis and tumor suppression to antiviral infection (Fu et al., 2015 ▸; Schwamborn et al., 2009 ▸; Hillje et al., 2013 ▸; Bawa et al., 2021 ▸). Structural information on the NHL module may provide an underlying molecular basis for distinct solutions to different partners and specific recognitions that may form a key to diverse biological roles of human TRIM–NHL proteins. In this study, we therefore sought to determine the crystal structures and provide comparative analyses of the intrinsic properties of TRIM NHL domains.
The cDNA of the NHL domains of human TRIM2 (aa 466–744; MGC:18215, IMAGE:4156234) and human TRIM3 (aa 466–744; MGC:111679, IMAGE:6108991) were subcloned into pGTVL2, and the proteins were recombinantly expressed as a His6-GST fusion in Escherichia coli. In brief, the bacteria cultured in TB media were initially grown at 310 K until OD600 reached 1.6–1.8. The cultures were then cooled to 291 K, and at an OD600 of ∼2.6–2.8, cells were induced with 0.5 mM IPTG overnight. The recombinant proteins were initially purified by Ni2+-affinity chromatography. The His6-GST tag was removed by TEV treatment and the cleaved proteins were separated by passing through Ni2+ beads. The proteins were further purified by size-exclusion chromatography using a Superdex s75 column with the buffer containing 20 mM HEPES pH 7.5, 200 mM NaCl and 0.5 mM TCEP.
The cDNA of the NHL domain of human TRIM71 (aa 590–868; MGC:190511, IMAGE:100062428) was subcloned into pSUMO-Lic, and the His6-Sumo tagged protein was recombinantly expressed in E. coli, of which the expression was performed as that described above for TRIM2 and TRIM3 NHLs. The recombinant protein was initially purified by Ni2+-affinity chromatography. The expression tag was removed by SENP1 treatment. The cleaved protein was purified by passing through Ni2+ beads, and subsequently size-exclusion chromatography using a Superdex s200 column with the buffer containing 20 mM HEPES pH 7.5, 200 mM NaCl and 0.5 mM TCEP.
The recombinant TRIM–NHL domains were concentrated to ∼7.5–10 mg ml−1. Crystallization was performed using the sitting-drop vapor-diffusion method at 293 K. The conditions used for growing crystals were (i) for TRIM2, 30%(w/v) PEG 5000 MME, 0.2 M ammonium sulfate and 0.1 M MES, pH 6.5; (ii) for TRIM3, 2.4 M ammonium sulfate; and (iii) for TRIM71, 20%(w/v) PEG 3350, 0.2 M potassium thiocyanate, 10%(v/v) ethylene glycol and 0.1 M bis-tris propane, pH 8.5.
Viable crystals were cryo-protected with mother liquor supplemented with 20%(v/v) ethylene glycol for TRIM2 and TRIM71 or 25%(v/v) glycerol for TRIM3. Diffraction data were collected at the Swiss Light Source, and were processed and scaled with XDS (Kabsch, 2014 ▸) and AIMLESS (Evans & Murshudov, 2013 ▸), respectively. Molecular replacement was performed using Phaser (McCoy et al., 2021 ▸) and the coordinates of the NHL of Drosophilla melanogaster Thin [PDB ID 6d69 (Bawa et al., 2020 ▸)]. The structures were subjected to manual model rebuilding alternated with refinement in Coot (Casañal et al., 2020 ▸) and REFMAC5 (Kovalevskiy et al., 2018 ▸), respectively. Geometric correctness of the final models was verified by MolProbity (Prisant et al., 2020 ▸). The data-collection and refinement statistics are summarized in Table 1 ▸.
The NHL domains of TRIM2 and TRIM3 were highly expressed as a fusion protein with an N-terminal His6-GST tag in E. coli. The same tag was also used for TRIM71, albeit with no success due to a remarkably lower yield and protein instability. An N-terminal His6-Sumo tag was instead exploited leading to an improvement of expression levels that enabled successful recombinant-protein production of the TRIM71 NHL domain. We observed that all three recombinant TRIM NHLs without the expression tags behaved as a monomer in gel filtration. With the aim to provide a structural model, we attempted crystallization and gratifyingly obtained the crystals of all three proteins. Crystals of TRIM2 NHL were obtained within 1–2 d, while TRIM3 crystals grew within one week and TRIM71 crystals formed after approximately one month. All crystals showed good X-ray diffraction quality, enabling high-resolution structure determination. For TRIM2, the structure was refined to a high resolution of 1.45 Å, and the crystals belonged to the monoclinic P21 space group with four molecules in the asymmetric unit. The TRIM3 NHL structure was determined at 1.7 Å resolution from the tetragonal crystals that contained a single molecule in the asymmetric unit, whereas the monoclinic crystals of TRIM71 NHL diffracted to 2.2 Å resolution had an asymmetric unit consisting of two protein molecules. All three TRIM NHLs shared a highly similar tertiary structure by adopting the canonical β-propeller topology, which was previously described for these protein modules in the homologues Danio rerio Lin41 (DrLIN41) [PDB ID 6fpt (Kumari et al., 2018 ▸)], D. melanogaster Thin (DmThin) (Bawa et al., 2020 ▸) and D. melanogaster Brain tumor (DmBrat) (Edwards et al., 2003 ▸). In brief, the TRIM–NHL propellers were built from six β-sheet blades, each having an identical construction consisting of four β strands (Fig. 1 ▸). The N-terminal starting point and the C-terminal end of the propeller were located at a similar position and were a part of the sixth β sheet. Such highly similar architecture resulted in similar dimensions for all three NHL domains with a diameter of ∼42 Å and a thickness of ∼26 Å. The high structural homology was unexpected considering the low level of sequence similarity of only 19–41% among the NHL motifs of the four members of the TRIM–NHL family (TRIM2, TRIM3, TRIM32 and TRIM71) and the NHL-like domain of TRIM56 [Figs. 2 ▸(a) and 2 ▸(b)]. Nonetheless, an exception was noted when comparing TRIM2 and TRIM3 that were most similar with ∼82% sequence identity. In contrast, high similarity was observed when comparing TRIM family paralogues from different species, exemplified by, for instance, an 88% identity between human TRIM71 and zebrafish DrLIN41 [Fig. 2 ▸(b)]. This suggests that, barring the TRIM2–TRIM3 pair, each NHL domain of human TRIMs might emerge from different ancestors and paralogues, and remain evolutionarily conserved based on phylogenetic relationships [Fig. 2 ▸(b)]. At a three-dimensional structural level, despite the high sequence differences, the β-propeller architectures of the TRIM2, TRIM3 and TRIM71 NHLs remained highly conserved, revealed by highly superimposable structures with pair-wise r.m.s.d. values of 0.75–1.33 Å [Fig. 2 ▸(b)]. Nonetheless, some structural variations were still observed, and these were located mainly at the rim of the binding pockets. Notable differences included the lengths and conformations of the blade-connecting loops, especially those that linked blades 2 and 3, 3 and 4, and 5 and 6 [Fig. 2 ▸(c)], of which some degrees of variation were also seen among the NHL domains of homologues DrLIN41, DmBrat, DmMei-P26 (Salerno-Kochan et al., 2022 ▸) and DmThin (Bawa et al., 2020 ▸) (see Fig. S1 of the supporting information). However, based on the RNA-complexed structures of DrLIN41 and DmBrat, the parts of these loops with structural differences did not directly involve the binding of the substrate (Fig. S1). We speculated therefore that such conformational alterations may play a role in the maintenance of intrinsic structural integrity rather than directly participating in intermolecular interactions. Further comparative sequence conservation analyses indeed confirmed high diversity around these blade-connecting loops as well as at the top opening with distinct amino acid compositions that constitute the binding site [Figs. 2 ▸(a), 2 ▸(d) and 2 ▸(e)]. Such differences resulted in an overall high diversity in shape and electrostatic properties of the putative intermolecular interface [Fig. 2 ▸(e)]. A less polar shallow groove surrounded by mixed positively and negatively charged patches was observed for the binding sites of TRIM2 and TRIM3, whereas a strong positively charged surface with a deeper central hole unveiled a unique characteristic of TRIM71 [Fig. 2 ▸(e)]. It is tempting to speculate that such distinct properties were probably constructed by coevolution of diverse NHL binding partners. For example, the highly positively charged interface with a deep central groove suggests potentially a similar function of TRIM71 NHL to that of the homologue DrLIN41 as an interacting protein module that accepts RNA substrates harboring a stem-loop motif (Kumari et al., 2018 ▸) (Fig. S2). In contrast, the rather shallow flat surface of the TRIM2/3 NHLs resembles that of their homologue DmBrat, which has been shown to recognize a linear RNA (Loedige et al., 2015 ▸). However, comparative analyses suggest that similar binding of an RNA observed previously in DmBrat would be unlikely in TRIM2/3 due to the lack of a central channel as well as low sequence conservation within the pockets (Fig. S2). All protein domains of TRIMs are known to serve as essential intermolecular interaction modules required for E3 ligase activity. This includes a RING domain for E2 binding and B-box and coiled-coil domains for intermolecular interactions and/or oligomerization (D’Amico et al., 2021 ▸). The C-terminal domains, such as the PHD–Bromodomain (Tsai et al., 2010 ▸), SPRY (James et al., 2007 ▸) and NHL (Kumari et al., 2018 ▸; Edwards et al., 2003 ▸), have been reported as recognizable protein interacting modules, which may likely be utilized for substrate recruitment. Dysfunctions of these domains potentially lead to an impairment of TRIM E3 ligase functions and deregulation of ubiquitin-mediated signaling, which could form a cause of multiple diseases including neurological disorders and cancers, as well as many rare diseases (Meroni, 2020 ▸; Hatakeyama, 2011 ▸; Balastik et al., 2008 ▸). In line with this, genetic studies have unveiled a number of mutations in the NHL motifs of TRIMs with a link to diverse pathological outcomes, in particular, diverse neurological disorders. This includes a link of the mutations in TRIM2 NHL to Charcot-Marie-Tooth disease (CMT), congenital bilateral vocal-cord paralysis (BVCP) and axonal neuropathy (Pehlivan et al., 2015 ▸; Ylikallio et al., 2013 ▸; Magri et al., 2020 ▸; van Diepen et al., 2005 ▸). In addition, the mutations in TRIM71 NHL have been associated with congenital hydrocephalus (Welte et al., 2019 ▸; Furey et al., 2018 ▸), while those in TRIM32 NHL have been linked to myopathy such as limb-girdle muscular dystrophy type 2H (LGMD2H) and sarcotubular myopathy (STM) (Schoser et al., 2005 ▸; Frosk et al., 2002 ▸, 2005 ▸; Kudryashova et al., 2011 ▸; Yu et al., 2017 ▸; Saccone et al., 2008 ▸; Panicucci et al., 2019 ▸; Neri et al., 2013 ▸). Strikingly, most of the genetic mutations in the NHL-containing TRIMs are located within the NHL domain, and we summarize these known mutations in Fig. 3 ▸(a). Several types of mutations were identified, including amino acid substitutions and deletions as well as nonsense mutations that lead to an early transcription termination, hence a loss of the NHL protein domain. We used the crystal structures of TRIM2 and TRIM71 as well as the AlphaFold model of TRIM32 (Jumper et al., 2021 ▸) to map the locations of the mutations onto the NHL domains. Although these disease-linked mutations are distributed throughout the propeller structure, the putative intermolecular interaction surface interestingly forms a hotspot [Fig. 3 ▸(b)]. For TRIM2 NHL, two nonsense mutations were reported. A frameshift within blade 2 (K567Rfs7X) undoubtedly leads to a loss of the NHL domain, whereas the other nonsense mutation (R741X) results in the loss of four amino acids at the C terminus on the bottom surface. The other mutations include a substitution, D640A, and a deletion, N594del, both of which are located in the proximity of the central groove on the top surface, thus they could affect directly the integrity of the intermolecular interface. Such similar effect would also likely be anticipated for all three amino acid substitutions in TRIM71 [Fig. 3 ▸(b)]. These disease-linked mutations involving the changes of three positively charged arginine residues at the top surface peripheral to the central channel to a shorter hydrophobic alanine or histidine would alter the characteristics of strong positively charged electrostatic potentials of the binding interface, probably built for the interaction with the phosphate backbone of nucleic acids as seen in the DrLIN41 homologue (Kumari et al., 2018 ▸). Among the TRIM–NHL members, TRIM32 has the highest number of reported genetic mutations, all of which have been associated with rare muscle disorders. Although we were not successful in determining the crystal structure of TRIM32 NHL, we used an AlphaFold model to map the locations of the mutations. We found that, consistent with TRIM2 and TRIM71, the reported mutations are located in the vicinity of the rim surface of the central cavity [Fig. 3 ▸(b)]. Sequence alignment showed similarly that these mutated residues in TRIM32 clustered within the groups of amino acids that were found to line the binding interfaces of TRIM2, TRIM3 and TRIM71 [Fig. 2 ▸(a)]. Analyses of the mutations suggested that the nonsense mutations in blade 4 (T520TfsX) and blade 5 (R613X) would result in a loss of the NHL module, whereas substitutions and deletion leading to the changes both in charge properties such as R394H, D487N and D588del and in sizes such as P374L and S594N could alter the physical properties that may affect the function of this protein domain.
The NHL motif is an evolutionarily conserved protein domain that has been found in many proteins. NHL domains are also present at the C termini of four human E3 ligase TRIMs, including TRIM2, TRIM3, TRIM32 and TRIM71, as well as TRIM56 that harbors an NHL-like domain. This protein module folds into a β-propeller architecture that mediates intermolecular interaction, with a function as a bona fide RNA-binding module established for the homologues from various eukaryotes (Kumari et al., 2018 ▸; Loedige et al., 2015 ▸). We have presented here the crystal structures of the NHLs from TRIM2, TRIM3 and TRIM71, providing structural insights for this domain in human TRIM–NHL proteins. Despite sharing a highly conserved three-dimensional topology, our structural models revealed significant differences in the central NHL binding pockets, comprising a high degree of variation in shape, amino acid compositions and electrostatic potentials. The highly diverse rim surface of the binding cavity probably serves as a binding site for highly diverse interaction partners. We found that this region was also a hotspot of genetic mutations linked to the development of diseases including several neurological and muscle disorders. Overall, our structural information highlights evolutionary divergence that differentiates intrinsic properties and potentially recognition functions of this conserved protein domain, diversifying the biological functions of TRIM–NHL proteins. In addition, these structures may serve as a template for further study to identify the interaction partners as well as the functions of these TRIM NHLs and potentially the development of small molecule binders that, in a similar manner to other β-propeller protein modules (Wei et al., 2021 ▸), might find applications in the development of protein-targeting chimeras and molecular glues.
Supporting information. DOI: 10.1107/S2052252522008582/lz5059sup1.pdf PDB reference: NHL domain of TRIM71, 7qrx PDB reference: NHL domain of TRIM3, 7qrw PDB reference: NHL domain of TRIM2 (full C terminal), 7qrv | true | true | true |
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PMC9634977 | 36074076 | Jingjing Ruan,Dongdong Liang,Wenjing Yan,Yongwang Zhong,Daniel C. Talley,Ganesha Rai,Dingyin Tao,Christopher A. LeClair,Anton Simeonov,Yinghua Zhang,Feihu Chen,Nancy L. Quinney,Susan E. Boyles,Deborah M. Cholon,Martina Gentzsch,Mark J. Henderson,Fengtian Xue,Shengyun Fang | A small-molecule inhibitor and degrader of the RNF5 ubiquitin ligase | 01-11-2022 | RNF5 E3 ubiquitin ligase has multiple biological roles and has been linked to the development of severe diseases such as cystic fibrosis, acute myeloid leukemia, and certain viral infections, emphasizing the importance of discovering small-molecule RNF5 modulators for research and drug development. The present study describes the synthesis of a new benzo[b]thiophene derivative, FX12, that acts as a selective small-molecule inhibitor and degrader of RNF5. We initially identified the previously reported STAT3 inhibitor, Stattic, as an inhibitor of dislocation of misfolded proteins from the endoplasmic reticulum (ER) lumen to the cytosol in ER-associated degradation. A concise structure–activity relationship campaign (SAR) around the Stattic chemotype led to the synthesis of FX12, which has diminished activity in inhibition of STAT3 activation and retains dislocation inhibitory activity. FX12 binds to RNF5 and inhibits its E3 activity in vitro as well as promoting proteasomal degradation of RNF5 in cells. RNF5 as a molecular target for FX12 was supported by the facts that FX12 requires RNF5 to inhibit dislocation and negatively regulates RNF5 function. Thus, this study developed a small-molecule inhibitor and degrader of the RNF5 ubiquitin ligase, providing a chemical biology tool for RNF5 research and therapeutic development. | A small-molecule inhibitor and degrader of the RNF5 ubiquitin ligase
RNF5 E3 ubiquitin ligase has multiple biological roles and has been linked to the development of severe diseases such as cystic fibrosis, acute myeloid leukemia, and certain viral infections, emphasizing the importance of discovering small-molecule RNF5 modulators for research and drug development. The present study describes the synthesis of a new benzo[b]thiophene derivative, FX12, that acts as a selective small-molecule inhibitor and degrader of RNF5. We initially identified the previously reported STAT3 inhibitor, Stattic, as an inhibitor of dislocation of misfolded proteins from the endoplasmic reticulum (ER) lumen to the cytosol in ER-associated degradation. A concise structure–activity relationship campaign (SAR) around the Stattic chemotype led to the synthesis of FX12, which has diminished activity in inhibition of STAT3 activation and retains dislocation inhibitory activity. FX12 binds to RNF5 and inhibits its E3 activity in vitro as well as promoting proteasomal degradation of RNF5 in cells. RNF5 as a molecular target for FX12 was supported by the facts that FX12 requires RNF5 to inhibit dislocation and negatively regulates RNF5 function. Thus, this study developed a small-molecule inhibitor and degrader of the RNF5 ubiquitin ligase, providing a chemical biology tool for RNF5 research and therapeutic development.
RNF5 is a RING finger ubiquitin ligase involved in endoplasmic reticulum (ER)-associated degradation (ERAD) of misfolded proteins, including the premature degradation of cystic fibrosis transmembrane conductance regulator (CFTR) and its most common mutant, deletion of phenylalanine 508 (ΔF508 CFTR) (Younger et al., 2006). ΔF508 CFTR degradation leads to a deficiency of CFTR function at the cell surface, which is the primary cause of cystic fibrosis (CF) (Ward and Kopito, 1994; Ward et al., 1995; Younger et al., 2006; D’Antonio et al., 2019). Wild-type (wt) CFTR degradation has been linked to the development of chronic obstructive pulmonary disease (COPD) (Patel et al., 2020; Grand et al., 2021). The RNF5 gene is among the high-priority genes associated with COPD (Wain et al., 2017). Additionally, RNF5 is frequently hijacked by viruses to evade the immune response and facilitate viral replication. Paramyxoviruses, such as Newcastle disease virus, utilize their V protein as an adaptor to bring the mitochondrial antiviral-signaling protein (MAVS) to RNF5 for ubiquitination and degradation (Sun et al., 2019). Loss of MAVS diminishes the innate immune response against RNA viruses, as it is a signaling adaptor for the cytosolic RNA sensors RIG-1 and MDA5-mediated, microbial RNA-induced, and TLR-independent interferon response (Tan et al., 2018). RNF5 also regulates the stimulator of interferon genes (STING) during various viral infections. STING is a critical component of the cytosolic double-stranded DNA (dsDNA)-sensing cGAS-cGAMP-STING pathway. This pathway is activated in response to viral infection to induce the expression of type I interferons and thus mediates host defense against a range of DNA and RNA viruses (Ma and Damania, 2016; Li and Chen, 2018). Viral infection enhances RNF5 interaction with STING, leading to STING ubiquitination and proteasomal degradation, thus contributing to evasion of innate immune responses (Liu and Gao, 2018; Sun et al., 2019). During RNA virus infection, the JMJD6 protein recruits RNF5 to promote IRF3 ubiquitination and degradation, thereby attenuating the type I interferon response (Zhang et al., 2021). RNF5 has also been implicated in nonviral infections, including group A streptococcus infection, by regulating the level of the ATG4B protein, which is essential for LC3 maturation and autophagosome formation and thus controls the degree of infection (Kuang et al., 2012). RNF5 has also been associated with cancer progression. In cancer, cGAS-cGAMP-STING signaling is activated by the accumulation of cytosolic dsDNA, a consequence of nuclear DNA damage, and induces tumor suppressor functions, including immune surveillance, cellular senescence, and cell death (Ma and Damania, 2016; Khoo and Chen, 2018; Li and Chen, 2018; Tan et al., 2018). However, some studies have reported that activation of the cGAS-cGAMP-STING pathway by chromosomal instability can conversely drive metastasis (Bakhoum and Cantley, 2018; Bakhoum et al., 2018). In breast cancer, increased expression of RNF5 is correlated with decreased survival. Knockdown of RNF5, which is highly expressed in several breast cancer cell lines such as MCF-7, produces increased sensitivity to DNA damage–inducing chemotherapeutics (Bromberg et al., 2007). In addition to inhibition of STING signaling, RNF5 has been linked to other cancer-related events such as the regulation of cell migration, an essential process in metastasis, through ubiquitination of the focal adhesion protein paxillin (Didier et al., 2003). An increased abundance of RNF5 contributes to acute myeloid leukemia development and survival (Khateb et al., 2021). RNF5 has also been implicated in pancreatic cancer, where it targets the tumor suppressor PTEN for degradation and accelerates tumor growth (Pitarresi et al., 2018). A recent study reported that altered unfolded protein response (UPR) signaling in RNF5 knockout (KO) mice coincided with altered gut microbiota composition and consequently limited tumor expansion (Li et al., 2019). RNF5 also controls the stability of the glutamine carrier proteins SLC1A5 and SLC38A2, which limit glutamine uptake, and renders tumor cells more sensitive to ER stress–inducing chemotherapy (Jeon et al., 2015). The various roles exhibited by RNF5 in numerous disease pathologies highlight the importance of identifying small-molecule modulators of its E3 activity for research and assessment of RNF5 as a potential drug target. In this study, we report the synthesis and characterization of FX12, a selective small-molecule inhibitor and degrader of RNF5, which directly binds to RNF5, inhibits RNF5 E3 activity, and targets RNF5 for degradation via ERAD.
Transport of misfolded proteins from the ER lumen to the cytosol, known as retrotranslocation or dislocation, is crucial during ERAD. Previously, we established a dislocation-induced reconstituted green fluorescent protein (drGFP) reporter system to quantify the dislocation of misfolded protein substrates in living cells (Zhong and Fang, 2012). Using drGFP reporter screening of small-molecule libraries, we have previously identified two dislocation inhibitors that possess anti-flavivirus (Dengue and Zika viruses) activity (Rothan et al., 2019; Ruan et al., 2019). In this study, we identified Stattic, a previously characterized small-molecule inhibitor of STAT3 activation (Schust et al., 2006), as a dislocation inhibitor from the Library of Pharmacologically Active Compounds (LOPAC) (Figure 1A).
A concise SAR campaign around the Stattic chemotype was performed to diminish the STAT3 inhibitory activity while retaining activity in dislocation. The previous study showed that the nitro group in Stattic is important for STAT3 inhibitory activity (Schust et al., 2006). Therefore, the SAR effort was focused on benzo[b]thiophene 1,1-dioxide–based analogues (Figure 1, B–D, and Table 1). Eleven analogues exhibit excellent potencies in inhibiting the misfolded null Hong Kong variant of α-1-antitrypsin (NHK) dislocation in the drGFP assay, among which FX12 has the lowest cytotoxicity (Figure 1, B–D, and Table 1). FX12 inhibited NHK dislocation with an IC50 value of 2.7 μM, similar to that of Stattic (IC50 = 2.8 μM) (Figure 1, B and D). FX12 inhibited HepG2 cell growth with an IC50 value of 32.2 μM, which was higher than that of Stattic (10.8 μM) (Figure 1, C and D). Some other analogues showed better dislocation inhibition activity than FX12, such as FX40 and FX41, but all of those compounds had higher cytotoxicities (Figure 1D). On the basis of these results, we chose to focus on FX12 for further characterization. Time-lapse, live-cell imaging of drGFP confirmed that FX12 inhibited NHK dislocation (Figure 2A). ER–luminal substrates, such as NHK, require dislocation to the cytosolic surface of the ER to be ubiquitinated by an E3 ubiquitin ligase for subsequent proteasomal degradation (Ye et al., 2001; Hebert et al., 2010; Olzmann et al., 2013; Christianson and Ye, 2014; Sun and Brodsky, 2019). Therefore, inhibition of dislocation is expected to inhibit the ubiquitination and degradation of luminal ERAD substrates. We examined the effect of FX12 on NHK ubiquitination in cells. HeLa cells stably expressing HA-tagged NHK were treated with the proteasome inhibitor bortezomib (BTZ) alone or combined with FX12 to accumulate ubiquitinated NHK. Anti-HA reimmunoprecipitation revealed that FX12 efficiently inhibited NHK ubiquitination (Figure 2B). Consistently, cycloheximide (CHX) chase showed that NHK degradation was inhibited by FX12 (Figure 2C). In addition, FX12 did not affect the degradation of the cytosolic protein GFPu, whose degradation does not require dislocation (Bence et al., 2001) (Figure 2D). These results provide additional evidence that FX12 is a bona fide inhibitor of NHK dislocation. FX12 is a derivative of benzo[b]thiophene 1,1-dioxide that does not have the nitro group required for Stattic activity (Schust et al., 2006). Therefore, we determined the effects of FX12 on IL6-stimulated STAT3 activation. Immunoblotting showed that FX12 at concentrations up to 20 μM did not inhibit IL6-induced phosphorylation of endogenous STAT3, compared with ∼75% inhibition by Stattic at 10 μM concentration (Figure 2E). Thus, FX12 is a dislocation inhibitor with diminished effects on STAT3 activation.
We determined the effects of FX12 on ERAD complex protein expression to seek the potential mechanism underlying its activity in dislocation. HeLa cells were treated with increasing amounts of FX12 for 16 h. A dose-dependent down-regulation of RNF5 protein was observed after the treatment (Figure 3A). Conversely, the levels of other ERAD components increased in a dose-dependent manner, such as Derlin1, Ube2j1, and BiP (Figure 3A), possibly resulting from the activation of adaptive UPR due to inhibition of dislocation. In support of UPR activation, PERK and Ire1α exhibited subtle changes in mobility and the activated ATF6 was increased (Figure 3A). Up-regulation of the C/EBP homologous protein (CHOP), an indicator of apoptotic UPR, was observed only in positive control cells treated with tunicamycin, but not in FX12-treated cells, suggesting that FX12 does not cause severe ER stress to activate apoptotic UPR even at concentrations that fully inhibit NHK dislocation (10–20 μM) (Figures 1C and 2A). A time-course study showed that the RNF5 protein was markedly down-regulated by FX12 9 h after the treatment (Figure 3B). Reverse transcription PCR (RT-PCR) showed that FX12 treatment caused little to no changes in RNF5 mRNA levels (Figure 3C), indicating that FX12-induced RNF5 protein down-regulation is not due to inhibition of its gene transcription. Next, we determined whether RNF5 down-regulation is due to degradation. HeLa cells were treated with FX12 or inhibitor of one of the two major protein degradation pathways: proteasome or lysosome, or in combination. RNF5 protein was stabilized in cells treated by either the proteasome inhibitor BTZ or the lysosome inhibitor chloroquine (CQ) (Figure 3D, lane 1 vs. lanes 3 and 5), suggesting that both pathways are normally involved in RNF5 degradation. Interestingly, FX12-induced down-regulation of RNF5 was inhibited only by BTZ but not CQ, indicating that FX12 binding targets RNF5 to the proteasome for degradation (Figure 3D, lane 2 vs. lanes 4 and 6). Moreover, FX12 treatment directed all RNF5 protein to proteasomes for degradation (Figure 3D, lane 5 vs. lane 6). To determine whether the proteasome-dependent ERAD pathway degrades RNF5, we examined whether p97/VCP, a central regulator of ERAD, is involved in RNF5 degradation. The role of p97/VCP in ERAD is the extraction of ubiquitinated ERAD substrates from the ER for delivery to the proteasome for degradation. As such, HeLa cells were treated with FX12 in combination with the p97/VCP inhibitor NMS-873 (Magnaghi et al., 2013). As previously reported (Magnaghi et al., 2013; Walworth et al., 2016; Xia et al., 2016), inhibition of p97/VCP accumulated total ubiquitinated proteins (Figure 3E, lane 4) and FX12 treatment enhanced the accumulation (Figure 5E, lane 5), possibly by inducing ER stress due to inhibition of ERAD (Figure 2). Increased RNF5 ubiquitination was observed with NMS-873 treatment alone (Figure 3E, lane 9), consistent with a previous report that RNF5 autoubiquitinates and targets itself to ERAD (Huang et al., 2018). We did not see increases in RNF5 ubiquitination in FX12-treated cells (Figure 3E, lane 8), which is likely due to its rapid degradation. Importantly, RNF5 ubiquitination was increased by NMS-873 and further increased by simultaneous treatment with FX12 and NMS-873 (Figure 3E, lanes 9 and 10). In addition, NMS-873 also inhibited FX12-induced RNF5 down-regulation (Figure 3F). These results suggest that FX12 promoted RNF5 degradation by the p97/VCP-dependent ERAD pathway.
FX12 is selectively targeting RNF5 for degradation, suggesting that RNF5 may directly bind to FX12. Surface plasmon resonance (SPR) was used to assess FX12 and RNF5 binding in vitro. RNF5 is predicted to have two transmembrane domains in its C-terminal region and an N-terminal cytosolic domain (amino acid [aa]1–117, RNF5N) harboring a RING finger (aa27–67) that confers its E3 activity (https://www.uniprot.org/uniprot/Q99942). If FX12 targets RNF5, one would expect that they directly bind in vitro, and thus we determined whether FX12 binds to the cytosolic domain of RNF5 (RNF5N). Two other benzo[b]thiophene 1,1-dioxide derivatives (FX41 and FX36) were analyzed as controls (Figure 4, A–D). The results showed that FX12 binds to RNF5N with a KD of 594 nM (Figure 4A). FX41 also showed a similar KD of 666 nM, whereas FX36 had no detectable binding (Figure 4, B and C), consistent with their activity in the drGFP assay (Figures 1, B and D, and 4D). These results indicate that FX12 and its active analogue FX41 directly bind to the N-terminal cytosolic domain of RNF5 in vitro. We next assessed whether FX12 binding to RNF5 affected its E3 activity by in vitro ubiquitination assay reconstituted with glutathione S-transferase (GST)-RNF5N. GST-fusions of the cytosolic domain of another ERAD RING finger E3 Hrd1 (Hrd1C, aa235–617) and a non-ERAD RING finger E3 praja1 were tested as E3 controls. FX12 markedly inhibited the E3 activity of RNF5N but did not affect Hrd1C and Praja1 (Figure 4E). Importantly, FX12 did not affect ubiquitin charging to either ubiquitin-activating enzyme (E1) or ubiquitin-conjugating enzyme (E2) (Figure 4, F and G). These results suggest that FX12 binds to RNF5 and inhibits its E3 activity in vitro. FX12 inhibited NHK dislocation and degradation (Figure 2, A–C). If inhibition of RNF5 E3 activity by FX12 also occurs in cells, we would expect that the E3-inactive RNF5 inhibits NHK degradation. Therefore, we transiently expressed an E3-inactive RING finger mutant RNF5 (RNF5 RINGm) and determined its effects on NHK degradation. CHX chase showed that overexpression of RNF5 RINGm indeed inhibited NHK degradation (Figure 4, H and I). These results suggest that FX12 inhibits NHK dislocation and degradation by generating E3-inactive RNF5.
The cellular thermal shift assay (CETSA) was used to determine the potential FX12-target engagement (Martinez Molina et al., 2013). CETSA is based on the biophysical principle of ligand-induced thermal stabilization of target proteins. A ligand-stabilized protein target can be detected in the soluble cellular fraction by immunoblotting or mass spectrometry (Martinez Molina et al., 2013; Jafari et al., 2014). Among major dislocation regulators examined, only RNF5 and Derlin1 showed increased thermal stabilities upon FX12 treatment (Figure 5, A–D). This may not be surprising because previous studies have shown that RNF5 and Derlin1 are interacting partners and stabilizing the target protein can lead to changes in the thermal stability of target-associated proteins (Younger et al., 2006; Morito et al., 2008). Next, a CETSA was performed in Derlin1 KO HEK-293T cells to determine whether FX12 could still stabilize RNF5 in cells lacking Derlin1. The results showed that FX12 stabilized RNF5 independent of Derlin1 (Figure 5, E–G). Interestingly, coimmunoprecipitation revealed that FX12 increased RNF5 interaction with Derlin1 but decreased its interaction with Derlin2 (Figure 5H). These results suggest that RNF5 is a molecular target for FX12 in cells and the increased thermal stability of Derlin1 is due to the FX12-enhanced RNF5-Derlin1 interaction.
RNF5 is a well-established E3 involved in the degradation of both misfolded wt-CFTR and ΔF508 CFTR (Younger et al., 2006). ΔF508 CFTR is a major cause of CF and exists in approximately 90% of CF patients (Younger et al., 2006). Nearly 99% of newly synthesized ΔF508 CFTR cannot fold correctly and is rapidly degraded by ERAD (Ward and Kopito, 1994). Moreover, only ∼25% of wt-CFTR reaches its correct localization at the cell surface, and the remaining protein is degraded, analogous to mutant CFTR, by ERAD (Ward and Kopito, 1994; Farinha and Amaral, 2005). We hypothesized that inhibition and degradation of RNF5 by FX12 should have a stabilizing effect on ΔF508 CFTR. BHK cells stably expressing HA-ΔF508 CFTR (Gentzsch et al., 2004) were used to test this hypothesis. As predicted, FX12 induced a dose-dependent degradation of RNF5 in BHK cells (Figure 6A), as seen in HeLa cells (Figure 3A). FX12 dose-dependent increases in both the ER core–glycosylated immature form (B form) and the complex-glycosylated mature form (C form) of ΔF508 CFTR were observed (Figure 6, A and C). The C form of CFTR represents the mature form trafficked through Golgi, suggesting that inhibition of RNF5 enhances the stability and improves cell surface trafficking of ΔF508 CFTR. The current U. S. Food and Drug Administration (FDA)-approved therapies for CF rescue the function of CFTR using correctors (e.g., VX809, VX661, and VX445) to improve ΔF508 CFTR folding and a potentiator (e.g., VX770) to increase CFTR channel activity (Dekkers et al., 2016; Davies et al., 2018; Keating et al., 2018). By slowing the degradation of misfolded CFTR through inhibition and degradation of RNF5, FX12 may cooperate with VX809 or VX661 to further improve cell surface trafficking of both wt-CFTR and ΔF508 CFTR. This possibility was tested by treating BHK cells stably expressing either HA-wt-CFTR or HA-ΔF508 CFTR (Gentzsch et al., 2004) with increasing amounts of FX12 with or without the folding correctors VX809 or VX661. Both VX809 and VX661 induced increases in the C form of wt-CFTR and ΔF508 CFTR, which is consistent with their activities in improving CFTR folding (Figure 6, B and C). Importantly, cotreatment with FX12 further increased the C form of both wt-CFTR and ΔF508 CFTR in a dose-dependent manner (Figure 6, B and C). As a negative control, FX36 that had no activity in drGFP also did not affect ΔF508 CFTR (Figures 4, C and D, and 6D). These results suggest that inhibition of RNF5 provides more wt-CFTR and ΔF508 CFTR for folding correctors to increase their trafficking to the surface of BHK cells. To further investigate whether the effect of FX12 on CFTR is correlated with its dislocation inhibitory activity, we tested the effects of FX12 analogues on ΔF508 CFTR. Among 10 FX12 analogues showing inhibitory activities in NHK drGFP, seven increased the B form of ΔF508 CFTR, which was improved by cotreatment with VX809 (Figure 6E). The other three active analogues (FX33, FX34, and FX50) did not increase ΔF508 FTR, possibly due to their higher cytotoxicity (Figure 1, C and D). FX12 inhibits RNF5 E3 activity in vitro (Figure 4E). Consistently, we demonstrated that it inhibited ΔF508 CFTR ubiquitination and degradation in BHK cells (Figure 6, F and G). Next, we tested the effect of FX12 on ΔF508 CFTR function in primary HBE cells obtained from a CF patient homozygous for ΔF508. Cells were grown at an air–liquid interface until differentiated. Then the well-differentiated ΔF508/ΔF508 HBE cultures were exposed for 24 h to basolaterally added corrector VX809 (5 µM) and/or FX12 (5 µM) in the presence and absence of potentiator VX770 (1 µM). CFTR function was then evaluated in Ussing chambers (Figure 7, A and B). Amiloride was added to inhibit the epithelial sodium channel. Treatment with VX809 significantly increased maximal CFTR (stimulated by subsequent addition of forskolin and acute addition of VX770) (Figure 7B). As previously observed, VX809+VX770–treated cells showed a smaller CFTR response than VX809-treated cells caused by destabilization of rescued ΔF508 CFTR in the presence of chronic treatments with VX770 (Cholon et al., 2014). Unlike VX809, FX12 did not enhance CFTR responses and did also not further enhance responses of VX809- or VX809+VX770–rescued cells. Biochemical rescue of ΔF508 was assessed by immunoprecipitation of CFTR followed by Western blotting (Figure 7C). Robust rescue of formation of mature ΔF508 (Figure 7C, band C) was detected when cells were treated with VX809, whereas a minor amount of band C was observed when cells were treated with FX12 (Figure 7C). Further studies are needed to optimize FX12 effects on CFTR function in ΔF508/ΔF508 HBE cells via adjusting drug dosage and treatment times and/or simultaneous targeting of alternative degradation pathways.
If RNF5 is a target for FX12 to prevent dislocation, knocking it down should reduce FX12 activity. To test this possibility, the effects of RNF5 knockdown on dislocation were determined in HeLa cells that stably express the NHK-drGFP reporter (Zhong and Fang, 2012). RNF5 knockdown decreased FX12-mediated suppression of NHK dislocation (Figure 8, A–C), indicating that RNF5 is a molecular target for FX12 in cells. Next, the effects of FX12 on HA-ΔF508 CFTR stability and trafficking in RNF5 knockdown BHK cells were investigated for more proof (Figure 9). Derlin1, which forms a complex with RNF5 to recruit CFTR, was knocked down as a control (Figure 5H). Interestingly, knockdown of RNF5 stabilized ΔF508 CFTR more in the C form than in the B form, which was not seen in Derlin1 knockdown cells (Figure 9, C–F, lane 1). These results suggest that RNF5 not only is an E3 of misfolded CFTR but, in this system, may also play a role in its ER retention. Unlike in control and Derlin1 knockdown cells where FX12 treatment caused dose-dependent increases of both B and C forms of ΔF508 CFTR (Figures 9, C–F), FX12 treatment of RNF5 knockdown cells did not further increase ΔF508 CFTR (Figure 9, C–F). An FX12 dose-dependent shift from the C form to the B form of ΔF508 CFTR may be due to residual RNF5 protein in RNF5 knockdown cells (Figure 9, C and E). Next, we determined the effects of FX12 on paxillin, another substrate of RNF5. RNF5 ubiquitinates paxillin and inhibits paxillin localization to focal adhesion (Didier et al., 2003). Consistent with the previous report, FX12 treatment increases paxillin localization to focal adhesion (Figure 8D). These findings contribute to the growing body of evidence indicating that RNF5 is a molecular target for FX12 in cells.
To date, more than 20 ER membrane–spanning E3s have been identified, with some having a known role in ERAD (Neutzner et al., 2011; Fenech et al., 2020). The ERAD E3s, such as Hrd1, gp78, RNF5, and RNF185, act by organizing ER-membrane–anchored protein complexes (Fang et al., 2001; Amano et al., 2003; Sever et al., 2003; Kikkert et al., 2004; Younger et al., 2006; Morito et al., 2008; Bernardi et al., 2010; Fenech et al., 2020; van de Weijer et al., 2020) and have the potential to form different subcomplexes (Hwang et al., 2017). Cell type and disease–specific expression of ERAD complex proteins has also been observed (Amano et al., 2003; Bhattacharya and Qi, 2019). The variety of ERAD complexes may have evolved to dispose of a structurally diverse set of misfolded proteins or control protein levels associated with maintaining physiologic functions. The existence of cell type and disease–specific ERAD complexes suggests that small-molecule ERAD E3 inhibitors, if developed, could modulate specific cellular functions or halt disease progression (Bhattacharya and Qi, 2019). However, this promise has yet to be realized, as there are few reports of such inhibitors in the literature. The novel RNF5 inhibitor and degrader FX12 described in this paper could be a valuable tool for studying the roles of RNF5 in physiology and disease and evaluating RNF5 as a therapeutic target. Previous studies supported the importance of ERAD E3 inhibitors. Hrd1 was the first ERAD E3 with a reported small-molecule modulator, LS-102, identified as a selective inhibitor of its autoubiquitination activity with an in vitro IC50 of 35 μM (Yagishita et al., 2012). LS-102 treatment improves rheumatoid arthritis and alleviates obesity in mouse models (Amano et al., 2003; Yagishita et al., 2012; Fujita et al., 2015), which agrees with the known roles of Hrd1 in pathogenesis (Amano et al., 2003; Bournat and Brown, 2010). Recently, a small-molecule inhibitor of RNF5, inh-2, was identified by ligand docking and virtual screening of the RING finger of the RNF5 structure obtained by homology modeling (Sondo et al., 2018). Inh-2 modulates the known downstream pathways of RNF5, including stabilization of ΔF508 CFTR and improves ΔF508 CFTR activity. However, it is not known whether inh-2 exerts its activities by directly binding to RNF5 (Sondo et al., 2018). We have provided evidence that FX12, as a newly identified RNF5 E3 inhibitor and degrader, binds directly to RNF5 and inhibits RNF5 E3 activity in vitro (Figure 4, A and E). CETSA experiments support a direct FX12-RNF5 engagement in a cellular environment, as indicated by thermal stabilization of RNF5 in HEK-293T cells (Figure 5, A–G). Moreover, RNF5 knockdown tests show that FX12 targets RNF5 to produce its biological effects (Figures 8 and 9). CETSA and in vitro ubiquitination assays have also demonstrated a certain degree of target selectivity of FX12 (Figures 4E and 5, A–G). Consistent with the role of RNF5 as an E3 for misfolded CFTR, FX12 stabilizes ΔF508 CFTR and decreases its ubiquitination as well as increasing paxillin localization to focal adhesions (Figures 6, 7C, 8D, and 9). Moreover, FX12 enhances the FDA-approved CF therapeutics VX809 and VX661 in rescuing cell surface expression of ΔF508 CFTR in BHK cells (Figures 6 and 8F). However, unlike with inh-2, to date we could not demonstrate the improvement in ΔF508 CFTR channel activity by FX12 in patient-derived HBE cells (Figure 7). This discrepancy warrants further investigation. Inh-2 is a RNF5 inhibitor, but FX12is not only an inhibitor but also a degrader of RNF5. This differential activity may affect the channel activity of stabilized ΔF508 CFTR. In addition, the potential differential expression of RNF5 and/or its complexes could also contribute to the different responses between BHK and HBE cells. As multiple pathways have been shown to be involved in ΔF508 CFTR degradation in primary cells, additional maneuvers may be required to rescue the misfolded protein in these cultures. Because wt-CFTR has a longer half-life than ΔF508 CFTR, it might be a more suitable target in diseases such as COPD than ΔF508 CFTR in CF for which highly effective modulator combinations have become available recently. In cancer, inhibition of RNF5 has either beneficial or detrimental effects depending on cancer types (Bromberg et al., 2007; Jeon et al., 2015; Pitarresi et al., 2018; Gao et al., 2019; Li et al., 2019; Khateb et al., 2021). FX12 could be a useful chemical biology tool to study the role of RNF5 in different types of cancers and evaluate RNF5 as a therapeutic target for certain types of cancer. Studies using the drGFP reporter demonstrated dislocation of NHK, a well-characterized substrate for Hrd1. Surprisingly, RNF5 was identified as an FX12 target because it has not been shown to regulate NHK degradation. Our data suggest that FX12 may inhibit NHK dislocation by an indirect mechanism. drGFP assays were performed in cells treated with FX12 for up to 6 h (Figures 1B and 2A). During the 6 h treatment, RNF5 protein was not down-regulated (Figure 3B), but its E3 activity is likely inhibited by FX12 based on an in vitro assay (Figure 4E). We speculated that the E3-inactive RNF5 might inhibit Hrd1-mediated ERAD by its abnormal interactions with Hrd1 complex proteins. In support of this possibility, we did see that FX12 enhanced RNF5-Derlin1 interaction (Figure 5H). Moreover, overexpression of the RNF5 E3–inactive mutant markedly inhibited NHK degradation (Figure 4, H and I). FX12 significantly down-regulated RNF5 protein 9 h after FX12 treatment (Figure 3B), but the underlying mechanism is not fully understood. FX12 binding might alter the normal structure of RNF5. Thus, FX12-bound RNF5 may be recognized as a misfolded ER protein and degraded by ERAD mediated by an E3 ubiquitin ligase that remains to be identified. RNF5, like other ERAD E3s, forms protein complexes with membrane-spanning proteins. Although we demonstrated a direct interaction between FX12 and RNF5 in vitro, we cannot rule out the possibility that the FX12 binding site may be formed by RNF5 and its interacting proteins in cells. This binding model may explain why FX12 retains residual effects on ΔF508 CFTR stabilization in RNF5 knockdown cells (Figure 9, C and E), although the residual activity may also be explained by incomplete knockdown of RNF5. FX12 hijacks ERAD to initiate degradation of RNF5, rather than only inhibiting RNF5 E3 activity, which has important implications. In drug discovery and development, small-molecule–induced degradation of pathogenic proteins is actively being pursued, exemplified by proteolysis-targeting chimeric molecules (PROTACs) and molecular glues (Gadd et al., 2017; Gu et al., 2018; Smith et al., 2019; Isobe et al., 2020; Lv et al., 2020; Slabicki et al., 2020; Zeng and Han, 2020). A PROTAC is a designed, bifunctional small molecule that links its target protein to a known E3, hijacking the E3 to degrade the target protein. This study indicates that FX12 hijacks the protein quality control mechanism to degrade RNF5. Theoretically, this approach may be used to target other critical pathogenic proteins that access the secretory pathway by directly targeting them to ERAD for degradation without needing to design a bifunctional PROTAC molecule. In support of this possibility, a previous study reported that metformin hijacks ERAD to degrade PD-L1 (Cha et al., 2018). Because protein quality control degradation also occurs in the cytoplasm and the nucleus, the same approach may also be used to remove disease-causing proteins in these subcellular compartments. The advantage of hijacking protein quality control degradation to remove disease proteins is that there is no need to design bimodal PROTACs. Instead, small-molecule monovalent degraders can be identified by screening compound libraries using cell lines that stably express the disease protein reporters.
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Cells from human cell lines including HeLa, HepG2, and HEK-293T were purchased from the American Type Culture Collection (ATCC; https//www.atcc.org). Previously published cell lines used in this study include a HeLa cell line stably expressing a drGFP reporter for NHK dislocation (NHK-drGFP) (Zhong and Fang, 2012) and BHK cells stably expressing extope HA-tagged wt-CFTR or ΔF508 CFTR (Gentzsch et al., 2004). Primary HBE cells were purchased from Scott H. Randell (Marsico Lung Institute, The University of North Carolina at Chapel Hill). The obtained HBE cells were prepared from explant lungs from patients homozygous for the ΔF508 mutation under protocols approved by the University of North Carolina at Chapel Hill Biomedical Institutional Review Board (Fulcher and Randell, 2013). These cells were expanded in BEGM (Lonza) and then cultured at air–liquid interface on 12 mm Millicell inserts (Millipore) in modified BEBM (Lonza) for 24 d until differentiated.
The following antibodies were purchased from MilliporeSigma: mouse monoclonal antibodies, anti-HA (H3663, Clone HA7), mouse monoclonal anti–β-actin−peroxidase (A3854, clone AC-15), rabbit polyclonal anti-Sel1L (S3699), rabbit polyclonal anti-Derlin1 (D4443), rabbit polyclonal anti-PDI (P7372), mouse monoclonal anti-FLAG (F3165, clone M2), and ANTI-FLAG M2 Affinity Gel (A2220). Antibodies from Santa Cruz Biotechnology include mouse monoclonal anti–ubiquitin-HRP (sc-8017-HRP, clone P4D1), mouse monoclonal anti-GRP94 (sc-393402, clone H-10), mouse monoclonal anti-paxillin (sc-365379, clone B-2), mouse monoclonal anti-RNF5 (sc-81716, clone 22B3), mouse monoclonal anti-ATF6α (sc-166659, clone F-7), and mouse monoclonal anti-Ube2j1 (sc-377002, clone B-6). Other antibodies were from different sources, including mouse monoclonal anti-HA.11 (clone 16B12) (ENZ-ABS120; Enzo Lifesciences), rabbit polyclonal anti-RNF5 (PA5-71703; ThermoFisher), rabbit monoclonal anti-Hrd1 (clone D3O2A) (14773; Cell Signaling Technology), mouse monoclonal anti-BiP/GRP78 (clone 40/BiP) (610979; BD Biosciences), rabbit monoclonal anti-OS9 (clone EPR4272(2)) (ab109510; Abcam), and anti-HA Affinity Matrix (11815016001; Roche). Mouse monoclonal anti-gp78 (clone 2G5) was previously published (Ballar et al., 2006). CFTR antibodies 596 and 217 were obtained from Tim Jensen through the University of North Carolina CFTR antibody program.
MG132 (474787), bortezomib (5043140001), Stattic (S7947), CHX (C7698), tunicamycin (T7765), hygromycin B (H3274), and isopropyl β-d-1-thiogalactopyranoside (I6758) were purchased from MilliporeSigma; CFTR modulators VX809 (HY-13262; MedChemExpress; S1565; Selleck), VX661 (HY-15448; MedChemExpress), and VX770 (9582; BioVision; S1144; Selleck).
Interleukin-6 (human) (rec) (RDX-RCP9298; Axxora), UbcH5b (E2-622-100; R&D Systems), UBA1 (E-305-025; R&D Systems).
SuperSignal West Pico PLUS Chemiluminescent Substrate (34578; ThermoFisher) and Lipofectamine RNAiMAX Reagent (13778150; ThermoFisher); and small interfering RNA (siRNA) targeting Derlin1 and RNF5 synthesized based on previous reports (Lilley and Ploegh, 2005; Delaunay et al., 2008).
pFLAG-Derlin-1 and pFLAG-Derlin-2 were constructed by inserting the cDNA fragments encoding the open reading frames (ORF) of human Derlin-1 and Derlin-2, respectively, amplified by PCR and cut with HindIII/XhoI, to the HindIII/SalI sites of pFLAG-CMV-6a. pFLAG-RNF5 was constructed by inserting the cDNA fragments encoding the ORF of human RNF5 to the BglII/SalI sites of pFLAG-CMV-6C. In the pGEX-RNF5N plasmid, the cDNA fragment encoding the N-terminal 117 aa of human RNF5 was amplified by PCR, cut with BglII/XhoI, and then inserted into the BamHI/SalI sites of pGEX-4T-3. In the pGEX-Hrd1C plasmid, the BclI/NotI fragment of human Hrd1 cDNA, encoding the C-terminal portion (aa235–617) of human Hrd1 was inserted into the BamHI/NotI sites of pGEX-5X-1. pGEX-praja1 was previously published (Lorick et al., 1999).
Each of pGEX-RNF5N, pGEX-Hrd1C, and pGEX-praja1 was transformed into BL21(DE3). Single clones of BL21(DE3) transformed were cultured overnight in Lysogeny Broth (LB) medium. Then the overnight culture was inoculated (1:100) to fresh LB medium and cultured at 37°C until the OD600 reached 0.4–0.6. The expression of GST-tagged proteins was induced with 0.2 mM isopropyl β-d-thiogalactoside (IPTG) at 37°C for 1 h. The bacteria were lysed in lysis buffer (50 mM Tris-HCl, pH 8.0, 1 mM EDTA, 1% Triton X-100, 5 mM dithiothreitol) containing 200 µg/ml lysozyme with sonication. The lysates were cleared by centrifugation at 20,000 × g for 20 min. To purify the GST-tagged proteins, the cleared lysates were bound to Glutathione Sepharose 4B (GE Healthcare) for 2 h at 4°C with rotation. The beads were washed with 20 column volumes (CV) of lysis buffer and then with 5 CV of 50 mM Tris-HCl, pH 8.0. The GST-tagged proteins were eluted from the beads with 10 mM reduced glutathione in 50 mM Tris-HCl, pH 8.0. The elution fractions containing proteins were dialyzed against 1× phosphate-buffered saline (PBS) (137 mM NaCl, 2.7 mM KCl, 4.3 mM Na2HPO4, 1.47 mM KH2PO4). To prepare GST-cleaved RNF5N, GST-RNF5N was bound to Glutathione Sepharose 4B and the beads were washed with 20 CV of lysis buffer and then with 10 CV 1× PBS. Then the beads were incubated with 10 U thrombin per milligram of GST-RNF5N at 4°C overnight with gentle rotation. Thrombin was removed from the supernatant by incubating the latter with p-aminobenzamidine–agarose for 30 min.
HeLa cells stably expressing HA-tagged NHK were established as previously reported (Zhong and Fang, 2012). HeLa cells were seeded at 5 × 105 in 100 mm dishes in complete DMEM supplemented with 10% fetal bovine serum (FBS) and cultured in 37°C, 5% CO2. The next day, 10 µg of pCIneo-NHK-HA together with 0.5 µg of pBABE-puro was transfected into the cells with Lipofectamine 2000 (Invitrogen). Twenty-four hours after transfection, cells were selected with puromycin (1 μg/ml) for 14 d. Single stable clones surviving in the selection were trypsinized and transferred to individual wells in 24-well plates containing the culture medium for further culture. The single clones were screened for expression of HA-tagged NHK by immunoblotting with an anti-HA tag antibody. Derlin1 KO cell lines were generated in HEK-293T cells using CRISPR-Cas9. The CRISPR single-guide RNA expression plasmids targeting Derlin1 (NM_001134671.2) were obtained commercially (HCP255023-SG01-3; GeneCopia.com). Derlin1-targeting plasmids were transfected into HEK-293T cells cultured in DMEM supplemented with 10% FBS in a 100 mm dish. Twenty-four hours after transfection, cells were selected with hygromycin B (250 µg/ml) for 7 d. Derlin1 KO was determined by screening the single clones for Derlin1 expression with an anti-Derlin1 antibody in immunoblotting.
We screened LOPAC small-molecule library (SigmaAldrich.com) for dislocation inhibitors using the HeLa cells stably expressing the NHK-drGFP reporter (Zhong and Fang, 2012). NHK-drGFP reporter cells were seeded at 2 × 104/well in a black wall, clear-bottom 96-well plate (3603; Costar.com) and cultured overnight. The next day, culture medium containing the proteasome inhibitor BTZ (1 µM) alone or together with each compound (10 μM) was added to the cells for an additional 4 h. Wells containing medium alone served as background control. After replaced the medium with 1× PBS, drGFP fluorescence intensity was measured on a TECAN F200 Pro multimode microplate reader using excitation = 488 nm and emission = 525 nm. Compounds that exhibited >70% inhibition compared with BTZ-alone samples after the background extraction were subject to measurement of auto green fluorescence, and fluorescent compounds were eliminated. The remaining compounds were retested in an NHK-drGFP assay and were denoted as dislocation inhibitors when confirmed. All dislocation inhibitors were further verified independently in the InCell Analyzer 2200 wide-field imaging system at the National Center for Advancing Translational Sciences (NCATS) using a different batch of compounds at 10 μM concentration.
NHK-drGFP reporter cells were seeded at 2 × 104/well in 96-well plates. After overnight culture, the cells were washed once with PBS and then treated with BTZ (1 μM) alone or BTZ (1 μM) together with increasing concentrations of Stattic or its analogue. Live-cell images were acquired immediately after the addition of the inhibitors and then every 30 min on the IncuCyte Live-Cell Analysis System under a 20× objective lens (Sartorius.com), and the fluorescence intensities were quantified in IncuCyte software or on a TECAN F200 Pro Multimode microplate reader.
FX12 was synthesized using methyl benzo[b]thiophene-2-carboxylate as the starting material. Briefly, a solution of methyl benzo[b]thiophene-2-carboxylate (192 mg, 1 mmol) in dichloromethane (10 ml) was added 3-chloroperbenzoic acid (m-CPBA, 2 mmol) in portions over 20 min. The reaction was stirred at 50°C for 5 h and then cooled to room temperature. To the reaction mixture was added 0.5 M sodium hydroxide (20 ml). The solution was stirred for another 15 min. The aqueous layer was removed. The organic phase was washed using brine, dried over Na2SO4, and concentrated. The crude material was purified by flash chromatography to give compound FX12: 1H NMR (Varian INOVA 400 MHz, CDCl3): δ 7.99 (d, J = 0.8 Hz, 1H), 7.78 (d, J = 8.0 Hz, 1H), 7.71-7.63 (m, 2H), 7.53 (dd, J = 1.6 Hz, J = 7.2 Hz, 1H) and 13C NMR (Varian INOVA 100 MHz, CDCl3): δ 154.4, 140.3, 134.9, 134.1, 133.4, 127.4, 127.2, 122.2, 53.4; and HRMS (ESI, JEOL AccuTOF with ESI/APCI ion sources coupled to an Agilent 1100 HPLC system): The exact mass calculated for C10H9O4S [M+H]+ 225.0222, found 225.0224.
To a solution of the benzo[b]thiophene-2-carboxylic acid (2 mmol), phenol (2 mmol), and 4-dimethylaminopyridine (1.0 equiv.) in dichlormethane (0.5 mmol/ml) was added N, N-dicyclohexylcarbodiimide (1 equiv.) at 0°C. After the reaction mixture had been stirred at room temperature overnight, it was filtered through a pad of silica gel and purified by flash chromatography to afford FX36. 1H NMR (400 MHz, CDCl3): δ 8.31 (s, 1H), 7.98 (t, J = 7.6 Hz, 2H), 7.56-7.49 (m, 4H), 7.37-7.31 (m, 3H); 13C NMR (100 MHz, CDCl3): δ 161.3, 150.6, 142.6, 138.6, 132.7, 131.9, 129.5, 127.3, 126.1, 125.8, 125.1, 122.8, 121.6.
Step 1: To a solution of the benzo[b]thiophene-2-carboxylic acid (2 mmol), 2-phenylethan-1-ol (2 mmol), and 4-dimethylaminopyridine (1.0 equiv.) in dichlormethane (0.5 mmol/ml) was added N, N-dicyclohexylcarbodiimide (1 equiv.) at 0°C. After the reaction mixture had been stirred at room temperature overnight, it was filtered through a pad of silica gel and purified by flash chromatography to afford precursor phenethyl benzo[b]thiophene-2-carboxylate. 1H NMR (400 MHz, CDCl3): δ 8.06 (s, 1H), 7.897.87 (m, 2H), 7.49-7.27 (m, 7H), 4.57 (t, J = 6.8 Hz, 2H), 3.11 (t, J = 7.2 Hz, 2H); 13C NMR (100 MHz, CDCl3): δ 162.7, 142.2, 138.7, 137.7, 133.6, 130.6, 129.0, 128.6, 126.9, 126.7, 125.6, 124.9, 122.8, 66.0, 35.2. Step 2: This step was carried out entirely in accordance with general method A. 1H NMR (400 MHz, CDCl3): δ 7.92 (s, 1H), 7.75 (d, J = 6.8 Hz, 1H), 7.68-7.60 (m, 2H), 7.51 (d, J = 7.2 Hz, 1H), 7.35-7.25 (m, 5H), 4.56 (t, J = 7.2 Hz, 2H), 3.10 (t, J = 6.8 Hz, 2H); 13C NMR (100 MHz, CDCl3): δ 158.5, 139.9, 138.1, 137.1, 134.7, 133.9, 133.2, 129.2, 128.6, 128.1, 127.4, 126.8, 121.9, 66.8, 34.9. HRMS (ESI): Exact mass calculated for C16H16NO4S [M+NH4]+ 332.0957, found 332.0964.
The CHX chase was performed as previously reported (Fang et al., 2001). HeLa cells stably expressing HA-tagged NHK or -CD3δ were seeded (2 × 105 cells/well) in a 12-well plate and cultured overnight. Cells were then incubated with CHX (50 μg/ml) alone or along with FX12 for the indicated time (Figure 2C) and then were harvested and processed for immunoblotting with anti-HA and anti–β-actin antibodies.
HEK-293T cells were transfected with pFLAG-Derlin-1 or pFLAG-Derlin-2 or vector as control. The cells were treated with FX12 for 2 h as indicated the next day. Cells were lysed in 0.2% NP-40 in cell lysis buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA [ethylene glycol-bis(β-aminoethyl ether)-N,N,N′,N′-tetraacetic acid)]. Proteins (total 700 µg) were incubated with 10 µl of ANTI-FLAG M2 Affinity Gel for 2 h at 4°C with rotation. The beads were washed three times with cell lysis buffer containing 0.2% NP-40 before being processed for immunoblotting. Immunoprecipitation and immunoblotting of CFTR from HBE cells were performed as described previously (Cholon et al., 2014; Gentzsch et al., 2016, 2017; McCravy et al., 2020; He et al., 2021).
NHK ubiquitination was analyzed as previously reported (Zhong and Fang, 2012). Briefly, HeLa cells expressing NHK-HA were treated with BTZ (1 μM) or BTZ + FX12 (10 μM) to prevent degradation of ubiquitinated NHK. After the treatment, cells were lysed in cell lysis buffer containing 0.2% NP-40 followed by immunoprecipitation with the Anti-HA Affinity Matrix. To remove any protein that may associate with NHK-HA, the immunoprecipitates were denatured with 2% SDS, and the beads were removed by centrifugation. The supernatants were then diluted 20 times in native lysis buffer from which NHK-HA was reimmunoprecipitated, followed by immunoblotting for HA and ubiquitin. To analyze RNF5 ubiquitination, HeLa cells were transfected with pFLAG-RNF5. The next day, transfected cells were treated with FX12 and NMS873 as indicated. After the treatment, cells were lysed in cell lysis buffer containing 0.2% NP-40 followed by immunoprecipitation with µl of ANTI-FLAG M2 Affinity Gel for 2 h at 4°C with rotation. The immunoprecipitates were denatured with 2% SDS, and the beads were removed by centrifugation. The supernatants were then diluted 20 times in native lysis buffer from which FLAG-RNF5 was reimmunoprecipitated, followed by immunoblotting for HA and ubiquitin.
The CETSA was performed following a previously reported protocol (Martinez Molina et al., 2013; Reinhard et al., 2015). HeLa cells were treated with 10 μM FX12 or dimethyl sulfoxide (DMSO) control at 37°C for 2 h. After the treatment, cells were trypsinized and then neutralized with culture medium. After brief centrifugation at 1000 × g for 5 min, the cells were washed once and then resuspended with 1× PBS. Multiple aliquots of cell suspension were heated at increasing temperatures for 3 min before cooling to room temperature (25°C) for 3 min. An equal volume of 1× PBS containing 0.8% NP-40 was then added to the cells to lyse them. The samples were centrifuged at 20,000 × g for 20 min to separate soluble fractions from precipitated proteins. The soluble fractions were incubated with a 4× SDS sample buffer for 30 min at 37°C before being processed for immunoblotting. The following ERAD complex proteins were examined, including the major candidate target proteins in ERAD complexes, including RNF5, gp78, Hrd1, Sel1L, Derlin1, Derlin2, VIMP, Ube2j1, BiP, grp94, PDI, and OS9.
The binding of FX12 and another Stattic analogue, FX41, with the purified recombinant cytosolic domain of RNF5N (aa1–117) was measured using Biacore. Sensorgrams were obtained for 62.5, 125, 250, or 500 nM compounds against RNF5N immobilized on the CM5 sensor chip. Purified RNF5(aa1–117) was coupled to the surface of Biacore CM5 sensor chips by direct immobilization. Ligands were performed at a flow rate of 30 µl/min in HBS running buffer. The association was recorded by SPR with a Biacore T200 (Biacore, USA).
In vitro ubiquitination was performed following our previously reported protocol (Fang et al., 2001). Briefly, 500 ng of GST-RNF5, GST-Hrd1c, or GST-Praja1 was immobilized on glutathione beads. Ubiquitination assays were carried out by adding 20 ng each of human E1 and E2 (Ube2j1) and ubiquitin (2 μg) in ubiquitination buffer containing 50 mm Tris-HCl, pH 7.4, 2 mM ATP, and 5 mM MgCl2 with an increasing amount of FX12 (0, 5, 10 μM). Reactions were carried out in 20 μl for 30 min at 30°C followed by processing for immunoblotting. FX12 does not affect ubiquitin charging to E1 and E2. E1 assay: 0.2 μg of recombinant UBA1 (E1) was mixed with ubiquitin, reaction buffer with or without ATP, and DMSO or FX12 (10 μM final) on ice as indicated. The reactions were incubated at 37°C for 30 min. E2 assay: 0.5 μg of recombinant UbcH5b (E2) was mixed with ubiquitin, reaction buffer, and DMSO or FX12 (10 μM final) on ice as indicated. Recombinant UBA1 (E1) (0.2 μg) was added to the reactions as indicated. The reactions were incubated at 37°C for 30 min. UbcH5b was detected by Ponceau S staining.
Ion transport was measured as short-circuit current (Isc) in a modified Ussing chamber system using Acquire and Analyze software (Physiological Instruments) as previously described (Cholon et al., 2014; Gentzsch et al., 2016, 2017; He et al., 2021) in a bilateral Krebs bicarbonate–Ringers (KBR) solution. Bridges were equilibrated in KBR bubbled with CO2 and maintained at 37°C, and each chamber was zeroed against a blank insert. Changes in Isc were measured. Amiloride (100 μM; Sigma-Aldrich) was added to the apical bath to inhibit the epithelial sodium channel ENaC. Bilateral addition of forskolin (10 μM; Sigma-Aldrich) was followed by apical addition of potentiator compound VX770 (1 μM; Selleck Chemicals) to stimulate CFTR channel activity. CFTR inhibitor-172 (10 μM; Sigma-Aldrich) was then apically introduced to inhibit CFTR. Transepithelial resistance (in Ω·cm2) was monitored to assess monolayer integrity. Traces were plotted using Origin Graphic software. Changes in Isc were calculated using Microsoft Excel. Bar graphs were plotted in Prism.
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PMC9635169 | Mengxing Guo,Jingyao Lian,Yaqing Liu,Bo Dong,Qianyi He,Qitai Zhao,Hongyan Zhang,Yu Qi,Yi Zhang,Lan Huang | Loss of miR-637 promotes cancer cell stemness via WASH/IL-8 pathway and serves as a novel prognostic marker in esophageal squamous cell carcinoma | 03-11-2022 | Cancer stem cells,Esophageal squamous cell carcinoma,Interleukin-8,miR-637,WASH | Background Esophageal carcinoma is the highly lethal cancer in the world, predominantly in some areas of East Asia. We previously reported that overexpression of cytoskeleton regulator Wiskott-Aldrich syndrome protein and SCAR Homolog (WASH) associates with poor prognosis of patients with esophageal squamous cell carcinoma (ESCC). However, the molecular mechanism and clinical significance involved in WASH overexpression have not been fully elucidated. Methods Bioinformatics analysis and luciferase reporter assay were used to predict and validate miR-637 as a regulator of WASH in ESCC cell lines. qRT-PCR, Western blotting and ELISA assays were performed to examine RNA expression and protein levels, respectively. Next, the biological functions of miR-637 were explored by tumor sphere formation assay in vitro and nude mouse tumor xenograft in vivo. Finally, we evaluated the association of miR-637 levels with clinical features in ESCC patients. Results We identified miR-637 as a WASH-targeting miRNA. miR-637 mimic strongly attenuated the downstream IL-8 production and tumor sphere formation in esophageal cancer cells, whereas miR-637 inhibitor displayed an opposite effect. IL-8 could facilitate stem-like properties and partially rescue the phenotypes induced by miR-637 mimic. Furthermore, miR-637 inhibitor dramatically promoted IL-8 expression and cancer stemness properties in a WASH-dependent manner. Ectopic expression of miR-637 also inhibited tumor growth in a mouse model. Clinically, low expression of miR-637 was observed in tumor tissues and the low expression levels of miR-637 were correlated with poor survival of ESCC patients. In particular, plasma miR-637 could be used as a noninvasive biomarker for ESCC patients. Conclusions These results implicate the potential application of miR-637 for diagnosis and prognosis of esophageal cancer. Supplementary Information The online version contains supplementary material available at 10.1186/s40364-022-00424-x. | Loss of miR-637 promotes cancer cell stemness via WASH/IL-8 pathway and serves as a novel prognostic marker in esophageal squamous cell carcinoma
Esophageal carcinoma is the highly lethal cancer in the world, predominantly in some areas of East Asia. We previously reported that overexpression of cytoskeleton regulator Wiskott-Aldrich syndrome protein and SCAR Homolog (WASH) associates with poor prognosis of patients with esophageal squamous cell carcinoma (ESCC). However, the molecular mechanism and clinical significance involved in WASH overexpression have not been fully elucidated.
Bioinformatics analysis and luciferase reporter assay were used to predict and validate miR-637 as a regulator of WASH in ESCC cell lines. qRT-PCR, Western blotting and ELISA assays were performed to examine RNA expression and protein levels, respectively. Next, the biological functions of miR-637 were explored by tumor sphere formation assay in vitro and nude mouse tumor xenograft in vivo. Finally, we evaluated the association of miR-637 levels with clinical features in ESCC patients.
We identified miR-637 as a WASH-targeting miRNA. miR-637 mimic strongly attenuated the downstream IL-8 production and tumor sphere formation in esophageal cancer cells, whereas miR-637 inhibitor displayed an opposite effect. IL-8 could facilitate stem-like properties and partially rescue the phenotypes induced by miR-637 mimic. Furthermore, miR-637 inhibitor dramatically promoted IL-8 expression and cancer stemness properties in a WASH-dependent manner. Ectopic expression of miR-637 also inhibited tumor growth in a mouse model. Clinically, low expression of miR-637 was observed in tumor tissues and the low expression levels of miR-637 were correlated with poor survival of ESCC patients. In particular, plasma miR-637 could be used as a noninvasive biomarker for ESCC patients.
These results implicate the potential application of miR-637 for diagnosis and prognosis of esophageal cancer.
The online version contains supplementary material available at 10.1186/s40364-022-00424-x.
Esophageal carcinoma ranks the seventh most common cancer and the sixth most deadly cancer worldwide, according to global cancer statistics in 2018 [1]. There are two main histological types of esophageal cancer, esophageal adenocarcinoma and esophageal squamous cell carcinoma (ESCC) [2, 3]. ESCC accounts for more than 90% of all esophageal cancer cases in China, which are often diagnosed at an advanced stage with a poor prognosis [3–5]. Despite intensive research for newly developed treatment strategies including novel immunotherapy, the improvement in survival of patients with esophageal cancer remains unfavorable [6]. Therefore, there is an urgent need to explore novel prognostic biomarkers and therapeutic targets for esophageal cancer patients. MicroRNAs (miRNAs), a family of short non-coding RNAs (~ 22 nucleotides), can modulate gene expression via specifically binding to the 3′-untranslated region (3′-UTR) of target mRNA, thereby inducing mRNA degradation [7–9]. Aberrant expression of miRNAs has been associated with various mechanisms of cancer progression, including oncogenesis, metastasis, drug resistance and immune escape [10]. Recently, an increasing number of studies have revealed that miRNAs play a key role in regulating cancer stem cells (CSCs) [11]. Depending on particular target genes, miRNAs function as either oncogenes or tumor suppressors, even in one type of cancer. For instance, down-regulation of miRNA-30e has been reported to increase cancer cell proliferation, invasion and tumor growth through targeting RPS6KB1 in esophageal cancer [12]. In contrast, miR-196b was significantly overexpressed and served as an oncogene to promote drug resistance in ESCC by targeting EPHA7 [13]. Furthermore, high expression of miR-17-5p and miR-4443 were closely correlated with tumor stages, suggesting a prognostic vale of the two miRNAs in ESCC [14]. Recently, emerging studies focused on the feasibility of blood miRNAs as noninvasive biomarkers for diagnosis and prognosis of cancers [15, 16]. WASH is a member of Wiskott-Aldrich syndrome protein (WASP) family and acts as nucleation-promoting factors for actin-related protein 2/3 complex (ARP2/3) to drive the generation of branched actin filaments [17]. WASH plays a pivotal role in distinct cellular biological processes, such as autophagy [18], mitosis [19], endosomal recycling [20] and phagosome maturation [21]. In our previous study, WASH was reported to enhance cancer stemness and associated with poor prognosis in ESCC patients [22]. However, the underlying mechanism of WASH expression remains largely unknown. Therefore, identification of miRNAs regulating the expression of WASH may offer new insights into prognostic biomarkers or therapeutic targets for ESCC. In this study, we identified miR-637 as an important miRNA that directly targets WASH expression and inhibits cancer cell stemness via IL-8 signaling in vitro. We further demonstrated the therapeutic potential of miR-637 in xenograft tumor models of ESCC. Furthermore, loss of miR-637 in tumor tissue as well as peripheral blood was closely associated with poor prognosis of patients with ESCC. Overall, our findings provide significant evidence for miR-637 as a valuable diagnostic and prognostic biomarker for ESCC.
Human ESCC cell lines KYSE70 and KYSE450 were obtained from Cancer Hospital of Chinese Academy of Medical Sciences and Peking Union Medical College. All cancer cell lines were grown in RPMI 1640 medium (Sigma) supplemented with 10% heat-inactivated fetal bovine serum (FBS) (HyClone), 100 units/mL penicillin and 100 μg/mL streptomycin. Cells were cultured at 37 °C in a humidified incubator with 5% CO2.
The mimic and inhibitor of miR-637, as well as the corresponding negative control (NC), were purchased from GenePharma (Shanghai, China). Cells were transfected with miR-637 mimic (100 nM) or inhibitor (100 nM) by using Lipofectamine 3000 transfection reagent (Invitrogen) according to the manufacturer’s instruction.
Total RNA was extracted from cancer cell lines, tumor tissue and plasma respectively, using RNAiso Plus (Takara Bio) according to the manufacturer’s instructions. The isolated total RNA was quantified by a NanoDrop spectrophotometer (Thermo Fisher Scientific). To quantify mRNA expression, total RNA was used to generate cDNA using PrimeScript RT reagent Kit with gDNA Eraser (Takara Bio). To measure the expression level of miR-637, total RNA was transcribed to cDNA using Mir-X miRNA First-Strand Synthesis Kit (Takara Bio). qRT-PCR was carried out using specific primers and SYBR Green Master Mix (Roche) on a Stratagene Mx3005P qPCR System (Agilent Technologies). The primer sequences are listed in Supplementary Table S1.
Cells were lysed with RIPA buffer (Solarbio, China) containing protease inhibitor cocktail (Sigma-Aldrich). Cell lysates were sonicated on ice, resolved on SDS-PAGE and transferred to a nitrocellulose membrane. After blocking with 5% non-fat milk, the membranes were probed with primary antibodies for the detection of WASH (ab157592, Abcam) and β-actin (3700S, Cell Signaling Technology) followed by horseradish peroxidase-conjugated secondary antibodies (Cell Signaling Technology). Protein expression was detected using enhanced chemiluminescent HRP substrate (Thermo Fisher Scientific) and photographed with a Chemiluminescence Imaging System (Bio-Rad Laboratories).
The 3′-UTR of WASH gene containing the predicted miR-637 binding site was synthesized by Sango Biotech (Shanghai, China) and cloned into the pmirGLO vector (Promega). The mutant construct with altered binding sites of miR-637 was generated by using the QuickChange Site-Directed Mutagenesis kit (Agilent Technologies). 293 T cells were transfected with the constructed pmirGLO vector (200 ng) and 50 nM miR-637 or NC mimic using Lipofectamine 3000 (Invitrogen). After 48 h of transfection, cells were collected and luciferase activity was measured using Dual-Luciferase Reporter Assay System (Promega). The firefly luciferase activity was normalized to Renilla luciferase activity.
Supernatant was collected from cell culture and plasma was isolated from peripheral blood, respectively. The IL-8 production was measured by using human IL-8 ELISA kit (BioLegend) according to the manufacturer’s instruction.
For enrichment of CSCs, cancer cells were seeded into ultra-low attachment 24-well culture plates (Corning) at a density of 20,000 cells/well and cultured in serum-free DMEM/ F12 medium (Gibco), containing 20 ng/mL human recombinant epidermal growth factor (Sigma-Aldrich), 20 ng/mL human recombinant basic fibroblast growth factor (Sigma-Aldrich), 1:50 dilution of B27 (Gibco) and 5 μg/mL insulin (Sigma-Aldrich). After 5 ~ 7 days of culture, the cells formed CSCs-like aggregates and the number of tumor spheres was counted under microscope.
The lentiviral plasmid overexpressing IL-8 (OE-IL-8) was purchased from GeneChem (Shanghai, China). To produce lentiviral particles, the plasmid was transfected into 293 T cells with pMD2.G and psPAX2 using Lipofectamine 3000. Forty eight hours after transfection, viral supernatant was harvested and then transduced into KYSE450 cells with 8 μg/ml polybrene. After culture for several days, stable IL-8 overexpressing cells were obtained upon culture in the presence of 1 μg/ml puromycin (Invitrogen). For stable overexpression of miR-637, the miRNA lentiviral stocks carrying miR-637 and GFP were purchased from Genechem (Shanghai, China) and transduced KYSE450 cells according to the manufacturer’s instructions. The successfully transduced cells were sorted for GFP-positive cells by Moflo-XDP cytometer (Beckman Coulter).
Cell proliferation was assessed using a Cell Counting Kit (CCK-8; Dojindo) according to the manufacturer’s protocol.
Cells were seeded in a 6-well plate and incubated for 24 h. The monolayer was scratched by a sterile pipette tip and cultured in serum-free medium for 0, 24 and 48 h. The area of wound closure was measured by microscopic photography with Image J software.
Transwell migration assays were performed using uncoated inserts (8-μm pore size; Corning). Cells were placed in serum-free media into the upper chamber. The lower well contained medium 10% FBS. After 24 h incubation, cells that had migrated through the pores were fixed, stained with crystal violet solution, photographed and counted.
Female 6–8 weeks old NTG mice (NOD-scid IL2Rγ−/−) were purchased from SPF Beijing Biotechnology (China) and housed in specific pathogen-free animal laboratory. NTG mice were subcutaneously injected with KYSE450 cells stably overexpressing miR-637 or NC (3 × 106 cells /mouse). Tumor size and body weight were measured twice a week. Tumor volume was calculated with the following formula: volume = (length × width2)/2. After 5 weeks, tumor xenografts were collected from the mice for qRT-PCR assays. The animal experiments were approved by the Animal Care and Use Committee of Zhengzhou University Animal Facility (Approval number: ZZU-LAC20200807).
Paraffin-embedded mouse tumor xenografts were used to IHC analysis. After deparaffinization, rehydration and antigen retrieval, the tissue slides were incubated with primary anti-human IL-8 (Proteintech, 27,095–1-AP) or anti-human Ki67 (Servicebio, GB121141) antibodies, respectively. Immunological reactions were detected by horseradish peroxidase-conjugated secondary antibodies and diaminobenzidine substrate (Dako). The slides were quantitatively evaluated according to staining percentage and intensity, ranged from 0 (no staining) to 12 (100% of cells with intense staining).
Cells were re-suspended in PBS containing 2% FBS and stained with PE-conjugated anti-human CD44 antibody (BioLegend) at 4 °C for 30 min. PE-conjugated mouse IgG1 isotype control (BioLegend) was used as the control. The expression of surface CD44 was analyzed by FACS Canto II cytometer (BD Biosciences).
All clinical samples were obtained from the First Affiliated Hospital of Zhengzhou University, China. The tumor tissue samples and adjacent normal tissue samples were collected from ESCC patients who underwent surgical resection at the Department of Thoracic Surgery. The clinical information of the patients from which these tissue samples were derived is shown in Supplementary Table S2. Peripheral blood samples were collected from ESCC patients and healthy donors (HD). The clinical information of the patients from which these peripheral blood samples were derived is shown in Supplementary Table S3. This study was approved by the Institutional Ethics Committee of the First Affiliated Hospital of Zhengzhou University (Approval number: 2019-KY-255).
Data were analyzed by paired or unpaired Student’s t-test. Correlation analysis was performed with Pearson’s test. miR-637 expression with overall survival was analyzed using Log-Rank test. All statistical analyses were performed by using GraphPad Prism 7 software. ROC curves were plotted by the R language packages pROC and glmnet. p < 0.05 was considered statistically significant. Data were presented as the mean ± standard deviation from 2 to 3 independent experiments.
We have previously reported that high WASH expression in ESCC tissue is significantly related to poor prognosis [22]. To dissect the molecular mechanism of WASH overexpression and the potential prognostic biomarker, we utilized TargetScan and miRanda to analyze miRNA-binding sites in the 3′-UTR of WASH. As a result, a total of eleven predicted miRNA candidates were screened out (Fig. 1A). To determine WASH-targeting miRNAs, specific miRNA mimics and a NC mimic were transfected into KYSE70 cells, respectively. Compared to the NC mimic, only miR-637 significantly suppressed WASH expression (Fig. 1B). We further confirmed that miR-637 mimic down-regulated WASH expression in two ESCC cell lines, at both mRNA and protein levels (Fig. 1C, D). In contrast, transfection of a miR-637 inhibitor resulted in a significant increase in the expression of WASH mRNA and protein (Fig. 1E, F). The successful regulation of miR-637 expression was shown in two ESCC cell lines upon transfection with miR-637 mimic or inhibitor, respectively (Fig. 1G, H). To validate whether WASH is a direct target of miR-637, we constructed the luciferase reporter plasmids containing wild-type or mutant 3′-UTR of WASH at the miR-637 binding site (Fig. 1I). The luciferase activity of 293 T cells transfected with the plasmid encoding wild-type 3′-UTR of WASH was remarkably reduced by miR-637 mimic, whereas the luciferase activity in 293 T cells transfected with the plasmid encoding mutant 3′-UTR of WASH was not affected (Fig. 1J). Collectively, these results clearly indicated that miR-637 down-regulates WASH expression through directly binding to the 3′-UTR of WASH.
Our previous data showed that WASH promoted the cancer stemness properties of ESCC through induction of IL-8 [22]. To identify the downstream effector of miR-637, the expression levels of IL-8 were analyzed by qRT-PCR and ELISA assays, respectively. The expression of IL-8 was found to be noticeably decreased after transfection of miR-637 mimic in two tested ESCC cell lines (KYSE70 and KYSE450), at transcription level (Fig. 2A) and protein level (Fig. 2B). Furthermore, treatment with miR-637 inhibitor consistently induced the increase of IL-8 production in KYSE70 and KYSE450 cells, at transcription level (Fig. 2C) and protein level (Fig. 2D). Next, the impact of miR-637 expression on the cancer stemness of ESCC cell lines was assessed by sphere formation assay in vitro. After incubation for 5–7 days, both KYSE70 cells and KYSE450 cells transfected with miR-637 mimic exhibited an inhibitory effect on tumor sphere formation (Fig. 2E). In contrast, transfection of miR-637 inhibitor resulted in a significant increase of the sphere formation capacity in the two cell lines (Fig. 2F). Consistently, the expression levels of stemness-related genes, including SOX4, SOX9, Nanog, CD44 and ABCG2, were also affected by miR-637 mimic (Supplementary Fig. S1A) or miR-637 inhibitor (Supplementary Fig. S1B), respectively. To confirm the role of IL-8 in miR-637-regulated tumor sphere formation, we generated IL-8 overexpressing KYSE450 cells (Supplementary Fig. S1C). As expected, ectopic expression of IL-8 promoted the formation of tumor spheres (Fig. 2G) and increased the expression of multiple stemness markers in KYSE450 cells (Fig. Supplementary S1D). Furthermore, IL-8 overexpression was able to rescue the miR-637-induced inhibition of tumor sphere formation (Fig. 2G) and stemness-related gene expression (Supplementary Fig. S1D). Similarly, addition of exogenous IL-8 also enhanced the CSCs properties and partially reversed the effect of miR-637 mimic in both KYSE70 cells (Supplementary Fig. S2A, B) and KYSE450 cells (Supplementary Fig. S2C, D). These data strongly indicated that miR-637 possesses a tumor suppressive role in ESCC through inhibition of IL-8 and cancer stemness.
To further determine the role of WASH in miR-637-induced tumor suppression, a lentiviral-mediated approach was used to construct stable WASH knockdown ESCC cell line [22]. The successful knockdown of WASH expression was detected in KYSE70 cells at the mRNA (Supplementary Fig. S3A) and protein levels (Supplementary Fig. S3B). Furthermore, WASH knockdown decreased the expression of IL-8 in KYSE70 cells (Supplementary Fig. S3C). As expected, transfection of miR-637 inhibitor resulted in WASH expression in control cells but not WASH-knockdown cells both at the mRNA level (Fig. 3A) and protein level (Fig. 3B). IL-8 is a downstream target of WASH and promotes the stemness of ESCC cells. Similarly, miR-637 inhibitor increased the expression of IL-8 in control cells but not WASH-knockdown cells (Fig. 3C), indicating that miR-637 regulates IL-8 expression by targeting WASH. Finally, we analyzed miR-637 inhibitor-induced tumor sphere formation and stem cell markers after modulation of WASH expression. Again, transfection with miR-637 inhibitor failed to increase the number of tumor spheres and up-regulate the expression of stemness-related genes (SOX4, SOX9, Nanog, CD44 and ABCG2) after stable knockdown of WASH in KYSE70 cells (Fig. 3D, E). These results demonstrate that down-regulation of miR-637 facilitates the maintenance of cancer stemness in ESCC cells likely through WASH/IL-8 pathway.
To evaluate the preclinical impact of miR-637 in ESCC, we established stably miR-637-overexpressing KYSE450 cells by using lentiviral expression system. Consistently, stable overexpression of miR-637 dramatically reduced the expression of WASH, IL-8 and stemness genes, as well as the number of spheres formed by KYSE450 cells (Supplementary Fig. S4A-G). In addition, miR-637 overexpression slightly suppressed cell proliferation, but had no effect on cell migration (Supplementary Fig. S4H-J). We examined the anti-tumor activity of miR-637 in a xenograft model. KYSE450 cells stably overexpressing miR-637 or control miRNA were subcutaneously injected into the flanks of NTG mice. Over a period of 5 weeks, ectopic expression of miR-637 leaded to an obvious attenuation in tumor growth curve (Fig. 4A), tumor size (Fig. 4B) and tumor weight (Fig. 4C) compared to control miRNA. Next, we detected the expression of miR-637 in KYSE450 xenografts by qRT-PCR (Fig. 4D). Consistent with our in vitro findings, miR-637 significantly inhibited the expression levels of WASH (Fig. 4E) and IL-8 (Fig. 4F) in KYSE450 xenografts. Similarly, the decreased levels of SOX4, SOX9, Nanog，CD44, KLF4 and ABCG2 were observed from the harvested KYSE450 tumors related to miR-637 overexpression, indicating the inhibition of cancer cell stemness by miR-637 (Fig. 4G). In addition, the expression of Ki67, a cell proliferation marker, decreased noticeably in miR-637 overexpressing KYSE450 xenografts (Fig. 4H). Furthermore, IHC analysis of tumor xenografts revealed that overexpression of miR-637 resulted in a significant reduction of IL-8 and Ki67 in protein level (Fig. 4I). Taken together, our data suggested the therapeutic potential of miR-637 in tumor bearing mice.
To determine the expression levels of miR-637 in human ESCC tissues, we performed qRT-PCR assay in 49 paired ESCC tumor and adjacent normal tissues. The expression level of miR-637 was significantly down-regulated in the tumor samples compared with adjacent normal tissues, revealing a possible tumor suppressive role of miR-637 in ESCC (Fig. 5A). Furthermore, the connection between miR-637 expression and pathological characteristics of ESCC was evaluated in 49 patients with ESCC. Low miR-637 expression was positively associated with late tumor-node-metastasis (TNM) stage (Fig. 5B), poor differentiation status (Fig. 5C) and lymph node metastasis (Fig. 5D). In Kaplan–Meier survival analysis, low miR-637 expression was positively correlated with poor overall survival of ESCC patients (Fig. 5E). Collectively, these data indicated that miR-637 was markedly down-regulated in tumor tissues and associated with poor prognosis of ESCC patients.
To explore the potential of miR-637 as a noninvasive biomarker for ESCC patients, the plasma samples from 50 ESCC patients and 25 healthy donors were used for qRT-PCR assays. We found that miR-637 expression was significantly decreased in the plasma from ESCC patients compared to that from healthy donors (Fig. 6A). Conversely, the levels of plasma IL-8 from ESCC patients were significantly higher than those from healthy donors (Fig. 6B). Pearson’s correlation analysis further confirmed that miR-637 and IL-8 expression were inversely correlated in ESCC plasma samples (Fig. 6C). Next, we analyzed the pathological relevance of plasma miR-637 and IL-8 in ESCC patients. Low levels of plasma miR-637 was positively correlated with advanced TNM stage (Fig. 6D) and lymph node metastasis (Fig. 6E). However, no connection between plasma IL-8 and TNM stage (Supplementary Fig. S5A) or lymph node metastasis (Supplementary Fig. S5B) was observed. To evaluate the diagnostic potential of plasma miR-637 and IL-8 levels, we calculated the area under the curve (AUC) using a receiver operating characteristic (ROC) curve. The AUC of miR-637 and IL-8 was 0.911 and 0.772, respectively (Fig. 6F, G), indicating that plasma miR-637 is a better diagnostic biomarker for ESCC patients. In summary, loss of plasma miR-637 may serve as a useful noninvasive marker for diagnosis and prognosis of ESCC patients.
In recent years, the discovery of miRNAs has deepened our understanding of human cancers. Numerous studies have demonstrated the importance of miRNAs in cancer development and clinical application. In this present study, we demonstrated that miR-637 as a direct regulator of WASH promoted IL-8 production and cancer stemness properties of ESCC cells in vitro and in vivo. More importantly, miR-637 expression was down-regulated in ESCC tumor tissues and exhibited a negative correlation with patient survival. Plasma miR-637 was also lower in ESCC patients than healthy donors, indicating a potential novel biomarker of ESCC. The members of the WASP family are primarily involved in actin polymerization and cytoskeleton reorganization, which is crucial for cancer development and metastasis [23]. Indeed, the WASP family proteins have been reported to be associated with adhesion, migration, invasion and colonization of malignant cells [24]. For instance, inhibition of N-WASP has been shown to reduce cancer stemness [25]. Data from our group also supports the participation of WASH in maintenance of cancer stemness [22]. However, there is little information about the regulation of WASH expression. Here, we identified that miR-637 could directly target the 3′-UTR of WASH and suppress its expression. Previously, miR-637 has been reported to inhibit tumor progression in several types of cancers though various mechanisms [26–28]. In glioma, miR-637 represses tumor cell proliferation and migration by targeting Akt1 [26]. In addition, miR-637 retards glioblastoma progression via the ZEB2/WNT/beta-catenin cascades [27]. Moreover, miR-637 has been observed to inhibit tumor cell proliferation and invasion by targeting AKT1 in liver cancer [28]. Other studies also find the role of miR-637 in suppressing tumorigenesis and drug resistance [29, 30]. In line with these studies, we observed that inhibition of miR-637 greatly promoted IL-8 production and cancer stemness in a WASH-dependent manner, while overexpression of miR-637 showed an inverse effect both in vitro and in vivo. Interestingly, miR-637 slightly inhibited the cell proliferation but not migration of ESCC cell line KYSE450. Although IL-8 was reported to play multiple roles in tumor progression, recent studies demonstrated that IL-8 may promote CSCs-like properties [31, 32]. Here our work demonstrates IL-8 as a linker between miR-637 and cancer stemness properties, suggesting that miR-637 may inhibit cancer cell stemness through suppression of IL-8 in ESCC. These results indicate that miR-637 might serve as a negative regulator of cancer progression through inactivating several oncogenic pathways. The present findings showed that miR-637 was markedly down-regulated in ESCC tissues and significantly correlated with TNM stage, tumor differentiation, lymph node metastasis and poor overall survival. In consistent with our study, miR-637 was reported significantly decreased in glioma tissues and positively associated with the prognosis of patients [26]. Recently, another study demonstrated that low expression of miR-637 was highly correlated with poor prognosis in patients with advanced breast cancer [33]. These findings support reduced expression of miR-637 as a novel biomarker for predication of advanced tumor features in ESCC patients. Many studies have demonstrated that miRNAs circulate in the blood and can be easily detected by qRT-PCR [34]. Therefore, blood miRNAs may reflect the condition of tumor tissue and serve as a convenient noninvasive biomarker for cancer diagnosis and prognosis [35, 36]. Recently, microRNA array-based approaches have been widely used to explore circulating miRNAs as a biomarker for many types of cancers including ESCC [37–39]. In our study, miR-637 was significantly decreased in the plasma of ESCC patients compared to that in the plasma of healthy donors. Furthermore, the levels of plasma miR-637 was also found to be correlated with TNM stage and lymph node metastasis. This result suggests that miR-637 may be associated with tumor progression. Of note, we did not observe obvious correlations between plasma IL-8 and pathological prognostic parameters in ESCC. In addition to cancer cells, many other cell types including macrophages, lymphocytes and fibroblasts have also been shown to secrete IL-8 in tumor microenvironment [40]. As a diagnostic marker of ESCC, plasma miR-637 thus showed a better sensitivity compared to plasma IL-8. However, the clinical significance of plasma miR-637 should be interpreted cautiously since the sample size is relatively small in this study. To verify the diagnostic ability of plasma miR-637, further investigations need to be conducted on a larger number of patient samples and healthy donors.
In summary, we found that miR-637 plays an important role as a tumor suppressor in ESCC cancer stemness through targeting WASH/IL-8 pathway. Additionally, decreased miR-637 expression is associated with poor prognosis. Thus, miR-637 may be a potential prognostic marker and therapeutic target for esophageal carcinoma.
Additional file 1: Supplementary Table S1. Primer Sequences for qRT-PCR assays.Additional file 2: Supplementary Table S2. Clinicopathological features of ESCC patients for tissue miR-637 analysis.Additional file 3: Supplementary Table S3. Clinicopathological features of ESCC patients for plasma miR-637 analysis.Additional file 4: Supplementary Fig. S1. Expression of stemness-related genes detected by qRT-PCR assays. A, B Regulation of SOX4, SOX9, Nanog, CD44 and ABCG2 expression in KYSE70 cells and KYSE450 cells after treatment with miR-637 mimic (A) or miR-637 inhibitor (B), respectively. C, D KYSE450 cells were stably transduced with lentivirus overexpressing (OE) negative control (NC) or IL-8. Identification of IL-8 overexpression (C) and stemness-related gene expression after treatment with miR-637 mimic (D). Data are presented as mean ± standard deviation. ns, not significant, *p < 0.05, **p < 0.01, ***p < 0.001.Additional file 5: Supplementary Fig. S2. IL-8 is involved in miR-637-regulated cancer stemness of ESCC cell lines. KYSE70 cells (A, B) and KYSE450 cells (C, D) were transfected with negative control (NC) or miR-637 mimic in the presence of control or recombinant human IL-8 (100 ng/ml). Tumor sphere formation (A, C) and qRT-PCR (B, D) assays were performed respectively. Scale bar, 200 μm. Data are presented as mean ± standard. *p < 0.05, **p < 0.01, ***p < 0.001.Additional file 6: Supplementary Fig. S3. Characteristics of KYSE70 cells with stable WASH knockdown. A, B Knockdown of WASH expression in KYSE70 cells through stable transduction with WASH shRNA (shWASH) or negative control shRNA (shNC) was validated by qRT-PCR (A) and Western blotting (B) assays, respectively. C The expression level of IL-8 was determined by qRT-PCR assay in KYSE70 cells with shRNA-mediated stable knockdown of WASH. Data are presented as mean ± standard deviation. ***p < 0.001.Additional file 7: Supplementary Fig. S4. Characteristics of KYSE450 cells stably overexpressing miR-637. KYSE450 cells were stably transduced with lentivirus (LV) expressing miR-637 or negative control (NC). A-D The expression levels of miR-637 (A), WASH mRNA (B), IL-8 mRNA (C) and secreted IL-8 (D) were determined by qRT-PCR or ELISA assays. E-G The effects of stable miR-637 overexpression on cancer stemness were examined by tumor sphere formation (E), qPCR assay (F) and flow cytometry analysis (G). Scale bar, 200 μm. H-J CCK8 (H), wound healing (I) and Transwell migration (J) assays were used to detect cell proliferation and migration, respectively. Data are presented as mean ± standard deviation. ns, not significant, *p < 0.05, **p < 0.01, ***p < 0.001.Additional file 8: Supplementary Fig. S5. Plasma IL-8 has no relationship with TNM stage and lymph node metastasis in ESCC patients. The expression levels of IL-8 in the plasma of ESCC patients were detected by ELISA assay and analyzed with TNM stage (A) and lymph node metastasis (B). ns, not significant. | true | true | true |
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PMC9635184 | Yanan Chen,Hao Zhang,Yue Li,Shuli Ji,Peilu Jia,Tian Wang | Pterostilbene attenuates intrauterine growth retardation-induced colon inflammation in piglets by modulating endoplasmic reticulum stress and autophagy | 04-11-2022 | Autophagic flux,Colon inflammation,Endoplasmic reticulum stress,Intrauterine growth retardation,Piglets | Background Endoplasmic reticulum (ER) stress and autophagy are implicated in the pathophysiology of intestinal inflammation; however, their roles in intrauterine growth retardation (IUGR)-induced colon inflammation are unclear. This study explored the protective effects of natural stilbene pterostilbene on colon inflammation using the IUGR piglets and the tumor necrosis factor alpha (TNF-α)-treated human colonic epithelial cells (Caco-2) by targeting ER stress and autophagy. Results Both the IUGR colon and the TNF-α-treated Caco-2 cells exhibited inflammatory responses, ER stress, and impaired autophagic flux (P < 0.05). The ER stress inducer tunicamycin and the autophagy inhibitor 3-methyladenine further augmented inflammatory responses and apoptosis in the TNF-α-treated Caco-2 cells (P < 0.05). Conversely, pterostilbene inhibited ER stress and restored autophagic flux in the IUGR colon and the TNF-α-treated cells (P < 0.05). Pterostilbene also prevented the release of inflammatory cytokines and nuclear translocation of nuclear factor kappa B p65, reduced intestinal permeability and cell apoptosis, and facilitated the expression of intestinal tight junction proteins in the IUGR colon and the TNF-α-treated cells (P < 0.05). Importantly, treatment with tunicamycin or autophagosome-lysosome binding inhibitor chloroquine blocked the positive effects of pterostilbene on inflammatory response, cell apoptosis, and intestinal barrier function in the TNF-α-exposed Caco-2 cells (P < 0.05). Conclusion Pterostilbene mitigates ER stress and promotes autophagic flux, thereby improving colon inflammation and barrier dysfunction in the IUGR piglets and the TNF-α-treated Caco-2 cells. Supplementary Information The online version contains supplementary material available at 10.1186/s40104-022-00780-6. | Pterostilbene attenuates intrauterine growth retardation-induced colon inflammation in piglets by modulating endoplasmic reticulum stress and autophagy
Endoplasmic reticulum (ER) stress and autophagy are implicated in the pathophysiology of intestinal inflammation; however, their roles in intrauterine growth retardation (IUGR)-induced colon inflammation are unclear. This study explored the protective effects of natural stilbene pterostilbene on colon inflammation using the IUGR piglets and the tumor necrosis factor alpha (TNF-α)-treated human colonic epithelial cells (Caco-2) by targeting ER stress and autophagy.
Both the IUGR colon and the TNF-α-treated Caco-2 cells exhibited inflammatory responses, ER stress, and impaired autophagic flux (P < 0.05). The ER stress inducer tunicamycin and the autophagy inhibitor 3-methyladenine further augmented inflammatory responses and apoptosis in the TNF-α-treated Caco-2 cells (P < 0.05). Conversely, pterostilbene inhibited ER stress and restored autophagic flux in the IUGR colon and the TNF-α-treated cells (P < 0.05). Pterostilbene also prevented the release of inflammatory cytokines and nuclear translocation of nuclear factor kappa B p65, reduced intestinal permeability and cell apoptosis, and facilitated the expression of intestinal tight junction proteins in the IUGR colon and the TNF-α-treated cells (P < 0.05). Importantly, treatment with tunicamycin or autophagosome-lysosome binding inhibitor chloroquine blocked the positive effects of pterostilbene on inflammatory response, cell apoptosis, and intestinal barrier function in the TNF-α-exposed Caco-2 cells (P < 0.05).
Pterostilbene mitigates ER stress and promotes autophagic flux, thereby improving colon inflammation and barrier dysfunction in the IUGR piglets and the TNF-α-treated Caco-2 cells.
The online version contains supplementary material available at 10.1186/s40104-022-00780-6.
Intrauterine growth retardation (IUGR), which presents as a failure of fetuses to achieve their intrinsic growth potential during pregnancy, has become one of the most common and challenging issues in animal production [1, 2]. IUGR alters the intestinal morphology, impairs intestinal barrier function, and creates feeding intolerance, all of which increase the occurrence of intestinal diseases and cause postnatal retardation of growth and development, resulting in high rates of mortality and morbidity [3–5]. IUGR neonates experience a high incidence of colitis since they show deficiencies in mucosal immunity and imbalances in T lymphocyte subpopulations and lack an efficient colonic barrier [6–9]. Therefore, protecting the colon against inflammation may be a pivotal requirement for the health of IUGR animals. The colon is one site that is vulnerable to endoplasmic reticulum (ER) stress, a physiological response induced by protein aggregation or misfolding within the ER lumen [10]. A variety of physiological and pathological stressors, including bacteria and inflammation tone from the intestinal lumen and host, can induce colonic ER stress [11]. In addition, the highly secretory cells that occur in the colonic epithelium manufacture a range of complex proteins and antimicrobial molecules that are currently recognized as crucial targets for protein misfolding in the ER [12]. To counteract these negative effects, the conserved signal transduction pathways unfolded protein responses (UPR) have evolved. They are initially activated as adaptive responses that strengthen protein folding capacity, re-establish ER function, and restore intestinal barrier homeostasis [13]. However, once these adaptive mechanisms fail to resolve the folding defects, the UPR will switch to programmed cell death by triggering pro-apoptotic signaling cascades and even induce inflammatory responses [13, 14]. Indeed, ER stress has been identified as part of the intrinsic machinery of intestinal inflammation [11, 15]. Autophagy, a pro-survival mechanism for cells and organisms suffering from starvation or other diverse pathologies, plays an important physiological role in intestinal health [16]. Autophagy modulates the relationship between gut microflora and host immunity and thereby maintains intestinal barrier function, whereas impaired autophagy in intestinal epithelium can induce intestinal inflammation [17, 18]. Interestingly, autophagy is recognized as an emerging effector mechanism that regulates ER homeostasis [19]. The misfolded proteins and damaged ER fragments can be degraded and recycled by autophagy [19]. The UPR branches and UPR-associated transcription factors, such as activating transcription factor 4 (ATF4) and CCAAT/enhancer binding protein homologous protein (CHOP), have also been demonstrated to modulate the autophagic process [20]. Based on these findings, exploring the exact interaction between ER stress and autophagy in the context of inflammation may help to uncover the mechanism and identify prospective strategies for the IUGR-induced colon inflammation. Stilbenes are a class of polyphenolic compounds that are widely distributed in blueberries, grapes, and other medicinal plants [21]. Their multiple biological activities, especially antioxidant and anti-inflammatory properties, suggest the potential value of stilbenes in the treatment or prevention of intestinal disorders [22–24]. Recently, considerable attention has focused on pterostilbene, a dimethylated analogue of resveratrol, due to its excellent metabolic stability, intestinal absorption features, and bioavailability [25, 26]. Our previous work indicated that pterostilbene had strong protective effects against intestinal barrier defects and small intestinal injuries in animals under the conditions of oxidative stress or immunological stress [27–29]. Pterostilbene was also found to mitigate colon inflammation in mice fed high-fat diets or treated with dextran sulfate sodium [30]. However, the potential for attenuation of the IUGR-induced colon inflammation by pterostilbene was not documented. Whether pterostilbene-mediated protection on intestine health is associated with its regulation of ER stress and/or autophagy is not known. The current study explored the mechanism by which pterostilbene protected against the IUGR-induced colon inflammation using a naturally occurring IUGR piglet model. We also revealed the crosstalk between ER stress and autophagy under inflammation conditions and the regulation of pterostilbene in these two events in the tumor necrosis factor-alpha (TNF-α)-treated human colonic epithelial cells (Caco-2).
Pterostilbene was obtained from BOC Science (Shirley, NY, USA). TNF-α was purchased from Sino Biological, Inc. (Beijing, China). Tunicamycin, 4-phenylbutyric acid (4PBA), and FITC-dextran were purchased from Sigma-Aldrich (St. Louis, MO, USA). Rapamycin and 3-methyladenine (3MA) were purchased from CSNpharm (Arlington Heights, IL, USA). Chloroquine was purchased from MedChemExpress (Shanghai, China). Adenovirus expressing mCherry-GFP-LC3B fusion protein recognizing CD46 (AdPlus-mCherry-GFP-LC3B) was purchased from Beyotime (Haimen, Jiangsu, China). Dulbecco’s modified Eagle’s medium (DMEM), fetal bovine serum (FBS), and 1% penicillin and streptomycin were obtained from Thermo Fisher Scientific Inc. (Grand Island, NY, USA). Antibodies used in Western blot analysis included: anti-occludin (1:3000; Proteintech; Chicago, IL, USA), anti-zonula occludens 1 (ZO-1; 1:1000; Proteintech), anti-glyceraldehyde phosphate dehydrogenase (GAPDH; 1:25,000; Proteintech), anti-nuclear factor kappa B p65 (NF-κB p65; 1:3000; Proteintech), anti-Lamin B1 (1:5000; Proteintech), anti-glucose-regulated protein 78 (GRP78; 1:3000; Proteintech), anti-CHOP (1:1000; Proteintech), anti-cleaved caspase 12 (c-Casp12; 1:2000; Bioss Biotechnology; Beijing, China), anti-phosphorylated protein kinase RNA-like ER kinase (PERK) (p-PERK; 1:2000; Affinity Biosciences; Cincinnati, OH, USA), anti-total-PERK (t-PERK; 1:1000; Affinity Biosciences), anti-phosphorylated inositol-requiring kinase 1 alpha (IRE1a) (p-IRE1a; 1:1000; Affinity Biosciences), anti-total-IRE1a (t-IRE1a; 1:2000; Affinity Biosciences), anti-activating transcription factor 6 (ATF6; 1:5000; Proteintech), anti-phosphorylated eukaryotic translational initiation factor 2 alpha (eIF2α) (p-eIF2α; 1:1000; Bioss Biotechnology), anti-total-eIF2α (t-eIF2α; 1:1000; Bioss Biotechnology), anti-Beclin1 (1:3000; Proteintech), anti-LC3 I/II (1:2000; Proteintech), anti-p62 (1:4000; Proteintech), anti-Rab7 (1:2000; Affinity Biosciences), and anti-lysosomal associated membrane protein 2 (LAMP2; 1:1000; Proteintech).
All animal experiments were reviewed and approved by the Institutional Animal Care and Use Committee of Nanjing Agricultural University (SYXK-2017-0027). Approximately 72 healthy sows (Landrace × Yorkshire) having same parity (second or third) and expected dates of confinement (< 4 d) were preselected during pregnancy. At delivery, only sows with about 11–13 live-born piglets were retained. The birth weights (BWs) of newborn piglets (Duroc × (Landrace × Yorkshire)) were recorded. Piglets were identified as normal birth weight (NBW) if their BWs were close to the mean litter BW (within 0.5 standard deviations), whereas those with BWs at least 2 standard deviations lower than the mean litter BW were defined as IUGR [27, 31]. All piglets were permitted to suckle naturally up to 21 days of age. After weaning, a total of 36 litters that met the selection criteria for NBW and IUGR piglets were reserved, and one IUGR and one NBW male piglet were selected from each litter. Half of the selected NBW and IUGR piglets received a basal diet (NBW-CON and IUGR-CON groups) for 14 d and the other half were fed a basal diet supplemented with 300 mg/kg pterostilbene (NBW-PTS and IUGR-PTS groups) over the same period. Each treatment group consisted of 6 replicate pens with 3 piglets per pen. The basal diet (Table S1) was formulated according to the nutrient requirements of swine (National Research Council, 2012) [32]. The level of supplemented pterostilbene was determined based on our previous publication [27, 28]. Diet and water were offered ad libitum during the entire experiment. The diarrhea rates of piglets were determined as reported by Liu et al. [33]. At the endpoint of the experimental period, the piglets were weighed, and one piglet whose BW was close to the average weight of each replicate was chosen for sampling. Heparinized blood samples were withdrawn by anterior vena cava puncture, centrifuged at 2500 × g for 10 min at 4 °C, and stored at − 80 °C for analysis. The piglets were then euthanized, and the colon tissues were removed. After flushing with ice-cold PBS, approximately 1 cm of colon samples taken from the middle of the colon were immersed in 4% paraformaldehyde fixative solution for histological analysis. The colonic mucosa was scraped from the remainder with a glass microscope slide, snap-frozen in liquid nitrogen, and stored at − 80 °C for further analysis.
Colon tissues fixed in 4% paraformaldehyde were dehydrated in an ascending alcohol series, cleared with xylene, embedded in paraffin blocks, cut into 5 μm sections, and mounted on glass slides. After deparaffinizing and rehydrating, the sections were stained with hematoxylin and eosin (H&E) buffer, and the histologic alterations were observed using a light microscopy (Olympus Corp; Tokyo, Japan). The Chiu scoring system was employed to evaluate the colon mucosal injury. The colon goblet cell numbers were determined by incubating the slices with Alcian blue/periodic acid-Schiff stains as described previously [31]. The colon goblet cell density was calculated as the goblet cell count divided by the corresponding villus length.
Plasma LPS levels were measured with an enzyme-linked immunosorbent assay (ELISA) kit obtained from CUSABIO Biotech (Wuhan, Hubei, China).
The colon mucosa was homogenized and centrifuged at 5000 × g for 5 min, and the concentrations of TNF-α, interleukin (IL)-1β, IL-4, IL-10, mucin 2, and trefoil factor 3 (TFF3) of the supernatants were determined with porcine-specific ELISA kits (CUSABIO Biotech). The colon myeloperoxidase (MPO) activity was measured with a commercial kit (Jiancheng Bioengineering Institute; Nanjing, Jiangsu, China). The protein levels of the supernatants were quantified using a bicinchoninic acid protein assay kit (Beyotime) for normalization of these parameters.
Caco-2 cell line was kindly gifted by Dr. Xiang Hou (Jiangsu Academy of Agricultural Sciences; Nanjing, Jiangsu, China). Caco-2 cells at passage numbers 28–36 were grown in DMEM medium supplemented with 10% FBS and 1% penicillin and streptomycin at 37 °C in a humidified atmosphere containing 5% CO2. The optimal treatment concentrations of TNF-α and pterostilbene were determined by exposing Caco-2 cells to TNF-α (0–50 ng/mL) with or without pterostilbene (0–250 μmol/L) for 24 h. ER stress in Caco-2 cells was induced and inhibited by tunicamycin (0.5 μg/mL for 24 h) and 4PBA (1 mmol/L for 24 h), respectively. Rapamycin (1 μmol/L for 24 h) and 3MA (5 μmol/L for 24 h) were used to induce and inhibit autophagy in Caco-2 cells, respectively. Chloroquine (20 μmol/L for 24 h) was used to obstruct the fusion of autophagosomes and lysosomes in Caco-2 cells. The Cell Counting Kit-8 (CCK-8; Dojindo Molecular Technologies; Shanghai, China) was used to evaluate the viability of Caco-2 cells. Briefly, Caco-2 cells were seeded in 96-well microplates at a density of 1 × 104 cells/well. After 24 h, Caco-2 cells were treated with a range of TNF-α or pterostilbene for 24 h. Then, 10 μL CCK-8 reagent was added to each well for 1.5 h, and the absorbance was measured by a microplate reader (Thermo Fisher Scientific Inc.) at 450 nm.
The autophagic flux was monitored by transfecting Caco-2 cells with AdPlus-mCherry-GFP-LC3B. The Caco-2 cells were seeded in confocal dishes at a density of 2 × 105 cells/well until confluence reached 50%. After infection with AdPlus-mCherry-GFP-LC3B dissolved in a complete DMEM medium (multiplicity of infection = 80) for 24 h, the cells were co-incubated with TNF-α and tunicamycin, 4PBA, rapamycin, or pterostilbene for another 24 h. The expression of GFP and mCherry was visualized and captured with a laser scanning confocal microscope (Carl Zeiss; Oberkochen, Germany).
Caco-2 cells (2 × 105 cells/well) were grown in 12-well permeable culture chambers (12 mm diameter inserts and 0.4 μm pore size) with a complete DMEM medium until they formed a tight monolayer with a transepithelial electrical resistance (TER) of 500 Ω. Thereafter, the cells were subjected to various treatments, and the TER and FITC-dextran flux were analyzed to determine the permeability of Caco-2 cell monolayers. The TER of each culture chamber was tested at 3 different sites using the Millicell-ERS resistance system (Millipore; Bedford, MA, USA) and corrected against the blank control. The net resistance was multiplied by the membrane area to give the resistance in Ω cm2. For FITC-dextran flux analysis, a complete DMEM medium containing 500 μg/mL FITC-dextran was placed in the upper chamber of the transwells. The FITC-dextran flux was measured by collecting the basal medium every 30 min for 3 h with replacement of the sampled volume with fresh medium without FITC-dextran at each sampling time and expressed as the flux into the basal chamber as a percentage of the total FITC-dextran initially added to the apical chamber.
Caco-2 cells were seeded in 6-well plates at a density of 5 × 105 cells/well for 24 h and then exposed to TNF-α and pterostilbene with or without tunicamycin and chloroquine for 24 h. The supernatants of Caco-2 cells were collected to determine the contents of IL-1β and IL-6 with ELISA kits (Multi Sciences Biotech; Hangzhou, Zhejiang, China). All procedures strictly followed the manufacturer’s protocols.
Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining and flow cytometry assays were carried out to determine the apoptotic index of the colon tissues and Caco-2 cells, respectively. The methods can be found in our previous study [28].
Caco-2 cells were plated in 12-well plates at a density of 2 × 105 cells/well for 24 h and then subjected to the study treatments. Subsequently, the cells were fixed in 4% paraformaldehyde, permeabilized with 0.1% Triton X-100, and incubated with 3% bovine serum albumin (BSA). The cells were then incubated overnight with the primary body against NF-κB p65 (1:200) at 4 °C, followed by Alexa Fluor 594-conjugated secondary antibody (1:200) for 1 h and 2-(4-amidinophenyl)-6-indolecarbamidine dihydrochloride (DAPI) for 1 min at room temperature. Images were recorded with a confocal laser scanning microscope (Carl Zeiss).
All reagents were purchased from Vazyme (Nanjing, Jiangsu, China). Total RNA from colon samples and Caco-2 cells was isolated with the FastPure Cell/Tissue Total RNA Isolation Kit and reverse-transcribed into cDNAs with the HiScript III 1st Strand cDNA Synthesis Kit. The ChamQ SYBR qPCR Master Mix and the StepOnePlus PCR System (Applied Biosystems; Carlsbad, CA, USA) were used to detect the expression levels of target genes by qRT-PCR analysis. Thermal cycling was initiated at 95 °C for 30 s, followed by 40 cycles of 95 °C for 10 s and 60 °C for 30 s. The specific primers for GRP78, glucose-regulated protein 94 (GRP94), CHOP, ATF4, spliced X-box binding protein-1 (sXBP-1), TNF-α, IL-1β, IL-6, and GAPDH were obtained from Sangon Biotech (Shanghai, China). The primer sequences were provided in Table S2. The results were calculated using the 2−ΔΔCT method and normalized to GAPDH.
Real-time PCR with T100 Thermal Cycler (Bio-Rad; Hercules, CA, USA) was carried out to determine the mRNA levels of sXBP-1 in Caco-2 cells. The PCR products were separated on 1.8% agarose gel and visualized with GelRed DNA stain. The primer sequences can be found in Table S2.
All reagents for protein extraction and determination of protein concentration were obtained from Beyotime. Total protein from colon samples and Caco-2 cells was extracted using the lysis buffer containing a cocktail of phosphatase and protease inhibitors. A nuclear protein extraction kit was employed to isolate the nuclear protein in colon tissues and Caco-2 cells. After measurement of the protein contents, 30 μg of protein from each sample was separated by SDS-PAGE and electro-transferred to the polyvinylidene fluoride membranes. Subsequently, the membranes were blocked with 5% BSA for 1 h at room temperature and then incubated with specific primary antibodies overnight at 4 °C with gentle rocking. After washing with the Tris-buffered saline containing 1% Tween 20, the corresponding secondary antibodies were added and incubated for 1.5 h at room temperature. Immunoreactive bands were captured by the ChemiDoc™ imaging system (Bio-Rad) and analyzed using the Gel-Pro Analyzer software.
All statistical analyses were performed using SPSS software version 26 (Chicago, IL, USA). Data from at least three independent experiments were shown as means with their standard errors (SE). For comparison of multiple datasets, one-way analysis of variance (ANOVA) with Tukey’s multiple-range test was employed. Values with P < 0.05 were considered statistically significant.
To evaluate the protective potential of pterostilbene on intestinal health of the IUGR piglets, we recorded the diarrhea rates and determined the contents of circulating LPS, a bacterially generated endotoxin that penetrates the intestinal epithelial barrier and reaches the bloodstream when the intestinal permeability is increased [34]. As shown in Fig. 1a and b, the diarrhea rates and plasma LPS levels were significantly higher in the IUGR piglets than in their NBW counterparts, whereas these negative effects caused by IUGR were attenuated after pterostilbene treatment (P < 0.05). We also assessed the colon morphology, apoptosis, and tight junction protein expression of piglets to determine the effects of pterostilbene on colon barrier function. H&E staining indicated extensive damage to the colon epithelium of IUGR piglets, with injuries including hemorrhage, crypt abscesses, and lymphocyte infiltration to the lamina propria (Fig. 1c). The histological scores and apoptotic rates were also higher in the colon of the IUGR piglets than in those of the NBW piglets (P < 0.05; Fig. 1d-f). Moreover, IUGR downregulated colon protein levels of occludin and ZO-1 of piglets (P < 0.05; Fig. 1g). In contrast, dietary pterostilbene administration improved the colon architecture and blocked hemorrhage and lymphocyte infiltration to the lamina propria, thereby reducing the histological scores of the IUGR colon (P < 0.05). The increased number of TUNEL-positive cells and the reduced protein levels of occludin and ZO-1 in the IUGR colon were also mitigated by pterostilbene (P < 0.05). These data suggest that pterostilbene potentially maintains colonic barrier function of the IUGR piglets.
The possible inhibition of the IUGR-induced colon inflammation by pterostilbene was evaluated by measurement of several markers of inflammatory responses. Compared with the NBW piglets, the IUGR piglets had higher concentrations of TNF-α and IL-1β but lower concentrations of IL-10 in the colon (P < 0.05; Fig. 2a-d). The MPO activity and nuclear NF-κB p65 protein expression in the colon were also increased by IUGR (P < 0.05; Fig. 2e and f). Further assessment of colon immune function revealed marked reductions in the contents of mucin 2 and TFF3 and the number of goblet cells in the IUGR piglets compared to their NBW counterparts (P < 0.05; Fig. 2g-j). Conversely, treatment with pterostilbene decreased the concentrations of TNF-α and IL-1β, the MPO activity, and the translocation of NF-κB p65 from the cytoplasm to the nucleus, while increasing the IL-10 levels in the IUGR colon (P < 0.05). Pterostilbene also promoted the secretion of mucin 2 and TFF3 and increased the number of goblet cells in the IUGR colon (P < 0.05). These observations indicate that pterostilbene suppresses inflammatory responses and overcomes defective immune function in the IUGR colon.
ER stress and autophagy are the crucial mechanisms implicated in inflammatory bowel diseases [14, 16]. However, an association between ER stress or autophagy and the IUGR-induced colon inflammation remains unconfirmed. Our results suggested that IUGR piglets showed apparent ER stress in the colon, as indicated by the increases in the mRNA abundance of GRP78, GRP94, CHOP, ATF4, and sXBP-1 and the protein levels of GRP78, CHOP, and c-Casp12 (P < 0.05; Fig. 3a and b). We also determined the expression of the three arms of the UPR (i.e., IRE1, PERK, and ATF6) and found that IUGR induced the phosphorylation of PERK and IRE1a and elevated ATF6 protein expression in the colon (P < 0.05). In contrast, pterostilbene treatment significantly downregulated the mRNA expression of GRP78, CHOP, and sXBP-1 and the protein expression of GRP78, CHOP, and c-Casp12 in the IUGR colon (P < 0.05). The phosphorylation of IRE1a in the IUGR colon was also significantly inhibited by pterostilbene (P < 0.05). The measurement of autophagy-associated proteins in the colon showed that the protein levels of Beclin1 and p62 and the ratio of LC3 II/I were higher in the IUGR piglets than in the NBW piglets (P < 0.05; Fig. 3c), indicating that IUGR may disturb the process of autophagosomal degradation in the colon. Pterostilbene treatment further upregulated the protein expression of Beclin1 and the ratio of LC3 II/I but markedly suppressed the protein expression of p62 in the IUGR colon (P < 0.05). To summarize, these results suggest that pterostilbene inhibits colon ER stress and reverses impaired autophagic flux in the IUGR colon.
Having determined the involvement of ER stress and autophagy in the IUGR-induced colon inflammation, we investigated the potential crosstalk between these in the context of inflammation using an in vitro system, the Caco-2 cell line. CCK-8 and qRT-PCR assays were conducted to assess the effects of TNF-α on the cell viability and inflammatory responses in Caco-2 cells, respectively. TNF-α stimulation at up to 50 ng/mL developed no obvious differences in cell viability (P > 0.05) but a concentration-dependent upregulation (5–50 ng/mL) of the mRNA abundance of TNF-α, IL-1β, and IL-6 (P < 0.05) in Caco-2 cells (Fig. 4a-d). Thus, an intermediate dosage of TNF-α (10 ng/mL) was chosen to induce inflammation in Caco-2 cells. To assess the effects of ER stress and autophagy on inflammatory responses and cell apoptosis, Caco-2 cells were co-incubated with TNF-α and the inducers or inhibitors of ER stress and autophagy. Treatment with the ER stress inducer tunicamycin and the autophagy inhibitor 3MA aggravated the TNF-α-induced increases in the mRNA abundance of TNF-α, IL-1β, and IL-6, and cell apoptosis in Caco-2 cells, whereas the ER stress inhibitor 4PBA and the autophagy inducer rapamycin had reversed actions (P < 0.05; Fig. 4e-h). To clarify the regulation of ER stress on autophagy activity under inflammation conditions, autophagy-related proteins and autophagic flux in the TNF-α-treated Caco-2 cells were determined after the combined treatment with tunicamycin or 4PBA. TNF-α treatment notably increased the ratio of LC3 II/I and the protein expression of Beclin1 and p62 but reduced the protein expression of Rab7 and LAMP2 (P < 0.05; Fig. 4i). All of these changes were further aggravated by the combined treatment with tunicamycin (P < 0.05). Although the ratio of LC3 II/I and Beclin1 protein in the TNF-α-exposed cells were upregulated by 4PBA, the protein levels of p62, Rab7, and LAMP2 in the TNF-α-exposed cells were recovered to the control levels by 4PBA (P < 0.05). The analysis of autophagic flux of Caco-2 cells illustrated basal autophagy in the control cells, which displayed only weak GFP and mCherry signals (Fig. 4j). TNF-α exposure induced the accumulation of autophagosomes (yellow puncta merged by mCherry and GFP fluorescence) and few autolysosomes (mCherry puncta) in Caco-2 cells, suggesting a blockage of autophagosome clearance. Treatment with tunicamycin further increased the number of autophagosomes but did not alter the number of autolysosomes in the TNF-α-exposed cells. In contrast, 4PBA treatment of the TNF-α-treated cells increased the number of autolysosomes but reduced the number of autophagosomes. These results suggest that the induction of ER stress worsens the TNF-α-induced obstruction of autophagic flux in Caco-2 cells, whereas the inhibition of ER stress exerts opposite effects. The effects of autophagy on ER homeostasis in the context of inflammation were ascertained by measurement of the ER stress markers and the UPR sensors. The mRNA levels of sXBP-1 and the protein levels of GRP78, CHOP, and c-Casp12 in Caco-2 cells were increased by TNF-α treatment (P < 0.05; Fig. 4k and l). TNF-α exposure also increased the phosphorylation of PERK, IRE1a, and, eIF2α, and the protein expression of ATF6 in Caco-2 cells (P < 0.05; Fig. 5m). Treatment with 3MA aggravated the TNF-α-induced elevations in ER stress markers and UPR sensors; however, rapamycin treatment mitigated these negative effects observed following TNF-α treatment (P < 0.05).
We next assessed the effects of pterostilbene on ER stress and autophagy activity in the TNF-α-treated Caco-2 cells with 4PBA and rapamycin as positive controls. The cell viability assay was first conducted to determine the effect of pterostilbene on cell viability in Caco-2 cells. Compared with the untreated cells, treatment with pterostilbene at increasing concentrations (0–50 μmol/L) showed no significant cytotoxicity in Caco-2 cells (P > 0.05; Fig. 5a). Thus, lower than 50 μmol/L pterostilbene was selected to determine the anti-inflammatory action of pterostilbene in the TNF-α-treated Caco-2 cells. As shown in Fig. 5b-d, pterostilbene (0–5 μmol/L) dose-dependently blocked the TNF-α-induced elevation of the mRNA abundance of TNF-α, IL-1β, and IL-6 in Caco-2 cells (P < 0.05). Therefore, 2.5 μmol/L of pterostilbene was chosen for subsequent in vitro studies. As indicated in Fig. 5e and f, pterostilbene was as effective as 4PBA at suppressing the splicing of XBP-1 mRNA and downregulating the protein levels of GRP78, CHOP, c-Casp12, and phosphorylated IRE1a in the TNF-α-exposed cells (P < 0.05). In addition, treatment with pterostilbene had similar effects on autophagy activity to those seen with rapamycin, as pterostilbene increased the ratio of LC3 II/I and the protein levels of Beclin1, Rab7, and LAMP2 and decreased p62 protein in the TNF-α-exposed cells (P < 0.05; Fig. 5g). Moreover, pterostilbene promoted the formation autolysosomes and consumption of autophagosomes in the TNF-α-exposed cells (P < 0.05; Fig. 5h). Taken together, these findings indicate that pterostilbene mitigates ER stress and accelerates autophagic flux in the TNF-α-treated cells.
To investigate the roles of ER stress and autophagic flux in pterostilbene-mediated protection on colonic inflammation, Caco-2 cells co-incubated with TNF-α and pterostilbene were treated with the ER stress inducer tunicamycin or the autophagosome-lysosome binding inhibitor chloroquine. Treatment with pterostilbene improved the TNF-α-induced intestinal barrier dysfunction in Caco-2 cells, as indicated by the increases in TER and the protein levels of occludin and ZO-1 and the decrease in FITC-dextran flux (P < 0.05; Fig. 6a-c). Pterostilbene also significantly inhibited cell apoptosis, the release of IL-1β and IL-6, and the translocation of NF-κB p65 from the cytoplasm to the nucleus in the TNF-α-treated cells (P < 0.05; Fig. 6d-h). However, both tunicamycin and chloroquine counteracted the pterostilbene-mediated beneficial effects on intestinal barrier function, cell apoptosis, and inflammatory responses in the TNF-α-treated Caco-2 cells (P < 0.05), suggesting that the attenuation of pterostilbene on inflammatory response and barrier dysfunction depends on the inhibition of ER stress and the promotion of autophagosome-lysosome fusion.
ER stress and autophagy have been recognized as the crucial mechanisms involved in inflammatory bowel disease [12, 17, 18]; however, a role in IUGR-induced colon inflammation has not been established. The present study revealed that IUGR caused the upregulation of ER stress indicators (GRP78, CHOP, and c-Casp12) and the activation of UPR sensors (IRE1a, PERK, and ATF6), and altered the expression of the proteins responsible for the formation and degradation of autophagosomes (LC3 II, Beclin1, and p62) in the colon. These changes implicated ER stress and impaired autophagy in the IUGR-induced colon inflammation. In addition, our investigation of the crosstalk between ER stress and autophagy in the TNF-α-treated Caco-2 cells indicated that the autophagy inhibitor 3MA intensified the TNF-α-induced ER stress, and the ER stress inducer tunicamycin, in turn, augmented the abnormal accumulation of impaired autophagosomes caused by TNF-α. In particular, tunicamycin and 3MA exacerbated the TNF-α-induced inflammatory responses and apoptosis in Caco-2 cells, whereas the ER stress inhibitor 4PBA and the autophagy activator rapamycin had opposite effects. These data imply that the regulation of ER stress and autophagy may be the potential therapeutic approaches for the IUGR-induced colon inflammation. Pterostilbene is a naturally occurring stilbene with various biofunctionalities and its effects on animal growth performance have been investigated [29, 35, 36]. We previously reported that pterostilbene could act as a feed additive in the broiler diet to prevent the growth performance descent caused by immunological stress and oxidative stress [29, 35]. Pterostilbene (300 mg/kg) also afforded protection against the diquat injection-induced body weight loss of piglets [36]. In this research, however, the compromised growth performance, including body weight, average daily gain, and average daily feed intake, of the IUGR piglets was not significantly improved by pterostilbene (300 mg/kg; data no shown). These conflicting findings are probably associated with the physiological conditions of animals. Although the growth performance of the IUGR piglets was not significantly altered, pterostilbene induced marked reductions in diarrhea rates and colon inflammation of the IUGR piglets. These benefits may result from the potent anti-inflammation action of pterostilbene. The available evidence from in vivo and in vitro experiments has indicated that pterostilbene prevents inflammatory responses by suppressing NF-κB signals [37–39]. Consistent with those findings, we observed that pterostilbene significantly downregulated the expression of pro-inflammatory mediators and prevented the nuclear accumulation of NF-κB p65 in the IUGR piglet colons and the TNF-α-treated Caco-2 cells. In addition, the pterostilbene-mediated protection against ER stress may provide another explanation for the attenuation of the IUGR-induced colon inflammation. IRE1, a conserved ER transmembrane protein, can be activated when the ER homeostasis is perturbed. Notably, the kinase domain of IRE1 complexes with the IkB kinase via interaction with TNF-receptor activating factor 2, which expedites the degradation of IkBα and triggers NF-κB signals, resulting in inflammatory responses [40]. A previous study with HK-2 cells has reported that resveratrol, the parent compound of pterostilbene, prevented the LPS- and tunicamycin-induced overproduction of inflammatory factors and NF-κB activation by inactivation of IRE1 [41]. Liu et al. [42] have also verified that the knockdown of IRE1 had parallel therapeutic effects to those seen with pterostilbene on the TNF-α-induced inflammatory responses in endothelial cells. In this study, pterostilbene treatment decreased the phosphorylation of IRE1a in both the IUGR colon and the TNF-α-treated Caco-2 cells, which may further inhibit the nuclear translocation of NF-κB p65 and the release of inflammatory factors. Intriguingly, the beneficial roles of pterostilbene in inactivation of NF-κB signals and downregulation of inflammatory cytokines in the TNF-α-treated Caco-2 cells were abrogated under a combined treatment with the ER stress inducer tunicamycin. These data suggest the protective effects of pterostilbene on colon inflammation depend on its suppression of ER stress. Autophagy is an indispensable pro-survival process under ER stress conditions that allows the misfolded proteins and damaged cellular components to undergo lysosome-dependent self-digestion and recycling [16]. Also, autophagy is strictly interconnected with inflammatory responses, as the damaged and non-functional mitochondria and aggregated inflammasome structures mainly removed through the autophagic process are the crucial mediators for inflammasome activation [43, 44]. Wang et al. [45] have demonstrated that pterostilbene prevented the activation of NLR family, pyrin domain containing three inflammasome (NLRP3) by inducing autophagy in immortalized rat kidney proximal tubular epithelial cells. In the current study, pterostilbene promoted the protein expression of LC3 II and Beclin1 and recovered the protein levels of Rab7 and LAMP2 in the TNF-α-treated Caco-2 cells, which not only reveals the strong regulation of pterostilbene in autophagy but also provides a possible mechanism by which pterostilbene alleviates the TNF-α-induced inflammatory responses in Caco-2 cells. Notably, autophagy is a dynamic biological process involving autophagosome formation, autophagosome-lysosome fusion, and final degradation. Disruption of any one of these steps may invoke dysregulated autophagy and cause damage to cells and tissues [16]. LC3 is a protein located in the autophagosomal inner membrane and it, along with Beclin1 protein, plays a pivotal role in the formation of autophagosomes [46]. The elevations in LC3 II in a stable state may result from autophagy activation or downstream blockage of autophagic vacuole processing. P62 is an autophagy substrate, and its reduction is associated with the promotion of autolysosome degradation [47]. Therefore, pterostilbene-mediated increases in the protein levels of LC3 II and Beclin1 and decrease in p62 protein in the IUGR colon and the TNF-α-exposed Caco-2 cells confirmed that pterostilbene enhanced autophagic activity and accelerated autophagic flux in this study. Emerging evidence has indicated that the protective effects of natural phytochemicals on autophagy are related to their regulation of the fusion of lysosomes with mature autophagosomes, a crucial step for the degradation of autophagic cargo [48–50]. Wang et al. [50] have reported that resveratrol mitigated oxidative stress-induced autophagic dysfunction by promoting the expression of autophagosome-lysosome fusion-promoting protein Rab7. Pterostilbene was also demonstrated to restore impaired autophagic flux and prevent acetaminophen-induced liver injury [51]. However, the protective responses were abolished by treatment with chloroquine, the autophagosome-lysosome binding inhibitor. The data presented herein substantiated the results from the previous studies showing that pterostilbene improved the TNF-α-induced impairment of autophagic flux by upregulating the protein expression of LAMP2 and Rab7 and the number of autolysosomes in Caco-2 cells. In addition, chloroquine treatment abrogated the positive roles of pterostilbene in inflammatory responses and cell apoptosis in the TNF-α-treated cells. These findings suggest that autophagosome-lysosome fusion plays a pivotal role in the pterostilbene-mediated protection against colonic inflammation. As the first physical and immunological protective barrier, intestinal epithelial cells separate the host from the external environment and prevent the invasion of bacteria, viruses, and endotoxins [52]. Defects in the intestinal barrier may therefore allow these undesired antigens to cross the intestinal epithelium and increase the risk of intestinal inflammation. Numerous studies have substantiated the potency of pterostilbene in protecting against intestinal barrier damage [22, 30, 53]. Here, pterostilbene treatment greatly alleviated the deficiencies in goblet cell quantity and the secretion of mucin 2 and TFF3 in the IUGR colon. It also upregulated the protein levels of occludin and ZO-1 and consequently led to a decrease in intestinal epithelial permeability in both the IUGR colon and the TNF-α-exposed Caco-2 cells. It is worthwhile to mention that ER stress inducer tunicamycin and the autophagic flux inhibitor chloroquine largely suppressed the pterostilbene-mediated beneficial effects on the intestinal epithelium permeability and intestinal tight junction protein. These findings indicate that the inhibition of ER stress and the promotion of autophagic flux are key mechanism by which pterostilbene prevents colon barrier dysfunction.
This study highlights the roles of ER stress and impaired autophagic flux as part of the mechanism of the IUGR-induced colon inflammation. Importantly, pterostilbene effectively overcomes ER stress, restores autophagic flux, and further mitigates inflammatory responses and intestinal barrier dysfunction in both the IUGR colon and the TNF-α-treated Caco-2 cells. These findings may broaden the understanding of how pterostilbene protects against colon inflammation and help in development of novel nutrition strategies for IUGR animals to improve intestinal health.
Additional file 1: Table S1. Composition and nutrient levels of the basal diet.Additional file 2: Table S2. Primer sequences for quantitative real-time PCR and real-time PCR analyses. | true | true | true |
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PMC9635553 | Changjiang Lei,Shaoting Li,Ying Fan,Li Hua,Qingyun Pan,Yuan Li,Zhixiong Long,Rui Yang | LncRNA DUXAP8 induces breast cancer radioresistance by modulating the PI3K/AKT/mTOR pathway and the EZH2-E-cadherin/RHOB pathway CANCER BIOLOGY & THERAPY | 03-11-2022 | LncRNA DUXAP8,AKT,EZH2,breast cancer,radioresistance | ABSTRACT Radiation resistance poses a major clinical challenge in breast cancer (BC) treatment, but little is known about how long noncoding RNA (lncRNA) may regulate this phenomenon. Here, we reported that DUXAP8 was highly expressed in radioresistant BC tissues, and high expression of DUXAP8 was associated with poor prognosis. We found that the overexpression of DUXAP8 promoted radioresistance, while the knockdown of DUXAP8 expression increased radiosensitivity. Further studies revealed that DUXAP8 enhanced the radioresistance of BC cells by activating the PI3K/AKT/mTOR pathway and by repressing the expression of E-cadherin and RHOB through interaction with EZH2. Together, our work demonstrates that the overexpression of DUXAP8 promotes the resistance of BC cells toward radiation through modulating PI3K/AKT/mTOR pathway and EZH2-E-cadherin/RHOB axis. Targeting DUXAP8 may serve as a potential strategy to overcome radioresistance in BC treatment. | LncRNA DUXAP8 induces breast cancer radioresistance by modulating the PI3K/AKT/mTOR pathway and the EZH2-E-cadherin/RHOB pathway CANCER BIOLOGY & THERAPY
Radiation resistance poses a major clinical challenge in breast cancer (BC) treatment, but little is known about how long noncoding RNA (lncRNA) may regulate this phenomenon. Here, we reported that DUXAP8 was highly expressed in radioresistant BC tissues, and high expression of DUXAP8 was associated with poor prognosis. We found that the overexpression of DUXAP8 promoted radioresistance, while the knockdown of DUXAP8 expression increased radiosensitivity. Further studies revealed that DUXAP8 enhanced the radioresistance of BC cells by activating the PI3K/AKT/mTOR pathway and by repressing the expression of E-cadherin and RHOB through interaction with EZH2. Together, our work demonstrates that the overexpression of DUXAP8 promotes the resistance of BC cells toward radiation through modulating PI3K/AKT/mTOR pathway and EZH2-E-cadherin/RHOB axis. Targeting DUXAP8 may serve as a potential strategy to overcome radioresistance in BC treatment.
Breast cancer (BC) is the most frequent cancer among women, with an estimated 1.5 million new cases per year. Radiation therapy plays an important role in the multidisciplinary management of BC. However, the intrinsic and acquired radioresistance remains a major challenge undermining the treatment outcome of radiotherapy in BC patients. Therefore, the exploration and clarification of molecular mechanisms implicated in the development of BC radioresistance would provide insights into the formulation of novel strategy in the management of radiotherapy resistance. Non-coding RNAs (ncRNAs), which can be classified into long ncRNAs (lncRNAs, ˃ 200 nt) and small ncRNAs (˂ 200 nt), have emerged as critical regulators of gene expression. As one major class of small ncRNAs, microRNAs (miRNAs) regulate the stability and translation of target mRNA by binding to the 3′-untranslated region. Previous studies have shown that miRNAs regulate a myriad of cellular and developmental processes, including the initiation and progression of cancer. Recent reports further showed that many lncRNAs are aberrantly expressed in human tumor tissues, which contributes to the malignant phenotypes of cancers, including the hyper-proliferation, cell invasion, metastasis and radioresistance. For instance, lncRNA NEAT1 acts as an oncogenic factor in various cancers, and functions as competitive endogenous RNAs to sponge downstream miRNAs. Certain lncRNAs could serve as bridge to target polycomb factor EZH2 (Enhancer Of Zeste 2 Polycomb Repressive Complex 2 Subunit) to its target gene loci, causing epigenetic silencing of tumor suppressors. In addition to ncRNAs, the dysregulation of many signaling pathways play crucial roles in the progression of BC. For example, the constitutive activation of PI3K/AKT/mTOR pathway confers a competitive growth advantage, metastatic competence and drug resistance in BC cells. LncRNA DUXAP8 is located on chromosome 20q11, and the upregulation of DUXAP8 has been reported in multiple cancers, including gastric cancer, lung cancer, bladder cancer, renal cell carcinoma, hepatocellular carcinoma, neuroblastoma and colorectal cancer. DUXAP8 acts as an oncogene in several cancer types through an EZH2-dependent mechanism or by regulating PTEN expression. However, the expression pattern and impact of DUXAP8 on development of BC radioresistance remain unknown. In this study, we aimed to figure out whether DUXAP8 modulates the radiosensitivity of BC cells and investigate the underlying mechanisms of DUXAP8-dependent radioresistant phenotype. We found that the overexpression of DUXAP8 could enhance the radioresistance of BC cells through activating PI3K/AKT/mTOR pathway and repressing the expression of EZH2 target genes (E-cadherin and RHOB). These data indicate that targeting DUXAP8 might serve as a therapeutic approach for the management of the radiotherapy efficacy in BC.
In this study, BC specimens and the matched noncancerous tissues were obtained from 50 patients who were diagnosed with BC and underwent surgery at the Fifth Hospital of Wuhan (China). All the cancer tissues and adjacent normal tissues were confirmed by experienced pathologists. No patient received any chemotherapy or radiotherapy before surgery. Tissue samples were immediately frozen in liquid nitrogen after surgical resection and stored at −80°C until use. A total of 60 BC patients who received radiotherapy after surgery were also enrolled in the study. These tissue specimens were divided into radiosensitive group (n = 30) and radioresistant group (n = 30) based on short-term response to radiotherapy, as previously described. Clinicopathological data were retrieved from patient medical records. This study was approved by the institutional ethical review committee of the Fifth Hospital of Wuhan (China), and written informed consent was obtained from each patient.
Human BC cell lines, including MCF-12A, MCF-12 F, MCF-7, T47D, ZR-75-1, HCC-1806, MDA-MB-468, BT-549, and MDA-MB-231, and the normal mammary epithelial cell line MCF-10A were obtained from the Cell Bank of Type Culture Collection (Chinese Academy of Sciences, Shanghai, China). The cells were maintained in DMEM/F12 medium (Thermo Fisher Scientific, USA) supplemented with 10% fetal bovine serum (Thermo Fisher Scientific, USA) in a humidified incubator containing 5% CO2. To study the impact of indicated signaling pathways, cells were treated with target-selective inhibitor of PI3K, NVP-BKM120 (4 µM, Novartis, Basel, Switzerland) or DMSO (Sigma, St. Louis, USA) for 24 h before subsequent experiments.
Total RNA was extracted from cultured cells or tissues using the TRIzol Reagent (Invitrogen, USA), and then reverse-transcribed into cDNA with the PrimerScript RT Reagent Kit (Invitrogen, USA). qRT-PCR analysis was performed using the SYBR Green PCR kit from Takara Biotechnology (Takara, Dalian, China) in a 7500 Real Time PCR System (Applied Biosystems, CA, USA). GAPDH was used as an internal control. Primers used for qRT-PCR assay were obtained from GenePharma (China, Shanghai) and shown as follows: human DUXAP8, forward: 5′-ACCAGCCTCACTAGCACTCT-3′ and reverse: 5′-GGCTTAGCTTGCACTTTTGGA-3′; human EZH2, forward: 5′-AATCAGAGTACATGCGACTGAGA-3′ and reverse: 5′-GCTGTATCCTTCGCTGTTTCC-3′; human p21, forward: 5′-TGTCCGTCAGAACCCATGC-3′ and reverse: 5′-AAAGTCGAAGTTCCATCGCTC-3′; human Bax, forward: 5′-CCCGAGAGGTCTTTTTCCGAG-3′ and reverse: 5′-CCAGCCCATGATGGTTCTGAT-3′; human Caspase-8, forward: 5′-TTTCTGCCTACAGGGTCATGC-3′ and reverse: 5′-GCTGCTTCTCTCTTTGCTGAA-3′; human Caspase-9, forward: 5′-CTTCGTTTCTGCGAACTAACAGG-3′ and reverse: 5′-GCACCACTGGGGTAAGGTTT-3′; human PTEN, forward: 5′-TGGATTCGACTTAGACTTGACCT-3′ and reverse: 5′-GGTGGGTTATGGTCTTCAAAAGG-3′; human E-cadherin, forward: 5′-CGAGAGCTACACGTTCACGG-3′ and reverse: 5′-GGGTGTCGAGGGAAAAATAGG-3′, human RHOB, forward: 5′-CTGCTGATCGTGTTCAGTAAGG-3′ and reverse: 5′-TCAATGTCGGCCACATAGTTC-3′; and human GAPDH, forward: 5′-GGAGCGAGATCCCTCCAAAAT-3′ and reverse: 5′-GGCTGTTGTCATACTTCTCATGG-3′.
The full-length cDNA sequence of human DUXAP8 was amplified by PCR using forward primer: 5’-GCGTGGTCAGAGCGAGCTT-3’; reverse primer: 5’-GCTTAGCTTGCACTTTTGGAAGA-3’. The resulting PCR fragment was cloned into the pcDNA3.1 vector (Invitrogen, USA). Stable BC cell lines overexpressing DUXAP8 were generated by the transfection of DUXAP8 expression vector or the control pcDNA3.1 vector into MCF-7 and T47D cells with low DUXAP8 expression. Stable BC cell lines with DUXAP8 silencing were generated by the transfection of DUXAP8-specific shRNA vector or the control shRNA vector (GenePharma, Shanghai, China) into BT-549 and MDA-MB-231 cells with high DUXAP8 expression. Transfection was performed using Lipofectamine 3000 (Invitrogen, L3000001) according to the manufacturer’s instructions. Transfected cells were selected with 1.0 μg/mL puromycin for two weeks to eliminate the uninfected cells as previously described.
Small interfering RNAs (siRNAs) against human EZH2, E-cadherin and RHOB, and scrambled siRNA were purchased from GenePharma (China, Shanghai). BC cells were transfected with 50 nm of above siRNAs using Lipofectamine 2000 (Invitrogen, USA) following the manufacturer’s guides.
To determine cell viability, cells were seeded in to a 96-well plate at a density of 5000 cell/well and cultured in a humidified cell culture incubator for 12 h. BC cells were exposed to the indicated radiation doses, and the cell viability was determined using a CCK-8 kit (Dojindo Laboratories, Kumamoto, Japan) according to the manufacturer’s instructions. The absorbance was determined at 450 nm using a microplate reader (Bio-Rad Laboratories, USA).
BC cells seeded into 6-well plates and incubated for 24 h, and then were irradiated with 0 to 8 Gy for 24 h. For apoptosis detection, BC cells were first digested by trypsin and re-suspended with binding buffer, and the staining for apoptotic cells was detected using an Annexin V-FITC Apoptosis Detection Kit (BD Biosciences, USA) as previously reported. The percentage of apoptotic cells was detected by BD FACS CantoTM II Flow Cytometer (BD Biosciences), and the data were analyzed using CellQuest software (BD Biosciences, USA).
The total protein of cells was extracted using RIPA lysis buffer (Solarbio, Shanghai, China). The protein concentration was quantified using the Pierce BCA protein assay kit (Thermo Fisher Scientific, USA). The protein extracts were subjected to SDS-PAGE, and then transferred to 0.22 μm polyvinylidene difluoride (PVDF) membrane (Millipore, USA). After blocking with 5% nonfat milk, the membranes were incubated at 4°C overnight with primary antibodies against γ-H2AX, cleaved caspase-7/8/9, Bax, PTEN, p-PI3K, PI3K, p-AKT, AKT, p-mTOR, mTOR, EZH2, E-cadherin, RHOB, Ki-67, and GAPDH (all antibodies from Cell Signaling Technology, USA, at 1:1000 dilutions). The membrane was then incubated with HRP-linked secondary antibody (1:3000; Cell Signaling Technology) at room temperature for 2 h. Signals were developed using an ECL Western Blotting Detection Kit from GE Healthcare (Amersham, UK). The protein bands were photographed on a gel imager system (Bio-Rad, CA, USA). The densitometry analysis was performed with Image J software (Bethesda, MD, USA).
The separation of nuclear and cytoplasmic fractions was performed using the PARIS Kit (Thermo Fisher Scientific, USA) according to the manufacturer’s instructions. The qRT-PCR analysis was performed to quantify the relative abundance of the target using the separated nuclear and cytoplasmic fractions. U6 served as the nuclear control, and GAPDH served as the cytoplasmic control.
RIP experiments were conducted according to the manufacturer’s protocol of the Magna RIP RNA-Binding Protein Immunoprecipitation Kit (Millipore, USA). In brief, magnetic beads were pre-incubated with anti-EZH2 or anti-rabbit IgG antibodies (Cell Signaling Technology) for 30 min, and the cell lysates were immunoprecipitated with beads overnight at 4°C with rotation. RNA was purified from RNA-protein complexes bounded to the beads using TRIzol reagent and was analyzed by qRT-PCR analysis.
ChIP experiments were performed using the Millipore EZ-Magna ChIP Kit (Millipore, USA) as previously reported. Briefly, BC cells were cross-linked with 1% formaldehyde for 10 min at room temperature. Then, chromatin was sonicated in the lysis buffer and the extraction of ChIP DNA was performed according to the kit’s protocol. The antibodies for ChIP were EZH2 and H3K27Me3 (Cell Signaling Technology, USA). The primer sequences for ChIP-qPCR analysis were previously described 2223.
The experiments involving animals were approved by the Animal Care and Use Committee of the Fifth Hospital of Wuhan and were performed in accordance with the Institutional Guide for the Care and Use of Laboratory Animals. Six-week-old nude mice were purchased from Shanghai Laboratory Animal Center, Chinese Academy of Sciences (Shanghai, China), and housed in specific pathogen-free conditions on a 12-h light/dark cycle with free access to food and water. 0.2 mL of cell suspension containing 1 × 106 cells were implanted into nude mice by subcutaneous injection (n = 6 mice in each group). Two weeks after injection, mice were irradiated with 8 Gy as previously reported. Tumor sizes were measured every 5 days for 7 weeks, and tumor volume was calculated according to the following formula: Volume = (length x width)/2. Seven weeks after tumor cell inoculation, all the mice were euthanized by CO2 asphyxiation, and the death was confirmed by t cervical dislocation. The xenograft tumors of terminally dead mice were removed for subsequent analysis.
All data were expressed as mean ± SD and were analyzed using SPSS 17.0 (SPSS Inc, USA). The differences were estimated using Student’s t-test (between two groups) or one-way ANOVA (among at least three groups). Fisher’s exact and chi-square tests were utilized to evaluate the correlations between DUXAP8 expression level and clinical parameter in BC patients. The correlation between DUXAP8 and EZH2 expression in BC tissues was determined by Pearson correlation analysis. The results were considered statistically significant if P < .05.
To understand the biological role of DUXAP8 in BC, we first investigated whether DUXAP8 was dysregulated in BC tissues. Using the GENT2 database (http://gent2.appex.kr/gent2/), we found that the expression level of DUXAP8 in BC tissues was much higher than that in normal tissues (P = .001, Figure 1a). We further employed the GEPIA database (http://gepia.cancer-pku.cn/) and the Kaplan Meier Curve plotter database (http://kmplot.com) to analyze the relationship between DUXAP8 level and the prognosis of BC patients. Kaplan-Meier survival analysis suggested that BC patients with higher levels of DUXAP8 were associated with an poorer overall survival compared with those with a lower level of DUXAP8 (Figure 1b). Subsequently, we examined the levels of DUXAP8 in 60 paired BC and adjacent normal breast tissues using qRT-CR analysis. The results also showed a significant upregulation of DUXAP8 in BC specimens compared with adjacent normal tissues (P < .0001; Figure 1c). Analysis of the relationship between DUXAP8 expression and clinicopathological characteristics (Table 1) revealed that elevated DUXAP8 expression was significantly associated with larger tumor size, more advanced tumor stage, and more lymph node metastasis. These results suggested that the upregulation of DUXAP8 may contribute to the BC progression. To explore whether the dysregulation of DUXAP8 is correlated with radiosensitivity in BC patients, we analyzed the expression of DUXAP8 in the BC tissues from radiosensitive and radioresistant patients using qRT-PCR analysis. Our results demonstrated that the expression levels of DUXAP8 were remarkably higher in the radioresistant group than those in the radiosensitive group (Figure 1d). The median level of DUXAP8 from qRT-PCR experiments was used the cutoff point to divide BC patients into high- (n = 30) and low- (n = 30) expression groups. Compared with DUXAP8-low expression group, the DUXAP8-high expression group exhibited a poorer short-term response to radiotherapy (Table 2). Overall, these results suggest that DUXAP8 might act as an oncogenic factor contributing to the BC progression and conferring resistance to radiation therapy in BC cells.
We next determined the expression levels of DUXAP8 in normal breast epithelial cell MCF-10A and several BC cell lines (MCF-12A, MCF-12 F, MCF-7, T47D, ZR-75-1, HCC-1806, MDA-MB-468; BT-549 and MDA-MB-231) by qRT-PCR assays. Compared to MCF-10A cells, BC cells showed a significant increase in the expression of DUXAP8 (Figure 2a). BT-549 and MDA-MB-231 cells expressed the highest levels of DUXAP8, while MCF-7 and T47D cells showed the lowest expression (Figure 2a). Thus, we selected MCF-7 and T47D with low DUXAP8 expression for overexpression experiments, and BT-549 and MDA-MB-231 cells with high DUXAP8 expression for knockdown experiments. To confirm the role of DUXAP8 in radiation resistance in BC cells, DUXAP8 was over-expressed by transfecting BC cells with DUXAP8 expression vector, or knocked down by transfecting BC cells with shRNA targeting DUXAP8 (Figure 2b). The cells were subjected to different dosage of radiation and CCK-8 viability assays and flow cytometry analysis were performed to examine cell viability and apoptosis. We found that the overexpression of DUXAP8 promoted cell survival and reduced apoptosis of MCF-7 and T47D cells after radiation treatment (0, 4, 8 Gy) (Figure 2 c and D). Conversely, the knockdown of DUXAP8 impaired cell survival and promoted the apoptosis of BT-549 and MDA-MB-231 cells after radiation treatment (Figure 2 c and d; Figure S1). Consistently, Western blot analysis showed that overexpression of DUXAP8 significantly decreased γ-H2AX (a marker for DNA damage) levels after irradiation, while the knockdown of DUXAP8 showed the opposite effects (Figure S2), which further indicates that overexpression of DUXAP8 reduces the level of DNA damage upon radiation in BC cells. Moreover, the overexpression of DUXAP8 led to a significant reduction in PTEN (negative regulator of PI3K signaling pathway), Bax (pro-apoptotic protein), and cleaved caspase-8/7/9 (apoptotic executors), while there were significant reductions in the phosphorylation levels of PI3K, AKT and mTOR. The knockdown of DUXAP8 showed the opposite effects (Figure 3 a and b; Figure S3, upper panel). Together, these data suggest that DUXAP8 mediate the radiosensitivity by modulating PI3K/AKT/mTOR pathway. To further confirm whether DUXAP8 mediates the radioresistance of BC cells by activating the PI3K/AKT/mTOR pathway, MCF-7/T47D cells overexpressing DUXAP8 were treated with a selective inhibitor of PI3K (NVP-BKM120), and the cell viability upon irradiation was examined determined by CCK-8 assay. As expected, overexpression of DUXAP8 promoted the survival of MCF-7 and T47D cells after the treatment of 8 Gy IR (Figure 3c). However, the treatment of PI3K inhibitor NVP-BKM120 significantly impaired the effects of DUXAP8 overexpression on cell viability (Figure 3c). Western blot analysis showed that the overexpression of DUXAP8 increased the phosphorylation levels of PI3K, AKT and mTOR, and decreased the levels of cleaved caspase-8/7/9 in MCF-7 and T47D cells (Figure 3d; Figure S3, bottom panel). However, the treatment of PI3K inhibitor significantly decreased the phosphorylation levels of PI3K, AKT and mTOR and increased the levels of cleaved caspase-8/7/9 in cells with DUXAP8 overexpression (Figure 3d; Figure S3, bottom panel). These findings indicate that the overexpression of DUXAP8 enhances the radioresistance of BC cells through activating PI3K/AKT/mTOR pathway and suppressing apoptosis.
To get more insight into the mechanism by which DUXAP8 promotes the radiosensitivity of BC cells, we examined the impact of DUXAP8 overexpression or DUXAP8 knockdown on genes involved in cell cycle regulation and apoptosis using qRT-PCR. The overexpression of DUXAP8 significantly decreased the expression of p21, Bax, caspase-8, caspase-9, PTEN, but increased the mRNA level of EZH2 in MCF-7 and T47D cells (Figure 4a). In contrast, the downregulation of DUXAP8 showed the opposite effects in BT-549 and MDA-MB-231 cells (Figure 4a). The effects of DUXAP8 overexpression and DUXAP8 knockdown on EZH2 protein levels were confirmed by Western blot (Figure 4b). We also determined the expression level of EZH2 in BC tissues and the adjacent normal tissues, and the results showed that there was an upregulation of EZH2 in BC tissues (Figure 4c). The mRNA levels of EZH2 were also significantly higher in BC cells than that of the normal breast epithelial MCF-10A cells (Figure 4d). Further, correlation analysis revealed that there was a positive correlation between DUXAP8 and EZH2 mRNA expression in the BC tissues (Figure 4e). A similar positive correlation between DUXAP8 and EZH2 expression was observed in the TCGA BC dataset using the ENCORI database (http://starbase.sysu.edu.cn) (figure 4f). These data suggest that DUXAP8 acts as an upstream activator of EZH2 in BC cells.
Then we prepared the nuclear and cytoplasmic fractions in BT-549 and MDA-MB-231 cells, and qRT-PCR results showed that DUXAP8 was predominantly localized in the nuclear fraction (Figure 5a). Using an online algorithm, RPISeq (http://pridb.gdcb.iastate.edu/RPISeq/), we found a possibility that DUXAP8 might directly target EZH2 protein since the DUXAP8-EZH2 interaction pair had a high score (interaction probability: RF = 0.75; SVM = 0.98). To investigate the potential interaction between DUXAP8 and EZH2 in BT-549 and MDA-MB-231 cells, RIP assays were performed. We found that DUXAP8 was significantly enriched with the EZH2 antibody when compared to the IgG control (Figure 5b). We therefore hypothesized that that DUXAP8 could bind to EZH2 to modulate its transcriptional regulation activity in BC cells. E-cadherin and RHOB are known as EZH2 target genes 1322, which can modulate the radioresistance in BC cells 2324. To investigate whether DUXAP8 regulates the expression of E-cadherin and RHOB by affecting the bindings of EZH2 to the target gene promoter, we carried out ChIP-qPCR assays using anti-EZH2 and H3K27me3 antibody in MDA-MB-231 cells with DUXAP8 knockdown. Our results showed that the knockdown of DUXAP8 not only decreased the binding ability of EZH2 to the promoter, but also attenuated the level of repressive epigenetic marker H3K27me3 at promoter regions of E-cadherin and RHOB in MDA-MB-231 cells (Figure 5c). On the contrary, the protein levels of E-cadherin and RHOB in BT-549 and MDA-MB-231 were downregulated following overexpression of DUXAP8, but showed upregulation upon DUXAP8 knockdown (Figure 5d). Consistently, qRT-PCR assays showed that E-cadherin and RHOB expression was significantly downregulated in BC tissues (with a higher DUXAP level) compared with adjacent normal tissues (Figure 5e). Furthermore, we found that there was a significant downregulation of E-cadherin and RHOB, and an upregulation of EZH2 in radioresistant BC tissues compared with radiosensitive BC tissues (figure 5f). Pearson’s correlation analysis further revealed the negative correlations between DUXAP8 and E-cadherin or RHOB in BC tissues (Figure 5g). Taken together, these data suggest that DUXAP8 functions as a scaffold lncRNA to facilitate the targeting of EZH2 to the promoter regions of E-cadherin and RHOB and their epigenetic silencing.
We next sought to explore whether DUXAP8 regulates the radioresistance of BC cells by regulating the EZH2-E-cadherin/RHOB axis. To this end, we transfected MCF-7 and T47D cells overexpressing DUXAP8 with EZH2 siRNA to silence EZH2, and the cells were irradiated with a dose of 8 Gy X-radiation. Using qRT-PCR and Western blot analysis, we showed that the expression of E-cadherin and RHOB was deceased by DUXAP8 overexpression, which was partially restored by EZH2 silencing (Figure 6a). This was also accompanied by the increase of caspase-7/9 and Bax upon EZH2 silencing. Furthermore, CCK-8 assays demonstrated that the promoted cell survival by DUXAP8 overexpression was largely impaired upon silencing EZH2 in MCF-7 and T47D cells (Figure 6b). These results suggest that DUXAP8 promotes the radioresistance of BC cells by upregulating EZH2. Next, we transfected BT-549 and MDA-MB-231 cells with DUXAP8 knockdown with E-cadherin or RHOB-siRNA, and the cells were subjected to a dose of 8 Gy X-radiation. The expression of E-cadherin or RHOB was increased by DUXAP8 knockdown, but was reduced by the transfection with E-cadherin or RHOB-siRNA in BT-549 and MDA-MB-231 cells (Figure 6c). We noted that the suppression on cell survival by DUXAP8 knockdown was partially alleviated upon the silencing of E-cadherin or RHOB in BT-549 and MDA-MB-231 cells (Figure 6d). The rescued cell survival phenotype upon the silencing of E-cadherin or RHOB was accompanied by the downregulation of Bax and cleaved caspase-7/9 in BT-549 and MDA-MB-231 transfected with E-cadherin or RHOB-siRNA (Figure 6e). These data suggest that DUXAP8 regulates the radioresistance of BC cells by targeting the EZH2-E-cadherin/RHOB axis.
To evaluate the effects of DUXAP8 on radiosensitivity in vivo, MDA-MB-231 cells with or without DUXAP8 knockdown, and MCF-7 cells with or without DUXAP8 overexpression, were injected into the nude mice to establish the xenograft tumorigenesis model. The knockdown of DUXAP8 sensitized MDA-MB-231 cells to irradiation in vivo, as evidenced by the reduced tumor volume and weight in DUXAP8 knockdown group (Figure 7 a and b). In contrast, the overexpression of DUXAP8 increased the radioresistance of BC cells in vivo (Figure 7 a and b). Collectively, these results suggest that a high level of DUXAP8 expression contributes to the radioresistance of BC in the xenograft model.
Recently, lncRNAs have been identified as critical regulators of radiosensitivity in several types of cancer, including BC. However, little is known about the role of lncRNA DUXAP8 in regulating the radioresistance of BC cells. In this study, we provided convincing evidence that DUXAP8 expression is increased in radioresistant BC tissues, and the upregulation of DUXAP8 dramatically enhances the radioresistance of BC cells in both in vitro and in vivo models. These data suggest that DUXAP8 overexpression confer survival advantages under irradiation, and the development of the therapeutic strategies targeting DUXAP8 may offer new opportunities to overcome the resistance of BC cells to radiation. DUXAP8 is frequently overexpressed in human tumors 12131415161718, and the upregulation of DUXAP8 is associated with more aggressive phenotypes of different cancers. Increased DUXAP8 expression was reported to be associated with larger tumor size, and advanced pathological stage of pancreatic cancer. Furthermore, a high level of DUXAP8 expression was associated with a poorer prognosis in many cancers. In agreement with these findings, our data validated that the overexpression of DUXAP8 predicted worse survival in patients with BC, indicating that DUXAP8 may serve as a biomarker for unfavorable prognosis BC patients. The oncogenic roles of DUXAP8 have been reported in gastric cancer, lung cancer, bladder cancer, renal cell carcinoma, hepatocellular carcinoma, neuroblastoma, and colorectal cancer. DUXAP8 can promote cell proliferation, migration, invasion, and metastasis 12131415161718. In addition, the knockdown of DUXAP8 significantly represses epithelial-mesenchymal transition (EMT) in lung cancer and hepatocellular carcinoma. However, the biological functions of DUXAP8 in BC have not been elucidated. Our gain-of-function and loss-of-function experiments revealed the critical role of DUXAP8 in enhancing the radioresistance of BC cells. However, the mechanisms underlying the upregulation of DUXAP8 in BC remain to be determined. LncRNAs are implicated in the regulation of tumor cell radioresistance via different mechanisms, including DNA damage repair, cell cycle arrest, apoptosis, autophagy, EMT, and cancer stemness 58. In our study, we provided evidence that DUXAP8 is capable of modulating DNA damage levels and radiation-induced apoptosis in BC cells. Furthermore, EZH2 has been shown to silence the expression of E-cadherin and RHOB through histone H3K27 trimethylation 132229. The overexpression of E-cadherin and RHOB were reported to sensitize BC cells to radiotherapy 2324. Consistent with these previous findings, we have demonstrated that DUXAP8 could promote radioresistance by epigenetically repressing the expression of E-cadherin and RHOB through targeting with EZH2. EZH2 can also regulate EMT by repressing the expression of E-cadherin, and the downregulation of EZH2 expression reduced the stemness of cancer cells in BC. Further studies are required to explore the possibility that DUXAP8 affects the EMT process and cancer stem cell properties in an EZH2-dependent manner, thereby leading to the development of radioresistance in BC. It has been reported that lncRNAs can interplay with miRNAs and antagonize the repressive activity of miRNAs on gene expression in tumor cells. For instance, DUXAP8 was shown to function as a sponge for miR-577 to promote the migration and invasion of colorectal cancer cells. Moreover, DUXAP8 enhances the progression of renal cell carcinoma via downregulating miR-126. Interestingly, miR-126 has been shown to increase chemosensitivity in drug-resistant gastric cancer cells by targeting EZH2. We found that forced expression of DUXAP8 induced the levels of EZH2 in BC cells. Whether DUXAP8 regulates EZH2 expression through intermediate miRNAs remain to be further explored. Abnormal EZH2 expression and the constitutive activation of PI3K/AKT/mTOR signaling contribute to the malignant progression of BC. In this study, we found that DUXAP8 could enhance the radioresistance in BC cells via recruiting EZH2 to the target genes and by activating the PI3K/AKT/mTOR pathway. Interestingly, a previous study showed that lncRNA UCA1 promotes gastric cancer cell proliferation by inducing EZH2 expression and activating AKT signaling. EZH2 and AKT signaling could also regulate the expression of each other in gastric cancer cells, and the treatment with EZH2 inhibitor (EZP005687) could decrease the phosphorylation and activity of AKT in gastric cancer cells. On the other hand, the administration of a specific PI3K inhibitor (LY49002) resulted in a significant reduction in EZH2 expression. Based on these results, it is likely that EZH2 and PI3K/AKT/mTOR signaling might constitute a positive feedback loop to reinforce the radioresistance in BC cells upon DUXAP8 overexpression. However, the relationship between EZH2 and PI3K/AKT/mTOR pathway, as well as the potential impact of their interplay on the aggressiveness and radioresistance of BC cells warrant further studies.
In conclusion, our study uncovered a novel role of lncRNA DUXAP8 in augmenting the radioresistance in BC by activating PI3K/AKT/mTOR pathway and repressing E-cadherin and RHOB expression through targeting EZH2. These findings suggest that DUXAP8 represents a promising therapeutic target for the clinical management of radiosensitivity in BC patients.
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PMC9635631 | 36063049 | Christopher J Sottolano,Nicole T Revaitis,Anthony J Geneva,Nir Yakoby | Nebulous without white: annotated long-read genome assembly and CRISPR/Cas9 genome engineering in Drosophila nebulosa | 05-09-2022 | genome sequencing,CRISPR/Cas9,genome editing,PacBio | Abstract The diversity among Drosophila species presents an opportunity to study the molecular mechanisms underlying the evolution of biological phenomena. A challenge to investigating these species is that, unlike the plethora of molecular and genetics tools available for D. melanogaster research, many other species do not have sequenced genomes; a requirement for employing these tools. Selecting transgenic flies through white (w) complementation has been commonly practiced in numerous Drosophila species. While tolerated, the disruption of w is associated with impaired vision, among other effects in D. melanogaster. The D. nebulosa fly has a unique mating behavior which requires vision, and is thus unable to successfully mate in dark conditions. Here, we hypothesized that the disruption of w will impede mating success. As a first step, using PacBio long-read sequencing, we assembled a high-quality annotated genome of D. nebulosa. Using these data, we employed CRISPR/Cas9 to successfully disrupt the w gene. As expected, D. nebulosa males null for w did not court females, unlike several other mutant strains of Drosophila species whose w gene has been disrupted. In the absence of mating, no females became homozygous null for w. We conclude that gene disruption via CRISPR/Cas9 genome engineering is a successful tool in D. nebulosa, and that the w gene is necessary for mating. Thus, an alternative selectable marker unrelated to vision is desirable. | Nebulous without white: annotated long-read genome assembly and CRISPR/Cas9 genome engineering in Drosophila nebulosa
The diversity among Drosophila species presents an opportunity to study the molecular mechanisms underlying the evolution of biological phenomena. A challenge to investigating these species is that, unlike the plethora of molecular and genetics tools available for D. melanogaster research, many other species do not have sequenced genomes; a requirement for employing these tools. Selecting transgenic flies through white (w) complementation has been commonly practiced in numerous Drosophila species. While tolerated, the disruption of w is associated with impaired vision, among other effects in D. melanogaster. The D. nebulosa fly has a unique mating behavior which requires vision, and is thus unable to successfully mate in dark conditions. Here, we hypothesized that the disruption of w will impede mating success. As a first step, using PacBio long-read sequencing, we assembled a high-quality annotated genome of D. nebulosa. Using these data, we employed CRISPR/Cas9 to successfully disrupt the w gene. As expected, D. nebulosa males null for w did not court females, unlike several other mutant strains of Drosophila species whose w gene has been disrupted. In the absence of mating, no females became homozygous null for w. We conclude that gene disruption via CRISPR/Cas9 genome engineering is a successful tool in D. nebulosa, and that the w gene is necessary for mating. Thus, an alternative selectable marker unrelated to vision is desirable.
Morphological and patterning diversities are common in nature. However, the mechanisms underlying these evolutionary differences have been studied only in a limited number of animals. High-throughput tools have been created to study development and evolution, yet the absence of high-quality genome sequences for many organisms of interest has been an obstacle to the exploration of mechanisms controlling diversity in nature. Here, we generated the first high-quality genome sequence of Drosophila nebulosa and employed genome engineering to test whether vision is necessary for mating.
The fruit fly Drosophila melanogaster has been a leading model system to study genetics and developmental biology. The large mutational screens performed in the 1980s (Lewis et al. 1980; Nusslein-Volhard and Wieschaus 1980; Spencer et al. 1982; Schupbach and Wieschaus 1986; St Johnston 2002), together with the plethora of effective genetic tools (Duffy 2002; del Valle Rodriguez et al. 2011), revealed the functions of many genes, gene regulatory networks, as well as demonstrated how organisms that are phenotypically unrelated share a large proportion of their genes and fundamental molecular and cellular functions (Holley et al. 1995; Pearse and Tabin 1998). The introduction of the CRISPR/Cas9 system for targeted and precise genome editing of D. melanogaster (Gratz et al. 2013) provided an efficient new system to directly manipulate genes and study their influence on organismal phenotypes without the tedious mutation and screening cycles. At the same time, there are thousands of other Drosophila species with interesting differences in behavior, chromosomal arrangement, gene expression, pigmentation, diverse cell signaling, and fascinating morphologies (Spieth 1952; Nakamura et al. 2007; Kagesawa et al. 2008; Schaeffer et al. 2008; Markow et al. 2009; Niepielko et al. 2011; Werner et al. 2018, 2020). Studying the evolution of species at the molecular level is restricted by the availability of their high-quality genome assemblies. A few sequenced Drosophila species have limited tools for genetic analyses (i.e. Holtzman et al. 2010; Werner et al. 2010; del Valle Rodriguez et al. 2011; Niepielko and Yakoby 2014; Stern et al. 2017), which presents an impediment to understanding the evolutionary mechanisms responsible for common and unique organismal traits. The improvement of sequencing technologies, including long-read sequencing via Pacific Biosciences (PacBio) and Oxford Nanopore, have allowed for massive efforts to sequence the genomes of many different species, including Drosophila (Kim et al. 2020), as well as updating and improving the contiguity and completeness of existing assemblies (Paris et al. 2020). These advances are monumental in furthering the development of biological systems in other Drosophila species, which are the stepping stone to study mechanisms of evolutionary diversity. Since the discovery of a white-eyed fruit fly in 1910 by Thomas Hunt Morgan (Morgan 1910), the w gene has been extensively studied in D. melanogaster. The gene encodes an ATP-binding cassette transporter, which forms heterodimers with the Scarlet or Brown proteins to deliver pigment precursors into pigment cells, consequently making red eyes in wild-type flies (Sullivan and Sullivan 1975; Sullivan et al. 1979). Mutation of this gene may result in the alteration of the protein structure and lead to loss of function, resulting in white-eyed flies (Mackenzie et al. 1999). Due to the simplicity of identification, eye-color of w-disrupted flies is frequently used as a selectable marker for transgenic flies. However, deleterious effects due to the loss of w have been uncovered in the past decades. Several studies have documented alterations in courting, copulation success, exploratory behavior, visual acuity, learning and memory of thermal stress, and sexual preference in D. melanogaster overexpressing or deficient of the White protein (Anaka et al. 2008; Sitaraman et al. 2008; Krstic et al. 2013; Ferreiro et al. 2017; Xiao et al. 2017). Behavioral changes, such as these have been shown to be the result of altered levels of specific neurotransmitters, such as serotonin and dopamine (Becnel et al. 2011; Ries et al. 2017), whose precursors are transmitted by the White protein (Krstic et al. 2013; Xiao et al. 2017). Other studies have reported that w-disrupted D. melanogaster lack optical insulation provided by eye pigment and thus show impaired visual acuity (Kalmus 1943), increased light sensitivity (Wu and Wong 1977), deficient contrast perception (Wehner et al. 1969), atypical phototactic response and electroretinogram (Pak et al. 1969; Stark and Wasserman 1972), as well as progressive retinal degeneration (Ambegaokar and Jackson 2010). At the same time, there are numerous white-eyed lines of Drosophila species that are viable and used in genetic studies (Holtzman et al. 2010). The fly species D. nebulosa belongs to the willistoni group (Pavan 1946; Schaeffer et al. 2008). This fly has been an attractive system to study the evolution of mating behavior (Spieth 1952; Gleason et al. 2012), cell signaling, gene patterning, and eggshell morphology (Niepielko et al. 2011, 2014; Niepielko and Yakoby 2014). In D. nebulosa, male fruit flies court by producing an anal droplet as a nuptial gift to the female, and subsequently fanning it in their direction with one wing (Spieth 1952; Steele 1986). Unlike other Drosophila species, D. nebulosa requires vision to locate females in order to initiate mating (Spieth 1952; Keesey et al. 2019). In addition, D. nebulosa males placed in constant darkness were incapable of inseminating any females (Gleason et al. 2012). Since w participates in the vision process in flies, we hypothesize that decreased visual acuity, caused by the disruption of w (Xiao et al. 2017) will impair D. nebulosa males’ ability to recognize potential mates, rendering them unable to reproduce. As a first step to testing the visual requirements underlying mating success in D. nebulosa on a molecular level, we generated a high-quality long-read genome assembly using PacBio sequencing. We produced de novo preliminary assemblies using 4 different programs, corrected with short-read Illumina sequencing data, and subsequently merged them into a single hybrid assembly. Gene annotation was then carried out on the assembly, and chromosome synteny was mapped, using D. willistoni as a reference. Based on the genomic information, we utilized CRISPR/Cas9 to successfully target the w gene in D. nebulosa. Independent white-eyed transgenic flies were then validated to ascertain that w was disrupted via nonhomologous end-joining. While responding to phototaxis, we observed that, unlike the many other Drosophila stocks with white eyes, w-disrupted D. nebulosa males did not attempt to mate with females.
The wild-type D. nebulosa stock #14030-0761.06 (Isoteca-48) was obtained from the National Drosophila Stock Center at Cornell University. Oregon R (OreR) Bloomington #25211 was used as a wild-type D. melanogaster stock. Stocks were kept at room temperature (∼22–24°C) and standard cornmeal fly food.
Genomic DNA (gDNA) was extracted using a modified protocol provided by the VDRC Stock Center (https://stockcenter.vdrc.at/images/downloads/GoodQualityGenomicDNA.pdf). Male and female D. nebulosa heads were used for gDNA extractions bound for PacBio sequencing, and whole male flies were used for Illumina sequencing. In short, tissues (heads or whole flies) were homogenized and incubated in a 0.1-M Tris–HCl/0.1 M EDTA/1% SDS solution and 10 µg RNase A at 70°C for 30 min. Then, 8 M KAc was added, and heads were incubated for another 20 min. Supernatant was phenol–chloroform extracted twice, pelleted using isopropanol and ethanol, in series, and then eluted in nuclease-free water. The gDNA was evaluated for quality on a 0.9% agarose gel (run for 45 min at 100 V) and quantified using a NanoDrop 2000 spectrophotometer (Thermo Scientific). Pacific Bioscience single molecule sequencing (PacBio) was carried out at the Waksman Genomics Core Facility, Rutgers, The State University of New Jersey. The DNA was quantitated using the Qubit 2.0 instrument and Fragment Analyzer with a DNF-467 Genomic DNA 50 kb Analysis Kit according to the manufacturer’s instructions (Agilent Technologies). Samples were purified using AMPure XP Clean beads (Agencourt Bioscience Corp., Austin, TX, USA). Sequencing libraries were constructed following the manufacturer’s protocol and sequenced on single-molecule real-time (SMRT) cells within a PacBio Sequel System, using version 3.0 chemistry and 10-h runs. Raw reads were generated by combining outputs of 4 sequencing runs, which were carried out using this method. Reads shorter than 3 kb were filtered out using cutadapt v1.8 (Martin 2011). Genomic DNA was prepared for short-read sequencing, using the NEBNext Ultra II FS DNA Library Prep Kit for Illumina (New England Biolabs). The samples were sequenced as paired-end 2 × 100 nt reads on the Illumina MiSeq platform at the Lewis-Sigler Genomics Core Facility, Princeton University. The FASTQ file was generated, using Illumina MiSeq Control Software under default settings. Only pass-filter reads were used for further analysis.
To account for different biases in genome assemblers, preliminary de novo assemblies were constructed using 4 different programs. All assembly file names, with brief descriptions, can be found in Supplementary Table 1. First, raw reads were corrected, trimmed, and assembled with Canu v2.1 (Koren et al. 2017, 2018; Nurk et al. 2020) default parameters (except -genomeSize = 222m), to generate neb_c. For the second preliminary assembly, raw PacBio reads were initially self-mapped (setting -x ava-pb), using minimap2 to detect overlaps (Marçais et al. 2018), and then concatenated into unitigs using miniasm (Li 2016) to generate neb_m1. Raw reads were then mapped back against neb_mi using minimap2, generating unpolished and uncorrected contig sequences, neb_m2. Racon (Vaser et al. 2017) was then used to generate genome consensus of the uncorrected assembly (with inputs <sequences>=Raw pacbio reads, <overlaps>=neb_m2, <target sequences>=neb_m1), generating neb_r1. Raw reads were additionally mapped against neb_r1 using minimap2, creating neb_m3. Once again, racon was run (with inputs <sequences>=Raw pacbio reads, <overlaps>=neb_m3, <target sequences>=neb_r1) to generate the final corrected a genome consensus, neb_m. The third assembly used Flye (Kolmogorov et al. 2019) to assemble and polish raw reads on default settings (except –pacbio-raw), generating neb_f. Finally, raw reads were also assembled using wtdbg2 (Ruan and Li 2020) with default settings (except -x sq, -g 222m) to create neb_w1. The final consensus, neb_w, was then generated using wtpoa-cns (Ruan and Li 2020) with default settings.
Preliminary assemblies were then polished using short-read Illumina sequencing data from D. nebulosa. Short-read data were aligned to each individual preliminary assembly, using BWA (Li and Durbin 2009). The resulting SAM file was converted to the BAM format, sorted, and indexed using Samtools (Li et al. 2009) with default settings. This resulting file, as well as its respective preliminary assembly, were then input into Pilon (Walker et al. 2014) for polishing. Parameters were set as diploid, but otherwise kept default. As a note, a “p” was appended to the end of Pilon-corrected preliminary and composite D. neb assemblies (e.g. Pilon-corrected neb_w was named neb_wp) (Supplementary Table 1).
Preliminary assemblies were then combined into hybrid assemblies, using quickmerge (Chakraborty et al. 2016) and MUMmer (Marçais et al. 2018). This was done by assigning the query assembly as the most contiguous, and the reference assembly as the second most contiguous. For example, out of the 4 preliminary assemblies, neb_wp was the most contiguous, and neb_fp was the most complete. As such, they were selected as the query and reference when generating the initial composite assembly (neb_q1p), respectively. The specific order of merging is detailed in Fig. 1a. Minimum seed contig length to be merged (length cutoff) was set to 500. Composite assemblies were again polished using short-read Illumina data in Pilon between each merge, as described in the previous section.
Assembly statistics (Supplementary Table 1) were calculated, using the abyss-fac function from ABySS v2.1.5 (Jackman et al. 2017) and stats function from BBMap v38.87 (Bushnell 2014). Assembly completeness (Supplementary Table 2) was evaluated using BUSCO v5.1.2 (Simao et al. 2015) to compare gene content in preliminary and composite assemblies to the diptera_odb10 lineage dataset (specifically, -l diptera_odb10 and -m genome). The diptera_odb10 lineage dataset set contains 3,285 orthologs found to be present and single copy across 56 dipteran genome assemblies performed to date. The presence of orthologous genes in their complete form, and without duplication, allows us to assess how complete our assemblies are with respect to gene content. Contiguity and completeness of reference Drosophila assemblies were assessed using the same methods.
We used the Maker v2.31.11 pipeline (Cantarel et al. 2008; Campbell et al. 2014) to annotate the polished final composite assembly (dneb_q3p) (Fig. 4a). For the initial run, parameters we edited in the maker_opts.ctl data file as described below, otherwise set to 0, or left blank. The polished composite D. nebulosa assembly was used for annotation in FASTA format (<genome>=neb_q3p, <organism_type>=eukaryotic). Proteomes obtained from UniProt of species D. melanogaster (GenBank reference: GCA_000001215.4), D. pseudoobscura (GCF_009870125.1), and D. willistoni (GCA_000005925.1) were provided as protein homology evidence in FASTA format (<protein>=mel_prot, pse_prot, wil_prot). The Repbase repeat library from D. willistoni (Jurka 1998, 2000) was used as a model organism for soft repeat masking (<model_org>=Drosophila_willistoni, softmask = 1). Gene prediction was inferred only using protein homology (<protein2genome = 1). Lastly MAKER behavior settings were set (with inputs <alt_peptide>=C, <cpus>=1, <max_dna_len>=200,000, <min_contig>=2,000, <pred_flank>=200, <AED_threshold>=1, <split_hit>=10,000, <tries>=5). We ran MAKER in a Singularity Biocontainer distributed by Bioconda (https://bioconda.github.io/). Repeat masking was performed using RepeatMasker v4.1.1 (Smit et al. 2013–2015). Initially, MAKER was used for ab initio gene prediction, as well as aligning protein evidence to neb_q3p (with MAKER flags -fix_nucleotides and -nodatastore and Singularity flags –no-home and –cleanenv). MAKER then used 2 gene annotation programs to integrate evidence and produce gene models: SNAP (Korf 2004), and Augustus v3.4.0 (Stanke et al. 2008). Both were then trained using the resultant predictions, with SNAP specifying an AED and amino acid length of 0.25 and 50 or greater, respectively (maker2zff -x 0.25 -l 50). BUSCO was used to train Augustus (with inputs -l diptera_odb10 -m genome -c 30 –augustus –augustus_species fly –long –augustus_parameters=’—progress=true’) on mRNA annotated regions flanked on either side by an additional 1,000 bp. MAKER was then rerun to improve on the existing gene models by replacing previous evidence with the newly generated SNAP and Augustus models (retraining parameters). Additionally, tRNAscan-SE (Chan and Lowe 2019) was enabled for the detection and annotation of tRNAs. The maker_opts.ctl file was altered in the following ways: <protein_gff>=rnd1.protein2genome.gff, <rm_gff>=rnd1.repeats.gff, <snaphmm>=dneb1.l50.aed24.hmm, <est2genome>=0, <protein2genome>=0, <trna>=1. MAKER was run a total of 4 times, each time replacing the repeat GFF file and SNAP HMM with that of the previous run. After each iteration, the models were evaluated for BUSCO completeness, number of gene models, and AED distribution. BUSCO was run using the transcript FASTA and Augustus retraining parameters of each respective MAKER iteration (with inputs -l diptera_odb10 -m transcriptome -c 8 –augustus_species Dnebulosa –augustus_parameters=’—progress=true’) (Supplementary Table 3). Specifically, the AED distribution (Supplementary Table 4 and Fig. 4e) was calculated using AED_cdf_generator.pl (https://github.com/mscampbell/Genome_annotation/blob/master/AED_cdf_generator.pl), by using the master GFF file as the input, and specifying the bin size (-b 0.025). Gene model IDs were renamed and mapped using MAKER’s maker_map_ids, map_gff_ids, and map_fasta_ids functions, following a protocol described in the section Renaming Genes for GenBank Submission in Campbell et al. (2014). The proteome of D. melanogaster was used as a BLAST reference to obtain names for D. nebulosa orthologs, using protocol described in section Assigning putative gene function (Campbell et al. 2014). Gene names were mapped to model IDs via Annie (Tate 2014), using the aforementioned D. melanogaster proteome and BLAST results (Supplementary Table 4). Finally, Genome Annotation Generator (GAG) (Geib et al. 2018) was used to rename gene models, as well as to pull annotation statistics. Drosophila nebulosa gene models were protein-aligned, via blastp, against the D. willistoni proteome as an additional measure of ortholog homology.
We used the BUSCO_phylogenomics pipeline (McGowan et al. 2020; McGowan and Fitzpatrick 2020) to assess the phylogenomic position of our D. nebulosa assembly with respect to related species with genome assemblies available. This pipeline involves first running BUSCO on any genomes to be included in the phylogeny to find orthologous genes for phylogenomic tree building. Using the same BUSCO settings described above, we analyzed 26 genome assemblies from 23 species (Supplementary Table 8) (Clark et al. 2007; Zimin et al. 2008; Hoskins et al. 2015; Chakraborty et al. 2017; Zhang, Yu, et al. 2018; Liao et al. 2019; Paris et al. 2020; Pinharanda et al. 2020; Reilly et al. 2020). Assemblies from the D. willistoni and D. saltans subgroups were also included to ensure adequate phylogenomic comparison within and to adjacent monophyletic branches (Kim et al. 2020). Results for each BUSCO run were then used by the BUSCO_phylogenomics pipeline to create trimmed alignments for each gene that was present and single copy in every queried genome. Alignments were performed using MUSCLE v 3.8.31 (Edgar 2004a, 2004b) and trimmed using TrimAl v 1.4 (Capella-Gutierrez et al. 2009). These alignments were then used to infer phylogenies by either concatenation and phylogenetic analysis in IQ-Tree v1.2.12 (Nguyen et al. 2015) (supermatrix method) or by inferring individual gene trees with IQ-TREE and performing species tree inference using ASTRAL v. 5.7.7 (Zhang, Rabiee, et al. 2018) (supertree method). We ran BUSCO_phylogenomics with default settings, except that IQ-TREE (Nguyen et al. 2015) was run using the -safe flag. All trees were rooted, using Scaptodrosophila lebanonensis and visualized using iTOL (Letunic and Bork 2021).
We next placed D. nebulosa scaffolds to predicted chromosomes by comparing conserved loci to a reference genome. D. willistoni was chosen as a reference due to the genome's similar chromosomal arrangement to D. nebulosa. As a note, the D. willistoni caf1 (GCA_000005925.1) and D. willistoni 17 (GCA_018903445.1) assemblies will hereby be referenced as wil_caf1, and wil_17, respectively. First, we identified chromosomal locations of wil_caf1 assembly scaffolds, using data from previous studies (Supplementary Table 9) (Schaeffer et al. 2008; Garcia et al. 2015). In an attempt to use a more contiguous reference, we used Satsuma2 (https://github.com/bioinfologics/satsuma2) (Grabherr et al. 2010) to find syntenic scaffolds between the wil_caf1 and wil_17 assemblies (Supplementary Table 10). This was done specifically by comparing only chromosome-annotated wil_caf1 scaffolds to the 20 largest wil_17 scaffolds. From this output, wil_17 scaffolds syntenic to those of wil_caf1 were selected, renamed according to chromosome (Supplementary Table 11), and then compared with 11 largest D. nebulosa scaffolds using Satsuma2 (Supplementary Table 12). As an additional evaluation, annotated wil_caf1 scaffolds were also compared with the 11 largest D. nebulosa scaffolds using Satsuma2 (Supplementary Table 13). All Satsuma2 comparisons were run with default settings. The R circlize package was used to visualize chromosome synteny between D. nebulosa and D. willistoni, using a representative subset of alignments with identities greater than 0.75. Only D. willistoni scaffolds with over 2,000 aligned regions with D. nebulosa were visualized (Fig. 5 and Supplementary Fig. 1).
Transposable element density was mapped to the 11 largest scaffolds to predict centromeric locations. To map transposable elements in the D. nebulosa genome, we used the Bedtools v2.50.0 (Quinlan and Hall 2010) makewindows function to divide the largest 11 scaffold lengths in the assembly into 10 kb windows (setting -w 10000 -s 10000), generating neb_win.10k. We then used LINE, LTR, DNA transposable elements, and helitron MAKER annotated repeats, as well as neb_win.10k, as inputs for the Bedtools coverage function, to calculate the number of genes within each 10 kb window. Statistics were visualized for the largest 11 scaffolds using the circlize package (Gu et al. 2014) in RStudio.
To develop a white-eyed Cas9-expressing transgenic D. nebulosa fly, we used homology-directed repair to disrupt w, while simultaneously inserting Cas9 under a nanos promoter. The CRISPR/Cas9 system works by creating a double-strand break proximal to a specified 20 bp target site adjacent to a protospacer adjacent motif (PAM) sequence, facilitated by a guide plasmid (Gratz et al. 2013). Point mutation w alleles in D. melanogaster from Mackenzie et al. (1999) were mapped to exons 3–6 in D. nebulosa. The locus was targeted (Fig. 6a) by aligning both sequences in MEGA (Kumar et al. 2016) and choosing PAM sites which flank the predicted region. Target cut sites were determined using CRISPR Optimal Target Finder (Gratz et al. 2013), and cleavage efficiency was predicted using CRISPR Efficiency Predictor (Housden et al. 2015). The formerly mentioned program was used to find target sequences adjacent to PAM sites, and to compare them to a reference genome to look for similar off-target cut sites. Since the D. nebulosa genome was not listed on the site, we attempted to account for off-target cut sites using the D. willistoni genome. The selected target sequences were then aligned via BLASTn to our D. nebulosa genome to look for matches. To repair the double-strand break(s) by the guide(s), a donor vector was designed featuring the Cas9 gene under the nanos promoter (see CRISPR constructs section in Materials and methods). The vector insert was flanked by two 1,000 bp, arms which are homologous to the D. nebulosa w loci surrounding the target region, as described in Gratz et al. (2013). Three separate guide injections (Rainbow Transgenics, CA) (Fig. 6c) were used increase the likelihood of a unique target, as well as to the test the efficiency of 1 vs 2 guides. All injections included the Cas9-containing donor plasmid (1.12 μg/μl) and Cas9 protein (5 μg/μl) (ThermoFisher #A36498). Injections differed in the combination of guide plasmids, where injection #1 contained the neb_w_guide1 plasmid (2.58 μg/μl), injection #2 contained neb_w_guide2 plasmid (1.33 μg/μl), and injection #3 contained neb_w_guide1 and neb_w_guide2 (1.76 μg/μl) (Fig. 6c). All oligonucleotides are shown in Supplementary Table 14.
Two different guide oligonucleotides were ligated into individual pU6-BbsI-chiRNA plasmids (Melissa Harrison, Kate O'Connor-Giles, and Jill Wildonger; Addgene plasmid # 45946), as described in Gratz et al. (2013). The donor vector (Fig. 6a) was designed using a modified Gratz et al. (2013) procedure. Left and right homology arms were amplified from genomic DNA of whole D. nebulosa flies. The donor vector backbone and nos-Cas9 locus were amplified from a pnos-Cas9-nos plasmid (Addgene plasmid # 62208) (Port et al. 2014). A complete list of primers used is listed in Supplementary Table 14 under Primers used for CRISPR. All donor vector fragments were ligated into a circular plasmid via New England Biolabs HiFi Assembly Master Mix Gibson Assembly (E2621). The 4-fragment Gibson assembly used a 1:1 vector: insert ratio, containing 148.85 ng pnos-Cas9-nos backbone (28.3 ng/μl), 163.84 ng nos-Cas9 insert (28.2 ng/μl), 31.68 ng right homology arm insert (57.6 ng/μl), and 32.70 ng (54.5 ng/μl) right homology arm insert, for a total reaction volume of 22.23 μl. All plasmids were cloned in DH10β E. coli bacteria, and screened by PCR amplification using T3 and T7 primers. Plasmids were extracted and purified using the ZymoPURE II Plasmid Midiprep Kit (Zymo Research). Guide plasmids were all sequence-validated with T3 primers. The donor vector was sequenced using primers w_insF, w_ins2R, and nebRHAwR (independent reactions). Plasmids were digested with SapI exonuclease (CutSmart R0569S) and validated using restriction fragment mapping.
CRISPR injected (G0) male and female flies were separated immediately after eclosion from the pupa and mated to wild-type D. nebulosa. Progeny (F1) were then screened for white eyes. Wild-type virgin females D. nebulosa were then crossed to white-eyed D. nebulosa males and left to self-cross in an attempt to establish a white-eyed stock. Red-eyed D. nebulosa males were selected against during this process. To validate the CRISPR locus, genomic DNA was extracted from white-eyed males of each positive line, and compared with wild-type D. nebulosa. Primers flanking the target region (w_insF, w_ins2R) were used for DNA amplification (Supplementary Table 14, Primers used for sequencing). The PCR products were then sequenced and aligned to the D. nebulosa w reference locus using MEGA. Mutations/deletions in the w gene as well as the presence/absence of Cas9 were determined.
Wild-type and white-disrupted D. nebulosa males were individually paired to virgin a female. Vials were video recorded for a span of 4 h, and the footage was analyzed using BORIS (Friard and Gamba 2016) to annotate instances of courting. We next tested whether white-eyed D. nebulosa could sense and respond to light. Flies were enclosed in a 28-cm plastic cylinder, segmented into thirds (labeled 1–3), and left to adjust to darkness for 30 min. Each phototaxis trial was initialized by placing 19 flies into the end of tube 1, leaving them in darkness for 15 min, and then recording the quantity of flies in each segment. Next, a Leica KL 200 LED cold light source set to 0.5 brightness was shone into the distal end of segment 3 for 15 min. To limit the brightness even further, the light was covered by one layer of a paper towel. The quantities of flies in each segment were again recorded. Four trials were conducted for both wild-type, as well as white-eyed male D. nebulosa. The experiment was carried out at room temperature (23°C). The experimental setup is visualized in Fig. 7a. Results were analyzed for significance in the dataset, using 1-way ANOVA, and subsequent Tukey HSD tests for all-verses-all comparison of treatment means were performed.
Long-read sequencing generated 13 Gb of sequence from 1,717,740 subreads above 3 kb with a read N50 of 8.4 kb. Short-read Illumina sequencing generated 29,211,787 forward reads, 100 bp in length. To account for different biases in genome assemblers we used 4 separate programs to generate preliminary assemblies, and subsequently merged them in a step-wise fashion (Fig. 1a). Composite assembly neb_q1p (merge of neb_wp and neb_fp) showed considerable improvements in contiguity, as well as completeness similar to neb_fp. Subsequent merges with neb_cp, and then neb_mp, improved the resultant composite assemblies (neb_q2p and neb_q3p, respectively), though only marginally (Fig. 1b). Contiguity of neb_q3p also compares favorably with other available Drosophila assemblies in the willistoni group (Fig. 2). The final assembly (neb_q3p) has a total of 1,600 scaffolds, with an N50 of 20.9 Mb, and a total size of 177 Mb. Statistics for each preliminary and composite assembly are listed in Supplementary Table 1. Analysis of 3,285 universal single copy Dipteran orthologs (BUSCO diptera_odb10 dataset) in neb_q3p revealed 98.2% (3,229) to be present and full length (97.7%, 3,211 single-copy; 0.5%, 18 duplicated), 0.9% (28) were present but fragmented, and 0.9% (28) of these genes were missing from our assembly (Fig. 3). Comparative BUSCO scores for our preliminary and final composite assemblies are listed in Supplementary Table 2.
Gene models were retrained via the MAKER pipeline a total of 4 times (Fig. 4a). Since the fourth run of the pipeline produced little improvement based on number and average length of gene models, BUSCO scores, and annotation edit distance (AED) (Fig. 4, b and e; Supplementary Table 3), the third iteration of gene predictions was chosen as the final annotation and will thus be reported on in this section. Genome annotation through de novo prediction and homology with D. melanogaster produced 13,067 gene models (Table 1). Protein BLAST alignments of D. nebulosa models with D. melanogaster and D. willistoni generated 12,548 and 12,578 alignments, respectively, indicating orthology with both species (Supplementary Table 4). BUSCO analysis of the transcriptome revealed 92.5% (3,040) completed (92.0%, 3,023 single-copy; 0.5%, 17 duplicated), 1.7% (57) fragmented, and 5.8% (188) missing orthologs (Fig. 4d) suggesting that our annotation includes the vast majority of genes present in our assembly. AED, a measurement of how well an annotation agrees with overlapping protein homology evidence (scores 0 and 1, denoting perfect and no agreement to aligned evidence, respectively) (Holt and Yandell 2011), shows 97% of the annotation with a score of 0.5 and under (Fig. 4e; Supplementary Table 5).
Scaffolds aligned between D. nebulosa and D. willistoni (wil_17) assemblies were found to be highly syntenic, allowing identification of homology between D. nebulosa and D. willistoni chromosomes. However, we observe considerable internal reorganization within chromosomes (Fig. 5). Drosophila nebulosa scaffolds dneb_sca_0 and dneb_sca_1 are each syntenic with D. willistoni scaffolds belonging to a single chromosome arm (Chr2L and Chr2R_1-4, respectively). Others, such as dneb_sca_3, 6, and 10 all appear to constitute the D. willistoni chromosome 3 scaffold (Chr3). Consistent with these data, the genes eyeless (ey) and cubitus interruptus (ci), which are known to be located on chromosome 3 in D. nebulosa (Pita et al. 2014), can be found on scaffold dneb_sca_3 in our assembly. The X chromosome appears to be less contiguous with dneb_sca_2, 5, and 9 syntenic to the D. willistoni left arm (ChrXL), and dneb_sca_2, 4, 7, and 8 syntenic to the D. willistoni right arm (ChrXR1-4). Of note, dneb_sca_2 appears to span across both arms of D. willistoni Chromosome X, with the right arm syntenic to position 22,716–8,714,232, and left to 8,775,313–21,991,806. Syntenic alignment of D. nebulosa and D. willistoni (wil_caf1) assemblies (Supplementary Fig. 1) were largely consistent with the aforementioned data, with the exception of a rearrangement between the right arm of chromosomes X and 2, which differ slightly in position and size. As a method of determining potential centromere location within the assembly, we mapped all annotated class I and II transposable elements to the largest 11 scaffolds. We see considerable enrichment of transposable elements at the start of dneb_sca_3 and 9, and at the end of dneb_sca_0, 1, and 4 (Fig. 5). A few smaller spikes of transposable elements are interspersed throughout the scaffolds, however, the higher density regions at the scaffold ends indicate these locations as likely centromeres.
Following genome assembly and gene annotation, we aimed to test the requirement of vision in D. nebulosa mating by generating a white-eyed D. nebulosa, as a proof of concept. If mutation in w can be tolerated, as in other species, we intended to insert the Cas9 gene into the w gene to obtain a stock that can potentially be used for future CRISPR/Cas9 genome engineering. Briefly, the D. nebulosa w locus was targeted by a combination of 2 guides, Cas9 protein, and a homology directed repair vector with the Cas9 gene (more details can be found in the Materials and methods) (Fig. 6a). Since the w gene is on the X chromosome, the expectation was to obtain white-eyed male flies hemizygous for disrupted gene. In total, 13 F1 lines of white-eyed D. nebulosa males were selected positive for the CRISPR disruption (Fig. 6, b and c). Despite repeated attempts, females remained heterozygous for the w null allele, thus the white-eye phenotype was only found in males. Although the initial intent was to insert the Cas9 gene into the w gene, PCR validation of white-eyed D. nebulosa CRISPR target loci revealed that neither insert integration, nor complete deletion occurred in any of the lines (Fig. 6, c–h). Interestingly, lines successful for disruption of w were all from embryos injected with both guide plasmids (Fig. 6c). While the 2 guides promoted gene disruption, deletions were only found to be present around one PAM site per line (7 out of the total of 10 achieved from guide 1, Fig. 6g), but never at both. Instead, both guide plasmids created asynchronous 1–14 bp deletions on or upstream of the PAM sites (Fig. 6, d–h). In one case, a 565-bp deletion was characterized adjacent to guide 1 (Fig. 6f).
A cross between wild-type females and white-eyed males failed to produce developing embryos. As a result, white-eyed females were never observed in any of the lines. This is supported by the fact that a white-eyed male in this cross was not observed courting the wild-type female even once over a period of 4 h. At the same time, a control cross of a wild-type male and female displayed 13 instances of distinctive courtship. The courtship was observed in varying intervals (42.0 ± 30.6 s) for ∼9.1 min, cumulatively. To assess phototaxis in wild-type and white-eyed D. nebulosa, we placed flies in a plastic tube segmented into 3, and quantified the average number of flies in each segment of the tube after dark and light conditions. All flies were initially placed in the proximal end of segment 1, and a light source was placed facing the distal end of segment 3 (Fig. 7a). We expected that flies kept in darkness would not show a tendency to travel to any specific part of the tube. Consequently, the distribution of the fly population would be random, and the number of flies at the distal end (segment 3) would not be significantly different between initial conditions and 15 min of darkness. However, flies with a positive phototactic response would be expected to travel toward the light source. Thus, the number of flies in segment 3 should be significantly greater after 15 min of light than compared with the amount after 15 min of darkness. The number of flies in segment 2 before (wt = 0, w− = 0) and after 15 min of darkness (wt = 3.3, w− = 1.0) showed no significant difference for both wild-type and white-eyed D. nebulosa (P = 0.58, P = 0.97). The same was true for number of flies in segment 3, before (wt = 0, w− = 0) and after 15 min of darkness (wt = 2.0, w− = 0.3). However, the number of flies in segment 3 was significantly greater (P = 0.02, P = 0.00) after 15 min of light (wt = 7.8, w− = 6.8), compared with the same segment after 15 min of dark conditions (Fig. 7b; Supplementary Table 6). This indicated that both wild-type and white-eyed D. nebulosa males respond to light.
In order to generate tools for genetic and genomic analyses in D. nebulosa, we assembled a long-read annotated D. nebulosa genome. This assembly is highly complete and contiguous and compares favorably to other genome assemblies in the willistoni clade. Using the genome of D. willistoni as a reference, we predicted that near-entire chromosomal arms can be reconstructed with ∼1–4 scaffolds from the D. nebulosa assembly. Scaffolds also span intergenic regions, facilitating the design of molecular experiments within the species. In particular, the assembly contiguity was outstanding relative to many available Drosophila genome assemblies. Comparison of N50 values across 26 Drosophila assemblies shows our assembly as the highest of the species sampled within its clade, with a contig size comparable to the current D. melanogaster assembly (Fig. 2). Assembly completeness was evaluated via BUSCO, with D. nebulosa showing scores in line with the other assembly references (Fig. 3; Supplementary Fig. 8). Annotation BUSCO scores are comparable to that of the genome, indicating that our annotation likely captures the majority of protein coding genes in this species. The number of D. nebulosa genes (Table 1) annotated in the final version (iteration 3) is comparable to the number of D. willistoni protein coding genes in the current assembly annotation (GCF_000005925.1) (Clark et al. 2007; Zimin et al. 2008), which is expected for 2 species of the same subgroup. This comparison provides further confidence in our de novo assembly. Phylogenomic trees inferred using supermatrix (Fig. 2) and supertree approaches (Supplementary Fig. 2) recovered identical tree topologies and placed D. nebulosa as sister to a clade containing D. willistoni, D. paulistorum, D. tropicalis and D. insularis, and within the monophyletic willistoni group (van der Linde and Houle 2008). We recover a nonsister relationship between D. nebulosa and D. sucinea. This finding supports previous work suggesting paraphyly of the bocainensis subgroup (Gleason et al. 1998; Tarrio et al. 2000; Zanini et al. 2018). In the several species of the willistoni group, the dot chromosome does not exist alone and is instead fused to chromosome 3 (fusion of Muller elements E + F). Previous studies have used fluorescence in situ hybridization to demonstrate that the ey, ci, and Ankyrin (Ank) genes, which are present on chromosome 4 in D. melanogaster, are part of chromosome 3 in a number of species in the willistoni and bocainensis subgroups, D. willistoni and D. nebulosa included (Papaceit and Juan 1998; Pita et al. 2014). Hence, D. nebulosa has 3 chromosomes: X, 2, and 3; with the X and 2 consisting of a left and right arms (Pavan 1946; Valente et al. 1996; Schaeffer et al. 2008). As an attempt to correlate some of the larger scaffolds to their potential chromosome, we searched for syntenic regions between D. nebulosa and D. willistoni assemblies (Fig. 5). Altogether, this evidence suggests that the D. nebulosa assembly succeeded in generating scaffolds that are congruent with known D. willistoni chromosomal arms 2L (dneb_sca_0), 2R (dneb_sca_1), 3 (dneb_sca_3, 6, and 10), XL (dneb_sca_2, 5, and 9), and XR (dneb_sca_2, 4, 7, and 8). These data show that the largest 11 scaffolds from the assembly account for all 3 D. nebulosa chromosomes (5 chromosome arms). Additionally, the ey, ci, and Ank genes are all on sca_3 in the D. nebulosa assembly. This reflects the fusion of the dot chromosome and chromosome 3 and is in agreement with experimental results of previous studies (Papaceit and Juan 1998; Pita et al. 2014). Scaffolds constituting the X chromosome appear to be less contiguous, most likely due to homologous, but divergent X and Y gametologs from the mix of male and female D. nebulosa used for sequencing. As of note, the assignment of D. willistoni chromosome 2 arms have been debated (Rohde et al. 1995; Schaeffer et al. 2008; Garcia et al. 2015), but for the purposes of this discussion, the study by Garcia et al. (2015) was mainly referenced. Accordingly, it should be noted that assigning scaffolds to chromosomes is based on synteny with D. willistoni. The genome of D. nebulosa has long been established to contain many instances of chromosomal rearrangements (Pavan 1946; Valente et al. 1996; Papaceit and Juan 1998; Pita et al. 2014). In addition, genetic recombination in the X chromosome is more frequent than in autosomes (Rius et al. 2016). This may account for the syntenic variation we see in reference to the D. willistoni assemblies, such as the rearrangement of specific regions between chromosome arms XL and XR, or XR and 2R (Fig. 5). Although this variation could be due to contig mis-joining in the reference assemblies, interspecies chromosomal variation has been previously characterized in D. willistoni (Rohde and Valente 2012). Overall, the latter possibility is favored since both reference D. willistoni assemblies (wil_caf1 and wil_17) are from different isolates (Supplementary Table 8). As a final metric of assembly contiguity and completeness, we set to assess how well scaffolds can recapitulate D. nebulosa chromosomal arms. One measure of this is whether the assembly scaffolds include centromeric regions at the end of the chromosome. In drosophilids, transposable elements have been shown to be distributed more densely in centro- and telomeric regions, as well as other regions of low recombination rate (Thomas et al. 2015; Rius et al. 2016). Indeed, we see high density regions of transposable elements at the ends of 5 D. nebulosa scaffolds that are predicted to each comprise different chromosome arms (Fig. 5). Furthermore, MAKER repeat annotation did not show a high density of HeT-A, TART, or TAHRE retrotransposable elements, which are known to constitute Drosophilid telomeres. Altogether, it provides evidence that scaffolds include sequence up to centromeric regions. Using CRISPR/Cas9 genome editing has a great potential to develop new and powerful model organisms to address evolutionary processes related to cell signaling, tissue patterning, morphogenesis, and behavior. In addition, it would bypass years of mutation-induced screens, as was done for many alleles found in D. melanogaster. In D. melanogaster, white-eyed flies are commonly used for transgenic experiments. To our knowledge, this is the first time CRISPR/Cas9 was successfully utilized in D. nebulosa by targeting w in the genome (Fig. 6b). At the same time, characterization of the disrupted locus revealed that although flies injected with both guides were positive for disrupted w, only one of the 2 PAM targets was cut in every case (Fig. 6c). Interestingly, using both guide RNAs generated 9 different deletions in the gene (Fig. 6, g and h). This strategy can potentially serve as a tool to generate different alleles and let selection act on a viability scale. While these were different types of deletions, none could produce a viable white-eyed female fly. Short indels proximal to the targeted PAM sites are indicative of nonhomologous end joining, as opposed to homology-directed repair (Gratz et al. 2013). This is further supported by the fact that the nos-Cas9 cassette, designed to integrate within the w gene, was not present in any of the tested lines (Fig. 6, d–f). The role of the donor vector is to repair double strand breaks created by Cas9. One possible reason for this is that the homology-directed repair pathway is known to be less efficient than the nonhomologous end joining (Roy et al. 2018). Therefore, breaks in the genome may have been ligated together before the donor vector was able to repair them. Previous studies have used various methods to increase CRISPR efficiency, such as piggyBac-mediated integration of the nos-Cas9 locus (Gratz et al. 2014; Nishizawa-Yokoi and Toki 2021), inhibition of nonhomologous end joining pathway (Maruyama et al. 2015), and timed embryo injection with in vivo sgRNA efficiency (Kotwica-Rolinska et al. 2019), providing several options for future improving homology-directed repair efficiency in D. nebulosa. Altogether, we show that CRISPR/Cas9 can work in D. nebulosa. However, additional considerations will need to be implemented prior to becoming an efficient genetic model system. Several Drosophila species are available as viable white-eyed stocks and used in transgenic experiments (Holtzman et al. 2010; Stern et al. 2017). However, most of these species court via acoustic, chemical, and tactical modalities and are not solely dependent on vision. In contrast, mating in several species has been found to require vision (Jezovit et al. 2017; Keesey et al. 2019). For example, D. nebulosa males initiate courtship by uppercutting the female with their legs, standing perpendicularly and angling their posterior toward her, and silently fanning an extruded anal droplet in her direction via flicking motions with one wing. In the absence of light, D. nebulosa males were unable to orient themselves toward the female, resulting copulation failure (Gleason et al. 2012). In species such as D. nebulosa and others like it, we expected that impairing visual acuity and optical insulation through disruption of w (Ferreiro et al. 2017) would jeopardize courtship rituals, and thus copulation. While commonly used, white-eyed D. melanogaster demonstrated reduced courtship. The phenotype was shown to be alleviated in these flies with the introduction of the mini-w gene (Xiao et al. 2017). It is possible that copulation was still successful in white-eyed D. melanogaster, since males court using wing-vibrations to produce a species-specific “song” (Spieth 1952). Conversely, disruption of w in D. suzukii, whose courtship rituals are more similar to D. nebulosa, resulted in no attempts at courtship or copulation (Yan et al. 2020). In our study, pairing white-eyed males with virgin wild-type D. nebulosa females produced eggs, but never any larva. Consequently, white-eyed females were never observed in any of the lines. We predicted that male and female crosses failed to copulate, thus leading to unfertilized eggs. To support this, we compared single crosses of a white-eyed and wild-type D. nebulosa male paired with a virgin female. A clear difference in courtship display was prevalent between the crosses of wild-type D. nebulosa, where males frequently attempted courting females and exploring the vial. This observation is in contrast to the white-eyed males, which did not attempt any courting, even when approached by females. In fact, these males rarely move at all in the vials. In light of these observations, and the established mating behavior of D. nebulosa, it is possible that white-eyed males are unable to visually locate the female. One way to assess the extent of lowered visual acuity is to examine the effects of the disrupted w gene on phototactic response in D. nebulosa. The expectation would be that wild-type D. nebulosa capable of perceiving light would be attracted to it and cluster near the source. Conversely, D. nebulosa that are completely blind would be expected to be ignorant to the light source, and thus disperse randomly. Furthermore, phototactic success in white-eyed flies due to perception via ocelli is also unlikely, since w is required for pigmentation in the eyes, as well as the ocelli (Levis et al. 1985; Caldwell et al. 2007). Our findings showed that similarly to wild-type D. nebulosa, white-eyed flies were attracted to light and traveled toward the source (Fig. 7b). These data indicate that white-eyed D. nebulosa can perceive light but perhaps lack the visual acuity to locate potential mates. It should also be noted that impaired vision may not fully account for the failure to court in white-eyed D. nebulosa. White protein also is responsible for transporting precursors of neurotransmitters across cell membranes, such as serotonin and dopamine. As such, studies have suggested that abnormal levels of neurotransmitters underlie mating irregularities, such as decreased copulation rate (Xiao et al. 2017) and enhanced male–male courtship (Krstic et al. 2013) in D. melanogaster null for and ectopically overexpressing w, respectively. The D. nebulosa species provides a compelling model system to investigate a variety of biological phenomena, such as evolution of cell signaling, patterning, morphology (Niepielko et al. 2012), chromosomal arrangements (Valente et al. 1996), mating behavior (Gleason et al. 2012), and even radiation resistance (Kratz 1975). Here we suggest that, given the fundamental evolutionary differences in D. nebulosa’s courtship, this species is an attractive organism to develop genetic tools to study the visual requirements underlying mating success. Unlike the challenges to rear D. willistoni (Holtzman et al. 2010), D. nebulosa is simple to rear in the same conditions as D. melanogaster. However, the disruption of the visual system, which is required for mating, should be avoided, and other phenotypic markers should be considered that are not involved with vision. A potential solution is to choose a selectable marker which is not involved in vision and behavior. One such possibility is the wing marker, crossveinless (cv) {CG12410, FBgn0000394}. This gene is known to be on the X chromosome in D. melanogaster. In our D. nebulosa assembly, cv has CDS length of 633 bp, and is found on dneb_sca_5 (predicted to belong to the chromosomal arm XL). The gene’s small size would make it easier to clone into vectors for phenotypic rescue (Shimmi et al. 2005), and like white, sex-linkage would allow us to screen for males of the F1 generation.
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PMC9635637 | 36124949 | Luqi Wang,Lei Ji,Hao Li,Deshun Xu,Liping Chen,Peng Zhang,Weibing Wang | Early evolution and transmission of GII.P16-GII.2 norovirus in China | 19-09-2022 | norovirus,genomic epidemiology,transmission analysis,phylodynamics,phylogeography,TransPhylo | Abstract Norovirus is the most common cause of acute gastroenteritis worldwide. During 2016–2017, a novel recombinant GII.P16-GII.2 genotype of norovirus suddenly appeared and over the next several years became the predominant strain in both China and worldwide. To better understand the origin and diffusion of the GII.P16-GII.2 genotype in China, we conducted molecular evolutionary analyses, including phylodynamics and phylogeography. Moreover, to trace person-to-person transmission of GII.P16-GII.2 norovirus, we applied the novel method, TransPhylo, to a historical phylogeny using sequences obtained from a publicly available database. A time-scaled phylogenetic tree indicated that the time to the most recent common ancestor of the GII.P16-GII.2 major capsid protein (VP1) gene diverged from the GII.P2-GII.2 VP1 gene at 2,001.03 with an evolutionary rate of 3.32 × 10−3 substitutions/site/year. The time to the most recent common ancestor of the GII.P16-GII.2 RNA-dependent RNA polymerase region diverged from the GII.P16-GII.4 RNA-dependent RNA polymerase region at 2,013.28 with an evolutionary rate of 9.44 × 10−3 substitutions/site/year. Of these 2 genomic regions, VP1 gene sequence variations were the most influenced by selective pressure. A phylogeographic analysis showed that GII.P16-GII.2 strains in China communicated most frequently with those in the United States, Australia, Thailand, and Russia, suggesting import from Australia to Taiwan and from the United States to Guangdong. TransPhylo analyses indicated that the basic reproductive number (R0) and sampling proportion (pi) of GII.P16-GII.2 norovirus were 1.99 (95% confidence interval: 1.58–2.44) and 0.76 (95% confidence interval: 0.63–0.88), respectively. Strains from the United States and Australia were responsible for large spread during the evolution and transmission of the virus. Coastal cities and places with high population densities should be closely monitored for norovirus. | Early evolution and transmission of GII.P16-GII.2 norovirus in China
Norovirus is the most common cause of acute gastroenteritis worldwide. During 2016–2017, a novel recombinant GII.P16-GII.2 genotype of norovirus suddenly appeared and over the next several years became the predominant strain in both China and worldwide. To better understand the origin and diffusion of the GII.P16-GII.2 genotype in China, we conducted molecular evolutionary analyses, including phylodynamics and phylogeography. Moreover, to trace person-to-person transmission of GII.P16-GII.2 norovirus, we applied the novel method, TransPhylo, to a historical phylogeny using sequences obtained from a publicly available database. A time-scaled phylogenetic tree indicated that the time to the most recent common ancestor of the GII.P16-GII.2 major capsid protein (VP1) gene diverged from the GII.P2-GII.2 VP1 gene at 2,001.03 with an evolutionary rate of 3.32 × 10−3 substitutions/site/year. The time to the most recent common ancestor of the GII.P16-GII.2 RNA-dependent RNA polymerase region diverged from the GII.P16-GII.4 RNA-dependent RNA polymerase region at 2,013.28 with an evolutionary rate of 9.44 × 10−3 substitutions/site/year. Of these 2 genomic regions, VP1 gene sequence variations were the most influenced by selective pressure. A phylogeographic analysis showed that GII.P16-GII.2 strains in China communicated most frequently with those in the United States, Australia, Thailand, and Russia, suggesting import from Australia to Taiwan and from the United States to Guangdong. TransPhylo analyses indicated that the basic reproductive number (R0) and sampling proportion (pi) of GII.P16-GII.2 norovirus were 1.99 (95% confidence interval: 1.58–2.44) and 0.76 (95% confidence interval: 0.63–0.88), respectively. Strains from the United States and Australia were responsible for large spread during the evolution and transmission of the virus. Coastal cities and places with high population densities should be closely monitored for norovirus.
Norovirus, a member of the Norovirus genus and Caliciviridae family, is the leading cause of acute gastroenteritis worldwide (Ahmed et al. 2014). Globally, norovirus causes approximately 677 million cases and 200,000 deaths each year (Pires et al. 2015). It is common in both developed and developing countries and causes approximately 50,000 deaths in developing countries each year (Nguyen et al. 2017). In general, norovirus circulates in colder weather and causes gastrointestinal symptoms such as vomiting, diarrhea, and abdominal pain. Outbreaks are frequently reported in semiclosed institutions, such as hospitals, nursing homes, and schools (Hall et al. 2013). The genome of norovirus consists of 3 open reading frames (ORFs), ORF1, ORF2, and ORF3, encoding RNA-dependent RNA polymerase (RdRp), major capsid protein (VP1) and minor capsid protein (VP2), respectively (Hardy 2005). Norovirus is highly diverse and is divided into 10 genogroups (GI–GX), which are further divided into at least 40 genotypes (Atmar et al. 2019; Li et al. 2021). Commonly isolated norovirus genogroups among cases of acute gastroenteritis are GI and GII (Chhabra et al. 2019). In the past 2 decades, the GII.4 genotype accounted for the majority of adult outbreaks of gastroenteritis and often swept across the globe (Noel et al. 1999; Hoa Tran et al. 2013). However, GII.P16-GII.2, a genotype that was rarely reported previously, suddenly reappeared in the winter of 2016–2017 in China, Japan, Germany, France, the United States, and Australia and, within just a few years, became the main genotype in China and other countries (Ao et al. 2017; Barreira et al. 2017; Niendorf et al. 2017; Nagasawa et al. 2018). According to monitoring data, most norovirus outbreaks reported in China from 2016 to 2018 were GII.P16-GII.2 (Jin et al. 2020). The genotype distribution, molecular evolution, and amino acid substitution profile of common norovirus genotypes like the GII genogroup group were widely studied in early epidemics (Chan et al. 2015; Qiao et al. 2017; Ozaki et al. 2018, 2019), but few studies have investigated this new recombinant GII.P16-GII.2 norovirus. A previous study concluded that the virus could be traced back to areas around the Pearl River delta in China (Ao et al. 2018). Nevertheless, how this genotype evolved worldwide and how it entered China and spread domestically remain unclear. Based on the previous studies, we applied phylodynamics, phylogeography, and TransPhylo methods to better understand the origin, evolution, and transmission process of the GII.P16-GII.2 norovirus and investigated a series of associated epidemic parameters. We found that the GII.P16-GII.2 norovirus may have evolved from GII.P16-GII.4 or previously circulating GII.P16-GII.2 strains, and Guangdong province seems to be the origin of this reemerging GII.P16-GII.2 norovirus in China.
All sequences used in this study were downloaded from the National Center for Biotechnology Information (NCBI) database (https://www.ncbi.nlm.nih.gov) (as of October 12, 2021). Sequences used included those with associated geographic location and collection time, and a linear time relationship; in cases of sequences submitted on the same day showing high similarity after sequence alignment, 10 sequences were randomly selected. Ultimately, a total of 164 of 609 GII.2 VP1 complete sequences and 163 of 504 GII.P16 RdRp complete sequences were included in this study. Detailed information is summarized in Supplementary Table 1. A total of 121 complete GII.P16-GII.2 norovirus sequences were downloaded from NCBI (as of October 12, 2021), including 56 sequences from China and 65 sequences from other countries, for analysis of the early origin and diffusion process of GII.P16-GII.2 norovirus in China. Detailed information is summarized in Supplementary Table 2. All norovirus genotypes were also confirmed using an online genotyping tool (https://www.rivm.nl/mpf/typingtool/norovirus) (as of October 12, 2021).
All sequences were aligned using MUSCLE implemented in MEGA-X (Kumar et al. 2018). Regression of root-to-tip distances of sequences was performed using TempEst software (v.1.5.3) (Rambaut et al. 2016). After removing sequences that affected the temporal signal, we used RDP4 (v.4.101) to confirm the absence of recombination signals in the dataset (Martin et al. 2015). The best-fit HKY + I + G nucleotide substitution model was determined based on the value of LnL in jModelTest (v.2.1.10) (Darriba et al. 2012). Time-scaled phylogenetic trees were constructed in BEAST (v.1.8.4) using tip dates (Suchard et al. 2018), a strict clock model, and a constant size coalescent model with a Markov chain Monte Carlo (MCMC) sample chain (108 steps with sampling every 1,000 steps). The convergence of parameters was tested using Tracer (v.1.6) software (Rambaut et al. 2018), using an effective sample size (ESS) of greater than 200 as an acceptance criterion. A maximum clade credibility tree was constructed using Treeannotator (v.1.8.4) (Drummond and Rambaut 2007), with burn-in of the first 10% of samples, and visualized with FigTree (v.1.4.4) (http://tree.bio.ed.ac.uk/software/figtree). The discrete phylogeography analysis was performed in SpreaD3 (v.0.9.6) software (Bielejec et al. 2016). After converting to a keyhole markup language file, we calculated Bayes factors (BFs) in SpreaD3 using a Bayesian stochastic search variable selection (BSSVS) file to obtain statistically significant migration routes. The reliability of the analysis was verified by running the BSSVS independently 3 times, and posterior probability and BF cutoffs were used to define significance.
To estimate sites under positive or negative selection pressure in the VP1 gene among all GII.2 strains and in the RdRp region of all GII.P16 strains, we calculated the rates of nonsynonymous (dN) and synonymous (dS) substitutions at every codon position with Hyphy (v2.3) (Pond et al. 2005) using a mixed effects model of evolution (MEME) and fixed effects likelihood (FEL) methods, with a P-value threshold of 0.1 (Murrell et al. 2012).
Because the phylogenetic approach only reveals the relationship between lineages and not transmission links, we used TransPhylo (Didelot et al. 2017) to reconstruct the transmission dynamics of the GII.P16-GII.2 genotype. TransPhylo, a software tool implemented as an R package, is designed to reconstruct infectious disease transmission using genomic data based on a combined model of transmission between hosts and pathogen evolution within each host (Didelot et al. 2021). Because this tool requires knowledge of Gamma distribution parameters representing the generation time (from infection to transmission), we input previous estimates of 3.35 and 1.09 for shape and scale parameters, respectively (Heijne et al. 2009). Transmission is inferred based on MCMC sampling; 500,000 iterations were performed, simultaneously yielding posterior probability, sampling proportion, within-host coalescent rate, and basic reproductive number. Before exploring inference results, we tested convergence and mixing properties based on the trace of each parameter (Supplementary Fig. 1) and ESS, computed with the CODA package. ESS values greater than 500 were accepted. A color-coded tree (Supplementary Fig. 2) containing all information about an outbreak was generated from input of a dated phylogeny, then the transmission tree was extracted from this tree. A series of transmission inference parameters were estimated based on this transmission tree using the TransPhylo method. The probability of direct transmission between cases was computed and visualized using Gephi software (v0.9.2) (Bastian 2009).
Figure 1a shows that VP1 gene sequences of GII.2 norovirus evolved at a rate of 3.32 × 10−3 [95% confidence interval (CI): 3.30 × 10−3–3.33 × 10−3] substitutions/site/year. A time-scaled phylogenetic tree based on full-length GII.2 VP1 gene sequences (Fig. 1b) indicates that there have been 3 major evolutionary clades worldwide since 2000, including 2 clades of GII.P16-GII.2 and 1 clade of GII.P2-GII.2. Of the 2 GII.P16-GII.2 norovirus clades, 1 consists mainly of isolates from Japan, whereas the other consists of isolates from China, Germany, and the United States. The most recent common ancestor (MRCA) of GII.P16-GII.2 occurred at about 2,001.03 [95% highest posterior density (HPD): 2,000.16–2,001.88]. The GII.P2-GII.2 clade consists mainly of isolates from Japan. The MRCA for GII.P16-GII.2 and GII.P2-GII.2 occurred at about 2,000.51 (95% HPD: 1,999.67–2,001.27). Figure 1c shows that GII.P16 has evolved at a rate of 9.44 × 10−3 substitutions/site/year (95% CI: 9.16 × 10−3–9.72 × 10−3). Figure 1d, which shows a time-scaled phylogenetic tree based on the RdRp region of GII.P16 norovirus, indicates that GII.P16-GII.2 isolates from China were similar to those from Germany and shared an MRCA at about 2,014.00 (95% HPD: 2,013.19–2,014.68). GII.P16-GII.2 isolates from China and Germany shared an MRCA with GII.P16-GII.4 isolates from Korea at about 2,013.28 (95% HPD: 2,012.29–2,014.06) and then shared an MRCA with GII.P16-GII.13 and GII.P16-GII.3 genotypes at about 2,007.32 (95% HPD: 2,005.5774–2,008.3171). In contrast, GII.P16-GII.2 isolates from China and Germany separated early in 1,995.48 (95% HPD: 1,982.3258–2,002.8254) from those from Japan, Korea, and Australia. Table 1 shows that 11 and 2 positive selection sites were detected by the MEME method in the GII.2 VP1 gene and GII.P16 RdRp region, respectively. The FEL method detected 3 diversifying positive and 281 purifying selection sites in the GII.2 VP1 gene and no diversifying positive and 93 purifying selection sites in the GII.P16 RdRp region. Sites 61 and 285 were detected as positively selected sites using both methods. These results suggest that, of the 2 genomic regions, sequence variations in the capsid gene (VP1) may be under greater selective pressure.
The animation shown in Supplementary Video 1 tracks the migration history of GII.P16-GII.2 norovirus between 2013 and 2018 in China and worldwide, revealing that GII.P16-GII.2 in China mainly came from Australia, the United States, Thailand, and Russia instead of neighboring Japan and South Korea. The earliest GII.P16-GII.2 norovirus strains were imported from Australia to Taiwan and from the United States to Guangdong, after which both domestic and foreign GII.P16-GII.2 norovirus disseminated throughout most areas of China. Domestically, there were roughly 3 epidemic centers that spread divergently to other parts of China: in the north, represented by Beijing; in the south, represented by Guangdong province and Shenzhen city; and in southeast coastal areas, represented by Zhejiang and Jiangsu provinces. Bayes modeling demonstrated a total of 20 well-supported dispersal routes (posterior probability >0.5), as summarized in Supplementary Table 3.
TransPhylo analyses showed that the basic reproductive number (R0), representing the number of secondary infections caused by each case, for GII.P16-GII.2 norovirus was 1.99 (95% CI: 1.58–2.44), and the sampling probability of each case was estimated to be 0.76 (95% CI: 0.63–0.88). A network analysis of transmission probability between cases, calculated using TransPhylo analysis, showed that the modularity of the network structure was 0.776 (Fig. 2a). The statistical parameters generated by Gephi, including indegree, outdegree, closeness centrality, betweenness centrality, and hub nodes, are summarized in Supplementary Table 4, which shows that the most highly exported strains were from the United States, Australia, and Germany, and the most imported strains were from Hong Kong and Shenzhen. Strains from the United States, Shenzhen, and Australia are hub nodes. As shown in Fig. 2b, the outbreak of GII.P16-GII.2 norovirus was initiated with a single index case, with infection occurring before 1995, and the large-scale transmission of GII.P16-GII.2 norovirus started in 2010. USA1, USA5, and AUS10 caused relatively large transmissions. Compared with transmission events in recent years, early years were characterized by a limited number of infected cases in which 1 case usually infected 1 or 2 other patients. In later transmission chains, unsampled cases were also observed. Figure 2c shows that most transmission occurred during high infectiousness (proportional to line intensity) and that most sampling (open red circles) occurred after transmission when infectiousness started to decline. Mean generation and sampling times, determined from the realized distributions of generation time (from infection to transmission) and sampling time (from infection to sampling) (Fig. 2, d and e), were approximately 1 and 0.76 days, respectively.
GII.P16-GII.2 norovirus was a rarely detected genotype before 2016, with only limited cases reported. However, epidemics caused by this genotype are currently increasing in China and elsewhere in the world. In this study, we performed comprehensive analyses of the molecular evolution and transmission of GII.P16-GII.2 norovirus. Our findings suggest that (1) this new variant may have evolved from GII.P16-GII.4 or previous GII.P16-GII.2 strains; (2) more positive selection sites were found in the VP1 gene of GII.2 norovirus, possibly helping the virus to evade the host immune system and persist; (3) the earliest GII.P16-GII.2 norovirus in mainland China might have been imported from Australia to Taiwan or from the United States to Guangdong; and (4) the basic reproductive number (R0) of GII.P16-GII.2 norovirus was estimated to be 1.99 (95% CI: 1.58–2.44). Norovirus shows high genetic diversity, and evolutionary and phylogenetic discrepancies between the VP1 gene and RdRp region have frequently been reported (Nagasawa et al. 2018; Ozaki et al. 2018; Matsushima et al. 2019). One study found differences in the VP1 gene and RdRp region of GII.P16-GII.2 norovirus and suggested that these 2 regions should be the focus of phylodynamic studies (Hernandez et al. 2018). Based on the GII.2 VP1 gene, we estimated that the MRCA of GII.P16-GII.2 from China appeared in strains from Japan in 2001. That this coalescence dates to an earlier time may be explained by the absence of obvious differences from previous GII.P16-GII.2 strains circulating in 2011–2012, as supported by a study from China suggesting that this was not a novel recombinant but instead evolved from these previously circulating GII.P16-GII.2 strains (Tohma et al. 2017). GII.P16-GII.2, in turn, shared a MRCA with GII.P2-GII.2 in June 2000, as supported by a study from China reporting that the first reemerging GII.P16-GII.2 strain in China shared a MRCA with GII.P2-GII.2 (Ao et al. 2018). Based on the GII.P16 RdRp region, we estimated that the MRCA of GII.P16-GII.2 from China appeared in strains from Germany in 2014 and that GII.P16-GII.2 shared a MRCA with GII.P16-GII.4 strains from South Korea in February 2013. Considering the time at which GII.P16-GII.2 norovirus first reemerged in China in 2016–2017, the downward trend in GII.4 in Asia and worldwide since 2016 (Tohma et al. 2017; Van Beek et al. 2018), and the observation of more positive selection sites in the VP1 gene, we speculate that the GII.P16-GII.2 strain likely evolved from GII.P16-GII.4. The findings of the current study are consistent with those of a study from Japan reporting that the reemergence of GII.P16-GII.2 norovirus might reflect a new recombination of GII.P16-GII.4 and the previous GII.P16-GII.2 (2010–2012 type) (Nagasawa et al. 2018). However, more positively selected sites found in VP1 rather than RdRp could also be due to a higher degree of evolutionary constraints on amino acid change, thus allowing for greater sensitivity to detect adaptive change. Meanwhile, more neutral noise in RdRp reduces the power to detect adaptive signal. We found no obvious differences in evolutionary rates of the GII.2 VP1 gene and GII.P16 RdRp region compared with those reported in previous studies. Both were estimated to be greater than 10−3 substitutions/site/year, but the GII.2 VP1 gene evolves slower than the RdRp region, reflecting the fact that the capsid protein (VP1) has a more stable structure than the RNA polymerase (RdRP) and may play an important role in the evolutionary process of the norovirus (Lindesmith et al. 2012; Lu et al. 2016). About other parts of norovirus (VP2), we found that it is a less-studied HuNoV protein (Hassard et al. 2017) and only 1 reported that it evolved at a rate of 8.99 × 10−3 (95% CI: 6.59 × 10−3–11.6 × 10−3) in GII.4 not in GII.2 (Cotten et al. 2014). Because phylogenetic analyses are unable to provide details on transmission between individuals, we applied a novel method, TransPhylo, to reconstruct transmission and extract several parameters of GII.P16-GII.2 norovirus detected in China and other countries. Two key parameters, R0 and pi, converged in the analysis, whereas neg diverged. The definition of neg is within-host coalescent rate, equaling the within-host effective population size (Ne) times generation duration g (days) divided by 365 (Didelot et al. 2021). Norovirus lives in the host for a short time, probably resulting small neg values that could not be estimated accurately. As expected, almost all unsampled cases occurred earlier, reflecting the inadequacies of surveillance and public health investigations at the time. At these earlier time points, 1 case usually infected 1 or 2 patients, whereas current cases are able to cause large outbreaks, indicating possible increased infectivity during the evolution of GII.P16-GII.2 norovirus. It is also possible that early sampling capacity was so weak that cases simply went undetected. In China, the earliest GII.P16-GII.2 norovirus might have entered through 2 routes—1 from Australia to Taiwan and the other from the United States to Guangdong—rather than from Japan and South Korea, which is geographically closer to China and where more cases were found. Phylodynamics analyses of VP1 and RdRp genomic regions showing that Chinese, Japanese, and South Korean strains were not divided into the same cluster but instead were neighboring clusters appeared to confirm this. This suggests that, because norovirus is highly contagious and can spread through contaminated water, air, food, and other items, geographic isolation is not a barrier to its spatial and temporal dispersal. Moreover, because this novel GII.P16-GII.2 norovirus spread from Guangdong to neighboring regions and northern China, including Zhejiang, Henan, Hong Kong, and Beijing, Guangdong may be the origin of reemergent GII.P16-GII.2 norovirus in China. Finally, our data suggest that GII.P16-GII.2 norovirus spread divergently from 3 epidemic centers—North (Beijing), South (Guangdong) and Southeast (Zhejiang and Jiangsu provinces)—to other parts of China. In these 3 areas, which are responsible for most exported cases, population mobility is a common feature, and Guangdong, Shenzhen, Zhejiang, and Jiangsu are coastal areas. Among them, Guangdong has been reported to be associated with the evolution and transmission of multiple genotypes of norovirus, including the first reemerging GII.P16-GII.2 norovirus (Lu et al. 2016; Ao et al. 2018). The reason why sporadic cases and community outbreaks of norovirus are common in coastal areas is that it has been often associated with the consumption of shellfish contaminated with fecal pollution (Campos and Lees 2014; Hassard et al. 2017). Overall, considering the infection risk, monitoring of shellfish safety and densely populated cities should be strengthened. There are some limitations to this study. First, the sampling scheme was based on all available complete sequences that include origin country and sampling year, considering some cases were not sampled, which may bias origin and transmission results of GII.P16-GII.2 norovirus. Second, we excluded some sequences because they affect the temporal signal, perhaps it would be better to compare results using all included samples. Last but not the least, sequence quality, algorithms, and parameter settings could also bias the results because this study is mainly based on bioinformatics analyses. Although we used the constant size coalescent model after a selection process, perhaps using an exponential population model also is appropriate considering GII.P16-GII.2 suddenly appeared and became the main genotype. Finally, we recommend that there could be a study to reconstruct GII.P16-GII.2 evolution using smaller samples like outbreak within China. Overall, while there is a risk theoretically, we have discussed and compared results in our study with published articles mentioned above, so that we believe our results were reliable.
Our results provide new insights into the early evolution and transmission of the reemerging GII.P16-GII.2 norovirus in China and worldwide. This reemerging strain may have evolved from GII.P16-GII.4 or previously circulating GII.P16-GII.2 strains, and the VP1 gene might play a key role in this evolutionary process. Guangdong province is likely the origin of this reemerging GII.P16-GII.2 norovirus in China, and coastal and densely populated areas are responsible for its successful spread.
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PMC9635640 | 36102801 | Hui Li,Elizabeth R Gavis | Drosophila FMRP controls miR-276-mediated regulation of nejire mRNA for space-filling dendrite development | 14-09-2022 | Fragile X syndrome,FMRP,nejire,dendritic arborization,Drosophila,neuron | Abstract MicroRNAs are enriched in neurons and play important roles in dendritic spine development and synaptic plasticity. MicroRNA activity is controlled by a wide range of RNA-binding proteins. FMRP, a highly conserved RNA-binding protein, has been linked to microRNA-mediated gene regulation in axonal development and dendritic spine formation. FMRP also participates in dendritic arbor morphogenesis, but whether and how microRNAs contribute to its function in this process remains to be elucidated. Here, using Drosophila larval sensory neurons, we show that a FMRP-associated microRNA, miR-276, functions in FMRP-mediated space-filling dendrite morphogenesis. Using EGFP microRNA sensors, we demonstrate that FMRP likely acts by regulating miR-276a RNA targeting rather than by modulating microRNA levels. Supporting this conclusion, miR-276a coimmunoprecipitated with FMRP and this association was dependent on the FMRP KH domains. By testing putative targets of the FMRP–miR-276a regulatory axis, we identified nejire as a FMRP-associated mRNA and, using EGFP reporters, showed that the nejire 3′ untranslated region is a target of miR-276a in vivo. Genetic analysis places nejire downstream of the FMRP–miR-276a pathway in regulating dendrite patterning. Together, our findings support a model in which FMRP facilitates miR-276a-mediated control of nejire for proper dendrite space-filling morphology and shed light on microRNA-dependent dendrite developmental pathology of fragile X syndrome. | Drosophila FMRP controls miR-276-mediated regulation of nejire mRNA for space-filling dendrite development
MicroRNAs are enriched in neurons and play important roles in dendritic spine development and synaptic plasticity. MicroRNA activity is controlled by a wide range of RNA-binding proteins. FMRP, a highly conserved RNA-binding protein, has been linked to microRNA-mediated gene regulation in axonal development and dendritic spine formation. FMRP also participates in dendritic arbor morphogenesis, but whether and how microRNAs contribute to its function in this process remains to be elucidated. Here, using Drosophila larval sensory neurons, we show that a FMRP-associated microRNA, miR-276, functions in FMRP-mediated space-filling dendrite morphogenesis. Using EGFP microRNA sensors, we demonstrate that FMRP likely acts by regulating miR-276a RNA targeting rather than by modulating microRNA levels. Supporting this conclusion, miR-276a coimmunoprecipitated with FMRP and this association was dependent on the FMRP KH domains. By testing putative targets of the FMRP–miR-276a regulatory axis, we identified nejire as a FMRP-associated mRNA and, using EGFP reporters, showed that the nejire 3′ untranslated region is a target of miR-276a in vivo. Genetic analysis places nejire downstream of the FMRP–miR-276a pathway in regulating dendrite patterning. Together, our findings support a model in which FMRP facilitates miR-276a-mediated control of nejire for proper dendrite space-filling morphology and shed light on microRNA-dependent dendrite developmental pathology of fragile X syndrome.
MicroRNAs (miRNAs) are ∼22-nt small noncoding RNAs that posttranscriptionally regulate gene expression (Iwakawa and Tomari 2015). miRNA biogenesis typically starts with synthesis of primary miRNAs (pri-miRNAs) which are processed by Drosha/DGCR8 to produce short hairpin precursor miRNAs (pre-miRNAs). Pre-miRNAs are exported from the nucleus to the cytoplasm by exportin 5, followed by cleavage by Dicer. One strand from the remaining duplex is subsequently loaded onto Argonaute (Ago), forming a miRNA-induced silencing complex (miRISC), which in turn promotes translational repression and/or degradation of target RNAs by base pairing with complementary sequences in 3′ untranslated regions (3′ UTRs) (Lai 2002; Ha and Kim 2014). miRNAs are highly expressed in the nervous system where they play important roles in neuronal development and function (Kosik 2006; Fineberg et al. 2009; Schratt 2009; McNeill and Van Vactor 2012; Rajman and Schratt 2017). They are distributed to dendrites (Sambandan et al. 2017) and contribute to spine development and synaptic plasticity by locally regulating protein synthesis (Schratt et al. 2006; Siegel et al. 2009). Dysfunction of the miRNA pathway has been linked to many neurodevelopmental disorders, such as autism spectrum disorder, Rett syndrome, fragile X syndrome (FXS), and Tourette’s syndrome (Kosik 2006; Fineberg et al. 2009; McNeill and Van Vactor 2012; Rajman and Schratt 2017). However, the regulatory roles of miRNAs in the development of complex dendritic arbors are still poorly understood. miRNA biogenesis and function are highly regulated by RNA-binding proteins (RBPs) (Ha and Kim 2014; Connerty et al. 2015). For example, TDP-43 interacts with Drosha and pri-miRNAs to facilitate pre-miRNA production (Kawahara and Mieda-Sato 2012). Xrn1, an exonuclease, regulates the turnover of mature miRNAs (Bail et al. 2010). In addition, recognition of RNA targets by miRNAs is controlled by a wide variety of RBPs (Kim et al. 2021). Pumilio (Kedde et al. 2010), IMP2 (Degrauwe et al. 2016), FUS (Zhang et al. 2018), and Dnd1 (Kedde et al. 2007) have been reported to bind to and/or change 3′ UTR RNA structures to promote or suppress miRNA targeting for translational repression. Fragile X mental retardation protein (FMRP) is a highly conserved RBP that has been implicated in miRNA-mediated gene regulation. FMRP was found to be associated with key components of the miRNA biogenesis pathway, including Ago1 and Dicer (Jin et al. 2004), and to bind to several miRNAs, such as bantam, let-7, miR-125b, miR-132, and miR-181d (Yang et al. 2009; Edbauer et al. 2010; Wang et al. 2015), suggesting that miRNA dysfunction may contribute to FXS. In neuronal development, FMRP was previously reported to regulate translation of synaptic mRNAs by interacting with individual miRNAs and promoting the formation of miRISC for proper synaptic structure and function (Edbauer et al. 2010; Muddashetty et al. 2011), to mediate axonal transport of miR-181d and local regulation of map1b and calm1 mRNAs for axonal elongation (Wang et al. 2015), and to control mature miR-124 levels during dendrite development (Xu et al. 2008). Despite the known functions of FMRP in miRNA-mediated synaptic and axonal regulation, whether and how FMRP regulates dendrite patterning through the miRNA pathway remain to be explored. In this study, using the Drosophila larval class IV da (C4da) sensory neurons as a model system, we have identified miR-276 as a regulator of dendritic arbor patterning and field coverage. We further show that miR-276 activity in C4da neuron dendrite development depends on FMRP, which most likely functions in miR-276 RNA targeting rather than by regulating mature miR-276 levels. Consistent with this, FMRP associates with mature miR-276a in a KH domain-dependent manner. By testing previously identified FMRP target transcripts that are also predicted to be miR-276a targets, we discovered potential RNA targets of the FMRP–miR-276a regulatory axis for proper C4da dendritic field coverage. Detailed analysis of one target, nejire (nej) mRNA, showed that it is enriched in FMRP immunoprecipitates from S2 cells and genetic interaction analyses placed nej downstream of FMRP-miR-276 activity. Finally, we show that miR-276 can regulate a nej 3′ UTR EGFP reporter when its target site is present. Collectively, these results uncover a mechanism by which FMRP participates in miR-276-mediated regulation of nej mRNA to ensure proper space-filling dendrite morphology.
The following transgenic stocks were used: ppk-GAL4, UAS-CD4::tdGFP (Bhogal et al. 2016); UAS-mCherry.scramble.sponge (Bloomington Stock 61501); ppk-GAL4 (Bloomington Stock 32079); UAS-mCherry.miR-276a.sponge (Bloomington Stock 61406); UAS-mCherry.miR-276b.sponge (Bloomington Stock 61407); UAS-mCherry.mir-9c.sponge (Bloomington Stock 61376); UAS-mCherry.mir-125.sponge (Bloomington Stock 61393); UAS-Fmr1.Z (Bloomington Stock 6931); UAS-fmr1-RNAi (Bloomington Stock 34944; TRiP HMS00248); UAS-Nf1-RNAi (Bloomington Stock 53322; TRiP HMC03551) (validated by Moscato et al. 2020); UAS-nej-RNAi (Bloomington Stock 37489; TRiP HMS01507) (validated by Jia et al. 2015); UAS-inaE-RNAi (Bloomington Stock 64885; TRiP HMC05758) (validated by Shieh et al. 2021); UAS-Hers-RNAi (Bloomington Stock 61858; TRiP HMJ23347); UAS-Ric-RNAi (Bloomington Stock 82973; TRiP HMC06651); UAS-Mkp3-RNAi (Bloomington Stock 57030; TRiP HMS04475); and UAS-Axn-RNAi (Bloomington Stock 62434; TRiP HMJ23888) (validated by Nye et al. 2020). ppk-GAL4 was used to drive expression of UAS transgenes specifically in C4da neurons. To enhance GAL4/UAS efficiency, the experiments using UAS-RNAi lines and UAS-mCherry-miRNA-SP lines were performed at 29°C. All other crosses were performed at 25°C.
The UAS-pre-miR-276a transgenes were generated using the same strategy as previously described (Xu et al. 2008). A 112-bp fragment containing 98 nt of the pre-miR-276a sequence was amplified from genomic DNA with the following pairs of primers: Fwd_pre-miR-276a (5′-GATCCTGAATTCTTTTTTACCTGGTTTTTGCC-3′) and Rev_pre-miR-276a (5′-GGCTATTCTAGAGCATTCACTTGGTTGTTTTTTG-3′). The PCR products and pattB-UASt vector were digested with EcoRI and XbaI and ligated together to generate pUASt-pre-miR-276a.
A fragment containing 2 copies of perfectly complementary sequence to miR-276a was generated by overlap extension PCR with the following pairs of primers: Fwd_XbaI_2x-miR-276a (5′-GCTATCTAGAAGAGCACGGTATGAAGTTCCTATAACGTTAACGTAACGTTAAAGAGCACGGTATGAAGTTCCTACTCGAGAGGATCGCGC-3′) and Rev_XhoI_2x-miR-276 (5′-GCGCGATCCTCTCGAG-3′). The PCR products and pCaSpeR4_Tub-nuc-EGFP (by courtesy of E. Lai) were digested with XbaI and XhoI then ligated together to produce pCaSpeR4_Tub-nuc-EGFP_2x-miR-276a. Tub-nuc-EGFP and Tub-nuc-EGFP_2x-miR-276a fragments were amplified from pCaSpeR4_Tub-nuc-EGFP and pCaSpeR4_Tub-nuc-EGFP_2x-miR-276a, respectively, using Fwd_EcoRI_Tub-nuc-EGFP (5′-GATCCTGAATTCGATATCAAGCTTGCACAG-3′) and Rev_BglII_Tub-nuc-EGFP (5′-GACAGTAGATCTGTCGACCTCGACATACATTG-3′) primers. The PCR products were digested with EcoRI and BglII and ligated individually into the pattB vector digested with EcoRI and BglII to produce pTub-nuc-EGFP and pTub-nuc-EGFP-2x-miR-276a.
To generate the intact reporter, a 493-bp fragment of the nej 3′ UTR was amplified from genomic DNA using Fwd_XbaI_nej-3′UTR (5′-GGCTATTCTAGAGTGCAACAAAATAGCAATAGCC-3′) and Rev_XhoI_nej-3′UTR (5′-GATCCTCTCGAGCGTTTAAGCCTAAAAGTCTATAGC-3′) primers and digested with XbaI and XhoI. The fragment was then ligated into pCaSpeR4_Tub-nuc-EGFP digested with XbaI and XhoI to produce pCaSpeR4_Tub-nuc-EGFP_nej-3′UTR. To generate the mut reporter, the miR-276a seed sequence was deleted from the nej 3′ UTR fragment by amplifying 2 regions of the nej 3′ UTR fragment using Fwd_XbaI_nej-3′UTR (5′-GGCTATTCTAGAGTGCAACAAAATAGCAATAGCC-3′) and Rev_Esp3I_nej-3′UTR_frag1 (5′-ATTACGTCTCAGCGCACAGACACACTCG-3′) and Fwd_Esp3I_nej-3′UTR_frag2 (5′-CCGTCGTCTCGGCGCATTCTTCGATTATTATACATTCATTTAATTTTCGATC-3′) and Rev_XhoI_nej-3′UTR (5′-GATCCTCTCGAGCGTTTAAGCCTAAAAGTCTATAGC-3′) primers. Two PCR products were digested with indicated enzymes and then ligated into pCaSpeR4_Tub-nuc-EGFP digested with XbaI and XhoI to produce pCaSpeR4_Tub-nuc-EGFP_nej-3′UTR-mut. Tub-nuc-EGFP_nej-3′UTR and Tub-nuc-EGFP_nej-3′UTR-mut were amplified from pCaSpeR4 plasmids using Fwd_EcoRI_Tub-nuc-EGFP (5′-GATCCTGAATTCGATATCAAGCTTGCACAG-3′) and Rev_BglII_Tub-nuc-EGFP (5′-GACAGTAGATCTGTCGACCTCGACATACATTG-3′) primers and digested with EcoRI and BglII for insertion into the pattB vector to produce pTub-nuc-EGFP_nej-3′UTR and pTub-nuc-EGFP_nej-3′UTR-mut.
Drosophila S2 cell culture, transfection, and RNA immunoprecipitation were conducted as previously described (Li and Gavis 2022). FMRP variants were detected by immunoblotting with 1:2,000 DYKDDDDK tag monoclonal antibody (Invitrogen, Cat # MA1-91878) and 1:2,000 HRP sheep anti-mouse antibody (VWR, Cat # 95017-332). Ten nanograms of total RNA was reverse transcribed using the TaqMan MicroRNA Reverse Transcription Kit (Applied Biosystems, Cat # 4366596). Real-time PCR was then performed with TaqMan Fast Advanced Master Mix (Applied Biosystems, Cat # 4444556) and dme-miR-276a TaqMan miRNA Assays (Applied Biosystems, Cat # 4440886). Poly(A) mRNA was reverse transcribed using SuperScript III First-Strand Synthesis System (Invitrogen, Cat # 18080051) and real-time PCR analysis was performed with SYBR Green PCR Master Mix (Thermo Fisher, Cat # 4309155). For −RT controls, nuclease-free water was added instead of reverse transcriptase. rp49 was used as endogenous control for real-time PCR. The primers listed below were used: chic_Fwd (5′-TGCACTGCATGAAGACAACA-3′) and chic_Rev (5′-GTTTCTCTACCACGGAAGCG-3′); nej_Fwd (5′-GTGGGCACTCAGATGGGTATG-3′) and nej_Rev (5′-CATGCCTGGTATGGCGTTCA-3′); nf1_Fwd (5′-CTTTTGGCACGTTTCGAGGAT-3′) and nf1_Rev (5′-GGTAGCGCGATATGTGGATCA-3′); 14-3-3ζ_Fwd (5′-CGACAGTCGATAAGGAAGAGC-3′) and 14-3-3ζ_Rev (5′-TCTCTGTGACGGACTTCATGG-3′); and rp49_Fwd (5′-CGGATCGATATGCTAAGCTGT-3′) and rp49_Rev (5′-GCGCTTGTTCGATCCGTA-3′).
Late 3rd instar larva preparation and staining were performed as previously described (Bhogal et al. 2016). For better immunostaining efficiency, the larval body wall muscles were removed as described (Tenenbaum and Gavis 2016). FMRP expression was detected with anti-FMRP monoclonal antibody (1:100, Abcam, ab10299) and AlexaFluor 568 goat anti-mouse (1:500, Life technologies, A-11004) secondary antibody. Neuronal membranes were visualized using Alexa 568-conjugated anti-HRP (1:200, Jackson, 123-585-021) and Alexa 647-conjugated anti-HRP (1:200, Jackson, 123-605-021). All antibodies were incubated in blocking buffer containing PBS/0.3% TritonX-100 with 5% normal goat serum either overnight at 4°C (primary antibodies) or for 1.5 h at room temperature (secondary antibodies). Larvae fillets were mounted between a coverslip and slide with VECTASHIELD Antifade Mounting Medium (Vector Laboratory, H-1000-10) and were imaged using a Leica SP5 laser scanning confocal microscope with a 63×/1.4 NA oil objective and sequential scanning. All images are confocal z series projections. Relative nuclear EGFP intensity values were measured by ROI with IntDen function in ImageJ software (https://imagej.nih.gov/ij/) and background was subtracted.
C4da (ddaC) neurons from live larvae were mounted individually in 80% glycerol between a slide and a coverslip and imaged by a Leica SP5 laser scanning confocal microscope (40×/1.25 NA oil objective). All images are confocal z series projections. For consistency, class IV ddaC neurons from abdominal segments A3–A5 were imaged. At least 10 neurons from 5 or more larvae were imaged and analyzed for each genotype. Quantitative analysis of dendrite morphology was performed with ImageJ software. The dendritic arbor field coverage was quantified by overlaying a grid of 20 × 20 squares on the image of interest and counting the number of empty squares. The dendritic field coverage ratio = # empty squares/400.
All data were analyzed and plotted using GraphPad Prism 9 (https://www.graphpad.com/). Comparisons between 2 groups were performed with the unpaired Student’s t-test. For 3 or more groups, 1-way ANOVA with Dunnett’s or Tukey’s multiple comparisons test was used. Values are mean ± SD; ns, not significant; *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.
Several miRNAs identified in FMRP immunoprecipitates from wild-type Drosophila ovaries, including miR-9c, miR-125, and miR-276a (Yang et al. 2009), have been implicated in the regulation of mammalian dendritic growth and spine formation (Edbauer et al. 2010; Giusti et al. 2014) and Drosophila olfactory memory formation (Li et al. 2013). To determine if these potential FMRP-associated miRNAs play a role in dendrite morphogenesis in Drosophila, we disrupted their activity in C4da neurons using miRNA sponges, which act as competitive inhibitors by sequestering miRNAs (Fulga et al. 2015). Each miRNA sponge was expressed selectively in C4da neurons using ppk-GAL4 (Fig. 1, a′–e′). As compared to the control scrambled sequence sponge (Fig. 1a), expression of the miR-276a and miR-276b sponges resulted in increased coverage of the dendritic field (Fig. 1, b–e and l), suggesting that miR-276a and/or miR-276b is required to limit the space-filling behavior of C4da dendritic arbors. Notably, this phenotype resembles loss of FMRP (Li and Gavis 2022) (also see Fig. 4, a, i, and n).
FMRP has been previously shown to regulate C4da dendrite arborization (Lee et al. 2003; Li and Gavis 2022) and to associate with miR-276a in the Drosophila ovary (Yang et al. 2009). miR-276a and miR-276b, which differ by a single nucleotide at position 10, are members of the Drosophila dme-miR-276 family (Supplementary Fig. 1, a and b). We therefore asked if miR-276 is involved in FMRP-mediated dendritic regulation. Overexpression of fmr1, which encodes FMRP, in C4da neurons (fmr1OE) results in a sparse dendritic arbor and dramatically reduced field coverage (Li and Gavis 2022). Expression of either the miR-276a or miR-276b sponge, but not the scrambled sponge, in fmr1OE neurons partially rescued this dendritic field coverage defect (Fig. 1, f–h and l). Together, these results support the idea that FMRP function in space-filling dendrite morphogenesis is mediated in part by its interaction with miR-276. The single nucleotide difference between miR-276a and miR-276b falls within the bulge that forms between each miRNA sponge and the bound miRNA (position 9-11, Supplementary Fig. 1c). Thus, miR-276a and miR-276b should each be sequestered by both the miR-276a and miR-276b sponges. miR-276a has functions in regulation of olfactory memory formation in mushroom body neurons (Li et al. 2013) and circadian rhythms in the central nervous system (Chen and Rosbash 2016). Because of its known activity in the nervous system, we focused on miR-276a in subsequent experiments, although we cannot rule out the possibility that miR-276a and miR-276b function redundantly in C4da dendrite regulation.
We next sought to determine how FMRP and miR-276a are mechanistically linked in regulating dendritic patterning. One possibility is that FMRP functions in miRNA maturation and/or stability to regulate steady-state levels of miR-276a (Fig. 2a). Alternatively, FMRP might function in miR-276a RNA targeting (Fig. 2b). To test the first possibility, we generated transgenic flies ubiquitously expressing nuclear EGFP sensors to monitor relative miR-276a levels in vivo (Fig. 2, c and d). EGFP sensors fused to the SV40 3′ UTR, with or without 2 copies of perfectly complementary sequences to miR-276a, were expressed in wild-type larvae or in larvae with C4da neuron-specific fmr1 RNAi or overexpression. If miR-276a levels are regulated by FMRP, EGFP expression should depend on the level of FMRP. With the control sensor lacking miR-276a complementary sequences, EGFP was detected in the nuclei of epidermal cells and all 4 classes of da neurons (Fig. 2e). By contrast, EGFP expression was dramatically reduced throughout larvae expressing the miR-276a sensor (Fig. 2f), indicating ubiquitous expression of miR-276a at the late 3rd instar larval stage. Expression of the miR-276a sponge, but not the scrambled sponge, in C4da neurons together with the miR-276a sensor significantly restored EGFP expression in the neurons (Supplementary Fig. 2), confirming the effectiveness of both the miR-276a sponge and the miR-276a EGFP sensor. More importantly, decreasing or increasing FMRP levels, as confirmed by anti-FMRP immunofluorescence, did not affect EGFP expression in C4da neurons (Fig. 2, g–h, g′–h′, and g″–h″), indicating that FMRP does not regulate levels of miR-276a.
Since FMRP does not control miR-276a levels, we asked whether it might instead function in miR-276a target RNA interaction. We first tested whether FMRP interacts with miR-276a by RNA coimmunoprecipitation. Flag-tagged FMRP (FMRP-3xFlag; Fig. 3a) was expressed in Drosophila S2 cells and immunoprecipitated with anti-DYKDDDDK antibody (Fig. 3b). RT-qPCR analysis of RNA extracted from the immunoprecipitates showed that amount of mature miR-276a was similar in S2 cells with or without induction of FMRP-3xFlag expression (Fig. 3c), which is consistent with results from the EGFP sensor experiments showing that miR-276a levels were unaffected by overexpression of FMRP in C4da neurons (Fig. 2, f and h). Mature miR-276a was enriched 2-fold in the FMRP-3xFlag immunoprecipitate compared to the control (Fig. 3d), indicating that FMRP associates with miR-276a in vivo, either directly or indirectly (see Discussion). FMRP has 3 RNA-binding domains (RBDs)—KH1, KH2, and RGG. The KH domains were previously shown to facilitate miRNA: mRNA complex formation in vitro (Plante et al. 2006). To assess the involvement of FMRP’s RBDs in its association with miR-276a, we generated a set of constructs to express Flag-tagged FMRP variants with individual RBD deleted in S2 cells (Fig. 3a). Following immunoprecipitation of the FMRP variants (Fig. 3b), the amount of coimmunoprecipitated miR-276a was quantified by RT-qPCR. Deletion of either the KH1 or the KH2 domain resulted in the loss of miR-276a enrichment in FMRP immunoprecipitates (Fig. 3d), indicating that both KH domains are indispensable for FMRP to bind to miR-276a.
To identify potential targets of the FMRP–miR-276a regulatory axis, we focused on the overlap between previously identified FMRP targets (Darnell et al. 2011; Ascano et al. 2012; Maurin et al. 2018) and predicted miR-276 targets (TargetScan 7.2), which includes nej, Neurofibromin 1 (Nf1), Mitogen-activated protein kinase phosphatase 3 (Mkp3), Ras-related protein interacting with calmodulin (Ric), inactivation no afterpotential E (inaE), Histone gene-specific Epigenetic Repressor in late S phase (Hers), and Axin (Axn). To determine if these putative targets function in C4da dendrite arborization, we specifically knocked them down in C4da neurons using RNAi driven by ppk-GAL4. miRNAs typically downregulate their targets by promoting mRNA degradation and/or translational repression (Iwakawa and Tomari 2015). However, because miRNAs often have only modest regulatory effects, we expected that depleting targets of FMRP–miR-276a regulatory pathway would at least partially mimic fmr1 overexpression in C4da neurons. Knockdown of Nf1, inaE, Hers, and Axn had no obvious phenotypic consequences (Fig. 4, a, d–g, and n). Ric and Mkp3 RNAi each resulted in increased dendritic field coverage, which resembles fmr1 knockdown rather than overexpression (Fig. 4, a–c, i, and n). On the contrary, nej RNAi led to a dramatic decrease in the dendritic coverage ratio, to an extent comparable to fmr1OE neurons (Fig. 4, h, l, and n). nej encodes Drosophila CREB-binding protein, which acts as a transcriptional coactivator and acetylates histones to regulate gene expression (Akimaru et al. 1997; Das et al. 2009). Overexpression of precursor miR-276a (pre-miR-276a) in C4da neurons resulted in similar dendritic coverage defects (Fig. 4, k–m and o), suggesting that nej might act in C4da dendritic morphogenesis through the FMRP–miR-276a regulatory pathway.
We further tested if nej is controlled by FMRP using genetic and biochemical analyses. Double RNAi of fmr1 and nej rescued both the increased dendritic field coverage caused by fmr1 RNAi and the decreased dendritic field coverage caused by nej RNAi (Fig. 4, h–j and n), suggesting that nej genetically interacts with fmr1 in regulating C4da space-filling dendrite arborization. To determine if nej physically interacts with FMRP, we performed RT-PCR for RNAs that coimmunoprecipitated with FMRP-Flag from S2 cells. nej, as well as other 2 putative RNA targets, Nf1 and 14-3-3ζ, coimmunoprecipitated with FMRP (Fig. 4p). Along with the phenotypic analysis above, our results suggest that nej mRNA is a target of FMRP in C4da dendrite patterning.
nej has a predicted miR-276a recognition element in its 3′ UTR (positions 1,567–1,573; TargetScan7.2) (Fig. 5a). To confirm that nej 3′ UTR is a target of miR-276a in C4da neurons, we generated a ubiquitously expressed nuclear EGFP reporter with a 493-bp fragment from the nej 3′ UTR containing the predicted miR-276a recognition site (intact reporter) and a corresponding reporter with the recognition site deleted (mut reporter) (Fig. 5b) inserted in the SV40 3′ UTR. EGFP expression from the mut reporter was significantly increased relative to that from the intact reporter (Fig. 5, c–e), consistent with the idea that the reporter expression is regulated by miR-276a. However, EGFP expression from the mut reporter was much lower than that of the EGFP control sensor with only the SV40 3′ UTR (Fig. 2, a and c), indicating that other factors may target this 493-bp region to control nej expression. As a further test that nej acts downstream of miR-276a, we knocked down nej by RNAi in C4da neurons expressing either of the miR-276 sponges. The increase in dendritic field coverage observed for the sponges was rescued by nej RNAi, consistent with a role for miR-276 in downregulating nej (Fig. 1, i–l). In sum, our results provide evidence that regulation of nej by binding of miR-276 to its 3′ UTR is necessary for proper C4da dendritic field coverage.
miRNAs contribute to the regulation of synaptic structure and axon elongation by FMRP, a highly conserved RBP that functions in different aspects of neuronal development (Edbauer et al. 2010; Muddashetty et al. 2011; Wang et al. 2015). However, their roles in FMRP-mediated dendrite patterning remained unclear. Here, we uncover a role for a FMRP-associated miRNA, miR-276, in FMRP-dependent regulation of space-filling dendrite morphology. FMRP has been shown to regulate the steady-state levels of miRNAs including miR-124 in Drosophila larvae (Xu et al. 2008). However, our results are most consistent with a role for FMRP in regulating RNA targeting by miR-276 rather than mature miR-276a levels, indicating multiple regulatory roles of FMRP in miRNA-mediated gene expression control of neuronal development. Given the wide range of RNAs identified as FMRP targets (Darnell et al. 2011; Ascano et al. 2012; Maurin et al. 2018), association with miR-276a might contribute to FMRP’s target selectivity and/or provide yet another point of control in addition to translation initiation (Napoli et al. 2008) and elongation (Darnell et al. 2011; Chen et al. 2014). Our findings support a model in which FMRP facilitates miR-276a targeting of nej mRNA for posttranscriptional regulation. nej-associated FMRP may help recruit miRISC to the transcript for regulation of gene expression. Deletion of the KH domains significantly alleviated FMRP-mediated enhancement of miRNA:mRNA complex formation in vitro (Plante et al. 2006). Consistent with this, mature miR-276a coimmunoprecipitated with FMRP from Drosophila S2 cells and this interaction was dependent on both the KH1 and KH2 domains, suggesting that FMRP may interact directly with miR-276a or the miR-276a-nej complex through the KH domains. Alternatively, since FMRP was found to coimmunoprecipitate with Ago (Jin et al. 2004), it might function in miR-276a targeting via KH-dependent interactions with protein components of miRISC. Lastly, FMRP could indirectly facilitate miR-276a binding to target mRNAs. For example, FMRP might function together with other proteins, such as the RNA helicase MOV10, to help unwind RNA secondary structure and expose miRNA-recognition elements for miRISC targeting (Kenny et al. 2014). In addition to their synergistic effects, miR-276a and FMRP might independently contribute to the regulation of dendrite space-filling morphogenesis. A previous study demonstrated that phosphorylation of FMRP inhibits miR-125a-mediated translational regulation of PSD-95 mRNA (Muddashetty et al. 2011); therefore, as an added layer of complexity, posttranslational modifications could affect FMRP’s functions in miRNA-mediated gene regulation. Since individual miRNAs often have modest regulatory effects on their targets (Baek et al. 2008; Selbach et al. 2008), and removal of the miR-276a-recognition element from the nej 3′ UTR did not completely restore expression of the EGFP reporter, it is likely that other factors also contribute to regulation of nej expression. For example, the 493-bp nej 3′ UTR fragment used in our analysis contains a predicted recognition element for dme-miR-2/5/6/11/13/308 (positions 1,651–1,657; TargetScan 7.2), indicating an involvement of these miRNAs in nej 3′ UTR regulation. Moreover, given the critical roles of RBPs in RNA posttranscriptional regulation (Glisovic et al. 2008), they are also likely to contribute to 3′ UTR-mediated regulation of nej. Interestingly, knockdown of 2 FMRP target RNAs—Ric and Mkp3—that are also predicted to be miR-276a targets led to increased dendritic field coverage, similar to that of fmr1 knockdown neurons. This effect could be explained by an overlap in the binding sites for FMRP and miR-276a, resulting in competition between FMRP and miRISC for the target RNAs (Kim et al. 2021). Thus, FMRP might function to prevent Ric and Mkp3 mRNAs from being targeted by miR-276a-induced silencing complex, thereby upregulating their expression. Whether these RNAs are controlled by FMRP and/or miR-276a or act independently in dendrite morphogenesis warrants further study.
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PMC9635648 | 36047852 | Kevin Batcher,Scarlett Varney,Verena K Affolter,Steven G Friedenberg,Danika Bannasch | An SNN retrocopy insertion upstream of GPR22 is associated with dark red coat color in Poodles | 01-09-2022 | pheomelanin,coat color,canine,retrogene,GWAS,inherited,dog | Abstract Pigment production and distribution is controlled through multiple genes, resulting in a wide range of coat color phenotypes in dogs. Dogs that produce only the pheomelanin pigment vary in intensity from white to deep red. The Poodle breed has a wide range of officially recognized coat colors, including the pheomelanin-based white, cream, apricot, and red coat colors, which are not fully explained by the previously identified genetic variants involved in pigment intensity. Here, a genome-wide association study for pheomelanin intensity was performed in Poodles which identified an association on canine chromosome 18. Whole-genome sequencing data revealed an SNN retrocopy insertion (SNNL1) in apricot and red Poodles within the associated region on chromosome 18. While equal numbers of melanocytes were observed in all Poodle skin hair bulbs, higher melanin content was observed in the darker Poodles. Several genes involved in melanogenesis were also identified as highly overexpressed in red Poodle skin. The most differentially expressed gene however was GPR22, which was highly expressed in red Poodle skin while unexpressed in white Poodle skin (log2 fold change in expression 6.1, P < 0.001). GPR22 is an orphan G-protein-coupled receptor normally expressed exclusively in the brain and heart. The SNNL1 retrocopy inserted 2.8 kb upstream of GPR22 and is likely disrupting regulation of the gene, resulting in atypical expression in the skin. Thus, we identify the SNNL1 insertion as a candidate variant for the CFA18 pheomelanin intensity locus in red Poodles. | An SNN retrocopy insertion upstream of GPR22 is associated with dark red coat color in Poodles
Pigment production and distribution is controlled through multiple genes, resulting in a wide range of coat color phenotypes in dogs. Dogs that produce only the pheomelanin pigment vary in intensity from white to deep red. The Poodle breed has a wide range of officially recognized coat colors, including the pheomelanin-based white, cream, apricot, and red coat colors, which are not fully explained by the previously identified genetic variants involved in pigment intensity. Here, a genome-wide association study for pheomelanin intensity was performed in Poodles which identified an association on canine chromosome 18. Whole-genome sequencing data revealed an SNN retrocopy insertion (SNNL1) in apricot and red Poodles within the associated region on chromosome 18. While equal numbers of melanocytes were observed in all Poodle skin hair bulbs, higher melanin content was observed in the darker Poodles. Several genes involved in melanogenesis were also identified as highly overexpressed in red Poodle skin. The most differentially expressed gene however was GPR22, which was highly expressed in red Poodle skin while unexpressed in white Poodle skin (log2 fold change in expression 6.1, P < 0.001). GPR22 is an orphan G-protein-coupled receptor normally expressed exclusively in the brain and heart. The SNNL1 retrocopy inserted 2.8 kb upstream of GPR22 and is likely disrupting regulation of the gene, resulting in atypical expression in the skin. Thus, we identify the SNNL1 insertion as a candidate variant for the CFA18 pheomelanin intensity locus in red Poodles.
In dogs, as with other mammals, coat color patterns are the result of varied production of the yellow-red pigment, pheomelanin, and the black pigment, eumelanin. While most dogs produce a mixture of both pigments, loss of function mutations in the pigment-type switching genes melanocortin 1 receptor (MC1R) and agouti signaling protein result in production of only 1 pigment type (Newton et al. 2000; Kerns et al. 2004; Berryere et al. 2005). Among pheomelanin-based dogs, pigment intensity can vary greatly within and between breeds, from white to deep red (Sponenberg and Rothschild 2001). Multiple genetic variants that modify pheomelanin pigment intensity have been identified in dogs, highlighting the complex, multigenic nature of coat color phenotypes. A missense variant in the MFSD12 gene and a copy number variant near KITLG have both been associated with pheomelanin intensity in a variety of breeds (Hédan et al. 2019; Weich et al. 2020). An across breed analysis of pheomelanin intensity that was published while the current study was being performed identified that genetic variants at 5 loci explained 70% of pheomelanin intensity in dogs, which included the variants at MFSD12 and KITLG as well as 3 novel loci on canine chromosomes (CFA) 2, CFA18, and CFA21 (Slavney et al. 2021). However, it is unclear how much each of the loci contribute to pheomelanin intensity within individual breeds. The Poodle breed has 3 size varieties (toy, miniature, and standard) and 11 coat colors that are officially recognized by the American Kennel Club, 4 of which are pheomelanin-based: white, cream, apricot, and red (www.akc.org). While the MFSD12 dilution variant was present in the white Poodles, it alone does not explain the range of pheomelanin intensity between the cream, apricot, and red Poodles (Hédan et al. 2019). Additionally, the copy number variant near KITLG, which was associated with pigment intensity in the pheomelanin-based Nova Scotia Duck Tolling Retrievers and the eumelanin-based silver and black Poodles, was not found to be associated with pigment intensity between the pheomelanin-based white and red Poodles, indicating that additional genetic factors affecting pheomelanin intensity exist within the Poodle breed (Weich et al. 2020). In this study, the genetics of pheomelanin intensity was analyzed within a single breed, the Poodle. A quantitative genome-wide association study (GWAS) was performed and a single associated locus on CFA18 was identified. An SNN gene retrocopy insertion was then identified as the most likely causative variant behind pheomelanin intensity in Poodles.
Collection of all Poodle samples (N = 225) was approved by the University of California, Davis Animal Care and Use Committee (protocol #18561). Breed, date of birth, sex, weight, and color were reported by the owner. Owners provided whole blood or buccal swabs from their privately owned dogs (58 white, 17 cream, 3 apricot, 6 red) in collaboration with the Poodle Club of America Foundation (grant #A182159001), and DNA was extracted using a Gentra Puregene DNA extraction kit (Qiagen, Valencia, CA, USA). Additional Poodle DNA samples from the Bannasch lab DNA repository at UC Davis were also included in the study (67 white, 9 cream, 23 apricot, 42 red). RNA from 8 red and 9 white Poodles was extracted from neonatal canine dewclaw samples using an RNeasy Fibrous Tissue Mini Kit (Qiagen, Valencia, CA, USA).
In order to perform a quantitative GWAS, Poodles were designated from 1 to 4 based on owner described coat color (also the AKC registered coat color), with white as “1,” cream as “2,” apricot as “3” and red as “4.” Genome-wide SNV genotyping was performed on the Illumina Canine HD BeadChip array. All dogs were confirmed homozygous for the recessive yellow “e” allele at MC1R with the exception of 2 cream Poodles which were heterozygous and thus excluded from further analysis (Newton et al. 2000). Variants with a minor allele frequency of less than 5% or less than 90% total genotyping rate were excluded using PLINK, resulting in 163,753 total variants (Purcell et al. 2007). The Bonferroni-corrected genome-wide significance threshold was set at P = 3.05 × 10−7. A multidimensional scaling plot showed that standard Poodles clustered separately from the toy and miniature Poodles, highlighting population stratification in the dataset (Supplementary Fig. 1). To control for this, the GWAS was performed using a univariate mixed model with a standardized relatedness matrix in GEMMA v.0.97 (Zhou and Stephens 2012). A similar GWAS using only the miniature and toy Poodles (N = 57) was also performed using GEMMA.
Whole-genome sequencing (WGS) data from standard Poodles (7 white, 1 apricot, and 1 red) were aligned to UU_Cfam_GSD_1.0 (Wang et al. 2021) using BWA v0.717 and converted to BAM files using samtools v1.14, both with default parameter settings (Li 2009). Variant calling across the critical interval was performed using bcftools mpileup (Danecek et al. 2021). Based on the GWAS results, the assumed inheritance pattern was alternate homozygotes for the red and white Poodles and heterozygous for the apricot Poodle. The WGS samples were confirmed to match this pattern at the top 4 GWAS SNV. Variants were tested for function using the Ensembl variant effect predictor (McLaren et al. 2016) with the UU_Cfam_GSD_1.0 annotation. Missense variants were tested for function using SIFT and Polyphen-2 (Ng and Henikoff 2003; Adzhubei et al. 2010). The region was also analyzed for structural variants through visual analysis of the alignment files of a single red Poodle in comparison to a single white Poodle using Integrative Genomics Viewer (IGV) (Robinson et al. 2011). To maintain consistency with the variants reported from the GWAS, all genomic locations were reported as their location in the CanFam3.1 reference.
A 3 primer PCR assay was developed for genotyping SNNL1, with a forward and reverse primer flanking the insertion site and 1 primer internal to the retrocopy. Internal primers were then used for sequencing the entire retrocopy. Primers were also designed for genotyping the SLC26A4 chr18:12,910,382 C/T variant. All primers were developed using Primer3 software (Untergasser et al. 2012). The primers used in this study are available in Supplementary Table 1. Sanger sequencing was performed on an Applied Biosystems 3500 Genetic Analyzer using a Big Dye Terminator Sequencing Kit (Life Technologies, Burlington, ON, Canada). Additional genotyping of SNNL1 from WGS data was performed visually in IGV. Samples which had no reads crossing either of the breakends at the insertion site were considered homozygous for the retrocopy insertion.
Submitted skin samples from the dewclaws of a white Poodle (SNNL1 0 copies), a cream Poodle (SNNL1 1 copy), and a red Poodle (SNNL1 2 copies) were used for histopathological analysis. The samples were fixed in 4% buffered formalin, bisected and embedded in paraffin. Five-micron paraffin sections were used for both histopathology and immunohistochemistry. Presence of melanin granules within matrical cells of the hair follicles as well as within hair shafts was assessed by Fontana-Masson’s stain. Anti-Sox10 antibody (mouse monoclonal, Abcam Ref. ab212843), which recognizes cells of neural crest origin, was used to identify melanocytes among the matrical cells within hair bulbs of anagen hair follicles, which are actively forming a new hair shaft. For immunohistochemistry, sections were deparaffinized (xylene: 10 min 2×, followed by 100% ethanol: 1 min 3×, 95% ethanol: 1 min and 70% ethanol: 1 min), followed by quenching of endogenous peroxidase (500 µl 10% sodium azide; 500 µl 30% hydrogen peroxide in 50 ml PBS; 25 min at room temperature) and 3 rinses in PBS. Antigen retrieval was performed by immersing slides in preheated antigen retrieval solution (1× Dako Target Retrieval Solution; stock solution S1699, pH6 at 95–100°C; 5 min). Slides were then cooled down to room temperature and washed 3 times in PBS. After exposing slides to 10% horse serum in PBS (15 min), the anti-Sox10 antibody (mouse monoclonal, Abcam Ref. ab212843) was applied at a 1:100 dilution for 1 h. After 3 rinses in PBS, the following steps were performed: (1) application of ImmPRESS HRP Horse Anti-Mouse IgG Polymer Reagent (Vector Cat. #MP-7402; 30 min), (2) thorough PBS rinses, and (3) addition of substrate (Vector, SK-4800). Development was monitored microscopically and reaction was stopped by immersing the slides in Milli-Q/distilled water. Counterstain in Gill’s hematoxylin #2 (RICCA, 3536-16; 15–30 s) was stopped by washing slides in running tap water. Slides were then cover slipped using Shandon-Mount media (Thermo Scientific, 1900331).
Poly(A) capture RNAseq Library preparation and NovaSeq S4 Illumina paired end sequencing were performed in 3 red and 1 white Poodle at the UC Davis Genome Center. RNAseq data were aligned to UU_Cfam_GSD_1.0 (Wang et al. 2021) using minimap v2.21 (Li 2018). Alignment files were analyzed for evidence of chimeric transcripts using IGV. Batch 3′ TagSeq library preparation and HiSeq 4000 Illumina single-end sequencing were performed on 8 red and 9 white Poodles at the UC Davis Genome Center. TaqSeq generates a single initial library molecule per transcript which is ideal for differential gene expression analysis (Meyer et al. 2011). Unique molecular identifiers were removed from the TagSeq data using UMI-tools (Smith et al. 2017), and reads were also trimmed to remove Illumina adaptors and polyA read through using bbduk (Bushnell 2014). Reads were aligned to UU_Cfam_GSD_1.0 (Wang et al. 2021) using STAR v2.7.9a (Dobin et al. 2013). The UU_GSD_1.0 annotation was used to perform gene counting with htseq-count (Anders et al. 2015). Genes with overlapping 3′ UTR in the annotation resulted in reads not being counted due to ambiguity; therefore, the “–nonunique all” option was used, which counts ambiguous reads to all overlapping features. Differential gene expression was performed using Limma-Voom (Law et al. 2014) and is reported as log2 fold change (FC) increase in expression in the red Poodles, where a negative FC indicates higher expression in white Poodles. Genes which had fewer than 5 normalized read counts across all samples were filtered. Different individuals were used in the RNAseq and TagSeq analyses than those used for variant discovery in the WGS analysis.
To identify regions of the genome associated with pheomelanin intensity specific to Poodles, a quantitative GWAS was performed in white (N = 51), cream (N = 5), apricot (N = 15), and red (N = 8) Poodles (Fig. 1a). A single locus on chromosome 18 reached genome-wide significance (Fig. 1b). A Q-Q plot of expected and observed chi-squared values indicated that population stratification was successfully controlled for (λ = 1.014; Supplementary Fig. 2). The top 4 associated variants, shown in Table 1, were in near perfect linkage disequilibrium (LD). Analysis of LD between the top associated SNV (chr18:16,968,786) and nearby variants revealed a large region of LD in Poodles (Fig. 1c). To determine if population structure within the dataset was affecting the association, a separate GWAS using only the miniature and toy Poodles (N = 57) was performed, which identified the same top 4 SNVs and confirmed the CFA18 association with pheomelanin intensity (Supplementary Fig. 3).
Variants within the interval of CanFam3.1 chr18:10–20 mb were analyzed from WGS data in 9 standard Poodles (7 white, 1 apricot, 1 red). Out of 47,095 total variants identified, 5,603 segregated by phenotype, including 4,834 SNV and 762 short indels. Variant effect prediction identified missense variants in 5 genes (Table 2), including a previously reported variant in the SLC26A4 gene (chr18:12,910,382 T > C) that was associated with pheomelanin intensity across breeds (Slavney et al. 2021). While the 2 missense variants in ARMC10 and GSAP were predicted to be deleterious by both SIFT and Polyphen-2, none of the missense variants affected genes known to be involved in any pigment pathways. Therefore, visual analysis of the aligned sequence data was also performed to identify larger structural variants. A cluster of discordant reads was observed in the red and apricot Poodles at approximately chr18:13,134,000–13,134,500 which mapped to the Stannin (SNN) gene locus (chr6:31,137,750–31,147,848), highlighting a putative retrocopy insertion (Fig. 2a).
The putative SNN retrocopy was investigated using primers flanking the insertion site to PCR amplify the region in a red Poodle. Sanger sequencing confirmed the insertion as a full-length SNN retrocopy (Supplementary File 1), referred to here as SNNL1. SNNL1 is inserted within the intron of COG5 and 2.8 kb upstream of and in the same orientation as GPR22. The SNNL1 retrocopy sequence contains 2 SNV in the 3′ UTR (chr6:31,139,403 C > A and chr6:31,140,045 G > A), but is otherwise identical to the parent gene sequence. SNNL1 has a 3′ poly (A) tail approximately 27 bp in length, and a 17-bp target site duplication (TGTGAAATACTGAAGTT) was also observed flanking the insertion, putting the exact insertion location at chr18:13,134,248–13,134,264. The syntenic region in humans for SNNL1 was viewed to determine its location relative to regulatory elements. SNNL1 inserted 2.8 kb upstream of GPR22, nearby multiple predicted GPR22 enhancers (Fig. 2b).
A 3 primer PCR genotyping assay was developed for SNNL1 (Fig. 2c). The retrocopy was then genotyped in a larger dataset of white, cream, apricot, and red Poodles to test the association with coat color (N = 224). SNNL1 copy number was highly predictive of red coat color in the breed (adjusted R2 = 0.840, P = 2.17 × 10−90) (Table 3). All (N = 125) white Poodles had 0 copies of SNNL1, and all red Poodles (N = 48) had at least 1 copy of SNNL1, with 38/48 of them having 2 copies. Most (19/25) apricot Poodles had 1 copy of SNNL1. The allele frequencies were 0.096 in cream, 0.500 in apricot, and 0.896 in red Poodles, indicating an additive effect on pheomelanin intensity. The nearby missense variant in SLC26A4 (chr18:12,910,382 T > C) was also genotyped in the same set of dogs to access LD in the region, and the “C” allele and the SNNL1 insertion were found to be in complete LD in the white, apricot, and red Poodles, however, 1 cream Poodle was identified with 0 copies of SNNL1 that was heterozygous for the SLC26A4 variant. The linkage between the chr18:12,910,382 T > C variant and SNNL1 was further assessed in a publicly available WGS dataset (Plassais et al. 2019). The “C” allele was observed in Tibetan Mastiffs, Chow Chows, village dogs, and a Xoloitzcuintli, Qingchuan, and Chongqing dog (Supplementary Table 2). SNNL1 was also genotyped in these same breeds through visual analysis of the aligned WGS data, and while SNNL1 was in strong LD with chr18:12,910,382 T > C, 8 village dogs and 1 Tibetan Mastiff were identified that have the SNV but do not appear to have SNNL1, indicating that linkage between the 2 is incomplete (Supplementary Table 2). Although Tibetan Mastiffs, Chow Chows, Xoloitzcuintli, Qingchuan, and Chongqing dogs all have deep red pheomelanin segregating within the breeds, we did not have access to phenotype data for the dogs from the WGS to confirm any associations.
Histopathological analysis was performed in skin tissue from a white (0 copies SNNL1), cream (1 copy SNNL1), and red (2 copies SNNL1) Poodles. Expression of Sox10, identifying melanocytes within the hair bulb, was observed in all Poodles irrespective of coat color or SNNL1 copy number (Fig. 3, a, d, and g). However, the white Poodle with 0 copies of SNNL1 lacked melanin within the hair bulbs and hair shaft cuticle (Fig. 3, b and c). Some melanin was observed in a cream Poodle with 1 copy of SNNL1 (Fig. 3, e and f), but melanin was most prominent in the red Poodles with 2 copies of SNNL1 (Fig. 3, h and i). The equivalent melanocytes and differential melanin indicated that the red coat color was occurring due to an increase in pigment synthesis.
Gene expression was analyzed in red and white Poodle skin. Poly(A) capture RNAseq was first performed in 3 red Poodles and 1 white Poodle to determine if SNNL1 was forming novel chimeric transcripts with the nearby genes GPR22 and COG5, and no novel chimeric transcripts were observed in any samples. Overall differences in expression were then analyzed in 8 red and 9 white Poodle skin samples using TagSeq (Supplementary Table 3). Among the most highly overexpressed genes in the red Poodles were several genes involved in melanogenesis, including TYR, PMEL, MLANA, SLC24A5, and MC1R (Fig. 4). Notably, among genes involved in the production of eumelanin, TYRP1 was not expressed in either red or white poodle skin, and no changes in expression were observed for DCT. The most differentially expressed gene in red Poodle skin was GPR22, which was unexpressed in the white Poodles and highly expressed in the red Poodles (FC 6.1; adjusted P = 0.00042). SNN also had small but significantly increased expression in the red Poodle skin (FC 0.49; adjusted P = 0.0315), as did COG5 (FC 0.41; adjusted P = 0.0433). Among the genes with missense variants in the red Poodles, neither SLC26A4 nor CDHR3 had sufficient expression in either the red or white Poodle skin to allow for differential expression analysis. However, while low levels of differential expression were observed in LAMB4 (FC 1.2; adjusted P = 0.0024) and GSAP (FC-1.1; adjusted P = 0.0006). GPR22, with a FC of 6.1, was the only gene within the chr18:10–20 mb interval that had greater than 2 FC in expression in the red Poodles.
Here we report the discovery of a recent SNN gene retrocopy insertion (SNNL1) which is 2.8 kb upstream from GPR22 and is strongly associated with pheomelanin intensity in Poodles. SNNL1 was part of a large LD block in Poodles which was identified through quantitative GWAS of pheomelanin colors in Poodles. Missense variants in 5 genes were identified in red Poodles within this region, including a variant in SLC26A4 which had previously been reported as a candidate for pheomelanin intensity across breeds. However, SLC26A4 was not expressed in Poodle skin, whereas GPR22 was identified as the most differentially expressed gene between red and white Poodles. SNNL1, which has inserted just upstream of GPR22, appears to interfere with regulation of the gene, resulting in ectopic expression of GPR22 in skin tissue. This study implicates GPR22 as being involved in the pigment production pathway, and the misregulation of GPR22 via the insertion of SNNL1 is likely the causal genetic influence behind red coat color in the Poodle breed. Retrocopy insertions are a type of large structural variant which can be expressed directly, form chimeric transcripts with nearby genes, or otherwise interrupt the typical expression patterns of nearby genes (Kubiak and Makałowska 2017). The SNN retrocopy insertion, SNNL1, is a full length copy of the parent gene. Additionally, it has no coding sequence variants differentiating it from the parent gene sequence, indicating that it is likely a recent insertion. SNN codes for Stannin, a metal ion binding mitochondrial membrane protein which may be involved in response to toxic substance as well as cell growth and apoptosis (Buck-Koehntop et al. 2005; Billingsley et al. 2006). No other SNN retrocopies are present in an across species database of reference genome retrocopies, indicating that SNN is not a commonly retrotransposed gene (Rosikiewicz et al. 2017). Interestingly, overall expression of SNN was also increased in the red Poodles, which may indicate that SNNL1 is capable of expression. Notably, multiple recent FGF4 retrocopy insertions have also been reported in dogs, several of which are expressed and involved in skeletal dysplasias (Parker et al. 2009; Brown et al. 2017; Batcher et al. 2020). In red and apricot Poodles, the insertion of SNNL1 immediately upstream of GPR22 is likely affecting regulation of the GPR22 gene, resulting in its atypical expression in skin tissue. Structural variants, which include retrocopy insertions, have been identified as a major source of gene expression differences which often affect multiple nearby genes (Scott et al. 2021). SNNL1 is inserted within an intron of COG5, and while no chimeric reads were observed between the genes, a small increase in expression was observed for COG5 in the red Poodle skin which may also be a consequence of the retrocopy. While several genes involved in pheomelanin intensity have been identified across dog breeds, the single-breed GWAS presented in this study only identified the CFA18 locus as significant within the Poodle breed. One of the top SNVs was a missense variant in SLC26A4 which has previously been associated with pheomelanin intensity across dog breeds, where it was hypothesized to be the causative variant (Slavney et al. 2021). The researchers found that the CFA18 locus explained a relatively small % of the total variance in pheomelanin across dog breeds (adjusted R2 = 0.047), whereas loci on CFA2 and CFA20 explained over 50% of the variance across breeds. However, when looking within a single breed, the Poodle, the CFA18 locus, identified herein as SNNL1, explained the majority of the variance between white, cream, apricot, and red Poodles (adjusted R2 = 0.840). Notably, 70 out of 73 apricot or red poodles had at least 1 copy of SNNL1, while none of the 125 white Poodles tested had any copies of SNNL1. Only 19% of the cream poodles had 1 copy of SNNL1, while the rest had 0 copies. Likely other genes involved in pheomelanin intensity, such as the MFSD12 dilution variant, explain the differences between white and cream coat colors (Hédan et al. 2019). Analysis of the SLC26A4 missense variant and the SNNL1 insertion in a larger WGS dataset revealed that they are rare across breeds and were only observed in East Asian dog breeds and village dogs, such as Tibetan Mastiffs, Chow Chows, and, notable for their rich pheomelanin appearance, Qingchuan and Chongqing dogs. It is possible that the CFA18 locus explains a relatively small proportion of the variance in pheomelanin intensity across breeds due to this breed exclusivity. Whereas, within breeds, SSNL1 may actually mask the effects of other genes involved in pheomelanin intensity. While the CNV upstream of KITLG was associated with pheomelanin intensity in Nova Scotia Duck Tolling Retrievers, the KITLG CNV was not significantly associated with pheomelanin intensity in Poodles, possibly due to the effects of SNNL1 within the breed (Bannasch et al. 2021). Pigment production is canonically regulated through the MC1R-tmAC-MITF pathway, which induces changes in expression of pigment genes such as TYR, PMEL, MLANA, SLC24A5, TYRP1, and DCT (Kawakami and Fisher 2017; Bang and Zippin 2021). The MC1R-tmAC-MITF pathway uses the second messenger molecule cyclic adenosine monophosphate (cAMP), and loss of function mutations in the MC1R gene lead to impairments in downstream cAMP signaling, resulting in impaired eumelanogenesis and the phenotype known as recessive yellow in dogs (Newton et al. 2000). In addition to cAMPs role as a second messenger molecule in the MC1R-tmAC-MITF pathway, tyrosinase itself is also affected by cAMP; reduction in cAMP within the melanosome results in a higher melanosomal pH, leading to greater tyrosinase activity and increased melanogenesis (Zhou et al. 2018). The sex hormones estrogen and progesterone have been found to regulate melanin synthesis through the alteration of cAMP signaling, showing that external factors which affect cAMP concentrations can have a downstream effect on melanogenesis (Natale et al. 2016). While the pheomelanin-based Poodles used in this study were homozygous for the recessive yellow mutation in MC1R, several pigment genes were still observed to be highly overexpressed in red Poodle skin, including TYR, PMEL, and MLANA, indicating upregulation of the melanogenesis pathway in red Poodles. Histopathological analysis in Poodle skin found that melanocytes were present within the hair bulbs of all Poodles, however melanin granules were present in high amounts in the red Poodles, consistent with an upregulation in pigment production in red Poodles. Notably, however, the main drivers behind eumelanogenesis, TYRP1 and DCT (Slominski et al. 2004), were not overexpressed in the red Poodles, suggesting specific upregulation in pheomelanogenesis. While SLC26A4 was not expressed in either red or white Poodle skin, differential expression was observed in several other genes within the red Poodle-associated region on chr18:10–20 mb. However, the degree of differential expression observed in GPR22 was much higher than any other genes, and was more comparable to the differential expression observed in genes involved in the pigment production pathway. Most notably, a near identical increase in expression was observed for GPR22 and TYR (FC 6.05 and 5.96), which were also the top 2 most differentially expressed genes between red and white Poodle skin. GPR22 is an orphan G-coupled protein receptor with a highly restrictive expression pattern in the heart and brain (Adams et al. 2008). While GPR22 knockout mice are viable and grossly indistinguishable from wild-type mice, they may be more susceptible to functional cardiac decompensation following aortic banding (Adams et al. 2008). Deregulation of GPR22 within the zebrafish embryo lead to defects in left-right patterning and resulted in abnormal cilia structure and length, indicating a possible developmental role for GPR22 (Verleyen et al. 2014). GPR22 has also been implicated in osteoarthritis in humans through GWAS (Kerkhof et al. 2010; Evangelou et al. 2011). While GPR22 was absent from healthy cartilage, it was found expressed in damaged cartilage, and overexpression of GPR22 was also shown to accelerate chondrocyte hypertrophy (Guns et al. 2016, 2018). While the GPR22 ligand is unknown, overexpression of GPR22 in HEK-293 cells identified that the GPR22 protein signals through the G inhibitory pathway, resulting in inhibition of adenylyl cyclase and a reduction in cAMP (Adams et al. 2008). The second messenger cAMP regulates numerous functions in melanocytes, and the abnormal expression of GPR22 in the melanocytes of red Poodles may have effects on pigmentation through this G inhibitory pathway. GWAS in dogs often succeed at identifying genomic regions, yet due to the extensive LD in dog breeds, it can be difficult to pinpoint causative mutations. In this study, several nearby missense variants within the pheomelanin associated region on chr18 could not be ruled out by segregation analysis alone. While the expression analysis in the skin was able to rule out the SLC26A4 and CDHR3 missense variants, further analysis including quantitative phenotyping in other breeds with the SNNL1 insertion such as Chow Chows and Tibetan Mastiffs may be required to rule out the other missense variants. Still, the differential expression analysis in Poodle skin highlighted the SNNL1 insertion and its effects on the expression of GPR22 as the likely causal mutation behind the red coat color phenotype in Poodles. Further analysis may also quantify the effects of SNNL1 on eumelanin-based coat patterns, as the upregulation of numerous genes involved in melanogenesis observed in the red Poodles might indicate an effect on eumelanin as well. The identification of another recent, functional retro copy insertion further serves to highlight the often complex nature behind genomic associations, and also shows that novel retrotransposition events continue to contribute to genomic and phenotypic diversity in dogs.
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PMC9635863 | 36321789 | Chang-Kyu Heo,Won-Hee Lim,Inseo Park,Yon-Sik Choi,Kook-Jin Lim,Eun-Wie Cho | Serum BRD2 autoantibody in hepatocellular carcinoma and its detection using mimotope peptide-conjugated BSA | 01-11-2022 | autoantibody biomarker,bromodomain-containing protein 2,hepatocellular carcinoma,cyclic peptide mimotope,human serum ELISA | Tumor-associated (TA) autoantibodies are considered to be promising biomarkers for the early detection of cancer, prior to the development of clinical symptoms. In the present study, a novel TA autoantibody was detected, which may prove to be useful as a diagnostic marker of human HCC using an HBx-transgenic (HBx-tg) hepatocellular carcinoma (HCC) mouse model. Its target antigen was identified as the bromodomain-containing protein 2 (BRD2), a transcriptional regulator that plays a pivotal role in the transcriptional control of diverse genes. BRD2 was upregulated in HCC tissues of the H-ras12V-tg mouse and human subjects, as demonstrated using western blotting or immunohistochemical analysis, with the BRD2 autoantibody. In addition, the truncated BRD2 reactive to the BRD2 autoantibody was detected in tumor cell-derived exosomes, which possibly activated TA immune responses and the generation of autoantibodies. For the detection of the serum BRD2 autoantibody, epitope mimicries of autoantigenic BRD2 were screened from a random cyclic peptide CX7C library with the BRD2 autoantibody. A mimotope with the sequence of CTSVFLPHC, which was cyclized by one pair of cysteine residues, exhibited high affinity to the BRD2 autoantibody and competitively inhibited the binding of the autoantibody to the cellular BRD2 antigen. The use of this cyclic peptide as a capture antigen in human serum enzyme-linked immunosorbent assay allowed the distinction of patients with HCC from healthy subjects with 64.41% sensitivity and 82.42% specificity (area under the ROC curve, 0.7761), which is superior to serum alpha-fetoprotein (AFP; 35.83% sensitivity; 100% specificity; area under the ROC curve, 0.5337) for the diagnosis of HCC. In addition, the detection of the BRD2 autoantibody combined with other autoantibody biomarkers or AFP has increased the accuracy of HCC diagnosis, suggesting that the combinational detection of cancer biomarkers, including the BRD2 autoantibody, is a promising assay for HCC diagnosis. | Serum BRD2 autoantibody in hepatocellular carcinoma and its detection using mimotope peptide-conjugated BSA
Tumor-associated (TA) autoantibodies are considered to be promising biomarkers for the early detection of cancer, prior to the development of clinical symptoms. In the present study, a novel TA autoantibody was detected, which may prove to be useful as a diagnostic marker of human HCC using an HBx-transgenic (HBx-tg) hepatocellular carcinoma (HCC) mouse model. Its target antigen was identified as the bromodomain-containing protein 2 (BRD2), a transcriptional regulator that plays a pivotal role in the transcriptional control of diverse genes. BRD2 was upregulated in HCC tissues of the H-ras12V-tg mouse and human subjects, as demonstrated using western blotting or immunohistochemical analysis, with the BRD2 autoantibody. In addition, the truncated BRD2 reactive to the BRD2 autoantibody was detected in tumor cell-derived exosomes, which possibly activated TA immune responses and the generation of autoantibodies. For the detection of the serum BRD2 autoantibody, epitope mimicries of autoantigenic BRD2 were screened from a random cyclic peptide CX7C library with the BRD2 autoantibody. A mimotope with the sequence of CTSVFLPHC, which was cyclized by one pair of cysteine residues, exhibited high affinity to the BRD2 autoantibody and competitively inhibited the binding of the autoantibody to the cellular BRD2 antigen. The use of this cyclic peptide as a capture antigen in human serum enzyme-linked immunosorbent assay allowed the distinction of patients with HCC from healthy subjects with 64.41% sensitivity and 82.42% specificity (area under the ROC curve, 0.7761), which is superior to serum alpha-fetoprotein (AFP; 35.83% sensitivity; 100% specificity; area under the ROC curve, 0.5337) for the diagnosis of HCC. In addition, the detection of the BRD2 autoantibody combined with other autoantibody biomarkers or AFP has increased the accuracy of HCC diagnosis, suggesting that the combinational detection of cancer biomarkers, including the BRD2 autoantibody, is a promising assay for HCC diagnosis.
Cancer is one of the leading causes of mortality worldwide, as it accounted for almost 10 million deaths in 2020, or nearly one in six deaths, with the most common cancers being breast, lung, colon and rectum and prostate cancers (1). Primary liver cancer is also a challenging global health concern, as more than one million individuals are estimated to be affected annually by the year 2025. The most common type of liver cancer is hepatocellular carcinoma (HCC), the incidence of which has been increasing worldwide, mostly due to chronic viral hepatitis B infection (2). Recently, non-alcohol-related steatohepatitis has also rapidly emerged as another etiological concern (3,4). In common clinical practice, HCC is diagnosed using non-invasive criteria, including a serum alpha-fetoprotein (AFP) or ultrasound test, and the treatment applied may vary, depending on the overall tumor burden and underlying liver disease severity (2). However, novel evidence points towards the importance of histology and of the characterization of the molecules that drive pathogenesis, to identify druggable targets (5,6). Moreover, immunotherapies that instigate the host immunity to induce a systemic response against tumors currently offer much clinical promise (7). Therefore, the clinical classification of HCC using appropriate biomarkers accompanied by treatment is important for the improvement of of the prognosis of patients with HCC. The majority of malignant tumors can be recognized by the host immune-surveillance defensive system, namely, natural killer (NK) and T-cells; however, cancer cells evolve to acquire genetic instabilities and other associated 'hallmarks' that can enable persistent growth and immune evasion from NK or T-cells, in spite of their presence near tumors (8). Unlike T-cells, relatively few B-cells are found in tumor infiltrates and have been rarely studied. However, their existence and functionality have been recently suggested as important prognostic factors for the response to immunotherapy (9). Furthermore, the plasma cells present in tumor infiltrates produce large amounts of cytokines and tumor-associated (TA) antibodies, even with reduced cell counts, and these soluble factors can serve as significant biomarkers (9). TA antibodies have been examined in patients with cancer for several decades. The levels of serum autoantibodies against TA self-antigens have been proposed as early markers of cancer (10-12). Moreover, serum autoantibodies can serve as potent prognostic markers at later stages of disease (13), which may be related to the prognostic significance of tumor-infiltrating B-cells (14,15). Therefore, screening significant autoantibody biomarkers related to cancer and identifying appropriate combined therapies may be necessary for precision cancer medicine in the future. In the present study, a bromodomain-containing protein 2 (BRD2) autoantibody was reported as a novel TA autoantibody biomarker for HCC. B-cell hybridoma pool obtained from the HBx-transgenic HCC tumor-bearing mouse model (16-18) was screened using human liver cancer cells, and one TA autoantibody, XC246, was obtained. Using the purified XC246 autoantibody, its target antigen was identified as BRD2, a transcriptional regulator of diverse genes, and the neo-antigenic properties of BRD2 in liver cancer cells were analyzed. The present study also screened the mimicries of the epitope on the autoantigen BRD2 from a random cyclic peptide library and used it to develop a serum autoantibody enzyme-linked immunosorbent assay (ELISA). Finally, the significance of the BRD2 autoantibody as a cancer diagnostic marker was evaluated using cyclic peptide ELISA and compared with other serum biomarkers.
A monoclonal TA auto-antibody, XC246, was prepared from a B-cell hybridoma generated from the splenocytes of HBx-transgenic mice bearing HCC tumors (18). The isotype of the XC246 autoanti-body was determined as IgM using an isotyping kit (Thermo Fisher Scientific, Inc.). The XC246 autoantibody was purified from hybridoma-cell culture media using protein L agarose (Thermo Fisher Scientific, Inc.) and applied in further studies after analysis, using SDS-PAGE and western blotting.
The following human cancer cell lines were obtained from the American Type Culture Collection (ATCC) or the Korean Cell Line Bank (KCLB): The liver cancer cells, HepG2 (ATCC HB-8065), Hep3B (ATCC HB-8064), PLC/PRF/5 (ATCC CRL-8024), Huh7 (KCLB 60104) and SK-HEP1 (ATCC HTB-52); the gastric cancer cells, SNU638 (KCLB 00638); the lung cancer cells, A549 (ATCC CRM-CCL-185); the colorectal cancer cells, HT-29 (ATCC HTB-38); the prostate cancer cells, LNCaP/LN3 (KCLB 80018); the cervical cancer cells, HeLa (ATCC CRM-CCL-2); and the breast cancer cells, SK-BR-3 (ATCC HTB-30). The cells were cultured in Dulbecco's modified Eagle's medium (DMEM) or RPMI-1640 (Invitrogen; Thermo Fisher Scientific, Inc.) supplemented with 10% inactivated fetal bovine serum (FBS; Sigma-Aldrich). Conditioned media containing exosomes were prepared from 70 to 80% confluent HepG2 cells, which were cultured in complete DMEM for 48 h with 10% exosome-free FBS (System Biosciences, LLC). The cell lines (HepG2, HT-29 and SK-HEP1) were authenticated using short tandem repeat (STR) analysis by the Korean Cell Line Bank (KCLB), and the STR profile results are presented in Data S1.
For the expression of full-length and a series of truncated BRD2 proteins (hBRD2: NM_005104.4) with N-terminal 3xFLAG-tag [(DYKDDDDK)x3] were cloned into the pE28a(+) vector (Novagen, Sigma-Aldrich) using NdeI and XhoI. Expression vectors were transformed into E. coli strain SHuffle® T7 (New England Biolabs, cat. no. C3029J). The E. coli SHuffle® T7 transformants were grown at 30°C for 16 h in 2xYT broth (16 g Bacto Tryptone, 10 g Bacto Yeast Extract, 5 g sodium chloride in 1 l distilled water, pH 7.0) containing kanamycin (50 µg/ml). The culture was diluted 100-fold into fresh 2xYT medium and grown in a shaking incubator at 30°C. When the OD600 of the cultures reached 2.0, isopropyl-β-D-thiogalactopyranoside (IPTG; Sigma-Aldrich cat. no. 16758) was added to a final concentration of 1 mM and incubation was continued at 25°C for 22 h. Cells were harvested by centrifugation at 3,000 x g for 30 min at 4°C. The cell pellets were solubilized in 5X SDS-PAGE sample buffer and used for further analysis.
The purified antibody or epitope-conjugated bovine serum albumin (BSA) was analyzed by using 10% SDS-PAGE and Coomassie brilliant blue staining. Western blot analysis of cell or tissue lysates was performed as previously described (18). Total cell or tissue lysates were prepared in radioimmunoprecipitation assay (RIPA) buffer containing a protease inhibitor cocktail (Sigma-Aldrich). The liver tissues from the H-ras mice were already available as part of a previous study (18). For subcellular fractionation, cells were lysed using NE-PER™ Nuclear and Cytoplasmic Extraction Reagents kit (Thermo Fisher Scientific, Inc. cat. no 78833). Exosomes were collected from the conditioned media using an exosome isolation kit ExoQuick-TC (System Biosciences, LLC, cat. no. EXOTC50A-1) or by ultra-centrifugation at 100,000 x g for 1 h at 4°C, and lysed in RIPA buffer (17). The protein concentration was determined using Bradford reagent (Bio-Rad Laboratories, Inc.). Equal amounts of protein (10 µg per lane) were analyzed using western blotting with the indicated primary antibodies. Briefly, proteins were separated by 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and transferred onto PVDF membranes (MERCK, cat. no. IPVH00010). After blocking with 5% BSA in Tris-buffered saline with 0.1% Tween-20 (TBST) at 25°C for 60 min, the membranes were probed with primary antibodies at 25°C for 2 h. After washing with TBST, the membranes were probed with HRP-conjugated secondary antibodies. The membranes were then washed with TBST and antigens were detected with enhanced chemiluminescence reagent (MERCK, cat. no. GERPN2106). β-actin or GAPDH was probed as loading controls. The primary antibodies used in this study were as follows: BRD2 (Novus Biologicals, cat. no. NBP1-84310, NBP1-30475; 1:1,000 dilution), AICAR transformylase/inosine monophosphate cyclohydrolase (ATIC; Thermo Fisher Scientific, cat. no. MA1-086; 1:500 dilution), programmed cell death 6-interacting protein (ALIX; exosomal marker; Merck Millipore, cat. no. ABC1435; 1:500 dilution), calnexin (endoplasmic reticulum marker; Santa Cruz Biotechnology, cat. no. sc-46669; 1:1,000 dilution), GAPDH (Santa Cruz Biotechnology, cat. no. sc-47724; 1:5,000 dilution), and β-actin (Santa Cruz Biotechnology, cat. no. sc-8432; 1:5,000 dilution). Anti-mouse IgG-horseradish peroxidase (HRP; Cell Signaling Technology, cat. no. #7076S; 1:2,500 dilution) or anti-rabbit IgG-HRP (Cell Signaling Technology, Inc. cat. no. 7074S; 1:2,500 dilution) were used as secondary antibodies. Band intensities were quantified using ImageJ v1.52a (National Institutes of Health), and the relative intensity compared with that of β-actin or GAPDH was calculated. For the immunoprecipitation analysis of the XC246 antigen, 600 µg of HepG2 cell lysate was incubated with XC246 antibody-conjugated agarose beads (bead volume, 20 µl) for 16 h at 4°C; after a brief washing the beads in RIPA buffer by centrifugation at 1,000 x g for 30 sec at 4°C, the agarose beads were analyzed by western blotting. For the proteomics analysis of the XC246 antigen, 500 µl of antibody-conjugated beads was used for immunoprecipitation with 9.69 mg of SNU638 cell lysate. As a control, blank agarose beads without the immobilized antibody were used. XC246 antibody-conjugated agarose beads were prepared using a co-immunoprecipitation kit (Thermo Fisher Scientific, cat. no. 26149). For competitive western blot analysis, 5 µg of the XC246 primary antibody in 10 ml of 5% skim milk in TBS was pre-incubated with epitope-peptide-conjugated BSA (2 µg) at 25°C for 90 min and then used as the primary antibody for probing the blot.
Cells were fixed and permeabilized with BD Cytofix/Cytoperm solution (BD Biosciences) at 4°C for 30 min. The cells were then incubated with the XC246 primary antibody solution and secondary reagent goat anti-mouse IgG F(ab')2-PE (Thermo Fisher Scientific, cat no. A10543; 1:400 dilution) at 4°C for 40 min. The stained cells were analyzed on a FACSCalibur (BD Biosciences) instrument, and the obtained data were analyzed using the CellQuest software v6.0 (BD Biosciences). When determining whether the autoantibody-mimotope display phages could compete with the target cellular antigen for antibody binding, the XC246 primary antibody was pre-incubated with each mimotope-display phage at 25°C for 60 min and then used as primary antibody solution.
Cells were plated on glass coverslips and, after an additional 48 h, were used for immunofluorescence detection, as described previously (17). Cells were fixed and permeabilized with BD Cytofix/Cytoperm solution (BD Biosciences), washed with BD Cytoperm/wash solution (BD Biosciences), and incubated with the primary antibody in wash-solution (5 µg/ml) at 4°C for 16 h. After washing with wash-solution, the cells were treated with goat anti-mouse IgGAM-rhodamine (Abcam, cat. no. ab6004) or anti-rabbit IgG-FITC (Abcam, cat. no. ab6798) in wash-solution (1:1,000 dilution) at 25°C for 1 h, followed by mounting on a DAPI-containing mounting medium (Vector Laboratories, cat. no. H-1200). Confocal microscopic analysis was performed using a Zeiss LSM 510 Meta microscope (Zeiss AG). The staining with an anti-FBXO2 antibody (mouse IgM; Santa Cruz Biotechnology, cat. no. sc-393873) was performed under the conditions described above to confirm the accessibility of the IgM antibody to the nucleus.
A paraffin-embedded human HCC BioMax Array (cat. no. LV1201b) was purchased from US BioMax Inc. Biopsy features included age, sex, organ or anatomic site involved, grading and pathological diagnosis (H&E-stained sections). The HCC tissue array was comprised of HCC or HCC-related liver disease tissues and normal liver tissues as follows: i) 10 cases of benign tumor (cavernous hemangioma); ii) 14 cases of normal liver tissues; iii) 25 cases of hepatocellular carcinoma (stage I, n=1; stage II, n=16; stage III, n=5; stage IV, n=3); iv) 29 cases of cirrhosis; v) 22 cases of chronic hepatitis; and vi) 13 cases of fatty degeneration. US Biomax states that 'All tissue is collected under the highest ethical standards with the donor being informed completely and with their consent. We make sure we follow standard medical care and protect the donors' privacy. All human tissues are collected under HIPPA approved protocols. All animal tissues are collected under IACUC protocol. All samples have been tested negative for HIV and hepatitis B or their counterparts in animals, and approved for commercial product development'. The Public Institutional Review Board of the Ministry of Health and Welfare reviewed the contents of the present study and confirmed that research using commercialized human tissue falls under the exemption categories specified by Bioethics and Safety Act in Korea (IRB, approval no.: P01-202008-31-009; Republic of Korea). The immunohistochemical staining for XC246 antigen was performed using XC246 monoclonal autoantibody (2 µg/ml) as previously described (16). The IHC staining of all tissues was performed under identical conditions, as tissues with all pathological characteristics were present on one slide. The endogenous peroxidase activity was blocked by treatment with 4% H2O2/methanol for 10 min, and this was followed by remaining staining protocol. The photomicrographs were acquired at x200 or x400 magnification using a multi-view fluorescence microscope (BX51; Olympus Corporation). The DAB intensity of each staining using DAB kit (GBI Labs, cat. no. D22-6) was quantified by ImageJ v1.52i software (National Institutes of Health) and plotted.
For the enrichment of the antigen against the XC246 autoantibody, a SNU638 cell lysate prepared with RIPA cell lysis buffer was immunoprecipitated using XC246 antibody-conjugated beads as described above. Subsequently, the beads were treated with 0.1 M glycine buffer (pH 2.5) to elute the target antigen, and the eluate was neutralized with 1.0 M Tris buffer (pH 8.0). The eluates were then separated using 10% SDS-PAGE, followed by western blotting or Coomassie brilliant blue staining at 25°C for 2 h. The Coomassie-stained band corresponding to the XC246 antigen band was confirmed using western blotting. The XC246 antigen band was then excised and in-gel digested with trypsin (Promega Corporation). Protein identification was performed via ESI-TRAP mass spectrometry (LTQ Orbitrap XL Hybrid Ion Trap Mass Spectrometer; Thermo Fisher Scientific, Inc.) and Mascot database search at the Korea Basic Science Institute (Ochang, Korea).
To verify whether the candidate proteins identified by mass spectrometric analysis corresponded to the XC246 antigen, the HepG2 cells were transfected with siRNAs, targeting the candidate genes (Bioneer Corporation and Thermo Fisher Scientific Inc.) using Lipofectamine RNAimax reagent (Thermo Fisher Scientific Inc.), followed by western blot analysis using the XC246 anti-body. The sequences of the siRNAs were as follows: siRNA for the elongation factor 2 (si-EEF2) si-sense, 5′-GAG AUG UAU GUG GCC AAG Utt-3′ and antisense, 5′-ACU UGG CCA CAU ACA UCU Ctt-3′; siRNA for the isoform 3 of unconventional myosin 1c (si-MYO1C) sense, 5′-GAC CAA GAC AGC CCU CAG Utt-3′ and antisense, 5′-ACU GAG GGC UGU CUU GGU Ctt-3′; si-BRD2 sense, 5′-CUG GGA GUC UUG AGC CUA Att-3′ and antisense, 5′-UUA GGC UCA AGA CUC CCA Gga-3′; si-control sense, CCU ACG CCA CCA AUU UCG U(dTdT) and antisense, ACG AAA UUG GUG GCG UAG G(dTdT). The siRNA-treated cells were analyzed 72 h after transfection. RT-PCR was performed using primer pairs purchased from Bioneer Corporation, as follows: EEF2 sense, 5′-GTG GAG AAC GTG AAC GTC ATC-3′ and antisense, 5′-GAA GGT GCG TGG CAG CTT CT-3′; MYO1C sense, 5′-CGC TAC CGG GCG TCG GCC-3′ and antisense, 5′-CGT GCG CAG TGC TCG GT AC-3′; BRD2 sense, 5′-AGA GTC CTC CAG TGA GGA AAG-3′ and antisense, 5′-CCA CTG CCA CTT GCT TTC TTG-3′. GAPDH sense, 5′-CCA ATA TGA TTC CAC CCA TGG C-3′ and antisense, 5′-GCT GAT GAT CTT GAG GCT GTT G-3′. Briefly, total RNA was extracted from the siRNA transfectants using the RNeasy Plus mini kit (Qiagen, Germany, cat. no. 74134) following the manufacturer′s protocol. Reverse transcription (RT) was performed using the GoScript™ Reverse Transcription Mix kit (Promega Corporation, cat. no. A2791). PCR was carried out using 2X Taq PCR Pre-Mix (BIOFACT, cat. no. ST302-10h) using a Verti 96 well Thermal Cycler (Invitrogen; Thermo Fisher Scientific, Inc.) which was performed by an initial denaturing step at 95°C for 5 min followed by 27 cycles of denaturation at 95°C for 10 sec, 55-62°C for 30 sec for annealing and elongation at 72°C for 30 sec. At the end of additional elongation at 72°C for 5 min, PCR products were analyzed by 1% agarose gel electrophoresis. The relative levels of each PCR product were quantified using ImageJ v1.53k software and normalized to that of GAPDH.
For the selection of the mimotope specific to XC246 auto-antibody, the phage-display random cyclic peptide library Ph.D.™-CX7C (New England Biolabs, Inc.) was used, as previously described (16-18). Panning was repeated four times, and the mimotope sequences were determined by sequencing the selected phages according to the manufacturer's instructions. DNA sequencing was performed by Bioneer Corporation based on Sanger method using -96gIII sequencing primer (5′-CCC TCA TAG TTA GCG TAA CG-3′).
ELISA was performed as previously described, with some modifications (17). Briefly, ELISA plates (Nunc Maxisorp; Thermo Fisher Scientific, Inc.) were coated with the indicated amount of antigen [cyclic peptide epitope-display M13 phages or epitope-miniPEG2-conjugated BSA] in PBS (pH 7.4) overnight at 4°C and blocked with Protein-Free Blocking Buffer (PFBB; Thermo Fisher Scientific, Inc.). The CX7C peptide mimotopes were synthesized and cyclized via terminal cysteine residues. The cyclized peptides were then coupled to miniPEG2 spacer via the free amine residue at the amino terminus of the peptide. The PEG-conjugated cyclic peptides were synthesized and purified by Peptron. Cyclic peptide-miniPEG2-conjugated BSA was prepared using the EDC coupling method (Thermo Fisher Scientific, Inc.). The miniPEG2-conjugated BSA without the peptide epitope was used as the control antigen. The primary anti-body or secondary reagent solution was prepared in PFBB. Anti-mouse IgGAM-HRP (Thermo Fisher Scientific, Inc.) was used as the secondary reagent. To detect the human autoantibody in patients' sera, ELISA plates were coated with XC246p9-miniPEG-conjugated BSA at 500 ng/well. After blocking with a buffer containing 1% polyvinyl alcohol (molecular weight, 14-15k; MilliporeSigma) and 1% Ficoll P400 (MilliporeSigma) in TBS, the plates were treated with human sera (1:50 dilution in blocking buffer) at 37°C for 90 min and detected using HRP-conjugated anti-human IgGAM antibody (Thermo Fisher Scientific, Inc.; 1:2,000 dilution). Serum AFP was quantified using a human alpha-Fetoprotein Quantikine ELISA kit (R&D Systems, cat. no. DAFP00). The present study using human HCC and normal serum samples was conducted after receiving an exemption from the Public Institutional Review Board of the Ministry of Health and Welfare (IRB No.: P01-202008-31-009; Republic of Korea). Human serum samples were obtained from the National Biobank of Korea, which is supported by the Ministry of Health and Welfare. Serum samples were kept at −80°C until further use.
All data are presented as the mean ± standard deviation (SD) and were analyzed using a two-tailed unpaired Student's t-test or one-way ANOVA followed by the Bonferroni post-hoc test. The results of ELISA were evaluated using receiver operating characteristics (ROC) analysis, leading to estimates of the area under the ROC curve (AUC), with 95% confidence intervals (CIs). The correlation between the biomarkers was assessed using Pearson's correlation analysis. Statistical analyses were performed using the Prism 7 software (GraphPad Software, Inc.). P<0.05 was considered to indicate a statistically significant difference.
HBx-transgenic mice, which exhibit spontaneous generation of liver cancer at 10-13 months after birth, have been proven to be suitable for the study of human HCC (19-21). Previously, three HCC-associated autoantibodies were identified by the authors using B-cell hybridoma cells constructed using tumor-bearing HBx-transgenic mice (16-18). In the present study, a monoclonal TA autoantibody, which was termed XC246, was identified, and its antigenic characteristics were analyzed. Firstly, the presence of an antigen reactive to the XC246 autoantibody in human cancer cells was examined using flow cytometry. For the staining of intracellular antigens, cells were fixed and mildly permeabilized with a paraformaldehyde/saponin solution, to maintain the conformational epitopes on the target antigens. The XC246 TA autoantibody in hybridoma-cultured media reacted to human liver cancer cells, including HepG2 and Huh7 cells (Fig. 1A). The colorectal cancer HT-29 cells also exhibited reactivity to the XC246 autoantibody. These results indicated that TA autoantigen against XC246 TA autoantibody generated in a mouse HCC tumor model may also be expressed in human tumor cell lines. The isotype of the XC246 antibody was determined using an isotyping kit as being IgM with a kappa light chain (data not shown). For the identification and characterization of the target antigen of the XC246 TA autoantibody, the antibody was purified from hybridoma-cultured media using protein L agarose. SDS-PAGE analysis of the purified antibody showed the presence of IgM heavy and light chains (Fig. 1B). The target antigen of XC246 antibody was analyzed by western blot analysis of human tumor cell lines. As depicted in Fig. 1C, the XC246 antibody reacted to a specific antigen (termed XC246 Ag) with a molecular weight of about 110 kDa. XC246 Ag was ubiquitously and highly expressed in various human tumor cells, including liver cancer (HepG2), gastric cancer (SNU638) and colorectal cancer (HT-29); however, its expression was unusually low in Hep3B cells. XC246 Ag was noted to be mainly localized to the cytoplasm, as demonstrated by tumor cell immunofluorescence staining (Fig. 1D). Of note, XC246 antibody detected a target antigen of ~110 kDa in liver tumor tissues of H-ras12V transgenic mice (Fig. 1E). The same antigen was also detected in non-transgenic mice; however, its expression was lower than that in tumor tissues (P≤0.05; Fig. 1E), which implies that the upregulation of the XC246 antigen is associated with tumorigenesis. XC246 antigen expression was also higher in human HCC tissues than in normal liver tissues, as shown by immunohistochemical staining (P≤0.001; Figs. 1F and S1). Of note, its expression was significantly increased in patients with chronic hepatic liver diseases (P≤0.0001), including cirrhosis and chronic hepatitis. Overall, these results suggested that the HCC-associated autoantibody XC246 may react with a specific antigen expressed in the cytoplasm of human or mouse tumor cells with a molecular weight of ~110 kDa and that its expression was increased in HCC and chronic liver diseases.
The target antigen of the XC246 autoantibody was identified using MS analysis. The lysate of SNU638 cells was immunoprecipitated using XC246 anti-body-conjugated agarose beads, and its eluate was separated on preparative 10% SDS-PAGE. As a control, the immunoprecipitates using agarose beads without antibodies were used. A tenth of the immunoprecipitates was analyzed using western blot analysis, revealing that the XC246 antigen was enriched in the immunoprecipitated fraction (Fig. 2A). The protein band on the preparative SDS-PAGE that corresponded to the immuno-stained antigen was then excised, in-gel digested and analyzed via ESI-TRAP MS (Fig. 2A). The isoelectric point (pI) of the XC246 antigen was also examined by two-dimensional gel electrophoresis of a total HepG2 cell lysate, followed by western blot analysos using the XC246 antibody; the pI of the 110-kDa antigen was estimated to be ~9 (Fig. S2). The identified proteins in MS analysis that exhibited a high %mol/score are listed in Table I. Based on the protein data acquired, including molecular weight and pI, three candidate proteins (EEF2, BRD2 and MYO1C) were selected for further validation. The downregulation of the XC246 antigen following the knockdown of each candidate gene was examined. The knockdown of BRD2 in HepG2 cells clearly reduced the expression of the XC246 antigen, as demonstrated using western blot analysis with the XC246 antibody. By contrast, the knockdown of the remaining candidates (EEF2 or MYO1C) had no or only a minimal effect on XC246 antigen levels (Fig. 2B). BRD2 was also confirmed as the XC246 antigen by analysis of the immunoprecipitates obtained with the XC246 antibody (Fig. 2C). The commercial anti-BRD2 antibody detected the target antigen with a molecular weight of ~110 kDa in cell lysate input or flow-through fractions. It also detected the immunoprecipitates obtained with the XC246 antibody, the molecular weight of which was identical to that of the BRD2 protein. Collectively, these results confirmed that the XC246 antigen, which induced the expression of the TA autoantibody in the HCC mouse model, was BRD2. The BRD2 gene encodes a transcriptional regulator that belongs to the bromodomain and extra-terminal domain (BET) family of proteins. BRD2 associates with transcription complexes and acetylated chromatin during mitosis; it then selectively binds to the acetylated lysine 12 residue of histone H4 via its two bromodomains (22). Decreased BRD2 expression has been associated with longevity-promoting processes (23,24), whereas increased BRD2 expression can promote cancer in murine hematopoietic cells and B lymphocytes (25). The Cancer Genome Atlas (TCGA) has demonstrated that BRD2 expression is elevated across 32 distinct tumor types and established BRD2 as a promising drug target for human cancers (26). The downregulation of BRD2 in HeLa cells leads to a 60% increase in tumor-suppressing p53 levels (27), which also supports the notion that increased BRD2 promotes cancer growth. Hence, the overexpression of BRD2 in HCC can be oncogenic, whereas the inhibition of the activity of BRD2 limits cancer progression. More importantly, Kaplan-Meier analysis of overall survival in patients with HCC by their BRD2 protein expression levels revealed BRD2 as a poor prognostic marker for liver cancer (Fig. S3). The mouse BRD2 protein (NP_001191902) presents with 96% amino acid sequence identity (97% similarity) to human BRD2 (NP_005095), which is composed of 801 amino acids. The difference between the mouse and human BRD2 protein sequences was primarily found between the 590 and 630th amino acids of human BRD2, a linker region located between the nuclear localization signal and the extra-terminal (ET) domain. The sequences of other domains were well conserved (Fig. S4), on which the target sequence of the XC246 antibody may be located. The protein function of BRD2 mentioned above raised a question about its cellular localization. BRD2 is a transcriptional regulator that functions mainly in the nucleus. However, the XC246 antigen was immunostained with the XC246 antibody mainly in the cytoplasmic region, as demonstrated in Fig. 1D. To examine the cellular localization of the XC246 antigen or BRD2, intracellular staining with a commercial anti-BRD2 antibody or the XC246 autoantibody was performed. The accessibility of XC246 antibody, a mouse pentameric IgM, within the nucleus was confirmed by control staining with an anti-FBXO2 antibody, the isotype of which is IgM. As depicted in Fig. 2D, the commercial anti-BRD2 anti-body stained BRD2 mainly within the nucleus. This antibody also stained the cytoplasmic region, although at reduced levels. By contrast, XC246 autoantibody stained the cytoplasmic region mainly (Figs. 1D and 2D). However, the nuclear staining of XC246 is more evident in Fig. 2D compared with Fig. 1D, which may be caused by different staining conditions or image capture settings. The reactivity of XC246 autoantibody to the intracellular fractions was examined again using western blot analysis (Figs. 2E and S5). The XC246 autoantibody stained the 110-kDa antigen with a similar ratio in cytoplasmic or nuclear fractions of HepG2 cells separated on the blot; however, the commercial anti-BRD2 antibody stained cytoplasmic BRD2 at half the strength observed for nuclear BRD2 in the same blot. These results imply that these two antibodies against BRD2 have different epitopes on the BRD2 protein. In addition, the XC246 autoantibody is more reactive to cytoplasmic BRD2, which may possess specific post-translational modifications related to its cytoplasmic localization. Another important aspect of TA autoantigens is their exposure to immune cells. The increased intracellular antigen must be exposed to the immune cells to induce a specific immune response, ultimately leading to the production of TA autoantibodies. The release of cellular components after cell death or necrosis accompanying tumorigenesis has been assumed to be a mechanism to expose intracellular proteins because most of the TA autoantigens reported to date are intracellular proteins, including p53 (28,29). However, a previous study on TA exosomes revealed that the intracellular components included in exosomes can be exposed to immune cells without cell death (30). In addition, the tumor-derived exosomes can stimulate or suppress immune responses (30,31). As described above, the XC246 antigen appears to be a post-translationally modified BRD2 mainly localized in the cytoplasm. The exosomes secreted by HepG2 cells were examined using western blot analysis to confirm the secretion of BRD2 via exosomes. ATIC (16), another TA autoantigen previously studied, was detected in the exosomes, as shown in Fig. 2E, which confirms that the TA exosome is a supplier of TA antigens. However, the BRD2 protein of 110 kDa was not detected in the exosomal fraction of HepG2 cells using XC246 or a commercial anti-BRD2 antibody. Instead of the full-sized BRD2 form, the XC246 antibody detected a protein band of ~65 kDa (Fig. 2E), whereas the commercial anti-BRD2 antibody did not detect it. The immunogen of the commercial anti-BRD2 antibody has been reported as the 179-259 amino acids of BRD2. Epitope mapping analysis using a series of truncated recombinant BRD2 proteins revealed that the commercial anti-BRD2 antibody recognized the region between residues 179 and 205 of BRD2. It also revealed that the XC246 autoantibody recognized the region between residues 206 and 229 of BRD2 (Fig. S6). Based on these results, it was concluded that the exosomal BRD2, a truncated BRD2 with a molecular weight of about 65 kDa and containing 206-229 amino acid residues of BRD2, is a post-translationally modified form of BRD2 that confers a neo-epitope for generating autoantibodies.
While epitope mapping of XC246 antibody was performed by using recombinant BRD2 proteins and western blot analysis, these recombinant proteins are not appropriate for the detection of serum autoantibody using ELISA, due to their relatively low affinity to the anti-body, as demonstrated in a previous study by the authors (16). In addition, some post-translational modifications that confer neo-epitopes for generating autoantibodies were anticipated. Therefore, high-affinity mimotopes were screened, which are the mimics of the conformational antigenic determinants, from a random cyclic peptide CX7C library, as described in previous studies (16-18,32,33). The biopanning of the M13 phage library containing 1011 cyclic peptides was repeated four rounds with the XC246 auto-antibody, consequently enriching the cyclic peptide display M13 phages specific to the XC246 antibody (Fig. 3A). The cyclic peptide sequences displayed on selected phages were determined by sequencing 20 selected M13 phage clones, and 10 different epitopes were confirmed (Table II). These phages displayed high reactivity only to the XC246 autoantibody in ELISA, not to other TA autoantibodies, i.e., K94 (32) or XC90 (17) (Fig. 3B). Among the selected peptide epitopes, the XC246p2, XC246p5, XC246p6, and XC246p9 sequences showed high reactivity to the XC246 autoantibody (Fig. 3B, Table II). The XC246 antibody-specific epitopes had the consensus sequence of C-XSXXLPX-C (Table II). XC246-specific phages competitively blocked XC246 antibody binding to HepG2 cells, as demonstrated by FACS analysis (Fig. 3C), indicating that the selected cyclic peptide with high affinity to the XC246 autoantibody can mimic the specific epitope on the cellular antigen BRD2. However, linearization of the cyclic peptide by treatment with a reducing agent resulted in the loss of those antigenic properties (Fig. S7), indicating that the conformational properties of the mimotope are critical for antibody binding. To construct an ELISA for the detection of serum BRD2 autoantibodies, a sufficient supply of target antigen is needed. Epitope-display M13 phages can be used as coating antigens, as described in previous studies (32,33); however, phage amplification and purification are cumbersome processes. Therefore, synthetic cyclic peptide-conjugated BSA was prepared and used as a coating antigen to display the auto-antigen-mimic epitope peptides. For the synthesis of epitope peptide, the XC246p9 sequence was selected (Table II), which does not contain less stable amino acids than others, including Q, M, W, N and D, which are prone to side reactions. Peptide epitope was cyclized by disulfide bonding between the two cysteine residues located at the N-terminus and C-terminus of the 9-mer peptide. Two miniPEG were conjugated to the N-terminus of the peptide as a spacer, and miniPEG2-linked cyclic peptides were conjugated to BSA via the EDC reaction (Fig. 3D). MiniPEG2-conjugated BSA without a peptide epitope was also prepared and used as a control antigen (Fig. S8). BSA-miniPEG2-XC246p9 demonstrated high affinity to the XC246 autoantibody, as shown using ELISA (Figs. 3E). BSA-miniPEG2-XC246p9 also blocked the binding of the XC246 autoantibody to the endogenous antigen in tumor cells (HepG2 or SNU638), as revealed by competitive western blot analysis (Fig. 3F). Collectively, the TA autoantibody XC246, which was generated in the HCC model mouse via an autoimmune response to the TA antigen BRD2, reacted with the XC246p9 cyclic peptide epitope with high affinity, and XC246p9 peptide-conjugated BSA was prepared for the detection of the BRD2 autoantibody in human serum.
Human serum ELISA for the BRD2 autoantibody biomarker was performed using XC246p9 peptide-conjugated BSA under conditions optimized for each step as follows: BSA-mini-PEG2-XC246p9 was employed as a coating antigen in ELISA using a Maxisorp plate. BSA-mini-PEG2 was also used as a negative control antigen. For blocking non-specific reactions of human sera to the antigen-coated plates, the plates were treated with a blocking buffer containing PVP, Ficoll and PFBB, as described in the 'Materials and methods'. Human serum samples were heat-inactivated at 50°C for 5 min and diluted 50-fold in blocking buffer. The sera of 118 patients with HCC and 91 healthy controls were analyzed for the BRD2 autoantibody biomarker. The patients' sera with cirrhosis (n=32) and benign cancer (n=3) were also analyzed. The level of BRD2 autoantibody biomarker was determined as the difference in OD between the ELISA for BSA-mini-PEG2-XC246p9 and that for BSA-mini-PEG2. As demonstrated in Fig. 4A, the BRD2 autoantibody biomarker was significantly elevated in the sera of patients with HCC compared with healthy subjects. The sensitivity of ELISA was 64.41% and the specificity 82.42% for the cut-off value of 0.1514, with an AUC value of 0.7761 [95% CI: 0.7136-0.8386, P<0.0001] (Fig. 4B). AFP levels, a most widely used HCC biomarker, were also analyzed in the same HCC cohort. All healthy controls had a serum AFP levels below the cut-off value of 20 ng/ml (Fig. 4A). However, not all patients with HCC had a serum AFP level above the cut-off value. AFP levels >20 ng/ml were detected only in 35.6% (42/118) of the patients with HCC and in 6.25% (2/32) of those with cirrhosis. A ROC curve analysis for the serum AFP test yielded an AUC value of 0.5357 [95% CI, 0.4514-0.6200; P=0.3746]. The sensitivity was 35.83%, and the specificity was 100% for the cut-off value of 20 ng/ml AFP (Fig. 4B). The detection patterns of these markers were dependent on the tumor stage. The AFP levels progressively increased in patients with HCC as the tumor stage advanced (Fig. 4C). However, BRD2 autoantibody levels were significantly high even in tumor stage I (P≤0.0001), maintaining those levels throughout all tumor stages (Fig. 4C). The presence or absence of viral infection in patients with HCC was not associated with these two markers in this cohort (Fig. 4D).
AFP is a typical protein biomarker of HCC that is secreted into the blood via a signal peptide from liver tissue. By contrast, TA autoantibody biomarkers appeared to be induced by TA anti-gens, that are released as components of the tumor exosome. The release mechanism of the tumor exosome is different from that of secretory proteins, which may reflect other aspects of tumor characteristics. The correlation between the anti-BRD2 response and AFP was analyzed using Pearson's correlation analysis, expecting that AFP and TA autoantibody biomarkers would represent different characteristics of tumors. The correlation between the BRD2 autoantibody and AFP in sera was very low (Fig. 5A left panel; Pearson's coefficient, r=0.04240; P=0.5097), suggesting that the appearance of these two serum markers is regulated by unrelated mechanisms. However, the BRD2 autoantibody responses in patients with HCC were highly correlated with another HCC autoantibody biomarker, the ATIC autoantibody (Fig. 5A right panel; Pearson's coefficient, r=0.7749; P<0.0001). The ATIC autoantibody (16) was detected in the same cohort, using the BSA-miniPEG2-XC154p1 antigen which was prepared according to the procedures used for the generation of the BSA-conjugated XC246p9 antigen. This biomarker was observed to be significantly elevated in the serum of patients with HCC as compared with that of healthy subjects (Fig. S9A), with an AUC value of 0.8262 [Fig. S9B; 95% CI, 0.7700-0.8824, P<0.0001]. The sensitivity of the ELISA for ATIC autoantibody was 64.41%, and the specificity was 78.02% for the cut-off value of 0.6125 (Fig. S9B). The correlation between the ATIC autoantibody and the BRD2 autoantibody detection in sera was strong (Pearson's coefficient, r=0.7749; P<0.0001), although their responses were not identical (Fig. 5A). Pearson's correlation analysis also was performed on the individual cohorts. As shown in Fig. S10, the AFP and anti-BRD2 autoantibody biomarker pairs exhibited correlation coefficients of -0.025, 0.105, 0.217 and -0.226 in the HCC, normal, cirrhosis and benign cohorts, respectively, indicating that there was no significant correlation between the two biomarkers in all cohorts. By contrast, the autoantibody biomarker pair (anti-BRD2 and anti-ATIC) demonstrated positive correlation coefficients (0.795, 0.602, 0.706, and 0.460) in all cohorts, confirming the correlation between autoantibody biomarkers. The simultaneous detection of cancer biomarkers representing the different properties of cancer would enhance diagnostic accuracy. To examine the effects of the combined analysis of BRD2 or ATIC autoantibody biomarker and AFP, the response of each biomarker was simplified to positive or negative and scored as 1 or 0, depending on whether the detection value was higher or lower than the cutoff value. The combined analysis of these biomarkers was performed by the simple addition of each score, which resulted in values of 0, 1, 2, or 3 (Fig. 5B). The proportion of AFP-positive patients with HCC in the present cohort was 35.6%, and the percentage of BRD2 autoantibody-positive patients with HCC was 64.4%. The simultaneous detection of the two autoantibody biomarkers (anti-BRD2 and anti-ATIC) increased the proportion of biomarker-positive HCC patients (score 1 and 2) to 89.8%. In addition, the combined detection of AFP and two TA autoantibody biomarkers (score 1, 2 and 3) could be detected in up to 92.4% of the patients with HCC. However, a notable proportion of autoantibody-biomarker-positive cases (score 1 and 2) was also observed among the normal healthy participant cohort (44.0%). The diagnosis of HCC is straightforward when significantly increased serum AFP levels and definitive imaging features are present. However, AFP-negative hepatic cancer (ANHC) is not as easily diagnosed, as the majority of ANHCs are early and small HCCs, often without typical imaging characteristics (34). The diagnosis of ANHC is crucial in clinical practice, because ANHC accounts for nearly half of HCC cases and has a better prognosis compared to AFP-positive HCC (APHC). The BRD2 autoantibody biomarker was analyzed in patients with ANHC or APHC from the present HCC cohort (Fig. 5C). In the APHC group, 66.6% of the patients with HCC exhibited a positive BRD2 autoantibody biomarker response. The BRD2 autoantibody biomarker was also detected as positive in 63.2% of ANHC cases. The ATIC autoantibody response in APHC or ANHC cases was similar to that of the BRD2 autoantibody. However, ~20% of all patients with HCC were determined to be double-negative for these biomarkers (Fig. 5C).
Patients with HCC are known to be frequently asymptomatic, and the appearance of symptoms can signal the development of severe disease. Therefore, the early diagnosis of HCC followed by effective treatment is currently critical for improving the prognosis and reducing the associated economic burden (34). AFP is by far the most widely used serum HCC biomarker. However, its sensitivity for HCC diagnosis is only 41-65% with a specificity of 80-94% (34). The present study demonstrated that the anti-BRD2 response can be used to diagnose liver cancer that is not be screened by AFP (Fig. 5C). In the cohort used in the present study, AFP was detected only in 35.6% of the patients with HCC, whereas the BRD2 autoantibody was present in 64.4% of the patients with HCC. Among the AFP-positive patients with HCC, 66.6% were positive for the BRD2 auto-antibody and 63.2% of the AFP-negative patients with HCC were also positive for the BRD2 autoantibody biomarker. Non-responders to AFP, as well as the anti-BRD2 antibody biomarker comprised the remaining 23.7% of the patients with HCC. However, the fraction of non-responders to the HCC biomarkers was decreased to 7.6% by additional detection with the ATIC autoantibody (Fig. 5B). TA autoantibodies are biologically amplified signals, corresponding to TA antigens and hence may be measurable early on, designating them as promising early biomarkers. However, the antibody response to each antigen depends on the individual's immune system, and the amount of antibody will not be exactly proportional to the amount of antigen. Therefore, in cancer diagnosis using auto-antibody biomarkers, it is necessary to concurrently measure various autoantibodies in order to increase accuracy (11,12). The majority of autoantibody cancer biomarker studies intend to propose a multiple diagnostic autoantibody panel (11,12). From the results of the present study, it can also be confirmed that the diagnostic efficiency is improved by simultaneously measuring two autoantibodies and AFP and the potential of multiplex detection of autoantibody biomarkers with AFP as a liver cancer diagnosis. Additional verification is required of whether TA auto-antibodies are detected even in in individuals classified as normal without HCC. In the present study, the corresponding individuals appeared to be exposed to abnormal antigens released from tissues, which induce antigen-specific auto-antibodies, although their association with HCC is not evidenced. Autoantibody detection in normal subjects can be an important signal predicting inflammations or liver diseases. Follow-up of the serum donor may be required to confirm the disease-related characteristics of the serum donor. TA autoantigens, which confer neo-epitopes to the immune system, can be oncogenic drivers or support tumorigenesis. The overexpression of the TA antigen ATIC (16) can function as an oncogenic gene that promotes survival, proliferation, and migration by targeting AMPK-mTOR-S6 K1 signaling (35). The genetic deletion of fatty acid synthase (FASN), which is another TA antigen (33), suppresses the hepatocarcinogenesis driven by AKT and AKT/c-Met proto-oncogenes in mice (36), which implicates the crucial role of FASN during tumorigenesis. The abundance of BRD2, as well as its high genetic alternation rate (~19%) in patients with HCC have been shown to be associated with a worse overall survival (37). However, the oncogenic properties of upregulated BRD2 in liver cancer have not been directly confirmed, which can be examined in further studies. Although the characteristics of the neo-epitope that induce BRD2 autoantibody, including genetic alteration, post-translational modification, abundance, or localization, were not defined in detail in the present study, evidence was provided, suggesting the presence of a neo-epitope on oncogenic BRD2. The BRD2 autoantibody obtained from the HCC mouse model, XC246, exhibited an unexpected pattern of intracellular staining for BRD2 in tumor cells. In contrast to the commercial anti-BRD2 antibody, which stains BRD2 mainly in the nucleus, the BRD2 autoantibody, XC246, stained the cytoplasmic BRD2. Furthermore, the XC246 antibody detected the exosomal BRD2, which was not detected using the commercial antibody. It is anticipated that these characteristics of BRD2 are related to the neo-epitope, which generates the autoantibody. To develop a detection method for the serum BRD2 auto-antibody, the structural features of the neo-epitope must be reflected in the capture antigen used in ELISA. Therefore, the epitope mimicries from a conformational peptide library were screened, using the BRD2 autoantibody, and then applying it as an antigen in serum TA autoantibody ELISA, instead of the recombinant BRD2 protein. It has been previously demonstrated that cyclic peptide epitopes fused to streptavidin are sufficient to capture serum autoantibodies (16-18). In the present study, a synthetic cyclic peptide epitope-conjugated antigen was prepared, in order to simplify the preparation process of the coating antigen. In addition, BSA was used as a carrier protein to reduce non-specific binding in the human serum ELISA. Synthetic cyclic peptide epitopes fully mimicked the cellular antigenic property, as revealed by ELISA and competitive western blotting and were proven to be appropriate for serum ELISA. The ATIC autoantibody was also detected using peptide epitope-conjugated BSA instead of the streptavidin conjugate used in a previous study (16). These results demonstrated that BSA-conjugated mimotope peptides are sufficient for detecting specific autoantibodies, offering the possibility of its application to the detection of other autoantigenic epitope mimics (16-18,32,33). In conclusion, the present study suggested that the BRD2 autoantibody can be used as an HCC-associated biomarker and the feasibility of serum BRD2 autoantibody ELISA using a specific conformational epitope against the BRD2 autoantibody. Serum BRD2 autoantibody ELISA was also proposed as an accompanying test to serum AFP detection for the enhancement of HCC diagnostic efficiency. Further studies on the serum BRD2 autoantibody using large and well-defined cohorts are necessary to corroborate its usefulness for cancer diagnosis. | true | true | true |
PMC9635976 | Tingting Yu,Hong Yu,Dong Xiao,Xiangyan Cui | Human Bone Marrow Mesenchymal Stem Cell (hBMSCs)-Derived miR-29a-3p-Containing Exosomes Impede Laryngocarcinoma Cell Malignant Phenotypes by Inhibiting PTEN | 18-10-2022 | Although microRNA-29a-3p was reported to inhibit laryngocarcinoma progression, the potential mechanisms have not been explored clearly. Laryngocarcinoma tissues were collected for analyzing the levels of miR-29a-3p and phosphatase and tensin homolog (PTEN). The miR mimics or inhibitor was transfected into laryngocarcinoma cell lines M4E and Hep2 for the investigation of the biological functions (proliferative, invasion, migratory rates, and apoptotic rates) of this miRNA. The exosomes (Exo) from human bone marrow mesenchymal stem cells (hBMSCs) after the transfection of miR mimics/inhibitor/si-PTEN were isolated and used to stimulate M4E and Hep2 cells. The in vivo mouse model was constructed to verify our findings. The miR-29a-3p level was decreased, and PTEN level was elevated in laryngocarcinoma tissues and the cancer cell lines. MiR mimics could inhibit proliferative, invasive migratory rates while promoting apoptotic rates of M4E and Hep2 cells. MiR-29a-3p was enriched in hBMSC-derived Exo, and the Exo from miR-29a-3p mimics transfected hBMSCs could inhibit laryngocarcinoma cell malignant phenotypes in vitro and prevent tumor progression in vivo. In addition, the direct binding relationship between miR-29a-3p and PTEN in this disease was determined. In conclusion, hBMSC-derived Exo with upregulated miR-29a-3p inhibited laryngocarcinoma progression via regulating PTEN, providing a potential diagnostic and therapeutic target in this disease. | Human Bone Marrow Mesenchymal Stem Cell (hBMSCs)-Derived miR-29a-3p-Containing Exosomes Impede Laryngocarcinoma Cell Malignant Phenotypes by Inhibiting PTEN
Although microRNA-29a-3p was reported to inhibit laryngocarcinoma progression, the potential mechanisms have not been explored clearly. Laryngocarcinoma tissues were collected for analyzing the levels of miR-29a-3p and phosphatase and tensin homolog (PTEN). The miR mimics or inhibitor was transfected into laryngocarcinoma cell lines M4E and Hep2 for the investigation of the biological functions (proliferative, invasion, migratory rates, and apoptotic rates) of this miRNA. The exosomes (Exo) from human bone marrow mesenchymal stem cells (hBMSCs) after the transfection of miR mimics/inhibitor/si-PTEN were isolated and used to stimulate M4E and Hep2 cells. The in vivo mouse model was constructed to verify our findings. The miR-29a-3p level was decreased, and PTEN level was elevated in laryngocarcinoma tissues and the cancer cell lines. MiR mimics could inhibit proliferative, invasive migratory rates while promoting apoptotic rates of M4E and Hep2 cells. MiR-29a-3p was enriched in hBMSC-derived Exo, and the Exo from miR-29a-3p mimics transfected hBMSCs could inhibit laryngocarcinoma cell malignant phenotypes in vitro and prevent tumor progression in vivo. In addition, the direct binding relationship between miR-29a-3p and PTEN in this disease was determined. In conclusion, hBMSC-derived Exo with upregulated miR-29a-3p inhibited laryngocarcinoma progression via regulating PTEN, providing a potential diagnostic and therapeutic target in this disease.
As a common malignant tumor in the upper respiratory tract, laryngocarcinoma accounts for approximately 25 to 30% of cancer cases worldwide [1, 2]. Although several efficient therapies for this disease including surgical operation, and the combination of radiotherapy and chemotherapy, have been developed in recent years, the prognosis of patients at advanced stage is still poor [3]. Therefore, the investigation of the underlying mechanisms during laryngocarcinoma progression may be helpful to find novel treatment strategies in this disease. microRNAs (miRNAs) have been emerged as a class of novel noncoding RNA regulator, and their length are approximately 22 nucleotides in length [4]. Increasing evidences determined that the occurrence and development of laryngocarcinoma were involved in the dysregulation of a large number of miRNAs. For example, miR-552 promotes cell proliferative rate of laryngocarcinoma cells by modulating p53 signaling [5]. MiR-125b participates in the oncogenic role of lncRNA MALAT1 in the phenotypes including cell proliferative and invasive rates in laryngocarcinoma [6]. MiR-29a-3p, a miRNA, was identified as a tumor-suppressing factor in various human cancers such as endometrial cancer [7], gastric cancer [8], and cervical cancer [9]. Recently, few studies revealed that miR-29a-3p could suppress the proliferative and invasive rates in laryngocarcinoma cells [10, 11]. Although the tumor-suppressive role of this miRNA in laryngocarcinoma is known, its potential mechanisms remain unclear. Exosome (Exo) (30–200 nm) is a small single-membrane vesicle and exists in almost all mammalian cells [12, 13]. It has been reported that exosomes always act as the cargo carrier for miRNAs and is a promising therapeutic tool [14]. Previous studies indicated that exosomal miR-29a-3p derived from human bone mesenchymal stem cells (hBMSCs) could efficiently suppress cell migrative ability and vasculogenic mimicry in glioma [15]. Due to its role in laryngocarcinoma, we speculated that the exosomal miR-29a-3p derived from hBMSCs might also exert a potential impact in cancer progression. Phosphatase and tensin homolog (PTEN) is a well-known tumor suppressor protein and has been identified to participate in various tumor progression including prostate cancer [16], osteosarcoma [17], non-small-cell lung cancer [18], and laryngeal carcinomas [19]. Previous studies revealed that inhibition of miR-744-3p could restore PTEN expression, and then suppressed laryngeal squamous cell carcinoma progression [20], suggesting that upregulation of PTEN could contribute to cancer development. More interestingly, PTEN was identified to be one of the downstream genes of miR-29a-3p and participated in the regulation of this miRNA during human diseases [21, 22]. This study aimed to investigate the role of exosomal miR-29a-3p in laryngocarcinoma, as well as the regulatory relationship between miR-29a-3p and PTEN. In this study, we further verified the downregulation of miR-29a-3p in laryngocarcinoma. Notably, exosomal miR-29a-3p-derived hBMSCs could prevent against the development of this disease, and the impact was involved in the change of PTEN level. The binding relationship between miR-29a-3p and PTEN was also elucidated in our study. Our results provided that hBMSC-derived exosomes might be a potential treatment target for laryngocarcinoma.
A total of 30 laryngocarcinoma patients were recruited at the First Hospital of Jilin University, and the tumor tissues as well as the adjacent normal tissues were collected and snap-frozen in liquid nitrogen for the gene expression analysis. All patients have signed the informed consents.
Human bone marrow mesenchymal stem cells (hBMSCs), laryngocarcinoma cell lines SUN-899, M4E, TU212, and the normal pharyngeal epithelial cell line NP69 were obtained from Bank of Chinese Academy of Sciences (Shanghai, China). The Hep-2 cell line was purchased from American type culture collection (ATCC; Manassas, VA, USA). All cell types were grown within Dulbecco's Modified Eagle's Medium (DMEM) with 10% fetal bovine serum (FBS) in a 37°C incubator. To manipulate the expression levels of miRNA and PTEN in cells, the small interfering RNA for PTEN (si-PTEN) and nonspecifc control (si-NC) were synthesized from Shanghai GenePharma Co., Ltd, and miR mimics/miR-NC and miR inhibitor/inhibitor NC were purchased from Guangzhou Ribobio Co., Ltd. The siRNAs/mimics/inhibitor were transiently transfected into Hep2 and M4E cells under the help of Lipofectamine 2000 (Invitrogen).
The surface markers of hBMSCs (CD14, CD29, CD34, CD45, D73, CD90, and CD105) were analyzed by flow cytometry. HBMSCs on passage 4-6 were cultured with serum-free medium for 72 h, and the supernatant was centrifuged through differential centrifugation as previously described [23]. After that, the pellet was washed by phosphate buffered saline (PBS) for twice, and the final pellet was collected in PBS through 2 h of ultrahigh speed (100,000 × g), that is, hBMSC-derived exosomes (BMSC-Exo). The concentration of BMSC-Exo was determined by BCA kit (Beyotime, China). The BMSC-derived Exo was visualized under a transmission electron microscopy (TEM; Hitachi-7500; Tokyo, Japan). Meanwhile, the exosomes were lysed by RIPA lysis buffer to extract the Exo protein, and the Exo-specific markers (CD9, CD63, and TSG101) were detected by western blot. To obtain the hBMSC-derived Exo with gene expression change of interest genes, hBMSCs were transfected with siRNA/mimics/inhibitor, followed by the isolation of Exo.
To investigate the impact of hBMSC-derived Exo on biological functions of laryngocarcinoma cell lines, Exo derived from hBMSCs transfected with mimics/inhibitor/siRNAs and named by Exo miR-NC, Exo miR mimic, Exo si-PTEN, Exo si-NC, Exo miR inhibitor, Exo inhibitor NC, or Exo miR inhibitor + si-PTEN. Then, 200 μg hBMSC-Exo (after transfection) or negative control PBS were added into Hep2 and M4E cells. Two days later, cells were harvested for the subsequent function analysis.
Cell counting kit (CCK)-8 (Dojindo, Tokyo, Japan) was used to evaluate cell viabilities as previously reported [24]. Absorbance values at time point of 24, 48, 72, and 96 h were detected at 450 nm. For colony formation, 1 × 103 cells were plated into 6-well plates and cultured for two weeks. Then cells were fixed with methanol for 10 min and stained by 0.1% crystal violet for 15 min. The images were photographed with a light microscope, and colony numbers were counted by naked eyes.
Cells were seeded into 6-well plates with 1 × 106 cells/well and cultured overnight, and the supernatant was removed by centrifugation. By using the Annexin V-FITC cell apoptosis detection kit (Biovision, USA), cell pellets were incubated with 500 μL loading buffer, 5 μL Annexin V-FITC, and 10 μL propidium iodide (PI) solution for 15–20 min. Apoptosis rate was measured using the flow cytometry (BD Biosciences).
An 8 μm well size Transwell chamber (Corning, N.Y., USA) was used for Transwell assay. For cell invasion, 100 μL of 50 mg/L Matrigel (1 : 40) was coated into the upper surface of chamber bottom membrane. 100 μL of cell suspension with 2 × 105 cells was added to the upper chamber, and the lower chamber was filled with 600 μL 20% FBS contained DMEM medium. After 24 h, cells were fixed by methanol for 10 min and stained with 0.1% crystal violet for imaging and counting under five random fields. Cell migration assay was performed as same to invasion assay without coating the Matrigel.
Total RNA was extracted by TRIzol reagent (Invitrogen), and the cDNAs were synthesized using specific Reverse Transcription Kit (Applied Biosystems). The subsequent PCR reactions were conducted with SYBR green Super-mix (Thermo Fisher) on ABI 7500 PCR fast detection system. The gene expression analysis was evaluated based on the 2–ΔΔCT method. GAPDH/U6 was used as the normalization control. The sequences of primers in this study are as follows: miR-29a-3p, F: 5′ AACAGGTGACTGGTTAGACAA 3′, R: 5′ GTGCAGGGTCCGAGGT 3′; U6, F: 5′ TCGCTTCGGCAGCACATATACT 3′, R: 5′ ACGCTTCACGAATTTGCGTGT 3′; GAPDH, F: 5′ GGCCCAGAATGCAGTTCGCCTT 3′, R: 5′ AATGGCACCCTGCTCACGCA 3′; PTEN, F: 5′ CTTACAGTTGGGCCCTGTACCATCC 3′, R: 5′ TTTGATGCTGCCGGTAAACTCCACT 3′.
Total protein samples were extracted by RIPA buffer. A total of 50 μg protein sample/lane was separated by 10% SDS-PAGE. After transferring into PVDF membranes, the membrane was incubated with the appropriate primary antibodies CD9 (1 : 1000), CD63 (1 : 1000), Tsg101 (1 : 1000), Calnexin (1 : 1000), and PTEN (1 : 1000) overnight at 4°C, followed by the incubation with HRP-conjugated secondary antibody (1 : 10000) for 1 h. All antibodies were purchased from Abcam. The images of protein bands were developed using chemiluminescence reagent, and Image J software was used to gray value analysis.
The full length of PTEN 3′UTR covering wild-type or mutant miR-29a-3p binding sites were subcloned into pmirGLO dual-luciferase reporter vectors (Promega). After that, the recombinant vectors (PTEN WT and PTEN MUT (the sequence of 3′UTR of PTEN that predicted to interact with miR-29a-3p was mutated into base complementary sequences)) were individually cotransfected into M4E cells with miR mimics or miR-NC. Two days later, the luciferase activity was tested by a dual-luciferase reporter assay system.
PTEN biotin and PTEN nonbiotin labelled probes (Bio-PTEN and Bio-NC) were constructed by Shanghai GenePharma Co., Ltd. and treated with M-280 streptavidin-magnetic beads (Invitrogen) for 2 h at 4°C. Then M4E cells were lysed, and the whole lysates were incubated with probe-coated beads overnight at 4°C. Finally, the RNA was extracted and subjected to qRT-PCR analysis for miR-29a-3p enrichment.
A total of 20 BALB/c nude mice (4–6 weeks) were used to construct the animal model. Approximately 1 × 106 M4E cells were injected into flank regions of mice via subcutaneous injection as previously described [25]. After the mean tumor volume reaching 100 mm3, all mice were randomly divided into four groups: Exo miR-NC, Exo miR-29a-3p mimics, Exo miR-29a-3p inhibitor, and Exo miR-29a-3p mimics/inhibitor. Five mice were in each group. A dose of 200 μg/mouse hBMSC-derived Exo was intravenously injected into mice every two days for a total of ten times. Four weeks later, mice were killed by asphyxiation in a CO2 chamber; tumors were collected and weighted. The tumor volume was evaluated by (length × width2)/2. Meanwhile, the immunohistochemistry (IHC) assay was performed to assess Ki67 positive cells in tumor tissues using anti-Ki67 primary antibody (1 : 200, Abcam) as previously reported [26]. Slides were observed under a light microscope (Olympus, Tokyo, Japan).
All data were shown as mean ± standard deviation (SD), and each experiment was repeated for three independent times. The differences between groups were analyzed by using the paired Student's t-test followed by Tukey's post hoc tests when only two groups were compared, or by one-way analysis of variance (ANOVAs) followed by Dunnett's post hoc tests when more than two groups were compared. Statistical significance was set at p < 0.05.
Firstly, qRT-PCR analysis confirmed that miR-29a-3p level was dramatically decreased in laryngocarcinoma tissues from patients and cancer cell lines (Figures 1(a) and 1(b), all p < 0.01). On the contrary, PTEN expression was significantly elevated in tumor tissues and cancer cell lines (Figures 1(c) and 1(d), all p < 0.01). The levels of miR-29a-3p and PTEN in tumor tissues were negatively correlated (Figure 1(e), R2 = 0.679). Although previous studies have reported the binding sites between miR-29a-3p and PTEN 3′UTR sequence (Figure 1(f)), we reverified their interaction. After transfected with miR mimics in M4E cells, the luciferase activity of PTEN WT was significantly reduced, while the PTEN MUT remained unchanged (Figure 1(g), p < 0.01). RNA pull-down assay further determined their direct binding M4E cells (Figure 1(h), p < 0.001). In addition, after transfected with miR mimic M4E cells, PTEN level was notably reduced compared to miR-NC (Figure 1(i), p < 0.01). It postulates that this axis may play potential roles in laryngocarcinoma.
To further confirm its function, we transfected miR mimics into M4E and Hep2 cell lines, and the level of miR-29a-3p was determined by qRT-PCR analysis (Figure 2(a), both p < 0.001). As shown in Figures 2(b)–2(e) , we observed that the transfection of miR mimics notably reduced cell proliferative, invasive, and migratory rates while enhanced cell apoptotic rate of two cell lines compared to miR-NC (cell viability, both p < 0.05; colony number, both p < 0.01; cell invasion and cell migration, all p < 0.01; cell apoptosis, both p < 0.001). It suggests that miR-29a-3p upregulation can inhibit laryngocarcinoma cell malignant progression in vitro.
To isolate the exosomes from hBMSCs, we observed the morphology of hBMSCs (Figure 3(a)) and hBMSC-derived Exo (Figure 3(b)). Meanwhile, western blot analysis revealed that Exo surface makers CD9, CD63, and Tsg101 exhibited a relative high level, while Calnexin level showed a low level in BMSC-Exo than that in hBMSCs (Figure 3(c)). In addition, flow cytometry analysis indicated that hBMSCs' surface makers CD29 (98%), CD73 (97.36%), CD90 (96.43%), and CD105 (95.73%) were highly expressed, while CD14 (0.28%), CD34 (0.92%), and CD45 (2.35%) were barely expressed (Figure 3(d)). The data suggest that hBMSC-derived Exo was successfully isolated and can be used for the subsequent studies.
Subsequently, we isolated hBMSC-derived Exo, then stimulated M4E and Hep2 cells with PBS as the control. As shown in Figures 4(a)–4(d), the proliferative, invasive, and migratory rates of two cell lines were significantly suppressed, while cell apoptotic rate was oppositely enhanced by hBMSC-derived Exo compared to control group (cell viability: Hep-2, p < 0.01, M4E, p < 0.05; colony number, both p < 0.01; cell invasion and migration, all p < 0.01; cell apoptosis, both p < 0.001). Based on these results, we think that hBMSC-derived Exo can attenuate laryngocarcinoma cell progression.
Interestingly, we observed that miR-29a-3p level in hBMSC-derived Exo was notably higher than that in M4E and Hep2 cells (Figure 5(a), both p < 0.001). Then we introduced miR-29a-3p mimics into hBMSCs and isolated miR mimics transfected hBMSC-derived Exo to stimulate M4E and Hep2 cells. MiR-29a-3p level in miR mimics transfected hBMSC-derived Exo was significantly higher than that in miR-NC transfected hBMSC-derived Exo (Figure 5(b), p < 0.001). As shown in Figures 5(c)–5(f), we found that Exo miR-29a-3p mimics notably reduced the proliferative, invasive, and migratory rates of two cell lines, while it enhanced the apoptotic rate of two cell types compared to Exo miR-NC (cell viability, both p < 0.01; colony number, both p < 0.001; cell invasion and migration, all p < 0.001; cell apoptosis, both p < 0.001). Exo miR-29a-3p mimics reduced PTEN expression compared with Exo miR-NC, and Exo miR-29a-3p inhibitor enhanced PTEN expression in comparison to Exo inhibitor NC (Figure 5(g), mimic, both p < 0.01; inhibitor, both p < 0.05). These data indicate that the inhibition of hBMSC-derived Exo on laryngocarcinoma cell malignant progression is associated with the upregulation of miR-29a-3p.
To confirm our findings, we cointroduced miR inhibitor and si-PTEN into hBMSCs and isolated hBMSC-derived Exo. The levels of miR-29a-3p and PTEN in Exo miR-29a-3p inhibitor and Exo si-PTEN were both reduced compared to that in Exo inhibitor NC and Exo si-NC, respectively (Figures 6(a) and 6(b), both p < 0.001). As shown in Figures 6(c)–6(e), Exo miR-29a-3p inhibitor enhanced the proliferative, invasive, and migratory rates of two cell lines (cell viability, both p < 0.05; colony number, both p < 0.01; cell invasion and migration, all p < 0.05); Exo si-PTEN reduced the proliferative, invasive, and migratory rates of two cell lines (cell viability, both p < 0.05; colony number, both p < 0.05; cell invasion and migration, all p < 0.05), while cotreatment of Exo miR-29a-3p inhibitor and Exo si-PTEN notably attenuated the impacts of Exo miR-29a-3p inhibitor on these cell malignant phenotypes (cell viability, both p < 0.05; colony number, both p < 0.05; cell invasion and migration, all p < 0.05). These data suggest that exosomal miR-29a-3p from hBMSCs inhibits the laryngocarcinoma progression via PTEN.
Finally, the in vivo animal model was constructed to verify our findings. As shown in Figures 7(a)–7(c), we found that Exo miR-29a-3p mimics inhibited tumor growth, tumor volume, and weight (all p < 0.05), while Exo miR-29a-3p inhibitor enhanced tumor progression (all p < 0.01). From Ki67 staining results, the number of Ki67 positive cells in tumor tissues was significantly reduced by Exo miR-29a-3p mimics and elevated by Exo miR-29a-3p inhibitor (Figure 7(d), all p < 0.01). In addition, western blot analysis showed that Exo miR-29a-3p mimics decreased PTEN level (p < 0.01), and Exo miR-29a-3p inhibitor increased PTEN level in tumor tissues (p < 0.05) (Figure 7(e)). It indicates that hBMSC-derived Exo can inhibit laryngocarcinoma progression via exosomal miR-29a-3p mediated suppression of PTEN.
In the past decades, stimulating literatures have pointed out the potential of stem cell-derived exosomes in the treatment for different human cancers [27, 28]. Despite of these important significances, more molecular mechanisms involved in exosome treatment have not been well studied. In this study, we revealed that hBMSC-derived exosomes could efficiently inhibit laryngocarcinoma cell malignant phenotypes in vitro and tumor growth in vivo. Moreover, our data demonstrated that miR-29a-3p and PTEN regulation might account for the suppressive role of hBMSC-derived exosomes in laryngocarcinoma. This study contributed us to understand the potential action mechanism of stem cell-derived exosomes in cancer progression and also develop exosome therapeutic strategy. How to deliver exosomes with changed miRNA level to body and how to make it work without no side effect is becoming a big challenge in laryngocarcinoma. MiR-29a-3p was observed to be downregulated in many human cancers and thought as a potential tumor-suppressive factor [29]. MiR-29a-3p inhibited the proliferative, migratory, and invasive rates of endometrial cancer cells [7]. Downregulated miR-29a-3p might account for the tumor promotion of the lncRNA KCNQ1OT1 in hepatocellular carcinoma [30]. In addition, miR-29a-3p was also shown to inhibit the proliferative ability of prolactinoma cells via modulating the β-catenin signaling pathway [31]. Although the oncogenic role of this miRNA in laryngocarcinoma has been reported, its deliver from hBMSCs and the complex mechanisms remains unclear. Here, we found that miR-29a-3p was significantly enriched in hBMSC-derived exosomes. HBMSC-derived exosomes and hBMSC-derived exosomes with upregulated miR-29a-3p could both inhibit laryngocarcinoma cell malignant phenotypes. The results in vivo animal model were keeping with that in vitro cell experiments. The hBMSC-derived exosomes carrying miRNA mimics might be a novel therapeutic strategy for laryngocarcinoma and other miR-29a-3p downregulated human cancers. PTEN is a tumor-related gene and was identified to be associated with the process of multiple malignant tumors including prostate cancer [32], acute myeloid leukemia [33], papillary thyroid cancer [34], and also laryngocarcinoma [35]. A series of previous studies indicated that PTEN was a downstream mRNA target of miR-29a-3p and participated in the regulation of this miRNA in tumor progression. For example, miR-29a-3p regulated the development and progression of abdominal aortic aneurysm via direct interaction with PTEN [21]. MiR-29-3p also regulated the progression of hepatocellular carcinoma through targeting PTEN followed by the NF-κB signaling pathway [36]. However, there were few related reports about the regulation of miR-29a-3p and PTEN in the development of laryngocarcinoma. Our study revealed the binding relationship between miR-29a-3p and PTEN, and further confirmed that this axis closely impacted the progression of laryngocarcinoma by performing the rescue experiments. As we known, one miRNA might target one or multiple downstream mRNAs simultaneously in eukaryotic cells. Except for PTEN, whether there were other potential target mRNAs that participated in regulation of miR-29a-3p in laryngocarcinoma, which should be well investigated in the subsequent experiments. Interestingly, several potential targets of miR-29a-3p have been reported in previous studies including STX17 [37], COL4A1 [38], CCNT2 [39], HDAC4 [40], and so on. We will explore these potential mechanisms in the future. The PTEN/PI3K/AKT axis is reported to implicate in diverse physiological and pathological conditions and plays an important role in the regulation of cell growth and apoptosis in diverse human cancer including prostate cancer, hepatocellular carcinoma, and pancreatic cancer [41–44]. As the key target of PTEN, PTEN is identified to negatively regulate PI3K/AKT signaling [45, 46]. Based on the regulation relationship between PTEN and PI3K/AKT signaling, we speculated that the effect of miR-2a-3p/PTEN regulation axis in laryngocarcinoma might be also mediated by PI3K/AKT signaling. This needed to be further investigated in the subsequent experiments.
In summary, our findings thought that hBMSC-derived exosomes could inhibit laryngocarcinoma progression via exosomal miR-29a-3p-mediated suppression of PTEN, suggesting that exosome therapy might be a promising choice for this disease. | true | true | true |
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PMC9636192 | Ryuji Owada,Yohei Kakuta,Kosuke Yoshida,Shinichi Mitsui,Kazuhiro Nakamura | Conditioned medium from BV2 microglial cells having polyleucine specifically alters startle response in mice | 04-11-2022 | Diseases of the nervous system,Neurological disorders | Repeat-associated non-AUG translation (RAN translation) is observed in transcripts that are causative for polyglutamine (polyQ) diseases and generates proteins with mono amino acid tracts such as polyalanine (polyA), polyleucine (polyL) and polyserine (polyS) in neurons, astrocytes and microglia. We have previously shown that microglia with aggregated polyQ led to defective differentiation and degeneration of neuron-like cells. However, it has not been determined whether only microglia containing a specific RAN product, but not other RAN products, is harmful in vitro and in vivo. Here we show that polyL-incorporating microglia specifically led to altered startle response in mice. Aggregated polyA, polyS and polyL induced aberrant differentiation of microglia-like BV2 cells. Differentiated PC12 cells treated with conditioned medium (CM) of polyS- and polyL- but not polyA-incorporating microglia-like BV2 cells showed retraction of neurites and loss of branch of neurites. Injection of the polyL-CM, but not polyA-CM and polyS-CM, into the lateral ventricle lowered startle response in mice. Consistently, polyL induced the highest expression of CD68 in BV2 cells. The lowered startle response was replicated in mice given the polyL-CM in the caudal pontine reticular nucleus (PnC), the key region of startle response. Thus, endogenous RAN proteins having polyL derived from polyQ diseases-causative genes in microglia might specifically impair startle response. | Conditioned medium from BV2 microglial cells having polyleucine specifically alters startle response in mice
Repeat-associated non-AUG translation (RAN translation) is observed in transcripts that are causative for polyglutamine (polyQ) diseases and generates proteins with mono amino acid tracts such as polyalanine (polyA), polyleucine (polyL) and polyserine (polyS) in neurons, astrocytes and microglia. We have previously shown that microglia with aggregated polyQ led to defective differentiation and degeneration of neuron-like cells. However, it has not been determined whether only microglia containing a specific RAN product, but not other RAN products, is harmful in vitro and in vivo. Here we show that polyL-incorporating microglia specifically led to altered startle response in mice. Aggregated polyA, polyS and polyL induced aberrant differentiation of microglia-like BV2 cells. Differentiated PC12 cells treated with conditioned medium (CM) of polyS- and polyL- but not polyA-incorporating microglia-like BV2 cells showed retraction of neurites and loss of branch of neurites. Injection of the polyL-CM, but not polyA-CM and polyS-CM, into the lateral ventricle lowered startle response in mice. Consistently, polyL induced the highest expression of CD68 in BV2 cells. The lowered startle response was replicated in mice given the polyL-CM in the caudal pontine reticular nucleus (PnC), the key region of startle response. Thus, endogenous RAN proteins having polyL derived from polyQ diseases-causative genes in microglia might specifically impair startle response.
Polyglutamine (polyQ) diseases include multiple neurodegenerative disorders such as dentatorubropallidoluysian atrophy, spinal and bulbar muscular atrophy, several types of spinocerebellar ataxia (SCA) and Huntington’s disease (HD). The region that is responsible for the pathogenesis is polyQ tract in the causative protein of each polyQ disease. When the polyQ repeats are expanded above the thresholds, the symptoms are observed. The extended polyQ repeats are brought by hereditarily expanded CAG repeats in the corresponding causative genes. Neuronal dysfunctions and cell death in polyQ diseases can be mainly explained in a cell-autonomous fashion. The extended polyQ in neurons is prone to form oligomers and fibrils in the cytoplasm and the nucleus where these aggregates exert toxicity to neurons. However, neural impairments in a non cell-autonomous fashion have been also proposed in polyQ diseases based on some observations. Microglia plays crucial roles in physiological brain functions via phagocytosis and scanning of the CNS. However, microglia can also give toxic effects to the brain. For instance, neurons in mice having mutant Huntingtin (HTT), the causative protein of HD, only in microglia had high cell death rate under sterile inflammation condition. Likewise, cultured microglial cells having aggregated peptide with 69 glutamine repeat (69Q) showed morphological changes and its conditioned medium (CM) induced degeneration of neuron-like cells. The non cell-autonomous effects by microglia having mutant polyQ might be associated with polyQ diseases because nuclear mutant HTT inclusions were detected in microglial cells in the frontal cortex of adult-onset HD and in the frontal cortex and striatum of juvenile-onset HD. Recently, repeat-associated non-AUG (RAN) translation from polyQ diseases-causative genes with CAG repeats has been reported. Upon the RAN translation, proteins containing polyalanine (polyA), polyserine (polyS), polyleucine (polyL) and polycystein (polyC) can be theoretically generated in addition to polyQ. PolyQ and polyA RAN proteins were generated from an ataxin 3 gene, a causative gene for SCA type 3. Similarly, polyA-, polyS-, polyL- and polyC-containing proteins were detected in the brain from patients with HD. The RAN proteins are likely toxic to the brain because injection of polyS and polyL aggregates into the lateral ventricle led to altered brain functions in mice. Notably, the RAN proteins were found in neurons, astrocytes and microglia in the caudate and putamen from patients with HD. Although contributions of polyQ- incorporating microglia to neural dysfunctions have been clarified as mentioned above, it is still elusive if brain functions are influenced by microglial cells having the RAN products. Aggregated misfolded proteins such as HTT have the general ability to propagate between cells. In line with this phenomenon, we reported that aggregated 13A, 13S and 13L entered PC12 cells and some of them changed their morphology. Especially, microglial cells are expected to facilitate such propagation because microglial cells phagocytose cell debris and damaged neurons. Indeed, we have previously shown that fluorescence-labeled polyQ was spontaneously taken up by BV2 microglia cells, which resulted in morphological changes of the cells. Thus, it is likely to predict that the RAN products are also taken up by microglial cells. In the present investigation, we particularly focused on polyL among the multiple RAN proteins. PolyL encoded by mixed DNA repeats was reported to be more toxic than polyQ in a mammalian cell line, although it is not known if that depends on cell types or that is a general phenomenon. We introduced aggregated polyL peptide into cultured microglia-like BV2 cells and employed morphological analysis. Then, degeneration of neuron-like cells in culture in the presence of the CM from the polyL-incorporating BV2 cells was studied. Finally, brain functions of the mice given the CM were studied.
Among the RAN proteins that are generated from polyQ disease-causative genes, polyC peptide with 13 cysteine repeat was difficult to synthesize, whereas, polyA, polyS, polyL having 13 repeats of the corresponding amino acids (13A, 13S and 13L) could be obtained and aggregated. Thus, we sought to study toxicity of microglia having either of the three RAN peptides inside. We added aggregated 13A, 13S and 13L to culture medium of BV2 microglia at a final concentration of 10 µg/ml. Four days after the addition of the aggregates, serial sectional images by confocal microscopy revealed localization of the 13A (Fig. 1a), 13S (Fig. 1b) and 13L (Fig. 1c) inside the cells (Fig. 1) as seen in PC12 cells. Ortho images of the XZ-axis cross section view verified that the 13A (Fig. 1d), 13S (Fig. 1e) and 13L (Fig. 1f) aggregates distributed between cytoplasmic phalloidin signals. These results confirmed that the three aggregates were taken up by microglial cells. Quantification of the cells revealed that 13A, 13S and 13L were taken up by 67%, 65% and 93% of BV2 cells, respectively. We previously reported that microglial cells were changed when the cells phagocytosed polyQ aggregates. To check if 13A, 13S and 13L were also the case, we added the aggregates to medium of BV2 microglial cells. Then, we excluded the aggregates from the medium and cultured the cells 3 days more (Fig. 2a). As the control, we also included TAMRA-treated cells because the three peptides are TAMRA-conjugated. Then, morphology of the cells was quantitatively estimated. For the quantification, we used three parameters: the area of cell body, total length of branches and thickness of proximal branches as previously reported. The area of cell body in 13S- but not 13A- and 13L-treated BV2 microglia was larger than TAMRA-treated cells (Fig. 2b,c). Similarly, total lengths of branches in 13A- and 13S- but not 13L-treated cells were significantly shorter than TAMRA-treated cells (Fig. 2b,d). However, proximal branches were thicker in 13A-, 13S- and 13L-treated cells than TAMRA-treated cells (Fig. 2b,e). Thus, the three aggregates led to changes in morphology but with different shapes each other. In contrast, viability of BV2 cells was not changed in the presence of 13A, 13S or 13L (Fig. 2f). We then examined state of BV2 cells using an another index. CD68 expression became higher in BV2 cells by addition of LPS, indicating that CD68 expression likely reflects pathological state of microglia. BV2 cells were stained with CD68 antibody and the signal intensities of the cells having visible aggregates were quantified. As shown in Fig. 3a,b, 13L led to significantly higher CD68 expression than 13A and 13S. Faint CD68 signals were also detected in cells without visible large aggregates, probably due to presence of invisible very small aggregates. Thus, 13L likely transformed BV2 cells into pathologically relevant cells.
CM includes the factor(s) secreted from cultured microglia-like BV2 cells having the aggregates inside. We previously reported that CM derived from polyQ-treated BV2 microglia induced neurite retraction in differentiated neuron-like PC12 cells. Hence, we decided to culture PC12 cells with CMs collected as shown in Fig. 2a. To study degeneration of neuron-like PC12 cells in the presence of CMs, neurites of PC12 cells were needed to be fully elongated before the addition of CMs. To this end, the cells were initially cultured for 5 days in the presence of NGF. NGF was removed on day 5 and the cells were further cultured for 4 days in the presence of CMs (Fig. 4a). To see degeneration of cells, retraction of neurites was estimated using 2 indices; total length of neurites of cells and number of branch point of neurites. 13S-CM and 13L-CM induced significantly shorter neurites than that by TAMRA-CM (Fig. 4b,c). However, 13A-CM did not lead to shorter neurites (Fig. 4b,c). Numbers of branch point of the neurites were significantly fewer in 13S-CM- and 13L-CM-treated PC12 cells than in TAMRA-CM-treated cells. However, 13A-CM did not alter the number (Fig. 4b,d). Again, viability of PC12 cells was not changed in the presence of 13A-CM, 13S-CM or 13L-CM (Fig. 4e).
Given the degeneration of neuron-like PC12 cells by CMs derived from 13S-treated and 13L-treated BV2 microglial cells, we sought to address the question whether the CMs also affect brain functions in mice. Since the ventricular system widely spreads in the brain, the CMs were injected into right lateral ventricle and did a battery of behavioral tests the next day. We recently showed that injection of 13S and 13L themselves into the lateral ventricle altered anxiety and depression or stress-coping behavior in mice, as proved by elevated plus maze and forced swim tests, respectively. Therefore, we carried out an elevated plus maze test using 13A-, 13S- and 13L-CMs. The mouse was first put on the center area and was allowed to freely enter the open and closed arms. The mice with 13A-, 13S- and 13L-CM in the ventricle spent time comparable to that in mice given TAMRA-CM (Fig. 5a). Similarly, spontaneous motor activities on the maze were not different among the groups because no differences in total number of entries into the arms were found (Fig. 5b). These results indicate that any CMs did not affect anxiety. As a behavioral test for a different kind of emotion, we tested active behavior using resident-intruder test. The male mouse that has been a resident in a cage shows active behavior (approaching, chasing or sniffing) against a male intruder mouse. The total time for the resident mice showing active behavior were not different among the 4 groups (Fig. 5c). The next test we employed was three chamber test. The time a subject mouse spent in the chamber with a mouse was not different among the groups (Fig. 5d), suggesting no difference in the social behavior. Then, a novel mouse was added to the cage that had been empty in the last trial, leaving the old mouse in another chamber. Likewise, the time in the chamber with the novel mouse was not different (Fig. 5e), indicating no difference in social memory. Injection of 13S and 13L themselves into the lateral ventricle did not essentially change open-field performances. Similarly, when we checked general motor activity in an open-field after injection of the CMs into the lateral ventricle, there were no differences in total moving duration (Fig. 5f), total walking distance (Fig. 5g), total number of movements (Fig. 5h), average speed of locomotion for 10 min (Fig. 5i), moving speed (Fig. 5j), distance per movement (Fig. 5k), duration per movement (Fig. 5l) and percentage of time spent in center area (Fig. 5m) among TAMRA-, 13A-, 13S- and 13L-CM injected mice. Startle response is a fast twitch of muscles elicited by sudden and intense stimuli. The physiological significance of this response is hypothesized to protects animals from injury by a predator. Acoustic, tactile and visual stimuli can evoke the response. We tested acoustic startle response after sound pressure level at 110 and 120 dB. There were no significant differences in startle response at 110 dB. However, 13L-CM, but not 13A- and 13S-CMs, evoked significantly smaller startle response than TAMRA-CM at 120 dB (Fig. 6a). The magnitude of acoustic startle response is diminished by preceding non-startling acoustic stimulation (prepulse). This is called prepulse inhibition (PPI), which theoretically reflects sensorimotor gating. The prepulse and startling sound levels used in the experiment were 74, 78, 82 dB and 110, 120 dB, respectively. No significant differences in PPI were detected by any combination of prepulses and startling sounds among the 4 groups (Fig. 6b). Collectively, only 13L-BV2-CM led to reduced startle response at 120 dB. Since 13L-CM in the lateral ventricle altered only startle response, we searched for the responsible brain region. The neuronal pathway controlling acoustic startle response includes the auditory nerve, the ventral cochlear nucleus, the dorsal nucleus of the lateral lemniscus, the caudal pontine reticular nucleus (PnC) and spinal motor neurons. Among them, neurons located in the PnC play key roles in primary acoustic startle pathway. We measured startle response 1 day after injection of 13L-BV2-CM into the PnC. As shown in Fig. 7a, magnitude of startle response at 120 dB was lower in 13L-CM-injected mice than TAMRA-CM-injected mice. In contrast, a sound level at 110 dB did not elicit the difference. However, the startle response at 120 dB was not different between the two groups when the CMs heated at 100 °C for 10 min were injected into the PnC (Fig. 7b). Therefore, the responsible factor(s) released from BV2 microglial cells might be proteins because the heat denaturation of proteins is essentially irreversible at temperatures higher than 80 °C. The results after injection of the CM into the PnC (Fig. 7a) were same as those after injection of the CM into the lateral ventricle (Fig. 6a). Using mice given 13L-CM in the PnC, we counted the number of the giant neurons in the PnC because the neurons are involved in neural circuit of acoustic startle response. The number was not different between TAMRA-CM-injected mice and 13L-CM-injected mice (Fig. 7c,d). Likewise, the size of the neurons was identical between the 2 groups (Fig. 7c,e). These results are in line with the finding that 13L-CM did not change viability of PC12 cells (Fig. 4e).
Theoretically, endogenous RAN proteins could be translated by polyQ-deaseses-causative genes in the microglial cells in the brain, and also the aggregated RAN products in damaged neurons could be phagocytosed by microglia. Therefore, we mimicked the situation where aggregated RAN proteins are located in the microglial cells by introducing pre-aggregated polyA, polyS and polyL peptides in microglia. The three peptides have common flanking sequences (KKW and KK) at both sides of the core repeats (A13, S13 and L13). Since the three peptides have a fluorophore TAMRA, we also treated microglial cells with TAMRA as the control. Scrambled peptides with different order of amino acids are generally used to determine the specific effects of the sequences of peptides. However, we could not prepare the single scrambled peptide because we used three different peptides. Nevertheless, we could suggest specific contribution of the sequences of polyL and polyS to functions of the microglia because CM from polyA-treated microglia did not show any morphological changes of PC12 cells. Therefore, polyA works as the negative control. Prion-like propagation of proteins is an intriguing phenomenon in which aggregated proteins spontaneously transmit from a cell to other cells. We found uptake of aggregated polyA, polyS and polyL peptides by BV2 microglial cells. Misfolded proteins that are responsible for neurodegenerative disorders including aggregated HTT with expanded polyQ have such ability. As the mechanisms of the transfer of polyQ-containing proteins, an interaction of polyQ aggregates with the cell membrane and clathrin-dependent endocytosis have been suggested. We have recently shown that aggregated polyA, polyS and polyL peptides were also taken up by neuron-like PC12 cells. The entry of the polyS and polyL into PC12 cells seems to use endocytosis because of the presence of the invaginated pits from the cell membrane containing the aggregates inside them, as proved by electron microscopic analysis. In neurodegenerative disorders, the microglial cells are changed and migrate to the lesion sites where the cells phagocytose damaged neurons and cell debris. Phagocytosis is a type of endocytosis but uses clathrin-independent mechanism. There are multiple types of receptors to initiate microglial phagocytosis such as Toll-like receptors with a high affinity to microbial pathogens and triggering receptor expressed on myeloid cells 2 (TREM-2). Regarding phagocytosis of polyQ-containing protein, prion-like transmission of neuronal HTT aggregates to phagocytic glia was observed in the Drosophila brain. Uptake of aggregated polyA, polyS and polyL by BV2 microglia in this study raises a possibility that not only polyQ proteins themselves but their RAN products have a potential to exert the prion-like transmission to phagocytic microglia. There is a need to specify the mechanisms that enable phagocytosis of polyA-, polyS- and polyL-containing proteins. It is of particular important to determine whether phagocytosis of polyA, polyS and polyL by microglia uses a common receptor. The nature of microglia is closely related to its morphology. Pathogenic insults change the appearance of microglia from a highly ramified morphology to an amoeboid shape. We therefore examined morphological changes of BV2 microglia after uptake of 13A, 13S and 13L. We applied three indices that reflect the shape of BV2 microglia. When microglial cells display amoeboid shape, large area of the cell, short length of branches and thick proximal branches are likely observed. The morphological changes by 13A, 13S and 13L were not identical. 13S led to the most prominent change in the morphology, showing larger area of cell body, shorter length of branches and thicker proximal branches. In contrast, 13A brought only shorter length of branches and thicker proximal branches with no difference in the area of the cell. Likewise, 13L induced only thicker proximal branches. Thus, biggest morphological changes were seen in 13S-incorporating BV2 microglia rather than 13A- and 13L-incorporating cells. However, CD68 expression level was highest in BV2 cells having 13L. Thus, the morphological changes and CD68 level seem not to reflect a same state of microglia. Consistently, toxicity of CMs from 13S- and 13L-incorporating microglia to PC12 cells was almost identical. 13S-CM and 13L-CM induced both shorter length of neurite and fewer number of branch point in PC12 cells, whereas, 13A-CM did not induce any morphological changes. Thus, degree of morphological changes of BV2 microglial cells seems not to be completely correlated with the toxicity of factor(s) released from BV2 microglial cells. Both short length of neurites and few number of branch point were observed in PC12 cells treated with CM from polyQ-incorporating microglia. Thus, 13L-incorporating microglia and those having polyQ might release same factor(s) that are toxic to neurons. It was reported that CM from microglia from human HD did not overtly affect the viability of striatal neurons from human pluripotent stem cell. In line with this observation, viability of PC12 cells was not changed in the presence of 13L-BV2-CM. However, we cannot exclude a possibility that enhanced new cell genesis by the CM hid the higher cell death in the in vitro viability assay. TNFα and IL-6 are candidates of the toxic factor(s) released from 13L-treated BV2 microglial cells based on the following observations associated with polyQ diseases. ATXN1[82Q] mice, a model for SCA type 1, showed significantly higher TNFα, MCP-1 and IL-6 mRNA levels in the cerebellum at 12 weeks old. Even at 4 weeks old, the mice showed significantly higher TNFα and MCP-1 mRNA levels, but that of IL-6 was not significantly higher. In humans, ELISA assay clarified elevated levels of TNFα and IL-6 in the plasma from patients with moderate HD. Moreover, IL-6 mRNA level was elevated in the striatum from HD individuals. As autonomous microglial changes by mutant HTT, increased TNFα and IL-6 mRNA levels were observed. Similarly, TNF and IL-6 were elevated in HD pluripotent stem cell-derived microglia with longer polyQ repeat after stimulation with LPS and IFN. However, the amount of TNFα in 13L-BV2-CM (117 ± 42 pg/ml) was not different from that in TAMRA-BV2-CM (107 ± 13 pg/ml) (p = 0.08) and IL-6 levels were below the detection limit. Thus, the toxic factor(s) that are released from 13L-treated BV2 cells have not been currently specified. The undefined factor(s) might finally induce reactive oxygen species. HD pluripotent stem cell-derived microglia released high levels of reactive oxygen species, which can directly cause damage to neurons by oxidative effects on the lipids, nucleic acids and proteins. Indeed, addition of a low concentration of hydrogen peroxide induced neurite degeneration without cell death in culture. Whatever the case, the finding that 13L-CM, but not 13A-CM and 13S-CM, in the ventricle specifically lowered startle response clearly indicates that the degree of toxicity of the released factors is differential among the three RAN products. Thus, inhibition of the release of polyL-specific factors by microglia likely prevents the effect on startle response. In BV2 microglial cells, part of phagocytosed 13L co-localized with a lysosome marker LAMP1 (Supplementary Fig. S1 online), indicating that 13L might potentially undergo lysosomal degradation. Nevertheless, abundant 13L aggregates were located in the cytoplasm of BV2 cells. Thus, 13L that did not undergo lysosomal degradation in endogenous microglia in the mouse brain likely changes the functions and promotes the release of the polyL-specific factors from microglia. The 13L-CM taken at early time point (from day 2 to day 4 after the addition of 13L) changed the functions in vitro and in vivo (Fig. 2). The 13L-CM collected at later time point (from day 5 to day 6) also induced same morphological changes in PC12 cells (Supplementary Fig. S2 online). Therefore, the factor(s) seem to be released at least until day 6 in BV2 cells. We recently reported that injection of 13L itself into the lateral ventricle changed brain functions in mice. The behavioral tests were carried out the next day after 13L injection. To compare the results with those after injection of 13L-BV2-CM, we also did behavioral tests the following day after injection of the CM in this study. One day after injection might not be enough to obtain full recovery from the operation. Therefore, we cannot exclude a possibility that behavioral results might be different at later time points after the injection. The application of 13L itself and that of 13L-BV2-CM led to different results in vitro and in vivo. An increased anxiety as evidenced by elevated plus maze test was recognized in mice to which 13L itself was injected into the lateral ventricle. By contrast, when 13L-BV2-CM was injected into the lateral ventricle of mice, their performances on the maze did not differ from TAMRA-BV2-CM-injected mice. Therefore, the increase in anxiety seen in 13L-injected mice could be ascribable to toxicity of aggregates to neurons in a microglia-independent manner. In contrast, addition of 13L itself to neuron-like PC12 cells did not induce neurite retraction, whereas, 13L-BV2-CM did cause the retraction in vitro. It is possible that resident microglial cells in the brain having endogenous polyL-containing RAN proteins impair functions of adjacent neurons in a non cell-autonomous manner. In line with this assumption, resident microglia in the PnC captured 13L in the mouse brain. Thus, it is plausible to predict that polyL and polyL-containing microglia differentially affect neuronal functions in the brain. Regarding startle response of model mice for polyQ diseases, the R6/2 transgenic mouse line, a model for HD, exhibited comparable startle levels after acoustic stimuli at 105 dB and 120 dB to wild-type mice during young stages. However, the levels became lower in the model mice at 12.5 weeks of age. Likewise, the startle magnitude was higher at some ages but was lower at the different ages in the HdhQ92 line, a knock-in model mouse for HD. We showed an involvement of CM from 13L-treated BV2 microglia in startle response. Our data implies that not only polyQ in neurons but its RAN product in microglia decreases acoustic startle response. However, the lower response was seen at 120 dB but not at 110 dB. Therefore, the functional defects by 13L-CM that are closely linked to startle response might be subtle, thereby making a difference specifically after a strong startling stimulus. Iba 1-positive microglia captured injected 13L in the PnC (Supplementary Fig. S3), indicating that endogenous microglia can recognize polyL. However, it has not been determined if the capture in vivo leads to lower startle response as seen by injection of 13L-BV2-CM. Subpopulation of giant reticulospinal neurons in the PnC receive direct acoustic input from the central auditory pathway. Then, the PnC neurons project to spinal motor neurons and are therefore regarded as sensorimotor interfaces for the components of acoustic startle response. The lower startle response observed when 13L-CM was injected into the PnC implies that either axon terminals of neurons of the central auditory system in the PnC or giant reticulospinal neurons themselves in the PnC was impaired. Although we could not find differences in the density and size of the giant neurons, the subtle changes might be present given the changes of neurites in PC12 cells. Inspection with electron microscope will clarify this point in future. Alternatively, neuronal activity of the giant neurons after the startle stimuli might be changed. Although R6/2 and YAC128 mice, models for HD, developed deficits in PPI of startle reflex, CM from 13L-treated BV2 microglia in the lateral ventricle did not lead to defective PPI. The brain regions responsible for modulation of startle response by prepulse are broadly distributed. The regions include multiple regions such as pedunculopontine tegmental nucleus, laterodorsal tegmental nucleus, locus coeruleus and substantia nigra, pars reticulate. Thus, the PnC neurons might be functionally vulnerable to 13L-BV2-CM than other regions. The sensitivity of each brain region other than the PnC to 13L-BV2-CM will be determined by behavioral tests using mice given the CM into each region in near future.
The sequences of the synthesized TAMRA-labeled 13A, 13S and 13L were 5-TAMRA-KKWAAAAAAAAAAAAAKK-NH2, 5-TAMRA-KKWSSSSSSSSSSSSSKK-NH2 and 5-TAMRA-KKWLLLLLLLLLLLLLKK-NH2 (GL Biochem, Shanghai, China), respectively. The purity of 13A and 13S were more than 95%. However, that of 13L was 75% because purification of the peptide was difficult. The stock solutions of the peptides were prepared by dissolving the powder in a 1:1 mixture of trifluoroacetic acid and hexafluoroisopropanol. The appropriate conditions to induce aggregation of the peptides were determined in our previous literature. 13L had highest aggregation property among the three peptides. It aggregated immediately after addition into culture media at a concentration of 10 µg/ml. By contrast, 13A and 13S needed long time incubation in aqueous solution for aggregation. Prior to addition to culture media, 13A and 13S were incubated in aqueous solution (1 mg/ml) for 7 days at 37 °C without shaking and for 2 days at 37 °C with shaking at 206 rpm/min., respectively, to induce aggregation.
BV2 microglial cell line was kindly provided by Dr. Choi (Korea University). The cell line was cultured in DMEM containing 10% fetal bovine serum (FBS) and 1% penicillin–streptomycin mixed solution at 37 °C with 5% CO2. BV2 cells were plated on micro cover glass in 6-well plates at density of 8.0 × 104 cells per well. 13A, 13S and 13L were applied to the culture medium to be a final concentration of 10 µg/ml. The next day, the medium was removed and cultured cells were washed with PBS once to remove the peptides. Then, the cells were further cultured for 3 days more in the same culture medium without peptide to collect CM. As a different protocol, the culture medium was changed 4 days after the addition of 13L to BV2 cells. Then, BV2 cells were cultured for 2 days more to collect CM (Supplementary Fig. S2a). ELISA assay was performed using ELISA MAX Deluxe Set Mouse TNFα and IL-6 (BioLegend, San Diego, CA) according to the manufacturer’s instruction.
PC12 cell was purchased from RIKEN BRC. PC12 cells were plated on micro cover glass coated with laminin in 24-well plates at density of 5 × 103 cells per well. Differentiation of the cells was induced for 5 days in the presence of 50 ng/ml NGF in DMEM containing 1% FBS and 0.25% BSA. Then, the cells were further cultured for 4 days in the presence of CM without NGF.
Morphology of cultured cells and mouse neurons were quantified using Image J software as previously reported. The original images were acquired by the software and the actual scale in the images was reflected to the acquired images. Then, length and area of interest were measured by tracing them using tools of the software. Because the length less than 1 µm was difficult to trace, the processes longer than 1 µm were measured. The area of cell body, total length of branches and thickness of proximal branches of BV2 microglia, the total length of neurites and number of branch points of the neurites of PC12 cells and area of cell body of the PnC neurons in mice were measured. Intensity of CD68 signals in BV2 cells was also measured using Image J software. Viability of cultured cells was determined using Cell Counting Kit-8 (Dojindo Molecular Technologies Inc., Rockville, MD) according to the manufacturer’s instruction.
All experimental protocols were approved by the Animal Resource Committees of Gunma University. All methods were carried out in accordance with relevant guidelines and regulations (NIH) and were reported in accordance with ARRIVE guidelines for the reporting of animal experiments. The number of mice used for experiments was bare minimum to obtain reliable data and we made every effort to minimize the suffering of mice during experiments. Mice were kept in specific pathogen-free conditions in a room where the temperature and light/dark cycle were constant (23 °C and 12 h, respectively). Male and female ICR mice 2–3 months of age were anesthetized with isoflurane and were fixed by stereotaxic instrument. Then, the mice received injection of CM. Two µl of CM or 13L (100 µg/ml) was injected into the PnC (AP − 5.35 mm; ML + 0.5 mm; DV − 5.6 mm). Five µl of CM was injected into the right lateral ventricle (AP + 0.2 mm; ML + 0.8 mm; DV − 2.5 mm). Behavioral tests were performed on the following day.
Fluorescent staining was done essentially as described previously. BV2 cells were fixed with 4% paraformaldehyde (PFA). Mice were also transcardially infused with 4% PFA. After postfixation with same fixative solution overnight and dehydration with 30% sucrose in PBS, coronal brain sections 25 µm in thickness were prepared using cryostat. The cytoplasm of BV2 cells was stained with Phalloidin-iFluor 488 or 647 conjugate (Cayman Chemical, Ann Arbor, MI) that binds to actin. For immunofluorescence staining for anti-CD68 antibody (Abcam, Cambridge, UK) and anti-LAMP1 (Merck, Boston, MA) antibody, BV2 cells were incubated with the primary antibodies for 1 h in RT and overnight at 4 °C, respectively. Then, the cells were incubated with Alexa fluor 488-labeled secondary antibody for 1 h in RT. When anti-Iba1 antibody (Gene Tex, Irvine, CA) was used as primary antibody, 2 N HCl was applied to the brain sections for 10 min prior to the addition of the primary antibody overnight at 4 °C. Then, the sections were incubated in HRP-labeled secondary antibody solution for 1 h in RT. Finally, tyramide 488 solution (Thermo Fisher Scientific, Waltham, MA) was applied for 10 min in RT. The fluorescent signals were detected using LSM 880 confocal microscope (Zeiss, Oberkochen, Germany). When the confocal images were acquired, the frame size was 512 × 512 pixels and the scan time was 7.45 s. Line mode and bit depth of 8 bit were applied for averaging. The serial z-stack images of BV2 microglial cells at every 1 µm were also obtained by the confocal laser scanning microscopy. Nissl staining of brain sections was also carried out essentially as reported previously. The sections were stained with 0.1% cresyl violet solution for 20 min at 60 °C. The visible images were taken by BZ-9000 microscope (Keyence, Osaka, Japan) or ECLIPS 80i microscope (Nikon, Tokyo, Japan).
Resident-intruder test was performed essentially as described previously. An intruder mouse that had not been a resident in the cage was put in the cage where a target mouse with the same sex has been a resident for 2 days. The total time the resident mice was showing active behavior (approaching, chasing, sniffing) against the intruder mouse during 5 min was measured. Size of the box used for three-chambered social approach task (O’HARA & CO., LTD., Tokyo, Japan) is 20 × 40 × 23 cm. There are three chambers (left, center, right) with an equal size in the box. Mice can enter each chamber through the entrances (5 × 3 cm) between the chambers. Each subject mouse was first put in the center chamber and was allowed to move for 5 min (first trial). After 5 min, the subject mouse was taken out. After a different mouse was placed in the cage in one side of the chamber, the subject mouse was again put in the center chamber and the mouse behavior was automatically measured for 5 min (second trial). Social approach was estimated by the time subject mouse spent in the chamber with the mouse. Then, third trial was conducted by adding a novel mouse in the cage that had been empty in the second trial for 5 min, leaving the old mouse in another chamber. Social memory was evaluated by the time subject mouse spent in the chamber with the novel mouse. The elevated plus maze test was performed as previously reported. Two open and two closed arms of the maze were 25 cm long and 5.5 cm wide. The closed arms but not open arms had transparent walls with 14.5 cm in height at both sides. The center area of the maze was 5.5 cm × 5.5 cm. The maze was elevated above the ground (55 cm). Mice were first placed on the center area facing an open arm. Entering into the arms was defined when all four paws were on the arms. The total time spent in the open arms and total number of entries into the 4 arms were measured during 10 min. The open field test was carried out as previously reported. The apparatus (50 cm × 50 cm × 50 cm, O’HARA & CO., LTD., Tokyo, Japan) automatically measured spontaneous locomotion of mice for 10 min. The floor of the open field was covered with black paper to detect movement of the white mouse. Mice were initially placed in the center area and were allowed to walk freely. The parameters measured were duration of movement during test session, total walking distance, total number of movements, average speed of locomotion during test session, speed during movement, walking distance per movement, duration per movement and the percentage of time the mice stayed in the center area. The acoustic startle response and PPI of the response were recorded using an acoustic startle reflex measurement system (O’HARA & CO., LTD., Tokyo, Japan). Mice were put into a cylinder which was placed on an acceleration sensor in a chamber. A loudspeaker in the chamber produced sounds as the startle stimulus (40 ms) and the software automatically detected the startle responses. For mice given CM into the lateral ventricle, prepulses (20 ms) with 74, 78 or 82 dB was applied 100 ms before the startling acoustic stimuli. Startle responses at 110 and 120 dB and six combinations of PPI (74–110, 78–110, 82–110, 74–120, 78–120, 82–120 dB) were presented 6 times in a pseudorandom order. The intensities of startling stimuli applied (110 and 120 dB) were chosen because the tone at 120 dB was extensively used for 11 inbred strains of mice and the startle response of ICR mice became big using tones at 110 dB and 120 dB compared to those at 80–100 dB. The prepulse levels (74, 78, 82 dB) were chosen because 74–90 dB were applied for the 11 inbred strains and 73–86 dB were used for ICR mice. The averaged value after each stimulus type was calculated for each mouse. The interval time of each trial ranged from 10 to 20 s. The behavioral tests using mice given CM in the lateral ventricle were done in the following order with intervals of 10 min; resident-intruder test, three chamber test, elevated plus maze test, open field test, startle response and PPI. Different sets of mice were used for startle responses of mice given 13L-CM in the PnC. All behavioral tests after injection into the lateral ventricle were done using 5 males and 5 females, each group. For startle responses after injection into the PnC, 5–10 males and 5 or 6 females per groups were used.
The values expressed are the mean in the graphs. The error bars represent SE. Statistical significances were examined using one-way ANOVA except three chamber test to which two-way ANOVA was applied. Tukey and Tukey–Kramer posthoc tests were applied when the sample sizes were same and different, respectively. p values less than 0.05 were considered as statistically significant.
Supplementary Information 1.Supplementary Information 2.Supplementary Information 3. | true | true | true |
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PMC9636259 | Jun Matsuda,Dina Greenberg,Sajida Ibrahim,Mirela Maier,Lamine Aoudjit,Jennifer Chapelle,Cindy Baldwin,Yi He,Nathalie Lamarche-Vane,Tomoko Takano | CdGAP maintains podocyte function and modulates focal adhesions in a Src kinase-dependent manner | 04-11-2022 | RHO signalling,Podocytes | Rho GTPases are regulators of the actin cytoskeleton and their activity is modulated by GTPase-activating proteins (GAPs) and guanine nucleotide exchanging factors (GEFs). Glomerular podocytes have numerous actin-based projections called foot processes and their alteration is characteristic of proteinuric kidney diseases. We reported previously that Rac1 hyperactivation in podocytes causes proteinuria and glomerulosclerosis in mice. However, which GAP and GEF modulate Rac1 activity in podocytes remains unknown. Here, using a proximity-based ligation assay, we identified CdGAP (ARHGAP31) and β-PIX (ARHGEF7) as the major regulatory proteins interacting with Rac1 in human podocytes. CdGAP interacted with β-PIX through its basic region, and upon EGF stimulation, they both translocated to the plasma membrane in podocytes. CdGAP-depleted podocytes had altered cell motility and increased basal Rac1 and Cdc42 activities. When stimulated with EGF, CdGAP-depleted podocytes showed impaired β-PIX membrane-translocation and tyrosine phosphorylation, and reduced activities of Src kinase, focal adhesion kinase, and paxillin. Systemic and podocyte-specific CdGAP-knockout mice developed mild but significant proteinuria, which was exacerbated by Adriamycin. Collectively, these findings show that CdGAP contributes to maintain podocyte function and protect them from injury. | CdGAP maintains podocyte function and modulates focal adhesions in a Src kinase-dependent manner
Rho GTPases are regulators of the actin cytoskeleton and their activity is modulated by GTPase-activating proteins (GAPs) and guanine nucleotide exchanging factors (GEFs). Glomerular podocytes have numerous actin-based projections called foot processes and their alteration is characteristic of proteinuric kidney diseases. We reported previously that Rac1 hyperactivation in podocytes causes proteinuria and glomerulosclerosis in mice. However, which GAP and GEF modulate Rac1 activity in podocytes remains unknown. Here, using a proximity-based ligation assay, we identified CdGAP (ARHGAP31) and β-PIX (ARHGEF7) as the major regulatory proteins interacting with Rac1 in human podocytes. CdGAP interacted with β-PIX through its basic region, and upon EGF stimulation, they both translocated to the plasma membrane in podocytes. CdGAP-depleted podocytes had altered cell motility and increased basal Rac1 and Cdc42 activities. When stimulated with EGF, CdGAP-depleted podocytes showed impaired β-PIX membrane-translocation and tyrosine phosphorylation, and reduced activities of Src kinase, focal adhesion kinase, and paxillin. Systemic and podocyte-specific CdGAP-knockout mice developed mild but significant proteinuria, which was exacerbated by Adriamycin. Collectively, these findings show that CdGAP contributes to maintain podocyte function and protect them from injury.
The Rho family of small GTPases (Rho GTPases) are master regulators of the actin cytoskeleton organization. Rho GTPases are molecular switches that shuttle between active (GTP-bound) and inactive (GDP-bound) forms. They are regulated by three families of Rho regulatory proteins: activating proteins called guanine nucleotide exchange factors (GEF) that exchange GDP for GTP, inactivating proteins called GTPase-activating proteins (GAP) that enhance intrinsic GTPase activity, and GDP dissociation inhibitors (GDI) that bind to and stabilize the inactive GDP-bound Rho GTPase forms in the cytoplasm. Deciphering the molecular mechanisms regulating Rho GTPase activity is crucial for our understanding of cellular morpho-dynamics in many disorders, including kidney glomerular diseases. The kidney glomerulus functions as a filtration barrier that retains large proteins and cells in the blood while allowing water and small molecules to pass into the urine. Proteinuria is a leakage of serum proteins into the urine and occurs when the glomerular filtration barrier is impaired. Proteinuria is one of the hallmarks of chronic kidney disease (CKD) and the degree of proteinuria often correlates with the rate of decline of kidney function. Thus, proteinuria is an important prognostic factor in kidney diseases. Podocytes are highly specialized epithelial cells in the glomerulus, where they act as a critical component of the filtration barrier; injury to podocytes underlies many proteinuric glomerular diseases. Podocytes have an intricate structure characterized by primary, secondary and tertiary projections. Finger-like tertiary projections, commonly known as “foot processes”, from adjacent podocytes interdigitate with one other and tightly surround glomerular capillary walls. The unique morphology of foot processes is supported by their well-organized actin cytoskeleton, and is critical for podocyte function. Indeed, Rho GTPases play a central role in podocyte morphology and attachment to the glomerular basement membrane (GBM). Of the 20 members, RhoA, Rac1, and Cdc42 are known as prototypical Rho GTPases and are the best studied. The importance of the Rho GTPases has been well demonstrated by previous studies using podocyte-specific transgenic mice. For example, hyperactivation of Rac1 in podocytes causes proteinuria, while loss of Rac1 can be situationally pathogenic or adaptive. Meanwhile, gene deletion of Cdc42 in podocytes results in congenital nephrotic syndrome. To date, 82 GEFs, 69 GAPs and 3 GDIs have been identified in humans, which act in concert to achieve cell-type and context-dependent dynamic balance of Rho GTPases. However, which and how Rho regulatory proteins regulate Rho GTPase activities in podocytes remains largely unknown. In this study, we sought to investigate which Rho regulatory proteins interact with Rac1 in podocytes using proximity ligation combined with proteomics (BioID), and investigated their role in podocyte function and morphology. We reported recently that ARHGEF7 (β-PIX) is a predominant GEF that activates Cdc42 in podocytes, and here we found that it is also an important Rac1 interactor. Furthermore, we identified Cdc42 GTPase-activating protein (CdGAP, also named ARHGAP31) as one of the major RhoGAPs that interacts with Rac1, but also with β-PIX, in podocytes. Given previous examples of CdGAP and GEF cooperation, we explored the functional interaction of CdGAP and β-PIX in modulation of cell migration and adhesion of podocytes, and found that CdGAP facilitates β-PIX localization and activation at the cell membrane in a Src kinase-dependent manner upon EGF stimulation. Furthermore, we showed that CdGAP is required for maintenance of podocyte morphology and normal glomerular barrier function. These findings are pertinent to the identification of targets for small molecule therapeutics of kidney disease.
Rho GTPases interact dynamically with their regulators and effectors. To identify activators (GEFs) and inhibitors (GAPs) of Rac1 in podocytes, we performed proximity-based ligation and proteomics, BioID, using the G15A mutant of Rac1 (Rac1G15A) as bait. Rac1G15A is a nucleotide-free mutant of Rac1 that binds to its GEFs with high affinity and, to a lesser extent, to its GAPs. Rac1G15A was fused with a promiscuous E. coli biotin ligase (BirA) and expressed in immortalized human podocytes. Podocytes expressing BirA alone were used as controls (Fig. 1A). In the presence of exogenous biotin, proteins in close proximity to Rac1G15A were biotinylated, captured by streptavidin beads and subjected to mass spectrometry analyses (Fig. 1B). One hundred and twenty-five proteins were identified in BirA-Rac1G15A expressing podocytes at significantly higher levels compared to control podocytes expressing BirA alone (Fig. 1C). Cross-referencing these interactors to the list of known Rho regulatory proteins, we identified three GEFs (ARHGEF7, FGD6, and DOCK9) and two GAPs (ARHGAP31 and ARHGAP29) (Fig. 1D). Adhesion of foot processes to the GBM via the adhesion molecules such as α3β1-integrins is critical for morphology and function of podocytes. Therefore, of the two GAPs, CdGAP was of particular interest because of its known functions in the regulation of cell migration, adhesion and focal adhesions (FAs). Similarly, among the three GEFs, β-PIX is known to modulate FA maturation via Rac1 or Cdc42, and to maintain podocyte architecture and glomerular function.
CdGAP is expressed widely in human and mouse tissues including the kidney. To assess its expression in the glomerulus, we first performed immunofluorescence staining of a transplant donor kidney biopsy and confirmed CdGAP staining in podocytes, as well as in other glomerular cells and tubular epithelial cells (Fig. 2A). To study the role of CdGAP in human podocytes in vitro, we next established immortalized cultured human podocytes with CdGAP knockdown (KD) and their controls using short hairpin RNA (shRNA) lentiviruses. Immunoblotting confirmed a significant KD efficiency of 73% (Fig. 2B,C). We first assessed the effect of CdGAP depletion on the levels of Rac1 activity in differentiated podocytes by performing a GST-CRIB pull down assay. Loss of CdGAP resulted in a significant 1.33-fold increase in basal Rac1-GTP levels compared to control cells, without affecting the total levels of Rac1 (Fig. 2D–F). In control podocytes, Rac1 was activated after 5 min of EGF stimulation. However, further Rac1 activation by EGF was absent in CdGAP KD podocytes (Fig. 2D,E). Similarly, basal Cdc42 activity in CdGAP KD podocytes was significantly higher (1.47-fold) than in control cells, but CdGAP KD cells showed no EGF-induced Cdc42 activation (Fig. 2G,H). Curiously, total Cdc42 (normalized to tubulin) was significantly decreased in CdGAP KD podocytes (0.61-fold) compared with controls (Fig. 2I). When podocytes were plated onto a laminin-coated surface, both CdGAP KD and control podocytes attached similarly at 2 h (Fig. 2J), however, their cell morphology differed. In contrast to the archetypical rounded podocyte morphology, CdGAP-depleted podocytes showed an elongated cell shape with an increased aspect ratio (the ratio of the longest to the shortest axis of the cell) and enriched F-actin lamellipodia at the cell periphery (Fig. 2K,L, Supplementary Fig. S1), as previously published. Furthermore, CdGAP KD podocytes were significantly slower to migrate compared to control cells in a wound healing assay over a period of 24 h (Fig. 2M, Supplementary Fig. S1). These results show that CdGAP acts as a GAP for Rac1 and Cdc42 in regulating podocyte morphology and migration but does not affect short-term cell adhesion.
We have previously reported that the Cdc42 GEF intersectin (ITSN) interacted with CdGAP and inhibited its GAP function. By analogy, we questioned if β-PIX and CdGAP, both found in the proximity of Rac1G15A in podocytes by BioID (Fig. 1), interacted with each other. When myc-CdGAP and GFP-β-PIX were transiently transfected in HEK293 cells and β-PIX was immunoprecipitated using anti-GFP antibodies, CdGAP co-immunoprecipitated, indicating that CdGAP and β-PIX bound one another, either directly or indirectly (Fig. 3A,B). We found that co-immunoprecipitation of CdGAP and β-PIX was not altered by EGF treatment of HEK293 cells (Supplementary Fig. S2). Attempts to demonstrate interaction between endogenous CdGAP and β-PIX in podocytes were unsuccessful due to nonspecific interactions detected in the rabbit immunoglobulin G negative control (Supplementary Fig. S2). CdGAP consists of a polybasic region (PBR) preceding the GAP domain, a basic central region (BR), a proline-rich domain (PRD), and an extended C-terminal regulatory domain (Fig. 3C). To determine which part of CdGAP is required for the interaction with β-PIX, we tested a series of CdGAP deletion constructs in co-immunoprecipitation assays with β-PIX (Fig. 3C). CdGAP constructs containing the BR domain [(BR alone (181–515), BR + PRD (181–820), and PBR-GAP + BR + PRD (1–820)] co-immunoprecipitated with the full-length β-PIX, while the PRD alone (515–820) and the C-terminus domain (1160–1425) failed to do so (Fig. 3D–G). Therefore, the BR domain of CdGAP is required and sufficient for the interaction with β-PIX.
The above findings indicate that there is a protein–protein interaction, either direct or indirect, between β-PIX and CdGAP. CdGAP KD human podocytes have some phenotypic similarity (e.g. impaired motility; Fig. 2) to that of β-PIX KD mouse podocytes. Thus, we hypothesized that CdGAP may modulate the function of β-PIX in podocytes. First, we studied whether CdGAP impacts subcellular localization of β-PIX by immunocytochemistry. In control podocytes, β-PIX and CdGAP were predominantly localized in the cytosol (Fig. 4A). When cells were stimulated with EGF for 30 min, translocation of both CdGAP and β-PIX to the plasma membrane was observed (Fig. 4A, quantified in Fig. 4B–D). When the overlap of CdGAP and β-PIX was quantified using the Pearson correlation coefficient, a modest but significantly increased overlap at the cell periphery (10 μm within the plasma membrane) was observed after EGF stimulation, while overlap in the whole cell was unchanged (Supplementary Fig. S3). Of interest, in CdGAP KD podocytes, the peripheral β-PIX ratio did not increase by EGF stimulation in contrast to control podocytes (Fig. 4E). Therefore, these results suggest that upon EGF stimulation, CdGAP translocates to the plasma membrane and facilitates membrane translocation of β-PIX.
FA is an integrin-based multi-protein complex and is essential for podocyte attachment to the GBM and its motility. Pro-migratory and pro-invasive functions were ascribed to CdGAP, which was shown to regulate directional membrane protrusions of migrating osteosarcoma cells, breast cancer cells, and prostate cancer cells. Consistently, CdGAP KD podocytes showed decreased motility (Fig. 2M). Thus, we next studied FA size in CdGAP KD podocytes. When FAs were visualized by immunostaining of vinculin, FAs in CdGAP KD podocytes were generally larger in size than those of control cells (Fig. 5A,B), while the number of FAs per cell area was comparable (Fig. 5C). It has previously been shown that tyrosine phosphorylation of β-PIX by EGF leads to its activation and increases FA turnover. To assess tyrosine phosphorylation of β-PIX, β-PIX was immunoprecipitated from control and CdGAP KD podocytes and immunoblotted for phospho-tyrosine (pY). Basal tyrosine phosphorylation of β-PIX was observed in unstimulated podocytes, and increased further by EGF stimulation (Fig. 5D,E). Depletion of CdGAP in podocytes did not alter basal tyrosine phosphorylation of β-PIX but rendered KD cells unresponsive to further stimulation (Fig. 5D,E). In fact, β-PIX tyrosine phosphorylation after EGF stimulation was significantly lower in CdGAP KD podocytes compared with control podocytes (Fig. 5E), while total amounts of β-PIX were unchanged between control and CdGAP KD podocytes (Fig. 5F). These results indicate that EGF-induced β-PIX tyrosine phosphorylation and activation is inhibited in the absence of CdGAP. Like β-PIX, many FA proteins become active when tyrosine-phosphorylated and this in turn results in FA turnover. Thus, we studied the tyrosine phosphorylation of the FA molecules, focal adhesion kinase (FAK) and paxillin. In control podocytes, basal pY of FAK (pY397) and paxillin (pY118) was observed, which significantly increased upon EGF stimulation (Fig. 5G,H). In CdGAP KD podocytes, basal pY was unchanged but the response to EGF was absent (Fig. 5G,H). The lack of increase of pY397-FAK and pY118-paxillin in response to EGF in CdGAP KD cells, which correlated with the ablated tyrosine phosphorylation of β-PIX (Fig. 5E), was specific since phosphorylation/activation of ERK in response to EGF was intact in these cells (Supplementary Fig. S4). These results demonstrate that activation of FA proteins (such as β-PIX, FAK and paxillin), in response to ligand stimulation (e.g. by EGF), is impaired in CdGAP KD podocytes, and this likely results in the increase of larger FAs. The above findings suggest that the absence of CdGAP blocks a signaling pathway that leads from EGF/EGFR interaction to β-PIX/FAK/paxillin tyrosine phosphorylation downstream of or in parallel with ERK activation, most likely implicating protein tyrosine kinase(s). A strong candidate is the Src-family tyrosine kinase because of its well-known role in tyrosine-phosphorylation of β-PIX, FAK, and paxillin. Src activity requires autophosphorylation of its Y416. Basal pY416-Src, representing active Src, was significantly decreased in CdGAP KD cells compared to controls, and did not increase in response to EGF stimulation (Fig. 6A,B). Thus, both basal and EGF-stimulated Src activity are suppressed in the absence of CdGAP. We next treated podocytes with the Src inhibitor, SU6656. Basal tyrosine phosphorylation of β-PIX, FAK, and paxillin were significantly decreased in treated group, and while all three responded to EGF stimulation, EGF-stimulated pY of β-PIX, FAK, and paxillin was lower in SU6656 treated cells compared to controls (Fig. 6C,D), phenocopying CdGAP KD podocytes (Fig. 5D–H). Our findings suggest that CdGAP modulates FAs through (1) its GAP activity towards Rac1/Cdc42, and (2) facilitation of Src kinase activity, which contributes to the tyrosine phosphorylation and subsequent activation of FA proteins such as β-PIX, FAK, and paxillin.
Since CdGAP is required to maintain podocyte function in vitro, we next interrogated the role of CdGAP in the glomerular barrier function using systemic CdGAP knockout (KO) mice. We reported previously that mice with systemic CdGAP KO have 44% embryonic/perinatal mortality resulting from vascular defects, but survivors grow normally and are fertile. To assess the role of CdGAP in the glomerular barrier function, we collected urine from systemic CdGAP KO mice and littermate controls and quantified the albumin-to-creatinine ratio (ACR). Male systemic CdGAP KO mice developed mild but significant proteinuria starting at around 6 months old (Fig. 7A). Female mice did not develop proteinuria up to 12 months old (data not shown). The susceptibility of males to proteinuria has been well recognized both in humans and mice and we have observed a similar sex difference of proteinuria in podocyte specific Ste20-Like Kinase (SLK) transgenic mice. Proteinuria can occur from defects of any of the three components of the glomerular filtration barrier; namely glomerular endothelial cells, GBM, and podocytes. To determine whether loss of CdGAP in podocytes is responsible for proteinuria, we next generated podocyte-specific CdGAP knockout (Pod KO hereafter) mice by crossing Podocin-Cre mice with CdGAPflox/flox mice. PCR of genomic DNA extracted from isolated glomeruli confirmed a truncated gene deletion product of CdGAP in Pod KO mice (Fig. 7B). Since we could not identify the antibody that reliably detects CdGAP expression in podocytes in the mouse kidney by immunostaining, we assessed the expression of CdGAP mRNA in the glomerulus using in situ hybridization. CdGAP mRNA was observed predominantly in the endothelial cells, consistent with previous reports. CdGAP mRNA was also observed in the glomerular podocytes in control mice, but not in KO mice (Fig. 7C), consistent with the successful podocyte-specific deletion of CdGAP. Male, but not female Pod KO mice developed significant proteinuria at around 7 months of age, phenocopying systemic CdGAP knockout mice (Fig. 7D). While Pod KO mice did not show discernible abnormalities by kidney histology (PAS staining) and serum biochemistry (blood urea nitrogen [BUN] and creatinine) up to 12 months old (data not shown), electron microscopy demonstrated that foot process width was significantly increased in 12-month-old male Pod KO mice compared with controls, indicating foot process effacement (Fig. 7E). To study whether young Pod KO mice are more susceptible to injury, we challenged 14–16-week-old male Pod KO and control mice with a podocyte toxin, Adriamycin (ADR). Urine ACR was increased after ADR injection in both groups, but Pod KO mice showed significantly higher ACR than control mice at 1 and 2 weeks (Fig. 7F). In addition, we found significantly more foot process effacement in Pod KO mice compared with controls at 4 weeks (Fig. 7G). Altogether, these findings indicate that CdGAP in podocytes is required for normal glomerular barrier function, and its absence makes podocytes susceptible to external injury.
Previous studies, including ours, have established that Rac1 plays pathogenic or adaptive/protective roles in podocytes depending on the context. Thus, our study aimed to ascertain which GAPs and GEFs regulate Rac1 activity in podocytes. Using Rac1 as bait in BioID, we identified CdGAP as one of the GAPs that interacts with Rac1. CdGAP is one of the causative genes associated with Adams-Oliver syndrome, a disorder characterized by scalp defects and terminal transverse limb abnormalities. CdGAP localizes at FAs and participates in the regulation of cell motility and adhesion dynamics, and its roles in cancer cells have been well described. While CdGAP was initially discovered as a GAP for Cdc42, it has been since shown that it acts on both Cdc42 and Rac1. In the current study, CdGAP KD podocytes showed increased basal activities of Rac1 and Cdc42, indicating that CdGAP suppresses both Rac1 and Cdc42 activities under basal conditions. This is consistent with previous reports using U2OS cells with CdGAP KD or an inactive CdGAP mutant. GEFs and GAPs act in concert in a cell-/context-dependent manner. We reported recently that β-PIX is a predominant GEF for Cdc42 in podocytes and facilitates its downstream YAP activity. The present study demonstrated that β-PIX interacts not only with Cdc42 but also with Rac1, and furthermore with CdGAP in podocytes. The existence of such GEF-GAP interactions is supported by a recent study showing that Rho regulatory proteins can form both homotypic and heterotypic interactions, suggesting that these complexes can potentiate the ability of GEFs and GAPs to coordinate downstream Rho GTPase activities. One study identified a GAP-GEF interaction between SrGAP and β-PIX that facilitates crosstalk between Cdc42 and RhoA in a collagen-dependent manner. Similarly, we previously showed that CdGAP interacts with another GEF, intersectin (ITSN), in fibroblasts via its BR domain, which is the same domain that is required for binding to β-PIX in the current study. In the case of the ITSN-CdGAP interaction, the binding of ITSN inhibits the GAP activity of CdGAP, facilitating Cdc42 activation. Here, we also observed a synergy between β-PIX and CdGAP. When CdGAP was depleted in podocytes, basal Rac1 and Cdc42 activities were increased compared to CdGAP intact podocytes, likely reflecting the diminished GAP activity of CdGAP toward Rac1 and Cdc42. However, the most striking phenotype was that CdGAP KD podocytes did not respond to EGF in the activation of Rac1 nor Cdc42. Similarly, EGF-induced membrane translocation and tyrosine phosphorylation of β-PIX were absent in CdGAP KD podocytes. Thus, CdGAP is required for the functional EGF-mediated translocation and activation of β-PIX. Our previous study showed that CdGAP translocated to the plasma membrane in response to platelet-derived growth factor and this was dependent on the N-terminal PBR domain. It is possible that EGF also triggers membrane translocation of CdGAP in a similar manner and β-PIX in complex with CdGAP moves together, where it gets tyrosine phosphorylated. Whether CdGAP-β-PIX interaction is direct or indirect is yet to be determined. β-PIX and other FA proteins such as FAK and paxillin are known to be tyrosine-phosphorylated by the Src-family kinases. A previous study clearly demonstrated that Src, in turn, is activated by mechanical stimuli via integrin at the FA. CdGAP is a key mediator of FA-based mechanosensing of extracellular matrix, thus it appears reasonable to postulate that CdGAP plays an important role in activating Src family kinases especially in FAs. Indeed, we observed a low Src kinase activity in CdGAP KD cells, which was unresponsive to EGF stimulation. The consistent lack of response to EGF stimulation in CdGAP KD cells highlights the potential role of CdGAP in FA-based mechanosensing and consecutive signal transduction. Similar findings were observed in our previous study, which showed that CdGAP deletion in endothelial cells impaired VEGF-induced tyrosine phosphorylation of Gab1 and its downstream signaling pathways. However, given that pY-β-PIX/FAK/paxillin in Src inhibitor-treated podocytes was blunted basally but remained partially responsive to EGF stimulation, it is likely that Src is not the sole regulator of the signaling pathway responsible for EGF-mediated tyrosine phosphorylation of β-PIX, FAK, and paxillin. Collectively, our findings support a model wherein CdGAP interacts with β-PIX in the cytoplasm and contributes to maintaining low Rac1/Cdc42 activity at basal conditions. In response to external stimuli, CdGAP and β-PIX are recruited to the plasma membrane, where they collaborate to activate Rac1/Cdc42 and regulate FA proteins in a Src kinase dependent-manner. In the absence of CdGAP, excess activation of β-PIX/Rac1/Cdc42 under basal conditions, or impaired response to external stimuli in pathological conditions, may be detrimental to podocyte health. Our findings in vivo support the importance of CdGAP in the maintenance of FAs in podocytes. We demonstrate that CdGAP deletion in glomerular podocytes exacerbated proteinuria in mice, both under basal conditions and within a podocyte injury model. The basolateral membrane of podocyte foot processes is anchored to the underlining GBM via a number of adhesion molecules represented by α3β1-integrins. The importance of the proteins that interact with the cytoplasmic domain of integrins (collectively called adhesion complex proteins) has been reported in podocyte function. Thus, it is likely that the absence of CdGAP in glomerular podocytes disrupts FA complexes, thereby impairing GBM-podocyte interaction and disrupting integrin-linked actin cytoskeleton regulation. In conclusion, CdGAP is required for podocyte function and is protective from injury in vivo. Improving our understanding of the CdGAP-mediated Src kinase activation that likely takes place downstream of integrins and FA complexes in podocytes may allow for the development of a novel approach to proteinuric kidney diseases.
Immortalized human podocytes (gifts from Dr. Moin Saleem) were cultured in RPMI1640 containing 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin (Gibco) at permissive conditions (33 °C in 5% CO2). Podocytes with CdGAP KD and their controls were established using MISSION Lentiviral shRNA (Sigma-Aldrich, TRCN0000047640 and SHC001, respectively). HEK293T cells were transiently transfected using the lentiviral packaging system (abm, LV003) according to the manufacturer’s instructions. Virus-containing supernatants were added to podocytes under permissive conditions for 16 h. Puromycin (Wisent Inc.) was added 48 h later and puromycin-resistant cells were pooled for further experiments. Unless stated otherwise, podocytes were differentiated under non-permissive condition (37 °C in 5% CO2) for 7 days, as described previously.
The antibodies and reagents used are summarized in Supplemental Table S1.
Myc-BirA-Rac1G15A (from Dr. Jean-Francois Cote, Montreal Clinical Research Institute) was subcloned into pTRE2 (Clontech) and expressed in immortalized human podocytes that stably express rtTA using nucleofection. As control, pTRE2-myc-BirA was used. BioID experiment was performed as described previously. Briefly, cells were incubated for 16 h with exogenous 50 μM biotin (BioShop, BIO302) and doxycycline (Sigma, D9891) and biotinylated proteins were captured by streptavidin beads (Thermo Scientific) and subjected to proteomics analyses. The results were analyzed by the Scaffold Q + Scaffold_4.9.0 software (Proteome Sciences). For statistics, t-test within the Scaffold was used after normalizing to total spectral counts. All proteins with a P-value of < 0.05 and a fold change of two over the BirA controls were considered as interactors of BirA-Rac1G15A. The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE (Perez-Riverol et al., 2019) partner repository with the dataset identifier PXD023718.
Cultured podocytes and HEK293 cells were lysed with ice-cold lysis buffer (10 mM Tris [pH 7.5], 1 mM EDTA [pH 8.0], 1 mM EGTA [pH 8.0], 125 mM sodium chloride, 10 mM sodium pyrophosphate, 25 mM sodium fluoride, 2 mM sodium orthovanadate, 1% Triton X-100 [Sigma-Aldrich], and protease inhibitor cocktail [Roche]). Protein concentrations were measured and quantitative densitometry was performed as previously described. Lysates were assayed by Western blot.
Histological analysis was performed as previously described. Human kidney sections were obtained from a kidney transplantation donor. Mice were transcardially perfused with PBS. Tissues were post-fixed with 4% paraformaldehyde (PFA) (Thermo Fisher Scientific) and embedded in paraffin. Paraffin sections were immunostained with the respective antibodies. Citrate treatment was performed for antigen retrieval. Tissue sections were counterstained with 4,6-diamidino-2-phenylindole (DAPI) (Invitrogen). Immunofluorescence images were obtained using a Zeiss LSM780 laser scanning confocal microscope with the Zeiss Plan Apochromat 63 × 1.40 Oil DIC objective. All imaging parameters were maintained constant throughout image acquisition of all samples.
Undifferentiated podocytes were plated on glass cover slips coated with 0.25 μg/cm2 laminin 521 (CORNING) and allowed to differentiate for 7 days. They were then fixed in 4% PFA before being permeabilized with 0.1% Triton. After blocking with 3% bovine serum albumin in PBS, cells were immunostained with the respective antibodies and stained with phalloidin. Immunofluorescence images were obtained using a Zeiss LSM780 laser scanning confocal microscope with the Zeiss Plan Apochromat 63 × 1.40 Oil DIC objective. All imaging parameters were maintained constant throughout image acquisition of all samples. Quantification of FA complex was done using ImageJ software, as described previously. Briefly, cell contour was traced using phalloidin-stained images with the ‘Freehand Line’ function and cell area was measured. After shifting to images with vinculin staining, the ‘Clear Outside’ function was used to erase the area outside the cell region. The number of particles between 1 and 8 μm2 was counted. FAs were classified into three groups: small (1–2 μm2), medium (2–6 μm2), and large (6–8 μm2). The proportion of the number of FAs in each group relative to total FAs was calculated.
Differentiated podocytes were serum starved (1% FBS) for 16 h prior to the experiment. Cells were stimulated with EGF (100 ng/ml) for 0, 2, or 5 min, washed once with ice-cold PBS, and lysates collected as described above. Active Rac1 and Cdc42 were pulled down using GST-CRIB beads (GE Healthcare Bio-Sciences), as described previously. Cells were lysed with the lysis buffer (above) and mixed with the beads for 1 h at 4 °C. Beads were washed three times, and proteins were eluted from beads into SDS loading buffer.
Undifferentiated podocytes were seeded onto a 96-well IncuCyte® ImageLock microplate coated with 0.25 μg/cm2 laminin 521 (CORNING) at a density of 40,000 cells per well. After cells reached a confluent state, they were serum starved in RPMI containing 1% FBS for 2 h at 37 °C. A scratch wound was made using the IncuCyte 96-well Wound Maker (Sartorius). Cells were then kept under non-permissive conditions up to 24 h and the migration rate was analyzed by IncuCyte Analysis Software (Sartorius).
Differentiated podocytes were seeded on a 96-well plate (10,000 cells per well) with laminin 521 (CORNING) and cultured under non-permissive conditions for 2 h. After 3 washes with PBS, adherent cells were fixed with 4% PFA for 15 min. Fixed cells were incubated with 0.1% crystal violet (Sigma-Aldrich) dissolved in 200 mM 3-(N-morpholino) propanesulfonic acid (BioShop) for 15 min at room temperature. After 3 washes with PBS, 10% acetic acid (Fisher Scientific) was added for 15 min. Dissolved crystal violet was quantified by the absorbance at 550 nm.
Transient transfection of HEK293 cells was performed using Lipofectamine 2000 (Invitrogen) at a 1:2 DNA/Lipofectamine ratio. Cell lysates were incubated with anti-GFP antibody overnight at 4 °C, and then with protein A agarose beads (Santa Cruz, sc-2001) for 1 h at 4 °C. Beads were washed three times, and proteins were eluted from the beads into SDS loading buffer. Normal rabbit IgG (CST, 2729) was used as a negative control.
CdGAPflox/flox mice, which have conditional floxed exon 1 allele (C57BL/6 background), systemic CdGAP deficient mice (C57BL/6 background), and Podocin-Cre mice (mixed ICR/129/B6 background) have been described previously. Male mice were used for experiments. Cre-negative mice were used as controls for CdGAPflox/flox;Pod-Cre (CdGAPPod-/-) mice. Spot urine samples were collected at the indicated time points. To determine the genotype, mice DNA was extracted from isolated glomeruli by DNA Isolation Kit for Cells and Tissues (Roche Diagnostics, 11814770001), and subjected to PCR. The genotyping primer sequences used were as follows: Cre-F 5′-gcttctgtccgtttgccg-3′. Cre-R 5′-actgtgtccagaccaggc-3′. CdGAP -F 5′-cctgcgctgtgcaaagagcct-3′. CdGAP -R 5′-cccaaagtttaagacccgagcctc-3′.
Myc-tagged and GFP-tagged full-length and their truncated CdGAP constructs have been described previously. Isoform 5 of human β-PIX was amplified by RT-PCR from mRNA from human podocytes using primers below, digested by BamHI (Cell Signaling Technology [CST], R3136) and NotI (CST, R3189), and ligated into pcDNA3.1 using T4 DNA Ligase (CST, M0202). F 5′-accgagctcggatccatgaccgataatagcaacaatcaactggtagtaagagcaaag-3′. R 5′-gactcgagcggccgcttatagattggtctcatcccaggcaggatcattca-3′. Next, the constructs were digested by EcoRI (CST, R3101) and KpnI (CST, R3142) or by PspOMI (CST, R0653) and KpnI, and ligated into pEGFP-C1 and pmCherry-C1 vectors, respectively. All constructs were verified by Sanger sequencing.
The urinary albumin and creatinine levels were measured as previously described. The former was normalized to the latter to obtain ACR. BUN and serum creatinine were measured at the Comparative Medicine and Animal Resources Centre (CMARC) of McGill University.
In situ hybridization assay was performed using RNAscope (Advanced Cell Diagnostics, Inc.) with the target probe for mouse CdGAP mRNA (Advanced Cell Diagnostics, Inc., 569971) according to the manufacturer’s instructions.
Transmission electron micrographs of 3 to 5 glomerular tufts per mouse were captured using an FEI Tecnai 12 BioTwin 120 kV transmission electron microscope. Analysis for foot process width has been described previously.
CdGAPPod-/- and control mice at the age of 14–16 weeks were intravenously injected with 12 mg/kg of ADR (Sigma-Aldrich, D1515). Urine was collected at indicated time points before and after ADR treatment, and analyzed for ACR. Kidneys were harvested at 4 weeks after treatment and subjected to electron microscopic analysis.
All results are expressed as the mean ± standard error (SE). Statistical analyses were conducted using JMP software (SAS Institute, Cary, NC) and GraphPad Prism 9 (GraphPad Software, San Diego, CA). Multiple-group comparisons were performed using analysis of variance (ANOVA) with post-testing using the Tukey–Kramer test. Differences between two experimental values were assessed by the Student’s t-test. P < 0.05 was considered statistically significant.
The human biopsy sample was obtained from the McGill University Health Centre Kidney Disease Biorepository and the study was approved by the McGill University Health Centre Review Board. Informed consent was obtained from all study participants, and research was performed in accordance with relevant guidelines and regulations. All procedures involving mice were approved by the Animal Care Committee, McGill University and were in accordance with relevant guidelines and regulations. This study is reported in accordance with ARRIVE guidelines.
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PMC9636380 | 36224345 | Michael J. Morgan,You-Sun Kim | Roles of RIPK3 in necroptosis, cell signaling, and disease | 12-10-2022 | Inflammatory diseases,Necroptosis | Receptor-interacting protein kinase-3 (RIPK3, or RIP3) is an essential protein in the “programmed” and “regulated” cell death pathway called necroptosis. Necroptosis is activated by the death receptor ligands and pattern recognition receptors of the innate immune system, and the findings of many reports have suggested that necroptosis is highly significant in health and human disease. This significance is largely because necroptosis is distinguished from other modes of cell death, especially apoptosis, in that it is highly proinflammatory given that cell membrane integrity is lost, triggering the activation of the immune system and inflammation. Here, we discuss the roles of RIPK3 in cell signaling, along with its role in necroptosis and various pathways that trigger RIPK3 activation and cell death. Lastly, we consider pathological situations in which RIPK3/necroptosis may play a role. | Roles of RIPK3 in necroptosis, cell signaling, and disease
Receptor-interacting protein kinase-3 (RIPK3, or RIP3) is an essential protein in the “programmed” and “regulated” cell death pathway called necroptosis. Necroptosis is activated by the death receptor ligands and pattern recognition receptors of the innate immune system, and the findings of many reports have suggested that necroptosis is highly significant in health and human disease. This significance is largely because necroptosis is distinguished from other modes of cell death, especially apoptosis, in that it is highly proinflammatory given that cell membrane integrity is lost, triggering the activation of the immune system and inflammation. Here, we discuss the roles of RIPK3 in cell signaling, along with its role in necroptosis and various pathways that trigger RIPK3 activation and cell death. Lastly, we consider pathological situations in which RIPK3/necroptosis may play a role.
The finding that cell death is a genetically encoded and evolutionarily conserved process in multicellular organisms was a highly significant discovery at the end of the last century; its discoverers were awarded the 2002 Nobel Prize for Physiology and Medicine. Typically referred to as “programmed cell death” (PCD) in cases where it is initiated in the case of a physiological setting (e.g., development) or “regulated cell death” (RCD) in cases where the program is initiated by an external stimulus (e.g., chemotherapy), the processes by which cells commit “cell suicide” are of vast and profound importance to the normal physiology of living things and to our ability to intervene in pathological situations. PCD allows tissues and organs to be shaped and organized during development and continues to play roles in adults, where it functions in tissue remodeling, organ and tissue homeostasis, immunity, and tumor suppression, among many other processes. Whereas coordinated PCD contributes to health homeostasis, inadequate PCD or overactivation of PCD frequently leads to pathological conditions and disease. While cell death happens through a number of mechanisms or modes, “apoptosis” is considered to be the most physiological form, and is primarily executed through the activation of cysteine proteases of the caspase family. These proteases target proteins involved in the cell cycle and DNA repair, structural proteins, transcription and survival factors, and other regulatory proteins, thus leading to the organized destruction of the cell. In the initial studies highlighting its discovery, receptor-interacting protein kinase-3 (RIPK3, also referred to as RIP3) was proposed to be a regulator of apoptosis downstream of the tumor necrosis factor (TNF) receptor 1 signaling complex since its overexpression led to caspase activation and cell death. This proposal was early in the cell death field, and a few years after the discovery that ligands of the TNF superfamily were capable of inducing the caspase-dependent apoptotic pathway, it became clear that alternative cell death pathways were also initiated in the absence of caspase activation that led to a form of death with a “necrotic-like” morphology that was later referred to as “necroptosis”. For a number of years, receptor-interacting protein kinase-1 (RIPK1, also referred to as RIP1) was the only downstream factor known to be involved in necroptosis. Almost a decade after the discovery of RIPK1, RIPK3 came to the forefront of necroptosis studies when it was determined that it interacts with RIPK1 during necroptosis and is an essential downstream partner for RIPK1 for this form of death. The kinase-dependent role of RIPK3 in necroptosis is now considered its prototypical role in cellular function; however, it is now clear that RIPK3 plays several different roles in cells and perhaps has more than one function within necroptosis itself.
To clarify the role of RIPK3 in necroptosis, let us first clarify the important differences between apoptosis and necroptosis; furthermore, since some facets of necroptosis resemble those of classic necrosis, let us also clarify how necroptosis differs from necrosis. As mentioned, the downstream consequence of apoptosis is the activation of the caspase proteases that cleave their different substrates to trigger cell death. The nature of these cleavage events results in a very organized process of cell death. Among the characteristics of this cell death are cellular shrinkage, chromatin condensation and fragmentation, nuclear condensation, and fragmentation, concluding in the formation of membrane blebs that break off and become membrane-bounded bodies that are rapidly phagocytosed by surrounding cells and professional phagocytes of the immune system. Because of this mechanism of action, apoptosis limits cell debris and content leakage that would trigger inflammation. In contrast, classic necrosis is a passive, nonprogrammed form of cell death that is not genetically encoded but is the result of direct cellular injury or other pathological trauma. As a passive form of cell death, it requires no energy, and it is characterized by the swelling of cells and/or organelles rather than shrinkage. Necrosis, therefore, results in (or is directly caused by) plasma membrane rupture, thus cellular contents are leaked, and inflammation is triggered. Necroptosis, on the other hand, is a mixture of apoptosis and necrosis. Like apoptosis, specific gene products are required for necroptosis, and the cell itself has a central role in initiating its own demise through a cellular “program”. Some energy is required for necroptosis, as kinase activity is essential. Like necrosis, necroptotic cell death results in plasma membrane permeability, cell leakage, and immune system activation. Thus, apoptosis, which has been thought to occur primarily without triggering inflammation (this is, in fact, an oversimplification, as in some contexts, apoptosis also activates the immune system), is largely perceived as having different physiological outcomes than necroptosis, which is highly proinflammatory. In addition, necroptosis processes have more recently been shown to activate the immune system somewhat differently than classic necrosis. Necroptotic cells may play multiple roles in innate immunity and shape subsequent adaptive immunity through the release of endogenous danger signals known as damage-associated molecular patterns (DAMPs), which interact with pattern recognition receptors (PRRs) of innate immune cells to prime immune cells to respond to pathogens and potentially harmful cells, such as those that are infected or tumorigenic. The de novo synthesis of cytokines and chemokines occurs especially in dying necroptotic cells, likely due in part to the types of signals that trigger necroptosis. Indeed, the activation of RIPK1/RIPK3 leads to the upregulation of inflammatory chemokines to promote the cross-priming of CD8+ T cells and/or promote the release of DAMPs; thus, necroptosis is believed to significantly contribute to antitumor immunity. As mentioned, necroptosis is highly dependent on RIPK3 as an essential part of the necroptotic machinery. The RIPK3 protein is characterized by an N-terminal kinase domain, with which it phosphorylates itself and other substrates, and a C-terminal domain that contains a receptor-interacting protein homotypic interaction motif (RHIM) through which it associates with other proteins to oligomerize. Once activated (as described below), RIPK3 autophosphorylates and then phosphorylates and activates a pseudokinase called MLKL, which is essential for membrane permeabilization during necroptosis. The phosphorylation of MLKL by RIPK3 causes a conformational change in the MLKL protein that exposes its N-terminal four- or five-helical bundle domain that is usually tightly bound to the pseudokinase domain, but that once released from its interaction with the pseudokinase domain mediates MLKL oligomerization. Reports have significantly varied as to how many MLKL subunits (3, 4, 6, 8, or more) are involved in the oligomers, perhaps because of differences between the mouse and human systems, or perhaps because the number of subunits may vary between where it is activated and where it is inserted. The oligomerization of MLKL promotes its membrane translocation, which is followed by membrane permeabilization. The exact mechanism for this permeabilization has been debated, but MLKL has been reported to bind to phosphoinositides and cause membrane leakage, perhaps through the formation of channels or pores or indirectly through interaction with ion channels that let various cations through. Regardless of whether this involves further osmotic pressure, the membrane is sufficiently permeabilized to let cellular contents out and kill the cell.
Necroptosis is initiated downstream of many cellular stressors, including the signaling events activated by death receptor ligands, such as TNF-α, FasL, or TRAIL, that act through their various death receptors. This is where necroptosis was discovered and where most research has been conducted. In actuality, the term “necroptosis” was initially applied specifically only to nonapoptotic death receptor-initiated cell death but was then redefined to include any cell death process “that critically depends on MLKL and on the kinase activity of RIPK1 (in some settings) and RIPK3”. Since the discovery of MLKL, many cell death stimuli have been added as initiators of necroptosis. Almost all of these can be classified as pattern recognition receptors (PRRs) of the innate immune system (see Fig. 1). These include Toll-like receptors 3 and 4 (TLR3 and TLR4, respectively) and ZBP1 (or DAI). Other pattern recognition receptors, such as retinoic acid-inducible gene I (RIG-I) (also referred to as DExD/H-box helicase 58, or DDX58), interferon-α and interferon-β receptor (INFAR1), and STING1, may induce necroptosis as well but are thought to initiate necroptosis indirectly through gene induction of one of the previously mentioned receptors. Necroptosis typically, but does not always, involves the sister kinase of RIPK3, RIPK1, which is required for many necroptotic signals, such as the prototypical necroptotic signaling pathway downstream of death receptors that are initiated by TNF-α through TNFRSF1B (TNF receptor 1). RIPK3 interacts with RIPK1 through its receptor-interacting protein homotypic interaction motif (RHIM). Although RIPK1 is a kinase, most of its signaling pathways do not actually require its kinase activity but rather its function as a scaffolding protein. An exception to this is its signaling role in necroptosis. The assembly and activation of the RIPK1-RIPK3 complex downstream of TNF-α are dependent on the activities of both of these kinases to autophosphorylate themselves but not apparently to phosphorylate each other. This complex, with its associated proteins, including FADD and caspase-8, is often referred to as the necrosome. The downstream RIPK1-RIPK3 complex is believed to form a large amyloid-type aggregate through the interaction of the two proteins, although it is probably the oligomerization of the proteins and not the amyloid nature of the complex that leads to necroptosis. When caspase-8 activity is high, apoptosis prevails by several mechanisms of action. Firstly, active caspase-8 activates downstream apoptosis factors, but secondly, caspase-8 and other caspases cleave proteins that are essential for necroptosis, including RIPK1, RIPK3, and the CYLD deubiquitinase (the last of which potentiates necroptosis by removing ubiquitin from RIPK1, allowing it to interact with RIPK3), thus stabilizing RIPK1/RIPK3 oligomerization and downstream activation of MLKL. As RIPK1 plays many important nonnecrotic proinflammatory and survival roles in death receptor signaling, the prosurvival roles of RIPK1, as well as its apoptotic roles, often must be prevented for necroptosis to proceed. For instance, in the TNF-α pathway, RIPK1 is essential for the efficient activation of the prosurvival transcription factor NF-κB, as well as the MAP kinases ERK, JNK, and p38, which sometimes may also result in prosurvival signals. RIPK1 not only positively regulates the activity of the necrosome complex after necrotic stimuli but also negatively regulates promiscuous basal RIPK3 induction of necrotic cell death. Thus, there are generally two main conditions that are important for necroptosis to prevail downstream of death receptors: a significant reduction in caspase activity and the inhibition of the various E3 ubiquitin ligases and other proteins that ubiquitinate or otherwise modify RIPK1 and drive it toward the induction of other pathways to prevent it from associating with RIPK3. The importance of apoptotic regulation of necroptosis is highlighted by the knowledge that the developmental defects/lethality of apoptotic gene deletions, including FADD knockout, caspase-8 knockout, cFLIP-FADD double knockout (but not cFLIP knockout alone), XIAP-cIAP1 double knockout and cIAP1-cIAP2 double knockout, are rescued completely or to some degree by RIPK1/RIPK3 deficiency.
The activation of necroptosis, as mentioned previously, involves RIPK1. Signaling within the TNF-R1 complex is mediated in large part by the recruitment of the death domain-containing proteins TRADD and RIPK1, which bind (via their death domains) to each other and to the internal death domain of the TNF-R1 receptor upon activation by TNF. RIPK1 can be recruited to TNF-R1 in the absence of TRADD, especially when cells express high levels of RIPK1; however, most studies show that RIPK1 ubiquitination is lost in the absence of TRADD. Some studies have found a significant reduction in RIPK1 recruitment to the activated TNF receptor in TRADD knockout cells, while other studies found no difference in the recruitment of unmodified RIPK1 in its absence. Once recruited to the complex, RIPK1 is modified by several post-translational modifications, including phosphorylation and polyubiquitination, through several mechanisms. K63 ubiquitination of TRADD recruits TRAF2, which then recruits RING finger E3 ligases cellular inhibitor of apoptosis protein-1 (cIAP1) and cIAP2, which promote K63 ubiquitination of RIPK1 on its internal domain. Further recruitment of the linear ubiquitin chain assembly complex (LUBAC) promotes linear M1 polyubiquitination. The modification of RIPK1 via K63 allows the recruitment of the TGFβ activated kinase-1 (TAK1) and IκB kinase (IKK) complexes, which results in the activation of ERK, JNK, p38 and NF-κB. A20, CYLD, and OTULIN ubiquitin hydrolases remove the K63-linked and linear ubiquitination of RIPK1. Other polyubiquitination events modify RIPK1 with K48-linked ubiquitin chains that promote the proteasomal degradation of RIPK1. While RIPK1 degradation would in itself downregulate cell death, K48-modified RIPK1 is also unable to trigger necroptosis. Correspondingly, some phosphorylation events promote the cell death activity of RIPK1, while other phosphorylation events (for instance, Ser25 phosphorylation by IKKs or Thr189 phosphorylation by TBK1) inhibit cell death and necroptosis. Whilst RIPK1 is associated with the TNF receptor I complex, it functions in cell signaling events, but does not appear to be involved in cell death. However, should RIPK1 lose its protective phosphorylation and polyubiquitination, it dissociates from the main complex and forms secondary complexes, with or without TRADD. It is believed that secondary TRADD-dependent complexes induce apoptosis independent of RIPK1 or its kinase activity, while complexes with RIPK1, FADD, and caspase-8 initiate apoptosis that is dependent on the kinase activity of RIPK1. Although FADD and caspase-8 are in the secondary complexes, these are not required for TNF-dependent necroptosis but are both inhibitory of the necroptotic process, in part through caspase-dependent cleavage of RIPK1 and RIPK3, as has been mentioned. Assuming that RIPK1 is not inactivated by caspase-8, the autophosphorylation of RIPK1 leads to the association of its RHIM domain with that of the RHIM domain of RIPK3, and the oligomerization of these components leads to the active necrosome, which resembles an amyloid fiber, and mediates the phosphorylation of MLKL that is required for necroptotic cell death. In contrast to when in the receptor complex, two different K63 ubiquitination events that occur to RIPK1 later actually promote necroptosis by promoting necrosome formation. The E3 ligase c-Cbl promotes K63-linked polyubiquitination of RIPK1 under conditions where TAK1 is inhibited, leading to a detergent-insoluble aggregation of RIPK1 and its binding partners and stimulating necroptosis. Pellino-1 (PELI1) mediates K63-linked polyubiquitination to kinase-active RIPK1, causing it to more strongly bind and activate RIPK3. Conversely, ubiquitination by the carboxy terminus of HSC70-interacting protein (CHIP) leads to lysosomal degradation of the cytosolic, nonactivated pool of RIPK1. Curiously, these last two E3 ligases also control the negative regulation of RIPK3 through ubiquitination. While CHIP appears to downregulate the basal levels of RIPK3 (also through lysosomal-dependent degradation), PELI1 downregulates kinase-active RIPK3 that has already been activated in the necrosome through K48 polyubiquitination and proteasomal degradation. Thus, these two E3 ligases may control the basal threshold of necroptosis in the cell. Other proteins are known to negatively control the RIPK1-RIPK3 interaction by interacting with one of the RIPK proteins. Among these are c-Myc (MYC) and Aurora kinase A (AURKA) and its substrate glycogen synthase kinase-3 beta (GSK3β). Last, there are some other mechanisms by which the RIPK1-RIPK3-MLKL complex is controlled. For instance, protein phosphatase 1B (PPM1B) suppresses necroptosis by dephosphorylating RIPK3, which then prevents MLKL from being recruited to the necrosome. Casein kinase family members, on the other hand, are known to reinforce the phosphorylation of serine 227, which is the same event that initially occurs via autophosphorylation, and therefore promotes RIPK1-RIPK3-MLKL complex activation. Finally, reactive oxygen species (ROS), perhaps including those directly induced in the TNF receptor complex via NADPH oxidases or downstream of RIPK3 or from the mitochondria, may affect the stability of RIPK1-RIPK3-MLKL, although ROS, especially mitochondrial ROS, may not be absolutely required for necroptosis to occur. It is proposed, for instance, that RIPK1 autophosphorylation is upregulated by ROS. Given that the thiol groups of cysteine residues within the active sites of enzymes are often reactive with ROS due to their low pKA, which is well established for inactivating not only classic protein tyrosine phosphatases but also dual specificity phosphatases, it is likely that ROS may amplify necrosome formation by inactivating phosphates that would remove the important activating phosphates on RIPK1, RIPK3, and MLKL. For instance, if PPM1B, which was mentioned above, was inactivated, RIPK3 would have a higher propensity to remain phosphorylated and to therefore activate MLKL.
FAS was the first receptor discovered to mediate RIPK1-dependent necroptosis. Shortly after this, however, the Fas system was largely abandoned for studying necroptosis in favor of using the TNF system as a model for studying necroptosis. A few studies have employed FAS as a control or second model system for the verification of necroptotic requirements [see, for instance, refs. ], but fewer actual mechanistic necroptosis studies have been performed using FAS than TNF-α. Therefore, many things about FAS-induced necroptosis have been largely inferred from the understanding of its well-known mechanisms for inducing apoptosis and comparing this with what is known about the TNF mechanism. The mechanism of FAS-induced necroptosis is believed to be somewhat similar to that induced through TNF-R1. Although secondary complexes also occur in response to FASLG, it is not completely clear whether secondary complexes are essential for FASLG-induced necroptosis, since FADD and RIPK1 are recruited via their death domains directly to the cytoplasmic death domain of FAS, and caspase-8 is brought along directly into the receptor signaling complex. Unlike the TNF pathway, FADD appears to be required for necroptosis in the FAS pathway, as FADD-deficient cells are completely resistant to both apoptotic and necroptotic cell death. This might be because FADD supports RIPK1 recruitment to the complex. Similar to the TNF pathway, the inhibition of caspase-8 and cIAPs is usually required to direct the pathway away from apoptosis to necroptosis. While little else is known about FAS-induced necroptosis (other than RIPK1 and RIPK3 are known to be essential for the process), it is assumed that necrosome function in FASLG-induced complexes functions downstream similarly to TNF-induced complexes, with RIPK1-RIPK3 oligomerization leading to MLKL phosphorylation.
TRAIL-induced cell death is mediated by different receptors, but TRAIL also initiates necroptosis upon cIAP inhibition or TAK1 deficiency and/or when apoptosis is blocked. While two receptors, DR4 and DR5, mediate TRAIL signaling in the human system, only a single TRAIL receptor exists in mice, which appears to be more similar to DR5 than to DR4. TRAIL-initiated necroptosis is predicted to be very similar to the FASLG-induced necroptotic pathway, given that (as in FAS signaling) FADD is essential for necroptosis to proceed because it is largely through FADD-dependent mechanisms that complex components are recruited to the receptor. RIPK1 is likewise essential for TRAIL-induced necroptosis, but unlike the mechanisms involved in the FAS, TNF-R1, and DR5 receptors, RIPK1 does not directly interact with the receptor but is recruited through interactions with FADD-recruited caspase-8, the FADD death domain itself, and possibly the FADD-recruited TRADD death domain. Unlike in FAS signaling, TRADD is also recruited to the TRAIL receptor complex via FADD, but in this case, it largely has a negative effect on cell death (at least upon apoptosis, necroptosis was not examined), possibly by reducing FADD recruitment to the receptor. Alternatively, TRADD may promote survival signaling through additional recruitment of TRAF2. Curiously, although less FADD is recruited to the receptor, more RIPK1 is recruited to the receptor complex in the presence of TRADD than in its absence. Similar to the TNF-R1 complex, ubiquitination negatively regulates necroptosis; for instance, linear ubiquitination of RIPK1 by receptor-recruited LUBAC blocks TRAIL-induced necroptosis. Similar to c-Cbl in TNF-initiated necroptosis, the E3 ubiquitin ligase TRIM21 is an upregulator of necroptotic cell death in response to TRAIL. As with FAS and TNF-R1 signaling, secondary complexes form downstream of the TRAIL receptor complex; however, whether these complexes are required for necroptosis and whether the main receptor complex is capable of mediating necroptosis have not been studied.
The next best-studied pathway that induces necroptosis is probably that which is downstream of TLR4, which is activated in immune cells in response to bacterial lipopolysaccharide (LPS). RIPK3 is required for LPS-mediated necroptosis; however, although RIPK1 is recruited to some of the activated complexes, it does not appear to be required Rather, RIPK3 is recruited to Toll receptors through the cytosolic adaptor Toll/IL-1 receptor domain-containing adaptor protein inducing interferon-β (TICAM-1, also referred to as TRIF). This is one of two main adaptors that are recruited to TLR4, the other being myeloid differentiation primary response protein 88 (MYD88). TRIF contains a RIP homotypic interaction motif (RHIM) similar to RIPK1, by which it interacts with RIPK3. Thus, the TLR4 necrosome components downstream of TRIF are TRIF itself, RIPK3, and MLKL. Like RIPK1, TRIF is cleaved by active caspase-8, allowing apoptosis to downregulate necroptosis in this context, and although TLR4 is not a potent mediator of caspase activation by itself, other pathways that are induced downstream, such as TNF, can activate caspase-8. For example, TLR3, which also utilizes TRIF, IS a strong inducer of caspase-8 activation. Likewise, cIAPs 1 and 2 limits the necroptosis induced by LPS, similarly to the TNF pathway. In addition to the noncanonical activation of necroptosis that occurs downstream of TRIF, the MYD88 arm of the TLR4 pathway can also induce the canonical RIPK1/RIPK3/MLKL necrosome, although this may be dependent on the induction of TNF and the TNF-R1 pathway.
TLR3 is a pattern recognition receptor that recognizes double-stranded (ds)RNA, such as poly(I:C), as well as UVB-damaged self-RNA. TLR3 can induce apoptosis, which, like the other receptors mentioned, is negatively modulated by cIAPs. Apoptosis requires RIPK1-mediated recruitment of FADD and caspase-8. Necroptosis induced by this receptor is mediated by TRIF and requires RIPK3 and MLKL. TLR3-dependent necroptosis does not require RIPK1 in most cells. However, there are clearly some cell-type differences in TLR3 signaling, as macrophages (but not fibroblasts or endothelial cells) require RIPK1 for TLR3-mediated necroptosis.
ZBP1/DAI is a nucleic acid pattern recognition receptor that binds and detects zDNA and zRNA from pathogens and induces necroptosis and apoptosis. ZBP1 recruits RIPK3 via its RHIM domain, which then recruits and activates MLKL to induce necroptosis. RIPK1 is not essential for necroptosis induction through ZBP1 but actually inhibits both apoptosis and necroptosis induced by the receptor.
While RIPK3 is not considered an essential molecule for death receptor apoptosis, RIPK3 is required for the full initiation of caspase-8 activity when LPS-treated macrophages are treated with IAP inhibitors. Moreover, while not absolutely essential, the presence of RIPK3 does contribute to TNF-induced apoptotic cell death under conditions of cIAP1/2 depletion or TAK1 inhibition.
Since caspase-8 activity leads to the processing of IL-1β and its secretion, TLR4-initiated RIPK3-mediated activation of caspase-8 activity in cIAP-depleted macrophages leads to the production of mature IL-1β . Perhaps more significantly, NLRP3 inflammasome-induced IL-1β activation by TLR4 requires RIPK3 along with ROS production. In the absence of caspase-8, this requires MLKL, but in its presence, only the expression RIPK3 is required for inflammasome activation. NLRP3 inflammasome activation occurs prior to or independently of necroptosis. RIPK3 is also required for TLR3-mediated late signals that activate the inflammasome, which also has a corequirement for MLKL. Therefore, RIPK3 can promote NLRP3 inflammasome and IL-1β inflammatory responses both dependent and independent of MLKL. In the absence of A20, LPS induces spontaneous NLRP3 inflammasome activation that is dependent on RIPK3. In this case, the ubiquitylation of pro-IL-1β is increased, which then further promotes IL-1β cleavage and activation. Importantly, pathogens may thus engage RIPK3-mediated signaling to activate NLRP3.
Elucidating a role for RIPK3 in NF-κB activation has been a back-and-forth story. When first discovered, overexpression studies indicated that RIPK3 activated the NF-κB pathway. Later, studies in cells from knockout mice concluded that it did not affect TNF-induced NF-κB. However, these studies based their observations on IκBα phosphorylation and degradation, which were similar between WT and KO mice. Closer examination revealed that although RIPK3 did not affect IκBα, LPS-induced and NF-κB-dependent cytokine expression were greatly hampered in bone marrow dendritic cells. Further examination revealed that nuclear translocation of the RelB-p50 heterodimer of NF-κB was impaired in RIPK3 knockout cells. Thus, noncanonical NF-κB activity requires RIPK3 in specific cell types.
RIPK3 may have additional alternative roles in regulating metabolic enzymes associated with glycolysis and the mitochondria. Zhang et al. identified several metabolic enzymes in screening for interactions with RIPK3, including glycogen phosphorylase (PYGL), glutamate-ammonia ligase (GLUL), glutamate dehydrogenase 1 (GLUD1), as well as fructose-1,6-bisphosphatase 2 (FBP2), fumarate hydratase (FH), glycosyltransferase 25 domain-containing 1 (GLT25D1), and isocitrate dehydrogenase 1 (IDH1). The interaction of PYGL, GLUL, and GLUD1 with RIPK3 was verified in overexpression systems. Later work by the same group further showed more convincingly that RIPK3 (and MLKL) activates the pyruvate dehydrogenase complex to increase aerobic respiration. This acts as a source of ROS during necroptosis but may also regulate metabolism outside of a cell death setting.
Very little has actually been studied with regard to RIPK3 substrates, other than those found in necroptosis. While there are some functions of RIPK3 (mostly adaptor complex functions) that can occur in the absence of kinase activity, most RIPK3 activities are due to its enzyme function as a serine-threonine kinase. In necroptosis, it is largely the phosphorylation of the MLKL activation loop at T357, S358, S345, and S347 in human MLKL or T349 and S352 in mouse MLKL that is necessary for necroptosis to proceed. This activity is considered to be the standard canonical kinase function. However, MLKL is definitely not the only substrate for RIPK3. As mentioned in the preceding paragraph, RIPK3 phosphorylates PYGL, GLUL, GLUD1, and other metabolic enzymes mainly associated with mitochondrial metabolic pathways to increase aerobic respiration, which may or may not be solely associated with necroptosis. Interestingly, Al-Moujahed et al. showed that the deletion of RIPK3 suppresses the reprogramming of MEFs into induced pluripotent stem cells (iPSCs), a phenomenon that, in association with other data indicating that the growth rate of RIPK3 KO MEFs is significantly lower than that of WT MEFs, led them to conclude that this was because RIPK3 affects the expression of cell cycle/cell division genes. Consistent with this, phosphoproteomic analysis of possible RIPK3 phosphorylated peptides concluded that many of them were functionally associated with the cell cycle. Therefore, while little is known about cell cycle-specific substrates, it is likely that RIPK3 has other functions of its kinase activity outside of necroptosis. Among the more recently identified substrates of RIPK3 is the autophagy protein ULK1, which regulates both canonical and alternative autophagy. In our 2015 paper, we found that cytotoxic chemotherapy, which induces DNA-damaging agents, induces RIPK1/RIPK3 activity and subsequent necroptosis. Torri et al. found that RIPK1-independent RIPK3 phosphorylation is also induced by the genotoxic stress associated with DNA-damaging agents, and RIPK3 then phosphorylates ULK1 on S746. This phosphorylation event thereby activates alternative autophagy.
In our work in cancer cell lines, we found that necroptosis was induced by chemotherapeutics; further analysis revealed that RIPK3 was silenced by methylation in cancer cell lines and primary cancers, suggesting that the expression of necrotic cell death molecules may play a role in tumor repression and chemotherapy resistance in cancers. Other investigations have concluded that necroptosis/RIPK3 has a role in cancer mitigation and control; it has also been suggested that necroptosis-mediated inflammation and cell death may alternatively contribute to tumorigenesis and an immunosuppressive tumor microenvironment. As mentioned previously, it has later become evident that necroptosis may play significant roles in immunosurveillance due to the de novo synthesis of cytokines and chemokines that occurs especially in dying necroptotic cells, along with the release of DAMPs, which promote efficient immunogenic responses to cancer cells. Over time, the list of diseases that involve necroptosis and/or RIPK3 function has grown (see Fig. 2). Originally, this list included a facilitative role in tissue damage, such as in ischemia-reperfusion injury, atherosclerosis and host defense against viral infections. The findings from recent research in cardiovascular diseases have continued to suggest roles for RIPK3/necroptosis. Multiple cardiovascular pathologies are affected, including atherosclerosis, myocardial infarction (see, for instance, ref. ), stroke and the accompanying (previously mentioned) ischemia-reperfusion injury, abdominal aortic aneurysm, myocarditis, and thrombosis. RIPK3/necroptosis is involved in lung disease and injury. This includes acute respiratory distress syndrome and both acute and chronic lung injuries, both infectious and sterile in nature. Pulmonary diseases in which necroptosis/RIPK3 plays a role are thought to include COPD, idiopathic pulmonary fibrosis, and asthma. In the liver, RIPK3/necroptosis has a role in many pathological conditions. Reports in which RIPK3/necroptosis is implicated include immune-mediated liver injury, nonalcoholic fatty liver disease (specifically nonalcoholic steatohepatitis), alcoholic hepatitis, liver fibrosis, and cirrhosis. RIPK3 may also play a role in Gaucher’s disease, which can have a heavy liver component. In kidney diseases, RIPK3/necroptosis has been reported to be involved in acute kidney injury and chronic kidney diseases, and the resulting renal fibrosis. Aside from the cardiovascular conditions previously mentioned above, RIPK3/necroptosis is implicated in many pathologies of the brain and nervous system. Among these are several neurodegenerative diseases, including Parkinson’s disease, Alzheimer’s disease, amyotrophic lateral sclerosis (ALS), and multiple sclerosis. Targeting traumatic brain injury (TBI) through RIPK3 inhibition is also currently a matter of investigation. RIPK3 is thought to play a role in other autoimmune and inflammatory diseases of various organs, including the skin. We have shown a role for RIPK3 in toxic epidermal necrolysis (TEN), a condition of the skin and mucous membranes that results from adverse drug reactions. Among other inflammatory diseases, RIPK1/RIPK3 are believed to play a role in psoriasis (an autoimmune condition of the skin), rheumatoid arthritis (RA), pancreatitis, Crohn’s disease, and inflammatory bowel disease (IBD). In addition to rheumatoid arthritis, we have found that RIPK3 contributes to osteoarthritis (OA), through roles at least partly independent of MLKL activation.
There is clearly much work to be done in the necroptosis field with respect to cell death mechanisms and the involvement of RIPK3 in signaling and disease. Due to the putative effects on disease and inflammation, further efforts to understand the roles of RIPK3 in signaling and disease are likely to be of significant benefit to science in general and especially have implications for therapeutic gains in the treatment of human diseases. | true | true | true |
PMC9636415 | Xin Tracy Liu,Long Hoa Chung,Da Liu,Jinbiao Chen,Yu Huang,Jonathan D. Teo,Xingxing Daisy Han,Yinan Zhao,Fiona H. X. Guan,Collin Tran,Jun Yup Lee,Timothy A. Couttas,Ken Liu,Geoffery W. McCaughan,Mark D. Gorrell,Anthony S. Don,Shubiao Zhang,Yanfei Qi | Ablation of sphingosine kinase 2 suppresses fatty liver-associated hepatocellular carcinoma via downregulation of ceramide transfer protein | 04-11-2022 | Cancer metabolism,Lipid signalling | Hepatocellular carcinoma (HCC) accounts for 90% of primary liver cancer, the third leading cause of cancer-associated death worldwide. With the increasing prevalence of metabolic conditions, non-alcoholic fatty liver disease (NAFLD) is emerging as the fastest-growing HCC risk factor, and it imposes an additional layer of difficulty in HCC management. Dysregulated hepatic lipids are generally believed to constitute a deleterious environment cultivating the development of NAFLD-associated HCC. However, exactly which lipids or lipid regulators drive this process remains elusive. We report herein that sphingosine kinase 2 (SphK2), a key sphingolipid metabolic enzyme, plays a critical role in NAFLD-associated HCC. Ablation of Sphk2 suppressed HCC development in NAFLD livers via inhibition of hepatocyte proliferation both in vivo and in vitro. Mechanistically, SphK2 deficiency led to downregulation of ceramide transfer protein (CERT) that, in turn, decreased the ratio of pro-cancer sphingomyelin (SM) to anti-cancer ceramide. Overexpression of CERT restored hepatocyte proliferation, colony growth and cell cycle progression. In conclusion, the current study demonstrates that SphK2 is an essential lipid regulator in NAFLD-associated HCC, providing experimental evidence to support clinical trials of SphK2 inhibitors as systemic therapies against HCC. | Ablation of sphingosine kinase 2 suppresses fatty liver-associated hepatocellular carcinoma via downregulation of ceramide transfer protein
Hepatocellular carcinoma (HCC) accounts for 90% of primary liver cancer, the third leading cause of cancer-associated death worldwide. With the increasing prevalence of metabolic conditions, non-alcoholic fatty liver disease (NAFLD) is emerging as the fastest-growing HCC risk factor, and it imposes an additional layer of difficulty in HCC management. Dysregulated hepatic lipids are generally believed to constitute a deleterious environment cultivating the development of NAFLD-associated HCC. However, exactly which lipids or lipid regulators drive this process remains elusive. We report herein that sphingosine kinase 2 (SphK2), a key sphingolipid metabolic enzyme, plays a critical role in NAFLD-associated HCC. Ablation of Sphk2 suppressed HCC development in NAFLD livers via inhibition of hepatocyte proliferation both in vivo and in vitro. Mechanistically, SphK2 deficiency led to downregulation of ceramide transfer protein (CERT) that, in turn, decreased the ratio of pro-cancer sphingomyelin (SM) to anti-cancer ceramide. Overexpression of CERT restored hepatocyte proliferation, colony growth and cell cycle progression. In conclusion, the current study demonstrates that SphK2 is an essential lipid regulator in NAFLD-associated HCC, providing experimental evidence to support clinical trials of SphK2 inhibitors as systemic therapies against HCC.
Primary liver cancer was estimated to result in 8.3% of all cancer-related deaths worldwide in 2020, making it the third leading cause following lung and colorectal cancer [1]. Hepatocellular carcinoma (HCC) accounts for >90% of primary liver cancer [2]. In recent years, non-alcoholic fatty liver disease (NAFLD) is emerging as the fastest-growing etiology of HCC in developed countries [2]. In HCC of all causes, cirrhosis is the most prevalent pathological event [3]. However, approximately 40% of NAFLD-associated HCC (NAFLD-HCC) can develop from non-cirrhotic livers [4], suggesting that NAFLD-HCC may represent a unique pathogenic route. In NAFLD-HCC, hepatic lipid dysregulation is believed to constitute a deleterious environment that cultivates cancer initiation and progression. However, the lipid risk factors in this condition have not yet been adequately explored [5]. So far, the management of HCC remains challenging. NAFLD creates an additional layer of difficulty in the systemic treatment [6], and, worse still, there is no FDA-approved drug for NAFLD [7]. As such, understanding the lipid metabolic causes of NAFLD-HCC is a fundamental step in developing effective treatment and a key topic in liver cancer research. Sphingolipids are essential lipids that act as cell membrane constituents and signaling molecules [8] and have been implicated in cancer development [9, 10]. Ceramide is the central metabolite in the sphingolipid metabolic network [8–10]. Once synthesized in the endoplasmic reticulum, the majority of ceramide serves as the substrate for the synthesis of more complex sphingolipids, mainly sphingomyelin (SM) [11]. In the ceramide-to-SM conversion, ceramide is delivered to the trans-Golgi by its unique intracellular transporter, ceramide transfer protein (CERT) [12], which enables its access to sphingomyelin synthase 1 and 2 (SMS1 and SMS2) for SM production [13, 14]. SM is subsequently distributed to the plasma membrane or other subcellular membrane compartments [15], where it can be hydrolyzed back to ceramide by neutral sphingomyelinase (nSMase) or acid SMase (aSMase) [16, 17]. The balance between SM and ceramide is a critical determinant of cell fate. SM is considered a pro-cancer factor, promoting cell survival, proliferation, and migration [14, 18–20], and it is increased in human HCC compared with para-tumorous tissues [21–23]. In contrast, ceramide has been well characterized as an anti-cancer factor, inducing apoptosis and cell cycle arrest [10, 24], and it is decreased in human HCC compared with adjacent non-tumorous tissues [21–23]. Therefore, efforts to understand and modulate SM/ceramide homeostasis may provide a new approach to HCC management. Sphingolipid levels are determined by their catabolism, where sphingosine kinase (SphK) is the rate-limiting enzyme [8]. In brief, ceramide is hydrolyzed into sphingosine and a free fatty acid (FFA), followed by SphK-mediated phosphorylation of sphingosine to form sphingosine 1-phosphate (S1P), and then hydrolysis of S1P into non-sphingolipid products [8, 9]. There are two mammalian isoforms of SphK, denoted SphK1 and SphK2. SphK2 is the predominant isoform in the liver, contributing to 90% of total SphK activity [25]. Only a few studies have investigated the role of SphK2 in HCC. Administration of the selective SphK2 inhibitor ABC294640 profoundly suppresses the growth of HepG2 or SK-HEP-1 HCC xenografts [26]. Knockdown or inhibition of SphK2 sensitizes HCC cells to the chemotherapeutic agent regorafenib in vitro [27]. These all suggest a pro-cancer role of SphK2 in HCC. In addition, ABC294640 has been tested for HCC treatment in a Phase I clinical trial (ClinicalTrials.gov Identifier: NCT01488513), was planned to be used as a monotherapy for advanced HCC in a Phase II study (NCT02939807), and is being tested for intrahepatic cholangiocarcinoma (NCT03377179). Surprisingly, SphK2 has never been studied in any primary liver cancer models in vivo. Furthermore, we and others have identified SphK2 as a critical regulator of NAFLD and hepatic insulin resistance in diet-induced obese mice [28, 29]. Therefore, investigating the role of SphK2 in the development of NAFLD-HCC is important. In this study, we examined the role of SphK2 in NAFLD-HCC using Sphk2 knockout (KO) mice on a high-fat, high-sugar diet (HFHSD). In addition to liver tumor incidence, we examined immune cell infiltration, fibrosis and cell proliferation in mouse livers upon ablation of Sphk2. We also assessed the anti-cancer effects of SphK2 deficiency on cell viability, clonogenicity and cell cycle in hepatic cells exposed to a high-fat environment. Through a near complete profiling of the lipidome in mouse livers, we identified that deletion of Sphk2 disrupted the balance between SM and ceramide via downregulation of CERT. We also visualized SM and ceramide levels in human HCC specimens using mass spectrometry imaging. We further showed that downregulation of CERT was responsible for the tumor-suppressive effects of SphK2 deficiency. Collectively, our study revealed a previously unknown link between SphK2 and CERT in sphingolipid homeostasis and a critical role of SphK2 in NAFLD-HCC.
The Cancer Genome Atlas (TCGA) data analyses showed that the hepatic SPHK2 level was significantly increased in HCC as compared with normal livers, and it was further increased in extremely obese HCC patients (Suppl Fig. 1). This suggests an association of SphK2 with NAFLD-HCC, as NAFLD is highly prevalent in obese subjects [30]. To examine the role of SphK2 in the development of NAFLD-HCC, we fed wild-type (WT) and Sphk2-KO mice an HFHSD for 46 weeks in the absence of any chemical carcinogens. Sphk2-KO mice exhibited significantly lower body weight gain than their WT littermates (Fig. 1A, B). The liver mass and epididymal white adipose tissue (eWAT) mass were also lower in Sphk2-KO mice (Fig. 1C, E). However, the percentages of liver mass and eWAT mass to body weight were indistinguishable between the two genotypes (Fig. 1D, F). The levels of plasma non-esterified fatty acid (NEFA), total cholesterol (TC) and triglyceride (TG) were decreased in Sphk2-KO mice (Fig. 1G–I). Visible liver tumors developed in 3 of 14 WT mice, and neoplastic lesions were found in the liver of an additional 2 WT mice by microscopic examination (Fig. 1J–L). In contrast, neither visible tumors nor neoplastic lesions were identified in the equivalent number of Sphk2-KO mice (Fig. 1J–L).
Extensive efforts have been made to elucidate cellular and histological disparities between tumorous and non-tumorous tissue in HCC, but the microenvironmental factors that determine HCC outcomes in non-tumorous tissues are understudied. In the following work, we mainly focused on analyzing the differences in non-tumorous liver tissues of WT and Sphk2-KO mice in order to understand why Sphk2-KO suppressed NAFLD-HCC. Comparable levels of steatosis were found in the parenchyma of WT and Sphk2-KO livers using hematoxylin and eosin (H&E) staining (Fig. 2A, B). However, Sphk2-KO significantly ameliorated immune cell infiltration, hepatocyte ballooning, NAFLD activity score (NAS) and hepatic fibrosis in non-tumorous tissues (Fig. 2C–F), which are classic hallmarks of pro-carcinogenic injury in NAFLD-HCC. In line with this, plasma alanine aminotransferase (ALT) activity was reduced by 66% in Sphk2-KO mice (Fig. 2G). In addition, the percentage of cells positive for proliferation marker Ki67 was decreased by 3.4-fold in Sphk2-KO livers (Fig. 2H), suggesting that SphK2 deficiency might exhibit anti-NAFLD-HCC effects at the cellular level.
We next examined whether SphK2 deficiency led to anti-cancer effects in hepatocytes. To simulate a high-fat environment in vitro, we treated Huh7 cells with a combination of palmitate and oleate. As expected, viable cell number doubled approximately every 24 h in the control group (shCtrl) over the three days of FFA treatment, whereas cell proliferation was impaired by shRNA-mediated knockdown of SphK2 (shSphK2, Fig. 3A). In the absence of FFA treatment, viable cell number increased at a lower rate, and SphK2 knockdown had minimal effects on cell proliferation (Suppl Fig. 2). Subsequently, we examined the clonogenicity of FFA-treated cells. The colony number was comparable between control and SphK2 knockdown cells, whereas colony size was decreased by 78–84% in SphK2 knockdown cells (Fig. 3B). This result also implies an anti-proliferative effect of SphK2 deficiency in a high-fat environment. To further this notion, we analyzed the cell cycle using flow cytometry. Knockdown of SphK2 significantly suppressed cell cycle progression to the G2/M phase (Fig. 3C).
To explicate the lipid metabolic basis underlying the anti-NAFLD-HCC effects of Sphk2-KO, we examined lipid changes in non-tumorous liver tissues using lipidomics. First, we determined 12 major FFAs. The hepatic levels of oleic acid and palmitic acid, the two most abundant FFA species, were significantly decreased in Sphk2-KO mice (Suppl Fig. 3A). Levels of cholesterol ester (CE), diglyceride (DG), TG, phosphatidic acid (PA), phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylglycerol (PG), phosphatidylinositol (PI) and phosphatidylserine (PS) were unaltered in Sphk2-KO livers (Suppl Fig. 3B–D). The hepatic content of neutral lipids, including CE and TG, confirmed that mice of the two genotypes developed a similar degree of hepatic steatosis (Fig. 2B). Ablation of Sphk2 resulted in a slight elevation of free cholesterol (FC) levels in the liver (Suppl Fig. 3B). In contrast, FC levels were significantly decreased in SphK2 knockdown hepatic cells, without prominent alterations in its subcellular localization (Suppl Fig. 4A, B). Since SphK2 is key to sphingolipid catabolism, we next focused on sphingolipids. Dihydro-sphingosine, dihydro-ceramide, ceramide, and sphingosine sit proximally upstream of SphK2-mediated sphingolipid catabolism [8]. When Sphk2 was ablated, the three most abundant hepatic dihydro-ceramide and ceramide species (C16:0, C22:0, and C24:1), as well as dihydro-sphingosine and sphingosine, were significantly increased (Fig. 4A–C and Suppl Fig. 3E). In contrast, hepatic levels of S1P and dihydro-S1P, the products of SphK2, were unchanged in these unperfused liver tissues (Fig. 4D and Suppl Fig. 3F). In the sphingolipid metabolic network, complex sphingolipids are distal to SphK2-mediated regulation. They are normally highly abundant and cannot be altered by SphK2 manipulation. Indeed, hexosyl-ceramide (HexCer) levels were unchanged (Suppl Fig. 3G). However, unexpected results were seen at SM levels. Remarkably opposed to ceramide and dh-ceramide, C16:0, C22:0, and C24:1 SM were significantly decreased in Sphk2-KO livers deficiency (Fig. 4E), resulting in a 64% reduction in the ratio of SM (Fig. 4F). We also examined ceramide and SM levels in FFA-treated Huh7 cells. Consistent with the in vivo findings, knockdown of SphK2 increased ceramide and decreased SM levels, leading to a significant 55% reduction in the ratio of SM to ceramide (Fig. 4G–I). To further test the notion that SM/ceramide ratio is associated with HCC development, we examined the levels of C24:1 SM and C24:1 ceramide, the most abundant hepatic SM and ceramide species in human HCC and para-tumorous tissues, using mass spectrometry imaging. We found that C24:1 SM levels were increased, whereas C24:1 ceramide levels were decreased, in the tumorous tissues as compared with para-tumorous tissues (Fig. 4J). Notably, the SM/cer ratios were increased in the tumorous tissues of all specimens (Fig. 4J).
Given the importance of SM/ceramide ratio in HCC, we next investigated the cause of SM/ceramide ratio change upon SphK2 deficiency. To this end, we examined key factors that primarily regulated the interconversion between ceramide and SM in the livers of HFHSD-fed mice. These factors include CERT, SMS1, aSMase, and nSMase. Ablation of Sphk2 led to a downregulation of CERT protein, while SMS1, aSMase and nSMase levels were unaltered (Fig. 5A). In line with this, Cert1 mRNA was significantly decreased in Sphk2-KO livers (Fig. 5B). These findings were confirmed in FFA-treated Huh7 cells, in which knockdown of SphK2 resulted in a downregulation of CERT protein and mRNA (Fig. 5C, D), suggesting that SphK2 regulated CERT expression at the transcriptional level. Nuclear factor-κB (NF-κB) is a primary transcription factor of CERT [31], and thus we examined the activation of NF-κB by determining the phosphorylation of its p65 subunit. Knockdown of SphK2 greatly reduced p65 phosphorylation in FFA-treated cells (Fig. 5E), associated with the decrease of CERT protein and mRNA levels (Fig. 5C, D). In contrast, p-p65 levels were relatively low, and shSphK2 marginally repressed p65 phosphorylation, in the absence of FFA loading (Suppl Fig. 5B). In accord, shSphK2 did not alter CERT protein and mRNA expression when no FFA was added (Fig. 5D and Suppl Fig. 5A, B). We further analyzed the correlation between SPHK2 and CERT1 genes in the human HCC dataset sourced from TCGA, focusing on NAFLD-HCC. Due to the lack of NAFLD diagnostic information, we extracted data from human subjects with body mass index (BMI) > 25 in the TCGA-LIHC dataset, as BMI is strongly associated with NAFLD risk [30]. We found that SPHK2 and CERT1 mRNA levels were positively correlated in overweight or obese HCC subjects (Fig. 5F).
Having demonstrated that ablation of Sphk2 simultaneously increased ceramide and decreased SM levels (Fig. 4), we examined whether the reduced hepatic cell proliferation after SphK2 knockdown was due to the ceramide and SM changes. We increased cellular ceramide levels by treatment with exogenous C6-ceramide. In response to this compound, viable cell number was decreased to a comparable extent in both shCtrl and shSphK2 cells (Suppl Fig. 6A). We next reduced cellular ceramide levels by treatment with ceramide synthase inhibitor fumonisin b1. Rather than rescuing cell proliferation in shSphK2 cells fumonisin b1 caused a further reduction in viable cell number in combination with FFA treatment (Suppl Fig. 6B). These data indicate that increased ceramide alone was not sufficient to cause the inhibition of cell proliferation in shSphK2 cells. To further explicate whether the anti-cancer effects of SphK2 deficiency could be attributed to a combinational effect of both SM and ceramide changes, we overexpressed CERT in Huh7 cells prior to FFA treatment (Fig. 6A). Although it had minor impacts on cell viability and colony size in control cells, overexpression of CERT significantly increased both of these parameters in SphK2 deficient cells (Fig. 6B, C). Consistent with this, enforced expression of CERT restored cell cycle progression to the G2/M phase in SphK2 knockdown cells to a level comparable with control cells (Fig. 6D). These data indicate that downregulation of CERT and the subsequent disruption of the SM/ceramide balance were, at least in part, responsible for anti-cancer effects of SphK2 deficiency in NAFLD-HCC.
The anti-cancer effects of SphK2 deficiency have been demonstrated in HCC cell lines and xenograft models [26, 27]. However, the in vivo role of SphK2 was never studied in any primary HCC models, which restricts the development of new anti-HCC treatments targeting SphK2. To define the exact role of SphK2 in NAFLD-related HCC in vivo, we fed Sphk2-KO mice an HFHSD to induce liver tumors. The HFHSD model recapitulates the development of spontaneous HCC in NAFLD livers and enables a focus on the underlying lipid metabolic causes. Long-term exposure to an HFHSD leads to liver tumors in mice with varied incidence rates up to 68.8% [32–35]. We identified visible liver tumors and neoplastic lesions in 35.7% of WT mice (Fig. 1J). In marked contrast, no tumors or lesions were found in Sphk2-KO mice (Fig. 1J), defining a requirement for SphK2 in NAFLD-HCC development in vivo. Microenvironmental changes and pro-cancer cellular alterations contribute synergistically to HCC development [36]. SphK2 deficiency abrogated HFHSD-induced body weight gain, hyperlipidemia, hepatic inflammation and fibrosis (Figs. 1 and 2), resulting in a tumor-suppressive microenvironment. Meanwhile, SphK2 deficiency inhibited hepatocyte proliferation under a high-fat condition both in vivo and in vitro (Figs. 2H and 3), which demonstrates another layer of its anti-NAFLD-HCC effects at the cellular level. In line with this, we and others have found that ablation of SphK2 impairs, whereas overexpression of SphK2 activates the signaling of Akt, a master regulator of cell proliferation [28, 29, 37]. Our results collectively indicate that SphK2 deficiency reduces HCC risk in fatty livers at both systemic and cellular levels, providing in vivo experimental evidence for clinical trials of SphK2 inhibitors as systemic therapies against NAFLD-HCC. The anti-HCC effects of SphK2 deficiency warrant further examination in more complex primary HCC mouse models, including HFHSD combined with DEN or fibrogenic agents. In addition, the hepatocyte-autonomous roles of SphK2 in HCC development should be further elucidated using cell type-specific knockout mice. Dysregulated lipids are believed to drive NAFLD progression to HCC, but exactly which hepatic lipids impose significant HCC risk remains elusive [5]. We found that both WT and Sphk2-KO mice developed a similar level of hepatic steatosis upon HFHSD feeding, as reflected in H&E staining (Fig. 2A, B) and hepatic TG and CE determination (Suppl Figs. 3B and 3C). This supports the notion that accumulation of neutral lipids in the liver represents the degree of simple hepatosteatosis but not the risk for carcinogenic injury [38]. In contrast, increased FC in hepatocytes promotes cytotoxicity and proinflammatory responses, predisposing to the development of NASH and HCC [39–43]. Correspondingly, dietary cholesterol reprograms genetic and metabolic signatures in the liver, in favour of NASH-HCC [44]. Adenoviral overexpression of SphK2 significantly reduces total hepatic cholesterol (FC + CE) in HFD-fed mice [29]. Consistently, Sphk2-KO mice exhibited slightly elevated hepatic FC levels, with unchanged CE levels (Suppl Fig. 3B). Sphk2-KO profoundly downregulates gene expression of many key regulators in hepatic cholesterol homeostasis, including low-density lipoprotein receptor, sterol 27-hydroxylase, farnesoid X receptor α and bile salt export pump [45]. This might lead to hepatic cholesterol disturbance with long-term HFD feeding. Contrasting with the FC changes in vivo, SphK2 deficiency decreased FC levels in hepatic cells in vitro (Suppl Fig. 4A), implicating SphK2 in non-hepatocyte-autonomous regulation of hepatic cholesterol homeostasis in vivo. Given the importance of FC in non-alcoholic steatohepatitis (NASH) and HCC, the role of SphK2 in hepatic cholesterol homeostasis should be investigated in further studies. In the present work, this minor hepatic FC increase was not sufficient to overwhelm the tumor-suppressive effects of Sphk2-KO. In addition, SphK2 did not alter the hepatic levels of phospholipids (Suppl Fig. 3D). Therefore, it was unlikely that SphK2 regulated HCC development via cholesterol or phospholipid metabolism in the liver. Accumulation of palmitic acid, the most abundant saturated FFA, in the liver can induce NASH characterized by hepatic fibrosis and inflammation, leading to HCC [46, 47]. In addition, the accumulation of oleic acid, the most abundant mono-unsaturated FFA, in the liver also contributes to HCC development by promoting hepatic cell proliferation [48]. Consistent with its HCC-suppressive phenotype, Sphk2-KO decreased hepatic levels of both palmitic acid and oleic acid (Suppl Fig. 3A). The decrease of hepatic FFA might result from the reduction of circulating NEFA in Sphk2-KO mice (Fig. 1G), as the latter is the primary source of the former in human fatty liver disease [49]. The regulation of hepatic FFA levels might, at least in part, explain the anti-NAFLD-HCC effects of Sphk2-KO. It was expected that ablation of Sphk2 would cause significant sphingolipid remodelling. Indeed, dihydro-ceramide, ceramide, dihydro-sphingosine and sphingosine were increased in Sphk2-KO livers (Fig. 4A–C and Suppl Fig. 3E), as all of them reside upstream of SphK2-mediated enzymatic regulation in the sphingolipid metabolic network [8]. However, S1P, the catalytic product of SphK2, was only marginally decreased in Sphk2-KO livers (Fig. 4D). This could be due to the presence of blood in the tissue samples. S1P levels are much higher in blood than in tissues, due to the lack of S1P-degrading enzymes, S1P lyase and S1P phosphohydrolase, in erythrocytes [50]. Any blood contamination in isolated tissues would cause inaccurate quantitation of S1P [51]. This is more problematic in Sphk2-KO mice. SphK2 is essential for the disposal of blood S1P in the liver, and thus ablation of Sphk2 ordinarily elevates blood S1P levels by 2-3 fold [51, 52]. We did not perfuse mice prior to the isolation of the liver tissues for this study, as it was important to examine the immune cell infiltration. Therefore, it is not surprising that S1P levels were only marginally reduced in unperfused Sphk2-KO livers (Fig. 4D). In support of this, we have previously demonstrated that S1P levels are significantly decreased in both Sphk2 global KO and hepatocyte-specific KO (LKO) mouse livers after perfusion [28, 53]. SphK1 redundancy after Sphk2 deletion might also contribute to the maintenance of hepatic S1P levels. However, SphK1 is expressed at low levels [29] and only accounts for ~10% of total SphK activity [25] in the liver. We found no changes in SphK1 levels after Sphk2 knockout (Fig. 5A). Although the unchanged protein expression cannot preclude possible redundancy in enzymatic activity, this data suggests that SphK1 redundancy, if present, would be minor. SM represents the most abundant sphingolipid subclass [13]. It is also an essential structural lipid in cellular membranes, particularly lipid rafts [54], where it promotes cell proliferation and growth [14, 19, 55, 56]. In marked contrast, ceramide is known as a tumor-suppressive factor in most cancer types via a variety of biological actions, including the inhibition of cancer cell proliferation [10, 24]. Consistently, we found that SM levels were increased, whereas ceramide was decreased in human HCC tissues, as compared with the para-tumorous tissue (Fig. 4J). C6-ceramide decreased cell viability (Suppl Fig. 6A), confirming the cytotoxic effects of this compound. However, the reduction of cell viability was comparable between control and SphK2 knockdown cells, indicating that increased ceramide alone could not explain the inhibition of cell proliferation induced by SphK2 deficiency (Suppl Fig. 6A). We also decreased endogenous ceramide levels by treatment with the ceramide synthase inhibitor fumonisin b1. If increased ceramide was a leading cause for the defects upon SphK2 deficiency, fumonisin b1 should restore cell proliferation in SphK2 knockdown cells. However, fumonisin b1 induced an additional decrease in cell viability by up to 15% in shSphK2 cells as compared with shCtrl cells (Suppl Fig. 6B). These data together suggest that increased ceramide was not the sole factor in SphK2 deficiency-mediated regulation. Instead, mounting evidence has demonstrated that the ratio of SM/ceramide might determine the outcome of cancer development [57]. In line with this notion, SphK2 deficiency differentially regulated ceramide and SM levels in HFHSD-fed mouse livers and FFA-treated Huh7 hepatic cells, leading to a profound decrease in SM/ceramide ratio (Fig. 4F, I), which was associated with its anti-HCC phenotype (Fig. 1). The ratio of SM/ceramide is dictated by three key regulators: CERT, SMS, and SMases, and the key step in ceramide-to-SM conversion is ceramide transport from the endoplasmic reticulum to the trans-Golgi, mediated by CERT [12]. It is well demonstrated that CERT can determine the ratio of SM/ceramide, independent of SMS and SMases [12, 58–61]. Sphk2-KO resulted in a significant downregulation of CERT but did not alter levels of SMS1, aSMase, and nSMase in the liver tissues, indicating that the change of CERT accounted for the decreased ratio of SM/ceramide (Fig. 5A, B). This was also observed in hepatic cells treated with FFAs (Fig. 5C, D). SphK2 deficiency decreased CERT mRNA levels both in vivo and in vitro (Fig. 5B, D), indicative of transcriptional regulation. It has been demonstrated that CERT gene transcription is regulated by NF-κB, and thus tumor necrosis factor α can increase CERT mRNA levels by 3-4 fold via NF-κB activation [31]. SphK2 knockdown profoundly suppressed NF-κB activation in cells loaded with FFA (Fig. 5E). This might contribute to the downregulation of CERT. In support of this, both knockdown and inhibition of SphK2 inhibit NF-κB activation in regorafenib-resistant HCC cells [27]. Exactly how SphK2 regulates NF-κB activation and whether other transcription factors are implicated in the regulation of CERT levels are worthy of further investigation. Multiple lines of evidence have demonstrated that CERT is highly expressed in drug-resistant human subjects with ovarian and breast cancers [60, 62, 63]. In addition, knockdown of CERT sensitizes HCT-116 human colon cancer cells, BT474, HCC1954 and SK-BR3 HER2 + breast cancer cell lines, MDA-MB-231 human triple-negative breast cancer cells and A549 human lung carcinoma cells to apoptosis induced by chemotherapeutic agents [60, 62]. Furthermore, knockdown or inhibition of CERT leads to cell cycle arrest in murine embryonic cells and paclitaxel-treated HCT-116 cells [60, 62, 64]. To examine if CERT downregulation was critical for the anti-HCC effects of SphK2 deficiency, we overexpressed CERT in control and SphK2 knockdown hepatic cells. We found that overexpression of CERT restored cell proliferation, colony formation, and cell cycle progression in SphK2-deficient hepatic cells (Fig. 6). Notably, SphK2-mediated regulation of CERT is dependent on a high-fat environment. Knockdown of SphK2 failed to downregulate CERT levels or suppress hepatic cell proliferation in the absence of FFA treatment (Fig. 5D, Suppl Figs. 2, 5A, B). The different effects of SphK2 knockdown on CERT expression and cell proliferation between untreated and FFA-treated hepatic cells might be attributed to the states of NF-κB activation (Suppl Fig. 5B). FFA treatment dramatically activated NF-κB, which was suppressed by SphK2 deficiency (Suppl Fig. 5B). In contrast, the active form of NF-κB (p-p65) stayed at a relatively low level in untreated cells (Suppl Fig. 5B). In accord, CERT protein levels were regulated in the same fashion (Suppl Fig. 5B). Similarly, NF-κB is activated in drug-resistant cancer cells [27], and CERT is highly expressed in drug-resistant human cancers [60, 62, 63]; whereas, a lower CERT expression level is reported in some human cancers in the absence of chemotherapies [59, 65, 66]. These studies suggest that CERT may play a more explicit pro-cancer role under stress, such as chemotherapies and overnutrition, when the conversion of pro-apoptotic/anti-proliferative ceramide to proliferative SM is more critical for cell proliferation and growth. In summary, the current study provides both in vivo and in vitro evidence demonstrating a pro-cancer role of SphK2 in the development of NAFLD-HCC (Suppl Fig. 7). Knockout of Sphk2 suppressed HFHSD-induced HCC in mice, associated with inhibition of hepatic cell proliferation in a tumor-suppressive microenvironment. Mechanistically, SphK2 deficiency resulted in downregulation of CERT, leading to a reduced ratio of SM/ceramide, which is unfavourable for HCC cell proliferation. Restoration of CERT expression substantially improved hepatic cell proliferation in SphK2 deficient cells. Our findings demonstrate the SphK2/CERT axis as a novel therapeutic target for NAFLD-HCC, although the intricate role of these coupled lipid regulators in different cancer contexts warrants further investigation.
WT and Sphk2-KO mice on a C57BL/6 J background were used according to protocols (#2019-033) approved by Research Ethics and Governance Office, Royal Prince Alfred Hospital, Sydney, Australia. Sphk2-KO mice were obtained from Dr Richard Proia, National Institutes of Health, USA [67]. Mice were housed in a temperature-controlled, pathogen-free environment on a 12-hour light/dark cycle, and allowed food and water ad libitum. No statistical methods were used to predetermine the sample size. The sample size was estimated based on similar published studies indicating an up to 68.8% incidence of HCC tumors after HFD feeding [32–35]. We adopted a sample size of n = 14 per group, which was sufficient to achieve statistically significant differences. Male mice aged 8 weeks with matched body weight were fed with an HFHSD, using the recipe detailed in [68] with no cholesterol, for 46 weeks. Liver tissue and plasma were collected after 16 h starvation at the endpoint of the project. Levels of plasma NEFA, TC, TG, and ALT activity were analyzed using colorimetric assays (NEFA, TC, and TG kits from Fujifilm Wako Diagnostics, Osaka, Japan; ALT kit from Sigma-Aldrich, St. Louis, USA), as described previously [69].
The histology of mouse liver tissues was examined by H&E (Sigma-Aldrich, St. Louis, USA) staining. The grading of NAS, encompassing steatosis, ballooning, and inflammation scores, was determined by three experienced researchers who were blinded to the experimental groups based on H&E staining following the scoring system established by Liang et al. [70]. Liver fibrosis was examined by Picro Sirius Red (PSR) staining (Sigma-Aldrich, St. Louis, USA). Immunohistochemical staining of Ki67 was performed with Ki67 antiserum (Abcam #ab15580, Cambridge, UK). Images were taken using a Nikon NiE microscope and quantified using ImageJ software (FIJI version 1.52).
The Huh7 hepatic cell line was obtained from and authenticated (Dec 2021) by CellBank Australia. Cells were tested mycoplasma-free by MycoAlert Detection Kit (Lonza Bioscience, Basel, Switzerland). Cells were maintained in Eagle’s minimal essential medium (DMEM) supplemented with 10% v/v fetal calf serum and 100 U/ml penicillin/streptomycin at 37 °C in a humidified incubator. Flag-tagged human CERT plasmid in pcDNA3.1/Zeo(+) vector was obtained from Genscript (Galaxis West Lobby, Singapore). Plasmid transfection was conducted using lipofectamine LTX PLUS reagent (Thermo Fisher, Waltham, USA). Short-hairpin RNAs (shRNAs) targeting SphK2 (#A, TRCN0000036973 and #B, TRCN0000359275) were constructed in pLKO.1 lentiviral vector (Sigma-Aldrich, St. Louis, USA). The lentivirus was generated in HEK293T cells using plasmids gifted from Dr. Didier Trono through Addgene, including pMD2.G, pMDLg/pRRE and pRSV-Rev [71]. Lentiviral transduction was carried out as described previously [28]. To simulate a high-fat environment, we treated cells with a combination of bovine serum albumin-coupled palmitate (200 µM, Sigma-Aldrich, St. Louis, USA) and oleate (400 µM, Sigma-Aldrich, St. Louis, USA). C6-ceramide and fumonisin b1 were supplied by Avanti Polar Lipids (Alabaster, USA) and Cayman Chemical (Michigan, USA), respectively.
Cell viability was examined by 3-(4,5-dimethyl-thiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt (MTS) assay (Promega, Madison, USA). The luminescence was determined at 490 nm on a TECAN Infinite M1000Pro plate reader.
Huh7 cells were seeded at 400 (Fig. 3B) or 800 (Fig. 6C) cells/well in 6-well plates. Cells were treated with FFA combination for 10 days. Colonies were fixed with 4% cold paraformaldehyde, followed by crystal violet (0.5% w/v, Sigma-Aldrich, St. Louis, USA) staining [72]. Images were captured using a ChemiDocTM Touch Imaging System (Bio-Rad Laboratories, Hercules, USA) and quantified using ImageJ software (FIJI version 1.52).
Cell cycle was examined in Huh7 cells fixed with 70% cold ethanol at 4 °C overnight. The fixed cells were then incubated with FxCycle™PI/RNase Staining Solution (Thermo Fisher, Waltham, USA) for 30 min at room temperature and analyzed using BD LSRII (BD Biosciences, Franklin Lakes, USA) [72]. G2/M phase was determined using FlowJo version 10 (BD Biosciences, Franklin Lakes, USA) in Waston (Pragmatic) mode.
Intracellular FC was probed by the transfection of mCherry-D4H [73, 74], while the nuclei were counterstained with ProLong Glass Antifade Mountant with NucBlue Stain. The confocal microscopy was performed using a Nikon C2 microscope, and the images were processed using FIJI ImageJ software.
Lipids were extracted from mouse liver tissues or Huh7 cells in 1 mL methanol containing a cohort of internal lipid standards (Avanti® Polar Lipids, Alabaster, USA). The liver tissues isolated from the first 8 animals of each genotype were analysed. Hepatic levels of TG, CE, and phospholipids were quantified using untargeted lipidomic profiling on a Q Exactive HF-X mass spectrometer, following lipid separation on a Waters Acquity C18 UPLC column [53]. LipidSearch software was used for lipid annotation, chromatogram alignment and peak integration [75]. In contrast, FFAs, DG, FC, and sphingolipids were determined by targeted lipidomics on a TSQ Altis triple quadrupole mass spectrometer, following lipid separation on an Agilent Eclipse Plus C8 column [28, 53]. Peaks were integrated using Xcalibur (Thermo Fisher, Waltham, USA) [28].
Human specimens encompassing both HCC and para-tumorous tissues were obtained from Royal Prince Alfred Hospital, Sydney, Australia, according to protocols (#2019/ETH13790) approved by the Sydney Local Health District Human Research Ethics Committee. Informed consent was obtained from all subjects. The specimens were fixed in 4% paraformaldehyde and cut into 40 µm-thick cryo-sections without embedding in paraffin or OCT. Lipid imaging was performed with a Prosolia 2D Desorption Electrospray Ionisation stage and a Synapt G2-Si QToF mass spectrometer with ion mobility using high and low fragmentation energy, denoted as DESI-HDMSE. The DESI solvent was 98:2 methanol:water, supplemented with 0.01% formic acid and 200 pg/µL Leu-enkephalin, and delivered at a flow rate of 2 µL/min. DESI imaging data were acquired using MassLynx (Waters, Milford, USA), and ion mobility drift time was processed with High Definition Imaging (HDI) software (Waters, Milford, USA) to calculate collision cross section (CCS) for lipid identification [76]. To verify lipid identifications, we scanned synthetic d18:1/24:1 ceramide and d18:1/24:1 sphingomyelin pure compounds (Sigma-Aldrich, St. Louis, USA) to confirm their mass and drift times. The obtained masses were compared against the LipidMaps database and the unified experimental CCS database. The signal intensities of these two lipids in tumor and non-tumor areas were analyzed using HDI software.
Proteins were extracted from liver tissue (isolated from the first 8 animals of each genotype) and Huh7 cells. Immunoblotting was conducted with the following antibodies: SphK1 (#12071), SphK2 (#32346, human-specific, used for Huh7 cells), FLAG (#14793), p-p65 (#3033), t-p65 (#4764) and GAPDH (#5174) from Cell Signaling Technology (Danvers, USA); nSMase (#ab131330) from Abcam (Cambridge, UK); SphK2 (#17096-1-AP, used for mouse liver tissues), CERT (#15191-1-AP), SMS1 (#19050-1-AP) and aSMase (#14609-1-AP) from Proteintech (Rosemont, USA). Chemiluminescence was detected with a BIORAD ChemiDoc TOUCH imaging system.
RNA extraction and reverse transcription were performed, as previously described [75]. RT-PCR was conducted on a Roche Lightcycler 480 machine using SensiFAST™ SYBR Lo-ROX Kit (Bioline, London, UK). Primer sets used for PCR were human/mouse CERT1/Cert1 F-CGATGTGTCCGTGCCAAAAT, R-CCATCCTCCAGGGTTCACATT (designed using Primer-BLAST, National Center for Biotechnology Information, US); mouse Gapdh F-GCCTGGAGAAACCTGCCAAG, R-TCATTGTCATACCAGGAAATG [77]; human GAPDH F-CGGAGTCAACGGATTTGGTC, R-CCATGGGTGGAATCATATTGG [78].
mRNA expression data of SPHK2 and CERT1 were extracted from the Liver Hepatocellular Carcinoma (LIHC) dataset generated by TCGA via https://portal.gdc.cancer.gov. We assessed 158 primary liver cancer cases that were overweight/obese subjects (BMI > 25). After determining both variables were not normally distributed by the Shapiro-Wilk test, SPHK2 and CERT1 data (transcripts per million) were log-transformed (natural log), followed by Pearson’s correlation analyses using Prism 9 (GraphPad, version 9).
Comparisons between two groups were analyzed by unpaired two-tailed t-tests, multiple comparisons were analyzed by one-way ANOVA with Tukey tests, and variance between groups were analyzed by F test, using GraphPad Prism version 9. The exact sample size and number of replicates (n) are indicated in the figure legends. Differences at values of p < 0.05 were considered significant.
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PMC9636497 | 35322581 | Ruizhen Huang,Chiyu Zhang,Xing Wang,Xin Zou,Zhengjie Xiang,Zewei Wang,Bin Gui,Tao Lin,Honglin Hu | Identification of FDFT1 as a potential biomarker associated with ferroptosis in ccRCC | 24-03-2022 | biomarker,FDFT 1,ferroptosis,renal cancer | Abstract Renal cell carcinoma (RCC) seriously threatens people's lives and health. The identification of some precise biomarkers during the process of RCC progression and the pathophysiologic procedure is critical for improving the diagnosis and management of RCC. Evidence suggests that ferroptosis may play a pivotal role in eradicating clear cell RCC (ccRCC, KIRC) tumor cells. We screened out the target prognostic ferroptosis‐associated genes and examined the functions of farnesyl‐diphosphate farnesyltransferase (FDFT1) in 786‐O cells by plasmid transfection. In our study, we identified FDFT1 as a potential marker correlating with ferroptosis in KIRC. Upregulated FDFT1 inhibited cell proliferation, migration, and invasion, and the underlying antitumor effects may occur via the AKT signaling pathway. Our study provides helpful evidence to study the complex physiopathology of KIRC. | Identification of FDFT1 as a potential biomarker associated with ferroptosis in ccRCC
Renal cell carcinoma (RCC) seriously threatens people's lives and health. The identification of some precise biomarkers during the process of RCC progression and the pathophysiologic procedure is critical for improving the diagnosis and management of RCC. Evidence suggests that ferroptosis may play a pivotal role in eradicating clear cell RCC (ccRCC, KIRC) tumor cells. We screened out the target prognostic ferroptosis‐associated genes and examined the functions of farnesyl‐diphosphate farnesyltransferase (FDFT1) in 786‐O cells by plasmid transfection. In our study, we identified FDFT1 as a potential marker correlating with ferroptosis in KIRC. Upregulated FDFT1 inhibited cell proliferation, migration, and invasion, and the underlying antitumor effects may occur via the AKT signaling pathway. Our study provides helpful evidence to study the complex physiopathology of KIRC.
Renal cell carcinoma (RCC) is a genitourinary system tumor that seriously threatens people's lives and health. The complex pathophysiological process of RCC, which makes up approximately 16% of patients, is metastatic at the time of diagnosis and has a low 5‐year relative survival rate, resulting in more than 65,000 new cases being diagnosed and almost 15,000 deaths every year in the United States. There are three major histological subtypes of RCC, including clear cell RCC (ccRCC), papillary RCC (pRCC), and chromophobe RCC (chRCC). Among these three histological subtypes, ccRCC accounts for 70%–80% of RCC cases. Surgical resection is the primary recommended therapeutic method for localized ccRCC; however, poor prognosis was also reported; approximately 30%–40% of ccRCC patients initially diagnosed with advanced‐stage disease developed metastatic recurrence, with 50%, 30%, and less than 11.2% survival rates at 1, 3, and 5 years, respectively, according to recent reports. , Herein, a comprehensive understanding of RCC is paramount to the development of improved patient management and treatment. In particular, the identification of some precise biomarkers during the process of RCC progression and pathophysiologic procedures is critical for improving the diagnosis and management of RCC. Thanks for the considerable development in transcriptome profiling, providing robust data that allow researchers to download cancer data for comprehensive analysis via bioinformatic technology. Furthermore, the application of the bioinformatics method may promote the identification of potential specific markers to encourage the early diagnosis or management of specific malignant tumors. For example, the Cancer Genome Atlas (TCGA) database provides publicly available cancer genomics data, including RNA sequence, copy number variation, DNA methylation, etc., which enables researchers to generate primary analysis before the comprehensive understanding of specific cancers. Ferroptosis is a novel form of cell death characteristic of iron‐dependent and reactive oxygen species (ROS)‐reliant and was first proposed by Dixon in 2012. In contrast to autophagy and apoptosis, ferroptosis is mainly triggered by iron‐dependent ROS accretion and subsequent extramitochondrial excessive lipid oxidation, which ultimately induces programmed cell death. Studies have shown that induction of ferroptosis can selectively eliminate a variety of tumor cells, which has been gradually regarded as a promising tumor treatment strategy. Emerging evidence suggests that ferroptosis plays a pivotal role in eradicating tumor cells deficient in key nutrients or damaged by ambient stress. , , Farnesyl‐diphosphate farnesyltransferase 1 (FDFT1, squalene synthase) has been identified as a ferroptosis‐related gene, and it is considered an important gene for prognosis prediction in colorectal cancer patients. , However, little is known about its mechanism in tumorigenesis and ferroptosis. Characterized by uncontrolled cell proliferation and partial resistance to radio‐ and chemotherapies, unsatisfactory outcomes are not uncommon in ccRCC patients. Traditional chemotherapeutic drugs are challenged by poor selectivity, strong side effects, and drug resistance. Despite great advances, novel therapeutics are still needed to address the poor outcomes in ccRCC. Ferroptosis is a promising strategy in renal cell carcinoma. The relationship between ferroptosis and ccRCC has attracted researchers' attention, and great efforts have been made to illustrate the molecular mechanism of ferroptosis in ccRCC. Yang et al. conducted a study on the effect of erastin in 60 tumor cell lines of eight tissues, and the representative RCC cell Lines 786‐O and Caki‐1 were used in their study. They found a high sensitivity of RCC cells in erastin‐induced cell death, which in other words, renal cell carcinomas were sensitive to ferroptosis. In addition, ccRCC cells were confirmed to be highly dependent on GSH synthesis in the processes of lipid peroxidation and ferroptosis, and any measures interfering with GSH generation may suppress renal tumor growth and restore normal renal tissue morphology. Our study was designed to screen some potential ferroptosis‐related genes and verify their role in ccRCC via in vitro experiments. The goal of our study was to elucidate their molecular mechanism and provide a theoretical basis for clinical treatment in ccRCC.
To conduct a comprehensive analysis of differentially expressed genes (DEGs) between ccRCC patients and normal tissues, the primary RNA sequencing (RNA‐seq) data and corresponding clinical information of ccRCC patients were downloaded from the TCGA cancer genomics program (https://portal. gdc.cancer.gov/repository). Then, the gene expression profiles were cleaned, sorted, and analyzed using the scale method provided in the “limma” R package. The data from TCGA are publicly available, and our study follows the TCGA data access policies and publication guidelines. Moreover, 60 ferroptosis‐related genes were obtained from previous studies. This study was assessed and approved by the Ethics Committee for Animal Experiments of the Second Affiliated Hospital of Nanchang University.
The DEGs between ccRCC and non‐cancerous samples were screened initially, adjusted p value <0.05 and |log2 FC| > 1 were set as the filter criteria, and all DEGs were selected and identified by using the “edgeR” R package and the “limma” R package. The clinical information and the expression of ferroptosis‐related genes in each sample were combined. Univariate regression analysis was performed to identify genes associated with prognosis, and a p value <0.05 was considered statistically significant to screen out prognostic genes. Candidate prognostic ferroptosis‐related genes were the intersection between prognostic genes and differentially expressed ferroptosis‐related genes.
The “clusterProfiler” R package was utilized to conduct Gene Ontology (GO), which was generated to annotate genes as well as identify featured biological properties of transcriptomic profiles and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses based on the prognostic ferroptosis‐related genes to systematically analyze gene functions. The visualization was performed by the “ggplot2” R package.
Using the online protein functional interaction database Search Tool for the Retrieval of Interacting Genes (STRING; http://string‐db.org), a protein–protein interaction (PPI) network was built, which may provide the perception of the pathogenesis or progression of diseases. Then, the possible predicted interactions among ferroptosis‐related molecules were indicated by the correlation plot, implemented through the “igraph” and “reshape2” R packages. The most significant modules in the PPI networks were identified using the plug‐in Molecular Complex Detection (MCODE). The selection criteria were as follows: MCODE scores ≥ 3, degree cutoff = 2, node score cutoff = 0.2, max depth = 100, and k‐score = 2. The results were regarded as the hub genes for subsequent studies.
The GEPIA database is a web platform that provides servers for cancer and normal gene expression profiling and interactive analyses. Based on the TCGA and GTEx gene expression profiles, this platform allows users to perform further survival analysis based on the gene expression levels of the specific selected genes. For example, by inputting a selected gene, a user can overview the overall or disease‐free survival (DFS) in their custom cancer types. We performed survival analysis based on the GEPIA platform and evaluated the relationship between hub gene expression and the survival rate in ccRCC patients.
786‐O and HK‐2 cells were purchased from the Cell Bank of Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences. 786‐O and HK‐2 cells were cultured in Dulbecco's modified Eagle's medium (DMEM) and minimum essential medium, respectively, containing 1% penicillin–streptomycin and 10% fetal bovine serum (FBS; Gibco) in 50‐mL culture flasks at 37°C under the condition of 5% CO2. The designed FDFT1 overexpression plasmid pcDNA3.1/KLF4‐HisB and the corresponding negative control (NC) plasmid pcDNA3.1‐EF1a‐mcs‐3flag‐CMV‐EGFP (Hanbio) were used for transfection. Cells were seeded into six‐well plates before transfection and were allowed to grow until 70%–80% confluence in each well. For transfection, Lipofectamine 3000 reagent (Invitrogen) was subsequently used to transfect cells with the FDFT1 overexpression plasmid and the corresponding control plasmid according to the manufacturer's instructions. Cells were collected 48 h after transfection for the subsequent studies.
TransZol Up reagent (TransGen Biotech) was used to extract total cellular RNA. Reverse transcription was performed to synthesize cDNA using the EasyScript® One‐Step gDNA Removal and cDNA Synthesis SuperMix reagent Kit (TransGen Biotech) according to the manufacturer's directions. Then, we used the SYBR Premix Ex Taq kit (Takara) for RT–PCR. Sequences for these genes are shown below: FDFT1 (sense: 5′‐GCAACGCAGTGTGCATATTTT‐3′, antisense: 5′‐CGCCAGTCTGGTTGGTAAAGG‐3′); GAPDH (sense: 5′‐ACCCAGAAGACTGTGGATGG‐3′, antisense: 5′‐TCAGCTCAGGGATGACCTTG‐3′), GPX4 (sense: 5′‐ACAAGAACGGCTGCGTGGTGAA‐3′, antisense: 5′‐GCCACACACTTGTGGAGCTAGA‐3′).
To evaluate the function of the FDFT1 gene in cancer cell migration, a wound healing assay was conducted in vitro. Transfected cells, NC group cells, and control group cells were inoculated into six‐well plates, and a wound line was created using a sterilized 200 μl plastic tip to scratch across the surface of cells when they grew to approximately 80%–90% confluence. The plates were washed twice with PBS to remove the suspended cells. The images of 0 h wound closure were photographed. After incubation with reduced serum culture medium for another 24 h, the cell wound closure was photographed again.
Transwell assays were performed to examine the cancer cell migration and invasion abilities. A total of 1 × 104,786‐O cells with 100 μl serum‐free DMEM cell suspension were seeded into the upper Transwell chamber, and 600 μl DMEM with 10% FBS was added into the lower chamber for the cell migration assay. For the cell migration analysis, we used Matrigel to mimic the extracellular matrix in the upper Transwell chamber before 1 × 104,786‐O cells were seeded into the surface of Matrigel. The same procedure was used for the migration assay in the bottom chamber. After incubation for 24 h (migration) and 48 h (invasion), the Transwell chambers were removed, and the cells were fixed with methanol. After the application of methyl violet staining solution, the penetrating cells were screened under a light microscope.
An iron assay kit (BioAssay Systems) was used to test the ferric iron level. The relative malondialdehyde (MDA) level was evaluated by a lipid peroxidation detection kit (Solarbio, Beijing, China) according to the manufacturer's instructions.
We first extracted total protein using RIPA lysis solution, and then a bicinchoninic acid protein determination assay (Solarbio) was implemented to examine the protein concentration. The cell proteins were separated by 10% SDS–PAGE, and the proteins were transferred to PVDF membranes in 1× Western blot transfer buffer. Five percent skimmed milk was used to block the PVDF membrane, and primary FDFT1 antibodies (1:5000; Proteintech), GAPDH antibodies (1:10,000; Proteintech), GPX4 antibodies (1:5000; Proteintech), and AKT antibodies (1:2000; Proteintech) were incubated with the membrane at 4°C overnight. After incubation with the secondary antibody, ECL (US Everbright Inc.) solution was used for further analysis.
To identify the function of FDFT1 in cancer proliferation, an EdU assay was performed. A total of 1 × 104 NC 786‐O cells and FDFT1 overexpression plasmid‐transfected 786‐O cells were plated in a 96‐well plate every well. The next day, the EdU assay was performed according to the manufacturer's instructions (EdU Imaging Kits; US Everbright Inc.). After the application of DAPI dye, cancer cell proliferation was examined by fluorescence microscopy.
A total of 500 cells/well were plated into a six‐well plate and incubated at 37°C in a humidified atmosphere with 5% CO2. After 14 days, the six‐well plate was removed and subsequently fixed with 4% paraformaldehyde and stained with methyl violet for colony counting.
All data were statistically analyzed using GraphPad Prism 8.0 software. Quantitative data are expressed as the mean (x) ± S.E. Differences of two groups were compared using the t test, and multiple groups were compared using one‐way ANOVA along with the Tukey post hoc multiple‐comparisons test; p < 0.05 was considered statistically significant.
The DEGs between normal tissues and KIRC were visualized by a volcano plot (Figure 1A). The significantly downregulated DEGs are displayed as a blue dot plot, and the red dot plot shows the upregulated DEGs. The prognosis‐related genes were screened by univariate Cox regression analysis based on the expression profile of ferroptosis‐associated genes in each sample (Figure 1B). The prognosis‐related ferroptosis‐associated genes were the intersection between ferroptosis‐associated genes and prognosis‐related genes (Figure 1C). The expression of these genes between normal tissues and tumors was visualized by the heatmap (Figure 1E), as well as the exhibition of prognostic risk among prognostic ferroptosis‐associated genes in the forest plots (Figure 1D).
Then, the PPI network of prognostic ferroptosis genes was constructed, and Cytoscape software was used to obtain this significant module (Figure 2A). The correlation analysis showed the correlation between the proteins. Cutoff = 0.2 was set as the filter criteria, red represents a positive correlation between proteins, and blue represents a negative correlation (Figure 2B). A total of five genes were identified as hub genes with degrees ≥3. KEGG pathway enrichment analyses and GO function enrichment analyses revealed that the biological classification of the prognostic ferroptosis‐associated genes was enriched in sulfur compound biosynthetic process, carboxylic acid biosynthetic process, organic acid biosynthetic process, and sulfur compound metabolic process in the term of biological processes. The cell projection membrane was the main change in the cell component. These genes may mainly exert their molecular function in lyase activity (Figure 2C). The top two changes in KEGG were ferroptosis and fatty acid metabolism (Figure 2D).
The exact expression of the five hub genes between normal tissues and the tumor was demonstrated in box plots accordingly (Figure 3). Although ACACA and FADS2 were differentially expressed between normal tissues and tumors, no significant difference was noted in overall survival (OS) or DFS. Furthermore, it seems that tumor tissues have low expression of ACSL3, FDFT1, and HMGCR, and the high expression of ACSL3, FDFT1, and HMGCR patients was shown to be significantly associated with better OS and DFS, which indicated the suppression roles in carcinogenesis.
To determine whether our hub genes exerted their functions as we predicted by the bioinformatics method, we selected FDFT1 for further study. Western blot and qPCR results showed that FDFT1 was downregulated in the KIRC cell Line 786‐O (Figure 4A,B). We further found that the overexpression of FDFT1 by plasmid transfection could downregulate the protein and mRNA levels of the ferroptosis biomarker GPX4 (Figure 4C,D), whose low expression level was associated with increased sensitivity to ferroptosis. , Iron assays and the lipid peroxidation product MDA were used to examine accumulation in FDFT1‐upregulated 786‐O cells (Figure 4E). These results suggested that FDFT1 participated in the metabolic processes of lipid metabolism and the biological process of ferroptosis. The Akt signaling pathway has been proven to participate in a series of biological mechanisms. Thus, we examined whether the Akt signaling pathway was involved in FDFT1‐regulated 786‐O cell development. Intriguingly, the Western blot results revealed that the Akt expression level was decreased when the FDFT1 level was upregulated (Figure 4F).
We focused on regulating FDFT1 expression to evaluate the potential mechanism of FDFT1 in the progression of KIRC. Upregulated expression of FDFT1 was realized by plasmid transfection. The transfection efficiency was examined by qPCR and western blot analysis. Our results illustrated that overexpression of FDFT1 remarkably attenuated 786‐O cell proliferation according to the EdU assay (Figure 5A). Similarly, our colony formation assay also revealed that the cell proliferation ability was significantly alleviated by the overexpression of FDFT1 (Figure 5B). The invasion and migration capacity after upregulation of FDFT1 and the corresponding NC plasmid was investigated by Transwell analysis (Figure 5C) and wound healing assays (Figure 5D). The results showed that in the FDFT1 overexpression group, cell migration and invasion were hampered compared with the corresponding NC group, indicating the potential cancer suppression ability.
Cholesterol is an important substance indispensable to human tissue cells and is involved in various aspects of human health and disease. It not only participates in the formation of cell membranes, but it is also the basic material of bile acid and vitamin D. It is generally believed that the accumulation of cholesterol is the culprit in the development of cardiovascular and cerebrovascular diseases in the elderly. However, multiple lines of evidence have identified that disordered cholesterol homeostasis could be a carcinogenic factor, thus leading to cancer progression, especially intracellular cholesterol levels. FDFT1 has been identified as the key enzyme for the synthesis of cholesterol. Furthermore, researchers have indicated that downregulated FDFT1 expression in kidney tumors could become a potential biomarker for kidney cancer diagnosis or therapies. Ferroptosis is a promising type of cell death pathway that is considered an essential link in the tumor suppression mechanism. Eliminating the key nutrient‐deficient malignant cells in the environment or cells that were damaged by infection or ambient stress led to the depression of tumorigenesis. In our study, the KEGG analysis results demonstrated that FDFT1 was mainly enriched in the ferroptosis pathway. Hence, we further investigated the role of FDFT1 in ferroptosis in KIRC. Our study provided evidence that FDFT1 overexpression could regulate GPX4 expression. The related indicators of ferroptosis, such as ferrous iron and MDA levels, in the KIRC cell Line 786‐O were also evaluated to determine whether FDFT1 was associated with ferroptosis. Our results indicated that FDFT1 may play a pivotal role in the development of KIRC by regulating ferroptosis. From a clinical perspective, TCGA datasets showed that high FDFT1 expression was associated with better prognosis in KIRC patients. Previous studies addressed that the inhibition of FDFT1 significantly inhibited prostate cancer cell proliferation, reflecting the antitumor effects in prostate cancer development and its aggressive phenotypes by suppression of FDFT1 expression. The important role of FDFT1 was also illustrated in ovarian cancer, colorectal cancer, and lung cancer. However, few studies have focused on FDFT1 in the field of KIRC research. Therefore, to further elucidate the function of FDFT1 in the physiopathologic mechanism of KIRC, we paid special attention to the biological behaviors of KIRC cells. Intriguingly, our study validated that upregulated expression of FDFT1 in 786‐O cells was correlated with poor proliferation, migration, and invasion abilities. This may, in other words, be related to FDFT1 overexpression inhibiting malignant progression and improving patient prognosis in KIRC. Akt is a serine/threonine kinase that can modulate many biochemical mechanisms once it is activated by a range of signals. Molecules that participate in cellular survival, proliferation, migration, metabolism, and angiogenesis have been reported to be downstream targets of Akt. There is growing evidence that Akt itself and its downstream effectors are highly related to tumor development in various types of cancers. A study on cholesterol deprivation in prostate cancer cells and xenografts found that tumor growth was suppressed and phosphorylation levels of Akt were reduced in the presence of alterations in lipid raft cholesterol content. In addition, FDFT1 is one of the key enzymes for the synthesis of cholesterol; therefore, the study of FDFT1 in cancer development is of great importance in cancer management. In our study, the overexpression of FDFT1 decreased the level of Akt expression, which was consistent with a previous study in colorectal cancer. Unfortunately, we failed to illustrate the specific molecular mechanisms of FDFT1 in the process of oncogenesis and its role in regulating ferroptosis via the Akt pathway. Our team will focus on studying the underlying mechanism and how FDFT1 exerts its antitumor effects in regulating cholesterol production and ferroptosis in the future. The protective mechanisms by which FDFT1 affects KIRC still need to be further explored.
In this study, we found that high expression of ACSL3, FDFT1, and HMGCR was associated with better OS and DFS. FDFT1 overexpression inhibited KIRC cell proliferation, migration, and invasion, and the ferroptosis and AKT signaling pathways were involved.
No conflict of interest exist in the submission of this manuscript, and the manuscript has been approved by all authors for publication.
This study was assessed and approved by the Ethics Committee for Animal Experiments of the Second Affiliated Hospital of Nanchang University.
This work was designed by Honglin Hu. Ruizhen Huang, Chiyu Zhang, Xing Wang, Xin Zou, Zhengjie Xiang, TL, BG, and ZW performed the whole study. RH, CZ, and XW analyzed and interpreted the data acquired by the bioinformatics method. RH drafted the paper and was responsible for the paper revision. | true | true | true |
PMC9636668 | Dandan Yu,Zhigang Zhao,Li Wang,Shishi Qiao,Zhen Yang,Qiang Wen,Guanghui Zhu | SOX21-AS1 activated by STAT6 promotes pancreatic cancer progression via up-regulation of SOX21 | 05-11-2022 | Pancreatic cancer,SOX21-AS1,SOX21,STAT6 | Background Pancreatic cancer (PC) is a highly malignant tumor which threatens human’s health. Long non-coding RNAs (lncRNAs) are implicated in many cancers, including PC, but their mechanisms in PC have not yet been entirely clarified. We focused on revealing the potential function of lncRNA SOX21-AS1 in PC. Methods Functional assays assessed SOX21-AS1 function on PC progression. Bioinformatics analysis, along with mechanism assays were taken to unmask the regulatory mechanism SOX21-AS1 may exert in PC cells. Results SOX21-AS1 possessed a high expression level in PC cells. SOX21-AS1 absence suppressed PC cell proliferation, migration, stemness and epithelial-mesenchymal transition (EMT) while elevated cell apoptosis. SOX21-AS1 positively regulated its nearby gene SRY-box transcription factor 21 (SOX21) at post-transcriptional level. Through mechanism assays, we uncovered that SOX21-AS1 sponged SOX21-AS1 to elevate SOX21 mRNA and recruited ubiquitin-specific peptidase 10 (USP10) to deubiquitinate and stabilize SOX21 protein. Moreover, signal transducer and activator of transcription 6 (STAT6) could transcriptionally activate SOX21-AS1 and SOX21 expression. Conclusions SOX21-AS1 aggravated the malignant development of PC, which might provide the utility value for PC treatment. Supplementary Information The online version contains supplementary material available at 10.1186/s12967-022-03521-5. | SOX21-AS1 activated by STAT6 promotes pancreatic cancer progression via up-regulation of SOX21
Pancreatic cancer (PC) is a highly malignant tumor which threatens human’s health. Long non-coding RNAs (lncRNAs) are implicated in many cancers, including PC, but their mechanisms in PC have not yet been entirely clarified. We focused on revealing the potential function of lncRNA SOX21-AS1 in PC.
Functional assays assessed SOX21-AS1 function on PC progression. Bioinformatics analysis, along with mechanism assays were taken to unmask the regulatory mechanism SOX21-AS1 may exert in PC cells.
SOX21-AS1 possessed a high expression level in PC cells. SOX21-AS1 absence suppressed PC cell proliferation, migration, stemness and epithelial-mesenchymal transition (EMT) while elevated cell apoptosis. SOX21-AS1 positively regulated its nearby gene SRY-box transcription factor 21 (SOX21) at post-transcriptional level. Through mechanism assays, we uncovered that SOX21-AS1 sponged SOX21-AS1 to elevate SOX21 mRNA and recruited ubiquitin-specific peptidase 10 (USP10) to deubiquitinate and stabilize SOX21 protein. Moreover, signal transducer and activator of transcription 6 (STAT6) could transcriptionally activate SOX21-AS1 and SOX21 expression.
SOX21-AS1 aggravated the malignant development of PC, which might provide the utility value for PC treatment.
The online version contains supplementary material available at 10.1186/s12967-022-03521-5.
Pancreatic cancer (PC) is an aggressive malignant tumor with high lethality [1]. It is estimated that the mortality of patients with PC is about 40,000 cases [2]. The prognosis and clinical outcomes of patients with PC are poor. Up to now, the major treatment methods for PC are surgical operation for early detection and chemotherapy [3]. In spite of various genetic and epigenetic changes recognized in PC, the accurate pathogenesis of PC remains indistinct [4]. Hence, understanding the occurrence and development of PC may be beneficial for us to identify novel and effective diagnostic and therapeutic targets for PC. As far as we know, at least 90% of the mammalian genome is transcribed as non-coding RNAs (ncRNAs). Accumulating studies have demonstrated that these ncRNAs are not transcriptional noise due to their important functions [5]. Long non-coding RNAs (lncRNAs), as a class of ncRNA, possess the length of over 200 nucleotides [6]. Recent evidences have proved that lncRNA modulates gene expression via various mechanisms [7]. The majority of well-studied lncRNAs are found to be important modulators in affecting cellular processes including cell cycle, growth, and apoptosis which make sure homeostasis [8]. As reported previously, lncRNAs can act as oncogenes or tumor repressors to regulate the development of PC [9]. LncRNA-H19 facilitates PC cell proliferation via modulating miR-194 and PFTAIRE protein kinase 1 (PFTK1) [10]. LncRNA PXN antisense RNA 1, namely PXN‐AS1 expresses at a low level in PC and inhibits PC progression [11]. Additionally, lncRNAs regulate genes activities via multiple mechanisms [12]. In parallel, lncRNAs also can participate in cancer regulation by acting as competing endogenous RNAs (ceRNAs) to competitively bind to microRNAs (miRNAs), thereby modulating the expression of miRNAs targets at post-transcriptional levels [13]. Up to now, there are still some unknown lncRNAs in PC to be further investigated. LncRNA SRY-box transcription factor 21 antisense divergent transcript 1 (SOX21-AS1) has been registered to exert regulatory functions in many types of cancer. SOX21-AS1 promotes breast cancer progression through the PI3K/AKT signaling pathway [14]. SOX21-AS1 aggravates glioma cell proliferation as well as cell invasion through elevating p21-activated kinase (PAK7) expression [15]. SOX21-AS1 accelerates the tumorigenesis of colorectal cancer by affecting myosin VI (MYO6) expression [16]. Moreover, SOX21-AS1 targets miR-24-3p and PIM2 to modulate lung cancer [17]. SOX21-AS1 serving as a diagnostic biomarker in cancer development has been demonstrated by many documents [18, 19]. However, how it may exert certain impact on PC is unclear. Through our investigation, SOX21-AS1 with high expression was firstly verified in PC cells. Therefore, we were intended to verify the detailed biological function as well as the potential mechanism SOX21-AS1 may have in PC.
ATCC (Manassas, VA, USA) supplied PC cells including CFPAC-1, Capan-1, BxPc3, PANC-1 and SW1990. Human normal pancreatic duct epithelial cells HPDE6-C7 were purchased from Shanghai Huzhen Biotechnology Co., LTD (Shanghai, China). CFPAC-1 and Capan-1 cells were grown in Iscove’s Modified Dulbecco's Medium (Gibco, USA). BxPc3 cells were grown in RPMI-1640 Medium (Gibco). PANC-1 and HPDE6-C7 cells were grown in Dulbecco's Modified Eagle's Medium (Gibco). SW1990 cells were grown in Leibovitz's L-15 Medium (Gibco). All the cells were supplemented with 10% fetal bovine serum (FBS), and the culture condition was set as: 37 °C, 5% CO2.
Two specific shRNAs were transfected into cells to stably silence SOX21-AS1 expression, and negative control (shRNA), pcDNA, and pcDNA-SOX21 overexpression vector, miR-576-5p mimics and NC mimics, miR-576-5p inhibitor and NC inhibitor, sh-STAT6#1/2 and shRNA were synthesized by Genepharma (Shanghai, China). The plasmids transfections were used Lipofectamine 3000 (Invitrogen, USA) for 48 h.
Total RNA was extracted from cells and tissues using Trizol reagent (Invitrogen, USA). Then RNA reverse transcription was applied via a PrimeScript RT master mix (Takara, Japan), followed by qPCR using SYBR Premix Ex TaqTM II (Takara, Japan). GAPDH and U6 were internal references. The gene expression was calculated using the 2−ΔΔCt method. The experiment was subject to three independent repeats.
600 cells were grown in plates for 14 days. Then cells were washed, fixed and subsequently stained. The colony numbers more than 50 cells were counted. The experiment was in triplicate.
The EdU (Ribobio, Guangzhou China) proliferation assay was performed conforming to the guidance. Cells were seeded into plates, and 100 μl medium containing 50 μM EdU was added. Then cells were fixed and counterstained by DAPI. Images were subject to the observation through a fluorescence microscopy (Nikon, Japan). The experiment was in triplicate.
Cells on slides were fixed, washed and permeabilized, followed by adding TUNEL reagent (12,156,792,910, Roche, Basel, Switzerland). DAPI was used to counterstain the cells and images were observed under a fluorescence microscope. The experiment was in triplicate.
Transfected PC cells were plated into 6-well plates for flow cytometry with Annexin V-FITC/PI double staining kit (Invitrogen) based on the guidance of suppler. Cells were double-stained in the darkroom for 15 min, and then subject to flow cytometer for cell apoptosis analysis.
8 μm transwell inserts were utilized in this assay. Cells (1 × 105) were cultured in a 200 μL serum-free medium and placed into the upper chamber. 600 μL of 10% FBS medium was added to the bottom chamber. After 24 h incubation, the non-migrated cells in the upper chamber were wiped and the migrated cells in the lower chamber were fixed and stained. The number of migrated cells was counted in five random views. The experiment was in triplicate.
Cells were plated in 6‐well plates and were cultured in the medium containing B27 (BD Pharmingen, USA), 20 ng/mL basic fibroblast growth factor (bFGF, Invitrogen, USA) and 20 ng/mL epidermal growth factor (EGF, Invitrogen, USA). After 14 days incubation, the cell spheroids were observed under an optical microscope. The experiment was in triplicate.
The proteins were extracted from cells using RIPA lysis buffer (Beyotime, Shanghai, China), followed by quantification using a BCA Protein Assay Kit (Abcam, UK). Then proteins were undergone SDS-PAGE electrophoresis and then transferred to PVDF membranes, followed by sealing with 5% skim milk. Subsequently, the membranes were hatched with primary antibodies at 4 °C overnight and then incubated with horseradish peroxidase-conjugated secondary antibodies. Immunoblots were detected using ECL western blotting substrate (Invitrogen, USA). The experiment was in triplicate.
Cells were planted into plates and grown on sterilized coverslips. Then cells were fixed with 4% paraformaldehyde and permeabilized with 0.5% Triton X-100, followed by sealing with 5% defatted milk. Next, the coverslips were incubated with primary antibodies against E-cadherin (Abcam, UK; 1/500) and N-cadherin (Abcam, UK; 1/500) at 4 °C overnight, and then incubated with fluorochrome-labeled secondary antibodies. Finally, the coverslips were stained with DAPI and imaged using a fluorescence microscopy (Nikon, Japan). The experiment went through three independent repeats.
GenePharma designed the FISH probes of SOX21-AS1. Cells were fixed and washed, and then were subject to permeabilization with 0.5% Triton X-100. Then, pre-hybridization buffer (Sigma-Aldrich, USA) was added with SOX21-AS1 probe. DAPI solution was used to redye cells and the fluorescent signal was observed under the microscope. The experiment was subject to three independent repeats.
PARIS kit was applied to measure the cytoplasmic and nuclear fractions based on instructions. Extracted RNAs were subject to RT‐qPCR analysis to determine the cellular distribution of SOX21-AS1. GAPDH and U6 were served as the cytoplasm control or the nucleus control. The experiment was in triplicate.
RIP assay was performed using the Magna RIP™ RNA-Binding Protein Immunoprecipitation Kit (Millipore, USA) in the light of provider’s descriptions. Cells were lysed in RIP lysis buffer, and immunoprecipitated with antibody against Ago2 (Abcam, UK; 1/50) or USP10 or negative control IgG (CST, USA; 1/20). Precipitated RNA was purified and analyzed by RT-qPCR. The procedure was subject to three independent repeats.
For RNA–RNA pull down assay, biotinylated miR-576-5p wild-type or mutant probe bought from Sangon Biotech (Shanghai, China) were utilized. Cell lysates was incubated with these biotinylated transcripts for 1 h at 37 °C. The RNA complexes were separated and analyzed by RT-qPCR. For RNA–protein pull down assay, the biotin-labeled SOX21-AS1 was transcribed in vitro. Cells were mixed with biotinylated SOX21-AS1. Then streptavidin agarose beads (Invitrogen, USA) was added. The associated complex was resolved by SDS-PAGE and went through western blot analysis. The experiment was run in three independent repeats.
Ubiquitin, SOX21, and the indicated plasmids were transfected into cells. The lysates were immunoprecipitated with the indicated antibodies on protein A/G beads with rotation. The eluted proteins were detected by western blot. This assay went through three independent repeats.
A ChIP assay kit (Beyotime, Shanghai, China) was used for the ChIP experiment based on the guidance of supplier. Cells were treated with paraformaldehyde for cross-links at room temperature. Cell lysates were then sonicated to get chromatin fragments of 200–300 bp. Then the cell lysates were hatched with anti-STAT6 (Abcam, UK; 1/50) or anti-IgG (CST, USA; 1/20). Precipitated chromatin DNA was purified and then for RT-qPCR analysis. The experiment went through three independent repeats.
pmirGLO dual luciferase vector (Promega, USA) was used to assess the direct binding sites of miR-576-5p on SOX21-AS1 or SOX21 3’UTR. The wild-type or mutant reporter constructs of SOX21-AS1 or SOX21 3’UTR was co-transfected with miR-576-5p mimics or NC mimics into cells for 48 transfection. The relative luciferase activity was measured using a Dual-Luciferase Reporter Assay kit (Promega, USA) and normalized to Renilla luciferase activity. The experiment was in triplicate.
The nude mice (4–6-week old) were purchased and maintained at the Experimental Animal Center of Shanghai Laboratory Animal Center, Chinese Academy of Sciences in SPF barrier facilities. PC cells stably transfected with sh-SOX21-AS1#1#1 or shRNA were re-suspended at 1 × 108 cells/ml. For subcutaneous tumorigenicity, a total of 100 μl of suspended cells were subcutaneously injected into the right bilateral hind legs of mice. The size of tumor volume was calculated every four days. Animals were sacrificed by cervical dislocation at 28 days post injection. The tumors were collected for further study. IHC was taken to detect the expression of E-cadherin, N-cadherin, Ki67 and PCNA of the tumor xenografts tissue to evaluate the proliferation area. The animal experiments were approved by the Animal Care Committee of Shanghai Municipal Hospital of Traditional Chinese Medicine, Shanghai University of Traditional Chinese Medicine (ethical code: T-No202204200S0720102 [202]).
This study was conducted with the application of many databases. GEPIA2 (http://gepia2.cancer-pku.cn/) database was applied to analyze the correlation of SOX21-AS1 expression and the overall survival of patients with PC. It was also utilized when we searched the co-expressed gene with SOX21-AS1 in PC. SOX21-AS1 expression in PC tissues was exhibited through TCGA database. UCSC (http://genome.ucsc.edu/) database was applied to confirm whether SOX21 was the nearby gene of SOX21-AS1. We applied starBase (http://starbase.sysu.edu.cn) to search potential miRNAs of SOX21-AS1 and SOX21. AnnoLnc (http://annolnc.gao-lab.org/) and JASPAR (http://jaspar.genereg.net/) databases were utilized to predict potential transcription factors combined with SOX21-AS1 promoter.
The experiments were undertaken independently for three times. Statistical analysis was analyzed using SPSS software. The analysis of data was performed using Student’s t-test and ANOVA between different groups. Experimental resells were exhibited as means ± SD. p value below 0.05 was referred as data with statistical significance.
At first, we applied GEPIA2 (http://gepia2.cancer-pku.cn/) database to analyze the correlation of SOX21-AS1 expression and the overall survival of patients with PC. As indicated in Fig. 1A, patients with high expression of SOX21-AS1 were accompanied with short survival time (p = 0.036, n = 89). Accordantly, data from TCGA database displayed a higher SOX21-AS1 expression in PC tissues than normal tissues (Fig. 1B). Consistently, PC cells including CFPAC-1, Capan-1, BxPc3, PANC-1 and SW1990 harbored high expression of SOX21-AS1 compared to human normal pancreatic duct epithelial cell HPDE6-C7 (Fig. 1C). We next transfected two specific shRNAs into two PC cells PANC-1 and SW1990 to silence SOX21-AS1 expression, and then conducted loss-of function experiments (Additional file 1: Figure S1A). As revealed in colony formation and EdU assays, SOX21-AS1 knockdown reduced the proliferative ability in PC cells (Fig. 1D-E). Besides, the apoptosis rate was elevated in SOX21-AS1 silenced PC cells, as manifested by TUNEL and flow cytometry assays (Fig. 1F-G). In parallel, SOX21-AS1 deletion also repressed the number of migrated PC cells (Fig. 1H). It was uncovered in sphere formation assays that SOX21-AS1 silence obviously inhibited the sphere formation efficiency of PC cells (Fig. 1I). Through IF assays, we found that the intensity of E-cadherin was strengthened after SOX21-AS1 interference, while N-cadherin displayed declined intensity (Additional file 1: Figure S1B). Additionally, western blot data showed that after SOX21-AS1 reduction, E-cadherin protein expression increased, while OCT4, Nanog and N-cadherin exhibited reduced expression (Fig. 1J). Same results were obtained from CFPAC-1 and BxPc3, another two PC cell lines (Additional file 2: Figure S2A-G). The above findings all demonstrated the oncogenic property of SOX21-AS1 in the regulation of PC cell malignancy.
We next searched the co-expressed gene with SOX21-AS1 via GEPIA2, and screened the top 4 genes (SOX21, VILL, PLCD3 and LMO7) for further screening (Fig. 2A). The data from GEPIA2 exhibited the positive correlation between SOX21-AS1 and these four genes in PC tissues (Fig. 2B). We further discovered that SOX21-AS1 silence declined the mRNA and protein levels of SOX21, whereas had no changes on those of the other genes (Fig. 2C, D). Subsequently, it was found that PC patients with high SOX21 expression had short survival time, and SOX21 displayed high expression in PC tissues (Fig. 2E). Furthermore, we confirmed that SOX21 was the nearby gene of SOX21-AS1 through UCSC (http://genome.ucsc.edu/) database (Fig. 2F). LncRNAs exerting their functions by cooperating with their nearby genes has been testified [20]. Thereby, to further confirm the regulatory model of which SOX21-AS1 on SOX21 in PC cells, we confirmed the cellular location of SOX21-AS1 in PC cells via FISH assay along with subcellular fractionation analysis, which confirmed the main location of SOX21-AS1 in the cytoplasm, suggesting the possibility of SOX21-AS1 regulating SOX21 at a post-transcriptional level (Fig. 2G, H). Collectively, SOX21-AS1 regulated its nearby gene SOX21 at a post-transcriptional level. We further investigated the interaction between SOX21-AS1 and SOX21 on PC progression, we up-regulated the level of SOX21 in PC cells (Additional file 1: Figure S1C), and found that SOX21 overexpression reversed the inhibited proliferation property in SOX21-AS1 silenced cells (Additional file 3: Figure S3A, B). The enhanced cell apoptosis led by SOX21-AS1 deletion was counteracted by co-transfection of pcDNA-SOX21 (Additional file 3: Figure S3C). Moreover, the suppressed migration capacity in SOX21-AS1 silenced cells was restored by co-transfection of pcDNA-SOX21 (Additional file 3: Figure S3D). The results from sphere formation assays indicated that SOX21 increase overturned the impaired effects of SOX21-AS1 knockdown on the stemness (Additional file 3: Figure S3E). Simultaneously, SOX21 overexpression could counteract the inhibited EMT process after SOX21-AS1 interference (Additional file 1: Figure S3F).
Through starBase (http://starbase.sysu.edu.cn), potential miRNAs that may combine with SOX21-AS1 and SOX21 were predicted. The results from Venn diagram displayed that only one miRNA (miR-576-5p) met the requirement (Fig. 3A). The low expression of miR-576-5p was verified in PC cells (Fig. 3B). The data from RIP assays showed that SOX21-AS1, miR-576-5p and SOX21 co-existence in the RNA-induced silencing complex (RISC), as demonstrated by the highly enrichment of these three RNAs in Ago2 groups (Fig. 3C). Meanwhile, we confirmed that SOX21-AS1 and SOX21 were both largely enriched in miR-576-5p WT probe groups, while had no changes in miR-576-5p MUT probe groups (Fig. 3D). Additionally, we separately predicted the binding sites of miR-576-5p on SOX21-AS1 and SOX21, and then we validated that miR-576-5p overexpression significantly reduced the luciferase activity of SOX21-AS1-WT and SOX21-WT, while the corresponding mutant groups displayed no difference (Additional file 1: Figure S1D and Fig. 3E, F). Furthermore, we carried out rescue experiments to explore whether SOX21-AS1 may regulate SOX21 expression and PC progression via sponging miR-576-5p. It was found that SOX21 expression and protein was reduced by SOX21-AS1 depletion, but this effect was partially offset by silencing miR-576-5p expression (Additional file 4: Figure S4A, B). Besides, we found that miR-576-5p down-regulation partly recovered the lessened proliferation ability caused by SOX21-AS1 silence (Additional file 4: Figure S4C, D). It was unveiled in TUNEL assays that miR-576-5p silence could counteract the elevated apoptosis in SOX21-AS1 silenced cells (Additional file 4: Figure S4E). Moreover, miR-576-5p inhibition could partly restore the repressed migration ability and stemness in SOX21-AS1 down-regulated PANC-1 and SW1990 cells (Additional file 4: Figure S4F, G). Additionally, the lessened EMT process caused by SOX21-AS1 deletion was partly rescued by miR-576-5p repression together (Additional file 4: Figure S4H).
We further treated two PC cells with the protein synthesis inhibitor CHX and measured the stability of the SOX21 protein. The results showed that the stability of SOX21 protein was decreased when SOX21-AS1 was down-regulated (Fig. 4A), and this effect was attenuated after treatment with proteasome inhibitor MG132 (Fig. 4B). Moreover, the ubiquitination of SOX21 protein was increased after SOX21-AS1 depletion (Fig. 4C). To uncover how SOX21-AS1 regulates the stability of SOX21 protein, we tried to identify the protein partners of SOX21-AS1 in PC cells using RNA pull down assay. One specific band exhibited on the electrophoretic gel at approximately 87 kDa in contrast to the antisense SOX21-AS1 (Fig. 4D). Then the gel was subjected to mass spectrometry and we finally identified SOX21-AS1-interacting protein USP10. Western blot and RIP analyses further confirmed the combination between SOX21-AS1 and USP10 (Fig. 4E, F). Also, the binding of SOX21 and USP10 was also verified by RIP assay (Fig. 4G). USP10 is a cytoplasmic ubiquitin-specific protease which can deubiquitinate and stabilize protein [21]. Thus we further supposed that SOX21-AS1 might interact with USP10 to deubiquitinate and stabilize SOX21 protein. To prove our assumption, we silenced USP10 expression and treated USP10-shRNA into two PC cells together with CHX to measure USP10 protein level. We could see that USP10 down-regulation inhibited the half-life of SOX21 protein (Fig. 4H, I), but this phenomenon was reversed after MG132 treatment (Fig. 4J). As expected, the ubiquitination of SOX21 protein was enhanced when USP10 expression was reduced (Fig. 4K).
Through AnnoLnc (http://annolnc.gao-lab.org/) and JASPAR (http://jaspar.genereg.net/) database, we predicted potential six transcription factors combined with SOX21-AS1 promoter (Fig. 5A). SOX21-AS1 expression was significantly declined when STAT6 was silenced, while the other candidates showed no variation (Fig. 5B–D), so STAT6 was chosen for further analyses. We found that STAT6 combined with SOX21-AS1 promoter and SOX21 promoter at four sites (Fig. 5E), and ChIP assays further validated that both SOX21-AS1 promoter and SOX21 promoter were enriched in STAT6 precipitates at P4 sites (Fig. 5F). Additionally, we found that P4-WT group displayed reduced luciferase activity after STAT6 silence, while the corresponding mutant group was barely affected (Fig. 5G). Conclusively, STAT6 transcriptionally activated the expression of SOX21-AS1 and SOX21.
In addition, we also performed in vivo experiments by establishing a xenograft tumor model to verify the impacts SOX21-AS1 may exert on tumor growth. According to the result, the sh-SOX21-AS1#1 group revealed an obviously lower speed of tumor volume and tumor weight compared with the empty vector group (Fig. 6A, B). Moreover, we found that SOX21-AS1 silence inhibited the EMT process according to western blot and immunohistochemistry analyses (Fig. 6C, D). Moreover, it was shown that after SOX21-AS1 silence, the apoptosis of tumor enhanced, while the expression of STAT6 exhibited no obvious change between different groups. Taken together, SOX21-AS1 silence inhibited tumor growth in PC.
In our study, we elucidated a new putative mechanism by which STAT6 transcriptionally activated SOX21-AS1 regulated its nearby gene SOX21 via acting as a ceRNA to target miR-576-5p and interacting with USP10 in a manner important for PC cell proliferation, apoptosis, migration and EMT (Fig. 7). In recent years, a large number of reports have demonstrated the close relationship lncRNAs possess with the tumorigenesis of PC [22]. The focus of our study, SOX21-AS1, is a relatively novel lncRNA. It has been elucidated in many cancers, such as oral cancer [23], hepatocellular carcinoma [24], lung adenocarcinoma [25], nephroblastoma [26] and osteosarcoma [27] in which SOX21-AS1 expression was testified to be higher in cancer cells. Consistent with these findings, we revealed the high expression pattern of SOX21-AS1 in PC cells, and SOX21-AS1 deletion obviously repressed PC progression in vitro and tumor growth in vivo. It was the first time that we had verified SOX21-AS1 as a potential regulatory molecule in the regulation of PC cells. LncRNAs have been described to interact with their nearby genes in the modulation of cancer cells [28, 29]. In our research, SOX21 was verified to be the nearby gene of SOX21-AS1, and it was positively regulated by SOX21-AS1 in PC cells. As reported previously, overexpression of SOX21 induces glioma cell apoptosis [30]. SOX21 promoter is a candidate noninvasive diagnostic biomarker for colorectal cancer [31]. Our study also proved that SOX21 was highly expressed in PC, and rescue experiments further validated that SOX21-AS1 aggravated PC cell malignancy via enhancing SOX21 expression. Cytoplasmic lncRNAs have emerged as ceRNAs in cancer development, including PC [32, 33]. Through bioinformatics analysis and related mechanism assays, miR-576-5p was proven to be the target miRNA of SOX21-AS1, and the ceRNA model was then uncovered in PC. MiR-576-5p has been documented to increase the cell migration and invasion in esophageal squamous cell carcinoma [34]. MiR-576-5p has been documented to aggravate colorectal cancer cell malignancy [35, 36]. Besides, miR-576-5p has been reported to be sponged by linc-PINT in esophageal cancer [37]. In line with these research outcomes, we verified the low miR-576-5p expression in PC cells. Since the experimental result of rescue assays in our study showed that miR-576-5p interference only partially offset the suppression on PC cell behaviors caused by SOX21-AS1 knockdown, we predicted that SOX21-AS1 may regulate PC cells via another pathway. Through mechanism experiments, we found that SOX21-AS1 could recruit USP10 to deubiquitinate and stabilize SOX21 protein. Furthermore, the ubiquitination of SOX21 protein was enhanced after USP10 expression was reduced in PC cells. USP10 is a member of the deubiquitinases (DUBs), and many studies have uncovered that USP10 can regulate protein stability by deubiquitination [38, 39]. USP10 has also been found to be targeted by miR-191 and thus contributing to the inhibition of PC [40]. What we revealed about USP10 on regulating SOX21 protein may help to provide some theoretical guidance for OC treatment in the future. At last, it was verified that STAT6 may be responsible for the up-regulation of SOX21-AS1 in PC as it could transcriptionally activate SOX21-AS1 and SOX21 expression in PC cells. STAT6 promotes the proliferation of colorectal cancer and breast cancer cells [41], but how STAT6 may exert certain functions on the biological properties of PC cells may need further exploration.
Our study elucidated that SOX21-AS1 played a tumor promoting role in PC, and a mechanism was further revealed whereby STAT6-activated SOX21-AS1 promoted PC cell malignancy via up-regulation of SOX21. Utilization of these results in clinical practice may contribute to the diagnosis and treatment for PC patients.
Additional file 1: Figure S1. Transfection efficiency of RNAs. A. SOX21-AS1 expression was reduced in PC cells via transfecting shRNAs targeting SOX21-AS1. B IF assays detected the intensity of EMT markers in sh-SOX21-AS1 transfected PC cells. C SOX21 expression was elevated in PC cells by pcDNA-SOX21 transfection. D MiR-576-5p expression was elevated by miR-576-5p mimics transfection. **P < 0.01.Additional file 2: Figure S2. SOX21-AS1 silence suppressed the progression of PC. A-G Loss-of-function assays were performed in another two PC cell lines (CFPAC-1 and BxPc3) to further verify the malignant cell behaviors including proliferation, migration, EMT as well as apoptosis upon SOX21 silence treatment. **P < 0.01.Additional file 3: Figure S3. SOX21-AS1 affected PC cell proliferation, apoptosis, migration, stemness and EMT via modulating SOX21 expression. Rescue experiments in PC cells transfected with shRNA, sh-SOX21-AS1#1 and sh-SOX21-AS1#1 + pcDNA-SOX21, respectively. A-B Cell proliferation detection. C Cell apoptosis detection. D Transwell assays detected the migration ability. E Sphere formation assays detected the stemness. F Western blot analyzed the protein levels of EMT markers and transcription factors. **P < 0.01.Additional file 4: Figure S4. SOX21-AS1 affected PC cell proliferation, apoptosis, migration, stemness and EMT via interacting with miR-576-5p. A MiR-576-5p expression was decreased in PC cells. Rescue experiments were conducted in PC cells transfected with shRNA, sh-SOX21-AS1#1 and sh-SOX21-AS1#1 + miR-576-5p inhibitor, respectively. B SOX21 mRNA along with protein levels. C, D Cell proliferation detection. E TUNEL assays detected the cell apoptosis. F The migration of PC cells was testified through Transwell assays. G Sphere formation assays detected the stemness. H Western blot analyzed the protein levels of EMT markers and transcription factors. *P < 0.05, **P < 0.01 | true | true | true |
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PMC9636673 | Shan Cong,Xin Di,Ranwei Li,Yingshu Cao,Xin Jin,Chang Tian,Min Zhao,Ke Wang | RBM10 regulates alternative splicing of lncRNA Neat1 to inhibit the invasion and metastasis of NSCLC | 05-11-2022 | NSCLC,RBM10,lncRNA Neat1,Alternative splicing,Invasion,Metastasis | Background Non-small cell lung cancer (NSCLC) accounts for more than 85% of the total cases with lung cancer. NSCLC is characterized by easy metastasis, which often spreads to bones, brains and livers. RNA-binding motif protein 10 (RBM10) is an alternative splicing (AS) regulator frequently mutated in NSCLC. We found that there were multiple peak binding sites between RBM10 and long non-coding RNA nuclear enriched abundant transcript 1 (LncRNA Neat1) by crosslinking-immunprecipitation and high-throughput sequencing (Clip-Seq). LncRNA Neat1 plays an indispensable role in promoting cancer in a variety of tumors and produces two splicing variants: Neat1_1 and Neat1_2. This study aims to explore the mechanism of RBM10 and LncRNA Neat1 in invasion and metastasis of NSCLC. Methods Through histological and cytological experiments, we assessed the expression level of RBM10 protein expression. The interaction between RBM10 and Neat1 was evaluated via Clip-Seq and RNA immunoprecipitation assay. The effect of RBM10 on Neat1 and its splicing variants was identified by RT-qPCR. The effect of RBM10 and Neat1 on invasive and metastasis phenotypes of NSCLC was analyzed using transwell invasion assay and scratch test. Additionally, downstream signaling pathway of RBM10 were identified by immunofluorescence and western blot. Results RBM10 exhibited low levels of expression in NSCLC tissues and cells. RBM10 inhibited the invasion and metastasis of NSCLC and recruited Neat1 and Neat1_2. Overexpression of RBM10 simultaneously inhibited Neat1 and Neat1_2, and promoted the expression of Neat1_1. On the other hand, silencing RBM10 promoted Neat1 and Neat1_2, and inhibited the expression of Neat1_1. From this, we concluded that RBM10 regulated AS of Neat1, and the tumor-promoting effect of Neat1 was mainly attributed to Neat1_2. RBM10 had a negative correlation with Neat1_2. In addition, RBM10 upregulated the expression of PTEN and downregulated the phosphorylation of PI3K/AKT/mTOR through Neat1_2, which ultimately inhibited the invasion and metastasis of NSCLC. Conclusion The RBM10 regulated AS of Neat1 to cause the imbalance of Neat1_1 and Neat1_2, and RBM10 suppressed the activation of the PTEN/PI3K/AKT/mTOR signal by downregulating Neat1_2, finally affected the invasion and metastasis of NSCLC. Supplementary Information The online version contains supplementary material available at 10.1186/s12935-022-02758-w. | RBM10 regulates alternative splicing of lncRNA Neat1 to inhibit the invasion and metastasis of NSCLC
Non-small cell lung cancer (NSCLC) accounts for more than 85% of the total cases with lung cancer. NSCLC is characterized by easy metastasis, which often spreads to bones, brains and livers. RNA-binding motif protein 10 (RBM10) is an alternative splicing (AS) regulator frequently mutated in NSCLC. We found that there were multiple peak binding sites between RBM10 and long non-coding RNA nuclear enriched abundant transcript 1 (LncRNA Neat1) by crosslinking-immunprecipitation and high-throughput sequencing (Clip-Seq). LncRNA Neat1 plays an indispensable role in promoting cancer in a variety of tumors and produces two splicing variants: Neat1_1 and Neat1_2. This study aims to explore the mechanism of RBM10 and LncRNA Neat1 in invasion and metastasis of NSCLC.
Through histological and cytological experiments, we assessed the expression level of RBM10 protein expression. The interaction between RBM10 and Neat1 was evaluated via Clip-Seq and RNA immunoprecipitation assay. The effect of RBM10 on Neat1 and its splicing variants was identified by RT-qPCR. The effect of RBM10 and Neat1 on invasive and metastasis phenotypes of NSCLC was analyzed using transwell invasion assay and scratch test. Additionally, downstream signaling pathway of RBM10 were identified by immunofluorescence and western blot.
RBM10 exhibited low levels of expression in NSCLC tissues and cells. RBM10 inhibited the invasion and metastasis of NSCLC and recruited Neat1 and Neat1_2. Overexpression of RBM10 simultaneously inhibited Neat1 and Neat1_2, and promoted the expression of Neat1_1. On the other hand, silencing RBM10 promoted Neat1 and Neat1_2, and inhibited the expression of Neat1_1. From this, we concluded that RBM10 regulated AS of Neat1, and the tumor-promoting effect of Neat1 was mainly attributed to Neat1_2. RBM10 had a negative correlation with Neat1_2. In addition, RBM10 upregulated the expression of PTEN and downregulated the phosphorylation of PI3K/AKT/mTOR through Neat1_2, which ultimately inhibited the invasion and metastasis of NSCLC.
The RBM10 regulated AS of Neat1 to cause the imbalance of Neat1_1 and Neat1_2, and RBM10 suppressed the activation of the PTEN/PI3K/AKT/mTOR signal by downregulating Neat1_2, finally affected the invasion and metastasis of NSCLC.
The online version contains supplementary material available at 10.1186/s12935-022-02758-w.
Lung cancer is one of the most common malignant tumors globally, of which non-small cell lung cancer (NSCLC) accounts for approximately 85% [1]. Due to the high rate of invasion, metastasis, and postoperative recurrence, the 5-year overall survival rate of NSCLC patients is less than 15% [2]. A greater understanding of the molecular mechanisms underlying NSCLC cell invasion and metastasis is crucial to prevent tumor metastasis and improve survival. RNA-binding proteins (RBPs), which are stably expressed in cells, bind to a variety of coding or non-coding RNAs (ncRNAs) and play an important role in the regulation of post-transcriptional gene expression, participating in splicing, processing, editing, methylation modification, and final decay [3]. RNA-binding motif protein 10 (RBM10) is a type of RBP located in the nucleus, which is frequently mutated in various types of cancers, especially lung adenocarcinoma (LA) [4]. RBM10 mutations were associated with tumor stage, lymphatic metastasis, and 5-year survival [5]. RBM10 inhibits the proliferation and invasion of lung cancer cells and promotes apoptosis by regulating tumor suppressor gene p53 [6] and Wnt/β-catenin signaling pathway [7]. RBM10 mutations are capable of changing the microenvironment of tumors and promoting their progression, rendering it a potential target for personalized cancer treatment [8]. Alternative splicing (AS) is an important mechanism by which cells regulate gene expression at the post-transcriptional level. Its products have a variety of spatial structures, which increase the diversity of transcriptomics. The expression levels of various splicing variants are also different, and their biological functions are also different. AS may have different or even opposing roles in tumor biologic behaviors, such as proliferation, apoptosis, angiogenesis, drug-resistance and metastasis [9]. RBM10 exerts a role in the regulation of AS similar to that of RBM5 [10]. NUMB is the most studied downstream effector of RBM10, and dysregulation of NUMB AS is frequently found in lung cancer [11]. RBM10 promotes the jump of NUMB exon 9, producing a NUMB isotype that leads to ubiquitination, and then the Notch receptor is degraded by the proteasome, thereby inhibiting the Notch signaling pathway to promote cell growth [12, 13]. To date, research on the AS function of RBM10 has mainly focused on coding RNAs. In the human genome, although nearly 75% of genes are transcribed into RNA, only about 2% of RNAs encode proteins; hence, the majority are ncRNAs [14]. Therefore, further research is necessary to clearly determine whether RBM10 affects ncRNAs via AS. Nuclear enriched abundant transcript 1 (Neat1), a long non-coding RNA (LncRNA), exerts a cancer-promoting effect in some tumors, such as thyroid carcinoma [15], colon cancer [16], liver cancer [17], and melanoma [18]. Using crosslinking-immunprecipitation and high-throughput sequencing (Clip-Seq), we found that RBM10 showed multiple peak binding sites on LncRNA Neat1. Two Neat1 isoforms sharing the same 5’ end have been described, the constitutive short isoform (Neat1_1, 3.7 kb) and the stress-inducible long isoform (Neat1_2, 22.7 kb) [19]. Neat1_1 transcripts are processed by 3′ end processing complex CPSF6–NUDT21 and are cleaved at the polyadenylation signal located upstream. processing of Neat1_1 is inhibited by Hnrnpk, which binds to sequences between the CFIm binding site and the PAS [20, 21]. Alternative poly A is also regulated by Tardbp, which binds to the GU-rich motifs upstream of the PAS [22]. The 3′ end of Neat1_2 is cleaved by RNase P, and the non-polyadenylated transcripts are stabilized by triple-helix RNA structures found in the terminal region of Neat1_2 [23]. Neat1_2 is a major component of paraspeckles [24]. Therefore, it is one of our research directions to determine whether RBM10 regulates the AS of Neat1 to affect the occurrence and development of lung cancer. In addition, PI3K/AKT/mTOR, as a classic signaling pathway in tumors, is often caused by abnormal PTEN gene function, resulting in inhibition of cell apoptosis, acceleration of cell cycle, promotion of angiogenesis and tumor invasion and metastasis [25–27]. The studies found that Neat1 can inhibit the expression level of PTEN in laryngeal cancer [28] and thyroid carcinoma [29] to promote malignant biological behavior of tumors. Previous research by our group also found that RBM10 can regulate RAP1/AKT/CREB to play a tumor suppressor role [30]. So whether RBM10 regulates the PTEN/PI3K/AKT/mTOR pathway through Neat1 is also one of the main objects of our research.
We collected 27 paired NSCLC and paracancerous tissues (> 3 cm away from tumor tissues) from patients who attended the Second Hospital of Jilin University between September 2019 and October 2021, including 14 cases of LA and 13 of squamous cell carcinoma (SCC). The patients did not receive chemotherapy or radiation before surgery. All patients were diagnosed according to the World Health Organization’s lung cancer criteria. This study was performed in accordance with the Declaration of Helsinki and was approved by the Ethics Committee of the Second Hospital of Jilin University (Changchun, China); all participants provided written informed consent. All tissue samples were assessed and confirmed by two pathologists independently, frozen in liquid nitrogen immediately after surgical resection, and then stored at − 80 °C until use.
The human NSCLC cell lines (A549 and H1299), and human bronchial epithelial cell line (BEAS-2B) were obtained from Shanghai Genechem Co., Ltd. (Shanghai, China). A549 and H1299 cells were cultured in Roswell Park Memorial Institute Medium 1640 medium (Gibco, Carlsbad, CA, USA) containing 10% fetal bovine serum (FBS), and BEAS-2B cells were cultured in Dulbecco’s modified Eagle’s medium (Gibco, Carlsbad, CA, USA) containing 10% FBS.
The overexpression RBM10 lentivirus, silencing RBM10 lentivirus, and negative vector were constructed by Shanghai Genechem Co., Ltd (Shanghai, China). A549 and H1299 cells were cultured in six-well plates, pooled to reach 70%–80% confluency, and cleaned with phosphate buffer solution (PBS). Lentiviruses and negative vector were added to the cells according to the manufacturer’s instructions. After 24 h of lentivirus transfection, cells were screened in puromycin medium, and cell lines with stable overexpression and silencing of the RBM10 gene were established. The Neat1_2 interference fragment was constructed by Suzhou GenePharma Co., Ltd. (Suzhou, China). A549 cells cultured in six-well plates were grown and pooled to reach 70%–80% confluency, and cleaned with PBS. The plasmid was added to the cell culture wells according to the instructions of Lipofectamine 2000 kit (Invitrogen, Carlsbad, CA, USA), and cultured for 24 and 48 h. The cells were used for subsequent experiments. si-Neat1_2a: Forward 5′-GCUUCCACCCUGGAAGAUATT-3′ Reverse 5′- UAUCUUCCAGGGUGGAAGCTT-3′ si-Neat1_2b: Forward 5′-GCUAGUUUCCUUCCAGUUATT -3′ Reverse 5′-UAACUGGAAGGAAACUAGCTT-3′
After cells and tissues were fully lysed in radioimmunoprecipitation assay buffer supplemented with protease and phosphatase inhibitors (Beyotime, Jiangsu, China), protein concentrations were detected using a BCA protein detection kit (Beyotime, Jiangsu, China). Protein samples (30 µg) were separated by electrophoresis on 8%–15% sodium dodecyl sulfate-polyacrylamide gels and transferred to polyvinylidene difluoride membranes (#PIVH00010, Merck Millipore, Burlington, MA, USA). After blocking for 2 h, the membranes were incubated with specific primary antibodies (diluted in PBS containing Tween-20) at 4 °C overnight, followed by the respective secondary antibodies at room temperature for 1.5 h. Protein bands were detected using enhanced chemiluminescence detection kit (Merck Millipore) and quantified using Image J software (http://rsb.info.nih.gov/ij/, Bethesda, MD, USA). The used primary antibodies were anti-RBM10 (#14,423‐1‐AP, 1:500), GAPDH (#60,004–1-lg, 1:3000) from Proteintech Group (Chicago, IL, USA); anti-PTEN (#9188 T, 1:1000), anti-PI3K (#4257, 1:500), anti-p-PI3K (#abs130868, 1:500), anti-AKT (#4691, 1:800), anti-p-AKT (#4060, 1:500), anti-mTOR (#2983, 1:1000), anti-p-mTOR (#5536, 1:1000) from Cell Signaling Technology (Danvers, MA, USA).
Total RNA was extracted from the tissues and cells using TRIzol reagent (Invitrogen, Carlsbad, CA, USA), and cDNA was obtained through reverse transcription using a kit (RR047A, Takara, Japan). RT-qPCR was performed using the SYBR Premix Ex TaqTM II (Perfect Real Time) kit (DRR081, Takara, Japan) on an ABI QuantStudio5 real-time PCR instrument (ABI, Foster City, CA, USA). Primers were synthesized by Shanghai Biotech Co., Ltd. (Shanghai, China) (Table 1). The Ct value of each well was recorded using β-actin as an internal reference. The 2−ΔΔCt formula was used to calculate the relative expression.
A microtome was used to slice 4-μm sections of NSCLC and paracancerous paraffin-embedded specimens. The sections were stained with a Bond-Max automated immunohistochemical staining device (Leica, Germany). Image-Pro Plus analysis software (version 7.0; Media Cybernetics, Inc., Rockville, MD, USA) was employed to determine the color integral optical density (IOD) value of high-power field images (200 × , 400 ×) taken at random.
A549 and H299 cells were seeded into confocal dishes at a density of 3 × 103 cells/dish. When the cell density reached 60%, the cells were fixed for 20 min with 4% paraformaldehyde. Cells were stained using the Immunol Fluorescence Staining Kit (#E-IR-R321, Elabscience Biotechnology Co., Ltd., Wuhan, China); after blocking with 10% goat serum at 37 °C for 30 min, PTEN antibody (#9188 T, 1:1000) from Cell Signaling Technology (Danvers, MA, USA) was added and incubated at 4 °C overnight. Incubation with CY3 fluorescent secondary antibody (1:100) at 37 °C for 60 min in the dark followed. Images were taken using an inverted fluorescence microscope.
Matrigel (#356,234, Corning Co., Ltd., Shanghai, China) was diluted with serum-free medium (1:8) and added to the upper part of the transwell chamber. The chamber was placed in a 24-well plate and incubated for 2 h at 37 °C in a humidified atmosphere containing 5% CO2. The cell concentration was adjusted to 105 cells/100 μl, and 100 μl were added to the upper chamber, whilst 500 μl of serum-containing (15%) medium were added to the lower chamber and incubated for 24 h. The small chamber was removed, and the Matrigel and upper chamber cells were wiped off with a cotton swab and fixed with 4% paraformaldehyde for 10 min. The cells were stained with 0.1% crystal violet for 10 min. Fields of view were randomly selected under a high-power microscope, and Image J software (http://rsb.info.nih.gov/ij/, Bethesda, MD, USA) was used to count the number of penetrating cells.
When the cell confluency reached approximately 90%, sterile pipette tips were used to apply a scratch to the middle of the well. The cells were cultured in serum-free medium for 24 h and cell migration was evaluated using Image J (http://rsb.info.nih.gov/ij/, Bethesda, MD, USA).
Clip-seq is a revolutionary technology to reveal the interaction of RNA molecules with RBPs at the genome-wide level. Clip-Seq technology refers to the coupling of the treated cells or tissue samples under 254 nm ultraviolet light irradiation, and the RNA is captured by the specific antibody of RBP to form a protein-RNA complex. The RNA fragments protected by protein molecules in the complex are retained, and the remaining fragments are degraded to protect the target fragments. The ends of the RNA fragments are labeled with radioactive phosphates, so that the complex of the protein and the RNA fragments can be separated by denaturing SDS-PAGE gel, and then radioactive imaging can be performed. Proteinase K degrades the protein component of the protein-RNA complex, thereby preserving the RNA component, extracting RNA fragments, and then performing high-throughput sequencing (Additional file 1: Fig. S1). The Clip-seq experiment was jointly completed by our research group and Appreciate The Beauty of Life, lnc (Wuhan, China). CLIP-Seq data of RBM10 generated by GSE48066. As for protocols, please refer to references [31, 32].
A549 cells were collected at the logarithmic growth phase, and dissociated at 4 °C for 1 h. The prepared magnetic beads were incubated with the antibody mixture for 2 h, and the final product was purified. RIP was performed using a RIP Kit (#P0101; Guangzhou Geneseed Biotechnology Co., Ltd., Guangzhou, China) in accordance with the manufacturer’s instructions. The antibodies used were anti-RBM10 (#14,423–1-AP, 1:500) and a secondary IgG antibody (ab205718, 1:50) from Abcam (Cambridge, MA, USA).
For all experimental results, statistical tests were performed using GraphPad Prism 6. Two-tailed Student’s t-tests were used for comparisons between the sample pairs. The correlation between RBM10 protein expression levels and LncRNA expression levels in tissues was examined using Pearson's Chi-squared test. Data are presented as mean ± standard deviation (SD), with a P value of < 0.05 indicating a statistically significant difference. All experiments were repeated at least three times.
We collected tissues from 27 patients with NSCLC, including cancerous and paracancerous tissues, 14 of which were LA tissues and 13 SCC tissues. We measured RBM10 protein expression using immunohistochemistry and western blot. The expression level of RBM10 in NSCLC tissues was lower than that in paracancerous tissues (Fig. 1A, B). Next, we compared the expression levels of RBM10 between NSCLC cell lines (A549 and H1299) and BEAS-2B cells, and found that the level was lower in A549 and H1299 cells compared to that in BEAS-2B cells (Fig. 1C).
We performed functional in vitro assays to explore the involvement of RBM10 in NSCLC progression. RBM10 was either overexpressed or silenced in A549 and H1299 cells through lentiviral transfection. Expression efficiency was determined by western blot (Fig. 2A). The results of the transwell invasion assay (Fig. 2B) and scratch test (Fig. 2C) indicated that RBM10 overexpression significantly attenuated cell invasion and metastasis, whereas silencing RBM10 markedly enhanced these processes. Epithelial-mesenchymal transition (EMT) is an important biological process in invasion and metastasis of tumor, and the upregulation of N-cadherin and Vimentin protein levels and downregulation of E-cadherin protein levels mark the occurrence of EMT. RBM10 overexpression decreased the expression of N-cadherin and Vimentin and increased the expression of E-cadherin. In contrast, RBM10 silencing reversely regulated these proteins (Fig. 2D).
Overexpression of RBM10 resulted increased the PTEN expression and decreased phospho‐PI3K, phospho‐AKT, and phospho-mTOR levels in NSCLC cells; in contrast, the PTEN expression was decreased and the phosphorylation of PI3K/AKT/mTOR was greatly increased in RBM10‐silenced NSCLC cells compared to the control group (Fig. 3A–C).
Clip-seq analysis showed that RBM10 could bind 353 lncRNAs, and among the top 10 maxHeight binding peaks, RBM10 had four binding sites with Neat1 (Fig. 4A). We confirmed the binding between RBM10 and Neat1 by RIP experiment (Fig. 4B). The expression level of Neat1 decreased after RBM10 overexpression, but increased after RBM10 silencing (Fig. 4C). We further explored the structure of Neat1 and its transcripts, and found that the main binding site of RBM10 was located at 65,426,044–65,434,501, and the binding site was mainly concentrated on NEAT1_2 (Fig. 4D). We confirmed that RBM10 could recruit Neat1_2 through RIP again (Fig. 4E). Moreover, we found that RBM10 overexpression inhibited Neat1_2 expression, while RBM10 silencing promoted Neat1_2 expression (Fig. 4F). In addition, the proportion of Neat1_2 in Neat1 decreased significantly in RBM10 overexpressed A549, while the proportion of Neat1_2 in Neat1 increased significantly after RBM10 silencing (Fig. 4G). Because Neat1_1 has no specific primer, based on the ratio of Neat1_2 in Neat1 and the expression of Neat1 and Neat1_2 we concluded that Neat1_1 increased after RBM10 overexpression and decreased after RBM10 silencing (Fig. 4H). Together, these results suggest that RBM10 regulates the balance between the two splicing variants of Neat1 through AS.
By RT-qPCR, the results showed that Neat1_2 expression was significantly upregulated in NSCLC cell lines compared to that in BEAS-2B cells (Fig. 5A). Furthermore, compared with the paracancerous tissue group, Neat1_2 expression was significantly higher in the NSCLC group on RT-qPCR analysis (Fig. 5B). Pearson correlation analysis indicated a negative relationship between RBM10 and Neat1 in LA tissues (R2 = 0.4390, p < 0.05) and SCC tissues (R2 = 0.4116, p < 0.05) (Fig. 5C).
We stably silenced Neat1_2 in RBM10-silenced A549 cells (Fig. 6A). The results of the transwell invasion assay (Fig. 6B), scratch test (Fig. 6C), and the expression of EMT relative proteins (Fig. 6D) illustrated that silencing of Neat1_2 reversed the impact of sh-RBM10 on cell invasion and metastasis. These results indicate that an inhibition of RBM10 promotes the invasion and metastasis of NSCLC cells by promoting the expression of Neat1_2.
RBM10 silencing resulted in decreasing PTEN protein level and increasing phospho‐PI3K, phospho‐AKT, and phospho-mTOR levels in NSCLC cells. Silencing Neat1_2 reversed these changes in expression in RBM10-silenced A549 cells (Fig. 7A–C). Together with the results presented above, these findings suggested that RBM10 promoted the expression of PTEN and suppressed the phosphorylation of PI3K, AKT, and mTOR via Neat1_2.
In recent years, several articles have reported the regulation of RBM10 in lung cancer [30, 33, 34]. Although there are conflicting conclusions about whether this protein promotes or inhibits lung cancer, most of the experimental results confirmed that RBM10 is a tumor suppressor factor [30, 34, 35]. We demonstrated by immunohistochemistry and western blot that RBM10 expression reduced in NSCLC tissues and cell lines. We subsequently investigated its effect on NSCLC phenotypes, and the results showed that RBM10 overexpression inhibited invasion and metastasis, whereas RBM10 silencing promoted these processes. The ability of tumor cells to invade and metastasize is enhanced through loss of epithelial features and acquisition of mesenchymal phenotypes in a process known as EMT [36]. EMT is often accompanied by upregulation of N-cadherin and Vimentin, and downregulation of E-cadherin. We found that RBM10 inhibited the expression of N-cadherin and Vimentin, and increased the expression of E-cadherin, further confirming that RBM10 inhibited invasion and metastasis of NSCLC. In further investigation of the mechanism of RBM10 in NSCLC, Clip-Seq confirmed that RBM10 binds 4040 RNAs, including 353 lncRNAs. LncRNAs regulate gene expression not only at the post-transcriptional level but also at the transcriptional and epigenetic levels [37]. Therefore, the mechanism of RBM10 and LncRNA in NSCLC is the focus of our study. Four of the top ten loci in lncRNA according to maxHeight are Neat1, and we further confirmed through the RIP experiment that RBM10 can recruit Neat1. One study showed that Neat1 expression increased in LA tissues, and that ATF2 and Neat1 form a positive feedback loop mediated by miR-26a-5p, coordinately contributing to LA progression [38]. Zhao et al. concluded that downregulation of Neat1 in NSCLC inhibits its growth, migration, and invasion through the miR-204/NUAK1 axis [39]. Neat1 was further found to affect NSCLC by elevating its malignant potential via the miR-582-5p/EIF4G2 axis [40]. As we expected, RBM10 overexpression suppressed Neat1 expression, whereas RBM10 silencing promoted Neat1 expression. From the structure of Neat1, we found two transcripts of Neat1 as Neat1_1 and Neat1_2. The latter is currently the only RNA molecule identified in paraspeckles, which are ribonucleoprotein bodies found in the interchromatin space of mammalian cell nuclei, coordinating various biochemical processes [23]. Overexpression of paraspeckles has been associated with some cancers and is often associated with poor prognosis [41, 42]. Two studies have shown that Neat1_2 promotes malignant biological behaviors in thyroid cancer through the competing endogenous RNA mechanism [43, 44]. The high expression of Neat1_2 in liver cancer is related to an increase in cisplatin resistance [45]. Knockdown of Neat1_2 increases the sensitivity of hepatocellular carcinoma cells to radiotherapy [46]. In contrast, Neat1_1 is not a key component of paraspeckles and has also been detected in several non-paraspeckle locations [47]. Carmen et al. showed that cancer cells lacking Neat1_1 did not exhibit cell cycle defects, and Neat1_1 specific knockout mice did not exhibit the phenotype observed in Neat1-deficient mice, suggesting that the function of Neat1 is mainly based on the Neat1_2 isoform [24]. And one study has shown that Neat1_2 has a stronger effect on RNA-RBP complex formation than Neat1_1 [48]. We demonstrated in RIP assay that RBM10 could recruit Neat1_2. Importantly, RBM10 overexpression significantly inhibited Neat1_2 expression, and RBM10 silencing significantly increased Neat1_2 expression. In addition, the proportion of Neat1_2 in Neat1 decreased significantly in RBM10 overexpressed A549, while the proportion of Neat1_2 in Neat1 increased significantly after RBM10 silencing. Based on the ratio of Neat1_2 in Neat1 and the expression of Neat1 and Neat1_2, we concluded that Neat1_1 increased after RBM10 overexpression and decreased after RBM10 silencing. RBM10 is a kind of AS factor that is enriched in the splicing sites at the 5′ and 3′ ends of introns and exons of pre-mRNA, with more abundant binding near the 3′ splicing site than those of the 5′ splicing site. By binding to small nuclear ribonucleoprotein and cleavage sites of pre-mRNA substrates, RBM10 can synergistically remove introns and increase exon jumping events by more than 74% [49]. The change in abundance and activity of RBM10 can lead to changes in some gene splicing patterns. Nicotine is a known risk factor for the development of lung cancer. A transcriptome sequencing study demonstrated that compared with the epithelial cells of normal controls, those of the nicotine exposure group showed a significant decrease in the expression level of Neat1, which was a result of AS [50]. Our data suggest that RBM10 regulates AS of Neat1, since RBM10 could upregulate Neat1_1 and downregulate Neat1_2. Also, we observed that RBM10 can also affect Neat1 through other mechanisms, because RBM10 inhibited total NEAT1 expression. In addition, previous studies have shown that RBM10 regulates the phosphorylation of the RAP1/AKT/CREB [30] and MAPK/PI3K/AKT [31] signaling pathways in lung cancer. The PI3K/AKT/mTOR signaling axis may be a key molecular pathway in lung tumorigenesis [51–54]. Accordingly, inhibitors of PI3K/AKT/mTOR signaling have been suggested as potential therapeutic agents for NSCLC [55–57]. PTEN is a tumor suppressor gene with phosphatase activity, which can inhibit the development of tumors by antagonizing the activity of phosphorylase, such as tyrosine kinase. PTEN inhibits the PI3K signaling pathway by dephosphorylating phosphatidylinositol-3,4,5-triphosphate to phosphatidylinositol-4,5-bisphosphate [58]. It has been reported that Neat1 promotes tumor development by inhibiting PTEN expression in laryngeal [28] and cervical cancer [59]. We investigated the relationship among RBM10, Neat1_2, and PTEN and the phosphorylation of downstream genes. We found that RBM10 promoted the expression of PTEN by inhibiting Neat1_2, which in turn inhibited the phosphorylation of the PI3K/AKT/mTOR signaling pathway.
In summary, RBM10 regulates AS of Neat1, leading to changes in the expression level of Neat1_2 and Neat1_1. RBM10 decreases Neat1_2 to inhibit the invasion and metastasis of NSCLC via PTEN/PI3K/AKT/mTOR signaling. The oncogenic effect of Neat1 is mainly attributed to Neat1_2. These data on multiple relationships of RBM10 and Neat1 in NSCLC contribute to our understanding of the detailed molecular mechanisms involved in the progression of NSCLC (Fig. 8).
Additional file 1: Figure S1. The flow chart of Clip-Seq. | true | true | true |
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PMC9636703 | Shirin Azizidoost,Ava Nasrolahi,Farhoodeh Ghaedrahmati,Bartosz Kempisty,Paul Mozdziak,Klaudia Radoszkiewicz,Maryam Farzaneh | The pathogenic roles of lncRNA-Taurine upregulated 1 (TUG1) in colorectal cancer | 04-11-2022 | Colorectal cancer,LncRNAs,TUG1,Tumorigenesis | Colorectal cancer (CRC) is a gastrointestinal tumor that develops from the colon, rectum, or appendix. The prognosis of CRC patients especially those with metastatic lesions remains unsatisfactory. Although various conventional methods have been used for the treatment of patients with CRC, the early detection and identification of molecular mechanisms associated with CRC is necessary. The scientific literature reports that altered expression of long non-coding RNAs (lncRNAs) contributed to the pathogenesis of CRC cells. LncRNA TUG1 was reported to target various miRNAs and signaling pathways to mediate CRC cell proliferation, migration, and metastasis. Therefore, TUG1 might be a potent predictive/prognostic biomarker for diagnosis of CRC. | The pathogenic roles of lncRNA-Taurine upregulated 1 (TUG1) in colorectal cancer
Colorectal cancer (CRC) is a gastrointestinal tumor that develops from the colon, rectum, or appendix. The prognosis of CRC patients especially those with metastatic lesions remains unsatisfactory. Although various conventional methods have been used for the treatment of patients with CRC, the early detection and identification of molecular mechanisms associated with CRC is necessary. The scientific literature reports that altered expression of long non-coding RNAs (lncRNAs) contributed to the pathogenesis of CRC cells. LncRNA TUG1 was reported to target various miRNAs and signaling pathways to mediate CRC cell proliferation, migration, and metastasis. Therefore, TUG1 might be a potent predictive/prognostic biomarker for diagnosis of CRC.
Colorectal cancer (CRC) is a gastrointestinal malignancy, ranked as the third most commonly diagnosed, and the second cause of cancer mortality worldwide [1]. This type of cancer originates from the colon, rectum, or appendix [2]. Pieces of evidence showed that the CRC incidence rate has risen during the past decades, specifically in developing countries [3]. Over 1.85 million new CRC cases are reported annually, with an increasing number of young people before the age of 50 [4]. Several factors such as genetics, epigenetics, and environment distributed across the CRC etiology and are responsible for disease heterogeneity [5, 6]. Etiologically, the three patterns of disease onset are sporadic, familial, and hereditary forms that affected 70%, 25%, and 5% of patients [7, 8]. It has been found that the age, environmental factors, dietary, lifestyle, gut microbiota, and genetic changes predispose persons to CRC [9]. Current treatment of CRC in primary and metastatic patients include laparoscopic surgery for primary disease, resection in case of metastatic tumors, radiotherapy for rectal neoplasm along with palliative, and neoadjuvant chemotherapies [10]. Besides, antibodies, probiotics, agarose tumor macrobeads, and gold-based drugs or their combinations are used for patients with CRC [11]. It has been found that the combination of chemotherapy and anti-EGFR (epidermal growth factor receptor) monoclonal antibodies cetuximab and panitumumab can prolong the median survival rate of these patients by 2 to 4 months compared with chemotherapy alone [12]. However, the impact of these therapies on the 5-year survival remains limited and very expensive [13, 14]. Several mutations in oncogenes, tumor suppressor genes, and genes associated with DNA repair have been identified as genetic risk factors for CRCs [15]. It has been reported that genetic mutations in SMAD4, BRAF, KRAS, PIK3CA, SMAD2, PTEN, and c-MYC play essential roles in patients with CRC [11]. Recent studies demonstrated that long non-coding RNAs (lncRNAs) as a subgroup of RNAs longer than 200 nucleotides presented important functions in the pathogenesis of CRC [16–18]. Aberrant expression of lncRNA is associated with several diseases, as well [17]. Previous investigations reported that lncRNA TUG1 showed tumor-suppressive or oncogenic functions in different types of cancers [19, 20]. Studies revealed that TUG1 expression was increased in CRC tumor tissues and promoted cell proliferation [21–23]. Further analysis revealed a significant negative correlation between the levels of TUG1 and the overall survival rate of patients with CRC [24]. In this review, we summarized functional roles of this lncRNA in the tumorigenesis of CRC cells.
An initial genomic screening for genes upregulated in response to taurine treatment in developing mouse retinal cells detected taurine-upregulated gene 1 (TUG1) (also known as TI-227 H, LINC00080, and NCRNA00080), a 7.1-kb lncRNA that in the human genome is located on chromosome 22q12.2 [25]. Functional studies revealed mice lacking TUG1 had impaired retinal development [26]. There is also evidence that TUG1 is transcriptionally regulated by p53 [27]. The polycomb-repressive complex 2 (PRC2) contains enhancer of zeste homologue 2 (EZH2), suppressor of zeste 12 (SUZ12), and embryonic ectoderm development (EED) [28] that catalyzes lysine residue 27 di- and trimethylation on histone 3 (H3K27me3) to repress gene expression [29]. TUG1 by recruiting and binding to the PRC2 complex functions as a dynamic scaffold [30, 31]. TUG1 knockdown induced an upregulation of the cell-cycle genes, suggesting that TUG1 is involved in both cell proliferation and apoptosis [22]. TUG1 as a potent epigenetic regulator can mediate histone modification and DNA methylation in target genes [24, 32]. Besides, TUG1 by acting as a competing endogenous RNA (ceRNA) could sponge and repress microRNA (miRNA) [33]. Figure 1 displays different functions of TUG1. TUG1 has recently been proposed as an oncogene in several types of cancer [34–36]., TUG1 is associated with large tumor size, advanced pathological stages, and distant metastasis [37, 38]. Experimental studies have disclosed that TUG1 significantly stimulated tumor cell proliferation, invasion, colony formation, and drug resistance in CRC cells (Table 1). Overexpression of TUG1 via mediating epithelial-mesenchymal transition (EMT)-associated gene expression, reduction of E-cadherin expression, and boosted the vimentin, N cadherin, and fibronectin expression promoted the invasion and metastasis of CRC cells [24, 39, 40].
There is growing evidence that TUG1 by targeting several signaling pathways plays critical roles in the progression of CRC cells [24]. Here, we described multiple miRNAs/transcription factors axes (Fig. 2) that can be regulated via this lncRNA in CRC.
High expression of TUG1 has been proved to be correlated with CRC pathogenesis including proliferative along with the migratory ability, cell viability, tumor growth, and subcutaneous tumor formation [41, 42]. TUG1 is known to interact with miR-145-5p and regulate CRC cellular processes. Moreover, miR-145-5p suppressed the expression transient receptor potential cation channel subfamily c member 6 (TRPC6) as its candidate target, which its overexpression brought back miR-145-5p function in CRC. Altogether, TUG1 induces progression of CRC through miR-145-5p/TRPC6 axis, thereby regarding as a possible diagnostic marker for CRC management [42].
It has been shown that high expression of TUG1 is correlated with increased proliferation and invasion along with reduced apoptosis of CRC cells [43]. TUG1 overexpression was also closely associated with the overall survival of CRC patients, indicating TUG1 as poor prognosis biomarker for CRC [44]. Zinc finger E-box binding homeobox 2 (ZEB2) is a binding protein that participated in CRC metastasis and associated with human CRC poor prognosis [45]. There was a positive and negative correlation between TUG1 with ZEB2 and miR-138-5p, respectively. Low expression of ZEB2 or overexpression of miR-138-5p reversed the induction of EMT which was caused by TUG1 overexpression. Therefore, TUG1 by suppressing the miR-138-5p/ZEB2 axis facilitated CRC occurrence and metastasis [3, 41, 43, 44].
High expression of TUG1 has been implicated in clinicopathological features of CRC including advanced tumor stage along with reduced overall survival and disease-free survival [24, 44, 46]. The Wnt/β-catenin signaling was found to transcriptionally regulate CRC proliferation [47]. TUG1 silencing resulted in low activity of Wnt/β-catenin and suppression of proliferation. In the CRC xenograft model, low expression of TUG1 inhibited both tumorigenicity and β-catenin nuclear localization. TUG1 through changing the nuclear localization of β-catenin reduced the Wnt/β-catenin signaling activity and subsequent induced CRC proliferation. Therefore, TUG1/Wnt/β-catenin could exert promising advancement for CRC prevention [44, 46].
Transforming growth factor-beta (TGF-β) is involved in CRC tumorigenesis [48]. A recent study reported that TGF-β induced migration of CRC and overexpressed TUG1 as its downstream molecule. Recent findings demonstrated that TUG1 blockade suppressed migration, invasion, in vitro EMT along with in vivo lung metastasis. Hence, TUG1 is necessary for TGF-β-promoted pathophysiological features of CRC [49]. Twist family BHLH transcription factor 1 (TWIST1) stands as a kind of transcriptional modulator which is activated by TGF-β, resulting in low expression of E-cadherin [50]. TWIST1 silencing using siRNA resulted in significant reduction of CRC migration and EMT. It can be concluded that TGF-β-induced metastasis of CRC is regulated through the TUG1/TWIST1/EMT network, highlighting TUG1 as a novel target to inactivate the TGF-β signaling [49].
Specificity protein 1 (SP1) has a positive-regulated manner with TUG1 in CRC cells [51]. SP1 as an oncogene was found to promote CRC progression and metastasis [51, 52]. TUG1 loss of function inhibited cell growth and induced apoptosis of CRC cells [51]. Growing evidence revealed a negative correlation between TUG1 and miR-421 as a CRC tumor suppressor factor [53, 54]. Lysine demethylase 2 A (KDM2A) is a CRC oncogenic gene and a target for miR-421. TUG1 by sponging miR-421 induced KDM2A expression [51, 55]. Moreover, TUG1 has been found to intensify in vitro progression of CRC through the ERK signaling. Also, SP1 promoted in vivo CRC tumorigenesis by miR-421 suppression and KDM2A induction through TUG1 overexpression. Altogether, TUG1 as an oncogene can interact with SP1 and the miR-421/ KDM2A/ERK axis to facilitate CRC progression [51].
Tribbles homolog 2 (TRIB2) is an atypical protein kinase that has been dramatically upregulated with TUG1 in CRC tissues and cells [56, 57]. High expression of TUG1 by suppressing miR-542-3p was associated with tumor stage, lymph node metastasis, and histological differentiation of CRC patients. TUG1 or TRIB2 loss of function prohibited proliferation, migration, invasion along with in vivo tumor growth but facilitated CRC apoptosis. Besides, upregulation of TRIB2 as a miR-542-3p target reversed the impact of TUG1 silencing on CRC progression [58]. Considering the role of the Wnt/β-catenin signaling in CRC development, miR-542-3p suppression or TRIB2 upregulation has been reported to partly bring back the inhibitory function of TUG1 knockdown on the Wnt/β-catenin signaling. ThereforeTUG1 is regarded as a tumor promoter that stimulated CRC pathogenesis and drug resistance through the miR-542-3p/TRIB2 axis [58, 59].
In contrast to highly-expressed TUG1 in CRC, miR-153-1 was under expressed. Depletion of TUG1 using si-TUG1 as well as ectopic expression of miR-153-1 repressed the proliferative and migratory capacity of CRC cells. Also, upregulated TUG1 reversed miR-153-1-mediated suppression of CRC cells [60]. Kruppel-like factor 4 (KLF4) is a zinc finger transcription factor that plays as a tumor suppressive gene in CRC [61]. Recent findings identified KLF4 as a direct transcription factor for miR-153-1 can suppress CRC pathogenesis but its expression negatively modulated by TUG1. Interestingly, TUG1-deficient mice demonstrated high and low expressions of E-cadherin along with N-cadherin as tumor metastasis-correlated EMT markers exerting the TUG1/miR-153-1/KLF4 axis in in vivo EMT of CRC cells. Such regulatory axis might provide great insights for either diagnostic or treatment possibility of CRC [60].
Chemotherapy in combination with targeted therapy has been found to impair tumor recurrence and increase survival rate of CRC patients, but chemotherapeutic resistance is considered as the leading cause of CRC therapy failure.Therefore, molecular knowledge of chemotherapeutic resistance is required for CRC tumor biology [62, 63]. 5-fluorouracil (5-FU) is regarded as an effective first-line drug for CRC patients, but unknown molecular approaches are still complicated its recovery features [17, 64]. Accumulating data demonstrated that TUG1 is overexpressed in 5-FU resistant CRC tissue and cells, which were associated with poor prognosis. TUG1 blockade was implicated to dramatically promote CRC cells sensitive to 5-FU through suppressing CRC cell apoptosis which is regulated by miR-197-3p and TYMS as a direct target of miR-197-3p. Such findings highlighted the possible significance of TUG1 as a predictive marker for exerting CRC response to 5-FU therapy and indicated TUG1 silencing as a novel therapeutic approach to reverse 5-FU resistance [65]. Besides, cancer stem cells are shown to be involved in CRC chemoresistance [66]. It has been found that TUG1 silencing inhibited CRC stem cell resistant to oxaliplatin through reducing GATA6 and targeting the BMP pathway. Altogether, TUG1 promoted CRC stem cell features and chemotherapeutic resistance via inducing the stability of the GATA6 protein, providing promising insights for CRC clinical therapy [67]. Improvement of drug resistant sensitivity remains an immediate necessity for CRC chemotherapies. CRC resistance to methotrexate (MTX) as the earliest cytotoxic drugs is still a main challenge to the physicians [62, 68]. A recent study indicated that TUG1 repressed CRC cell sensitivity to MTX through targeting its interaction with miR-186. TUG1 blockade re-sensitized CRC cell resistant to MTX. Indeed, a negative correlation between miR-186 and the cytoplasmic polyadenylation element binding protein 2 (CPEB2) protein has been shown in MTX resistant tumors. Therefore, TUG1 regulated CRC resistant to MTX through targeting miR-186 and consequent induction of CPEB2 expression, thereby holding TUG1 as a possible target for CRC management [69]. Insulin-like growth factor-2 mRNA–binding protein (IGF2BP) family members as a kind of RNA-binding proteins participated in tumorigenesis as well as chemoresistance via influencing either stability, translatability, or localization of lncRNA [70–72].IIt has been found that TUG1 and IGF2BP2 were both high-expressed in CRC cell resistant to cisplatin through autophagy activation. Low TUG1 expression decreased CRC chemoresistance to cisplatin and facilitated miR-195-5p expression. Therefore, the TUG1/IGF2BP2/miR-195-5p axis intensify CRC cell growth and induce such malignant cell resistance to cisplatin, regarding as underlying target for CRC therapy [73].
Along with the biological behaviors of TUG1 in regulating CRC pathogenesis, it is also emerging as a crucial substrate for the progress of CRC biomarkers for early detection, prognosis prediction, and anticipating therapy response to diverse chemotherapies and developing therapies [74]. Currently, a study proposed that TUG1 could play a vital function in CRC metastasis. following investigation of the TUG1 expression levels in 120 CRC patients, high TUG1 expression was observed in tumor tissue which was closely associated with the poor survival time of the CRC patients [24, 75, 76]. Further in vitro experiments revealed the oncogenic impact of TUG1 upregulation in CRC cell lines. In the xenograft animal model, increased expression of TUG1 stimulated colony formation, migratory ability, and metastatic potential. Indeed, the researchers observed that TUG1 activated EMT-correlated gene expression [24, 77]. Another study proposed that the highly-expression of TUG1 was a CRC convinced unfavorable prognosis marker [78]. Cumulatively, TUG1 may act as a prognostic biomarker and a curative target. With more attempts affirm to the study of lncRNA particularly TUG1, it is optimistic that TUG1 will finally attain clinical utility [79]. In contrast, a recent study on 47 CRC patients indicated that there were no remarkable correlations between TUG1 expression and clinicopathological features of CRC. Besides, TUG1 expression could not forecast the overall survival and progression-free survival in CRC patients [80].
LncNAs can be used as biomarkers for the diagnosis, prognosis, and monitoring of the progression of the disease because of their tissue-specific expression and high stability [81]. Several studies reported that lncRNAs are closely linked to a variety of cancer types and might function as oncogenes or oncogene suppressors depending on the type of cancer [82, 83]. Although the role of TUG1 in the characteristics and chemoresistance of CRC stem cells is still not well-defined, it has been already presented as an attractive potential biomarker because of its tumor-promotive function via diverse mechanisms, such as RNA-RNA and RNA/transcription factors interactions [46, 84]. It was investigated by the same group that TUG1 increased the characteristics and oxaliplatin resistance of CRC stem cells by enhancing GATA6 stability [14]. TUG1 is suggested to solve the problem of fluoropyrimidine (Fu)-based chemotherapy. TUG1 appears to mediate 5-Fu resistance in CRC through the miR-197-3p/TYMS axis [9]. Knockdown of TUG1 resensitized resistant cells to the exposure of 5-Fu and induced cell apoptosis. This lncRNA by targeting miR-186 stimulated CPEB2 to mediate methotrexate resistance in CRC [15]. Taken together, the role of lncRNA TUG1 in CRC drug resistance seems to be crucial and holds great promise as a potential therapeutic target. Current findings regarding TUG1 not only promote a better understanding of CRC pathogenesis and development but also facilitated the progress of cancer lncRNA therapy. However, many mechanisms remain poorly described suggesting a great need for further study. | true | true | true |
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PMC9636723 | Lei Wang,Na Yuan,Yuanli Li,Qinqin Ma,Ying Zhou,Zhifei Qiao,Shutie Li,Chunyan Liu,Liqian Zhang,Meng Yuan,Jianjing Sun | Stellate ganglion block relieves acute lung injury induced by severe acute pancreatitis via the miR-155-5p/SOCS5/JAK2/STAT3 axis | 04-11-2022 | Stellate ganglion block,Acute lung injury,Severe acute pancreatitis,miR-155-5p,SOCS5 | Acute lung injury (ALI), a prevalent complication of severe acute pancreatitis (SAP), is also a leading contributor to respiratory failure and even death of SAP patients. Here, we intended to investigate the function and mechanism of stellate ganglion block (SGB) in ameliorating SAP-induced ALI (SAP-ALI). We engineered an SAP-ALI model in rats and treated them with SGB. HE staining and the dry and wet method were implemented to evaluate pathological alterations in the tissues and pulmonary edema. The rats serum changes of the profiles of TNF-α, IL-6, IL-1β, and IL-10 were examined. The profiles of miR-155-5p and SOCS5/JAK2/STAT3 were detected. Functional assays were performed for confirming the role of miR-155-5p in modulating the SOCS5/JAK2/STAT3 pathway in pulmonary epithelial cells. Our findings revealed that SGB vigorously alleviated SAP rat lung tissue damage and lung edema and lessened the generation of pro-inflammatory cytokines TNF-α, IL-6, and IL-1β. SGB enhanced SOCS5 expression, hampered miR-155-5p, and suppressed JAK2/STAT3 pathway activation. As evidenced by mechanism studies, miR-155-5p targeted the 3′UTR of SOCS5 and repressed its expression, hence resulting in JAK2/STAT3 pathway activation. During animal trials, we discovered that SGB ameliorated SAP-ALI, boosted SOCS5 expression, and mitigated the levels of pro-inflammatory factors and miR-155-5p in the plasma. In vitro, miR-155-5p overexpression substantially facilitated pulmonary epithelial cell apoptosis, inflammation, and JAK2/STAT3 pathway activation and restrained SOCS5 expression. All in all, our work hinted that SGB could modulate the miR-155-5p/SOCS5/JAK2/STAT3 axis to alleviate SAP-ALI. | Stellate ganglion block relieves acute lung injury induced by severe acute pancreatitis via the miR-155-5p/SOCS5/JAK2/STAT3 axis
Acute lung injury (ALI), a prevalent complication of severe acute pancreatitis (SAP), is also a leading contributor to respiratory failure and even death of SAP patients. Here, we intended to investigate the function and mechanism of stellate ganglion block (SGB) in ameliorating SAP-induced ALI (SAP-ALI). We engineered an SAP-ALI model in rats and treated them with SGB. HE staining and the dry and wet method were implemented to evaluate pathological alterations in the tissues and pulmonary edema. The rats serum changes of the profiles of TNF-α, IL-6, IL-1β, and IL-10 were examined. The profiles of miR-155-5p and SOCS5/JAK2/STAT3 were detected. Functional assays were performed for confirming the role of miR-155-5p in modulating the SOCS5/JAK2/STAT3 pathway in pulmonary epithelial cells. Our findings revealed that SGB vigorously alleviated SAP rat lung tissue damage and lung edema and lessened the generation of pro-inflammatory cytokines TNF-α, IL-6, and IL-1β. SGB enhanced SOCS5 expression, hampered miR-155-5p, and suppressed JAK2/STAT3 pathway activation. As evidenced by mechanism studies, miR-155-5p targeted the 3′UTR of SOCS5 and repressed its expression, hence resulting in JAK2/STAT3 pathway activation. During animal trials, we discovered that SGB ameliorated SAP-ALI, boosted SOCS5 expression, and mitigated the levels of pro-inflammatory factors and miR-155-5p in the plasma. In vitro, miR-155-5p overexpression substantially facilitated pulmonary epithelial cell apoptosis, inflammation, and JAK2/STAT3 pathway activation and restrained SOCS5 expression. All in all, our work hinted that SGB could modulate the miR-155-5p/SOCS5/JAK2/STAT3 axis to alleviate SAP-ALI.
Pancreatin activated in the pancreas will trigger pancreatic autodigestion and thus result in acute pancreatitis (AP), which is usually correlated with cholelithiasis, binge-eating, and excessive drinking [1]. An aberrant hike in serum amylase is a typical indicator of AP. As per the severity, AP can be categorized into mild, moderate, and severe. Severe acute pancreatitis (SAP) features pancreatic necrosis, systemic inflammation, and even organ dysfunction [2]. Acute lung injury (ALI), a prevailing severe inflammatory reaction, is also an essential contributor to AP patients’ death. It is estimated that the mortality rate of AP elicited by ALI and acute respiratory distress syndrome (ARDS) can reach as high as 60% [3]. In the teeth of scanty efficacious intervention strategies for SAP-ALI with very sophisticated pathogenesis, we conducted the research in the hope that novel therapies could be uncovered to ameliorate the existing poor prognosis of SAP-ALI. The stellate ganglion comprises the sixth and seventh cervical vertebrae as well as the first thoracic sympathetic ganglion, functionally belonging to the sympathetic nerve. When stimulated by inflammation and serious pressure, the body will experience imbalances between the sympathetic nerve and the parasympathetic nerve, which then elicits diseases concerning sympathetic nerve regulation like arrhythmia [4, 5]. SGB blocks the sympathetic nerve and redresses the vegetative nerve imbalance through the injection of anesthetic drugs into stellate ganglion tissues, thus meeting its goal of stabilizing the internal environment of the body [6]. SGB, a novel therapy with great potential, has been demonstrated to greatly enhance the spatial learning and memory capabilities of rats stimulated by unpredictable chronic stress and alleviate their depression-like behaviors [7]. SGB vigorously hampers inflammatory factor release and mitigates the clinical symptoms of patients suffering from ulcerative colitis (UC) [8]. Concentrating on SGB, we probed whether it could be applied in SAP-ALI treatment in the research. microRNAs (miRNAs), endogenous non-coding small molecule RNAs, modulate multiple biological functions like cell proliferation, apoptosis, differentiation, angiogenesis, inflammation, and infection [9]. As demonstrated by many scholars, miRNAs are the underlying targets for AP treatment. miR-92b, miR-10a, and miR-7, which are down-regulated in AP patients, can be utilized for early AP diagnosis. miR-551b-5p, down-regulated in AP patients with complications or a low level of plasma calcium, enables us to differentiate the degrees of the disease [10]. miR-214-3p, whose expression is uplifted in the pancreas of hyperlipidemia pancreatitis (HP) rats, dampens PTEN and upregulates Akt to step up HP-elicited pathological alterations and inflammation [11]. miR-155-5p, another member of the miRNA family, displays a pro-inflammatory function in a few inflammatory diseases like hippocampal inflammation in acute seizures [12], Parkinson’s disease [13], and lipopolysaccharide-induced acute lung injury [14]. Suppressors of cytokine signaling (SOCS) can suppress their downstream signaling to regulate the signaling speed and time of various cytokine receptors [15]. For instance, SOCS5, a pivotal member of the SOCS family, boasts a central SH2 domain and a C-terminal conserved domain and has a special kinase-inhibitory region (KIR) domain, which can impede kinase activity [16]. miR-151a-3p overexpression hinders SOCS5 and initiates the JAK2/STAT3 pathway to abate MC3T3-E1 cell viability and spur postmenopausal osteoporosis progression [17]. The Janus kinase 2/signal transducer and activator of transcription 3 (JAK2/STAT3) pathway mediates proliferation, immunity, programmed cell death and other processes [18]. Interestingly, hydrostatin-SN10 cramps the IL-6-triggered activation of the JAK2/STAT3 pathway, lessens cell apoptosis, inflammation, and oxidative stress and mitigates lung damage induced by pancreatitis [19]. Therefore, a probe into the JAK2/STAT3 pathway mediated by SOCS5 helps us better understand the exact mechanism of lung damage elicited by pancreatitis. In a nutshell, the outstanding an-inflammatory function of SGB has been verified. Here, we went further into the therapeutic function of SGB in SAP-ALI and discovered that SGB considerably ameliorated ALI in the SAP SD rat model, suppressed miR-155-5p expression and JAK2/STAT3 pathway activation, and bolstered SOCS5 expression. Thus, we conjectured that SGB could modulate the SOCS5-JAK2/STAT3 regulatory axis mediated by miR-155-5p to alleviate SAP-ALI.
MLE-12 cells, lung epithelial cells derived from mice, were ordered from the American Type Culture Collection (ATCC, Rockville, MD, USA). Inoculated into an RPMI1640 medium incorporating 10% inactivated fetal bovine serum (FBS, HyClone, Logan, UT, USA), 1 × 105/mL cells were cultured with 5% CO2 at 37 ℃ and passed every two or three days. Cells in the logarithmic growth phase were digested, passed, and seeded into 12-well plates with a density of 5 × 106/well. As the cells met 80–90% confluence, miR-155-5p mimics, SOCS5 overexpression plasmid, and their corresponding negative controls were transfected into them as per the instructions of Lipofectamine®3000 (Invitrogen; ThermoFisher Scientific, Inc.). The cells were grown in an incubator with 5% CO2 at 37 ℃. LPS (1 μg/mL; Sigma-Aldrich, St. Louis, MO, USA) was administered to treat the MLE-12 cells for the construction of an in vitro pulmonary epithelial cell damage model.
MLE-12 cells, inoculated into 96-well plates with a density of 5 × 103/well, were dealt with LPS (1 μg/mL) for 24 h. Then, 10 μL of CCK8 reagent (Beyotime, Shanghai, China) was given for 2 h incubation at 37 ℃. A microplate reader was exploited to gauge the absorbance at 450 nm.
Thirty SD rats 220–250 g in weight, ordered from the Animal Experimental Center of Zhangjiakou Medical College (Zhangjiakou, China), were reared in an environment of 50–60% humidity at 25 ℃ for seven days under a 12 h light/dark cycle, given sufficient food and water in Laboratory animal management department of Zhangjiakou Medical College. The SGB model: eight hours after the SD rats were put into a fast, they were intraperitoneally injected with pentobarbital sodium (40 mg/kg) for anesthesia; they were then fixed on the operating table in a supine position; with a 1 cm incision made in the middle of their necks, the subcutaneous tissues, fascia, and muscle were isolated; when the right common carotid artery was seen, the right sympathetic nerve trunk was isolated and exposed under a microscope; the sympathetic nerve trunk of the broken neck was treated 3 mm below the superior cervical ganglion, the severed end was ligated, and the incision was sewed up. The rats manifested a drooping right eyelid, enophthalmos, narrowed pupils and other Horner symptoms after they woke up, which verified the success in the establishment of the SGB model. The AP model: 12 h after the fast, the rats were intraperitoneally transfused with pentobarbital sodium (40 mg/kg) for anesthesia; a cut was made right in the middle of the abdomen, and sodium taurocholate (3.5%, 1 ml/kg, Sigma, St. Louis, MO, United States) was retrograde-injected into the biliary pancreatic duct to elicit AP in the animals. As the rats displayed symptoms like drinking a lot of water, poor diet, lusterless and tangled hair, short breath, and lung noises, we confirmed the success. Twenty-four hours later, the rats were narcotized through the intraperitoneal injection of pentobarbital sodium (100 mg/kg) and euthanized. We harvested their abdominal aortic blood and lung tissues for the following analysis.
The harvested lung tissues were immobilized employing 4% paraformaldehyde at 4 ℃ for 24 h, embedded in paraffin, and sectioned in the thickness of 5 μm. Then, the slices were dewaxed, dehydrated with gradient alcohol, and dyed with hematoxylin and eosin solution (HE). A microscope (200 × magnification, Olympus Corporation, Tokyo, Japan) was deployed to monitor pathological alterations in the lung tissues. In accordance with the Hofbauer scoring system, we observed pathological changes like edema, hemorrhage, and inflammatory infiltration in the tissues. Zero indicates normality. One point suggests that the above alterations appear in 25% of the visual field. Two points mean the changes covering 50% of the field. Three points indicate the changes covering 75% of the field. Four points denote that those alterations are seen in the whole field.
The right pulmonary tissues of the rats were flushed in PBS and dried, with the wet weight (W) gauged. Then, the tissues were heated in a constant temperature oven at 70 ℃ for 72 h, with the dry weight (D) measured. The level of lung edema was examined with this formula: pulmonary edema index = W/D.
Uncoagulated rat abdominal aortic blood samples incorporating heparin were put into the blood gas analyzer (Beckman Coulter, Inc., USA) to examine the arterial partial pressure of oxygen (PaO2), the partial pressure of carbon dioxide (PaCO2), and the PH value.
Serum amylase is a typical indicator of acute pancreatitis diagnosis. Biocompare Company (CA, USA) supplied us with the amylase ELISA kit, which was adopted to examine serum amylase in the rats.
The bronchoalveolar lavage fluid (BALF) of the rats was obtained for quantifying inflammatory cells. After the rats were narcotized, as mentioned in 2.1, we put the front end of a bronchoscope into the opening at the lateral segmental bronchus in the middle lobe of the right lung, injected 2 mL of 37 ℃ normal saline, and conducted lavage three times. PMNs, extracted out of BALF, were purified and centrifuged at 1000 r/min for 10 min. With the supernatant harvested, RMPI-1640 was administered to resuspend the cells. A hematocyte counter (Beckman Coulter, Inc) was utilized to count inflammatory cells in BALF. Cytospin (Thermo Fisher Scientific, Waltham, USA) was harnessed to centrifuge 100 μl of BALF onto slides. After getting dried, the slides were dyed employing the Protocol HEMA-3 Cell Staining Kit (Fisher, Pittsburg, PA).
The rat lung tissues were lysed employing a lysis buffer supplemented with the protease inhibitor and centrifuged at 2500 r/min and 4 ℃ for 20 min. With the supernatant obtained, the ELISA kits (R&D Systems, Minneapolis, MN, USA) were utilized to determine the levels of inflammatory cytokines (TNF-α, IL-6, IL-1β, IL-10). We harvested the plasma and operated the ALT and AST kits (Nanjing Jiancheng Institute of Biotechnology, Nanjing, China) to gauge ALT and AST expression levels in the samples. A Power Wave microplate reader (Bio-TEK, USA) was exploited to examine the OD value at 450 nm.
Total RNA was extracted out of the cells with the use of TRIzol reagent (Invitrogen, Carlsbad, CA, USA), with the RNA purity determined. Then, the RevertAid First Strand cDNA Synthesis Kit (Thermo Fisher Scientific, Waltham, MA, USA) was adopted to reverse-transcribe the mRNA into cDNA, and the miScript II Reverse Transcription Kit (Qiagen, Hilden, Germany) was employed to reverse-transcribe the miRNA into cDNA. qRT-PCR was implemented with the assistance of SYBR Green PCR Master Mix (Roche) or the miScript SYBR® Green PCR Kit (QIAGEN, Dusseldorf, Germany) in the ABI Step-One PlusTM Real-Time PCR System (Applied Biosystems). GAPDH was taken as the internal parameter of SOCS5. The primer sequences are detailed in Table 1. The relative profile of each gene was calculated through the 2−ΔΔCT method.
The biological information website Targetscan (http://www.targetscan.org/vert_72/) discovered that SOCS5 was an underlying target of miR-155-5p. MLE-12 cells, inoculated into 24-well plates with a density of 5 × 105/well, were transfected along with SOCS5-WT, SOCS5-MT, miR-155-5p mimics, and their respective negative controls using Liposome 2000 reagent (Invitrogen; Thermo Fisher Scientific, Inc. Waltham, MA, USA). The luciferase activity was gauged as instructed by the manufacturer (Promega, Madison, WI, USA). The experiment was duplicated three times.
The rat lung tissues were subjected to homogenization through a homogenizer containing a reagent for cell nucleus and cytoplasm protein extraction (Sigma-Aldrich; Merck KGaA). We harvested MLE-12 cells. RIPA (Beyotime, Shanghai, China) was administered to the tissue homogenate and cells, and the total protein was separated. Bicinchoninic acid assay (Pierce, Rockford, USA) was carried out for protein quantification. Protein samples (50 µg) were isolated through 12% SDS-PAGE and then moved onto polyvinylidene fluoride (PVDF) membranes (EMD Millipore, Billerica, MA, USA). After being sealed with 5% skimmed milk for an hour at room temperature (RT), the membranes were incubated along with primary antibodies Anti-SOCS5 antibody (1:1000, sc-100858, Santa Cruz Biotechnology, USA), Anti-JAK2 (phospho Y1007) antibody (1:1000, Ab195055, abcam, USA), Anti-JAK2 antibody (1:1000, ab108596, abcam, USA), Anti-STAT3 (phospho Y705) antibody (1:1000, ab76315, Abcam, USA), Anti-STAT3 antibody (1:1000, Ab68153, Abcam, USA), Anti-Bcl2 antibody (1:1000, Ab194583, Abcam, USA), Anti-Bax antibody (1:1000, Ab32503, Abcam, USA), Anti-Caspase3 antibody (1:1000, Ab184787, Abcam, USA), and Anti-GAPDH antibody (1:1000, ab181602, Abcam, USA) overnight at 4 ℃. TBST was taken to flush the membranes twice, which were later incubated along with the fluorescein-labeled goat anti-rabbit IgG secondary antibody (1:3000, ab150077, abcam, USA) at RT for an hour and rinsed another three times. The protein bands were examined employing the ECL Western blot kit (Amersham Biosciences, UK), and the Image Lab v5.2.1 software (Informer Technologies, Inc.) was introduced for quantification.
The GraphPad Prism 8 software (GraphPad Software, Inc., city, state) was introduced to analyze statistical differences, with the measurement statistics presented as mean ± standard deviation (X ± S). One-way ANOVA was taken for comparison among multiple groups, while Tukey post hoc test was implemented to compare two groups. P < 0.05 was regarded as statistically meaningful.
To confirm whether SGB could ameliorate SAP-ALI, we engineered an SAP model in rats and treated the animals with SGB as per the experimental requirements. We discovered that the rats in the SAP-ALI group displayed pulmonary capillary hyperemia, plentiful inflammatory cytokine infiltration in the alveolar cavity, interstitial edema, destroyed alveolar structure, and thickened alveolar wall. In contrast with the control group, the SAP-ALI group gained a substantially higher pathological score. However, the SAP-ALI + SGB group exhibited no distinct pathological alterations, with a score much lower than that of the SAP-ALI group but higher than that of the SGB group (P < 0.05, Fig. 1A). As evidenced by the dry and wet method, the lung water content was obviously heightened in the SAP-ALI group as opposed to the sham group. Lung edema was alleviated in the SAP-ALI + SGB group, less than the SAP-ALI group but more than the SGB group (P < 0.05, Fig. 1B). Arterial blood gas analysis disclosed that by contrast to the sham group, the SAP-ALI group witnessed a notable drop in the levels of PH and PaO2 and a rise in the level of PaCO2. As compared with the SAP-ALI group, the SAP-ALI + SGB group experienced a rise in PH and PaO2 and a decline in PaCO2. When compared to the SGB group, the SAP-ALI + SGB group went through a decrease in PH and PaO2 and an increase in PaCO2 (P < 0.05, Fig. 1C–E). We examined serum amylase, ALT, and AST and uncovered that in contrast with the sham group, there was a remarkable uplift in serum amylase, ALT, and AST in the SAP-ALI group. The levels of these three indicators in the SAP-ALI + SGB group were evidently fewer than in the SAP-ALI group but more than in the SGB group (P < 0.05, Fig. 1F–H). These findings denoted that SGB could conspicuously abate ALI induced by SAP in rats.
We dug deeper into rat-associated inflammation induced by SAP. With the rat BALF harvested, we discovered that the PMN/total cell proportion was notably heightened in the SAP-ALI group against the sham group, and the proportion was much lower in the SAP-ALI + SGB group than the SAP-ALI group (P < 0.05, Fig. 2A). Immunohistochemistry examined neutrophils marked by MPO, indicating that in contrast with the sham group, the SAP-ALI group witnessed a substantial increase in positive MPO cells, but SGB attenuated the number of these cells (Fig. 2B). ELISA evaluated inflammatory cytokines, displaying that by contrast to the sham group, there was a rise in pro-inflammatory factors TNF-α, IL-6, and IL-1β and a decline in the anti-inflammatory factor IL-10 in the SAP-ALI group. As opposed to the SAP-ALI group, the SAP-ALI + SGB group distinctly hampered pro-inflammatory responses induced by SAP-ALI and upregulated IL-10. When compared to the SGB group, the levels of TNF-α, IL-6, and IL-1β were heightened, and the level of IL-10 was lowered in the SAP-ALI + SGB group (P < 0.05, Fig. 2C–F). Given these phenomena, SGB could dampen inflammatory factor release induced by SAP.
qRT-PCR determined miR-155-5p’s level in the plasma and lung tissues. As a result, miR-155-5p’s level was dramatically augmented in the plasma and lung tissues of the SAP-ALI rats, whereas SGB failed to alter its level (in contrast with the sham group). By contrast to the SAP-ALI group, SGB gave rise to a distinct drop in miR-155-5p’s level (Fig. 3A). Then, immunohistochemistry and Western blot confirmed the levels of SOCS5 and JAK2/STAT3 in the lung tissues. As opposed to the sham group, SAP-ALI vigorously restricted SOCS5’s level and boosted JAK2 and STAT3 phosphorylation, whereas SGB dramatically augmented SOCS5 and suppressed JAK2 and STAT3 phosphorylation (Fig. 3B, C). In light of these findings, SGB could repress miR-155-5p expression and JAK2/STAT3 pathway activation and upregulate SOCS5.
Through the biological information website (http://starbase.sysu.edu.cn/), we confirmed that SOCS5 was an essential downstream target of miR-155-5p (P < 0.05, Fig. 4A). Dual luciferase activity assay disclosed that miR-155-5p vigorously hindered the dual luciferase activity of the SOCS5-WT group but exerted little inhibitory impact on that of SOCS5-MT (P < 0.05, Fig. 4B). miR-155-5p mimics were transfected into MLE-12 cells dealt with LPS (P < 0.05, Fig. 4C). RT-PCR and Western blot displayed that miR-155-5p mimic transfection considerably restrained the protein profile of SOCS5 (P < 0.05, Fig. 4D, E). These discoveries denoted that miR-155-5p targeted SOCS5 and repressed its expression.
We transfected MLE-12 cells along with miR-155-5p mimics and miR-155-5p mimics + SOCS5 overexpression plasmid. Then the cells were dealt with LPS (1 μg/mL) for 24 h. qRT-PCR unveiled that miR-155-5p expression in the LPS group was much higher than in the control group, and it was further heightened by miR-155-5p overexpression. Nevertheless, no distinct differences were discovered in miR-155-5p expression in MLE-12 cells between the LPS + miR-155-5p group and the LPS + miR-155-5p + SOCS5 group (Fig. 5A). As unraveled by BrdU and CCK8, in contrast with the control group, LPS weakened MLE-12 cell proliferation and viability. By contrast to the LPS group, miR-155-5p overexpression gave rise to a reduction in MLE-12 cell viability and proliferation, which were strengthened by SOCS5 overexpression (in contrast with the LPS + miR-155-5p group) (Fig. 5B, C). As evidenced by flow cytometry, as compared with the control group, LPS contributed to a rise in MLE-12 cells’ apoptosis. Their apoptosis was evidently facilitated by miR-155-5p overexpression (in contrast with the LPS group, Fig. 5D) and vigorously hampered by SOCS5 overexpression (by contrast to LPS + miR-155-5p, Fig. 5D). Western blot confirmed the profiles of apoptosis-concerned proteins, signifying that by contrast to the control group, LPS culminated in a rise in Bax and c-Caspase3 expressions and a decline in Bcl2 expression in MLE-12 cells, while miR-155-5p overexpression boosted the increase of Bax and c-Caspase3 expressions and the reduction of Bcl2 expression (compared to the LPS group). As opposed to LPS + miR-155-5p, SOCS5 overexpression cramped Bax and c-Caspase3 expressions and augmented Bcl2 expression (Fig. 5E). ELISA reflected that in contrast with the control group, LPS uplifted the profiles of TNF-α, IL-6, and IL-1β and attenuated the profile of IL-10 in MLE-12 cells. miR-155-5p overexpression bolstered TNF-α, IL-6, and IL-1β expressions and lowered IL-10 expression in MLE-12 cells dealt with LPS (by contrast to the LPS group). As compared with the LPS + miR-155-5p group, SOCS5 overexpression resulted in the opposite situation (Fig. 5F, G). Western blot determined the profile of SOCS5/JAK2/STAT3 in MLE-12 cells. As a result, in contrast with the control group, LPS suppressed SOCS5 expression and strengthened JAK2 and STAT3 phosphorylation, whereas miR-155-5p overexpression lowered SOCS5 expression and uplifted JAK2 and STAT3 phosphorylation levels (by contrast to the LPS group). When compared to the LPS + miR-155-5p group, SOCS5 overexpression substantially enhanced SOCS5 expression and weakened JAK2 and STAT3 phosphorylation (Fig. 5H). These findings revealed that miR-155-5p modulated SOCS5/JAK2/STAT3 to bolster MLE-12 cell damage mediated by LPS, and SOCS5 overexpression weakened the promoting influence of miR-155-5p overexpression on MLE-12 cell injury.
miR-155-5p mimics were transfected into MLE-12 cells, followed by the administration of the STAT3 inhibitor Stattic. qRT-PCR displayed that no conspicuous differences in miR-155-5p expression were discovered in MLE-12 cells between the miR-155-5p group and the miR-155-5p + Stattic group (Fig. 6A). As supported by BrdU and CCK8, by contrast to the control group, miR-155-5p overexpression abated MLE-12 cell viability and proliferation, whereas Stattic enhanced the viability and proliferation of MLE-12 cells transfected along with miR-155-5p mimics (in contrast with the miR-155-5p group) (Fig. 6B, C). Flow cytometry disclosed that by contrast to the control group, miR-155-5p overexpression gave rise to an increase in MLE-12 cell apoptosis, which was vigorously frustrated by Stattic (against the miR-155-5p group, Fig. 6D). Western blot ascertained the profiles of apoptosis-concerned proteins and uncovered that as compared with the control group, miR-155-5p overexpression bolstered Bax and c-Caspase3 expressions and lowered Bcl2 expression, but Stattic inverted the phenomena (against the miR-155-5p group) (Fig. 6E). ELISA reflected that in contrast with the control group, miR-155-5p overexpression heightened TNF-α, IL-6, and IL-1β expressions and repressed IL-10 expression in MLE-12 cells, but such a situation was reversed by Stattic (against the miR-155-5p group) (Fig. 6F, G). Western blot examined the profile of SOCS5/JAK2/STAT3 in MLE-12 cells. It transpired that by contrast to the control group, miR-155-5p overexpression drove up JAK2 and STAT3 phosphorylation levels. However, STAT3 phosphorylation was impaired, while SOCS5 expression and JAK2 phosphorylation remained basically the same in the miR-155-5p + Stattic group vis-a-vis the miR-155-5p group (Fig. 6H). All these findings confirmed that STAT3 inhibition weakened the damaging function of miR-155-5p overexpression in MLE-12 cells.
As the economy develops rapidly and living standards improve, the intake of high-fat and high-protein food and drinks is also on the rise, which adds to the burden of the digestive system. Gastroenterology patients are usually hospitalized because of acute pancreatitis (AP). Reportedly, 33.74 of every 100,000 people would develop AP every year [20], and the incidence rate continues to increase. Around 10% of AP patients will evolve to severe acute pancreatitis (SAP) [21], which is often accompanied by severe organ dysfunctions, among them acute lung injury (ALI) a common one. SAP-ALI occurrence and progression may pertain to trypsin activation, inflammation, oxidative stress, regulatory functions of microRNAs in downstream pathways, and other mechanisms [22]. Stellate ganglion block, a prevailing treatment strategy, regulates abnormalities in the neuro-endocrine system through local blocking without incurring central nervous system impairment while upholding the integrity of the surrounding nerve. Through experiments, we discovered that SGB vigorously dampened rat lung damage elicited by SAP, which demonstrated that SGB might be utilized for SAP-ALI treatment. Stellate ganglion block (SGB) is known as a century-old technology that blocks the sympathetic nerve chains in the cervical spine, upper chest and other places for diagnosis and treatment. SGB has been substantiated to ameliorate trigeminal neuropathy and paresthesia following dental surgery [23], post-traumatic stress disorder syndrome [24], sleep disorders in patients with breast cancer, etc [25]. Moreover, it also exerts a good therapeutic function in lung damage resulting from multiple factors. SGB vigorously suppresses the sepsis-elicited release of inflammatory factors IL-6 and TNF-α, alleviating ALI in rats [26]. In the rabbit ALI model elicited by hydrochloric acid, SGB could attenuate stress responses, modulate homeostasis in the autonomic nervous system, substantially abate the profiles of pro-inflammatory factors IL-6 and TNF-α, and upregulates IL-10, thus enhancing the functions of lungs affected by ALI [4]. Unfortunately, it is still poorly understood whether SGB can function in SAP-ALI and how it exactly works. Given the above findings, we can know that the function of SGB to ameliorate pulmonary functions may be correlated with anti-inflammatory responses. Luckily, our experiments demonstrated that SGB vigorously repressed pathological lesions in the lungs of the SAP-ALI rats, efficaciously enhanced their ventilation functions, distinctly lowered the levels of serum amylase, ALT, and AST and the PMN/total cell proportion, and also conspicuously weakened pro-inflammatory cytokine release. miRNAs play a pivotal part in gene expression, mediating the occurrence and progression of umpteen diseases. Of note, many miRNAs now are extensively adopted as biomarkers for diagnosis, treatment, and prognosis. For instance, miR-216a-5p, miR-216b-5p, miR-217-5p, and miR-375-3p, whose sensitivity is higher than serum amylase and lipase, can perfectly serve as biomarkers for AP [27]. miR-22-3p, miR-1260b, miR-762, miR-23b, and miR-23a are all considerably upregulated in the context of SAP-ALI [28]. miR-155 targets SOCS1 to strengthen inflammation mediated by Th17 in AP, while miR-155 inhibition vigorously impedes inflammation and ameliorates pancreatic pathology [29]. miR-155 is also a pro-inflammatory regulator inextricably associated with exacerbated AP, but miR-155 inhibition can mitigate ALI in cerulein-elicited AP mice [30]. miR-155-5p targets DUSP14 and initiates the NF-κB and MAPKs signaling pathways to boost OGD/R-elicited SH-SY5Y cell apoptosis and inflammation [31]. lncRNA CTBP1-AS2 targets miR-155-5p and upregulates FOXO1 to abate HG-elicited proliferation, oxidative stress, ECM accumulation, and inflammation in human glomerular mesangial cells [32]. lncRNA XIST targets miR-155-5p and upregulates WWC1 to dampen inflammatory cytokine generation and cell apoptosis, hence attenuating acute kidney injury induced by sepsis [33]. Here, we discovered that miR-155-5p’s expression was greatly uplifted in the plasma and lung tissues of the SAP-ALI rats, but SGB notably drove down its expression. In vitro, miR-155-5p overexpression remarkably facilitated MLE-12 cells’ apoptosis and inflammation and weakened their proliferation and viability. miR-155-5p overexpression exacerbated MLE-12 cell injury mediated by LPS. All these findings confirmed that miR-155-5p displayed pro-apoptotic and pro-inflammatory functions in the context of SAP-ALI. SOCS5 is characterized as a specific inhibitor of IL-4 signaling [34]. During COPD progression, miR-132 overexpression targets and hampers SOCS5 to bolster EGFR protein expression and inflammatory cytokine generation in human monocyte-like cells (THP-1) [35]. The JAK2/STAT3 signaling pathway exerts an outstanding function in lung damage. For instance, miR-216a overexpression suppresses JAK2/STAT3 and NF-κB pathway activation to mitigate ALI induced by LPS [36]. SOCS3 overexpression represses JAK2/STAT3 pathway activation and inflammatory factor production to facilitate the repair of rat lung damage elicited by SAP [37]. Leonurine down-regulates miR-18a-5p and enhances SOCS5 expression to curb JAK2/STAT3 signaling pathway activation, impeding CML cells’ proliferation, migration, and colony formation, boosting their apoptosis, and thus exerting a prominent anti-leukemia function in chronic myeloid leukemia [38]. Notwithstanding, we are still in the dark about the mechanism of the SOCS5/JAK2/STAT3 pathway in lung damage elicited by pancreatitis. Here, we uncovered a notable drop in SOCS5 expression and immunohistochemical scores and a rise in JAK2 and STAT3 phosphorylation in SAP-ALI rat lung tissues. Nevertheless, SGB culminated in a distinct uplift in SOCS5 expression and immunohistochemical scores and a remarkable decline in JAK2 and STAT3 phosphorylation. The bioinformatics website denoted that SOCS5 might be a downstream target of miR-155-5p, which was substantiated by dual luciferase activity assay. In vitro, miR-155-5p overexpression restrained SOCS5 expression. SOCS5 overexpression abated the promoting influence of miR-155-5p overexpression on MLE-12 cell injury mediated by LPS and attenuated JAK2 and STAT3 phosphorylation. STAT3 inhibition weakened the damaging function of miR-155-5p overexpression in MLE-12 cells. Therefore, we speculated that SGB could down-regulate miR-155-5p and enhance SOCS5 expression to curb JAK2/STAT3 pathway activation, thereby ameliorating SAP-ALI.
To summarize, we have uncovered that SGB can prominently alleviate SAP-ALI through engineering an SAP-ALI rat model and treating it with SGB. SGB bolsters the profile of SOCS5, represses miR-155-5p expression, and impedes JAK2/STAT3 pathway activation. miR-155-5p induces JAK2/STAT3 pathway activation by directly targeting SOCS5 (Fig. 7). However, several limitations remain to be investigated in our future study: (1) lack of clinical trial; (2) lack of in vivo experiments on genetic modification of miR-155-5p/SOCS5 on stellate ganglion block-mediated effects. All in all, our finding provides a new reference in treating SAP-ALI by SGB. | true | true | true |
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PMC9636746 | Xiao Bai,Zaiwen Qi,Mingzhen Zhu,Zhuangzhuang Lu,Xin Zhao,Lining Zhang,Guangmin Song | The effect of lncRNA MIR155HG-modified MSCs and exosome delivery to synergistically attenuate vein graft intimal hyperplasia | 04-11-2022 | MIR155HG,Mesenchymal stem cells,Exosome,Vein graft,Intimal hyperplasia | Background The mesenchymal stem cells (MSCs) were used to repair tissue injury. However, the treatment effect was not satisfactory. We investigated whether lncRNA MIR155HG could promote survival and migration of MSCs under oxidative stress, which mimics in vivo environments. Furthermore, we studied the protective effect of exosomes secreted by MSCs transfected with MIR155HG on endothelial cells. This study aimed to determine whether exploiting MSCs and exosomes modified with lncRNA MIR155HG would exert synergistic therapeutic effect to attenuate vein graft intimal hyperplasia more effectively. Methods Lentivirus containing lncRNA MIR155HG overexpressing vector was packaged and used to infect MSCs. Then, CCK-8 assay, flow cytometry, Transwell assay, and Elisa assay were used to assess the functional changes of MSCs with overexpressed MIR155HG (OE-MSCs). Furthermore, the associated pathways were screened by Western blot. MIR155HG-MSCs-derived exosomes (OE-exo) were collected and co-cultured with human umbilicus vein endothelial cell (HUVEC). We validated the protective effect of OE-exo on HUVEC. In vivo, both MSCs and exosomes modified with MIR155HG were injected into a vein graft rat model via tail vein. We observed MSCs homing and intimal hyperplasia of vein graft using a fluorescent microscope and histological stain. Results Our study found that lncRNA MIR155HG promoted proliferation, migration, and anti-apoptosis of MSCs. NF-κB pathway took part in the regulation process induced by MIR155HG. OE-exo could enhance the activity and healing ability of HUVEC and reduce apoptosis. In vivo, OE-MSCs had a higher rate of homing to vascular endothelium. The combined treatment with OE-MSCs and OE-exo protected vascular endothelial integrity, reduced inflammatory cell proliferation, and significantly attenuated intimal hyperplasia of vein graft. Conclusion LncRNA MIR155HG could promote the survival and activity of MSCs, and reduce the apoptosis of HUVECs using exosome delivery. Exploiting MSCs and exosomes modified with MIR155HG could attenuate vein graft intimal hyperplasia more effectively and maximize the surgical effect. | The effect of lncRNA MIR155HG-modified MSCs and exosome delivery to synergistically attenuate vein graft intimal hyperplasia
The mesenchymal stem cells (MSCs) were used to repair tissue injury. However, the treatment effect was not satisfactory. We investigated whether lncRNA MIR155HG could promote survival and migration of MSCs under oxidative stress, which mimics in vivo environments. Furthermore, we studied the protective effect of exosomes secreted by MSCs transfected with MIR155HG on endothelial cells. This study aimed to determine whether exploiting MSCs and exosomes modified with lncRNA MIR155HG would exert synergistic therapeutic effect to attenuate vein graft intimal hyperplasia more effectively.
Lentivirus containing lncRNA MIR155HG overexpressing vector was packaged and used to infect MSCs. Then, CCK-8 assay, flow cytometry, Transwell assay, and Elisa assay were used to assess the functional changes of MSCs with overexpressed MIR155HG (OE-MSCs). Furthermore, the associated pathways were screened by Western blot. MIR155HG-MSCs-derived exosomes (OE-exo) were collected and co-cultured with human umbilicus vein endothelial cell (HUVEC). We validated the protective effect of OE-exo on HUVEC. In vivo, both MSCs and exosomes modified with MIR155HG were injected into a vein graft rat model via tail vein. We observed MSCs homing and intimal hyperplasia of vein graft using a fluorescent microscope and histological stain.
Our study found that lncRNA MIR155HG promoted proliferation, migration, and anti-apoptosis of MSCs. NF-κB pathway took part in the regulation process induced by MIR155HG. OE-exo could enhance the activity and healing ability of HUVEC and reduce apoptosis. In vivo, OE-MSCs had a higher rate of homing to vascular endothelium. The combined treatment with OE-MSCs and OE-exo protected vascular endothelial integrity, reduced inflammatory cell proliferation, and significantly attenuated intimal hyperplasia of vein graft.
LncRNA MIR155HG could promote the survival and activity of MSCs, and reduce the apoptosis of HUVECs using exosome delivery. Exploiting MSCs and exosomes modified with MIR155HG could attenuate vein graft intimal hyperplasia more effectively and maximize the surgical effect.
Coronary artery bypass grafting (CABG) is the most effective treatment for severe coronary heart disease. The autogenous veins are commonly used as bridging vessels in CABG. However, approximately 20–40% of vein grafts could become stenosed or even occluded two years after surgery [1, 2]. Intimal hyperplasia of vein graft seriously reduces the therapeutic effect of CABG [3, 4]. The pathological basis of intimal hyperplasia is endothelial cell injury [5], which induces oxidative stress reaction and delays vascular endothelial repair [6]. These pathological processes lead to intimal hyperplasia, lumen stenosis, and occlusion. Therefore, early repair of the vascular endothelium is essential in preventing graft remodeling [7, 8]. The mesenchymal stem cells (MSCs) are used to repair tissue injury [9, 10], and endothelial cells serve as a vascular barrier. The apoptosis of endothelial cells would destroy the barrier, directly exposing vascular smooth muscle cells to blood flow, leading to the inflammatory hyperplasia. Our previous studies confirmed that MSCs could be home in the endothelial injury location to promote endothelial repair, attenuating vein graft intimal hyperplasia. However, studies suggested a high level of oxidative stress caused by vein grafting in the blood circulation. The survival and activity of MSCs could be decreased under oxidative stress induced by tissue injury, weakening the effect of MSCs therapy [11]. Therefore, we enhanced the function of MSCs in the oxidative stress environment using long non-coding RNA (lncRNA) to improve the therapeutic effect of MSCs transplantation. lncRNA is a non‐coding RNA with more than 200 nucleotides in length. lncRNA MIR155 host gene (MIR155HG), also known as B‐cell integration cluster, located in chromosome 21q21, is considered the precursor of miR-155 [12, 13]. MIR155HG could promote the migration and invasion of cervical cancer cells [14], and MIR155HG knockdown suppresses cell proliferation, migration, and invasion in non-small cell lung cancer [15]. There is no evidence that MIR155HG effects migration and the anti-apoptotic ability of MSCs. In this study, we try to use MIR155HG to improve the migration and anti-apoptotic ability of MSCs. The increase in the homing MSCs in vivo may promote endothelial repair effect and better alleviate intimal hyperplasia of grafted veins. The exosomes secreted by MSCs are tiny membranous vesicles with lipid bilayers, 30–150 nm in diameter. Exosomes are rich in lncRNA, miRNA, and other genetic materials [16, 17] and worth studying their functional changes after MIR155HG transfection. Exosomes downregulate inflammatory response and reduce apoptosis [18, 19]. Therefore, we want to study the change of genetic materials in the exosomes derived from MIR155HG-MSCs and the protective effect of exosomes on endothelial cells. In this study, we attenuated vein graft intimal hyperplasia by exploiting both MSCs and exosomes modified with MIR155HG. We showed that MIR155HG promoted the migration and anti-apoptosis of MSCs. MIR155HG-exosomes enhanced the healing ability of HUVEC and reduced the apoptosis. MIR155HG could serve as a target for modifying MSCs and exosomes to prevent intimal hyperplasia of vein graft and improve the effect of vascular transplantation.
The MSCs were purchased from Cyagen Biosciences Inc. (Shanghai, China) and were cultured in DMEM containing 10% fetal bovine serum (Gibco, USA) in a humidified atmosphere at 37 °C and 5% CO2. MSCs were characterized by cell surface markers (CD29, CD34, CD45 and CD90) using flow cytometric analysis. The HUVECs were purchased from FuHeng Biology (Shanghai, China) and were cultured in ECM (ScienCell, USA) containing 5% fetal bovine serum and 1% endothelial cell growth supplements. Cells were maintained at 37 °C in a humidified atmosphere of 5% CO2. HUVECs were characterized by CD31.
The MIR155HG-expressing lentivirus vector was pCDH-CMV-MIR155HG-EF1-copGFP-T2A-Puro(rLv-MIR155HG) obtained from OLIGOBIO Co., Ltd. (Beijing, China). The rLv-MIR155HG vector and control vector were, respectively, co-transfected with packaging vectors PCDH-CMV-MCS-EF1-copGFP-T2A-puro, psPAX2 and p MD2.G into HEK-293T cells using Lipofectamine 2000 transfection reagent. The MIR155HG-knockdown lentivirus vector was plvx-shRNA2-Zsgreen-T2A-puro. Short-hairpin RNAs (shRNAs) against MIR-155HG were constructed in pcDNA3.1 by OLIGOBIO Co., Ltd. (Beijing, China). The target sequence of sh-MIR155HG was GCATTCACGTGGAACAAAT, and the control sequence was TTCTCCGAACG- TGTCACGT. Primary MSCs were incubated with recombinant MIR155HG-GFP or sh-MIR155HG-GFP lentivirus vectors at a multiplicity of infection (MOI) of 60. Cells were infected with the lentivirus medium. After 6 h, 2 ml fresh medium was added to dilute polybrene. Then, the lentivirus culture medium was replaced with a fresh medium for another 24 h. The green fluorescent protein signal was detected using a fluorescence microscope, and gene transfection efficiency was verified using PCR 48 h later. Primers were as follows: MIR155HG forward 5′-GCTTGCTGAAGGC TGTATGC-3′, MIR155HG reverse 5′-GTCTTGTCATCCTCCCACGG-3′; GAPDH forward 5′-GATTTGGCCGTATCGGAC-3′, GAPDH reverse 5′-GAAGACGCCA GTAGACTC-3′. Each reaction was replicated three times. Fold changes in cDNA relative to GAPDH endogenous control were calculated using the 2−ΔΔCt method.
NF-κB inhibitor BMS-345541 was purchased from MCE (New Jersey, USA). Cells were treated with 5 μM BMS-345541 for 2 h. The NF-κB pathway was significantly inhibited at this concentration, but no apoptosis was observed.
Cell viability was assessed using a Cell Counting Kit-8 (CCK-8) (MCE, New Jersey, USA). Cells were seeded onto 96 well plates and incubated for 24, 48, 72, and 96 h. Then, 10% CCK-8 solution was added to each well according to the manufacturer’s instructions. Two hours later, the absorbance at 450 nm was detected using enzyme micro-plate reader (Tecan F50, Switzerland).
Cell proliferation was assessed using a kFluor555 Click-iT EdU Kit (Keygen Biotech, Nanjing, China) according to the manufacturer’s instructions. KFluor555 stained the proliferating nuclei in red, and Hoechst 33342 stained the nucleus in purple. The proliferation rate equals the number of cells in proliferation state (red) divided by the number of total cells (purple).
Total RNA was isolated from samples using TRIzol reagent (Invitrogen, USA), and reverse transcription was performed using the PrimeScript RT reagent kit (Takara, Japan). QRT-PCR with SYBR Green was performed using a Bio-Rad real-time PCR system according to the manufacturer’s protocol. Melt curve analysis was conducted to verify that only one product was produced. RNA levels were calculated relative to GAPDH levels using the 2−ΔΔCt method.
Proteins were subjected to SDS-PAGE on polyacrylamide gels (8–10%) and transferred onto a PVDF membrane. After blocking with 5% non-fat milk in TBS containing 0.1% Tween-20, the membrane was incubated at 4 °C overnight with one of the following primary antibodies: anti-p-NF-κB P65, anti-NF-κB P65, anti-p-mTOR, anti-p-ERK, anti-ERK, anti-PDCD4, anti-GNA12 (Affinity, USA); anti-mTOR, anti-GAPDH, anti-TSG101, anti-CD63, anti-CD81, anti-Bax (Proteintech, USA); anti-Bcl-2 (Abclonal, China). Subsequently, the peroxidase-conjugated AffiniPure goat anti-rabbit or mouse IgG (Proteintech, USA) was added. Bound antibody was visualized via ECL plus TM Western blotting system detection kit (Amersham, USA).
A cell migration assay was performed using a transwell (8 μm pore size) (Corning, USA) to observe the migration function of MSCs. 200 μL transfected MSCs (1.5 × 105/ml) were seeded in the upper chamber, and 600 μl complete medium with SDF-1a (100 ng/ml, Proteintech, USA) were placed into the lower chamber. Cells on the upper side of the membrane were removed after 12 h. Cells on the bottom surface of the membrane were stained with 0.1% crystal violet and counted in 5 randomly selected microscopic fields.
Experimental groups of MSCs were stained with rabbit anti-rat CXCR4 antibody (1:250, abcam, UK). The blank control group was stained with the isotype control antibody. Cells were incubated at 4 °C for 1 h. Then, the secondary antibody Cy3 goat anti-rabbit IgG (H + L) (Abclonal, China) was added and incubated at 4 °C for 20 min. Flow cytometry was used to identify the phenotypes of MSCs.
The flow cytometry was performed using an Annexin V/PI apoptosis detection kit (Keygen Biotech, Nanjing, China) to quantify the apoptosis of the MSCs according to the manufacturer’s instructions.
The culture medium of each group of MSCs was collected and centrifuged at 1000 g for 20 min to obtain the supernatant. The HGF or VEGF concentration in the supernatant was quantified using an enzyme-linked immunosorbent assay (ELISA) kit (ExCellBio, Shanghai, China).
Exosomes were extracted from supernatants of MSCs cultures using density gradient ultracentrifugation. Morphology of exosomes was observed using a transmission electron microscope after uranyl acetate staining. Exosome particle size was detected using a nanometer particle size detector. The markers of exosomes, including CD63, CD81, and TSG101, were identified using western blot. Uptake of exosome by HUVECs was observed using confocal laser microscopy (Leica, Germany).
All the Sprague–Dawley (SD) rats weighing 250–300 g were purchased from Charles River (Beijing, China). The external jugular vein was harvested without damage from the neck of the rats. Small branches of blood vessels were ligated using thin silk threads. The length of the obtained vein was approximately 1.5 cm. Vein grafting was performed with the cuff technique and inserted into the infrarenal abdominal aorta in the same rat. Heparin (100U/100 g) was used before and after grafting. Transfected cells (5 × 107/ml × 0.2 ml) and exosome (400 μg protein suspended in 0.2 ml PBS) were injected into rat model through the caudal vein 24 h after grafting. Rats were humanely killed one week after operation to evaluate MSC homing using fluorescence microscopy. All remaining rats were killed four weeks after the operation for histological detection. All the experimental procedures were performed with the approval of the Ethical Committee of the Qilu Hospital of Shandong University (KYLL-2021(KS)-976) and followed the Institutional Animal Care and Use Committee guidelines.
Vascular specimens were fixed in 4% paraformaldehyde and then embedded in paraffin. Paraffin specimen sections (4-μm-thick) were prepared by dewaxing. Van Gieson (VG) stain was used to assess collagen infiltration according to the manufacturer’s instruction. The morphology of the vascular intima was observed using light microscopy. The nucleus was stained in blue, muscle fibers were stained in yellow, and collagen fibers appeared bright red. For the immunohistochemistry test, the sections were incubated with rabbit anti-rat PCNA (1:100) or NF-κB P65 (1:100) overnight at 4℃. Then, sections were incubated with biotinylated goat anti-rabbit IgG (1:200) for 30 min. Positive staining was identified using diaminobenzidine (DAB). Positive staining for PCNA or NF-κB P65 was observed as brown areas. We applied Image J software to calculate the integral optical density (IOD) of each field.
First, the sections were stained with anti-CD31 (Affinity, USA) overnight at 4°C. The secondary antibody was added after the primary antibody was washed with PBS. Sections were incubated for 45 min at room temperature. Nuclei were counterstained with DAPI. Images were observed using a fluorescent microscope (OLYMPUS, Japan).
Data were expressed as means and standard deviations (SD). SPSS version 20.0 software (SPSS, Chicago, USA) was used to analyze differences between samples, either using the two-sample student t test or one-way ANOVA for differences between selected pairs of samples. P value < 0.05 was considered statistically significant.
The MSCs cultured in vitro presented a homogeneous fibroblast-like form (Fig. 1A). Flow cytometry demonstrated that the cells were uniformly negative for the hematopoietic markers CD34 and CD45 and positive for the stem cell antigens CD29 and CD90 (Fig. 1B). Thus, the phenotype of the cell population used in our study was consistent with that of MSCs. MSCs were transfected with recombinant MIR155HG-GFP or sh-MIR155HG-GFP lentiviral vector (MOI = 60). GFP-labeled MSCs showed strong green fluorescence under a fluorescent microscope (Fig. 1C). GFP fluorescence intensity was detected by flow cytometry, and the result showed that the transfection efficiency of lentivirus to MSCs was approximately 90% (Fig. 1D). The high transfection efficiency could meet the experimental requirements. After transfection with MIR155HG, the level of MIR155HG in the OE-MIR155HG group was 40.14 times that in the vector group. After MIR155HG interfered with shRNA, the level of MIR155HG in the sh-MIR155HG group was 0.14 times that in the shRNA vector group (Fig. 1E). The overexpression or knock-down of MIR155HG was successfully achieved in this experiment.
We attempted to explore the molecular changes in MSCs under the impact of MIR155HG overexpression. According to literature and our previous studies, several pathway proteins, including ERK, NF-κB p65, and mTOR, and their phosphorylated forms were screened out for the test. These pathways were widely involved in cell activity, apoptosis, tumor metastasis, and angiogenesis. As indicated by the western blot, there was no significant change in the level of P-ERK/ERK (Fig. 2A, B) or P-mTOR/mTOR (Fig. 2E, F). However, the phosphorylation level of NF-κB p65 was significantly increased by MIR155HG overexpression (p < 0.05, Fig. 2C, D). The OE-MIR155HG group was treated with BMS-345541 (NF-κB inhibitor, 5 μM) to confirm that MIR155HG could regulate the NF-κB pathway in MSCs. BMS-345541 abolished the increased phosphorylation level of p65 caused by OE-MIR155HG (Fig. 2G, H). These results indicated that MIR155HG could regulate MSCs through the NF-κB pathway.
CCK-8 assay was applied to study the effect of MIR155HG on the viability of MSCs. The results indicated that sh-MIR155HG significantly inhibited the viability of MSCs (Fig. 3A). OE-MIR155HG increased total cell viability, which was reduced by BMS-345541 antagonizing the NF-κB pathway (Fig. 3B). The EdU assay suggested that sh-MIR155HG significantly inhibited the proliferation of MSCs (Fig. 3C, D). OE-MIR155HG promoted cell proliferation significantly. Besides, BMS-345541 could reverse that role, which confirmed that the NF-κB pathway was involved in the regulatory process (Fig. 3E, F). In addition, we observed the effect of MIR155HG on migration of MSCs. Transwell assay showed that OE-MIR155HG promoted the migration of MSCs, and NF-κB inhibitor could block the effect. NF-κB pathway regulated MIR155HG on the migration of MSCs (Fig. 4A, B). Flow cytometry indicated that CXCR4 expressed on the surface of MSCs was increased following OE-MIR155HG (Fig. 4C, D), causing enhanced migration of MIR155HG-MSCs. Several articles suggested that CXCR4 was the primary molecule mediating MSCs migration [20, 21].
We used hydrogen peroxide to simulate oxidative stress. Following treatment with H2O2 of different concentrations for 12 h, the viability of MSCs was markedly decreased. CCK-8 assay displayed that the half-maximal inhibitory concentration (IC50) was 226.5 μM (Fig. 5A). Therefore, we chose 220 μM as the experimental concentration. Flow cytometry demonstrated that H2O2-induced oxidative stress led to significant apoptosis of MSCs. OE-MIR155HG could effectively relieve the apoptosis caused by oxidative stress. The protective role of OE-MIR155HG was reversed by BMS-345541, antagonizing the NF-κB pathway (Fig. 5B, C). MSCs could secrete anti-apoptotic factors, such as hepatocyte growth factor (HGF) and vascular endothelial growth factor (VEGF). Elisa assay showed that OE-MIR155HG significantly elevated the level of HGF and VEGF in supernatant (Fig. 5D, E). MIR155HG regulated the secretion of pro-survival factors via the NF-κB pathway.
Exosomes were extracted from the culture medium of MSCs. The exosome particles were typical saucer-like shape under the transmission electron microscope (Fig. 6A). Western blot was used to identify exosome-specific phenotypic markers CD63, CD81 and TSG101 on the surface of the vesicles (Fig. 6B). Detection of nanoparticle size showed that the particle diameter was approximately 143.6 nm, consistent with exosome size (30–200 nm) (Fig. 6C). These results were consistent with previous reports, and these vesicles were confirmed as exosomes of MSCs origin. Exosomes are capable of delivering genetic materials. Based on qRT-PCR, we observed that the RNA level of MIR155HG in exosomes derived from MIR155HG-MSCs was 4.9-fold higher than that from control-MSCs (Fig. 6D). A similar genetic material change was observed in exosomes as in transfected MSCs. In the co-incubation experiment, we observed that PKH67-labeled exosomes could fuse with the cell membrane and get inside HUVECs. Exosomes could be taken up apparently by HUVECs after 12 h (Fig. 6E). Furthermore, the level of MIR155HG in HUVECs treated with MIR155HG-exosomes (OE-exo) was 2.1-fold higher than that with control-exosomes (Con-exo) (Fig. 6F). The results confirmed that exosomes derived from OE-MSCs could deliver MIR155HG into HUVECs.
HUVECs were identified using CD31 staining (Fig. 7A). CCK-8 assay was used to investigate the effect of OE-exo on the cell viability of HUVECs. Compared with the Con-exo group, the activity of HUVECs in OE-exo group was significantly enhanced. The activity of HUVECs in the sh-exo group was reduced (Fig. 7B). In addition, we used a wound healing assay to observe the function of OE-exo on the migration of HUVECs. The results showed that OE-exo could promote the migration of HUVECs and accelerate endothelial healing (Fig. 7C, D). The flow apoptosis assay showed that OE-exo significantly alleviated apoptosis of HUVECs caused by oxidative stress. However, the apoptosis was increased in the sh-exo group (Fig. 7E, F). We used western blot to investigate the molecular mechanism that led to these effects in HUVECs. The results showed that two anti-apoptotic proteins, including GNA12 and Bcl-2, were up-regulated in the OE-exo group. Furthermore, the pro-apoptotic proteins, including Bax and PDCD4, were down-regulated (Fig. 7G–L).
The autologous external jugular vein-abdominal aorta transplantation model in rats used in this experiment was skilled grasping. The external jugular vein was inserted into the infrarenal abdominal aorta in the same rat with the cuff technique (Fig. 8A). The model could well simulate autogenous vein intima hyperplasia clinically after coronary artery bypass graft (CABG). After transfected MSCs and exosomes were injected into the rat model through the caudal vein, the fluorescence microscope was used to observe the homing of MSCs to vein graft. The number of OE-MSCs migrated to the intima of vein graft was significantly increased compared to the control group (Fig. 8B, C). VG staining was performed to assess neointimal hyperplasia. The collagen fibers were stained in red, and the muscles were stained in yellow. The results demonstrated that neointima of the OE-MSCs group was thinner than that of the Con-MSCs group. The effect of the OE-MSCs + OE-exo group was better than that of the OE-MSCs group. Intimal hyperplasia in the grafted vein was significantly reduced in the OE-MSCs + OE-exo group (Fig. 8D, E). Endothelial integrity of the vessel wall was detected using CD31 staining. The results showed that endothelial integrity was impaired in the untreated group. Endothelial integrity was relatively better in the OE-MSCs + OE-exo group (Fig. 9A, B). Besides, proliferation and inflammation were assessed using PCNA and NF-κB P65 immunohistochemistry. The results showed that OE-MSCs + OE-exo administration significantly decreased the level of PCNA-positive cells (Fig. 9C, D) and NF-κB P65-positive cells (Fig. 9E, F). These results confirmed that OE-MSCs + OE-exo could effectively attenuate vein graft intimal hyperplasia by protecting endothelial integrity, synchronously inhibiting cell proliferation and inflammatory infiltration.
MSCs transplantation has been widely used to prevent atherosclerosis and promote angiogenesis in ischemic tissues [22, 23]. Using the autologous vein transplantation model in rats, we have confirmed that transplanted MSCs could home to the intima of the grafted vein, repair vascular endothelium and inhibit vascular remodeling [11]. However, some studies have reported that the homing or survival rate of transplanted MSCs was low, and the differentiation was uncertain [24, 25]. Therefore, we modified and regulated MSCs with lncRNA to improve the migration and survival of MSCs, improving the therapeutic effect of MSCs transplantation. Increasing evidence has confirmed that lncRNA exerts suppressive or promotion effects regulating various biological processes. MIR155HG plays an essential role in hematopoiesis, inflammation, and tumorigenesis [26–28]. To explore the biological activity of MIR155HG, we constructed MIR155HG up-regulated or down-regulated MSCs. The western blot results showed that MIR155HG could promote NF-κB P65 phosphorylation, suggesting that the NF-κB pathway was involved in MSCs regulation by MIR155HG. Our previous experiments have confirmed that TNF-α promoted the survival and migration of MSCs under oxidative stress via the NF-κB pathway. Further, one study reported that NF-κB/MIR155HG had a mutual regulating relationship [29]. Through a series of cell function experiments, we found that OE-MIR155HG promoted cell viability, proliferation, and migration of MSCs. These biological effects were all regulated by the NF-κB pathway. H2O2-induced oxidative stress led to significant apoptosis of MSCs. OE-MIR155HG could relieve apoptosis significantly. This may be related to the up-regulation of pro-survival factors in MSCs. The experiments showed that OE-MIR155HG significantly elevated the level of HGF and VEGF in the supernatant, confirming our hypothesis. The BMS-345541 antagonizing NF-κB pathway reversed the protective role of OE-MIR155HG. Through these experiments, we found that MIR155HG was an important lncRNA with a wide range of regulatory effects. MIR155HG could significantly improve cell function in many directions, including proliferation, migration, secretion, and anti-apoptosis. MSCs are the preferable source of therapeutic exosomes [30]. Exosomes have been reported to play a significant role in the paracrine effects. Exosomes are capable of delivering genetic materials. We observed that the RNA level of MIR155HG in exosomes derived from MIR155HG-MSCs was 4.9-fold higher than that of the control-MSCs. Similar genetic material changes were observed in exosomes as in transfected MSCs. In addition, the level of MIR155HG in HUVECs treated with OE-exo was 2.1-fold higher than that of con-exo. The results confirmed that exosomes derived from MSCs could efficiently deliver genetic materials into HUVECs. In our experiment, OE-exo could promote the migration of HUVECs and alleviate its apoptosis. Besides, in the sh-exo group, the activity of HUVECs was reduced, and apoptosis was increased. The exosomes could transmit the overexpressed genetic materials into target cells. One study reported that MSCs-derived exosomal MALAT1 could be transferred to osteoblasts and alleviate the symptoms of osteoporosis [31]. Therefore, we assume that it was not MIR155HG itself in sh-MIR155HG exosomes that affected the activity of HUVECs. Various miRNAs up-regulated by the ce-RNA mechanism may be the key to playing this role in sh-exo. There was sparse evidence on this. Through gene sequencing, we found that several miRNAs, including miR-133b, miR-206 and miR-675-3p, had significant negative feedback relationships with MIR155HG. These miRNAs could inhibit cell migration and promote apoptosis [32–34]. We will continue to explore this regulatory mechanism in subsequent studies. We constructed the rat model of autogenous vein transplantation to simulate the process of intima hyperplasia in patients undergoing CABG. We found that intima hyperplasia was significantly alleviated through injecting both OE-MSCs and OE-exo into autologous vein grafted rats. The number of OE-MSCs homed to the intima of vein graft was significantly increased compared to the Con-MSCs group. The result suggested that OE-MSCs had a better home function in vivo. Part of the reason may be that MIR155HG enhances the migration ability of MSCs. Another reason may be that MIR155HG alleviates the apoptosis of MSCs in vivo. The fluorescence intensity of CD31 was higher in the OE-MSCs + OE-exo group than Con-MSCs. Furthermore, the enhanced vascular CD31 expression indicated greater intima integrity. Besides, the immunohistochemical test suggested that the expression of PCNA and NF-κB p65 in the vascular wall were significantly reduced in the OE-MSCs + OE-exo group than Con-MSCs or OE-MSCs group. The NF-κB played an essential role in inflammation and cell proliferation [35, 36]. These data confirmed that OE-MSCs + OE-exo could alleviate intimal hyperplasia by reducing cell proliferation and inflammatory response. The effect was better than using OE-MSCs alone. At present, no articles have been published about the combined application of functionally improved stem cells and exosomes for treating intimal hyperplasia. We believe that this synergistic effect could achieve better therapeutic outcomes.
In summary, we confirmed that lncRNA-MIR155HG could promote the proliferation, migration, secretion, and anti-apoptosis of MSCs. Meanwhile, the NF-κB pathway participated in the regulation process. Exosome derived from MIR155HG-MSCs could delivery MIR155HG into endothelial cells and further inhibit endothelial cell apoptosis. Combining MIR155HG-MSCs and exosomes rich in MIR155HG could play a synergistic role in attenuating vein graft intimal hyperplasia more effectively. Then, we could provide a theoretical basis for improving the surgical treatment of coronary heart disease. | true | true | true |
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PMC9636846 | 35653895 | Brandon Johnson,Paulina Panek,Andy Yu,Elizabeth Fischer,Marli Koba,Daniel Mendoza Hermosillo,Christopher T. Capaldo | Interferon gamma upregulates the cytokine receptors IFNGR1 and TNFRSF1A in HT-29-MTX E12 cells | 30-05-2022 | Barrier function,TNF-α,IFN-γ,Colon,Inflammatory bowel disease | The intestinal mucosa protects the body from physical damage, pathogens, and antigens. However, inflammatory bowel diseases (IBDs) patients suffer from poor mucosal tissue function, including the lack of an effective cellular and/or mucus barrier. We investigated the mucus producing human colonic epithelial cell line HT29-MTX E12 to study its suitability as an in vitro model of cell/mucus barrier adaption during IBD. It was found that the proinflammatory cytokine interferon-gamma (IFN-γ), but not tumor necrosis factor-alpha (TNF-α), reduced cell viability. IFN-γ and TNF-α were found to synergize to decrease barrier function, as measured by trans-epithelial electric resistance (TER) and molecular flux assays. Cells cultured under an air-liquid interface produced an adherent mucus layer, and under these conditions reduced barrier function was found after cytokine exposure. Furthermore, IFN-γ, but not TNF-α treatment, upregulated the IFN-γ receptor 1 (IFNGR1) and TNF-α receptor super family 1A (TNFRSF1A) subunit mRNA in vitro. Co-stimulation resulted in increased mRNA expression of CLDN 2 and 5, two gene known to play a role in epithelial barrier integrity. Analysis of IBD patient samples revealed IFNGR1 and TNFRSF mRNA increased coincidently with guanylate binding protein 1 (GBP1) expression, an indicator of NFkB activity. Lastly, CLDN2 was found at higher levels in IBD patients while HNF4a was suppressed with disease. In conclusion, IFN-γ and TNF-α degrade epithelial/mucus barriers coincident with changes in CLDN gene and cytokine receptor subunit mRNA expression in HT29-MTX E12 cells. These changes largely reflect those observed in IBD patient samples. | Interferon gamma upregulates the cytokine receptors IFNGR1 and TNFRSF1A in HT-29-MTX E12 cells
The intestinal mucosa protects the body from physical damage, pathogens, and antigens. However, inflammatory bowel diseases (IBDs) patients suffer from poor mucosal tissue function, including the lack of an effective cellular and/or mucus barrier. We investigated the mucus producing human colonic epithelial cell line HT29-MTX E12 to study its suitability as an in vitro model of cell/mucus barrier adaption during IBD. It was found that the proinflammatory cytokine interferon-gamma (IFN-γ), but not tumor necrosis factor-alpha (TNF-α), reduced cell viability. IFN-γ and TNF-α were found to synergize to decrease barrier function, as measured by trans-epithelial electric resistance (TER) and molecular flux assays. Cells cultured under an air-liquid interface produced an adherent mucus layer, and under these conditions reduced barrier function was found after cytokine exposure. Furthermore, IFN-γ, but not TNF-α treatment, upregulated the IFN-γ receptor 1 (IFNGR1) and TNF-α receptor super family 1A (TNFRSF1A) subunit mRNA in vitro. Co-stimulation resulted in increased mRNA expression of CLDN 2 and 5, two gene known to play a role in epithelial barrier integrity. Analysis of IBD patient samples revealed IFNGR1 and TNFRSF mRNA increased coincidently with guanylate binding protein 1 (GBP1) expression, an indicator of NFkB activity. Lastly, CLDN2 was found at higher levels in IBD patients while HNF4a was suppressed with disease. In conclusion, IFN-γ and TNF-α degrade epithelial/mucus barriers coincident with changes in CLDN gene and cytokine receptor subunit mRNA expression in HT29-MTX E12 cells. These changes largely reflect those observed in IBD patient samples.
The ability of the intestinal mucosa to form a barrier against luminal antigens and pathogens is of primary importance to host innate immune defense. Failure of this barrier during inflammatory episodes is a core pathological feature of inflammatory bowel diseases (IBDs) such as Crohn’s Disease (CD) and Ulcerative Colitis (UC) [1,2]. Importantly, advances in our understanding of the intestinal mucosal barrier reveal that both mucus and cellular epithelial barriers must be maintained to prevent disease [3]. In vitro cell culture systems offer an experimentally tractable model for exploring how this dual barrier fails following an inflammatory stimulus. HT29-MTX E12 cells are an intestinal adenocarcinoma cell line distinct from the HT29 parental strain in that they produce mucus as well as form measurable cellular barrier [4]. However, functional studies detailing the properties of dual mucus/epithelial barrier models after proinflammatory stimulus are limited. Mucus has two essential roles in the intestine; it acts as a lubricant for fecal transit, and forms a barrier to intestinal microbes [5]. The ability to perform these functions is degraded in IBD [6]. HT29-MTX E12 cells produce both secreted and membrane-bound mucins [7]. Importantly, a semi-wet, air-liquid interface (ALI) cell culture environment is needed to produce an adherent mucus layer [8]. Tumor Necrosis Factor Alpha (TNF-α) and Interferon Gamma (IFN-γ) are proinflammatory cytokines commonly found at elevated levels in IBD intestinal tissues [9]. In addition to modifying immune functions, these cytokines also impact epithelial cell barrier capacity and viability in a complex temporal and concentration-dependent manner [10]. The effects of inflammatory stimuli on epithelial cells have been well documented, with cytokine treatment resulting in decreased barriers effectiveness [11,12]. This loss is thought to proceed via two mechanisms: apoptosis, and changes in the function of tight junctions structures that seal adjacent cells [10,13]. At the molecular level, the tight junctions seal is semipermeable due to heterogeneous claudin protein expression [13]. The transcription factor Hepatocyte nuclear factor 4a (HNF4a) has been shown to regulate claudin gene expression and has been linked to IBD by genome-wide association studies [14,15]. During inflammation, cytokine-induced changes in epithelial barrier function occur after ligand-receptor binding. IFN-γ and TNF-α cytokines bind to Interferon gamma receptor 1 (IFNGR1), which encodes the ligand-binding alpha subunit, and members of the Tumor necrosis factor receptor superfamily members (TNFRSF), respectively [16,17]. Receptor activation stimulates second messenger activity, altering a number of cellular processes, including gene transcription through the NFκB pathway [16,18]. NFκB regulates a number of inflammatory response genes in epithelial cells, including Guanylate binding protein 1 (GBP1), which functions as a stasis signaling molecule in intestinal epithelial cells [19]. The study below utilizes GBP1 mRNA expression as a positive control indicating cytokine signaling in cells or tissues. HT29-MTX E12 have been recently employed as a model system to study drug adsorption and host-microbe interactions, frequently in co-culture with the better characterized intestinal epithelial cell line, Caco-2 [20-23]. Given that under physiological circumstances the above processes likely take place in the presence of proinflammatory cytokines, a better understanding of HT29-MTX E12 cytokine responses would aid in the design of more robust invitro disease models. We examined HT29-MTX E12 barrier function under normal growth conditions, following cytokines stimulation, as well as under cell culture conditions that produce a mucus layer. These investigations revealed synergistic effects between TNF-α and IFN-γ that occur coincident with upregulation of IFNGR1 and TNFRSF cytokine receptors. Alterations of these receptors were also found to occur in IBD patients, as IFNGR1 and TNFRSF are overrepresented in tissue-derived cDNA samples that contain correspondingly high levels of the NFκB activity marker GBP1.
Caco-2 were purchased from ATCC (ATCC, USA). HT29-MTX E12 cells were obtained through Millipore/Sigma (Merck, USA) via the European Collection of Authenticated Cell Cultures and maintained in DMEM with 10% fetal bovine serum (FBS), 1% penicillin/streptomycin, 2 mM L-glutamate and 1% MEM amino acids solution (Cytiva, USA). Prior to cytokine exposure, cells were cultured at 8% CO2, 37 °C until 80% confluent with media changes every other day. HT29-MTX E12 cells were cultured on 0.4 μm pore Transwell® filter systems (Corning, USA). For standard liquid interface cultures (LI), 250 μL growth media was placed in the top chamber and 500 μL in the bottom. LI cultures treated with cytokine after monolayers achieved TER of ~ 300 Ω·cm2. ALI conditions were generated by removal of media from the top chamber once the above-mentioned TER was achieved. After transition to ALI, cells were then treated with TNF-α and IFN-γ at 2 ng/ml each for 48 h unless otherwise indicated (bottom chamber, BioVision, USA).
HT29-MTX E12 cells were grown on a 96-well plate and each cytokine treatment was performed in triplicate for each concentration. After ~14 days of incubation, growth media was removed and cells were stained with an Alcian blue dye for 15 min (Merck, Germany). The cells were rinsed with water twice. Excess water was removed by tapping the plate onto a clean paper towel. The absorbance of each set of cells was then measured using the Biotech Synergy HTX Multi-Mode plate Reader (Agilent, USA) at 595 nm as a measure of mucus and the number of mucus-producing cells present.
HT29-MTX E12 cells were grown on 0.4 μm pore Transwell® filter systems and monolayers were monitored for electrical resistance using an epithelial volt-ohmmeter (EVOM/EndOhm; World Precision Instruments, USA). Molecule flux assays utilized 25 μg 4 kDa FITC Dextran in the top chamber. This amount was delivered in 250 μL and 10 μL of PBS 2% FBS in LI and ALI conditions, respectively. Contents of the bottom chamber were assessed after 1 hr. 4 kDa FITC Dextran detection was performed using a BioTek Synergy HTX Multi-Mode plate reader (Agilent, USA).
HT29-MTX E12 cells treated as above were frozen at −80°C with 1 ml RiboZol RNA extraction reagent (VWR, USA) and processed according to the manufacturer’s instructions. The Bio-Rad iScript Reverse Transcription Supermix for RT-qPCR was used to produce cDNA; it was performed with the SsoAdvanced Universal SYBR Green Supermix on a C1000 Touch CFX96 Real-Time System (Bio-Rad Laboratories, USA). Human patient cDNA was obtained from Origene Tissue scan TSC10765-CCRT502 (Origene, USA). Full details concerning these samples can be found at: https://www.origene.com/catalog/tissues/tissuescan/ccrt502/tissuescan-crohnscolitis-cdna-array-ii). Primer sequences can be found in Supplemental Table 1.
Transcriptomic studies were selected based on data availability and inclusion of UC patients. GEO accession GSE109142, GSE59071, GSE48958, and GSE117993 are summarized in Table 1 after screening for TNFRSF genes and GBP1. Original data, analysis, and project details are available in the primary text. Statistics were performed using GraphPad Prism 9 (GraphPad Software, USA). Biological replicates are indicated, containing at least three technical replicates; error bars are SEM.
HT29-MTX E12 cells are a valuable system for the study of the colonic barrier, and serve as a model to investigate the function of a dual mucus/cellular barrier. However, the cytokine response of these cells is not well understood (24). To better understand cellular cytokine responses in these cells, HT29-MTX E12s were grown to ~90% confluence and treated with IFN-γ and TNF-α, two cytokines found at high levels in IBD patients and commonly used in in vitro cell models of inflammation [12,25]. After 48 h treatment with cytokines, cells were stained with Alcian blue in order to assess mucus production and cell viability. Fig. 1A shows that Alcian blue stained prominent mucus vacuoles within the cells, in addition to a light apical glycocalyx stain. Treatment with low concentrations of IFN-γ (48 h, 2 ng/ml) resulted in a reduction of mucus vacuoles within the monolayer (Fig. 1B). At higher IFN-γ concentrations, low power magnification revealed a stark contrast in Alcian blue staining between untreated control cells (Fig. 1C) and cells treated for 48 h with 100 ng/ml IFN-γ (Fig. 1D). In these IFN-γ treated cultures, an absence of stained vacuoles was again noted, however, the decrease in staining was primarily due to cell loss (Fig. 1C/D). As detailed in Methods above, stained cells are washed prior to imaging, thereby removing dead cells and debris. Therefore, Alcian blue uptake by HT29-MTX E12 cells allowed for the assessment of cell vitality and mucus production by absorbance measurements at 595 nm. The resulting dose response curves are shown in Fig. 1E/F. IFN-γ treatment reduced cell viability in a dose dependent manner as concentrations increased above 1.5 ng/ml (Fig. 1E). Remarkably, TNF-α treatment did not appreciably reduce cell viability under the conditions tested (Fig. 1E). Further studies demonstrated that the addition of TNF-α did not significantly alter IFN-γ-induced changes to cell viability below IFN-γ concentrations of 5 ng/ml (Fig. 1F). Together, these studies demonstrate a simple, novel, and effective methodology for assessing cytokine responses in HT29-MTX E12 cells. The epithelial cellular barrier is comprised of a cell monolayer joined together by tight junction protein structures (13). Alterations in cell barrier effectiveness can result from either increased cell death or changes in the composition of tight junction [10,11]. The experiments detailed in Fig. 1 allowed us to study barrier adaptation at sub-lethal concentration of cytokine (2 ng/ml for 48 h), thereby allowing for the investigation of cellular factors that regulate barrier function independent of apoptosis. Fig. 2A shows a schematic of the Transwell® system that was used to assess barrier function across a suspended semipermeable membrane. Growth media is present in both the top and bottom chamber; conditions forming a liquid interface (LI). The barrier integrity of HT29-MTX E12 cells grown in this fashion were monitored for Trans-epithelial resistance (TER) as a measure of barrier integrity (Fig. 2B). Cells were then treated with cytokines as indicated and barrier function was assessed following 48 h of treatment. As shown in Fig. 2B, both IFN-γ and TNF-α treatments suppressed barrier function as indicated by lowered TER. Additionally, cotreatment of IFN-γ and TNF-α resulted in further suppression of TER to values below those found with either individual treatment alone (Fig. 2B). In addition to the regulation of ion flow, tight junction structures form a barrier against the paracellular passage of small molecules, termed the leak pathway (26]. To determine how HT29-MTX E12 cells adapt to cytokine exposure, 4 kDa FITC Dextran was added to the apical surface of the Transwell® cell culture system and flux was monitored by sampling the bottom chamber after between 1 and 2 h following addition of the tracer. As shown in Fig. 2C, 48 hr treatment with IFN-γ, but not TNF-α, resulted in an increase in 4 kDa FITC dextran flux. However, in concordance with our findings in Fig. 2B, cotreatment of IFN-γ and TNF-α produced a dramatic increase in molecular flux. As a control, unseeded wells were assessed with 4 kDa FITC dextran to determine maximal rate of flux (max, Fig. 2C). Together, these findings demonstrate the both IFN-γ and TNF-α depress barrier function and that cotreatment elicits a synergistic suppression. HT29-MTX E12 cells produce an adherent mucus layer in addition to a polarized epithelial layer, and recent studies have used HT29-MTX E12 cells to model colonic mucosal tissue [4,8,26,27]. However, studies evaluating dual mucus-epithelial barrier adaptation to cytokines are limited [24]. To address this knowledge gap we altered cell culture procedures to provide an air-liquid interface (ALI), previously described as a semi-wet culture method [8]. In short, HT29-MTX E12 cells were grown on Transwell® filters until confluent and the TER had reached ~ 300 Ohms*cm2 (approximately 2 weeks). Growth media was then removed from the apical chamber, stimulating the accumulation of an apical mucus layer (Fig. 2D). To visualize the mucus layer, filters were excised and stained with Alcian blue. The image in Fig. 2E shows a stained filter with the mucus layer folded backwards to reveal the cells underneath. After one week in ALI conditions, cytokines were added to the bottom chamber, and barrier function was assessed following 48 h of treatment as in Fig. 2 (Fig. 2F). TER could not be evaluated in these cultures due to the lack of fluid in the apical chamber. Therefore, dextran flux was investigated by the addition of 4 kDa FITC dextran to the apical surface. After 1–2 h, the media in the bottom chamber was assayed for the presence of FITC dextran by fluorescence spectrometry (Fig. 2F). Similar to our findings in Fig. 2C, cytokine treatment increased barrier permeability. This was observed for both IFN-γ and TNF-α individually, as well as for co-stimulation of IFN-γ and TNF-α together, exhibiting trends that indicate co-stimulation results in reduced barrier integrity. Together these data show that IFN-γ and TNF-α decrease HT29-MTX E12 barrier function in both LI and ALI interface cultures. IBDs are caused by complex pathologies where multiple cytokines likely act simultaneously on epithelial cells to inhibit epithelial barrier function [28,29]. Previous studies in Caco-2 cells demonstrated that IFN-γ and TNF-α cotreatment reduce barrier function and upregulate TNF cytokine receptor expression [30,31]. To better understand our findings that IFN-γ and TNF-α cotreatment depresses barrier function to a greater extent than either cytokine alone, we examined the mRNA expression of cytokine receptor subunits after cotreatment (Fig. 3 A/B). Expression of Interferon gamma receptor 1 (IFNGR1), which encodes the ligand binding alpha subunit and Tumor necrosis factor receptor superfamily member 1A (TNFRSF1A) were examined by qPCR in HT29-MTX E12 cells 48 h after cotreatment with IFN-γ and TNF-α. As shown in Fig. 3A/B, both IFNGR1 and TNFRSF1A mRNA were upregulated after cytokine treatment. In contrast, no change in receptor mRNA was observed in Caco-2 cells at these cytokine concentrations, indicating greater cytokine sensitivity in HT29-MTX E12 cells. Given the functional enhancement of IFN-γ and TNF-α cotreatment, receptor mRNA expression was assessed after treatment with IFN-γ or TNF-α alone. As seen in Fig. 3C, IFN-γ exposure induced expression of both IFNGR1 and TNFRSF1A whereas TNF-α stimulation had no discernable effect on receptor mRNA. As a control to assess the level of cytokine stimulation on cellular transcription, we monitored guanylate binding protein 1 (GBP1), an interferon stimulated gene [32]. As shown in Fig. 3D, TNF-α did not upregulate GBP1 in HT29-MTX E12 cells at these cytokine concentrations, whereas IFN-γ or cotreatment resulted in large increases in GBP1 mRNA quantity. We conclude that IFN-γ/TNF-α reduction in barrier function is coincident with increases in cytokine receptor mRNA and increased expression GBP1. The above data indicate reduced barrier loss in HT29-MTX cells after co-treatment with IFN-γ and TNF-α. Previous studies have shown that cytokine treatment alters the mRNA expression of genes involved in regulating barrier tightness [12]. In order to better characterize HT29-MTX cells cytokine responses with respect to barrier function, the mRNA levels of the transcription factor HNF4a, an IBD-linked gene, was investigated. HNF4a mRNA can be produced from promoters P1 and/or P2, resulting in isoforms with distinct N-termini (Fig. 4A). Previous studies have shown that P2 is spatial restricted to the crypt proliferative zone and increases colitis susceptibility in mice [33]. Using RT-qPCR, we demonstrate that HNF4a is expressed in HT29-MTX cells primarily as the P2 isoform (Fig. 4B). Treatment with IFN-γ and TNF-α did not significantly alter HNF4a levels, however we note that PI levels trended higher after treatment in all tests. HNF4a is known to regulate CLDN gene expression. Claudin proteins compose the transcellular component of tight junctions and are vital for paracellular ion/antigen regulation [10]. We therefore investigated HT29-MTX cells to assess CLDN mRNA expression, limiting our analysis to the most abundant colonic CLDN gene family members. The CLDN family members detected are indicated in Fig. 4C; CLDN8 and 10 were assessed but not detected. Following cytokine treatment CLDN2 and CLDN5 were found to be unregulated relative to non-treated control (Fig. 4C). Enhanced CLDN2 and CLDN5 expression is associate with “leaky” tissues in vivo, and is consistent with our findings that HT29-MTX cells exhibit reduced barrier function. IFN-γ and TNF-α are commonly found expressed at high levels in the intestinal tissue in IBD patients [9]. Given our above findings that IFN-γ and TNF-α increase cytokine receptor mRNA levels, IBD patient samples were examined for relative mRNA levels of IFNGR1 and TNFRSF1A (Figure 5). RT-qPCR was performed using cDNA libraries form 47 patient samples. Patient sample arrays contained cDNA from control, UC, and CD patients collected from rectum, colon, or small intestine. RT-qPCR was performed using primers for IFNGR1 and TNFRSF1A, as well as GBP1 mRNA levels, which were assessed as an indicator of NFκB stimulation. As shown in Figure 5A, a robust positive correlation was discovered between IFNGR1 and GBP1 levels in all samples (p = 0.0013). This was not the case for TNFRSF1A, which did not demonstrate a relationship with GBP1 levels. A significant increase in IFNGR1 mRNA was observed in an analysis that include only colon tissues (Fig. 5B). Additionally, these same samples also expressed an increased level of GBP1 mRNA (Fig. 5C). IBD patient samples were then examined for the occurrence of HNF4a isoforms and CLDN genes (Fig. 5D/E). Unlike HT29-MTX cells, IBD patient samples exhibited lowered levels of HNF4a isoforms in comparison to histologically normal colon controls (Fig. 5D). Examination of CLDN 2, 5 and 15 revealed a dramatic upregulation of CLDN2 only, when compared to controls. Importantly, claudin 2 is known to form a cation channel in epithelial tissues [13]. Together, the findings demonstrate general concurrence between IBD samples and HT29-MTX responses to cytokine exposure, with the notable exception of reduced HNF4a levels. We next sought to investigate the discrepancy between our in vitro and in vivo findings with respect to TNFRSF1A, which is upregulated in HT29-MTX E12 cells but not in IBD samples. TNF receptors are composed of a large superfamily of 29 genes [34]. To assess the possibility that alternative TNFRSF genes were upregulated in IBD, we performed a meta-analysis of available transcriptomic studies (Table 1). Four studies were selected that analyzed rectal or colon biopsies were taken from healthy controls and IBD patients, encompassing both pediatric and adult IBD patients. Indeed, TNFRSF gene family members are commonly upregulated in IBD patients compared to controls, and this is coincident with increased levels of GBP1. Interestingly, TNFRSF1A was not reported in these studies, and not all upregulated receptors were found in all studies. These differences in detection may be attributable to the cellular heterogeneity in patient biopsies as well as our comparison to a clonal epithelial cell line. Combined with our previous data, these findings demonstrate increased IFNGR1 and TNFRSF receptor levels in IBD tissues that also express high levels of GBP1. IBDs are conditions of globally increasing incidence with an accelerating expected global health burden [35]. Our findings will allow for further investigation of mucosal barrier function in an in vitro model of mucus/cellular barrier during exposure to proinflammatory cytokines. Furthermore, future studies can focus on cellular contributions to mucus function, as barrier adaption was observed at cytokine concentrations below levels shown to increase apoptosis. Our studies demonstrate that HT29-MTX E12 cells respond to cytokines in a similar manner to the well-studied epithelia cell line Caco-2. As in Caco-2 cells, IFN-γ and TNF-α cooperate to alter HT29-MTX E12 barrier properties, likely through the upregulation of cytokine receptors. However, this occurs at lower cytokine concentrations than are commonly used in Caco-2 studies (IFN-γ 2 ng/ml vs. 10 ng/ml [30,36]. Therefore, studies of cytokine stimulation in mixed Caco-2/HT29-MTX E12 should consider the complication of divergent responses when utilizing these two cell lines simultaneously. Additionally, we find that both in vitro and in vivo cytokine receptors are upregulated during inflammatory stimulus, indicating that HT29-MTX E12 may be an effective model system for the study of epithelial cytokine exposure. Interestingly, while CLDN2 levels are altered both in vivo and in vitro, HNF4a expression levels are not suppressed in HT29-MTX cells after inflammatory stimulation. Given that HT29-MTX cells are cancer cells, incongruities between transcription factors in patient samples and in tumor-derived cell culture models are to be expected. However, further studies will be required to better understand these phenomena. In conclusion, our finds show synergy between IFN-γ and TNF-α coincident with upregulation of cytokine receptors and CLDN2. These findings add to our understanding of cellular cytokine response in this system. Furthermore, we demonstrate similarities between HT29-MTX E12 cytokine responses and those found in IBD; a correspondence indicative of a robust in vitro disease model. | true | true | true |
PMC9637042 | Hao Jianbing,Liu Xiaotian,Tang Jie,Chang Xueying,Jin Honge,Zhu Bo,Hao Lirong,Zhang Lei | The Effect of Allograft Inflammatory Factor-1 on Inflammation, Oxidative Stress, and Autophagy via miR-34a/ATG4B Pathway in Diabetic Kidney Disease | 29-10-2022 | Increasing evidence suggests that disorders of inflammation, oxidative stress, and autophagy contribute to the pathogenesis of diabetic kidney disease (DKD). This study attempted to clarify the effect of allograft inflammatory factor-1 (AIF-1), miR-34a, and ATG4B on inflammation, oxidative stress, and autophagy in DKD both in vitro and in vivo experiments. In vivo, it was found that the levels of AIF-1, miR-34a, oxidative stress, and inflammatory factors were significantly increased in blood and urine samples of DKD patients and mouse models and correlated with the level of urinary protein. In vitro, it was also found that the expressions of AIF-1, miR-34a, ROS, and inflammatory factors were increased, while ATG4B and other autophagy related proteins were decreased in human renal glomerular endothelial cells (HRGECs) cultured with high concentration glucose medium (30 mmol/L). When AIF-1 gene was overexpressed, the levels of miR-34a, ROS, and inflammatory factors were significantly upregulated, and autophagy-related proteins such as ATG4B were downregulated, while downregulation of AIF-1 gene had the opposite effect. In addition, miR-34a inhibited the expression of ATG4B and autophagy-related proteins and increased the levels of ROS and inflammation. Furthermore, the result of luciferase reporter assay suggested that ATG4B was the target gene of miR-34a. When ATG4B gene was overexpressed, the level of autophagy was upregulated, and inflammatory factors were downregulated. Conversely, when ATG4B gene was inhibited, the level of autophagy was downregulated, and inflammatory factors were upregulated. Then, autophagy inducers inhibited the levels of inflammation and ROS, whereas autophagy inhibitors had the opposite function in HRGECs induced by glucose (30 mmol/L). In conclusion, the above data suggested that AIF-1 regulated the levels of inflammation, oxidative stress, and autophagy in HRGECs via miR-34a/ATG4B pathway to contribute to the pathogenesis of diabetic kidney disease. | The Effect of Allograft Inflammatory Factor-1 on Inflammation, Oxidative Stress, and Autophagy via miR-34a/ATG4B Pathway in Diabetic Kidney Disease
Increasing evidence suggests that disorders of inflammation, oxidative stress, and autophagy contribute to the pathogenesis of diabetic kidney disease (DKD). This study attempted to clarify the effect of allograft inflammatory factor-1 (AIF-1), miR-34a, and ATG4B on inflammation, oxidative stress, and autophagy in DKD both in vitro and in vivo experiments. In vivo, it was found that the levels of AIF-1, miR-34a, oxidative stress, and inflammatory factors were significantly increased in blood and urine samples of DKD patients and mouse models and correlated with the level of urinary protein. In vitro, it was also found that the expressions of AIF-1, miR-34a, ROS, and inflammatory factors were increased, while ATG4B and other autophagy related proteins were decreased in human renal glomerular endothelial cells (HRGECs) cultured with high concentration glucose medium (30 mmol/L). When AIF-1 gene was overexpressed, the levels of miR-34a, ROS, and inflammatory factors were significantly upregulated, and autophagy-related proteins such as ATG4B were downregulated, while downregulation of AIF-1 gene had the opposite effect. In addition, miR-34a inhibited the expression of ATG4B and autophagy-related proteins and increased the levels of ROS and inflammation. Furthermore, the result of luciferase reporter assay suggested that ATG4B was the target gene of miR-34a. When ATG4B gene was overexpressed, the level of autophagy was upregulated, and inflammatory factors were downregulated. Conversely, when ATG4B gene was inhibited, the level of autophagy was downregulated, and inflammatory factors were upregulated. Then, autophagy inducers inhibited the levels of inflammation and ROS, whereas autophagy inhibitors had the opposite function in HRGECs induced by glucose (30 mmol/L). In conclusion, the above data suggested that AIF-1 regulated the levels of inflammation, oxidative stress, and autophagy in HRGECs via miR-34a/ATG4B pathway to contribute to the pathogenesis of diabetic kidney disease.
At present, diabetic kidney disease (DKD) as a serious complication of diabetes mellitus has become a leading cause of chronic renal failure on a global level [1–5]. It is very well recognized that excessive inflammation and oxidative stress in diabetes are an important cause of DKD [6–10]. Under physiological state, oxidative stress and inflammation are important to maintaining vital functions, but excessive oxidative stress and inflammation might cause diseases such as DKD. Increasing evidence suggests that treatment with antioxidative or anti-inflammatory drugs could inhibit the progress of DKD [11–13]. For example, SGLT2i or GLP-1R agonists attenuate inflammation and oxidative stress to contribute to reduce urinary protein in DKD patients [11]. In addition, it has been suggested that there is an interaction between inflammation and oxidative stress. Oxidative stress is activated by inflammation, and inflammation is also induced by oxidative stress. When the level of oxidative stress is increased, the expression of chemokines and cytokines which increase inflammation is also upregulated [14]. Moreover, autophagy as an essential process for normal cell homeostasis is considered to defend against oxidative stress and inflammation [15]. It has been reported that autophagy regulates the level of oxidative stress and inflammation in kidney injury [16]. However, the exact mechanisms still need to be fully elucidated. Autophagy as a self-protection response to inflammation and oxidative stress protects renal cells against injury in DKD, including podocytes, proximal tubular, mesangial, and endothelial cells. On the one hand, intact autophagic flux contributes to maintaining podocyte homeostasis. Dysfunction of autophagy in the podocyte is responsible for the progression of DKD [17–19]. On the other hand, high concentration glucose inhibits autophagy of mesangial cells by upregulating p62/SQSTMI and downregulating LC3 expression [20, 21]. Xu et al. also have confirmed that autophagy contributes to the survival of mesangial cells [22]. In addition, it has been confirmed that autophagy in renal tubular epithelial cells has a protective function in DKD [23]. Based on the reports mentioned above, although increasing evidence has suggested that dysregulated autophagy in glomerular and tubular cells is strongly associated with the pathogenesis of DKD, the exact mechanism of autophagy remains to be elucidated. An increasing number of studies have shown that there is an interplay between inflammation, oxidative stress, and autophagy which play an important role in DKD [24, 25]. Autophagy as an evolutionarily conserved cellular process protects renal cells from damage by inhibiting oxidative stress and inflammation in DKD [26, 27]. It is reported that the oxidative stress in glomerular endothelial cells induced by sustained high concentration blood glucose is a key link for DKD [28]. What is more, autophagy of glomerular endothelial cells protects glomeruli from oxidative stress and maintains the integrity of glomerular capillaries [29]. Enhancing endothelial autophagy may provide a novel therapeutic approach to improving glomerular diseases [30–32]. Therefore, regulation of autophagy in glomerular endothelial cells is helpful for the prevention of DKD. However, how to maintain autophagy of glomerular endothelial cells at the appropriate level is still unclear. MicroRNAs have a comprehensive regulatory effect, such as cell growth and apoptosis, blood cell differentiation, and homeobox gene regulation, which brings hope for accurate regulation of autophagy [33]. Wang et al. confirmed that many microRNAs, especially miR-34a, were significantly increased in serum and kidney of early DKD mice [34], but the biological functions in DKD are not clear. In our previous study on the role of miR-34a in chronic kidney disease-related vascular calcification [35], we found that autophagy-related gene 4B (ATG4B) was not only a potential target gene for miR-34a regulation but also a key gene for autophagy regulation. Liu et al. suggest that miR-34a via ATG4B regulates the level of autophagy in renal tubular epithelial cells in a mouse model of acute kidney injury [36]. However, whether miR-34a/ATG4B has the similar role in glomerular endothelial cells is not reported. In addition, our previous study has confirmed that AIF-1 could induce smooth muscle cell calcification and the expression of miR-34a in inflammation [37]. AIF-1 plays an important role in many chronic diseases, especially inflammatory responses, and DKD is also known as a chronic inflammatory disease. Our latest research shows that AIF-1 contributes to the pathogenesis of DKD [38]. However, the exact mechanism of AIF-1 in DKD remains unclear. Therefore, based on the previous studies, it is proposed that there might be interaction between AIF-1, miR-34a, and ATG4B to induce inflammation, oxidative stress, and autophagy in DKD (Figure 1). The study intends to explore the interactions of AIF-1, miR-34a, and ATG4B on inflammation, oxidative stress, and autophagy via in vivo and in vitro experiments.
In accordance with the clinical study program approved by the ethics committee of Southern University of Science and Technology Hospital and the ethical standards of the 1964 Helsinki Declaration, we randomly collected 5 mL samples of blood and urine from 60 diabetic kidney diseases (ACR: urinary albumin to creatinine ratio; group 1, 30 mg/g ≤ ACR ≤ 300 mg/g; group 2, ACR > 300 mg/g) and 30 diabetic patients (ACR < 30 mg/g). 30 healthy people without any underlying disease served as the control group. Then, the collected samples were centrifuged and used for further biochemical tests. All patients signed consent for the use of urine and serum specimens.
The db/db mice (4-week-old males and females are equally divided) as a spontaneous type 2 diabetic model were divided into five groups by random number table method as follows: model control group (no intervention), miR-34a agonist group (miR-34a agonist injected into caudal vein at an initial dose of 20 nmol and a maintenance dose of 5 nmol every 3 days), miR-34a agonist control group (negative control of miR-34a agonist was intravenously injected by caudal vein), miR-34a antagonist group (miR-34a antagonist was injected by caudal vein at an initial dose of 200 nmol and maintenance dose of 50 nmol every 3 days), and miR-34a antagonist control group (negative control of miR-34a antagonist was injected intravenously by caudal vein at the same dose of antagonist). The db/m mice were used as the control group. At the same time, to elucidate the effect of AIF-1 on DKD, another type 2 diabetic kidney disease model was established with AIF-1 transgenic mice which treated with unilateral renal artery ligation + low − dose intraperitoneal injection of streptozotocin (25 mg/kg, every two days for a week) + high − calorie diet [39, 40]. All mice were grouped according to the above method, when blood glucose > 16.5 mmol/L and urinary albumin/creatinine > 30 mg/g were a successful DKD model. At the end of the experiment, when urine samples were collected for 24 hours, all mice were anesthesia (pentobarbital sodium; 150 mg/kg) by intraperitoneal injection and euthanized. The kidney and blood specimens were collected from each mouse as soon as possible. After centrifugation, the serum was stored at -80°C for later use. The kidney samples were fixed in 4% paraformaldehyde for 24 h at room temperature or stored at -80°C after further study. The whole animal experiments were carried out according to research protocols approved by the Animal Ethics Review Committee of Harbin Medical University or Southern University of Science and Technology and adhered to the principles stated in the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health.
Human renal glomerular endothelial cells (HRGEC, USA) were purchased from ScienCell Research Laboratories Company. Cells were cultured with endothelial cell medium (ECM, ScienCell, USA) supplemented with 10% fetal bovine serum for 2-3 days and cultured with serum-free ECM when cells were fused up to 80% for 24 hours for standby use. Then, the cells received the following treatment: normal glucose (5.6 mmol/L glucose), high concentration glucose (10, 15, 20, and 30 mmol/L), miR-34a mimic, miR-34a inhibitor, rapamycin (autophagy inducer), and DC661 (autophagy inhibitor). According to the manufacturer's protocol, miR-34a mimic (50 nmol) and miR-34a inhibitor (100 nmol, RiboBio, China) were transiently transfected into HRGEC using riboFECT™ CP transfection agent and collected after culture for 48 hours. Incubation with the transfection reagent alone served as a control.
Total miRNAs from clinic specimens or cultured HRGEC were extracted using the total miRNAs Isolation Kit (Qiagen, Germany), according to the manufacturer's protocols. Templates for miR-34a were prepared with the miScript II RT Kit (Qiagen, Germany). Bulge-Loop primers of miR-34a (RiboBio, China) and SYBR Green Assays (TaKaRa, Japan) were used for qPCR in a Roche LightCycler480 II. U6 was used as an endogenous reference gene for normalization of the data. qPCR was also performed using SYBR Green Assays. The relative expression level was determined by LightCycler480 Software 1.5, as described by the manufacturer.
AIF-1 CRISPR Activation Plasmid (h, sc-400513-ACT), AIF-1 siRNA (h, sc-43857) Atg4b Lentiviral Activation Particles (h, sc-404173-LAC), Atg4b siRNA (h, sc-72584), and control siRNA-A (sc-37007) were purchased from Santa Cruz Biotechnology, Inc. According to manufacturer's instructions, all plasmids were transfected into HRGEC using riboFECT™ CP (RiboBio, Guangzhou, China) as the transfection agent. All plasmids were sequenced to ensure authenticity.
To construct a luciferase reporter containing wild-type ATG4B-3′UTR (pGL3-ATG4B-WT), a segment of human ATG4B was amplified by PCR using the following primers: Fwd: 5′- CCGCTCGAGGGCGCCGGCCGGATCGATCG-3′ Rev: 5′- CCCAAGCTTCCATCTTGC- GGTACGGACGT-3′ [41] from human genomic DNA and inserted into the pGL3 basic luciferase reporter vector (Promega, Madison, USA). Three-point mutations in the 3′UTR region of pGL3-ATG4B-WT were induced using a Quick Change site-directed mutagenesis kit (Strata-gene, La Jolla, CA, USA), resulting in mutant pGL3-ATG4B (pGL3-ATG4B-MUT). In addition, plasmid DNA was sequenced for authenticity. The luciferase reporters and miR-34a mimics or miRNA controls were cotransfected into HRGEC. Forty-eight hours after transfection, luciferase activities were detected using the Luciferase Assay System (Promega Biotech, USA).
Specimens of kidney in experimental mice were fixed with 10% buffered formalin and embedded in paraffin, and 4-micron tissue sections were prepared and treatment as our previous study [37]. Then, the tissue sections were incubated with primary antibodies against AIF-1 (1 : 1,000, Abcam, UK) at 4°C overnight. After rinsing with PBS, the tissue sections were incubated with goat antimouse antibody at 37°C for 1 h. Finally, the immunocomplexes were stained with DAB and observed under a microscope (Nikon Corp., Japan).
Protein concentrations were determined using the Bradford protein assay. For electrophoresis, 50 μg of total protein was loaded onto 10% SDS-PAGE gels using a Bio-Rad Mini Gel apparatus. After electrophoresis, the separated proteins were transferred onto a polyvinylidene difluoride membrane (Millipore, Billerica, MA, USA). The membrane was blocked in western blocking buffer for 1 h at room temperature and then incubated with primary antibodies against AIF-1, ATG4B (1 : 2000, Abcam), p62, LC3B, and β-actin (1 : 1000; Santa Cruz) for 2 h. Membranes were then incubated with goat antimouse/goat antirabbit secondary antibodies (1 : 10000; DyLight®800, Immuno Reagents, USA) for 1 h. The bands in the membrane were visualized and analyzed using the Odyssey Imaging System (LICOR Bioscience, USA). Protein levels were quantified and normalized to β-actin levels.
The intracellular ROS in HRGEC was detected by ROS assay kit (Beyotime Biotechnology Co., Ltd.) according to the manufacturer's protocols. Briefly, DCFH-DA (2,7-dichlorodi-hydrofluorescein diacetate) were diluted in serum-free medium at 1 : 1000 to a final concentration of 10 mmol/L. The medium was discarded and added DCFH-DA diluted in appropriate volume at 37°C for 20 minutes. The cells were washed three times with serum-free culture medium to fully remove the residual DCFH-DA. The fluorescence was read at 485 nm for excitation and 530 nm for emission with a fluorescence plate reader (Genios, TECAN). ROS level was quantified by measurement of fluorescence intensity.
The levels of AIF-1, ATG4B, NLRP3, IL-1β, and IL-18 in serum or urine were detected by human or mouse ELISA kit (Shanghai Jianglai Biotechnology Co., Ltd., China), according to the manufacturer's instructions. In simple terms, 50 μL sample of serum, urine, or cell supernatant was added into the enzyme label plate and incubated at 37°C for 30 min. After washing the plate 5 times, 50 μL enzyme standard reagent was added and incubated at 37°C for 30 min. After washing the plate 5 times, 50 μL color solution A or B was added and incubated at 37°C for 15 min. 50 μL stop solution was added and read by semiautomatic single channel microplate reader (Thermo Multiskan FC, USA) at 450 nm immediately. The software of Curve Expert 1.30 was used to draw the standard curve. According to the OD value of the standard sample, the regression equation was calculated from the standard curve. Then, the OD value of the sample was substituted into the equation to calculate the concentration of sample.
Physiological and biochemical indicators of all volunteers were collected from medical records. The levels of physiological and biochemical indicators in experimental mice were measured commercial assay kits. Basically, fasting plasma glucose (FPG) was measured by a glucose oxidase method using a glucose analyzer. Plasma insulin was measured using an automated chemiluminescence system (ADVIA Centaur Immunoassay System, Siemens Healthcare Diagnostics, USA). Blood lipids, nitrogen (BUN), and serum creatinine (Scr) were measured by commercial assay kits (Roche Diagnostics, Basel, Switzerland) on an automatic blood chemistry analyzer (Roche-Hitachi 7180, Roche Diagnostics, Basel, Switzerland). Estimated glomerular infiltration rate (eGFR) was calculated using the 2009 Chronic Kidney Disease Epidemiology Collaboration Equation. UACR was measured by immunonephelometric assay method.
All experimental results were presented as mean ± standard deviation. Analysis of variance together with the least significant difference post hoc test was used to assess differences between multiple groups using SPSS25.0 for Windows (SPSS, USA). All experiments were repeated for 3 times, and P < 0.05 was considered statistically significant.
It is well accepted that diabetes is a metabolic disease with many abnormal physiological indicators and excessive inflammation. To clarify the general condition of the enrolled patients, assay or qPCR was used to measure the physiological indicators and inflammatory factors of serum or urine. As the results showed in Table 1, physiological indicators such as blood glucose, islet function, blood lipids, kidney function, and urinary protein were abnormal, except liver function, compared with healthy people. Furthermore, the levels of AIF-1, miR-34a, NLRP3, IL-1β, and IL-18 in serum or urine were increased except ATG4B protein (Tables 1 and 2, P < 0.01). The data mentioned above showed that the levels of inflammatory factors were increased, and autophagy-related protein was decreased in DKD patients with albuminuria. It suggested that inflammation and autophagy were involved in DKD.
To determine the levels of autophagy and inflammation in DKD model, 4-week-old db/db mice which were spontaneous type 2 diabetic models and were fed with high-calorie diet for 4, 8, and 12 weeks. Compared with the control group (db/m mice), blood glucose, blood lipids, urinary albumin, AIF-1, miR-34a, NLRP3, IL-1β, and IL-18 in serum or urine were increased in db/db mice (Table 3, P < 0.01). In addition, the expression of AIF-1, miR-34a, p62, and NLRP3 in the kidney was significantly upregulated, whereas the expression of ATG4B and LC3II was significantly downregulated (Figure 2, P < 0.01). The above data showed that the level of autophagy was decreased, and the level of inflammation was increased in db/db mice.
It has been confirmed that many microRNAs such as miR-34a are significantly increased in serum and kidney of DKD patients and play an important role in regulating biological functions. To investigate the effects of miR-34a on autophagy and inflammation in DKD, miR-34a agonist or antagonist was used to regulate the level of miR-34a in db/db mice. Compared with the control group, treatment with miR-34a agonist reduced urinary albumin and miR-34a antagonist induced urinary albumin. At the same time, the expression of p62, NLRP3, IL-1β, and IL-18 was increased, whereas ATG4B and LC3II were decreased in miR-34a agonist group (Table 4, Figure 3(b), P < 0.01). However, miR-34a antagonist group had the opposite effect in Figure 3(b) (P < 0.01). In addition, the results mentioned in Figures 3(a) and 3(b) suggested that there was no significant difference in AIF-1 expression between the miR-34a agonist and inhibitor groups (P > 0.05). Therefore, miR-34a could aggravate urinary albumin in DKD by regulating the level of ATG4B, LC3IIp62, NLRP3, IL-1β, and IL-18 directly, except AIF-1.
To clarify the role of AIF-1 on autophagy and inflammation in DKD, AIF-1 transgenic mice were used to establish DKD models. Compared with wild mice, the levels of urinary albumin, inflammation, and autophagy were significantly increased in AIF-1 overexpression mice (AIF-1+/+) and decreased in AIF-1 knock-down mice (AIF-1−/−). The results showed that the expressions of miR-34a, p62, and NLRP3 were significantly upregulated in AIF-1+/+mice and downregulated in AIF-1−/− (Table 5 and Figures 3(c) and 3(d), P < 0.01). The above data showed that AIF-1 was involved in DKD via autophagy and inflammation pathway.
Inflammation, oxidative stress, and autophagy in glomerular endothelial cells play an important role in DKD. To clarify the effect of inflammation, oxidative stress, and autophagy in HRGECs, cells were cultured with different concentration glucose (5.6, 10, 15, 20, and 30 mmol/L) for 0 h, 12 h, 24 h, 36 h, 48 h and 60 h. As the results mentioned in Figure 4, glucose induced the expression of AIF-1, miR-34a, p62, NLRP3, and ROS and inhibited the expression of ATG4B and LC3II in HRGECs with a dose- and time-dependent manner (Figure 4, P < 0.01). These results suggested that high concentration glucose regulated the levels of AIF-1, miR-34a, inflammation, oxidative stress, and autophagy in HRGECs.
Allograft inflammatory factor 1 (AIF-1) is a highly conserved immunomodulatory inflammatory response calcium-binding protein and involved in various inflammatory reactions. The above data showed that the expression of AIF-1 was significantly upregulated and not affected by miR-34a in db/db mice and HRGECs. However, the expression of miR-34a was upregulated in AIF-1 overexpressing mice. To explore the exact effect of AIF-1 on miR-34a and ATG4B, AIF-1 plasmid or siRNA was transfected into HRGECs. When AIF-1 was overexpressed, the levels of miR-34a, p62, NLRP3, and ROS were increased, whereas the levels of ATG4B and LC3II were decreased in HRGECs exposed to high concentration glucose (Figures 5(a) and 5(b), P < 0.01). However, AIF-1 siRNA inhibited the expression of miR-34a, p62, NLRP3, and ROS and induced the expression of ATG4B and LC3II (Figures 5(a) and 5(b), P < 0.01). Therefore, the results mentioned above have shown that AIF-1 could induce autophagy and inflammation via miR-34a and ATG4B pathway.
Because miR-34a has a variety of biological functions, to further clarify the role of miR-34a in autophagy and inflammation induced by high concentration glucose, miR-34a mimics or inhibitors were transfected into HRGECs. Compared with the control group, the expression of p62, NLRP3, and ROS was upregulated, and ATG4B and LC3II were downregulated in miR-34a mimic group (Figures 5(c) and 5(d), P < 0.01). In addition, miR-34a inhibitors had the opposite function. However, miR-34a had no obvious effect on the expression of AIF-1. The results mentioned above suggested that miR-34a contributed to autophagy and inflammation in HRGECs cultured in high concentration glucose via regulating the expression of ATG4B, p62, LC3II, NLRP3, and ROS.
The results mentioned above suggested that miR-34a downregulated the expression of ATG4B protein in HRGECs exposed to high concentration glucose. To clarify the relationship between miR-34a and ATG4B, TargetScan.6.2 and http://microRNA.org/ were used to predict the coding sequence of ATG4B as a potential target of miR-34a. Luciferase assay results in Figure 2(d) showed that miR-34a mimic significantly inhibited the luciferase activity of wild-type 3′UTR of the ATG4B reporter gene (Figure 6(a), P < 0.05). However, there was no significant repressive effect of the MUT-ATG4B-3-UTR reporter genes (Figure 6(a), P < 0.05). The negative control of miRNA simulators had no significant effect on the activity of ATG4B luciferase of wild-type and mutants (Figure 6(a), P < 0.05). Furthermore, miR-34a overexpression significantly decreased the expression of ATG4B protein. It was suggested that miR-34a exerted its function via an effect at the posttranscriptional level. Consequently, these results indicated that ATG4B was an important target of miR-34a in HRGECs.
To further investigate whether ATG4B could regulate HRGECs autophagy, the ATG4B overexpression vector (pcDNA3.1-ATG4B) or ATG4B siRNA was transfected into HRGECs. ATG4B protein overexpression induced the expression of LC3II, whereas inhibited the expression of p62, NLRP3, and IL18 in HRGECs exposed to high concentration glucose (30 mmol/L) for 48 h (P < 0.05), as shown in Figures 6(b)–6(d). However, ATG4B siRNA inhibited the expression of LC3II and induced p62, NLRP3, and IL18 expression (P < 0.05), as shown in Figures 6(b)–6(d). Control empty vector (pcDNA3.1) and control siRNA did not cause any effects on HRGECs. These results demonstrated that ATG4B participated in autophagy and inflammation of HRGECs induced by high concentration glucose.
The results mentioned above suggested AIF-1 and miR-34a inhibited the level of autophagy in HRGECs cultured with high concentration glucose. In contrast, whether autophagy influenced the level of AIF-1, miR-34a, inflammation, and ROS is still unclear. To explore the effect of autophagy on the level of AIF-1, miR-34a, NLRP3, and ROS, HRGECs exposed to high concentration glucose (30 mmol/L) were treated with an autophagy inducers or inhibitors for 48 h. As shown in Figure 7, rapamycin (autophagy inducer) inhibited inflammation and ROS (Figure 7, P < 0.001) production, but it did not influence the expression of either AIF-1 or miR-34a (P > 0.05). In addition, DC661 (autophagy inhibitor) exerted the opposite effect (Figure 7, P < 0.001). Thus, these data suggested that autophagy inhibited the inflammation and ROS of HRGECs induced by high concentration glucose.
Diabetic kidney disease develops in approximately 40% of patients who are diabetic and has become the leading cause of CKD worldwide. Increasing evidence has shown that inflammation, oxidative stress, and autophagy contribute to the pathogenesis of DKD [31, 42], especially the role of autophagy in DKD has been paid increasingly attention all over the world [30, 43–46]. For example, autophagy significantly decreases in the kidney of early stage DKD and is involved in pathogenesis of DKD [47–49]. It might be a new direction of diagnosis and treatment in diabetic nephropathy. However, the exact pathogenesis of DKD and how to maintain the adaptive level of autophagy are still uncertain. It is well known that the glomerulus consists of parietal epithelial cells, podocytes, glomerular endothelial cells (GECs), and mesangial cells. Significantly, GECs cover the luminal surface of glomerular capillaries, expose to circulating high blood glucose levels, and are particularly vulnerable to injury induced by hyperglycemia in the early stage of DKD [50]. Furthermore, the pathogenic signals from GECs are transmitted into podocytes, as well as mesangial cells, and induce a phenotypic switch that modifies their intracellular signaling leading to dysfunction [51–53]. At the present, an increasing number of studies suggest that GECs dysfunction as a key event in the pathogenesis of DKD [54–57]. Thus, it is very important to elucidate the mechanism of GECs dysfunction induced by high glucose for the pathogenesis of diabetic nephropathy. In our previous study, our team has confirmed that AIF-1 facilitates glomerular endothelial cell inflammation and oxidative stress in DKD via the NF-κB signaling pathway [38]. Furthermore, in this study, we demonstrated for the first time that AIF-1 regulated the level of inflammation, oxidative stress, and autophagy in glomerular endothelial cells through miR-34a/ATG4B pathway in diabetic kidney disease. AIF-1 is an allograft inflammatory factor and plays an important role in regulating the inflammatory response. Our previous studies have confirmed that it is involved in the regulation of inflammatory reactions in various tissues and organs, including peritoneum [58], kidney [59], and blood vessels [37]. Although increasing evidence has confirmed that AIF-1 contributes to the pathogenesis of DKD [38, 60], the exact mechanism is still unclear. In this study, it was demonstrated that AIF-1 regulated the levels of miR-34a, ATG4B, autophagy, inflammation, and oxidative stress in glomerular endothelial cells induced by hyperglycemia via in vivo and in vitro studies. miRNA is a kind of small noncoding RNA molecule (containing about 22 nucleotides) with the function of RNA silencing and posttranscriptional regulation of gene expression [61] which acts both as a functional RNA and a potential biomarker for disease prediction [62]. As a class of small single-stranded noncoding RNA, miR-34a is involved in the pathological process of various diseases such as dietetic kidney disease by regulating the function of target genes such as Egr1, GAS1, and Sirt1/HIF-1α [63–67]. Wang et al. have found that miR-34a expression significantly increased in serum and kidney of early DKD mice via gene chips, but the specific mechanism of action is still unclear [34]. In addition, Xiao et al. and Opazo-Ríos et al. also have confirmed that miR-34a contributes to the pathogenesis DKD in rat or mouse models [67–69]. At the present, the mechanism that miR-34a/ATG4B regulated autophagy had already been described in tumor cells [63] and epithelial cells after kidney injury [36]. However, whether there is the similar effect in glomerular endothelia cells has not been investigated. In our study, we found that the levels of inflammation, oxidative stress, and autophagy in HRGECs were regulated via miR-34a/ATG4B pathway in DKD for the first time. In addition, we also clarified that ATG4B was the target gene of miR-34a in HRGECs. Koch et al. suggest that under normal blood glucose level, autophagy is an important protective mechanism in renal epithelial cells, including podocytes, proximal tubular, mesangial and endothelial cells. However, down regulation of autophagy in hyperglycemic condition, can contribute to the development and progression of diabetic kidney disease [45]. To date, the dysregulation of autophagy is still unclear. It has confirmed that autophagy-related gene (ATG) contributes to the regulation of autophagy, especially ATG4, which is covalently connected with phosphatidylethanolamine (PE) by cutting the c-terminal arginine of ATG8 to form ATG8-PE, which is anchored on the autophagy bubble membrane. Meanwhile, ATG4 removes the fate of ATG8-PE, promotes the fusion of autophagosomes and lysosomes, and induces the formation of autophagosomes [70]. In mammalian cells, ATG4 gene has four subtypes, ATG4A, ATG4B, ATG4C, and ATG4D. Particularly, ATG4B, as the main member of ATG4 family, exists in the cytoplasm and activates ATG8 family (LC3 family and GABARAP), especially LC3B, which activation efficiency is 1500 times of the other three subtypes [71]. Previous studies have identified that the basic level and hunger-induced autophagy significantly decreased in all organizations of ATG4B gene knock-down mice, but the existence of ATG4A/C/D cannot effectively make up for ATG4B lack. On the other hand, the experimental results mentioned above confirmed that there was a positive correlation between ATG4B and autophagy levels in HRGECs cultured with high concentration glucose medium. Therefore, it is conceivable that ATG4B may participate in HRGEC autophagy and oxidative stress. At present, the main accepted mechanism for DKD is dysregulated autophagy and oxidative stress in glomerular endothelial cells [72–74]. Autophagy protects cells from damage by eliminating damaged proteins and organelles. Clinically, blood and urine biomarkers of autophagic proteins are depressed in the patients with diabetic kidney disease [75, 76], and renal biopsy specimens of patients with insulin resistance exhibit molecular evidence of autophagy suppression [77]. In this study, we found that autophagy was negatively correlated with the level of inflammation and ROS, which was similar to previous research results. Intracellular oxidative stress, which induces autophagy, interacts with autophagy [78], which in turn inhibits oxidative stress and protects cells from damage. In conclusion, the present study confirmed that AIF-1 regulated the level of autophagy, oxidative stress, and inflammation via miR-34a/ATG4B in HRGECs induced by high concentration glucose. Therefore, regulating autophagy, oxidative stress, and inflammation via AIF-1/miR34a/ATG4B pathway would provide a new therapeutic target for the prevention of DKD. | true | true | true |
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PMC9637177 | Caiwang Peng,Fengyan Zhao,Hengli Li,Ling Li,Yantao Yang,Fang Liu | HSP90 mediates the connection of multiple programmed cell death in diseases | 05-11-2022 | Cell death,Diseases | Heat shock protein (HSP) 90, an important component of the molecular chaperone network, is closely concerned with cellular signaling pathways and stress response by participating in the process of maturation and activation of client proteins, playing a crucial role both in the normal and abnormal operation of the organism. In functionally defective tissues, programmed cell death (PCD) is one of the regulable fundamental mechanisms mediated by HSP90, including apoptosis, autophagy, necroptosis, ferroptosis, and others. Here, we show the complex relationship between HSP90 and different types of PCD in various diseases, and discuss the possibility of HSP90 as the common regulatory nodal in multiple PCD, which would provide a new perspective for the therapeutic approaches in disease. | HSP90 mediates the connection of multiple programmed cell death in diseases
Heat shock protein (HSP) 90, an important component of the molecular chaperone network, is closely concerned with cellular signaling pathways and stress response by participating in the process of maturation and activation of client proteins, playing a crucial role both in the normal and abnormal operation of the organism. In functionally defective tissues, programmed cell death (PCD) is one of the regulable fundamental mechanisms mediated by HSP90, including apoptosis, autophagy, necroptosis, ferroptosis, and others. Here, we show the complex relationship between HSP90 and different types of PCD in various diseases, and discuss the possibility of HSP90 as the common regulatory nodal in multiple PCD, which would provide a new perspective for the therapeutic approaches in disease.
HSP90 takes part in multiple PCD by regulating the stability and function of clients. HSP90 inhibitors are widely used in various diseases by affecting multiple PCD. HSP90 involves in the crosstalk of multiple PCD.
PCD is commonly found that relates to embryonic development, immune response, aging and other physiological processes, playing an important role in cellular homeostasis by removing damaged and senescent cells [1]. Besides the well-known apoptosis, some non-apoptotic PCD forms such as ferroptosis and necroptosis are gradually discovered, which have been demonstrated that may occur together to maintain a normal cell cycle for avoiding internal and external stimuli [2–4]. Therefore, it may bring a new trend for the therapy of diseases based on the special mechanism clarification. However, the complex crosstalk of multiple PCD processes are not independent of each other but sharing a coordinated system to mediate pathology, resulting in the difficulty to affect them as expected synchronously. For example, features of necroptosis and ferroptosis can be observed in the model of acute kidney injury which could be alleviated by necroptosis inhibitor Nec-1 and ferroptosis inhibitor Fer-1, though the inhibition of ferroptosis is beneficial to necroptosis [5]. The deep studies of relevant regulatory mechanisms focusing on the crosstalk and interaction among various PCD, caspase family proteins, HSPs and other proteins that acted as common regulatory nodal have attracted widespread attention [6, 7]. Among those, HSPs, as a molecular chaperones, are synthesized to maintain homeostasis when cells are under diverse physiological states, which are considered to involve in various pathways like hormone and cell cycle [8], including constituent proteins (HSP40 and HSP90), and inducible proteins (HSP70 and HSP27) [9]. The different classes of HSPs exert specific function, and also could work together to maintaining proteostasis. As the typical representative HSPs, HSP90 and HSP70 have received the most attention belongs to the important part of the chaperone network [10]. Therefore, their roles in the pathogenesis and treatment of diseases are revealed gradually, including the regulation of proteostasis, immune and cell death pathways [11]. And the HSP90 appears to be a critical regulator of PCD. HSP90 is the most abundant class in HSPs, whose expression could reach up to 4%-6% under stress conditions, and about 600 clients have been found in mammal [12, 13]. Generally, HSP90 fulfills the chaperone function by forming complexes with the co-chaperone and client to maintain the stability, processing and function of organisms [14]. For instance, binding to HSP90 is an indispensable part of the activation of some kinases and steroid receptors [15, 16]. As a wide range of clients, HSP90 is related to the development of lots of diseases by mediating key proteins of PCD, including receptor-interacting serine/threonine kinase (RIP) 1 in necroptosis, glutathione peroxidase (GPX) 4 in ferroptosis, Beclin-1 in apoptosis and autophagy [17–19]. The HSP90/co-chaperone/client complex is considered to be an emerging target for the treatment of diseases. So, how to block the interactions of HSP90 and client is gradually investigated, and inhibitors were proved to be the most commonly effective strategy [20–23]. Relevant genomics and experimental studies demonstrated that HSP90 inhibitors are potential antitumor agents, and some of them like geldanamycin (GA) and 17-allylamino-17-demethoxy-geldanamycin (17-AAG) have been developed and advanced into clinical trials [24, 25]. In consideration of the importance of PCD and the relation of HSP90 in the disease treatment, accumulating evidence indicates that HSP90 connects to multi-PCD such as apoptosis, necroptosis and ferroptosis [18, 26]. So, we summarized the latest researches on the correlation between HSP90 and PCD in diseases.
HSP90 is highly conserved during evolution, containing three conservative domains of C-terminal domain(CTD), middle domain(M-domain) and N-terminal domain(NTD) [13]. There is an ATP-binding site in NTD which is responsible for mediating the combination of co-chaperone and client based on ATPase activity [16]. The M-domain provides a site for ATP hydrolysis as well as binds to client [14], and CTD is related to the dimerization [27]. After dimerization of HSP90 connecting to ATP, the client binds to the M-domain, accompanied by NTD from open into closure [28] (Fig. 1). In general, there are four isoforms of HSP90 in cells which involves in various cellular functions, including HSP90α and HSP90β in the cytoplasm, tumor necrosis factor receptor-associated protein-1 (TRAP1) in the mitochondria and the glucose regulated protein 94 in the endoplasmic reticulum [8]. As an essential carrier of HSP90, the combination of client and HSP90 is decided by the modification of HSP90 and co-chaperone. It was reported that the moderate phosphorylation of HSP90 contributes to the maturation of most clients, but the hyperphosphorylation shows a negative effect on the chaperone machine [29]. In addition, the acetylation or the S-nitrosylation of HSP90 also blocks the interaction between HSP90 and client, which results in the inactivation and degradation of the client [12, 30]. Co-chaperone is also related to client activity, which assists with the function of HSP90 and provides selectivity for HSP90 by regulating its ATPase cycle, including p23, cell division cycle (CDC37), and so on [12, 31]. For instance, p23 is beneficial to stabilize the conformation of steroid receptors, but CDC37 is mainly responsible for kinase [32, 33]. Similarly, the inhibitors are also affecting clients selectively. The HSP90 inhibitor gedunin induces the degradation of steroid receptors in a dose-dependent manner, but has little effect on the stability of the kinase client [34]. Another inhibitor FW-04-086 regulates the cancer-related kinase to reduce proliferation and inducing apoptosis in breast cancer cells [35, 36]. Overall, the assembly of the HSP90/co-chaperone/client complex is closely relevant to various diseases, which promotes the application of HSP90 inhibitors. HSP90 inhibitors are mainly used in cancer research, divided into NTD inhibitors, M-domain inhibitors and CTD inhibitors according to the action sites [37]. Some NTD inhibitors like GA impact the ATP binding pocket of HSP90, to promote the degradation of clients through lysosomal and ubiquitinated protease pathways [38, 39]. However, the compensatory expression of HSP70 would be induced by NTD inhibitors, which acts as a cytoprotection [40]. On the contrary, inhibitors designed for interacting with M-domain or CTD will not induce HSP70-dependent cell survival [33]. An M-domain inhibitor KA blocks the interaction between HSP90 and CDC37 by combining Cys420 with HSP90 while reserving its ATPase activity [41]. Except for the interaction with three domains of HSP90, inhibitors can also impact the co-chaperone directly. As seen in the study of Patwardhan et al., gedunin induces the cleavage of p23 by recruiting caspase7 in NTD, which decreases the interaction between HSP90 and p23 and affects the client subsequently [34, 42].
The enhancive synthesis and release of HSPs are beneficial to maintain cellular homeostasis [43, 44]. Generally, highly expressed HSP90 shows a positive cytoprotection and the cell viability is decreased by the inhibition or knockdown of HSP90, which is considered to be a feasible therapeutic target in cancer such as colorectal cancer, lung cancer and gliomas [21, 26, 45]. The main mechanism HSP90 inhibitors acted on cancer cells is mediated by inflammation and PCD, and similar events also have been found in ischemic diseases, neurodegenerative diseases, and others [7, 8, 46] (Fig. 2). For example, apoptosis could be induced in ovarian carcinoma cells when HSP90 is knockout [47]. But in the model of cerebral ischemia, treating with GA is conductive to the inhibition of apoptosis. And further more relevant information is listed in the table below [48] (Table 1).
Compared with normal tissue, cancer cell shows a higher expression of HSP90 on mRNA and protein level [49]. It was reported that the concentration of HSP90 in plasma is associated with the malignant degree of the tumor, which is supported by the clinical data of more than 4000 patients with breast cancer [50]. Some clients may mutate or overexpress in pathological cells, which is alleviated by HSP90 inhibitors, indicating a therapeutic role in cancer. The cancer-related clients, including mitogen-activated protein kinase (MAPK) kinase, extracellular signal-regulated kinase (Erk) 1/2, and Akt [49, 51], take part in the proliferation, invasion and resistance of cancer cells [49, 52, 53]. For example, A synthetic inhibitor PF-4942847 exerts intensive effects on the viability of cancer cells by inducing apoptosis, delaying OS tumor growth and reducing lung metastasis [54]. Some recent studies shows that a higher level of HSP90 is found in plasma membrane and extracellular space of cancer cells, which participate in the invasion of tumor [52, 55]. For instance, the up-regulation of HSP90α enhances the aggressiveness of cancer cells by interacting with matrix metalloproteinase (MMP) 2 [56]. As antitumor drugs, triggering PCD is one of the most essential events, and the HSP90 inhibitors affect multiple PCD in various cancer have been verified [34]. Classical inhibitor GA has been reported that can induce autophagy and apoptosis in osteosarcoma cells by inhibiting the Akt signaling pathway [57]. Inhibitor 17-DMAG regulates the phosphorylation of Akt and Bcl-xl to increase apoptosis by targeting HSP90 whether in human or mouse lung cancer cells [49]. In addition, necroptosis is also an optional pathway for HSP90 inhibitor, which is related to the RIP1/RIP3/ mixed lineage kinase domain-like protein (MLKL) cascade [58]. Furthermore, the expressions of some resistance-related proteins like breast cancer resistance protein and survivin are regulated by HSP90 [47]. For example, multiple myeloma is a type of cancer with high recurrence and resistance, which is alleviated by blocking the interaction of HSP90 and CDC37 [59]. Due to the compatibility of unstable kinases with HSP90, inhibitors are beneficial to eliminate resistance after kinase inhibition in the treatment of cancer [60, 61]. 17-AAG affects radiosensitivity which is a pivotal point of clinical treatment by increasing the F-box protein 6 mediated polyubiquitination of CD147 [59]. HSP90 also is a key mediator of IFN-γ-induced adaptive immune resistance by regulating the expression of immune checkpoints like programmed death ligand 1, which exerts physiologic and pathologic effects in autoimmunity and immune escape [53]. In addition, some classical signaling pathways in cancer are affected by HSP90, such as Akt and NF-κB pathway [51, 62, 63]. In general, HSP90 inhibitors have been already demonstrated that against several cancers effectively due to its regulatory of key proteins in the development of cancer.
Protein homeostasis is one of the important factors of neuronal state, and its disorder may be the main reason of some conformational diseases. It was reported that the misfolding and aggregation of symbolic proteins are partly related to HSP90 chaperone machines [64]. The characteristic Tau tangles and β-amyloid deposition are co-locating with HSP90 in Alzheimer’s disease patients, whose aggregation and degradation are regulated by HSP90 [65]. According to the study of Chen et al., the neurotoxicity caused by β-amyloid could be reduced by the HSP90 inhibitor, further promoting the normalization of synaptic function [66]. In addition, the complex consisting of HSP90, p23 and PHD2 shows a significant increase both in vitro and vivo PD models, and the clinical symptomatic relief of PD can be obtained when the assembly of complex is inhibited [67]. And the HSP90/FK506-binding protein (FKBP) 51 machine is demonstrated that involves in the activity of GR, which is regarded as a regulatory of psychiatric diseases like depression [68, 69]. In general, HSP90 chaperone machine is widely participating in the development and treatment of a variety of neurodegenerative diseases by regulating the balance between HSP90 and different co-chaperones. Tau participates in the assembly and stabilization of microtubules and performed hyperphosphorylation and aggregation in neurodegenerative diseases [70]. HSP90 plays an essential role in the process of folding, degradation and aggregation of tau, and the relevant co-chaperones include FKBP51 and CDC37 [35, 65]. Oligomerization of Tau is synergistically triggered by the HSP90/FKBP51 machine, which is conductive to the accumulation of the toxic Tau [71]. The co-chaperone Aha1 also increases the aggregation and toxicity of Tau [72]. In contrast, protein phosphatase 5 and cyclophilin 40 promote phosphorylation and decomposition of the aggregating Tau [65]. Similarly, TAR DNA binding protein (TDP) 43 is also a client of HSP90, whose aberrant aggregation is a signature of amyotrophic lateral sclerosis [73]. As the study of Lin et al., the toxicity of TDP-43 is regulated by the specific interaction between HSP90/stress-inducible phosphoprotein (Sti) 1 machine and TDP-43 [74, 75]. The appropriate expression of Sti1 reduces the toxicity of TDP-43, while the abnormal Sti1 promotes the dysfunction of the neuron. In addition, HSP90/CDC37 is associated with the nuclear location of TDP-43 [76]. The all above suggest that the HSP90 machine plays a significant effect on neurodegenerative diseases.
The function of endothelial nitric oxide synthase, endothelial growth factor receptor and other vascular-related proteins are regulated by HSP90, which is related to the circulatory system [77]. It was reported that the Erk and Heme-Oxygenase-1 signaling are activated by suppressing the interaction of CDC37/HSP90, which could effectively reduce the infarct area, fibrosis and macrophage infiltration in the myocardial ischemia/perfusion model [78]. And inhibiting apoptosis is also the mechanism by which HSP90 inhibitors exert their protective effect [79]. GA reduces apoptosis in the process of myocardial injury by mediating the complement system and JNK signaling pathway [80]. HSP90 is also involved in cerebrovascular disease. Numerous studies demonstrated that HSP90 is significantly increasing in the model of cerebral ischemia-reperfusion injury [81]. The HSP90 inhibitor GA shows obvious neuronal protection both in the whole and focal cerebral I/R model, owing to the up-regulation of HSP70 and HSP25 in neurons [82]. In the model of four-vessel occlusion ischemic on rat, the intensive association of HSP90 and MLK3 is reversed by GA, which exerts a strong neuroprotection [83]. In addition to necroptosis, the inhibition of HSP90 is also related to apoptosis, autophagy and other PCD in stroke [48]. The another therapeutic mechanism of HSP90 inhibitor for cerebrovascular disease is maintaining the function of blood-brain barrier (BBB), which easily affects by inflammation under hypoxia condition [84]. In the study of Zhang et al., injecting siHSP90 alleviates oxidative stress and inflammation in the model of I/R[81]. And 17-DMAG is considered to play a protective role by down-regulating MMP9 to maintain BBB [85].
Some studies have been demonstrated that HSP90 is involved in pulmonary fibrosis, and 17-AAG could decrease fibrosis and MMP activity to alleviate idiopathic pulmonary fibrosis [86]. In addition, HSP90 is associated with the activation of glial cells in the spinal cord that are involved in the typical pain signaling cascade events [87]. Treating with 17-DMAG could relieve abnormal pain induced by exercise and monoarthritis, which is related to inflammatory cascade [88, 89]. In the case of low oxygen, HSP90α combines with the low-density lipoprotein receptor-related protein 1 cytoplasm tail to stabilize the receptor on the cell surface, and the Hsp90β is secreted into the cell space to increase cell movement, to promote wound healing by interacting with the low-density lipoprotein-related protein-1 receptor signaling [90]. Overall, the treatment strategy of targeting HSP90 has been widely studied, but its specific role in diseases remains not fully clarified. HSP90 is generally thought to be up-regulated in response to stress rather than cause disease. However, the up-regulation of HSP90 is beneficial to the survival of cancer cells and the mutation of cancer-related proteins, which is the reason for the use of inhibitors in cancer treatment. Furthermore, HSP90 in cancer cells is distinguished from the ordinary cells by the greater activities, extracellular localization and special post-translational modifications [45]. There are more than 30 different post-translational modifications of HSP90 in cells, and the change of modification induced by exogenous stimulus might be one of the mechanisms of its negative role in diseases. For example, alcohol induces the acetylation of HSP90 to decrease the interaction with eNOS, further leading to liver injury [91]. And the S-nitrification of HSP90 is observed in atherosis [30]. And for neurodegenerative diseases like AD, the imbalance of the chaperone system including HSP90 might be an important factor in its pathogenesis. In general, HSP90 has a unique mechanism under the disease state, but its specific mechanism still needs to be supplemented.
As a fundamental process of cells, there are complex connections among multiple PCD, and HSP90 is one of the common regulatory nodal in apoptosis, autophagy, necroptosis, ferroptosis and other PCD. (Fig. 3).
Apoptosis is the earliest cognitive PCD, which occurs ubiquitously in various diseases by eliminating aberrant cells through intrinsic and extrinsic pathways [92]. The intrinsic pathway is caused by internal stress signals like DNA damage, resulting in mitochondrial outer membrane permeabilization(MOMP) [93]. Then, the Cyt C released from mitochondria assembles the apoptotic bodies to facilitate the activation of the caspase cascade and further induce apoptosis ultimately [94]. And the extrinsic pathway can promote the assembly of the DISC to induce the maturation of caspase8 and triggers the caspase cascade to mediate apoptosis [95]. With further study of the mechanism, both the intrinsic and extrinsic pathways are believed to be regulated by HSP90. HSP90 is closely related to multiple processes of intrinsic apoptosis. For example, HSP90 decreases the release of Cyt C by interacting with Bcl-2 [96]. And the G-TPP could cause MOMP and the release of Cyt C by inhibiting TRAP-1 [97]. HSP90 also takes part in the assembly of apoptotic bodies by regulating apoptotic protease activating factor 1 and is associated with the cleavage and function of numerous caspases, including caspase3, 6, 9, and so on [98–101]. Some N-terminal inhibitors also could regulate apoptosis by activating HSF-1, which is beneficial for the up-regulation of HSP70 and HSP27 [102]. In the extrinsic pathway, c-FLIP is an essential negative regulatory which blocks the activation of caspase 8/10 in DISC [103]. 17-AAG induces apoptosis in lung cancer cells by decreasing the expression of c-FLIP, suggesting that HSP90 may mediate apoptosis by impacting the degradation of c-FLIP [39, 104, 105]. In addition, the MG132, a proteasome inhibitor, inhibits the down-regulation of c-FLIP in CALu-1 cells after 17-AAG treatment [39], which proves the regulation of 17-AAG in apoptosis owes to mediating the degradation of c-FLIP via the proteasome pathway.
Autophagy is defined as a process of removing damaged proteins and organelles by engulfing them into vesicles to form autophagosomes [106], including microautophagy, chaperone-mediated autophagy (CMA), and macroautophagy. CMA is described as a process that degrades specific clients by transporting them into lysosomes when recognized by lysosome-associated membrane protein type (LAMP) 2a [107]. And the stability of LAMP-2a is regulated by HSP90 [108]. Generally, autophagy is closely related to the degradation of clients which includes lysosome and ubiquitinated protease pathways. For example, IKK is selectively degraded when HSP90 is inhibited, and the inhibition of ATG5 can reverse IKK degradation [38], suggesting that HSP90 may lead to the degradation of IKK through the lysosomal pathway. In contrast, the protease inhibitor MG132 interrupts the degradation of RIP3, which is related to the ubiquitinated protease pathway [41]. In addition, HSP90 is associated with several key proteins in macroautophagy, such as ULK1, Beclin-1, ATG7, and so on [109]. ULK1, a mammalian homolog of ATG1, is involved in the nucleation and extension process of the autophagosome, whose function is maintained by forming a complex with HSP90 and CDC37 on its N-terminal kinase domain [110]. On one hand, the interaction between HSP90 and ULK1 contributes to the autophosphorylation of ULK1 at Ser1047, which is interrupted by the treatment of HSP90 inhibitor [110]. On the other hand, 17-AAG does not affect the mRNA level of ULK1, but it could decrease the homeostasis of ULK1, indicating that the interaction between HSP90 and ULK1 is in favor of the stabilization of ULK1. In addition, ATG13 is a substrate of ULK1 and could be phosphorylated by ULK1 at Ser318, whose phosphorylation needs HSP90/CDC37/ULK1 complex [110, 111]. HSP90 is also related to the localization of ATG13, which is described as translocating to damaged mitochondria to mediate its elimination [112]. Beclin-1 is a key regulatory in early autophagy whose function depends on HSP90 [113]. HSP90 forms a complex with Beclin-1 through an evolutionarily conserved domain to maintaining its stability and phosphorylation [69, 114]. GA separates Beclin-1 from HSP90, further promoting its degradation through the ubiquitinated protease pathway [114]. In addition, SNX-2112 could inhibit the formation of the ATG7/caspase9 complex, which is a key to the alteration of apoptosis and autophagy [115]. GA also affects the interaction between HSP90 and ATG7 by disrupting the stability of ATG7 [112]. It was also reported that the regulation of HSP90 inhibitor on autophagy is related to the rate of LC3II/I and the formation of autophagosomes [116, 117].
Compared to necrosis that had been considered as unregulated, increasing evidence indicates that there is a caspase-independent PCD defined as necroptosis, characterized by the loss of cell membrane integrity and release of cytoplasmic contents [3]. Necroptosis is strictly regulated by the RIP1/RIP3/MLKL pathway, and the HSP90 inhibitor involves in the stability, phosphorylation, and expression levels of RIP1, RIP3 and MLKL in necroptosis [17, 40]. For example, the higher expression and hyperphosphorylation of RIP1, RIP3 and MLKL in the model of heart failure would be reversed by HSP90 inhibitor [118]. The expression of RIP1 and RIP3 is inhibited by 17-AAG which could be reversed by CDC37 knockdown, suggesting that activation of RIP3 is related to the HSP90/CDC37 complex [119]. In addition to CDC37, p23 is also co-located with RIP3, affecting its phosphorylation [120]. The correlation between HSP90 and RIP3 expression remains controversial. Although most studies have shown that HSP90 inhibitors simultaneously reduce the phosphorylation of RIP3 in disease models [40], 17-AAG has no effect on the abundance of RIP3 and merely regulates its function [119]. The duration of HSP90 inhibitors effecting on cells is also critical to the function of RIP3, which demonstrated that short-term inhibition of HSP90 may lead to conformational changes of RIP3, and the degradation of RIP3 via ubiquitinated protease pathway is mediated by long-term inhibition of HSP90 [41, 121, 122]. Activity of HSP90 is also essential for the processes of phosphorylation, oligomerization and membrane translocation of MLKL [123, 124]. Previous studies show that MLKL is phosphorylated at Ser227 and Ser358 during necroptosis, which is strongly inhibited by 17-AAG [122]. HSP90 is also vital to promote the oligomerization and translocation of MLKL, though the interaction with HSP90 is weak or transitory [122–124]. Considering that MLKL is downstream of this cascade, the RIP3-deficient fibroblast cell is used for determining that HSP90 inhibitors can directly regulate MLKL to mediate necroptosis [122]. In general, HSP90 takes part in multiple processes of necroptosis regulated by different HSP90 inhibitors.
Ferroptosis is an emerging iron-dependent cell death way, which is characterized by membrane rupture and vesiculation, mitochondrial atrophy, decrease of the mitochondrial ridge, and an increase in membrane density [2]. Some studies show that HSP90 is a potential target for ferroptosis, while its role in ferroptosis remains controversial [18, 125]. Su et al. found that HSP90 inhibitor GA promotes the depletion of GSH to accelerates the occurrence of ferroptosis [125]. On the contrary, another HSP90 inhibitor CDDO is beneficial in reducing ferroptosis [18]. According to the study of Wu et al., CDDO significantly inhibits ferroptosis by affecting the expression of GPX4, which is one of the most classical biomarkers of ferroptosis [18, 126]. A previous study shows that GPX4 is the substrate of CMA, which is affected by LAMP-2a [108, 127]. The overexpressed LAMP-2a promotes CMA to decrease the expression of GPX4, which enhances the sensitivity to erastin-induced ferroptosis, and an obvious high expression of GPX4 is detected in LAMP-2a knockdown cells [128]. The interaction of HSP90 and Lamp-2a shows a significant increasing when the cells treat with erastin [18, 108]. Another latest study also demonstrates that HSP90 plays a positive role in ferroptosis by regulating ACSL4. As a key biomarker of ferroptosis, ACSL4 involves in lipid peroxidation, whose expression is related to the interaction of HSP90 and Drp1 [129].
In addition to the above, some studies have shown that HSP90 is also associated with other PCD, such as pyroptosis. Pyroptosis is a recently discovered PCD accompanied by an inflammatory response, which is characterized by rapid plasma membrane rupture, DNA damage, and the release of pro-inflammatory cytokine [130]. And the NLRP3/caspase-1/GSDMD pathway is considered to be the key to regulating pyroptosis. Inhibition of NLRP3 inflammasome is a reliable therapeutic target for a variety of inflammatory diseases, and the interaction of HSP90 and NLRP3 is related to its stability and activation, further regulates downstream IL-1β secretion and pyroptosis[131]. Normally, NLRP3 is inactivated when it binds to HSP90, and upon receiving an inflammatory signal, the interaction is blocking to prompting the activation of NLRP3 and the initiation of subsequent inflammatory cascades [132]. Finally, the complex correlations between HSP90 and emerging PCD, like Cuproptosis, NETosis and PANoptosis, remain to be further investigated.
In the studies of targeting for HSP90, some researchers have found that HSP90 may involve in the selection of multiple PCD. As reported, the inhibition of HSP90 could transform necroptosis induced by DD receptor into apoptosis [133]. And different HSP90 inhibitors selectively activate or inhibit multiple PCD, even in the same objects. For example, DHQ3 induces necroptosis by activating the RIP1/RIP3/MLKL pathway in human breast cancer cells, while 17-DR induces caspase-3 and caspase-8-dependent apoptosis [58]. In addition, HSP90 inhibitors also appear under different regulations when affecting multiple PCD simultaneously (Table 2). According to the research of Yan et al., HSP90 is a critical regulator of necroptosis and apoptosis, whose inhibitor alleviates necroptosis and promote the activation of apoptosis [97]. In another study, the inhibition of HSP90 could reduce both necroptosis and apoptosis in nucleus pulposus-derived stem cells [17]. The specific regulation is both related to types of inhibitors and pathological context of cell. For, example, 17-AAG, a widely used inhibitor, induces apoptosis in lung cancer cells but reduces apoptosis in a rat CCI model [39]. With the deepening of relevant researches, parts of complexes and proteins are regarded as key nodal in the complex regulatory network of PCD, selectively promoting cells towards different PCD, including complex II, Beclin1-Bcl 2, and some caspases [134].(Fig. 4). Mechanistically, necroptosis and extrinsic apoptosis share a common initiation, which is separated by the assemble of complex I and II. cIAP1/2 is one of the key regulatory, exerting anti-apoptotic function [135]. When cIAP1/2 is inhibited, the assembly of complex IIa is increasing to activate the caspase cascade and induce apoptosis, which is consisted of RIP1, FADD, and caspase-8 [136]. When RIP1 deubiquitinated and caspase-8 inhibited, the assembly of complex IIb induces necroptosis promoted after the binding of RIP3 and RIP1. Inhibitor CDDO could inhibit necroptosis by disrupting the formation of complex IIb [18]. Another determining element is the balance of c-FLIP and RIP1/RIP3/MLKL whose expression and function are regulated by HSP90 [39]. Beclin-1/Bcl-2 is also an important common regulatory nodal between apoptosis and autophagy, whose stability requires the involvement of HSP90 [137]. Gedunin targets HSP90 to mediate the interaction of Beclin-1/Bcl-2 and endoplasmic reticulum stress, then regulates the transformation between apoptosis and autophagy [19]. Beclin1 could also disrupt the Xc- system by interacting with SLC7A11, which may mediate the correlation between autophagy and ferroptosis [138]. In addition, mitochondrial complex I is an important nodal for autophagy, necroptosis and ferroptosis, which could be inhibited by celastrol [139, 140]. In addition to apoptotic activity, some caspases can also participate in other physiological activities [141]. For example, caspase 9 is involved in the initial autophagosome formation when its apoptotic activity is inhibited by interacting with ATG7, which could be regulated by an HSP90 inhibitor [6, 112, 142].
Since PCD is closely related to the process of disease development, drugs usually take effect by eliminating aberrant cells and protecting normal cells. The HSP90 inhibitors have been used in the research of diseases due to the excellent effect in this respect. However, there are still many problems need to be solved in this process. For example, some N-terminal inhibitors regulate PCD by binding to the NTD of HSP90, which would up-regulate the HSP70-related cytoprotection to cause unsatisfactory results [33]. This phenomenon promotes the development of CTD and MD inhibitors which would not induce this protection. Furthermore, although HSP90 inhibitors have shown promising therapeutic effects in related mechanistic studies, they have not performed as expected in clinical trials. The dissatisfactory specificity of inhibitor is one of the main limiting factors, which is caused by the sequence identity of the four isoforms, especially the 85% similarity between HSP90α and HSP90β [8]. So, the researches on isoform-selective inhibitors are still continually deepened, which is conductive to reducing the pan-inhibition of Hsp90. In the study of Chaudhury et al., the reported complex shows the greatest selectivity towards HSP90β, and even more than 370 fold compared with HSP90α [143]. In general, although the problems of targeting HSP90 in disease treatment have revealed, it still be a feasible approach because these problems could be gradually solved. Overall, according to relevant studies of targeting HSP90 for diseases therapy, the feasibility and prospects are as follows: [1] Due to the complex interactions of HSP90, co-chaperone and clients, HSP90 inhibitors are designed to regulating key proteins in diseases by blocking the combination. However, the current studies mainly focus on single PCD, and the effect of inhibitors on various PCD is complex. How to select the appropriate inhibitors to promote cell survival or inhibit cell activity as a whole may be one of the important problems for the situation of HSP90 as a therapeutic target. [2] As far as cancer treatment is concerned, the mechanism of targeting HSP90 to regulate apoptosis and necroptosis has already presented lots of relevant studies, but the relationship between HSP90 and some emerging cell death pathways should be further explored, including ferroptosis, pyroptosis, and cuproptosis. [3] N-terminal inhibitors of HSP90 can activate the heat shock response and increase the expression of HSP70, which exerts strong cellular protection. The low-dose inhibitors may acutely activate the heat shock response to alleviate disease without extensive cytotoxicity, so HSP90 inhibitor with an appropriate dose may be a vital event in disease treatment. In general, the role of HSP90 in disease and regulation in multiple PCD still has great prospects. | true | true | true |
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PMC9637195 | Hongwei Zhang,Yachong Liu,Wei Wang,Furong Liu,Weijian Wang,Chen Su,He Zhu,Zhibin Liao,Bixiang Zhang,Xiaoping Chen | ALKBH5-mediated m6A modification of lincRNA LINC02551 enhances the stability of DDX24 to promote hepatocellular carcinoma growth and metastasis | 05-11-2022 | Liver cancer,Oncogenes | As the most important RNA epigenetic regulation in eukaryotic cells, N6-metheyladenosine (m6A) modification has been demonstrated to play significant roles in cancer progression. However, this modification in long intergenic non-coding RNAs (lincRNAs) and the corresponding functions remain elusive. Here, we showed a lincRNA LINC02551 was downregulated by AlkB Homolog 5 (ALKBH5) overexpression in a m6A-dependent manner in hepatocellular carcinoma (HCC). Functionally, LINC02551 was required for the growth and metastasis of HCC. Mechanistically, LINC02551, a bona fide m6A target of ALKBH5, acted as a molecular adaptor that blocked the combination between DDX24 and a E3 ligase TRIM27 to decrease the ubiquitination and subsequent degradation of DDX24, ultimately facilitating HCC growth and metastasis. Thus, ALKBH5-mediated LINC02551 m6A methylation was required for HCC growth and metastasis. | ALKBH5-mediated m6A modification of lincRNA LINC02551 enhances the stability of DDX24 to promote hepatocellular carcinoma growth and metastasis
As the most important RNA epigenetic regulation in eukaryotic cells, N6-metheyladenosine (m6A) modification has been demonstrated to play significant roles in cancer progression. However, this modification in long intergenic non-coding RNAs (lincRNAs) and the corresponding functions remain elusive. Here, we showed a lincRNA LINC02551 was downregulated by AlkB Homolog 5 (ALKBH5) overexpression in a m6A-dependent manner in hepatocellular carcinoma (HCC). Functionally, LINC02551 was required for the growth and metastasis of HCC. Mechanistically, LINC02551, a bona fide m6A target of ALKBH5, acted as a molecular adaptor that blocked the combination between DDX24 and a E3 ligase TRIM27 to decrease the ubiquitination and subsequent degradation of DDX24, ultimately facilitating HCC growth and metastasis. Thus, ALKBH5-mediated LINC02551 m6A methylation was required for HCC growth and metastasis.
Hepatocellular carcinoma (HCC) is one of the most common cancer and the third leading cause of cancer-related deaths worldwide [1]. The high mortality rate results from late presentation at an advanced stage, a high incidence of tumor metastasis, and tumor recurrence after surgical resection [2]. Although significant progress has been achieved, very few approaches can be utilized in the clinic to prevent the recurrence and metastasis of HCC [3]. Hence, identifying the molecular mechanisms of HCC pathogenesis and metastasis is of great importance. LncRNAs are emerging as fundamental to cell biology because of their ability to reprogram gene expression and influence distinct cellular functions [4, 5]. And it has been shown that some lncRNAs serve as oncogenes or tumor suppressors [6, 7]. Mechanistically, lncRNAs are decoys for messenger RNAs and microRNAs (miRNAs) [8, 9]. Additionally, although named “non-coding RNAs”, some lncRNAs can encode small peptides [10, 11]. Because of their relatively large size enables them to form complex structures, lncRNAs can interact with proteins and regulate protein-protein interactions [12]. Reversible RNA modification leads to a new level of post-transcriptional regulation of gene expression that is involved in many physiological and pathological processes [13, 14]. N6-methyladenine (m6A) is a dynamic and reversible chemical modification and is the most abundant modification of mRNA [15, 16]. m6A is extensively involved in mRNA metabolism, affecting mRNA degradation and stability or translation [17, 18]. AlkB Homolog 5 (ALKBH5), an important “erasers”, plays significant roles in the regulation of gene expression [19, 20]. It has been proved that ALKBH5 specifically demethylates m6A-modified RNA and suppresses malignancy in HCC via m6A-guided epigenetic inhibition of LYPD1 [21]. m6A RNA deposition is initiated by “writers”, and the modification is recognized by “readers”, both of which are conserved and preferentially bind an RR (m6A) CU (R = G or A) consensus motif [22]. A kind of “readers” belong to the insulin-like growth factor (IGF) family, and they specially bind m6A-modified RNA and regulate RNA stability [23, 24]. However, the mechanisms through which m6A regulates lncRNA activity in HCC need to be further explored [25–27]. In this study we identified LINC02551 as the downstream target of ALKBH5, which regulated its expression in an m6A-dependent manner. Insulin-like growth factor 2 mRNA binding protein 1 (IGF2BP1) has been implicated in prolonging the LINC02551 lifespan. LINC02551 that is located in the nucleus can interact with DDX24 to decrease the TRIM27-induced ubiquitination-induced degradation of DDX24. We found that LINC02551 promoted HCC progression by stabilizing DDX24 expression. Overall, our study reveals that LINC02551 is a promising biomarker for prognostic prediction and a potential therapeutic target in HCC.
Human liver tumor tissues and corresponding adjacent normal tissues were obtained from patients undergoing hepatectomy between 2012 and 2015 at Tongji Hospital Liver Surgery Center, Tongji Medical College, Huazhong University of Science and Technology (HUST; Wuhan, China). Written informed consent was obtained from all patients and this study was approved by the Medical Ethics Committee of Tongji Hospital. All procedures conformed to the standards of the Declaration of Helsinki.
HLF cells were transfected with pcDNA3.1-ALKBH5 or pcDNA3.1-Vector plasmids. After 48 h, total RNA was extracted and transcriptome sequencing was performed by by GeneRead Biotechnology (Wuhan, China) to identify the differentially expressed lncRNAs. The cutoff value of differentially expressed lncRNAs was set as |log2[fold change]| > 1 and P < 0.05.
To generate lentivirus-based stable overexpression or knockdown cell lines, CDS of ALKBH5 was cloned into the BamHI/SalI sites of pLenti-CMV-Puro plasmid (Addgene #17448) and the short hairpin RNAs (shRNAs) targeting ALKBH5 and LINC02551 were cloned into the pLKO.1-TRC vector (Addgene #10879). For the luciferase reporter assay, Full-length of LINC02551 (wt) or m6A mutant (m1, m2, m3, m4) LINC02551 sequences were cloned into psiCHECKTM-2 Vector (Promega, USA). pcDNA3.1 vector was used for the construction of LINC02551, ALKBH5, ALKBH5-H204A, IGF2BP1, IGF2BP1-mut, DDX24 and TRIM27 plasmids. siRNAs targeting IGF2BP1 and DDX24 was synthesized by Ribo Biotech (Guangzhou, China). All sequences were verified by DNA Sanger sequencing. The sequences of shRNA and siRNA were listed as follows: shLINC02551-1: 5′- CCGGTGTCAACTCCACCTTTAGACTCGAGTCTAAAGGTGGAGTTGACATTTTTG-3′; shLINC02551-2: 5′-CCGGGTTCAACGGAAATTCACAACTCGAGTTGTGAATTTCCGTTGAACTTTTTG-3′; shLINC02551-3: 5′-CCGGTGCCTTGAATAAAGACGTACTCGAGTACGTCTTTATTCAAGGCATTTTTG-3′; shDDX24-1: 5′-CCGGCGCTCAAGAAAGATGAGGATACTCGAGTATCCTCATCTTTCTTGAGCGTTTTTG-3′; shDDX24-2: 5′-CCGGCCCACGTACCTCGGAGATTTACTCGAGTAAATCTCCGAGGTACGTGGGTTTTTG-3′; shDDX24-3: 5′-CCGGTTTCTGTTCTCTGGCTATTTGCTCGAGCAAATAGCCAGAGAACAGAAATTTTTG-3′. More detailed methods were described in Supplementary Materials.
It has been reported that downregulation of ALKBH5 was associated with poor prognosis in HCC. ALKBH5-mediated m6A demethylation led to the posttranscriptional inhibition of LY6/PLAUR Domain Containing 1 (LYPD1), which induced oncogenic behaviors in HCC. To explore how ALKBH5-mediated m6A-modified lncRNAs regulation of HCC progression, we applied RNA sequencing (RNA-seq) to HLF cells overexpressing ALKBH5 (Fig. 1a and Fig. S1a). Ten lncRNAs up- or downregulated by ALKBH5 overexpression in 97H and HLF cells were then verified via qRT-PCR (Fig. S1b, c). LINC02551 appeared to be the top hit because of its concurrent regulation by ALKBH5 in two these HCC cell lines. In 97H and HLF cells, LINC02551 was negatively regulated by ALKBH5 (Fig. 1b). An Me-RIP assay showed that the extent of m6A modification in LINC02551 was much lower in the ALKBH5 overexpression group (Fig. 1c). Since ALKBH5 is a vital “eraser” in the m6A modification process, an inactive ALKBH5-mut (mutant) was utilized to test whether ALKBH5-mediated LINC02551 regulation is m6A dependent [28] (Fig. 1d). When wild-type (ALKBH5-wt) or ALKBH5-mut was overexpressed in dose gradients, LINC02551 levels were downregulated only in the ALKBH5-wt group, not in the ALKBH5-mut group (Fig. 1e and Fig. S1d, e). According to SRAMP (http://www.cuilab.cn/sramp) analysis, four potential m6A sites were identified (with very high confidence) in LINC02551 (Fig. 1f). To confirm that these four sites are critical for LINC02551 regulation, point mutations were introduced into putative m6A sites: m1, m2, m3, or m4. Then, we constructed a psiCHECK2 plasmid containing LINC02551-wt or point mutants. The dual luciferase reporter assay suggested that ALKBH5 did not bind to LINC02551 when the m1 putative m6A site was mutated (Fig. 1g). These results indicate that ALKBH5 might regulate LINC02551 levels in an m6A-dependent manner. Previous studies have suggested that m6A modification mediates different RNA fates. We wondered how the m6A modification affected LINC02551 level. After using Actinomycin D to block RNA transcription, the degradation rate of LINC02551 decreased when ALKBH5 was knocked down and increased with overexpression of ALKBH5, suggesting that ALKBH5 regulates the decay of LINC02551 (Fig. 1h). Considering that “readers” directly and critically affect m6A-modified transcripts, we investigated potential effectors participating in the process. It has been reported that IGF2BP1 enhanced RNA stability [24]. When IGF2BP1 was knocked down, the LINC02551 level was decreased (Fig. 1i). RIP assays showed that less IGF2BP1was bound to LINC02551 in the ALKBH5-wt group but not in the ALKBH5-mut group, which was verified through RNA pull-down assays (Fig. 1j, k). Next, an inactive IGF2BP1-mut was used to verify that IGF2BP1-mediated LINC02551 regulation is m6A-dependent [24] (Fig. 1l). The transfection of IGF2BP1-mut did not lead to the upregulation of LINC02551 (Fig. 1m). The knockdown of IGF2BP1 partially attenuated the upregulation caused by the knockdown of ALKBH5 (Fig. 1n). Me-RIP assays indicated that the m6A modification was enriched to a greater extent on LINC02551 in the ALKBH5-mut and LINC02551-wt groups (Fig. 1o). An IHC analysis showed that the expression of ALKBH5 in HCC patient samples was negatively correlated with the expression of LINC02551 level (Fig. 1p). These data suggested that ALKBH5 downregulates LINC02551 in an m6A-dependent manner, and that this effect is mediated by IGF2BP1 recognition.
To explore the function of LINC02551 in HCC, we checked its expression in different HCC cell lines (Fig. 2a) and constructed stable knockdown cell lines (Fig. 2b) and overexpression cell lines (Fig. 2c). In Hep3B and HLF cells, shRNA-mediated knockdown of LINC02551 led to decreased cell migration and invasion (Fig. 2d and Fig. S2a). On the contrary, overexpression of LINC02551 in HLF and 97H cells increased cell motility (Fig. 2e and Fig. S2b). To further verify the effect of LINC02551 in cell metastasis, the cell wound healing assay was performed and the result indicated that knockdown of LINC02551 led to decreased cell motility in HLF and Hep3B cells (Fig. 2f and Fig. S2c) and overexpression of LINC02551 led to increased cell motility (Fig. 2g and Fig. S2d). To determine the effects of LINC02551 on the cell proliferation, we conducted plate clonality assay and a CCK-8 (Cell Counting Kit 8) assay. The results showed that knockdown of LINC02551 led to decreased cell colony formation (Fig. 2h) and slower cell growth (Fig. S2e); LINC02551 overexpression promoted the cell colony formation (Fig. 2i) and led to increased cell proliferation rate (Fig. S2f)
To evaluate the tumor-promoting role of LINC02551 in HCC in vivo, we established various xenograft models. Fist, we subcutaneously injected 97H cells overexpressing LINC02551 in nude mice. Four weeks post-injection, we found that LINC02551 overexpressing 97H cells caused larger (volume) and heavier (weight) tumor (Fig. 3a, b). We also injected 97H cells overexpressing LINC02551 in the left lobe of each mouse liver. Five weeks post-inoculation, we examined the intrahepatic metastasis of the 97H cells. Injection of LINC02551 overexpressing cells substantially increased the capacity of the HCC cells to form secondary lesions in the liver (Fig. 3c, d). IHC staining showed higher N-cadherin, Vimentin, Snail, Ki67, and PCNA signals and weaker E-cadherin signals in the livers of the LINC02551 overexpression group (Fig. 3e, f). In the lung metastasis model, control or LINC02551 overexpressing cells were injected into the tail vein of the mice. Six weeks after injection, more lesions were observed in the mice injected with the LINC02551 overexpressing cells than in those injected with the control cells (Fig. 3g). These results suggested that LINC02551 promoted the progression of HCC in vivo.
We next explored the underlying mechanism of LINC02551-induced promotion of HCC progression. RNA pull-down assays followed by mass spectrometry (MS) led to the identification of potential LINC02551 interaction partners (Table S1). DDX24, a member of the DEAD box-containing RNA helicases family, was verified through analysis (Fig. 4a). RIP assays coupled with RNA pull-down assays were performed to confirm the interaction between LINC02551 and DDX24 (Fig. 4b, c). Immunostaining showed that LINC02551 was associated with DDX24 in the nucleus (Fig. 4d). Further, a pull-down analysis showed that DDX24 interacted with an 800-1200 bp sequence of LINC02551 (Fig. 4e). LINC02551 overexpression caused an increase in the protein level of DDX24 but not its RNA level (Fig. 4f, g). Therefore, we speculated that LINC02551 increased DDX24 expression at the posttranscriptional level, probably by attenuating DDX24 degradation. Cycloheximide (CHX) chase results verified our hypothesis (Fig. 4h, i). The protein degradation process involves at least three main systems: the ubiquitin-proteasome, autophagosome and lysosome pathways. The LINC02551-induced increase in DDX24 was blocked by MG132 (a proteasome inhibitor) treatment but not by CQ (a lysosome inhibitor) (Fig. 4j). These data suggested that LINC02551 might attenuate the ubiquitination-mediated degradation of DDX24.
To further characterize the specific molecule that mediates DDX24 degradation, immunoprecipitation (IP) followed by MS was performed to identify potential DDX24 interaction proteins (Table S2). TRIM27 appeared to be the top hit in the MS results (Fig. 5a). The combination of DDX24 and TRIM27 was then verified in 293 T cells (Fig. 5b). MG132 treatment compensated for the downregulation of DDX24 caused by TRIM27 (Fig. 5c, d). Since TRIM27 is a E3 ubiquitin-protein ligase, an inactive TRIM27-mut was utilized to determine whether TRIM27-mediated DDX24 downregulation is ubiquitin dependent. When the TRIM27-mut was expressed in a dose gradient, the DDX24 protein level was not affected (Fig. 5e, f). IF coupled with IP showed that LINC02551 attenuated the interaction between TRIM27 and DDX24 (Fig. 5g, h). In vitro ubiquitination assays with Ub bearing mutations on all but one lysine residue indicated that lysine 48 was necessary for TRIM27-stimulated polyubiquitination of DDX24 (Fig. 5i). The ubiquitin enrichment on DDX24 was decreased in cells when transfected with TRIM27-mut (Fig. 5j). These data revealed that TRIM27 was the E3 ligase that mediated the ubiquitination leading to degradation of DDX24 and that LINC02551 blocked this process.
To explore the function of DDX24 in HCC, we measured its expression in different cell lines (Fig. 6a) and constructed stable overexpression and knockdown cell lines (Fig. 6b, c). In ALEX and HLF cells which express low levels of DDX24, forced overexpression of DDX24 led to increased cell motility (Fig. 6d and Fig. S4a), metastasis ability (Fig. 6e and Fig. S4b), and cell proliferation rate (Fig. S4c). To validate that the effects of LINC02551 are partially dependent on DDX24, shRNA-mediated knockdown of DDX24 was performed with 97H and Hep3B cells. In LINC02551 overexpressing cells, the knockdown of DDX24 partially decreased cell motility (Fig. 6f and Fig. S4d), metastasis ability (Fig. 6g and Fig. S4e), and cell proliferation rate (Fig. S4f). We next evaluated whether DDX24 exerts an effect on downstream signaling in metastasis or proliferation pathways. Microarray gene expression profiling was performed with DDX24-expressing or control 97H cells. A Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis indicated that genes regulated by DDX24 overexpression were more enriched in cell adhesion molecule pathway (Fig. S2a). Gene set enrichment analysis based on our own sequencing data and TCGA data indicated that genes induced by the epithelial mesenchymal transition (EMT) were enriched to a greater degree in DDX24-overexpressing cells than in control cells (Fig. 6j and Fig. S2b). To confirm this result, a set of genes regulated by the EMT was selected. Genes upregulated by the EMT were increased in the DDX24-expressing group. Among these upregulated genes, the top five most highly upregulated were selected for use in further experiments. In Hep3B and 97H cells overexpressing LINC02551, the knockdown of DDX24 inhibited the upregulation that had been induced by LINC02551 overexpression in these five genes (Fig. 6k). These results indicated that LINC02551 exerted its effects partially by stabilizing DDX24 expression.
An IHC analysis indicated that the expression of LINC02551 was positively correlated with the expression of DDX24 in a paraffin-embedded HCC sample array obtained from Tongji Hospital (n = 99) (Fig. 7a, b). Through qRT-PCR analysis, we found that LINC02551 levels were upregulated in HCC tumor tissues in another patient cohort (n = 120) (Fig. 7c). When combined with the specific information on HCC patients in the latter cohort, high LINC02551 levels were associated with a higher number of tumors (P = 0.039), incomplete tumor encapsulation (P = 0.027) and advanced BCLC stage (P = 0.021) (Table S3). A Kaplan-Meier analysis showed that patients with high LINC02551 levels and high DDX24 levels were associated with a poor overall survival rate and shorter disease-free survival (Fig. 7d, e). Ultimately, our research revealed that LINC02551 and DDX24 are potential biomarkers for the prognostic prediction of HCC because both of them indicate a poor prognosis.
In the present study, we identified a lncRNA downregulated by ALKBH5 in an m6A-dependent manner in HCC, and this regulatory effect was mediated by IGF2BP1 recognition of m6A. LINC02551 interacted with DDX24 in the nucleus, decreased TRIM27-induced ubiquitination-related degradation, thereby stabilizing its protein level, and ultimately promoting the EMT process. RNA m6A modification affects tumor progression through various mechanisms, and “writers”, “erasers”, and “readers” play vital roles in this process [15, 29, 30]. The effect of METTL3, an important “writer”, has been extensively studied in gastrointestinal tumors [31–33]. However, the effect of ALKBH5, an “eraser”, remains poorly understood. To elucidate the mechanism of ALKBH5-induced dysregulation of lncRNAs, we performed RNA-seq to screen lncRNAs that were dysregulated by ALKBH5 overexpression. LINC02551 appeared to be the most affected lncRNA and became our research target. An inactive mutant of ALKBH5 was used to verify that the regulatory effect was m6A dependent. When the enrichment of the m6A modification in LINC02551 increased due to the downregulation of ALKBH5, IGF2BP1, a “reader”, recognized m6A and stabilized the degradation of the modified lncRNA. Whether other “readers” participate in this process remains to be further explored. The function of lncRNAs depends on their subcellular location. When located in the cytoplasm, lncRNAs can act as decoys for miRNAs or combine with ribosomes and be translated into micropeptides [34, 35]. When in the nucleus, lncRNAs can combine with proteins and form functional RNA-protein complexes [36]. After determining the subcellular location of LINC02551, we found that nearly one-half of the LINC02551 content was located in the nucleus (data not shown). RNA pull-down followed by MS indicated that many proteins interacted with LINC02551. DDX24, a DEAD box protein, appeared to be the top hit as a LINC02551 interaction partners. Previous research demonstrated that DDX24 was a putative RNA helicase and played an inhibitory role in p53 transcriptional activity and cell proliferation arrest and senescence in breast cancer cells [37]. Emerging evidence has linked DEAD box proteins to cancer development and progression [38]. In our study, LINC02551 decreased the ubiquitination-induced degradation of DDX24, which was dependent on the recognition of TRIM27, a member of the E3 ligase family [39]. Overexpression of DDX24 influenced a set of cell adhesion molecules and was closely related to the EMT (epithelial-mesenchymal transition), which was in line with the results of our in vitro experiments. Moreover, knockdown of DDX24 partially impeded the effects caused by LINC02551 overexpression, suggesting that LINC02551 promoted the progression of HCC by stabilizing DDX24 expression. However, this axis explained only the prometastatic roles of LINC02551, and whether the mechanism of the LINC02551 proliferation effect in HCC is in line with that in breast cancer is unclear and remains to be further studied. In summary, we found that ALKBH5-downregulated lncRNA LINC02551, which was dependent on the demethyltransferase function of ALKBH5. When the m6A on LINC02551 was enriched, IGF2BP1 was recruited and recognized the m6A modification, leading to reduced degradation of LINC02551. In the nucleus, LINC02551 bound to DDX24 and decreased its TRIM27-induced ubiquitination-related degradation. Stabilized DDX24 promoted the EMT in HCC. Our study highlights the fact that LINC02551 is a newly discovered regulator in HCC and might serve as a potential prognostic biomarker for HCC (Fig. 7f).
Supplementary figure legends Supplementary materials and methods Supplementary table 1 Supplementary table 2 Supplementary table 3 Supplementary figure 1 Supplementary figure 2 Supplementary figure 3 Supplementary figure 4 Supplementary figure 5 WB (full length) A reproducibility checklist | true | true | true |
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PMC9637198 | Yangyi Xie,Hongsheng Lin,Wei Wei,Yinzhi Kong,Qiaoling Fang,Enran Chen,Jianghua Liu,Mingfen Li | LINC00839 promotes malignancy of liver cancer via binding FMNL2 under hypoxia | 05-11-2022 | Oncogenesis,Biomarkers,Molecular medicine | Liver cancer is one of the most common malignant tumors in the world and metastasis is the leading cause of death associated with liver cancer. Hypoxia is a common feature of solid tumors and enhances malignant character of cancer cells. However, the exact mechanisms involved in hypoxia-driven liver cancer progression and metastasis have not been well clarified so far. The aim of this study was to investigate the contribution of long non-coding RNA (lncRNA) in hypoxia promoting liver cancer progression. We screened and revealed LINC00839 as a novel hypoxia-responsive lncRNA in liver cancer. LINC00839 expression was up-regulated in liver cancer tissues and cell lines, and the patients with high LINC00839 expression had shortened overall survival. LINC00839 further overexpressed under hypoxia and promoted liver cancer cell proliferation, migration, and invasion. Mechanistically, LINC00839 bound multiple proteins that were primarily associated with the metabolism and RNA transport, and positively regulated the expression of Formin-like protein 2 (FMNL2). LINC00839 could promote hypoxia-mediated liver cancer progression, suggesting it may be a clinically valuable biomarker and serve as a molecular target for the diagnosis, prognosis, and therapy of liver cancer. | LINC00839 promotes malignancy of liver cancer via binding FMNL2 under hypoxia
Liver cancer is one of the most common malignant tumors in the world and metastasis is the leading cause of death associated with liver cancer. Hypoxia is a common feature of solid tumors and enhances malignant character of cancer cells. However, the exact mechanisms involved in hypoxia-driven liver cancer progression and metastasis have not been well clarified so far. The aim of this study was to investigate the contribution of long non-coding RNA (lncRNA) in hypoxia promoting liver cancer progression. We screened and revealed LINC00839 as a novel hypoxia-responsive lncRNA in liver cancer. LINC00839 expression was up-regulated in liver cancer tissues and cell lines, and the patients with high LINC00839 expression had shortened overall survival. LINC00839 further overexpressed under hypoxia and promoted liver cancer cell proliferation, migration, and invasion. Mechanistically, LINC00839 bound multiple proteins that were primarily associated with the metabolism and RNA transport, and positively regulated the expression of Formin-like protein 2 (FMNL2). LINC00839 could promote hypoxia-mediated liver cancer progression, suggesting it may be a clinically valuable biomarker and serve as a molecular target for the diagnosis, prognosis, and therapy of liver cancer.
Liver cancer is a major malignancy worldwide ranking as the fourth cause of cancer-related deaths. Hepatocellular carcinoma (HCC) accounts for about 90% of primary liver cancers. Currently, surgical resection and liver transplantation are the most effective therapeutic approaches for patients with early-stage tumors. However, majority of patients with HCC are not eligible for surgery at the time of diagnosis. Besides, the long-term prognosis remains poor due to high occurrence of local invasion and distant metastasis. Better understanding of the critical dependencies and underlying molecular mechanisms in HCC might enable more efficient therapeutic approaches for patients with HCC. Hypoxia is a common and critical feature of solid tumors, which is attributed to the excessive oxygen consumption due to rapid cell proliferation, and low oxygen supply from insufficient functional blood vessels. Hypoxia microenvironment facilitates cell proliferation, motility, metabolism, drug resistance and stem cell biology. In response to hypoxia, the cancer cells alter the transcription of numerous genes and activate multiple oncogenic signaling pathways to coordinate the malignant cell phenotypes. Hypoxia results in the stabilization of hypoxia-induced factor-1 (HIF-1), which helps cancer cells adapt to hypoxic stress by activating gene expression programs that control angiogenesis, glycolytic metabolism, invasion, migration, and erythropoiesis. Nevertheless, the molecular details leading to the hypoxic survival advantage of cancer cells have not been fully elucidated. The human transcriptomic studies demonstrate that the vast majority of genomic sequence is pervasively transcribed into a diverse range of protein-coding RNAs and non-coding RNAs (ncRNAs), of which protein-coding RNAs compose less than 3%. Long non-coding RNA (lncRNA) is defined as a type of transcript with a length of more than 200 nucleotides and no protein coding potential. Increasing evidence has uncovered that lncRNAs are specifically regulated and conserved rather than being the product of transcriptional noise. Their functional mechanisms are diverse, including lncRNAs that act as scaffolds, decoys or signals and can act through genomic targeting, regulation in cis or trans, and antisense interference. Recent discoveries have revealed that lncRNAs drive many important cancer phenotypes through their interactions with other cellular macromolecules including DNA, protein, and RNA. The number of hypoxia-responsive lncRNAs identified in cancer has risen sharply, illustrating the complexity of the hypoxia-induced gene reprogramming and the expanding roles of lncRNAs in hypoxia signaling cascade and responses. It is of great significance to characterize the long non-coding transcriptome involved in hypoxia adaptation for a comprehensive understanding of hypoxia-associated tumor biology. The high stiffness of the tumor or the surrounding extracellular matrix (ECM) and subsequent loss of mechanical tissue homeostasis are common hallmarks of cell invasion and tumor progression. Formins constitute a diverse protein family, which are recognized as potent nucleators of linear actinfilaments that control a large variety of cellular and morphogenetic functions. Formin-like protein 2 (FMNL2), a member of formins, which contributes to the formation of protrusive actin structures such as lamellipodia and filopodia at the leading edge of migrating cells, plays a key role in governing rapid cytoskeletal adaptation to environmental changes and supporting cell efficient adhesion and migration. In the present study, we investigated the contribution of lncRNA in hypoxia promoting liver cancer progression. We identified and verified hypoxia-responsive LINC00839 in liver cancer and systematically investigated the effects of LINC00839 on liver cancer cell proliferation, migration, and invasion under hypoxia. Additionally, mechanistic investigations were performed to explore the potential mechanism of LINC00839 and its connection with FMNL2, which may provide a basis for clinical diagnosis and treatment of liver cancer.
The RNA-seq and clinical data of Liver Hepatocellular Carcinoma (LIHC) were downloaded from The Cancer Genome Atlas (TCGA) database (https://cancergenome.nih.gov/). The Ensembl ID was converted to gene symbol, and the expression profile was divided into lncRNA and mRNA using gencode v22 annotation. The differently expressed lncRNAs were identified by DESeq2 package, and log2FoldChange > 1 and padj < 0.001 were set as the threshold for significantly different expression. The co-expression analysis was performed using starBase database. The Gene Ontology (GO) analysis was performed using GO database. The Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis was performed using KEGG database. We also labeled tumor samples as “high” or “low” according to whether the expression of FMNL2 was higher or lower than the median value among all samples, and the Kaplan Meier plot was made by R package.
Liver cancer cell lines Li-7, SNU-387 and SNU-182 were kindly provided by Stem Cell Bank, Chinese Academy of Sciences, and the human liver cell line HL-7702 was obtained from Beyotime (Shanghai, China). All cells were cultured in Roswell Park Memorial Institute (RPMI) 1640 medium supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin, and maintained in a humidified atmosphere containing 5% CO2 at 37 °C, which was considered as the normoxic condition. To simulate hypoxia, cells were cultured under 1% O2 hypoxic condition in a 3-gas incubator.
Total RNA was extracted from the cells using RNeasy Mini Kit (Qiagen, Germany). Complementary DNA (cDNA) synthesis was performed using the PrimeScript™ RT Master Mix (Takara, Japan). The qPCR was performed using the TB Green® Premix Ex Taq™ II (Takara, Japan) under standard conditions according to the manufacturer’s instructions and conducted with the LightCycler480 System (Roche, Switzerland). The primers were synthesized by Takara (Tokyo, Japan) and the β-actin gene was used as an endogenous control. The data were analysed using the 2-△△Ct method. The primers were listed in Table 1.
Cell proliferation was determined using CCK-8 (Dojindo, Japan). The 2 × 103 cells per well in 100 μL medium were seeded into 96-well plates and incubated under normoxia or hypoxia for indicated time period, then 10 μL CCK-8 solution was added into each well and incubated at 37 °C for an additional 2 h. Optical density (OD) value at 450 nm was measured.
Cell migration was determined using wound healing assay. The cells were plated to 100% density on 6-well plates and wounded with 1-mL pipette tip each well. The cells were continuously incubated with fresh medium without FBS under normoxia or hypoxia, and wound healing status of each group was observed and photographed at 0 h and 24 h.
Cell invasion was determined using transwell assay. Matrigel was dissolved at 4 °C overnight and diluted in medium without FBS at a volume ratio of 1:10, then 100 μL matrigel mixture was added into each tranwell chamber and placed at 37 °C for 2 h to allow the matrigel to solidify. The 1 × 105 cells in 100 μL medium without FBS were added to the upper transwell chamber and 600 μL medium with 10% FBS was added to the lower transwell chamber. After incubation under normoxia or hypoxia for 24 h, cells in the upper chamber were removed and the remaining cells were fixed with 4% paraformaldehyde, stained with 0.1% crystal violet, and photographed.
Cell apoptosis was determined using Annexin V-FITC Apoptosis Detection Kit (Solarbio, China). According to the manufacturer’s instructions, cells were collected and stained with Annexin V-FITC and PI, then analyzed by flow cytometry within 1 h.
Lentivirus that express full-length LINC00839 or LINC00839 shRNA, and their corresponding negative control (NC) lentivirus were constructed by Genechem (Shanghai, China). Lentivirus was infected into SNU-387 cell line respectively for 72 h and 1 μg/mL puromycin was utilized to select stably overexpressed or interfered SNU-387 cell lines.
The location of LINC00839 in SNU-387 cells was detected using FISH kit (GenePharma, China). Hybridization was carried out utilizing the Cy3 fluorescent dye conjugated probe of β-actin, LINC00839 or NC according to the protocol. The samples were placed under the confocal laser scanning microscope for observation.
The binding proteins of LINC00839 were collected using RNA pull down Kit (BersinBio, China). Biotinylated RNA probes were incubated with cell protein extract to form RNA–protein complexes. The complexes were separated from other components in the incubated solution through binding to streptavidin-labeled magnetic beads and the proteins were eluted to further analyzed.
All cells were lysed by RIPA Lysis Buffer (Beyotime, China). The proteins were separated by the sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene fluoride (PVDF) membranes (Millipore, America). The PVDF membranes were incubated with primary antibodies at 4 °C overnight, then incubated with secondary antibody at room temperature for 1 h, finally incubated with enhanced chemiluminescence (ECL) solution to expose. The antibodies were as followed: β-actin (Cell Signaling Technology, 1:1000), HIF-1α (Cell Signaling Technology, 1:1000), FMNL2 (Abcam, 1:5000).
The binding RNA molecules of FMNL2 were collected using RIP Kit (BersinBio, China). The cell extract was incubated with anti-FMNL2 antibody (Santa Cruz Biotechnology, America) or anti-IgG antibody at 4 °C overnight, then protein A/G magnetic beads were added into the samples to incubate at 4 °C for 1 h. Finally the RNA was eluted to subject to qPCR analysis.
Tissue microarray chips containing HCC tissues from 64 patients with HCC and adjacent nontumoral liver tissues from 26 patients with HCC were purchased from Shanghai Outdo Biotech (Shanghai, China), and the related clinicopathological and survival information were also provided.
The tissue samples came from National Human Genetic Resources Sharing Service Platform (2005DKA21300). Informed consent was obtained from each patient, and the study protocol was approved by the ethics committee of Shanghai Outdo Biotech Company. All research was performed in accordance with the Declaration of Helsinki.
All statistical analysis was conducted using SPSS 20.0 software. The experimental data was represented by mean ± standard deviation (SD) from at least three independent experiments. The expressions of FMNL2 in tumor and normal samples were compared using Welch’s t test. The survival analysis was performed using log-rank test. The intergroup comparison was carried out using Student’s t test. Differences with a P < 0.05 were considered statistically significant.
To confirm whether hypoxia promotes the malignant phenotypes of liver cancer cells, we cultured SNU-387 liver cancer cells under normoxic and hypoxic conditions respectively for 24 h. The CCK-8 assay, wound healing assay and transwell assay showed that hypoxia obviously accelerated liver cancer cell proliferation, migration and invasion, compared with that under normoxia (Fig. 1A–C). However, hypoxia did not affect liver cancer cell apoptosis (Supplementary Fig. 1). The WB analysis demonstrated significant up-regulation of HIF-1α in various liver cancer cell lines under hypoxia (Fig. 1D).
A total of 14,826 lncRNAs were extracted from TCGA database. We first identified 2296 differently expressed lncRNAs in liver cancer samples (n = 374) and normal samples (n = 50) (T vs. N, log2FoldChange > 1 and padj < 0.001). Then, we unearthed 13 differently expressed lncRNAs between patients with OS.event dead (n = 130) and OS.event alive (n = 235) (D vs. A, log2FoldChange > 1 and padj < 0.001). We screened two lncRNAs overlapped in two sets (Fig. 2A), LINC00839 and LINC00942, that were both up-regulated in liver cancer tissues and correlated with patient unfavorable survival (Table 2). Finally, we selected LINC00839 to carry out further research by co-expression analysis with HIF1A in LIHC (n = 374), whose coefficient-r was higher (Fig. 2B). We examined the expression of LINC00839 in liver cancer cell lines and normal liver cell line using qPCR to validate its expression pattern. The results showed that its expression levels in liver cancer cell lines were approximately three to eightfold higher than that in normal liver cell line (Fig. 2C). What's more, our data demonstrated that LINC00839 tended to increase in liver cancer cells under hypoxia compared with normoxia, with significant differences in SNU-387 and Li-7 liver cancer cells (Fig. 2D), which suggested that LINC00839 may be involved in the promoting effect of hypoxia on liver cancer progression.
To estimate the function of LINC00839 on the malignant phenotypes of liver cancer cells under hypoxia, we constructed LINC00839-overexpressing lentivirus and LINC00839 shRNA lentivirus, and then established stably overexpressed or interfered SNU-387 cell lines. We cultured cells from each group under hypoxia for 24 h and verified the expression of LINC00839 (Fig. 3A,B). First, the CCK-8 assay showed that when LINC00839 was overexpressed, the proliferation ability of SNU-387 cells was significantly increased under hypoxia, while interference of LINC00839 markedly decreased the proliferation ability of SNU-387 cells (Fig. 3C,D). Next, we performed wound healing assay and transwell assay to assess the migration and invasion abilities of SNU-387 cells under hypoxia. The results demonstrated that exogenous expression of LINC00839 dramatically facilitated migration and invasion in SNU-387 cells, in contrast, LINC00839 interference significantly inhibited the migration and invasion in SNU-387 cells (Fig. 3E–H).
To further investigate the oncogenic function of LINC00839 in liver cancer, we detected the location of LINC00839 in SNU-387 cells. The FISH assay displayed that comparing with β-actin, LINC00839 was located in both the nucleus and cytoplasm (Fig. 4).
We then performed RNA pull down assay using a biotin-labeled LINC00839 probe to identify potential binding proteins of LINC00839 in liver cancer. Mass spectrometry (MS) was used to analyzed the proteins pulled down by LINC00839 and the results were available via ProteomeXchange with identifier PXD034526. In order to predict the potential biological function and connection of the binding proteins of LINC00839, GO and KEGG pathway enrichment analysis were performed. According to the distribution of the binding proteins of LINC00839 in the GO enrichment analysis, the number of proteins was statistically analyzed with significant enrichment of each GO term to clarify protein function in biological process (BP), cellular component (CC) and molecular function (MF). It demonstrated that the proteins participated in various BP terms, especially metabolic process (Fig. 5A). And the most enriched CC term was related to membrane-bounded organelle while the most enriched MF terms were related to catalytic activity and small molecule binding (Fig. 5A). The KEGG pathway enrichment analysis showed that the related pathways were mainly involved in metabolic pathways and RNA transport (Fig. 5B).
The protein samples pulled down by LINC00839 probe and NC probe were separated via SDS-PAGE followed by silver staining. Compared with NC group, LINC00839 pulled down more proteins, especially a specific band around 130 kD (Fig. 6A). According to the MS results, we speculated it was FMNL2 that specifically bound to LINC00839, and used WB to examine. The results showed that FMNL2 was identified in the protein complexes pulled down by LINC00839 but not in those pulled down by NC probe (Fig. 6B). We compared the expression levels of FMNL2 in liver cancer and normal samples and its prognostic significance using the RNA-seq and clinical data from TCGA database. It was found that FMNL2 was highly expressed in liver cancer and elevated expression level of FMNL2 was associated with worse overall survival (OS) of liver cancer patients (Fig. 6C,D). Consistently, it was also observed that FMNL2 was significantly up-regulated in liver cancer cell lines compared with normal liver cell line (Fig. 6E). Besides, the protein expression levels of FMNL2 were determined and compared between the LINC00839 stably overexpressed and interfered cell lines. The results demonstrated a significantly increased FMNL2 expression in the oeLINC00839 group and a decline in FMNL2 in the shLINC00839 group (Fig. 6F). In addition, RIP assay was employed to enrich RNA molecules that bind to FMNL2, followed by examination of the enrichment of LINC00839 in each group through qPCR. However, the results showed that anti-FMNL2 antibody couldn’t enrich LINC00839, compared with IgG group (Supplementary Fig. 2).
The LINC00839 expression levels were further determined in 64 HCC tissue samples and 26 adjacent nontumoral liver tissue samples using qPCR. Unfortunately, LINC00839 expression data were obtained from only 37 HCC tissue samples and 15 nontumoral liver tissue samples. LINC00839 levels were substantially up-regulated in HCC (Fig. 7A). Furthermore, the correlations of LINC00839 levels with clinicopathological features of patients were investigated in the 37 HCC cases. The up-regulation of LINC00839 was positively associated with poor OS (Fig. 7B). The correlations of LINC00839 levels with other clinicopathological features, such as tumor size, number of lesions, and pathological grade were shown in Supplementary Fig. 3.
Approximately, 50–60% of patients with HCC are estimated to be exposed to systemic therapies in their lifespan. Systemic therapies based on immunecheckpoint inhibitor (ICI) were reported to increase overall survival and the quality of life of patients in the past 5 years, underscoring the key role of the tumor microenvironment in the progression of cancer. However, only 15–20% of responders were provided substantial clinical benefits. Understanding the interaction between cancer cells and their microenvironment will be crucial for developing new therapies and identifying biomarkers. The median oxygen partial pressure (pO2) in liver tumors is 6 mm Hg compared with 30 mm Hg in normal liver tissues, revealing that liver tumor oxygenation is heterogeneous and severely compromised. Hypoxia, one of the aberrant physical properties of the tumor microenvironment, can cause broad changes in the epigenome, which are beneficial to the phenotypic selection of hallmark capabilities, including the invasive growth capability of cancer cells. Even so, in preclinical studies, most of cancer cells are cultured in a relatively high ambient atmospheric oxygen concentration (approximately 21% oxygen concentration), which is usually presumed as normoxia, while reality tumors in vivo are usually in a hypoxic state (1–2% oxygen concentration). Recent studies demonstrated that in different oxygen concentration environments, the physiological state and the sensitivity to drugs of cancer cells were different. Therefore, evaluating cancer cells under hypoxia could more closely recapitulate their physiopathologic status in vivo microenvironment. Our results provided evidence that the malignant phenotypes were significantly enhanced in liver cancer cells under hypoxia, including proliferation, migration, and invasion. Besides, no significant change in cell apoptosis rate was occurred under hypoxia for 24 h, which was consistent with several previous studies, suggesting that there was hypoxia-adaptive response in liver cancer cell to attenuate apoptosis. For example, it was demonstrated that HCC cells survived under hypoxia by modulation of mitochondrial dynamics through activation of mitochondrial fission and mitophagy. Nevertheless, another study found that 10% oxygen concentration seemed to provide optimum conditions for long term growth in culture for human cell lines, while oxygen may be inhibitory for growth if its steady-state concentration is below or above optimal, which indicates the possibility that cumulative damage arising from oxygen and products of oxidative metabolism may limit the growth of cells. In fact, even small abnormalities in oxygen concentration can result in altered cellular function. The normoxic condition used in this study was not entirely consistent with the oxygen concentration in vivo, which may result in some results that were not sufficiently objective. Therefore, oxygen levels should be taken into account in genetic studies in vitro, which is likely to be critical to clearly defining and comparing what occurs in whole tissues or tumors. Recent largescale transcriptome sequencing approaches have identified thousands of lncRNAs that are differentially transcribed between normal tissues and tumors arising from the same organ. It is now recognized that lncRNAs are exquisitely regulated, and can identify clinically relevant cancer subtypes, predict tumor behavior and disease prognosis. Multiple therapeutic strategies have been developed to target lncRNAs. For instance, antisense oligonucleotides (ASOs) are under active investigation to be exploited as RNA inhibitors to treat various diseases. The development of RNA targeting therapeutics provides tremendous opportunities to modulate lncRNAs for anti-cancer purposes. Thus, further exploring the role of lncRNA in the occurrence and development of liver cancer contributes to develop ideal biomarkers for cancer diagnosis and potential drug targets. In this study, we screened two differently expressed lncRNAs with prognostic potential in TCGA database and focused our investigations on the role and potential mechanism of LINC00839 in liver cancer progression and metastasis under hypoxia. Our data revealed that LINC00839 was expressed at a higher level in liver cancer, and increased LINC00839 indicated poor prognosis, suggesting its tumor-promoting effect. LINC00839 was reported to be dysregulated in many kinds of cancer, including HCC. However, whether LINC00839 is involved in the hypoxic microenvironment of HCC remains largely elusive. The co-expression analysis revealed that LINC00839 expression levels exhibited a positive correlation with HIF1A expression levels and remarkably, LINC00839 further overexpression was also observed in liver cancer cells cultured under hypoxia. HIF-1 is the most crucial transcription factor under hypoxic stress and binds to the hypoxia response elements (HREs) within the promoter regions of target genes to coordinate cellular transcriptional response. The hypoxia-responsive lncRNAs often act as direct or indirect effectors of HIF-transcriptional cascade, and play pivotal roles in regulating hypoxic gene expression at chromatic, transcriptional, and post-transcriptional levels. The upstream regulator of LINC00839 is largely unknown. This remains the subject of further investigations. Functionally, this study demonstrated that LINC00839 promoted the proliferation, migration, and invasion of liver cancer cells under hypoxia, suggesting that LINC00839 may be a novel functional regulator of hypoxia-induced signaling. The key roles of lncRNA in gene regulation are linked with their specific subcellular localizations. The lncRNAs localized in the nucleus can modulate chromatin function and regulate the assembly and function of membraneless nuclear bodies, while localized in the cytoplasm, lncRNAs mostly regulate gene expression at the post-transcriptional level, and specific organelle-localized lncRNAs can participate in organelle function and metabolic regulation, such as mitochondrial oxidation and homeostasis. We observed that LINC00839 was localized in both the nucleus and cytoplasm, indicating its functional mechanisms are diverse. The interactions between lncRNAs and binding proteins are central to determine lncRNA functional effects. Therefore, we identified the binding proteins of LINC00839 and performed GO and KEGG pathway enrichment analysis. The proteins were found to be primarily associated with the metabolism and RNA transport. The capability to reprogram cellular metabolism in order to most effectively support neoplastic proliferation now is considered a core hallmark of cancer. LINC00839 responded to hypoxia and may function as a regulator of reprogramming energy metabolism. So the exact function of LINC00839 in liver cancer should be investigated further. FMNL2 has been disclosed to be required for migration and invasion by transformed cells and strongly implicated in driving tumorigenesis and metastasis of specific tumors. Bioinformatic analysis showed that FMNL2 was up-regulated in liver cancer and correlated with patient unfavorable survival..Our data revealed FMNL2 may be a functional partner of LINC00839. LINC00839 could pull down FMNL2, and overexpression of LINC00839 accumulated FMNL2. These experimental results indicated that LINC00839 may promote liver cancer progression by up-regulating FMNL2 expression. However, RIP assay demonstrated that anti-FMNL2 antibody did not enrich LINC00839. Previous studies have shown that FMNL2 binds directly many actin bundling proteins, such as fascin and cortactin, then facilitates cancer cell migration. We speculate that FMNL2 exists in the protein complexes pulled down by LINC00839 with other proteins participated in binding of LINC00839 and FMNL2. Further research is warranted to illustrate a more detail mechanism. We also found correlations of LINC00839 levels with clinicopathological features of patients. The higher LINC00839 expression was positively associated with worse prognosis and bigger tumor size. However, a study with a larger cohort is needed to verify the clinical significance of elevated LINC00839 expression. Additionally, this study lacks of in vivo experiments, which ought to be supplemented for a better understanding of the effects of LINC00839 on liver cancer. In conclusion, our study reported that LINC00839 was significantly up-regulated in liver cancer under hypoxia. LINC00839 promoted liver cancer cell proliferation, migration, and invasion under hypoxia. More importantly, LINC00839 may function as a regulator of reprogramming energy metabolism and can up-regulate the protein expression level of FMNL2. Hence, LINC00839 is implicated as a potential target for anticancer therapy.
Supplementary Information 1.Supplementary Information 2.Supplementary Information 3.Supplementary Information 4.Supplementary Legends. | true | true | true |
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PMC9637218 | Klaudia Klicka,Tomasz M. Grzywa,Alicja Klinke,Aleksandra Mielniczuk,Jarosław Wejman,Joanna Ostrowska,Agata Gondek,Paweł K. Włodarski | Decreased expression of miR-23b is associated with poor survival of endometrial cancer patients | 05-11-2022 | Gynaecological cancer,Gynaecological cancer,Biomarkers | Endometrial cancer (EC) is one of the most common types of cancer of the female reproductive system. EC is classified into two types (EC1 and EC2). MiRNAs are single-stranded RNA molecules that regulate gene expression posttranscriptionally. They have aberrant expression profiles in cancer, including EC. This study aimed to assess the level of expression of a panel of 16 miRNAs in both types of EC and healthy endometrium (HE). A total of 45 patients were enrolled into the study, 18 patients diagnosed with EC1, 12 diagnosed with EC2, and 15 HE controls. Tumor tissues or healthy endometrial tissues were dissected from archival formalin-fixed paraffin-embedded (FFPE) using laser capture microdissection (LCM). RNA was isolated from collected material and the expression of selected miRNAs was determined using the real-time qPCR. We found that miR-23b, miR-125b-5p, miR-199a-3p, miR-221-3p, and miR-451a were downregulated in EC in comparison to HE. Moreover, the expression of miR-34a-5p and miR-146-5p was higher in EC1 compared to EC2. Analysis of The Cancer Genome Atlas (TCGA) database confirmed decreased levels of miR-23b, miR-125b-5p, and miR-199a-3p in EC. Decreased miR-23b expression was associated with worse survival of EC patients. | Decreased expression of miR-23b is associated with poor survival of endometrial cancer patients
Endometrial cancer (EC) is one of the most common types of cancer of the female reproductive system. EC is classified into two types (EC1 and EC2). MiRNAs are single-stranded RNA molecules that regulate gene expression posttranscriptionally. They have aberrant expression profiles in cancer, including EC. This study aimed to assess the level of expression of a panel of 16 miRNAs in both types of EC and healthy endometrium (HE). A total of 45 patients were enrolled into the study, 18 patients diagnosed with EC1, 12 diagnosed with EC2, and 15 HE controls. Tumor tissues or healthy endometrial tissues were dissected from archival formalin-fixed paraffin-embedded (FFPE) using laser capture microdissection (LCM). RNA was isolated from collected material and the expression of selected miRNAs was determined using the real-time qPCR. We found that miR-23b, miR-125b-5p, miR-199a-3p, miR-221-3p, and miR-451a were downregulated in EC in comparison to HE. Moreover, the expression of miR-34a-5p and miR-146-5p was higher in EC1 compared to EC2. Analysis of The Cancer Genome Atlas (TCGA) database confirmed decreased levels of miR-23b, miR-125b-5p, and miR-199a-3p in EC. Decreased miR-23b expression was associated with worse survival of EC patients.
Endometrial cancer (EC) arises from the epithelial lining of the uterus (endometrium) and is the most common cancer of the female reproductive system in the USA. It is estimated that 65,950 new cases will be diagnosed in 2022, representing 3.5% of all new cancer cases in the USA, and the disease will be fatal to 12,550 patients, which is equivalent to 2.1% of all cancer deaths. The incidence and mortality are steadily increasing in the population which is associated with many factors, including the growing prevalence of obesity—one of the major risk factors for the development of the EC. The 5-year relative survival is estimated at 81.1%. EC can be classified based on two different classification systems. The traditional classification proposed in 1981 by Bokhman, divides EC into two types, where type 1 of EC (EC1) is defined as an estrogen-dependent tumor associated with endometrial hyperplasia, and type 2 of EC (EC2) as an estrogen-independent tumor which is associated with endometrial atrophy. Another classification of EC divides tumors based on the histopathological characteristics, where EC can be categorized into endometrioid carcinoma, serous carcinoma, clear-cell adenocarcinoma, carcinosarcoma, and other types. The Cancer Genome Atlas Research Network (TCGA) analysis revealed that EC can be classified based on their molecular features which enable better stratification of EC patients. MiRNAs (microRNAs) are endogenous, small single-stranded non-coding RNA molecules that regulate gene expression at the posttranscriptional level. They play a significant role in a broad range of biological processes such as cellular proliferation, differentiation, and apoptosis. The biogenesis of miRNAs involves multiple steps that occur at each step of the synthesis of the functional molecule. The expression of miRNAs is controlled by numerous transcription factors, two of which, p53 and c-Myc, appear to play a key role in this process. The dysregulation of miRNAs may result in the initiation of carcinogenesis, which can affect all hallmarks of cancer as defined by Hanahan and Weinberg, including replicative immortality, proliferative signaling, immune evasion, deactivation of growth suppressors, or inducing angiogenesis. Notably, miRNAs can act as both: oncogenes or tumor suppressors, as dictated by their target genes. Over the past decade, a body of evidence of dysregulation of miRNA expression in a variety of cancer types has been described. The focus on this subject by numerous research groups, including our, resulted in reports on the role of miRNAs as cancer biomarkers, as well as markers of cancer invasiveness and metastasis. This study aimed to further characterize the pathogenesis of EC by determining the level of miRNAs typically involved in cancer development. The study was conducted on microdissected tissue samples of both types of EC: EC1 and EC2 and the healthy endometrial tissue.
In this study, we enrolled 30 primary EC patients, previously untreated, and 15 control patients with healthy endometrium (HE) operated due to other gynecological pathologies (leiomyoma). The endometrium of these patients was histopathologically confirmed to be normal. To ensure that material obtained for miRNAs expression analysis contained only EC or HE tissue, we used Laser Capture Microdissection (LCM) to precisely dissect only specific fragments of tissue. From each FFPE sample of EC, only tumor tissue was dissected using LCM. In HE samples, only glandular endometrial tissue was dissected (Fig. 1). From the collected material we have determined the level of: miR-21-3p, miR-23b, miR-34a-5p, miR-96-5p, miR-125b-5p, miR-134-5p, miR-146-5p, miR-150-5p, miR-181b-5p, miR-182-5p, miR-199a-3p, miR-200b-3p, miR-211-3p, miR-221-3p, miR-410-3p, and miR-451a.
The analysis of 16 miRNAs revealed that the levels of expression of certain miRNAs were significantly downregulated in EC compared to HE. miR-23b was downregulated 4.54 times, (Fig. 2a, p < 0.0001), miR-125b-5p was downregulated 7.15 times (Fig. 2b, p = 0.0005), miR-199a-3p was downregulated 11.11 times (Fig. 2c, p < 0.0001), miR-221-3p was downregulated 4.54 times, (Fig. 2d, p = 0.0029), and miR-451a was downregulated 17.24 times (Fig. 2e, p < 0.0001). In contrast, there were no statistically significant differences between the expression of miR-21-3p, miR-34a-5p, miR-96-5p, miR-134-5p, miR-146-5p, miR-150-5p, miR-181b-5p, miR-182-5p, miR-200b-3p, miR-211-3p, and miR-410-3p in EC compared to HE (Fig. 3a–k, p > 0.05).
Next, we analyzed the differences in miRNAs expression profiles between EC1 and EC2. Out of 30 EC samples used in this study, 18 samples were EC1 type and 12 samples were EC2. The expression of two miRNAs was upregulated in EC1 compared to EC2. These were miR-34a-5p (Fig. 4a, upregulated 5.43 times, p = 0.031) and miR-146-5p (Fig. 4b, upregulated 3.50 times, p = 0.0479). There was no differences in expression of miR-21-3p, miR-23b, miR-96-5p, miR-125b-5p, miR-134-5p, miR-150-5p, miR-181b-5p, miR-182-5p, miR-199a-3p, miR-200b-3p, miR-211-3p, miR-221-3p, miR-410-3p, and miR-451a between EC1 and EC2 specimens (Fig. 5a–n, p > 0.05).
To confirm our findings, we analyzed the expression of investigated miRNAs in The Cancer Genome Atlas (TCGA) on the cohort of 418 EC tissues and 32 HE controls using OncomiR database. The levels of miR-23b (Fig. 6a, downregulated 1.87 times, p < 0.0001), miR-125b-5p (Fig. 6b, downregulated 2.34 times, p < 0.0001) miR-199a-3p (Fig. 6c, downregulated 1.26 times, p = 0.0067), and miR-221-3p (Fig. 6d, downregulated 1.45 times, p = 0.0110) were significantly downregulated in EC compared to HE. There were no differences in expression levels of miR-451a in EC compared to HE (Fig. 6e, p > 0.05).
Further, we checked whether the expression of downregulated miRNAs in the EC tissue correlated with the survival of patients. We analyzed the patients' survival and miRNAs level in TCGA EC cohort using OncoLnc. We found that worse survival was associated only with decreased expression of miR-23b (Fig. 7a, p = 0.0203). The decreased expressions of miR-125-5p, miR-199a-3p, miR-221-3p, or miR-451a were not correlated with the survival of EC patients (Fig. 7b–e, p > 0.05).
Decreased expression of miR-23b in EC tissue suggests its role as tumor-suppressor miRNA. To determine whether miR-23b may act as a tumor-suppressor, we transfected Ishikawa EC cells with synthetic mimic miR-23b, anti miR-23b (inhibitor of miR-23b), and corresponding scramble control and performed a proliferation assay. We observed that upregulation of miR-23b with mimic miR-23b potently suppressed the proliferation of Ishikawa cells (Fig. 8, p = 0.0065). Conversely, inhibition of miR-23b with anti miR-23b upregulated the proliferation of Ishikawa cells (Fig. 8, p = 0.0226). It suggests that miR-23b is a tumor suppressor miRNA in EC. Further, we analyzed The Encyclopedia of RNA Interactomes (ENCORI) to identify the enriched KEGG pathways of miR-23b targets (Table 1). It revealed that miR-23b regulates crucial pathways in cancer, including P53 signaling pathway, Wnt signaling pathway, mTOR pathway, cell cycle, and pathways regulating the actin cytoskeleton.
Aberrant expression of miRNAs has been observed in many types of cancers, including EC. Our recent systematic review revealed that miRNAs are crucial regulators of EC progression. By analyzing 115 articles, we identified 106 dysregulated miRNAs involved in the modulation of the EC invasiveness and metastasis. They regulate not only EC cells invasion and migration but also influence metastasis and tumor growth. Moreover, the expression of several miRNAs was correlated with clinical parameters of EC patients. In this study, we analyzed the expression of 16 miRNAs with a well-established role in tumor tissues of EC1 and EC2 as well as in healthy endometrium. We identified five miRNAs, miR-23b, miR-125b-5p, miR-199a-3p, miR-221-3p, and miR-451a, that are downregulated in EC compared to HE. MiR-23b plays contrary roles in different types of cancer but is a tumor suppressor miRNA in EC. MiR-23b targets metastasis-associated in colon cancer-1 (MACC1) inhibiting EC cells proliferation, invasion, and migration. Moreover, miR-23b suppresses EC metastasis in vivo in a murine model. The expression of miR-23b was downregulated in EC-derived cell lines compared to the normal fallopian epithelial cells It is also downregulated in FFPE EC tissues in the miRNA-profiling study. Moreover, the expression of miR-23b was lower in grade 3 EC1 compared to grade 1 tumors. We found that miR-23b is downregulated in EC tissues compared to HE regardless of the EC type. Additionally, analysis of the TCGA cohort revealed that decreased expression of miR-23b was correlated with the poor survival of EC patients. Upregulation of miR-23b in Ishikawa EC cells suppressed their proliferation while its inhibition potently upregulated it. Analysis of enriched pathways of miR-23b targets revealed that it regulates key signaling pathways in EC. Further studies are required to dissect the role of miR-23b as tumor suppressor miR in EC. MiR-125b-5p expression was downregulated in EC tissue in our study which was confirmed in the TCGA cohort. In EC, miR-125b-5p acts as a tumor suppressor miRNA and inhibits invasion of EC cells by directly targeting protooncogene ERBB2. However, the expression of miR-125b-5p was not correlated with patients' survival, even though the decreased level of miR-125b-5p was found to be associated with higher histological grade and myometrial invasion. MiR-199a-3p is another miRNA that was downregulated in EC compared to HE. Also, the increased level of miR-199a-3p was associated with better progression-free survival and overall survival of EC patients, however, it was not the case for TCGA cohort patients. Moreover, we found that miR-221-3p and miR-451a were downregulated in EC tissue compared to HE regardless of EC type. Notably, miR-221-3p was confirmed to be downregulated in the TCGA cohort of EC patients. Of note, miR-221 was identified as an oncomiRNA in other types of cancer, including breast cancer and liver cancer. Its overexpression promotes tumor cell migration, invasiveness, and proliferation. Our results suggest that the role of miR-221 is not critical for tumor growth in EC since its expression is decreased in this pathology. This is contrary to published data on other cancers, where miR-221 and miR-451a both act as tumor suppressor miRNAs, including ovarian cancer and cervical cancer. Further, we found that the expression of the majority of analyzed miRNAs was very similar in both types of EC. Only miR-34a-5p and miR-146-5p levels differed between both types, namely the expression of miR-34a-5p and miR-146-5p was upregulated in EC1 compared to EC2. There are discordant data concerning the level of expression of miR-34a-5p in EC compared to HE. Nonetheless, miR-34a-5p was identified to act as tumor suppressor miRNA in EC. It targets Notch1, L1CAM, MMSET and thus inhibits EC cells migration, invasion, and EMT in vitro as well as tumor growth in vivo. There are no studies regarding the role or the level of expression of miR-146-5p in EC. However, the expression of miR-146-5p was increased by estrogen in the plasma of rats with prostate cancer, which may suggest that upregulation of miR-146-5p may be related to estrogens in EC1. Notably, currently most of the studies on miRNA expression in EC are based on classical classification into two types. However, there is an effort to include molecular classification of EC patients in clinical practice. Therefore, studies are required to determine the profile of miRNAs expression in different types of EC. As miRNAs reveal different expression patterns in healthy and cancerous tissues, they have great potential to be diagnostically and prognostically valuable biomarkers as well as potential therapeutic targets. So far, a variety of miRNAs with different expression patterns in normal and malignant endometrial tissue have been identified. MiRNAs expression can be determined in FFPE tissues and this evaluation could be performed in addition to the standard histopathological examination. Moreover, using laser capture microdissection (LCM) it is possible to precisely dissect only tumor tissue without contamination of non-malignant cells surrounding tumor. In this study, we dissected only neoplastic tissues or glandular healthy endometrium, so the analyses were not disturbed by adjacent tissues. The main limitation of our study is a low number of specimens of EC tissue and a lack of complete clinical data of included patients. For this reason, we were unable to correlate the expression of analyzed miRNAs with e.g. survival of our patients. Therefore, further studies are required to assess the clinical relevance of studied miRNAs in EC, especially the role of miR-23b as a prognostic biomarker.
In this study, we found that miR-23b, miR-125b-5p, miR-199a-3p, miR-221-3p, and miR-451a were downregulated in endometrial cancer compared to healthy endometrium. Additionally, the expression of miR-34a-5p and miR-146-5p were higher in EC1 than in EC2. Decreased miR-23b expression is associated with worse survival of EC patients. There is a need for further studies assessing the potential clinical use of described miRNAs as biomarkers.
A total of 45 patients were enrolled into the study, 18 patients diagnosed with EC1, 12 diagnosed with EC2, and 15 HE controls. Tumor tissues or healthy endometrial tissues were dissected from archival formalin-fixed paraffin-embedded (FFPE) using laser capture microdissection (LCM). The FFPE samples have been obtained from the Department of Pathology, Medical Center of Postgraduate Education, Warsaw, Poland. Patients data are presented in Table 2. The study was conducted following the Declaration of Helsinki and was approved by the Bioethical Committee Medical University of Warsaw (AKBE/78/2021, 17 May 2021). The patient’s consent was waived due to the performed anonymization and retrospective character of the study.
Resected tumors were formalin-fixed and paraffin-embedded according to the standard protocol in the tissue processor. Thereafter the samples were cut on microtome and hematoxylin and eosin-stained for the pathologist examination. All samples were cut with a microtome to 10 µm slices and were mounted on glass slides with a drop of UltraPure DNAse/RNAse-free water. Then, samples were incubated in a fume hood at 56 °C for one hour to increase slices’ adherence. Mounted slices were hematoxylin and eosin-stained according to the standard protocol in a set of stains, alcohol solutions, and xylene. Slides were immediately subjected to LCM.
Stained and dehydrated sections of EC or HE were subjected to LCM-aided dissection of regions containing only neoplastic tissues or glandular healthy endometrium. Approximately 10 mm2 of each region was marked to dissect with LCM system (PALM Robo, Zeiss, Germany). These regions were selected by a board-certified pathologist. Each LCM was preceded by optimization of LCP energy and spot distance to provide a full dissection of marked areas. LCM was performed under the following conditions: LCP energy—80–90, LCP spot distance—25 μm, magnification—5×, tissue collected in 20 μl of Digestion Buffer (Norgen Biotek) in 500 μl sterile PCR-tube cap. Caps were sealed back with tubes, centrifuged briefly, and placed on wet ice until further steps.
Norgen Biotek FFPE RNA/DNA Purification Plus Kit was used for RNA isolation according to the manufacturer guidelines. RNA was eluted with 30 µl ultrapure, molecular-grade water, and stored at − 80 °C until the next steps. The purity and quantity of isolated RNA were assessed by the absorbance measurements at wavelengths of 260/280 nm using the NanoDrop2000 spectrophotometer (Thermo Fisher Scientific). Samples with 260/280 ratios between 1.8 and 2.1 were used for further analysis. RNA was then subjected to reverse transcription using Mir-X miRNA FirstStrand Synthesis (Takara, Clontech) followed by real-time qPCR using SYBR qRT-PCR (Thermo Fisher Scientific). Primers sequences used in the study are presented in Table 3. U6 (Takara, Clontech) was used as an endogenous control for the analysis of microRNA expression. The 2−ΔCt method was used to calculate relative expression using the mean Ct values of target genes and the control.
Analysis of TCGA database of Uterine Corpus Endometrial Carcinoma (EC) cohort was performed using OncomiR database. Analysis of the association of the miRNAs expression and EC patients' survival from the TCGA cohort (n = 533) was performed using OncoLnc. Patients were divided based on miRNA level low: high 75:25. Analysis of enriched signaling pathways was performed using starBase.
EC Ishikawa cells were cultured in Dulbecco's Modified Eagle Medium (DMEM) supplemented with heat-inactivated 10% (v/v) fetal bovine serum (FBS, Gibco), 100 U/ml penicillin and 100 μg/ml streptomycin (Sigma-Aldrich) at 37 °C in an atmosphere of 5% CO2 in the air. Cells were tested for Mycoplasma contamination using PCR technique and were confirmed to be negative. All transfections were performed using DharmaFECT (ThermoFisher) according to the manufacturer's protocol. miR-23b mimic (assay ID: MC12931), anti miR-23b (assay ID: MH12931), mimic miR-scramble (miR-scr, miRNA Mimic Negative Control, catalog number: 4464058), and anti miR-scramble (anti-miR scr, miRNA Inhibitor, Negative Control, catalog number: 4464078) were obtained from Invitrogen mirVan (Thermo Fisher Scientific). miRs were used at a final concentration of 50 nM. The efficiency of the transfection was determined by RT-qPCR method. For proliferation assay, 1 × 105 cells/well were seeded in 12-well plates 24 h after transfection and were incubated for 48 h. Then, cells were fixed with 4% PFA and stained with 0.1% crystal violet. Cells were photographed using Nikon Ti-U. The photos were analyzed using ImageJ (National Institutes of Health, Bethesda MD, USA) and ColonyArea plugin.
Data were collected and processed with Excel 2016 (Microsoft, USA). Statistical analyses were conducted with GraphPad Prism 8.4.3 (GraphPad Software Inc.) using the Mann–Whitney and log-rank tests. Data distribution was tested using the Shapiro–Wilk test. All values in Figs. 2, 3, 4 and 5 are represented as median and 95% CI. Data in Fig. 6. is presented as a violin plot with the median indicated as dashed line and quartiles indicated as solid lines. Data in Fig. 7 is presented as a Kaplan–Meier plot. A p value of < 0.05 was considered statistically significant. | true | true | true |
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PMC9637816 | Luis Alberto Bravo-Vázquez,Natalia Frías-Reid,Ana Gabriela Ramos-Delgado,Sofía Madeline Osorio-Pérez,Hania Ruth Zlotnik-Chávez,Surajit Pathak,Antara Banerjee,Anindya Bandyopadhyay,Asim K. Duttaroy,Sujay Paul | MicroRNAs and long non-coding RNAs in pancreatic cancer: From epigenetics to potential clinical applications | 01-11-2022 | Pancreatic cancer,MicroRNAs,lncRNAs,Biomarker,Therapeutics | Highlights • Both miRNA and lncRNA expression is dysregulated in pancreatic cancer. • Altered miRNA and lncRNA expression is related to pancreatic cancer progression. • miRNAs and lncRNAs affect tumor suppressor genes, oncogenes, and signaling pathways. • miRNAs and lncRNAs are prospective theragnostic targets for pancreatic cancer. • Further studies are required to boost the development of ncRNA-based therapeutics. | MicroRNAs and long non-coding RNAs in pancreatic cancer: From epigenetics to potential clinical applications
• Both miRNA and lncRNA expression is dysregulated in pancreatic cancer. • Altered miRNA and lncRNA expression is related to pancreatic cancer progression. • miRNAs and lncRNAs affect tumor suppressor genes, oncogenes, and signaling pathways. • miRNAs and lncRNAs are prospective theragnostic targets for pancreatic cancer. • Further studies are required to boost the development of ncRNA-based therapeutics.
Abbreviations(PFKFB3)6-Phosphofructo-2-Kinase/Fructose-2,6-Biphosphatase 3(ABHD11-AS1)ABHD11 antisense RNA 1(ANRIL)Antisense Non-Coding RNA in the INK4 Locus(AMF)Autocrine Motility Factor(ATG5)Autophagy related 5(AXIN2)Axis Inhibition Protein 2(BTG2)B-cell Translocation Gene 2(BLACAT1)Bladder Cancer Associated Transcript 1(BANCR)BRAF-Activated Non-Protein Coding RNA(BRCA2)Breast Cancer Type 2 protein(CASC2)Cancer Susceptibility Candidate 2(CA 19–9 and CA 199)Carbohydrate antigen 19–9(CAV1)Caveolin 1(cfDNA)Cell-free DNA(CERS6-AS1)Ceramide Synthase 6 Antisense RNA 1(CRNDE)Colorectal Neoplasia Differentially Expressed(HOST2)Competing Endogenous lncRNA 2 for microRNA let-7b(CPS1-IT1)CPS1 Intronic Transcript 1(CDKN2D)Cyclin Dependent Kinase Inhibitor 2D(CDKN2A)Cyclin Dependent Kinase inhibitor 2A(DLEU2L)Deleted In Lymphocytic Leukemia 2 Like(DNA)Deoxyribonucleic Acid(DANCR)Differentiation Antagonizing Non-Protein Coding RNA(ID4)DNA-Binding Protein Inhibition ID-4(DUXAP8)Double Homeobox A Pseudogene 8(E2F3)E2F Transcription Factor 3(PKM2)Enzyme Pyruvate Kinase M2(EIF5A2)Eukaryotic Translation Initiation Factor 5A2(ERK2)Extracellular Signal-Regulated Kinases 1(FEZF1-AS1)FEZ family Zinc Finger 1-Antisense RNA 1(FGD5-AS1)FGD5 Antisense RNA 1(FDA)Food and Drug Administration(FOXA1)Forkhead Box A1(GATA3-AS1)GATA Binding Protein 3 Antisense RNA 1(GLUT 1 and GLUT3)Glucose Transporter(GAPDH)Glyceraldehyde 3-Phosphaste Dehydrogenase(GOLM1)Golgi Membrane Protein 1(GAB1)GRB2-Associated Binding Protein 1(GAS5)Growth Arrest Specific 5(H19)H19 Imprinted Maternally Expressed Transcript(H19)H19 maternally expressed transcript(HK1 and HK2)Hexokinase(HMGB1)High Mobility Group Box 1(HCP5)HLA Complex P5(HOTAIR)HOX Transcript Antisense RNA(HOTTIP)HOXA Distal Transcript Antisense RNA(HIF-1α)Hypoxia Inducible Factor 1-Alpha(HIF1A-AS1)Hypoxia Inducible Factor 1-Alpha Antisense RNA 1(HIF-1α)Hypoxia-Inducible Factor 1-Alpha(ING5)Inhibitor of Growth Protein 5(JAK)Janus Kinase(KRAS)Kirsten Rat Sarcoma Virus(KFL12)Kruppel-like Factor 12(LDHA)Lactate Dehydrogenase A(LRP6)LDL Receptor Related Protein 6(ELAVL1)Like RNA Binding Protein 1(LIN28B)LIN-28 Homolog B(LINC01111, LINC00671, LINC00857, LINC01094)Long Intergenic Non-Protein Coding RNA(lncRNAs)Long non-coding RNAs(MEG3)Maternally Expressed Gene 3(MMP16)Matrix Metallopeptidase 15(mRNA)Messenger RNA(MALAT-1)Metastasis Associated Lung Adenocarcinoma Transcript 1(miRNAs)MicroRNAs(MAPK)Mitogen Activated Protein Kinase(MEK)Mitogen/extracellular Signal-related Kinase(MEK2)Dual Specificity Mitogen-Activated Protein Kinase 2(MAP3K10)Mitogen-Activated Protein Kinase 10(SMAD4)Mother Against Decapentaplegic Homolog 4(MEF2C)Myocyte Enhancer Factor 2C(NORAD)Non-coding RNA Activated by DNA Damage(ncRNAs)Non-coding RNAs(NOTCH1)Notch homolog 1(NF-κB)Nuclear Factor Kappa B(PC)Pancreatic Cancer(PDAC)Pancreatic Ductal Adenocarcinoma(PanIN)Pancreatic Intraepithelial Neoplasia(PARP)Poly-ADP-ribose Polymerase(PTEN)Phosphatase and Tensin Homolog(PGI)Phosphoglucose Isomerase(PI3K)Phosphoinositide 3-Kinases(PVT1)Plasmacytoma Variant Translocation 1(AKT)Protein Kinase B(PIM1 and PIM2)Proto-Oncogene Serine/Threonine-Protein Kinase(PACER)PTGS2 Antisense NFKB1 Complex-Mediated Expression Regulator RNA(RAF1)RAF-1 Proto-Oncogene, Serine/Threonine Kinase(Ras)Rat Sarcoma Virus(qRT-PCR)Real-Time Quantitative Reverse Transcription PCR(ROR)Retinoic Acid-Related Orphan Receptors(NOB1)RNA-Binding Protein NOB1(SAGE)Serial Analysis of Gene Expression(TOR)Serine/Threonine Protein Kinase(STAT)Signal Transducer and Activator of Transcription(SNHG7, SNHG12 and SNHG8)Small Nucleolar RNA Host Gene(SOX9)SRY-Box Transcription Factor 9(STARD13)StAR Related Lipid Transfer Domain Containing 13(TUG1)Taurin Up-Regulated 1(TEX10)Testis-Expressed Protein 10(THRIL)TNF and HNRNPL Related Immunoregulatory long non-coding RNA(TRIAP1)TP53-Regulated Inhibition of Apoptosis 1(TP73-AS1)TP73 Antisense RNA 1(TCF4)Transcription Factor 4(TGFBR3)Transforming Growth Factor Beta Receptor 3(TGF-β)Transforming Growth Factor Beta(TRAF6)Tumor Necrosis Factor Receptor-Associated Factor 6(TP53)Tumor Protein P53(TP53INP1)Tumor Protein p53-Inducible Nuclear Protein 1(TSLNC8)Tumor Suppressive lncRNA on Chromosome 8p12(YWHGA)Tyrosine 3-Monooxygenase/Tryptophan 5-Monooxygenase Activation Protein Zeta(YB1)Y-Box Binding Protein 1
Pancreatic cancer (PC) is considered the 12th most prevalent cancer with the 7th highest death rate worldwide [1], [2], [3]. According to the literature, in 2030, PC will have the second-highest cancer mortality rate around the world [4], and by 2040 the total worldwide incidence of PC will have increased by 30% [1]. One of the main symptoms that is significantly linked with PC is jaundice [5]; however, early diagnosis and prevention of PC represent two major concerns since patients hardly manifest symptoms, and there is still a lack of specific markers for the accurate detection of PC tumors [6]. Besides, many PC symptoms such as abdominal pain, diarrhea, constipation, and vomiting are also associated with other gastrointestinal diseases [5]. Consequently, most PC diagnoses occur only at an advanced stage [7], and hence novel biomarkers could be prospective tools for the early and precise diagnosis of this type of cancer [8], [9], [10], [11]. In addition, a number of studies have demonstrated that PC patients suffer from depression, anxiety, fatigue, sleep problems, and decreased quality of life; some of these problems are also attributed to the caregivers of such patients [12], [13], [14]. It is worth mentioning that the genetic background of each individual has a significant impact on the tendency of developing PC [15], [16], [17], [18]. In fact, genetic mutations in several proteins, such as CDKN2A, TP53, SMAD4, KRAS, BRCA2, produce abnormalities in the ductal cells of the pancreas, thus triggering the growth of papillary-like structures that can be transformed into a non-invasive microscopic preneoplastic lesion called pancreatic intraepithelial neoplasia (PanIN) [2,[19], [20], [21], [22]]. PanIN is a well-defined precursor of PC that might play a significant role in the progression of pancreatitis and can damage the cell repair cycles leading to the propagation of the neoplastic lesion process [2,23]. PanIN is divided into four grades based on the degree of both architectural and cytological alterations in pancreatic ducts: PanIN-1A (lowest grade), PanIN-1B and PanIN-2 (intermediate grade), and PanIN 3 (highest grade) [24]. Apart from genetic modifications, epigenetic factors, e.g., DNA methylation, RNA methylation, histone modifications, and non-coding RNAs (ncRNAs), promote PC initiation and progression [25], [26], [27]. DNA methylation is a DNA methyltransferase-mediated process that modifies cytosine residues and alters gene expression by adding a methyl side group, creating 5-methylcytosines [28]. Aberrant hypermethylation and hypomethylation have been shown to predispose patients to PC. Multiple genes show a hypomethylation status in PC, while hypermethylation is usually implicated in the downregulation of tumor suppressor genes, which leads to PC development [29]. Similarly, it has been noticed that the methylation of diverse microRNAs (miRNAs) and long non-coding RNAs (lncRNAs), such as miR‑29b‑3p, LINC00901, ANRIL, and LIFR-AS1 contributes to PC progression [30], [31], [32], [33]. Innovative protocols for the isolation and analysis of tumor-derived fraction of circulating cell-free DNA (cfDNA), such as liquid biopsies, have an optimistic potential for early diagnosis of cancers via detecting methylations, point mutations, gene fusions, among other cancer-related signatures [34], [35], [36]. In the pharmacological context, several types of therapeutic approaches for PC have been explored over the last decade, such as poly-ADP-ribose polymerase (PARP) inhibitors (e.g., olaparib, veliparib, rucaparib, and talazoparib), mitogen/extracellular signal-related kinase (MEK) inhibitors, EGFR inhibitors, KRAS targeting agents, JAK/STAT inhibitors, hydroxychloroquine, immunotherapy, and electrochemotherapy. Nonetheless, the number of patients who may benefit from these therapies is constrained due to the particular attributes of the pancreatic tumor microenvironment, i.e., unique immune structure and cell-to-cell communication [37], [38], [39], [40]. Thus, the current leading treatment for advanced PC is surgery followed by adjuvant chemotherapy; however, solely a few patients are identified with locally resectable, non-metastatic illness [41,42]. Accordingly, the accurate comprehension of the epigenetic regulation of the molecular mechanisms underlying PC pathogenesis might represent a novel source of next-generation molecular medicine and diagnostics for this disease [43,44]. In this regard, miRNAs and lncRNAs have been projected as encouraging epigenetic clinical targets for PC [45], [46], [47], [48], [49], [50], [51]. Both miRNAs and lncRNAs can be found in specialized tissues [52,53] as well as in other types of biological samples (e.g., blood, plasma, serum, urine, exosomes, and stool), in the form of circulating miRNAs and lncRNAs [54], [55], [56], [57]. Significantly, exosomal miRNAs and lncRNAs are involved in homeostasis regulation because they function as mediators of cell-to-cell communication. Additionally, their altered expression can cause tissue dysfunction, aging, and a myriad of diseases [58]. It has been evidenced that these exosomal ncRNAs play a fundamental role in the regulation of a wide range of cancer-associated events, such as angiogenesis, metastasis, drug resistance, and immune escape [59]. Besides, since exosomal ncRNAs are highly stable and protected from enzymatic and chemical degradation by the membrane of exosomes, they are prospective biomarkers for different types of cancer [60], [61], [62]. Such is the case of exosomal miRNAs miR‑21 and miR‑210, which have been suggested as candidate biomarkers for the diagnosis of PC [63]. Therefore, the quantification of the expression levels of these ncRNAs in such samples leads to the identification of dysregulated (upregulated or downregulated) miRNAs and/or lncRNAs in multiple pathological conditions [64,65]. In fact, miRNAs and lncRNAs might serve as prognostic, diagnostic, and predictive biomarkers for PC [66]. Consistently, analyzing their expression profile in patients with suspected PC could be consolidated as a molecular diagnostic method in the near future. In addition, deciphering which miRNAs and lncRNAs are crucially implicated in PC development and progression might help to lay the groundwork for the development of ncRNA-based drugs aimed to re-establish the physiological levels of miRNAs and lncRNAs [67,68]. Given this, a variety of total RNA isolation protocols have been devised to investigate the physiological and pathological roles of miRNAs and lncRNAs. For example, early published protocol for miRNA isolation based on guanidine thiocyanate-phenol-chloroform extraction followed by RNA precipitation, commonly performed with TRIzol reagent (Thermo Fisher Scientific, Waltham, MA, USA) [69,70]. Nonetheless, high contamination levels could be present when using the aforesaid method and miRNAs with small guanine-cytosine content may be lost during phenol-chloroform extraction. As a consequence, more efficient procedures for small RNAs isolation have been conceived, which consist of placing the aqueous phase obtained during the phenol-chloroform extraction on an RNA adsorption column and, subsequently, wash and elute the RNA [69]. Two examples of well-known column-based miRNA isolation kits are: miRVana (Thermo Fisher Scientific) and miRNeasy (Qiagen, Hilden, Germany) [71]. Solid-phase isolation techniques have also been designed to separate miRNAs from clinical samples. The rationale for those protocols is based on the fact that miRNAs can be retained by suitable solid sorbents (e.g., fibers or membranes) [70]. Isolate II (Bioline, London, UK) and Norgen Total (Norgen Biotek, Thorold, ON, Canada) are representative examples of solid-phase-based total RNA isolation kits for miRNA extraction [71]. Similar to miRNA isolation protocol, both guanidine thiocyanate-phenol-chloroform and column-based systems are the mainstay protocols for lncRNA isolation. Despite this, lncRNA samples are often contaminated with organic and phenolic compounds when using TRIzol reagent, so column-based approaches are the most appropriate for lncRNA isolation [72,73]. Additionally, methods based on immunoprecipitation are frequently used to enrich lncRNAs linked to particular proteins [74]. At the end, miRNA and lncRNA expression profiling is performed using different techniques and tools, such as microarrays, qRT-PCR, Serial Analysis of Gene Expression (SAGE), RNA sequencing, and northern blots [75,76]. The general overview of the design of miRNA- and lncRNA-based clinical approaches for PC is depicted in Fig. 1. MiRNAs are short (20–24 nucleotides), endogenous, highly conserved RNA molecules that regulate gene expression post-transcriptionally. The discovery of miRNAs occurred in 1993 when Lee et al. [77] studied the regulatory mechanisms of LIN-14 protein expression in the nematode Caenorhabditis elegans. Subsequently, this finding led to the identification of miRNAs in a wide range of species, including Homo sapiens, Mus musculus, Drosophila melanogaster, and Arabidopsis thaliana [78], [79], [80]. Besides, over the last decades, the altered expression of miRNAs has been found to be associated with many multifactorial human diseases, such as neuropsychiatric disorders, neurodegenerative diseases, parasitic diseases, hair loss, diabetes, cancer, osteoporosis, COVID-19, pediatric diseases [81], [82], [83], [84], [85], [86], [87], [88], [89]. Further, these master regulators of gene expression have been demonstrated to be involved in cystic diseases, irritable bowel syndrome, smoking-induced chronic diseases, gastrointestinal cancers, hepatocellular carcinoma, and regulation of stem cell populations [90], [91], [92], [93], [94], [95]. As a matter of fact, let-7 family, miR-200 family, miR-21, miR-155, miR-27a, miR-205, miR-34a, miR-106a, miR-506, miR-23a/b, miR-216a, miR-29a/c, miR-221, miR-372, miR-212, miR-196b, miR-191, miR-182, miR-375, miR-142, miR-455, miR-15a, miR-202, miR-300, amongst other miRNAs, own a remarkable potential as both diagnostic and therapeutic tools for PC [20,45,96,97]. On the other hand, lncRNAs are a class of endogenic ncRNAs whose average size is longer than 200 nucleotides. Interestingly, lncRNAs can interact with miRNAs, mRNAs, DNA, and proteins, thus regulating gene expression at multiple levels, i.e., epigenetically, post-transcriptionally, translationally, and post-translationally [98,99]. Remarkably, the study of lncRNAs has allowed a more in-depth understanding of the etiological principles of a variety of genetic diseases since their discovery [100,101]. Indeed, since lncRNAs are implicated in a vast range of biological functions, their dysregulation has been related to different human pathologies, including ischemic stroke, cancer, viral diseases, diabetes, and neurodegenerative diseases [86,[102], [103], [104], [105], [106]]. Under this premise, relevant studies have demonstrated that several lncRNAs, such as HOTTIP, RP11–567G11.1, HOTAIR, MALAT-1, H19, GAS5, FEZF1-AS1, BANCR, LINC01111, TUG1, and DUXAP8 have a noteworthy potential as theragnostic targets for PC [46,[107], [108], [109]]. Further, aberrant ncRNA biogenesis are commonly found in human cancers due to mutations or dysregulations that affect the components of such pathways [76,110]. According to the previous information, miRNAs and lncRNAs own an emerging potential as biomarkers and therapeutic targets for PC. Therefore, throughout this review, we present a general overview concerning the most recent experimental evidence of the functional implications of miRNAs and lncRNAs within the molecular pathophysiology of PC to shed light on their clinical significance. Furthermore, we discuss several challenges that should be addressed in forthcoming studies to design innovative ncRNA-mediated medications for PC.
PC is associated with multiple molecular pathways that stimulate its accelerated progression and intricate complexity. The inactivation of tumor suppressor genes, activation of oncogenes, and dysregulation of signaling pathways lead not only to the development of clinical symptoms [111], but also to the acquisition of chemoresistance in PC cells as well [112,113]. Particularly, studies have shown that miRNAs play a relevant role in pancreatic tumorigenesis since these RNA molecules regulate several cellular processes that comprise the expression and function of genes involved in cell proliferation, antitumor immune response, apoptosis, invasion, metastasis, and drug resistance [114]. Accordingly, the identification of abnormally expressed miRNAs related to PC pathological events could allow designing novel diagnostic protocols and medications for this disease. As a matter of fact, it has been stated that RNA-centered tools for prognostic, diagnostic, and predictive aims will experiment with remarkable growth in the following years, characterized by a forecasted investment of approximately 6.8 billion dollars by 2028 [115]. Even so, there are several challenges that must be resolved for these approaches to be successful in the future. Firstly, in the case of miRNAs as biomarkers, preservation of miRNA purity and integrity represents a major constraint during the isolation and purification of these nucleic acids [116]. In the same sense, storage conditions and time must be precisely defined to prevent changes in miRNA composition before their analysis. As well, the source of the sample is another factor that should be considered when studying miRNA implications in cancer since miRNA expression varies between samples of the same patient (e.g., serum and plasma), hence, the implementation of data normalization with internal controls should be mandatory in these assays [116]. Secondly, in relation to the use of miRNAs as therapeutic agents, a number of hurdles, including efficient delivery systems, administration routes, effective dosages, off-target and immunostimulatory effects, and toxicity, must be aborded in forthcoming studies so that miRNA-based drugs for cancer can enter the pharmaceutical market [117,118].
To begin with, Zou et al. [119] investigated serum-derived exosomes and tissues from PC patients and noticed that let-7b-5p, miR-19a-3p, miR-19b-3p, miR-25–3p, miR-192–5p, and miR-223–3p were significantly upregulated in such samples. Similarly, the outcomes of another study elucidated that miR‑126‑3p, miR‑139‑5p, miR‑424‑5p, miR‑454‑3p, miR‑1271‑5p, miR‑3613‑5p, and miR‑5586‑5p were downregulated in tissues derived from patients with an early stage of PC. Besides, this miRNA signature might be applied to classify PC patients into low-risk and high-risk groups [120]. As a result, all these miRNAs are proposed as biomarkers for the timely identification of PC; however, more studies are required to confirm their reliability in PC diagnostic. Later, miR-1290 and miR-1246 were found to be overexpressed in the serum of PC patients [121]; indeed, higher expression of miR-1290 was more frequent in patients at stage III and IV of PC, indicating that they were independent risk factors for PC and might be useful as biomarkers for PC diagnosis, along with the antigen CA19–9. Nonetheless, additional studies are required to validate the prognostic value of these miRNAs for the PC [121]. An investigation in which serum samples of PC patients were examined led to discover 13 PC signature miRNAs that can help to distinguish between PC patients and healthy individuals (i.e., miR-125a-3p, miR-125b-1–3p, miR-204–3p, miR-575, miR-1469, miR-4294, miR-4476, miR-4792, miR-6075, miR-6729–5p, miR-6820–5p, miR-6836–3p, and miR-6893–5p) [122]. The same study identified 432 serum miRNAs that could indicate whether a PC patient is operable (can go through surgery). Hence, these miRNAs profiles could be noteworthy to evaluate PC patients' surgical feasibility and develop better diagnoses for this type of cancer [122]. Likewise, Shams et al. [123] combined diverse serum expression profiles of miRNAs to identify the most relevant miRNA signatures for PC diagnosis. Subsequently, miR-92a-5p, miR-125a-3p, and miR-4530 were found to be the most significantly downregulated in PC. While, a substantial upregulation of miR-642b-3p, miR-663a, miR-1246, miR-1469, miR-5100, and miR-8073 was observed. Additional analysis revealed miR-125a-3p, miR-642b-3p, and miR-5100 as the most potent biomarkers for PC diagnosis. However, this dataset does not support the immediate clinical use of these biomarkers until further validation [123]. In addition, Lee et al. [124] analyzed serum samples of PC patients, reporting 39 circulating miRNAs as PC-specific diagnostic markers; among those, 15 miRNAs had already been reported as PC indicators in previous studies. qRT-PCR analysis revealed that miR-155–5p, miR-661, miR-4703–5p, and miR-7154–5p were significantly downregulated in the PC samples, whereas let-7b-5p, miR-22–3p, miR-4486, and miR-5100 were significantly upregulated; hence, these miRNAs may be applied for the early diagnosis of PC. Subsequently, a microarray-based analysis evidenced that hsa-miR-210 is significantly upregulated in PC, whereas hsa-miR-216a/b, hsa-miR-217, hsa-miR-375, and hsa-miR-634 are downregulated. Therefore, these miRNAs represent potential biomarkers for PC diagnosis [125]. Recently, Hata et al. [126] reported that miR-593–3p is significantly upregulated in peritoneal lavage fluids of patients with PC. Increased expression of this miRNA is correlated with the occurrence of micrometastasis even in patients with localized PC. Accordingly, miR-593–3p could be a promising prognostic indicator for PC patients subjected to staging laparoscopy.
Interestingly, an investigation in which PC cells were treated with the chemotherapeutic drug doxorubicin displayed downregulation of miR-137 and autophagy induction, while induced overexpression of miR-137 promoted the therapeutic effect of doxorubicin activating cell apoptosis, diminishing cell survival, and blocking autophagy. The positive effect of miR-137 was linked to the fact that such miRNA regulates the expression of ATG5, a protein implicated in autophagy. Since autophagy may be related to chemoresistance, miR-137 could be used in combination with doxorubicin to treat PC [127]. In a similar study, He et al. [128] suggested that miR-137 is a prospective clinical target for PC since it reduces PC stemness and tumorigenicity by targeting KLF12 in human PC cell lines, thus inhibiting β-catenin nuclear translocation as well as Wnt signaling activation. Fang et al. [129] detected that miR-106b, miR-125b, miR-148a, and miR-320a/c were upregulated in cancer-associated fibroblasts, while miR-29a, miR-378d, miR-422a, and miR-1285 were downregulated after treatment with gemcitabine (a chemotherapeutic agent for PC). They also demonstrated that miR-106b promotes gemcitabine resistance in PC via targeting TP53INP1, which is associated with oncogenesis and tumorigenesis. As a result, miR-106b could be a promising therapeutic target for gemcitabine resistance management in PC. In 2020, Wu et al. [130] suggested that the reduction of eIF5A2 expression to suppress autophagy and increase apoptosis in pancreatic ductal adenocarcinoma (PDAC) in vivo via plectin-1/miR-9 nanocomplexes greatly improves the anti-cancer impact of doxorubicin. Additionally, Meng et al. [131] investigated the miRNA profile in gemcitabine resistance pancreatic ductal adenocarcinoma (PDCA), and their results revealed that miR-146a-5p was significantly downregulated in PDCA tissues. Further experiments showed that miR-146a-5p inhibited PDAC cell growth and made PDAC cells more susceptible to gemcitabine treatment by targeting TRAF6, a signal transducer involved in regulating inflammation and immunity. MiR-146a-5p was also shown to suppress the miR-146a-5p/TRAF6/NF-κB p65 axis, which regulates PDAC cell proliferation and chemoresistance [131]. Furthermore, Panebianco et al. [132] demonstrated that overexpression of miR-217 (downregulated in human PC) boosts PC sensitivity to gemcitabine by inhibiting cell cycle progression in PDAC cells, thus representing a remarkable therapeutic target for PC. Liu et al. [133] noticed that miR-3662 is downregulated in PDAC cell lines and tissues. Besides, they also found that miR-3662 suppresses gemcitabine resistance and aerobic glycolysis in PDAC cells by regulating the expression of HIF-1α, which is related to chemoresistance and tumorigenesis. Hence, co-delivery of this miRNA with gemcitabine could be a promising approach to overcome PC gemcitabine resistance. Xu and Zhang [134] detected that miR-299–3p is downregulated in PC cells and tissues. Mechanistically, the downregulation of this miRNA occurs due to the high levels of expression of TUG1, which functions as a molecular sponge of miR-299–3p. TUG1 is an oncogene in many types of cancer that promotes cell proliferation, invasion, migration, and epithelial-mesenchymal transition, and in PC tissues, its expression was negatively associated with miR-299–3p expression. Moreover, researchers noticed that the inhibition of the interaction TUG1/miR-299–3p repressed PC malignant progression by suppressing the Notch1 pathway, a highly conserved cell signaling system. Accordingly, these results imply that blocking the Notch1 pathway by repressing the TUG1/miR-299–3p axis might be a potential therapy option for PC [134]. To further elucidate the roles of miRNAs in PC, Wu et al. [135] inquire about the miRNA profile of PC-1.0 derived exosomes, revealing 62 upregulated ones. Remarkably, miR-125b-5p was shown to be substantially overexpressed in highly invasive PC cells, increasing migration, invasion, and epithelial-to-mesenchymal transition via targeting the tumor suppressor STARD13. Besides, the upregulation of miR-125b-5p was associated with MEK2/ERK2 signaling activation. These outcomes suggest that miR-125b-5p has a major role in PC metastasis. The results obtained in another inquiry supported that hypoxia upregulates the expression of miR-616–3p and miR-4465 in pancreatic stellate cells-derived exosomes. These miRNAs were implicated in the proliferation and invasion of PC cells by targeting PTEN, activating the AKT pathway [136]. In another recent investigation, Zhou et al. [137] isolated PC stem cells from xenograft cells and detected that miR-146b-3p was significantly downregulated in such specimens. Additionally, they demonstrated that miR-146b-3p targets a protein, namely MAP3K10, involved in tumorigenesis and the survival of PCs. Finally, they concluded that miR-146b-3p might induce apoptosis and repress proliferation in PC stem cells downregulating MAP3K10, and therefore it has an outstanding potentiality for the development of miRNA-based therapies for PC. To further illustrate the roles of miRNAs in PC, Chang et al. [138] analyzed M2 macrophage-derived extracellular vesicles and noticed that miR-21a-5p was substantially upregulated in such structures. In the same study, they elucidated that miR-21a-5p promotes the differentiation and activity of PC stem cells through targeting KLF3, a transcription factor with putative antitumor activity. Compelling evidence obtained from another inquiry revealed that PC cell-derived exosomal miR-27a is linked with the angiogenesis of human microvascular endothelial cells via targeting BTG2, a protein involved in cell differentiation, apoptosis, antiproliferation, and DNA damage repair, and could be relevant for treating PC; nevertheless, further studies are required to fully understand the molecular crosstalk between this miRNA and angiogenic factors [139]. In a preclinical study, tumor xenograft-bearing mice were treated with a miR-24–3p mimics formulated within polymeric nanoparticles, and, as a consequence of the activation of necrosis and apoptosis, tumor inhibition was observed. Additionally, it was determined that the direct targets of miR-24–3p are PIM1 and PIM2, two proteins associated with oncogenesis [140]. In another investigation, miR-145 was detected to be downregulated in PC tissues and cells. Consistently, induction of the expression of miR-145 in vivo disrupted tumor growth by suppressing the TGF-β signaling pathway (which promotes cell differentiation, cell proliferation, and chemotaxis) and inhibiting epithelial-mesenchymal transition. This evidence indicates that miR-145 is a possible candidate for anti-cancer drug development [141]. Besides, an investigation in which PC cells were treated with baicalein (an active flavonoid present in Scutellaria baicalensis Georgi) supported that this therapeutic approach enhances apoptosis and cell cycle arrest by altering the expression of at least 59 miRNAs, where the most significantly affected miRNAs are miR-196b-5p (downregulated) and miR-139–3p (upregulated) [142]. Additional experiments evidenced that miR-139–3p stimulates apoptosis of PANC-1 cells by inhibiting NOB1 expression, whereas miR-196b-5p can suppress the apoptosis mechanism targeting ING5. Therefore, baicalein might serve as an innovative therapy for PC; nevertheless, more research is required to unveil the underlying molecular mechanism of this flavonoid [142]. A few of these clinical applications of miRNAs in PC are illustrated in Fig. 2. Long et al. [143] examined PC cells and tissue samples and detected that miR-409 was substantially downregulated. In fact, the decreased expression of this miRNA was linked with a poor survival rate of PC patients and tumor development. Thereafter, researchers evidenced that miR-409 might inhibit tumor progression by targeting GAB1, a protein with oncogenic implications in PC; however, additional tests are required to disclose the molecular interface and the theragnostic significance of miR-409 in PC. Similarly, Li et al. [144] noticed that miR-190b had been downregulated in PC cell lines and tissues. Additionally, they found that this miRNA targets factors involved in the malignant progression of PC (i.e., MEF2C and TCF4), and hence miR-190b is a prospective target for the diagnosis and management of PC. Very recently (in 2022), miR-802 was reported to attenuate KRAS-induced acinar-to-ductal metaplasia via suppressing SOX9 activity and F-actin reorganization, thus inhibiting PC initiation. Nonetheless, upcoming studies should validate the therapeutic use of miR-802 for PC [145]. Some of the most significant biological and clinical implications of miRNAs in PC are listed in Table 1.
LncRNAs are implicated in a wide range of molecular mechanisms related to the development of gastrointestinal cancers, such as resistance to apoptosis, chemoresistance, cell differentiation, division, migration, and invasion [146,147]. As a matter of fact, lncRNAs play crucial roles in cancer pathways since they act as biological sponges that modulate miRNA levels, thus affecting the regulation of tumor suppressors and oncogenes [148,149]. Under this premise, understanding the molecular crosstalk between lncRNAs and miRNAs might help pave the way for designing innovative ncRNA-centered therapies and diagnoses for PC.
Xu et al. [150] demonstrated that induced overexpression of the lncRNA DLEU2L (downregulated in PC tissues) hinders gemcitabine resistance via sponging miR-210–3p. Mechanistically, the suppressive effect of DLEU2L on miR-210–3p promotes the upregulation of BRCA2, thus stimulating apoptosis and repressing PC cell proliferation, migration, and invasion via blocking the Warburg effect (aerobic glycolysis) as well as AKT/mTOR signaling. Thereafter, in another study, the lncRNA ANRIL was detected to be overexpressed in PC tissues, depicting that ANRIL triggers HMGB1-induced cell autophagy (a pivotal process in oncogenesis) and augments resistance to gemcitabine via sponging miR-181a, the miRNA that targets HMGB1 protein [151]. Throughout an interesting investigation, the lncRNA HIF1A-AS1 was detected to be upregulated in gemcitabine-resistant PC cells. Systematically, this lncRNA promoted the interaction of serine/threonine kinase AKT with YB1, thus triggering the phosphorylation of the latter (pYB1) [152]. The outcomes of this study also indicated that the translation of HIF-1α was enhanced due to the recruitment of pYB1 to HIF-1α transcripts, which was mediated by HIF1A-AS1. As a result, the upregulation of HIF-1α promoted glycolysis and gemcitabine resistance in PC cells. Intriguingly, researchers noticed that HIF-1α enhances HIF1A-AS1 transcription as well. Therefore, the molecular interplay between HIF1A-AS1 and HIF-1α encloses an attractive clinical potential for targeting PC [152]. Additionally, SNHG7 has been reported as a substantially upregulated lncRNA in PC tissues. This lncRNA functions as a competing endogenous RNA to miR-342–3p (whose gene target is ID4) via sponging mechanisms, promoting PC cell proliferation and metastasis. Remarkably, SNHG7 knockdown repressed PC tumorigenesis in vivo [153]. An additional investigation revealed that the lncRNA DANCR (upregulated in PC cells and tissues) promotes tumor progression in PC since it acts as a sponge to miR-33b, positively regulating the expression of MMP16, a protein involved in PC cell migration and invasion [154]. Later, Cao and Zhou [155] studied the relationship of both lncRNA SNHG12 and miR-320b in PC, and their results showed an increased expression level of SNHG12 during the progression of PC with a proportional rate to that of PC cell invasion and cell growth. Mechanistically, miR-320b is repressed by the SNHG12’s absorbing effect. Since miR-320b is a negative regulator of TRIAP1 (protein implicated in apoptosis), as well as a suppression element in different carcinomas, the SNHG12-mediated downregulation of this miRNA stimulates epithelial-mesenchymal transition, proliferation, and invasion of PC cells [155]. These facts suggest that the effects of PC could be ameliorated via therapeutically silencing SNHG12. In contrast, lncRNA LINC00671 was noticed to be downregulated in PC patients and cell lines. Moreover, induced overexpression of LINC00671 was linked to the inhibition of cancer cell proliferation through the suppression of epithelial-mesenchymal transition, ERK, and AKT pathways [156]. Another lncRNA with promising medical implications in PC is LINC00857. Meng et al. [157] demonstrated that this lncRNA is upregulated in PC tissues and cells due to N6-Methyladenosine (m6A), which methylates LINC00857 and enhances its stability. As a consequence, the increased expression of LINC00857 foments the downregulation of miR-150–5p and upregulation of E2F3 by exerting a sponging effect on the former. Since E2F3 is an oncogene, its increased expression promotes PC tumorigenesis. Likewise, lncRNA TP73-AS1 was detected to be upregulated in PC cells and tissues and is linked with PC growth and metastasis since it promotes the expression of GOLM1 (a protein that participates in tumor progression) via downregulating miR-128–3p. Remarkably, TP73-AS1 silencing repressed tumor growth in vivo as well as PC cell proliferation, migration, and invasion in vitro, thus evidencing its therapeutic potential against PC [158]. In 2021, Liu et al. [159] found that GATA3-AS1 is substantially upregulated in PC tissues and cell lines. The effects of GATA3-AS1 knockdown were also investigated, resulting in an augment of apoptosis and inhibition of cancer cell viability, proliferation, and invasion. Moreover, a bioinformatic analysis demonstrated that GATA3-AS1 plays an important role in the downregulation of miR-30b-5p expression, implying that this lncRNA can act as a sponge to miR-30b-5p, thus leading to the release of miR-30b-5p targeted transcript: TEX10, which has carcinogenic roles. The GATA3-AS1/miR-30b-5p/TEX10 axis is believed to be related to Wnt/β-catenin signaling in PC cells [159]. In another study, lncRNA FGD5-AS1 was discovered to be upregulated in PC cells, enhancing both cancer cell proliferation and migration. Mechanically, FGD5-AS1 sponges miR-520a-3p, causing its low expression in PC cells. Since KIAA1522 is the target of such miRNA, low levels of miR-520–3p triggered the upregulation of this oncogene, which has been demonstrated to increase the tumorigenicity of various cancers (e.g., breast and lung cancers). Overall, this investigation might provide an RNA-based alternative to treat PC [160]. Subsequently, Xu et al. [161] found that lncRNA CERS6-AS1 is significantly overexpressed in PC tissues and cells. Consistently, CERS6-AS1 overexpression increased the expression of YWHGA via sponging miR-217 and enhancing cancer cell growth, proliferation, and invasion. In addition, researchers showed that YWHGA promotes the phosphorylation of RAF1, thus activating ERK signaling. These findings imply that the CERS6-AS1/miR-217/YWHGA/RAF1 axis is a promising medical target for the treatment of PC. Experimental evidence has also demonstrated that lncRNA NORAD (upregulated in PC cells and tissues) is a plausible therapeutic target for PC since it enables the expression of ANP32E via blocking the regulatory activity of miR-202–5p; therefore, enhancing self-renewal and proliferation of PC stem cells [162]. Additionally, Luo et al. [163] noticed that induced suppression of lncRNA LINC01094, which is usually overexpressed in PC, leads to the inhibition of metastasis and tumorigenesis in mouse xenografts and lessens both metastasis and proliferation of PC cells. They also clarified that this lncRNA acts as an endogenous sponge that downregulates miR-577, thus allowing the overexpression of LIN28B (the target of miR-577) and triggering the PI3K/AKT pathway, which in turn promotes PC progression [163]. Moreover, Zhang et al. [164] detected that the lncRNA FGD5‑AS1 is overexpressed in PC cell lines and tissues. FGD5‑AS1 was also related to cancer cell proliferation, migration, and invasion owing to the fact that it suppresses the regulatory activity of miR-577. Indeed, FGD5‑AS1-mediated downregulation of miR-577 alters the expression levels of β‑catenin, LRP6, cyclin D1, AXIN2, and c‑Myc, thus affecting the Wnt/β-catenin signaling pathway and contributing to the progression of PC. Another study established that the expression of lncRNA PVT1 was increased under hypoxia in PC cell lines. Remarkably, HIF-1α transcription was demonstrated to be promoted by PVT1, while PVT1 requires HIF-1α expression for its efficient transcription and transcript stabilization. As well, it was noticed that PVT1 increases HIF-1α post-translationally [165]. The findings of this investigation also showed that patients with high levels of PVT1 and HIF-1α presented worst survival rates than those overexpressing only one of the two. In addition, PVT1 knockdown impeded HIF-1α-mediated PC tumorigenesis. Therefore, the positive feedback loop PVT1-HIF-1α should be thoroughly examined in the future to design next-generation therapeutics for PC [165]. In 2021, Zhu et al. [166] showed that the lncRNA CRNDE is significantly upregulated in PC cells and tissues. Furthermore, they concluded that the overexpression of this lncRNA boosts the progression and angiogenesis of PC by sponging miR-451a, thus allowing the enhanced expression of protein CDKN2D implicated in the regulation of tumor growth. Accordingly, CRNDE-mediated modulation of the miR-451a/CDKN2D axis could be a reliable clinical target for PC. An additional study suggested that the lncRNA MIR99AHG, whose transcription is enhanced by FOXA1, sponges miR-3129–5p and recruits ELAVL1. As a result, MIR99AHG increases the progression of PC by regulating NOTCH2 expression and promoting the activation of the Notch signaling pathway [167]. Meng et al. [168] revealed that the lncRNA LINC01320 is upregulated in PC cell lines and sponges miR-324–3p, thus promoting PC cell growth and migration. However, LINC01320 downregulation represses the growth and migration of PC cells and mediates apoptosis, suggesting that such lncRNA may be a prospective target for PC therapy. The main results regarding the clinical and pathological implications of lncRNAs in PC are summarized in Table 2.
Over the last few years, a number of relevant studies have elucidated the crucial epigenetic regulatory role of both miRNAs and lncRNAs in the pathophysiology of PC. In fact, it has been evidenced that the altered expression of these ncRNAs affects a variety of biological processes implicated in PC development and progression, such as apoptosis, autophagy, tumor growth, tumor suppression, chemoresistance, cancer cell proliferation, migration, and invasion. Moreover, miRNAs and lncRNAs are suggested as prospective biomarkers for accurate PC diagnosis and prognosis. Therefore, the experimental evidence conferred in this current review implies that miRNAs and lncRNA own a noteworthy clinical potential against PC. Nevertheless, it is worth mentioning that further investigations are required to properly understand the regulatory functions of miRNA and lncRNA transcriptomes that remain elusive in the etiology of PC.
As stated throughout the previous sections, molecular biologists have been assiduously analyzing the biological implications of miRNAs and lncRNAs in PC pathophysiology. Nonetheless, there are still a number of concerns and subtle questions that should be addressed in forthcoming investigations. For instance, different reports have suggested that melatonin might have an important modulatory role in the progression of PC since this indoleamine could induce cancer cell apoptosis via regulating the activity of a variety of pathways, such as vascular endothelial growth factor, oxidative stress, and heat shock proteins [169]. Moreover, the therapeutic role of melatonin in various types of cancers, including breast, oral, gastric, colorectal, and prostate cancer, has been linked with the regulation of the expression of certain miRNAs (e.g., let-7i-3p, miR-21, miR-24, miR-155, miR-34b-5p, miR-319, miR-148a-3p, miR-3195, and miR-374b) [170,171]. In the same context, relevant reports have shown that lncRNAs H19, MEG3, CPS1-IT1, and lnc010561 also display biological interactions with melatonin in cancer which are related to apoptosis, pyroptosis, and metastasis [172]. Therefore, more research is required to unveil the molecular crosstalk between miRNAs, lncRNAs, and melatonin, as well as their clinical significance in PC. Besides, it is worth mentioning that diverse signaling pathways regulated by lncRNAs (e.g., DLX6-AS1, LINC00261, TSLNC8, SNHG1, LINC01133, LINC00462, DLEU2, PVT1, and H19) are implicated in PC progression. Some examples of these pathways are Wnt/β-catenin, NOTCH, TGFβ/SMAD, and JAK/STAT [173]. Similarly, miR-96, miR-193b, miR-206, miR-20a, miR-216a, miR-744, miR-940, miR-296, miR-615–5p, miR-301–3p, and miR-421 have been associated with JAK/STAT, MAPK/ERK, Wnt/β-catenin, TGFβ, and AKT/mTOR pathways [112]. Accordingly, future investigations should focus on continuing to illuminate the regulatory implications of the lncRNA and miRNA transcriptomes in the aforesaid PC-related pathways, especially those poorly studied in this research field, i.e., JAK/STAT and TGFβ/SMAD. On the other hand, since glycolysis enhances PC progression, invasion, metastasis, epithelial-mesenchymal transition, angiogenesis, metastatic colonization of remote organs, and chemoresistance [174,175], the development of ncRNA-based approaches for glycolysis regulation has been suggested as a promising alternative for PC suppression [176]. In this regard, studying the modulatory effects of miRNAs and lncRNAs on those glycolytic proteins linked with cancer progression, e.g., HK1, GAPDH, PKM2, GLUT1, GLUT3, HIF-1α, CAV1, Ras, HK2, LDHA, PGI/AMF, and PFKFB3 [174,175,177], might be very favorable for the design of novel treatments for PC. Some examples of miRNAs and lncRNAs that have been recently associated with glycolysis regulation in PC are miR-210–5p, miR-202, miR-135, miR-505, BLACAT1, LINC00941, MIR210HG, and LINC01448 [178], [179], [180], [181], [182], [183], [184]. Since chemotherapy is one of the standard treatments for PC, chemoresistance is another important concern implicated in this disease [42,185]. This type of drug resistance is very common in PC and is linked with genetic or epigenetic alterations, desmoplastic stroma, metabolic reprogramming, tumor microenvironment, and epithelial-mesenchymal transition [186]. Under this premise, miRNAs and lncRNAs could be reliable tools for better management of PC chemoresistance. For instance, miR-210, miR-124, miR-7, miR-205, miR-21, miR-221, miR-200b/c, miR-155, among others, have been correlated with chemoresistance, whereas miR-153, let-7a, miR-33a, miR-205–5p, miR-138–5p, miR-203, and additional miRNAs have been demonstrated to be implicated in chemosensitivity [187]. Similarly, CASC2, GAS5, HCP5, HOST2, HOTTIP, HOTAIR, MEG3, PVT1, ROR, SNHG8, and TUG1 are examples of lncRNAs involved in the regulation of drug resistance in PC [188]. Consistently, these ncRNAs should be studied in-depth to generate next-generation medications that could be co-delivered with chemotherapeutic drugs for PC. In addition, even though the pancreas does not have local microbiota, dysbiosis (alteration of gut microbiota balance) and intestinal bacteria overgrowth promote a leaky gut, which in turn facilitates the translocation of intestinal microbiota into the pancreas [189]. Many studies have indicated that host microbiota could play an important role in PC [190,191]. As a matter of fact, experts have suggested that microbiota could contribute to PC progression by triggering inflammatory pathways involved in carcinogenesis, overturning both innate and adaptative immune responses, mediating chemoresistance, and interacting with other factors, including food and diet, bile acids, and tumor microenvironment [192,193]. Nevertheless, to the best of our knowledge, the interplay between pancreas microbiota, miRNAs, and lncRNAs in PC remains unexplored; thus, assessing these matters in upcoming studies could be beneficial for the design of new medicines for this ailment (Fig. 3). It is worth mentioning that the identification of differentially expressed miRNAs during the different stages of PC could be helpful to overcome its progression [20]. Nonetheless, studies focused on this aim are still scarce. For instance, in 2015, Rachagani et al. [194] used a KrasG12D; Pdx1-Cre mouse model to examine the miRNA expression level from the precursor lesions to the final stage of PC (which is PDAC), and they noticed that a wide range of miRNAs had different expression levels at 10, 30, 40, and 50 weeks of PC progression. In fact, miR-216 and miR-217 levels reduced progressively in the mice models, whereas the expression levels of miR-21, miR-34c, miR-146b, miR-205, and miR-223 increased substantially. Hence, upcoming studies should be focused on unveiling the biological impact of those miRNAs and lncRNAs that are differentially expressed during PC development. Intriguingly, in the past years, several RNA therapeutic agents (e.g., fomivirsen, mipomersen, eteplirsen, pegaptanib, patisiran, lumasiran, and givosiran) have been approved for medical use by the FDA [195,196]. As a result, investors are paying much attention to this research field, and various biopharmaceutical corporations have arisen, intending to develop ncRNA-based drugs. Some of these companies are Regulus Therapeutics, miRagen Therapeutics Inc., Mirna Therapeutics Inc., EnGeneIC, Santaris Pharma, and InteRNA Technologies, which are conducting miRNA-centered programs for a variety of human ailments, including cancer [117,197,198]. Further, the increasing interest in ncRNA-mediated treatments for human diseases has led to the initiation of a variety of ongoing clinical trials in which the prospective use of miRNAs and lncRNAs is being analyzed for cancer management. Some of the most representative examples of these ncRNA molecules currently in clinical trials are miR-31 and miR-210 (oral cancer), miR-34a (melanoma, primary liver cancer, multiple myeloma, lymphoma, among other cancers), miR-100 (breast cancer), miR-155 (bladder cancer), miR-16 (non-small cell lung cancer and malignant pleural mesothelioma), miR-221 and miR-222 (hepatocellular carcinoma), THRIL and PACER (stomach cancer), and HOTAIR (thyroid cancer) [199]. To date, only one clinical trial is ongoing related to the application of a specific miRNA in PC detection (NCT03432624); such a study is centered on evaluating the use of miR-25 in the diagnosis of PC with a detection kit, but there is still no miRNA or lncRNA drug in clinical tests [48,200]. Thus, more comprehensive studies are needed to apply miRNA and lncRNA in PC theranostics. Under such assertions, many challenges should be aborded in future studies for miRNA- and lncRNA-mediated therapeutics to reach the pharmacological breakthrough. For example, it has been established that ncRNA expression is affected by age, sex, body mass, physical activity, smoking, alcohol consumption, and diet [201,202], and hence these factors should be taken into account when developing ncRNA-based drugs and diagnoses. As well, toxicity analyses, improved delivery systems, reduction of the immunostimulatory potentiality of synthetic RNA medications, enhancement of on-target specificity, and lack of undesired on-target and off-target effects are concerns that must be considered to develop ncRNA-centered therapeutics [203]. In conclusion, from our personal perspective and based on our current knowledge on ncRNA-focused therapeutics for diverse human diseases, we assume that, over the coming decades, both miRNA- and lncRNA-based cancer therapeutics will reach sky-high. Relevantly, the development of mRNA vaccines for COVID-19 during the past two years has provided a crystal-clear demonstration that next-generation RNA-centered molecular medicine could be a significant lifesaver. In fact, as these types of treatments gain more attention, more opportunities will come up to extrapolate their application to various health issues. Although further research on the mechanisms and behaviors of ncRNA-based drugs within the human system is required, we believe that the information presented in this review will strengthen this research arena to set up the ncRNA-focused therapeutic pipeline for PC.
LABV, SujayP conceived, performed the literature search, and wrote the manuscript. NFR, AGRD, SMOP, and HRZC performed the literature search and contributed to writing the manuscript. SP, AntaraB, AB, AKD critically revised the manuscript. All authors have reviewed and approved the final manuscript.
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. | true | true | true |
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PMC9637919 | Zhengyu Gao,Laixing Zhang,Zihao Ge,Hao Wang,Yourun Yue,Zhuobing Jiang,Xin Wang,Chenying Xu,Yi Zhang,Maojun Yang,Yue Feng | Anti-CRISPR protein AcrIF4 inhibits the type I-F CRISPR-Cas surveillance complex by blocking nuclease recruitment and DNA cleavage | 07-10-2022 | CRISPR/Cas,anti-CRISPR,cryo-EM structure,protein–protein interaction,protein complex,Acr, anti-CRISPR,Cas, CRISPR-associated,CRISPR, clustered regularly interspaced short palindromic repeats,Csy, crRNA-guided surveillance,EMSA, electrophoretic mobility shift assay,HB, helical bundle,NTS, nontarget strand,RBC, R-loop binding channel,TS, target strand | The clustered regularly interspaced short palindromic repeats (CRISPR)-Cas system provides prokaryotes with protection against mobile genetic elements such as phages. In turn, phages deploy anti-CRISPR (Acr) proteins to evade this immunity. AcrIF4, an Acr targeting the type I-F CRISPR-Cas system, has been reported to bind the crRNA-guided surveillance (Csy) complex. However, it remains controversial whether AcrIF4 inhibits target DNA binding to the Csy complex. Here, we present structural and mechanistic studies into AcrIF4, exploring its unique anti-CRISPR mechanism. While the Csy–AcrIF4 complex displays decreased affinity for target DNA, it is still able to bind the DNA. Our structural and functional analyses of the Csy–AcrIF4–dsDNA complex revealed that AcrIF4 binding prevents rotation of the helical bundle of the Cas8f subunit induced by dsDNA binding, therefore resulting in failure of nuclease Cas2/3 recruitment and DNA cleavage. Overall, our study provides an interesting example of attack on the nuclease recruitment event by an Acr, but not conventional mechanisms of blocking binding of target DNA. | Anti-CRISPR protein AcrIF4 inhibits the type I-F CRISPR-Cas surveillance complex by blocking nuclease recruitment and DNA cleavage
The clustered regularly interspaced short palindromic repeats (CRISPR)-Cas system provides prokaryotes with protection against mobile genetic elements such as phages. In turn, phages deploy anti-CRISPR (Acr) proteins to evade this immunity. AcrIF4, an Acr targeting the type I-F CRISPR-Cas system, has been reported to bind the crRNA-guided surveillance (Csy) complex. However, it remains controversial whether AcrIF4 inhibits target DNA binding to the Csy complex. Here, we present structural and mechanistic studies into AcrIF4, exploring its unique anti-CRISPR mechanism. While the Csy–AcrIF4 complex displays decreased affinity for target DNA, it is still able to bind the DNA. Our structural and functional analyses of the Csy–AcrIF4–dsDNA complex revealed that AcrIF4 binding prevents rotation of the helical bundle of the Cas8f subunit induced by dsDNA binding, therefore resulting in failure of nuclease Cas2/3 recruitment and DNA cleavage. Overall, our study provides an interesting example of attack on the nuclease recruitment event by an Acr, but not conventional mechanisms of blocking binding of target DNA.
Clustered regularly interspaced short palindromic repeats (CRISPR, for all abbreviations see the abbreviation footnote) and CRISPR-associated (Cas) genes in prokaryotes encode an adaptive immune system that provides protection against the invasive mobile genetic elements such as phages (1, 2). The CRISPR-Cas system is classified into two broad classes and further divided into six types (I–VI), based on a wide variation in the protein composition among different types (3). Class 1 systems (types I, III, and IV) account for ∼90% of the CRISPR-Cas systems observed in nature, which depend on CRISPR RNA (crRNA)-guided multisubunit complexes to recognize foreign nucleic acids. Class 2 systems (types II, V, and VI) deploy a single multidomain protein that serves as a crRNA-guided effector nuclease to target foreign DNA/RNA. Although CRISPR-Cas systems are diverse in their protein composition, they share similar working stages. In their immunity mechanism, integrating short foreign DNA fragments into the host CRISPR locus is used for recognizing and destroying previously encountered nonself nucleic acid sequences, which is the most distinctive feature of CRISPR-Cas system as an adaptive immune system (4). To evade this bacterial immunity, phages have evolved protein inhibitors called anti-CRISPR (Acr) proteins (5). These proteins differ greatly in their sequence and structure, providing diverse inhibitory mechanisms that act on different components of the CRISPR-Cas system and at distinct stages of the CRISPR-Cas immunity (6). The type I-F CRISPR-Cas system encodes a ∼350 kDa crRNA-guided ribonucleoprotein complex (named the Csy complex), which comprises four kinds of protein (1 Cas5f, 1 Cas8f, 1 Cas6f, and 6 Cas7f subunits). After the Csy complex recognizes target DNA, the transacting nuclease-helicase Cas2/3 will be recruited and degrades the DNA. Up to now, 24 Acrs have been identified to target the type I-F CRISPR-Cas system, out of which both structures and inhibition mechanisms of 14 Acr proteins have been determined, including AcrIF1/2/3/5/6/7/8/9/10/11/13/14/23/24. Among these, all Acrs except AcrIF3/5/23 act by inhibiting target dsDNA binding (7, 8, 9), mainly in two approaches, either inhibiting hybridization between target DNA and crRNA or imitating DNA substrates. There are also several novel mechanisms based on this canonical one. AcrIF11 is an ADP-ribosyltransferase that modifies the key Csy residue responsible for target DNA recognition (10). AcrIF9/14/24 are dual functional Acrs with an ability to not only inhibit DNA-crRNA hybridization but also induce nonspecific DNA binding to the Csy complex (11, 12, 13). AcrIF3 and AcrIF5 do not inhibit target DNA binding by the Csy complex but prevent subsequent Cas2/3 recruitment. Target dsDNA binding by the Csy complex will induce a ∼180° rotation of the helical bundle (HB) of Cas8f subunit, which exposes an α helix responsible for Cas2/3 recruitment (7). Both AcrIF3 and AcrIF5 exploit this process to achieve their inhibition but function in different ways. AcrIF5 specifically targets the dsDNA-bound Csy complex to reposition the Cas8f HB (9), while AcrIF3 is a mimic of Cas8f HB and directly binds Cas2/3 to prevent it from being recruited (7, 14). AcrIF4 was identified in 2013 among the first five identified Acr proteins (15) and was found to bind the Csy complex in 2015 (16). While the structure of Csy–AcrIF4 complex has been solved in 2021 (17), the inhibition mechanism of AcrIF4 remains controversial. The structure of the Csy–AcrIF4 complex showed that AcrIF4 is wrapped between the helical backbone formed by Cas7f and the tail composed of a Cas5f-Cas8f heterodimer of the Csy complex. Based on structural comparison, Gabel et al. (17) proposed that AcrIF4 will not prevent target DNA binding, which was further verified by the electrophoretic mobility shift assay (EMSA) in their study. However, a previous study in 2015 showed that AcrIF4 inhibits target dsDNA binding through in vivo experiments (16). Moreover, AcrIF4 binding has been proposed to prevent the rotation of Cas8f HB, which has not been experimentally verified (17). Taken together, the inhibition mechanism of AcrIF4 still remains enigmatic and also controversial about its effect on target DNA binding. In this study, we first repeated the EMSA experiment of AcrIF4, which showed that the presence of AcrIF4 on the Csy complex does weakly inhibit target DNA binding. Notably, structural and biochemical studies revealed that AcrIF4 decreases target DNA binding through an approach very different from the canonical ones of competing with target DNA. The Csy–AcrIF4 complex can still bind target DNA; however, the Cas8f HB is held by AcrIF4 and does not rotate upon DNA binding to the Csy–AcrIF4 complex, which results in prevention of Cas2/3 recruitment and DNA cleavage. In all, our study presents an unprecedented mechanism in which AcrIF4 decreases target DNA binding to the Csy complex and inhibits Cas2/3 recruitment by anchoring the Cas8f HB.
To clarify the present controversy about the effect of AcrIF4 on target DNA binding by the Csy complex, we repeated the EMSA experiment of AcrIF4 in the study of Gabel et al. (17). The results showed that AcrIF4 in fact exhibits a weak inhibition on target DNA binding but does not completely prevent dsDNA binding even at a concentration 64 times that of the Csy complex (Fig. 1A). In comparison, both AcrIF1 and AcrIF13 can achieve a complete prevention at a concentration 4 times that of the Csy (Fig. 1A). This suggests that AcrIF4 is an only weak inhibitor of target DNA binding by the Csy complex, not as efficiently as AcrIF1 or AcrIF13 which directly hinders crRNA–DNA hybridization or acts as DNA mimic, respectively. To further explore the effect on dsDNA binding of the Csy complex by AcrIF4, we characterized the binding affinities of the Csy and Csy–AcrIF4 complex to dsDNA by EMSA assays. The results showed that the Csy–AcrIF4 complex exhibits a dsDNA binding Kd of ∼20.1 nM compared to the dsDNA binding Kd of ∼9.46 nM by the apo Csy complex (Figs. 1B and S1), also suggesting a very weak inhibition by AcrIF4 binding. Interestingly, the binding of target DNA by the Csy–AcrIF4 complex was saturated only at a fraction DNA bound value of ∼0.75 (Fig. 1B), suggesting an unstable binding between them. Taken together, the presence of AcrIF4 on the Csy complex results in a decreased binding to the target DNA, which is consistent with the previous results of in vivo experiment (16).
The above results suggested that while showing a decreased binding, Csy-AcrIF4 does bind target DNA as proposed by Gabel et al. (17). To confirm this, we performed a pull-down assay to investigate whether AcrIF4 and target DNA can co-exist on the Csy complex to form a ternary complex. After the pull-down assay, the samples were subjected to both SDS-PAGE gel and native PAGE, respectively, to detect the presence of dsDNA in the protein complex (Fig. 1, C and D). Due to the negative charge of dsDNA, the electrophoretic mobility of DNA-bound protein samples will run faster than the apo protein samples in the native PAGE, so that the migration position of the band can be used to determine whether there is bound DNA in the protein complex. The results showed that AcrIF4 and target DNA do not compete with each other on the Csy complex (Fig. 1, C and D). Target DNA can bind the preformed Csy–AcrIF4 complex, and AcrIF4 can also bind the preformed Csy–dsDNA complex. Up to now, most Acrs inactivate the CRISPR-Cas complex before target DNA binding; however, we recently found that AcrIF5 exhibits its inhibition capacity only after the Csy complex binds its target DNA (9). Therefore, we tested whether AcrIF4 inhibits the type I-F system before or after target DNA binding to the Csy complex. The results showed that preincubation of AcrIF4 and the Csy complex potently inhibits the activity of CRISPR-Cas system, but adding AcrIF4 after incubation of Csy and target DNA almost eliminates its inhibition capacity (Fig. 1E). Taken together, AcrIF4 inhibits the CRISPR-Cas system by binding the Csy complex, and the Csy–AcrIF4 complex binds target DNA but does not trigger its cleavage.
To investigate why Csy-AcrIF4 binds DNA but does not trigger its cleavage by Cas2/3, we incubated Csy-AcrIF4 and target DNA, purified the complex to homogeneity (Fig. S2), and solved the structure of the Csy–AcrIF4–dsDNA complex using single-particle cryo-electron microscopy (cryo-EM) at a resolution of 3.37 Å (Figs. 2A and S3; Table S1). As shown in other Csy structures, the Csy complex is composed of an unequal stoichiometry of four different Cas proteins guided by a single 60-nt crRNA (Cas8f1:Cas5f1:Cas7f6:Cas6f1:crRNA1). Compared with the structure of Csy-AcrIF4 (PDB code: 7JZW), AcrIF4 binds the Csy complex at almost the same position in the Csy–AcrIF4–dsDNA complex, mainly surrounded by Cas8f and Cas5f subunits (Fig. 2B). In the Csy-AcrIF4 structure, AcrIF4 also has contacts with Cas7.4f-7.6f subunits (Fig. 2B). However, these contacts are all absent in the Csy–AcrIF4–dsDNA complex (Fig. 2B), because of the elongation of the Csy helical backbone (to be introduced below). Consistently, densities for residues 49 to 52 of AcrIF4 are lacking in the Csy-AcrIF4-dsDNA structure; however, the same region is ordered and contacts Cas7.4f in the Csy-AcrIF4 structure (Fig. 2C). Next, we compared the structures of Csy-AcrIF4-dsDNA and Csy-dsDNA (PDB code: 6NE0) complexes. Previous studies have revealed that after target dsDNA binding, the Csy complex undergoes marked conformational changes compared to the apo Csy or that bound to a partially duplexed DNA target (7), which include clamping onto the dsDNA with the N-terminal segment of Cas8f, ∼18 Å elongation of the Csy backbone due to the movement of Cas8f-5f and hybridization between the target DNA and crRNA, and most importantly, a ∼180° rotation of the Cas8f HB (7). Compared to the structure of Csy-dsDNA, dsDNA binding to the Csy–AcrIF4 complex also induces movement of the N terminal of Cas8f onto the dsDNA (Fig. 2D, region 1), as well as elongation of the Csy backbone (Fig. 2D, region two and Fig. S4). However, strikingly, the Cas8f HB is not rotated upon DNA binding to Csy-AcrIF4 (Fig. 2D, region three and Fig. 2E), and meanwhile no density for the displaced R-loop can be found in the Csy-AcrIF4-dsDNA structure (Fig. 2F). The detailed differences between the structures of Csy–AcrIF4–dsDNA and Csy–dsDNA complexes will be discussed below.
The most notable feature of Csy-AcrIF4-dsDNA structure is the lack of rotation of the Cas8f HB, compared to the structure of Csy-dsDNA (Fig. 2E). Previous studies proposed that rotation of Cas8f HB of the Csy complex by dsDNA binding is dependent on R-loop formation (7), since this rotation was not observed in the structure of the Csy bound to a partially duplexed DNA which cannot form an R-loop (18). Interestingly, while a 54-bp fully duplexed DNA was used in our experiment, electron density only allows modeling of 39-nt in the target strand (TS) and 12-nt in the nontarget strand (NTS) with no nucleotides in the R-loop region (Fig. 2F). Close inspection of the structural alignment between Csy-AcrIF4-dsDNA and Csy-dsDNA revealed severe clash between AcrIF4 and the 9-nucleotide R-loop region of NTS (Fig. 3A), suggesting a different path of the displaced NTS in the Csy–AcrIF4–dsDNA complex. Importantly, a previous study identified a positively charged channel formed by residues in Cas8f and Cas5f, named R-loop binding channel (RBC), which is important for the stabilization of the R-loop and therefore, dsDNA binding to the Csy complex (Fig. 3B) (7). Importantly, AcrIF4 engages Cas8f R207/R219 and Cas5f R77 of the RBC (Fig. 3C) and thus forces the R-loop to extend toward other positions. This may explain the lack of density of the R-loop, suggesting that it becomes flexible without the stabilization effect of the RBC. Moreover, rotation of the Cas8f HB will also expose several positively charged residues in Cas8f HB to form another part of the RBC (7), which is also inhibited in the Csy–AcrIF4–dsDNA complex. On the other hand, in addition to its role in Cas2/3 recruitment, rotation of the Cas8f HB upon DNA binding also contributes to a stable “locked” conformation of the crRNA-target DNA duplex (7). In this conformation, the ‘thumbs’ of Cas7.2f and Cas7.3f fold over the target DNA strand and further bind the HB of Cas8f from one side, and their ‘webs’ interact with the Cas8f HB from the opposite site of the target DNA, thus completely locking the target DNA strand (Fig. 3D). However, compared to the Csy-dsDNA structure, densities for the 5′-end five nucleotides of the TS are lacking in the Csy-AcrIF4-dsDNA structure (Fig. 3D), suggesting that the absence of the locking effect of the Cas8f HB further destabilize the crRNA-DNA duplex in the 5′-end orientation of the TS of dsDNA. Taken together, we hypothesized that lack of both the stabilization of the R-loop by the RBC and the locking effect of Cas8f HB in the Csy–AcrIF4–dsDNA complex may increase the possibility of reannealing of the DNA duplex, thus decreasing the DNA binding ability of the Csy–AcrIF4 complex. To test this hypothesis, we repeated the EMSA experiment in Figure 1A with a dsDNA substrate containing a noncomplementary ‘‘bubble”, which results in an R-loop incapable of reannealing (7, 9). Consistent with our hypothesis, the Csy-AcrIF4 complex binds to the “bubble” dsDNA with a binding affinity similar as that of the Csy complex and can achieve a 100% binding (Figs. 3E and S5). Moreover, distinct from AcrIF1 and AcrIF13, AcrIF4 does not decrease the binding of the “bubble” DNA by the Csy complex (Fig. 3F). Taken together, our results indicated that the existence of AcrIF4 on the Csy complex blocks the RBC of the Csy complex and prevents the rotation of Cas8f HB upon dsDNA binding, thus reducing the R-loop stability and dsDNA binding ability of the Csy complex.
It has been reported that rotation of the Cas8f HB of the Csy complex upon dsDNA binding will expose a “Cas2/3 recruitment helix” in Cas8f HB, which is essential for Cas2/3 recruitment and subsequent DNA cleavage (7). Therefore, we investigated whether the Csy–AcrIF4 complex has the ability to recruit Cas2/3 upon dsDNA binding through EMSA experiments. As shown in Figure 4A, the band of Csy–DNA complex shifted again with the addition of Cas2/3, indicating the formation of a ternary complex Csy–DNA–Cas2/3 (lanes 2–5). However, Cas2/3 recruitment was markedly inhibited when Csy-AcrIF4 was used instead of the Csy complex (Fig. 4A, lanes 7–10). Notably, the Csy–AcrIF4 complex displays a reduced binding to dsDNA (compare lanes two and seven in Fig. 4A), which may also lead to less recruitment of Cas2/3. To avoid this, we also used the “bubble” dsDNA instead of fully duplexed dsDNA in the experiment shown in Figure 4A, which also showed that the presence of AcrIF4 prevents Cas2/3 recruitment by the Csy–dsDNA complex (Fig. 4B). Since AcrIF4 can bind either the apo Csy complex or the Csy–dsDNA complex (Fig. 1, C and D), next we tested different incubation orders with an AcrIF4 concentration gradient. The results showed that AcrIF4 displays potent inhibitory effect on Cas2/3 recruitment when Csy was preincubated with AcrIF4 (Fig. 4C, lanes 4–6). However, AcrIF4 displayed no inhibitory effect on Cas2/3 recruitment when Csy and dsDNA were preincubated first (Fig. 4C, lanes 7–9). This is consistent with the results of the in vitro DNA cleavage assay (Fig. 1E), suggesting that prevention of Cas2/3 recruitment by binding the Csy is the major inhibition mechanism of AcrIF4.
Notably, AcrIF4 has extensive interactions with the apo Csy complex with a ∼2580 Å2 buried surface, engaging the middle region and HB of Cas8f and Cas5f, as well as Cas7.4 to 7.6f (17). After DNA binding, the interfaces with Cas7f subunits are interrupted because of the elongation of the Csy backbone (Fig. 2B). To investigate which interface is essential for the inhibitory effect of AcrIF4, we designed eight AcrIF4 mutants with mutations of residues involved in each interface (Fig. 5A) and purified these proteins to homogeneity. Interestingly, pull-down assay showed that none of the mutations impair the interactions between AcrIF4 and the Csy complex (Figs. S6A and 5B), suggesting that the Csy-AcrIF4 interface is too extensive to be broken by mutations in a single part of the interface. Consistently, all the AcrIF4 mutants can prevent Cas2/3 recruitment by the Csy–dsDNA complex as WT AcrIF4 in the EMSA experiments (Figs. S6B and 5C). Because prevention of rotation of Cas8f HB is the inhibitory mechanism of AcrIF4, we wonder whether AcrIF4 will still retain its inhibitory capacity when its interface with the Cas8f HB is completely interrupted. However, AcrIF4 L39G/F54G, a mutant designed to interrupt its interaction with the Cas8f HB (Fig. 5A, right panel), still inhibits recruitment of Cas2/3 (Fig. 5C, left panel), suggesting that the Cas8f HB may still be held by the AcrIF4 mutant. To completely interrupt the interaction between AcrIF4 and the Cas8f HB, we further designed a Csy mutant with Cas8f R299G/R302A mutation in the Cas8f HB-AcrIF4 interface (Fig. 5A, right panel). Pull-down assay showed that AcrIF4 L39G/F54G is still able to interact with this Csy mutant as WT Csy (Fig. 5B). However, while the Csy mutant can recruit Cas2/3 upon dsDNA binding similarly as WT Csy, the AcrIF4 L39G/F54G mutant cannot inhibit either the Cas2/3 recruitment by the Csy mutant–dsDNA complex (Fig. 5C, right panel) or DNA cleavage (Fig. 5D). This suggested that although the AcrIF4 L39G/F54G mutant still binds the Csy mutant through the middle region of Cas8f and Cas5f, complete loss of the interaction with Cas8f HB renders AcrIF4 uncapable to hold the Cas8f HB in place and lose its inhibitory capacity. Moreover, it indicated that interaction between AcrIF4 and Cas8f HB is not essential for AcrIF4-Csy binding. This also suggested that although the presence of AcrIF4 on the Csy complex keeps the R-loop away from the RBC on the middle region of Cas8f, the R-loop can still trigger the rotation of Cas8f HB when the AcrIF4-Cas8f HB interface is completely interrupted. Taken together, interaction with the Cas8f HB is essential for the inhibitory activity of AcrIF4 by holding the Cas8f HB in place.
Intense competition between bacteria and bacteriophages promotes microbial evolution, explaining why Acrs are highly different in their sequences, structures, and inhibitory mechanisms (6). This also provides exciting resources for both researches into microbial life processes and potential regulation tools of genome editing based on CRISPR-Cas systems. In this study, we describe the inhibitory mechanism of AcrIF4, to our knowledge the only known type I-F Acr that targets the apo Csy complex but does not use inhibition of target DNA binding as its major inhibitory mechanism. Our structural and functional data demonstrate that AcrIF4 prevents the Csy complex from completing the conformational changes necessary for Cas2/3 recruitment upon target dsDNA binding. Interestingly, AcrIF4 engages a very broad interface with the Csy complex to ensure that AcrIF4 can stably anchor the Cas8f HB. This resembles a “ship anchor” model, in which the extensive interface clamps AcrIF4 within the Csy complex, like an anchor wrapped in the sand, and the Cas8f HB is like a “ship” anchored by AcrIF4 and is prevented from moving (Fig. 6). Previous studies are controversial about whether the binding of AcrIF4 affects DNA binding of the Csy complex. While in vivo assays showed the presence of AcrIF4 decreases DNA binding to the Csy (16), the EMSA result by Gabel et al. (17) showed no effect of AcrIF4 on this binding. Our EMSA results indicated that AcrIF4 does weakly inhibit DNA binding to the Csy complex. However, the binding site of AcrIF4 on the Csy complex neither conflicts with the hybridization site of the target DNA strand on Cas7f subunits nor shields the PAM recognition region on Cas8f. This is reflected by the fact that Csy-AcrIF4 can still bind dsDNA to form a ternary complex, which is significantly different from the canonical competition approaches utilized by AcrIF1/2/6/7/8/9/10/13/14/24 (11, 12, 13, 17, 18, 19, 20, 21, 22, 23). We found that AcrIF4 shields some residues of the RBC and also disrupts the Cas8f HB part of RBC by preventing rotation of the Cas8f HB. The lack of both the stabilization of the R-loop by the RBC and the locking effect on target DNA strand by the rotated Cas8f HB may collectively decrease the DNA binding to the Csy–AcrIF4 complex. Up to now, out of the 15 Acrs including AcrIF4 with known inhibition mechanisms, only AcrIF4 and AcrIF5 can coexist with target DNA, which is bound at the right place, on the Csy complex. AcrIF4 is also an Acr that utilizes the conformational change of the Cas8f HB to exert its inhibitory effect. Unlike AcrIF5 which specifically binds the Csy-dsDNA complex to compete off the Cas8f HB and make it flexible, AcrIF4 interacts tightly with Cas8f HB to prevent its rotation upon dsDNA binding. In contrary to AcrIF5 which functions only after the formation of Csy–dsDNA complex, AcrIF4 functions by binding the Csy complex before target DNA binding. Therefore, elucidation of the mechanism of AcrIF4 also has evolutionary implications that targeting the same procedure of host immunity can be executed in different ways by phage proteins. In all, our study reveals an unprecedented anti-CRISPR mechanism and highlights the functional diversity of Acr proteins.
The full-length AcrIF4 gene was synthesized by GenScript and amplified by PCR and cloned into a modified pET28a vector which encodes a SUMO protein to produce a His-SUMO–tagged fusion protein with a Ulp1 peptidase cleavage site between SUMO and the target protein. The mutants of AcrIF4 or the Csy complex were generated by two-step PCR and were subcloned, overexpressed, and purified in the same way as the wildtype protein. The AcrIF4 protein was expressed in E. coli strain BL21 (DE3) and induced by 0.2 mM isopropyl-β-D-thiogalactopyranoside when the cell density reached an A600nm of 0.8. After growth at 16 °C for 12 h, the cells were harvested, re-suspended in lysis buffer (300 mM NaCl, 50 mM Tris-HCl pH 8.0, 30 mM imidazole and 1 mM PMSF), and lysed by sonication. The cell lysate was centrifuged at 20,000g for 45 min at 4 °C to remove cell debris. The supernatant was applied onto a self-packaged Ni-NTA affinity column (2 ml High Affinity Ni-NTA Resin; GenScript), and contaminant proteins were removed with wash buffer (300 mM NaCl, 50 mM Tris-HCl pH 8.0, 30 mM imidazole). The AcrIF4 protein was eluted from the Ni column after reaction with Ulp1 peptidase at 18 °C for 2 h. The eluant was concentrated and further purified using a Superdex-75 (GE Healthcare) column equilibrated with a buffer containing 10 mM Tris–HCl pH 8.0, 200 mM NaCl, and 5 mM DTT. The purified protein was analyzed by SDS–PAGE. The Csy complex and Cas2/3 were cloned, overexpressed, and purified as described previously (13). To obtain the Csy–AcrIF4 complex, the AcrIF4 gene was cloned into the pACYCDuet-1 vector together with the gene that encodes the pre-crRNA, and the Csy-AcrIF4 complex can be purified using the same purification method as the Csy complex. The Csy–AcrIF4–dsDNA complex for cryo-EM study was prepared as follows. The Csy–AcrIF4 complex was incubated with dsDNA in a molecular ratio of 1:2.5 (Csy-AcrIF4: dsDNA). Then the Csy–AcrIF4–dsDNA complex was separated using a Superdex-200 (GE Healthcare) column equilibrated with a buffer containing 10 mM Tris–HCl pH 8.0, 200 mM NaCl, and 5 mM DTT. Purified proteins were finally flash-frozen in liquid nitrogen.
Aliquots (4 μl) of Csy–AcrIF4–dsDNA complex (2.5 mg/ml) were applied to carbon grids (Quantifoil 300-mesh Au R1.2/1.3, Micro Tools GmbH). The grids were blotted for 1.5 s and plunged into liquid ethane in 100% humidity at 8 °C with Mark IV Vitrobot (Thermo Fisher Scientific). Eight hundred thirty-nine raw movie stacks were collected using a Titan Krios microscope (Thermo Fisher Scientific) operated at 300 kV by a K3 Summit direct electron detector using SerialEM software at a nominal magnification of 29,000× in super-resolution mode with a total dose of 50 e/Å2 and exposure time of 3 s, and the defocus range were set from −1.3 μm to −1.8 μm.
In general, movies were motion-corrected using MotionCor2 (24). Gctf (25) was used to determine the contrast transfer function parameter and produce the contrast transfer function power spectrum on basis of summed micrographs. Particles were auto-picked on dose-weighted micrographs using Laplacian of Gaussian in RELION 3.1 (26). Briefly, about 2000 particles were selected and generated 2D averages templates for particle auto-picking and 621,597 particles were extracted from manually selected micrographs. The previously reported model of Csy–AcrIF14–dsDNA complex (PDB: 7ECW) was low-pass filtered to 40 Å for initial model. All extracted particles using a box size of 240 and a binned pixel size of 7.76 Å were subjected to two rounds of 2D classification and 3D classification using C1 symmetry, and 238,361 particles were retained. 117,510 selected particles were subjected to 3D autorefinement without symmetry and performed an overall resolution of 3.37 Å. Meanwhile, Cas8f and Cas6f were signal subtracted based on the refined map with soft mask, then particles were subjected to 3D autorefine and 3D classification without image alignment. Finally, 49,498 particles and 93,188 particles were separately performed to 3D autorefine and yielded a map at 3.59 Å and 3.96 Å resolution. The composite map of Csy–AcrIF4–dsDNA complex was generated in UCSF Chimera (27) using ‘vop maximum’ command and used for model building and refinement. All reported resolutions are based on the gold standard FSC = 0.143 criteria, and the final FSC curves were corrected for the effect of a soft mask by using high-resolution noise substitution. The final density maps were sharpened by B-factors calculated with the RELION postprocessing program. The final maps for model building and figure presentation were performed using DeepEMhancer (28). Local resolution map was calculated using ResMap (29). Further information for all samples is provided in Table S1.
Atomic models of the Csy–AcrIF4–dsDNA complex were modeled using the PDB 6NE0. AcrIF4 was modeled using the PDB 7JZW. The initial model was manually refined using Coot (30) and subjected to real_space_refinement using PHENIX (31). All the figures were created in the PyMOL software.
For EMSA and in vitro DNA cleavage assay, various 5′-end FAM labeled single-stranded DNA molecules were synthesized from Sangon, Shanghai, and were hybridized with their complementary unlabeled single-stranded DNA with a molar ratio of 1:1.5 to obtain double-stranded DNA. Specifically, dsDNA molecules used in pull-down assay were generated through the same method described above except that both strands were unlabeled and added with a 1: 1 ratio. Target DNA strand (54 bp; 5′-FAM fluorescein labeled or unlabeled). GGAAGCCATCCAGGTAGACGCGGACATCAAGCCCGCCGTGAAGGTGCAGCTGCT. Nontarget DNA strand (54 bp; 5′-FAM fluorescein labeled or unlabeled). AGCAGCTGCACCTTCACGGCGGGCTTGATGTCCGCGTCTACCTGGATGGCTTCC. Nontarget DNA strand of the dsDNA bubble (54 bp; unlabeled). AGCAGCTGCACCAAGTGCCGCCGCTTGATGTCCGCGTCTACCTGGATGGCTTCC.
Duplexed DNA was prepared as above with 5′ FAM label at the TS. Reactions were performed by incubating 1.6 μM Csy complex with 0.4, 1.6, 6.4, 25.6, and 102.4 μM Acr for 30 min at 37 °C in a buffer containing 20 mM Hepes pH 7.5, 100 mM KCl, 5% glycerol, and 1 mM TCEP. And then 0.1 μM dsDNA (or dsDNA bubble) was added and incubated for another 30 min at 37 °C. The mixtures were separated using 5% native polyacrylamide gels and visualized by fluorescence imaging.
dsDNA (100 nM) or 0.8, 3.2, 12.8 μM AcrIF4 was preincubated with 1.6 μM Csy complex at 37 °C in the reaction buffer (20 mM Hepes pH 7.5, 100 mM KCl, 5% glycerol, and 1 mM TCEP) for 30 min and then AcrIF4 or dsDNA was added and incubated for another 30 min. Afterwards, Cas2/3 was added to a final concentration of 0.8 μM. The reaction was further incubated for 10 min. Products of the reaction were separated using 5% native polyacrylamide gels and visualized by fluorescence imaging.
The apo or the AcrIF4-bound Csy complex was incubated in a concentration gradient (0, 0.005, 0.05, 0.25, 0.5, 2.5, 5, 10, 100, 1000, 10,000 nM) with 16 nM 54 bp-dsDNA or bubble dsDNA (5′-FAM in the TS). Binding reactions were conducted at 37 °C for 30 min in the buffer containing 20 mM Hepes pH 7.5, 100 mM KCl, 5% glycerol, and 1 mM TCEP. Products of the reaction were separated using 5% native polyacrylamide gels and visualized by fluorescence imaging. The fluorescence signal was measured using ImageJ(32).
Csy complex (6 μM), 15 μM dsDNA, and 180 μM AcrIF4 or mutants were incubated in the required order (Csy and DNA first or Csy and AcrIF4 first) for 30 min at 37 °C, and then the mixtures were incubated with Ni-NTA beads for 30 min at 4 °C. The buffer containing 300 mM NaCl, 50 mM Tris-HCl pH 8.0, and 30 mM imidazole was used to wash the beads. Samples of input and pull-down were separated using SDS-PAGE and native PAGE after washing three times.
First, 6.4 μM AcrIF4 or mutant and 0.4 μM Csy complex were incubated at 37 °C for 30 min in the reaction buffer containing 20 mM Hepes pH 7.5, 100 mM KCl, 5% glycerol, and 1 mM TCEP. And then 0.04 μM dsDNA (5′-FAM in the not-target strand) was added and incubated for another 30 min. The incubation order of DNA and AcrIF4 is adjusted according to the purpose of the experiment. Afterward, Cas2/3 was added to a final concentration of 0.2 μM, along which 5 mM MgCl2, 75 μM NiSO4, 5 mM CaCl2, and 2 mM ATP were added into the buffer. The reaction was further incubated for 30 min and quenched with 1% SDS and 50 mM EDTA. The products were separated by electrophoresis over 14% polyacrylamide gels containing 8 M urea and visualized by fluorescence imaging.
Cryo-EM reconstruction of Csy-AcrIF4-dsDNA has been deposited in the Electron Microscopy Data Bank under the accession numbers EMD-33837. The coordinate for atomic model of Csy-AcrIF4-dsDNA has been deposited in the Protein Data Bank under the accession number 7YHS.
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PMC9637986 | Jun-sheng Tian,Peng-fei Qin,Teng Xu,Yao Gao,Yu-zhi Zhou,Xiao-xia Gao,Xue-mei Qin,Yan Ren | Chaigui granule exerts anti-depressant effects by regulating the synthesis of Estradiol and the downstream of CYP19A1-E2-ERKs signaling pathway in CUMS-induced depressed rats 10.3389/fphar.2022.1005438 | 24-10-2022 | chaigui granule,depression,CYP19A1-E2-ERKs signaling pathway,chronic unpredictable mild stress,xiaoyao san | Background: There is a significant gender difference in the prevalence of depression. Recent studies have shown that estrogen plays a crucial role in depression. Therefore, studying the specific mechanism of estrogen’s role in depression can provide new ideas to address the treatment of depression. Chaigui granule has been shown to have exact antidepressant efficacy, and the contents of saikosaponin (a, b1, b2, d) and paeoniflorin in Chaigui granule are about 0.737% and 0.641%, respectively. Some studies have found that they can improve depression-induced decrease in testosterone (T) levels (∼36.99% decrease compared to control). However, whether Chaigui granule can exert antidepressant efficacy by regulating estrogen is still unclear. This study aimed to elucidate the regulation of estrogen levels by Chaigui granule and the underlying mechanism of its anti-depressant effect. Methods: Eighty-four male Sprague-Dawley (SD) rats were modeled using a chronic unpredictable mild stress (CUMS) procedure. The administration method was traditional oral gavage administration, and behavioral indicators were used to evaluate the anti-depressant effect of Chaigui granule. Enzyme-linked immunosorbent assay (ELISA) was adopted to assess the modulating impact of Chaigui granule on sex hormones. Then, reverse transcription-quantitative PCR (RT-qPCR), and Western blot (WB) techniques were employed to detect extracellular regulated protein kinases (ERK) signaling-related molecules downstream of estradiol in the hippocampus tissue. Results: The administration of Chaigui granule significantly alleviated the desperate behavior of CUMS-induced depressed rats. According to the results, we found that Chaigui granule could upregulate the level of estradiol (E2) in the serum (∼46.56% increase compared to model) and hippocampus (∼26.03% increase compared to model) of CUMS rats and increase the levels of CYP19A1 gene and protein, which was the key enzyme regulating the synthesis of T into E2 in the hippocampus. Chaigui granule was also found to have a significant back-regulatory effect on the gene and protein levels of ERβ, ERK1, and ERK2. Conclusion: Chaigui granule can increase the synthesis of E2 in the hippocampus of CUMS-induced depressed rats and further exert antidepressant effects by activating the CYP19A1-E2-ERKs signaling pathway. | Chaigui granule exerts anti-depressant effects by regulating the synthesis of Estradiol and the downstream of CYP19A1-E2-ERKs signaling pathway in CUMS-induced depressed rats 10.3389/fphar.2022.1005438
Background: There is a significant gender difference in the prevalence of depression. Recent studies have shown that estrogen plays a crucial role in depression. Therefore, studying the specific mechanism of estrogen’s role in depression can provide new ideas to address the treatment of depression. Chaigui granule has been shown to have exact antidepressant efficacy, and the contents of saikosaponin (a, b1, b2, d) and paeoniflorin in Chaigui granule are about 0.737% and 0.641%, respectively. Some studies have found that they can improve depression-induced decrease in testosterone (T) levels (∼36.99% decrease compared to control). However, whether Chaigui granule can exert antidepressant efficacy by regulating estrogen is still unclear. This study aimed to elucidate the regulation of estrogen levels by Chaigui granule and the underlying mechanism of its anti-depressant effect. Methods: Eighty-four male Sprague-Dawley (SD) rats were modeled using a chronic unpredictable mild stress (CUMS) procedure. The administration method was traditional oral gavage administration, and behavioral indicators were used to evaluate the anti-depressant effect of Chaigui granule. Enzyme-linked immunosorbent assay (ELISA) was adopted to assess the modulating impact of Chaigui granule on sex hormones. Then, reverse transcription-quantitative PCR (RT-qPCR), and Western blot (WB) techniques were employed to detect extracellular regulated protein kinases (ERK) signaling-related molecules downstream of estradiol in the hippocampus tissue. Results: The administration of Chaigui granule significantly alleviated the desperate behavior of CUMS-induced depressed rats. According to the results, we found that Chaigui granule could upregulate the level of estradiol (E2) in the serum (∼46.56% increase compared to model) and hippocampus (∼26.03% increase compared to model) of CUMS rats and increase the levels of CYP19A1 gene and protein, which was the key enzyme regulating the synthesis of T into E2 in the hippocampus. Chaigui granule was also found to have a significant back-regulatory effect on the gene and protein levels of ERβ, ERK1, and ERK2. Conclusion: Chaigui granule can increase the synthesis of E2 in the hippocampus of CUMS-induced depressed rats and further exert antidepressant effects by activating the CYP19A1-E2-ERKs signaling pathway.
The high prevalence and the heavy social burden of depression have attracted increasing attention (Huang et al., 2019; Papathanasiou et al., 2020). Some studies have shown that women are more likely to suffer from depression than men, approximately 1.5–3 times more often than men (Nelemans et al., 2021). Among depressed patients, depressive episodes last longer and recur more frequently in women (Walf and Frye, 2006). While the reasons for vulnerability to such disorders in women remain to be fully understood, the strongest candidate is the disruption of gonadal steroid levels (Albert and Newhouse, 2019). Studies have found that estrogen therapy can produce anti-depressant effects (Saad et al., 2019). The use of estrogen can be accompanied by severe adverse reactions, including coronary heart disease, stroke, and breast cancer (Rozenberg et al., 2013). In recent years, traditional Chinese medicine has unique advantages in the treatment of depression, with multiple components, multi-target, multi-mechanism, fewer side effects, strong synergy, and overall regulation. Therefore, traditional Chinese medicine is expected to become an effective and safe treatment option for patients with depression. Chaigui granule is derived from the traditional Chinese medicine Xiaoyao San, which is composed of Mentha canadensis L. [Lamiaceae; Mentha canadensis herba], Bupleurum chinense DC. [Apiaceae; Bupleurum chinense radix], Glycyrrhiza uralensis Fisch. ex DC. [Fabaceae; Glycyrrhiza uralensis radix], Angelica sinensis (Oliv.) Diels [Apiaceae; Angelica sinensis radix], Atractylodes macrocephala Koidz. [Asteraceae; Atractylodis macrocephalae rhizoma], and Paeonia lactiflora Pall. [Paeoniaceae; Paeonia lactiflora radix] in the dose ratio of 2:6:3:6:6:6. Modern pharmacological studies have proved that Xiaoyao San has a significant anti-depressant effect (Gao et al., 2021). Based on the apparent anti-depressant efficacy of Xiaoyao San, researches on the Chaigui granule, including the screening of the prescription, the assumption of effectiveness and safety, and the chemical composition analysis, were performed systematically (Wu et al., 2019). At present, as the preparation process of Chaigui granule has been optimized and the anti-depressant effect has repeatedly been proved in our previous work (Chen et al., 2016; Wu et al., 2019), it has met the approval as a potential novel anti-depressant drug by the National Medical Products Administration (No. 2018L03149). The multicenter, randomized, placebo-controlled Phase IIa clinical trial is performed in the National drug clinical trial centers, including the Sixth Hospital of Peking University, Oriental Hospital of Beijing University of Traditional Chinese Medicine, and Wuhan mental health center. CYP19A1 is a cytochrome P450 family 19 subfamily 1 that is mainly found in gonadal and extragonadal tissues (others such as bone, adipose tissue, brain, etc.) (Yague et al., 2006). In recent years, numerous related studies have shown that CYP19A1 gene polymorphism is associated with depression (Ancelin et al., 2020). Experimental animal studies have shown that the elimination of CYP19A1 in female mice significantly decreases estrogen levels and depression-like behavior (Dalla et al., 2004). This may be related to the ability of CYP19A1 to convert T to E2 (Punjani et al., 2021). In addition, the hippocampus is the critical lesion in depression. It was reported that the granular cells of the dentate gyrus of the hippocampus carry an entire steroid production system that synthesizes steroid hormones from cholesterol (Rebourcet et al., 2020). Recent experiments provide evidence that hippocampal synthesis does occur, which suggests that hippocampal estrogen synthesis regulates estrogen-sensitive hippocampal responses (Scharfman and Maclusky, 2006). In our previous study on the anti-depressant effects of Chaigui granule, which has been confirmed that they restored the serum T levels of depressed rats (Chen et al., 2015). So, whether the antidepressant effect exerted by Chaigui granule is related to CYP19A1 promoting estradiol synthesis. However, there is no study on the effect of Chaigui granule on estradiol. Estradiol, an anabolic steroid hormone, is the most biologically active estrogen in the human body (Kuiper, 1997; Russell et al., 2019). Many studies have found that E2 is closely associated with depression. Patients with depression showed a significant decrease in estradiol levels compared with normal subjects. Serum estradiol levels were significantly higher in treated depressed patients than before treatment (Baischer et al., 1995; Young et al., 2000; Furuta et al., 2013). The effect of E2 on depression is complex, and its mechanism of action is mainly related to estrogen receptors (ER). It has been reported (Scharfman and Maclusky, 2006) that E2 in the brain can activate the ERK-cAMP-response element binding protein (CREB) signaling pathway through binding to ER, thus exerting an anti-depressant role. At the same time, many clinical trials have shown that the ERKs signaling pathway is involved in the occurrence of depression, which indicates that ERKs signaling pathways are closely related to mental illness (Dwivedi et al., 2006; Duman et al., 2007; Yuan et al., 2010). In order to further clarify the specific mechanism of Chaigui granule exerting antidepressant efficacy, we investigated the effect of Chaigui granule on the levels of related sex hormones in the serum and hippocampus of CUMS rats, as well as the downstream pathways that may exert antidepressant efficacy. This provides a new idea and basis for traditional Chinese medicine to exert antidepressant efficacy by regulating sex hormones.
Paeonia lactiflora Pall. (No.1806436111), Mentha canadensis L. (No.1802017131), Atractylodes macrocephala Koidz. (No.1809657151), Angelica sinensis (Oliv.) Diels (No.1803583111), Bupleurum chinense DC. (No.1805215131) and Glycyrrhiza uralensis Fisch. ex DC. (No.1810013171) as authenticated by Dr. Xuemei Qin from Modern Research Center for Traditional Chinese Medicine of Shanxi University. Chaigui granule were processed and prepared by the preparation center of Shanxi Academy of traditional Chinese medicine (No.20181009); HPLC analysis of Chaigui granule are shown in Supplementary Table S1; Venlafaxine Hydrochloride Capsule (No.181203) and Shuganjieyu Capsule (No.180101) were obtained from Chengdu Kanghong Pharmaceutical Group Co., Ltd.
84 male Sprague-Dawley (SD) rats, Specific Pathogen Free (SPF) grade, weight (180–200 g), were provided by Vital River Laboratory Animal Technology Co., Ltd. (Beijing) (SCXK-2016–0006). All animals were reviewed and approved by the Committee of Scientific Research at Shanxi University (CSRSX) in April 2018 (NO. SXULL2018015). Animal welfare and all experimental programs were carried out under the regulations of the State Committee of Science and Technology of the People’s Republic of China on the Administration of Experimental Animals. All animals were housed 5 per cage under controlled breeding room conditions (lights on at 8:00 a.m., 12 h light/dark cycle, temperature: 24 ± 2°C, humidity: 50% ± 20%.) with free access to food and water.
Rats were randomly selected as control group (Con), model group (Mod), venlafaxine-treated group (35 mg/kg) (Ven), Shuganjieyu group-treated group (0.15 g/kg) (Shu), and Chaigui granule with high dose group (6.29 g drug/kg) (Hig), medium dose group (3.15 g drug/kg) (Med), low dose group (1.59 g drug/kg) (Low) respectively. Volume: 1 ml/100 g (rat body weight). The control and model groups were given normal saline 1 ml/100 g/d. CUMS model is improved by referring to the methods of Willner et al. (Willner et al., 1987). A stimulus factor was randomly selected every day, and the same stimulus factor could not appear continuously to ensure the randomness and unpredictability of the stimulus factor. The detailed schedule of the CUMS model is shown in Figure 1. Stressors used in the Chronic Unpredictable Stress paradigm are shown in Table 1.
During the CUMS induction period, the body weights of rats were recorded regularly throughout the experiment, and all rats in each group were weighed at 0, 3, and 7 weeks. This was used to evaluate CUMS modeling and the effect on rat body weight after administration.
Rats were given a bottle of purified water and a bottle of 1% sucrose water. Purified water was fixed on the right side, and sucrose water was fixed on the left side. Sugar water preference training was then performed for 24 h, and the positions of the two bottles were exchanged at 12 h. After the sugar water training, a sugar water preference test was performed for 3 h. Sugar water preference was calculated by measuring the weight of the bottles before and after the experiment (Qi et al., 2018). Sugar water preference rate = (sucrose water consumption/total water consumption) × 100%.
A self-made open-field box (5 × 5) was used to place the rat pinto in the central grid and adapt for 1 min. Record the activity of the rat in the open field for 4 min, including recording the stay time in the central grid, the number of crossing grids, and the number of standing upright (Sturman et al., 2018). The whole experimental process is under normal lighting conditions, and noise interference is avoided. After the measurement of each rat, the open field box will be cleaned and sprayed with alcohol before the measurement of the next rat.
On the first day after the experiment (the 50th day), each rat was placed in a 10 L glass beaker containing clean water at 25 ± 1°C, filled to a depth of 20 cm. The process took 6 mins, and the rat’s immobile time was recorded for 4 min. Immobility was defined as no movement of limb or body, including staying afloat, except movements caused by staying afloat (Eid et al., 2020).
After the end of the experiment, the blood of rats was collected, placed in the blood collection vessel without heparin sodium, after standing for 30 min, centrifuge at 3500 r/min for 14 min at 4°C, and the serum was separated and divided into 2.5 ml EP tubes, and stored in the refrigerator at -80°C.
After the end of the experiment, the rat brain was quickly dissected on ice, and the hippocampus samples were separated and placed in a 1.5 ml cryopreserved tube. The samples were quickly frozen in liquid nitrogen and stored in a refrigerator at -80°C.
Rat testosterone ELISA kit (No.F16854), Rats estradiol ELISA kit (No.F15321), and Rat aromatase P450 19A1 ELISA kit (No.F11248) were obtained from Sangon Biotech (Shanghai) Co., Ltd. and performed according to the manufacturer’s instructions.
Total RNA was extracted from hippocampal tissues by referring to the specification of EZ-10RNA Miniprep kits (No.B618133, Sangon Biotech (Shanghai) Co., Ltd.). A multi-functional enzyme marker determined the OD260/280 value and OD260/230 value of RNA. The OD260/280 of RNA ranged from 1.8 to 2.0, and OD260/230 was more significant than 2.0, indicating that the purity of RNA was good. Then configure the reverse transcription system (20 μl reaction system) according to the instructions of the M-MuLV First Strand cDNA Synthesis Kit (No.b532435, Sangon Biotech (Shanghai) Co., Ltd.) to reverse transcribe mRNA into cDNA and perform the reaction on ice. Finally, prepare the PCR reaction solution on ice according to the instructions of the SGExcel FastSYBR Mixture (No. B532955, Sangon Biotech (Shanghai) Co., Ltd.). The PCR reaction procedure is pre-denaturation at 95°C for 3 min, then denaturation at 95°C for 5 s, annealing and extension at 60°C for 20 s, and a total of 40 cycles. Each sample of RT-qPCR was repeated in three replications. The primer sequence is shown in Table 2.
Western blot was used to analyze the levels of AR, CYP19A1, ERα, ERβ, ERK1, and ERK2 proteins in the hippocampus. Put 30 mg of hippocampus tissue into a 1.5 ml EP tube, add 150 μl of RIPA lysate mixture (PMSF: RIPA = 1:100), ground with tissue grinder for 2 min, then put it into ice water for 1 h. During this period, took it out and shook it several times to ensure that the tissue was fully lysed. After centrifugation at 13,000 rpm for 15 min at 4°C, take the supernatant to obtain the total protein solution. Then, that protein concentration was determined using a BCA protein detection kit (No.M1003, miniBio). Each sample of the protein concentration was quantitatively set at 2.5 mg/ml, and the prepared protein samples were boiled in a 99°C metal bath for 10 min and then stored at −80°C. The protein samples were separated by 8% SDS-PAGE (No.M1014, minibio) and transferred onto polyvinylidene difluoride membranes, and then blocked with TBST sealant (5% skimmed milk powder) at 37°C. Select the desired strips on the transferred PVDF membrane, and place them in the corresponding primary antibody diluent. After complete incubation, place the washed target strip in the diluent of the secondary antibody and react on a shaker at 37°C for 1 h. After total incubation, remove the target strip and wash it with TBST 3 times. Rabbit Anti-Estrogen Receptor alpha Polyclonal Antibody (No.bs-0122R, Bioss); Rabbit Anti-Androgen Receptor Polyclonal Antibody (No.bs-0118R, Bioss); Rabbit Anti-Estrogen Receptor beta Polyclonal Antibody (No.bs-0116R, Bioss); P44/42 MAPK (ERK1/2) Rabbit mAb (No.137F5, Cell Signaling); Phospho-P44/42 MAPK (ERK1/2) (Thr202/Tyr204) Rabbit mAb (No.D13.14.4E, Cell Signaling); Anit-Aromatase antibody (EPR4532-2) (No.ab124776, Abcam); GAPDH Rabbit mAb (No.D16H11, Cell Signaling); The mime color protein marker is 10–180kda (No.M1028, minibio);
The 2−ΔΔCt method was used to calculate the quantitative real-time PCR results (Livak and Schmittgen, 2013). The experimental data were expressed as mean ± standard deviation (mean ± SD). One-way ANOVA was used for comparison between groups, and SPSS 22.0 was used for statistical analysis. The results of ANOVA were p < 0.05 or p < 0.01.
The behavioral results indicated that the rat model of depression was successfully replicated after 3 weeks of modeling (Figure 2). Treatment was administered in the following 4 weeks of modeling, and analysis of behavioral data revealed that all treated groups could improve depression-like behavior in CUMS rats. Compared with the CUMS group, the sucrose preference rate (Figure 2A) was significantly increased after treatment with Chaigui granule, Ven (positive group), and Shu (positive group) at the 7 weeks, indicating significant improvement in the anhedonia and reward behavior dysregulation in CUMS model rats. The crossing number (Figure 2B) and the rearing number (Figure 2C) were also significantly increased after the treatment at the 7 weeks, indicating the improvement of mobility reduction, reduction of acting ability, and capability of exploring new things in the CUMS rats. However, the immobility time was significantly decreased in the FST (Figure 2D), indicating that desperate behavior in CUMS model rats was improved and regulated considerably. At the 7 weeks, the body weights of rats in the Chaigui granule, Ven, and Shu groups were significantly increased compared with the CUMS group (Figure 2E). These results indicated that the Chaigui granule remarkably improved the depressive-like behavior of CUMS-induced rats and had notably anti-depressant effects.
Previous studies have found that Chaigui granule can reverse the level of T in the serum of depressed rats (Chen et al., 2015). However, Sex hormone levels in the brain are not only related to self-synthesis but also to peripheral sex hormone levels, which is based on the fact that sex hormones can cross the blood-brain barrier (Cui et al., 2013). In order to investigate the potential antidepressant mechanism of Chaigui granule and whether it also regulates E2, we measured the contents of T and E2 in the serum and hippocampus of experimental rats. Compared with the control group, T and E2 were lower in both serum and hippocampus in the CUMS group (Figures 3A–D), suggesting that CUMS modeling could lead to disturbance of sex hormone levels in rats. In contrast, the levels of T and E2 in serum and hippocampus were significantly increased after gavage with Chaigui granule (Figures 3A–D). These results showed that Chaigui granule had a significant inhibitory effect on the decrease of T and E2 contents in serum and hippocampus induced by CUMS rats. Based on the above findings and previous literature research, we are very interested in how the Chaigui granule regulates E2 levels in the hippocampus of depressed rats. Next, we examined CYP19A1 content in the hippocampus of experimental rats. It was found that the content of CYP19A1 in the hippocampus of CUMS group was also lower than in the control group (Figure 3E); however, after treatment, Chaigui granule could significantly inhibit the decrease of CYP19A1 level in the hippocampus of rats induced by CUMS modeling. However, venlafaxine and Shuganjieyu capsule did not show regulatory effects on T, E2, and CYP19A1 in the serum or hippocampus of treated rats. Therefore, the regulatory impact of Chaigui granule on T, E2 and CYP19A1 content may be a potential mechanism of its antidepressant effect.
Hippocampus is the critical lesion in depression. According to previous studies (Prange-Kiel et al., 2003; Rune and Frotscher, 2005), the estradiol level in the brain can play an anti-depressant role. To further investigate whether Chaigui granule exerts antidepressant efficacy by regulating estradiol and its downstream signaling pathways, we measured ERKs signaling pathways downstream of E2 using RT-qPCR and WB techniques. It was shown that the mRNA levels of CYP19A1, AR, ERα, ERβ, ERK1, and ERK2 mRNA in the hippocampus of CUMS group were significantly reduced compared with the control group. In contrast, the levels of CYP19A1 mRNA in the hippocampus were significantly increased after administration of Chaigui granule compared with the CUMS group (Figure 4A). Positive drugs Ven and Shu had no ameliorating effect But had a regressive impact on hippocampal ERβ mRNA with no pronounced difference. The levels of AR and ERα and ERβ mRNA in the hippocampus were significantly increased after administration of Chaigui granule and Ven compared with the CUMS group (Figures 4B–D). Positive drugs Ven and Shu had no improvement effect on the AR or ERα mRNA in the hippocampus but had a regressive effect on hippocampal ERβ mRNA. The levels of ERK1 and ERK2 mRNA in the hippocampus were significantly increased after the administration of Chaigui granule, Shu, and Ven compared with the CUMS group (Figures 4E,F). Compared with the control group, the protein levels of CYP19A1, ERα, ERβ, ERK1/2, and p-ERK1/2 in the hippocampus of CUMS group were significantly reduced. The protein levels of CYP19A1, ERα, and ERβ in the hippocampus were significantly increased after the treatment (Figures 5B–D). The protein levels of ERK1/2 and p-ERK1/2 in the hippocampus were also considerably increased after the Chaigui granule and Ven administration compared with the CUMS group (Figures 5E,F). These results suggest that Chaigui granule can not only regulate the content of E2 in the hippocampus of CUMS rats but also regulate its downstream ERKs signaling pathway from two levels: gene and protein, thus exerting antidepressant effects.
Depression is a psychiatric disorder with a high prevalence and high disability rate. Although the current clinical estrogen therapy is effective, it is also associated with many side effects. Chaigui granule is a new type of anti-depressant drug. In the previous experiment, the composition of the Chaigui granule was further studied and 95 compounds were explicitly identified. This provides a basis for the study of the chemical composition of the Chaigui granule (Xg et al., 2020). Combined with the previous research, the Chaigui granule also had an excellent regulatory effect on the reduction of serum T levels caused by depression (Chen et al., 2015). However, the impact of Chaigui granule on estrogen and its anti-depressant effect has not been studied. In this study, the anti-depressant effect of Chaigui granule was evaluated by the therapeutic administration to CUMS depression rat model (i.e., modeling followed by drug administration while continuing modeling). Currently, the clinical treatment of depression patients is mostly post-diagnosis pharmacotherapy. Therefore, we used the therapeutic administration method to evaluate the therapeutic administration efficacy of the Chaigui granule to fit the clinical treatment mode better. It is worth noting that the dosage of Chaigui granule in this experiment was obtained based on previous research (Teng et al., 2020). Although the recognized modeling procedure for CUMS lasts for 4 weeks, the current modeling cycle used to establish the CUMS depression model varies (Fu et al., 2018; Li et al., 2019). In this study, behavioral tests were performed on rats on the basis of 3 weeks of modeling, and it was found that the CUMS depression rat model had been successfully replicated after analysis of behavioral data, and then the depressed rats were treated on this basis. By analyzing the final behavioral results, we can find that all the administration groups of Chaigui granule improved the depression-like behavior of CUMS depressed rats.
It is well-known that the hippocampus plays a vital role in depression. Depressed patients are accompanied by varying degrees of hippocampus damage. The effects of E2 in the hippocampus may alter the processing of emotional information and subsequent memory. Also the presence of E2 seems to provide conditions for neurons to create new synapses by increasing dendritic spines; estradiol treatment protects hippocampus synapses from the deleterious effects of acute cortisol elevation (Albert and Newhouse, 2019). The source of E2 in the hippocampus may be the entry of peripheral E2 through blood circulation or the catalytic synthesis of CYP19A1 in the hippocampus (Aizawa et al., 2016). Because sex hormones can enter the brain through the blood-brain barrier by free diffusion (Cui et al., 2013), the hippocampus has an intact steroidase system that generates estradiol (Rebourcet et al., 2020). However, experimental data showed that T levels in the hippocampus were two orders of magnitude lower than T in serum. In contrast, E2 levels in the hippocampus were twice as high as in serum. These results suggest that E2 in the hippocampus be converted mainly by T catalyzed by the CYP19A1 enzyme, which may also be one reason for the lower T levels in the hippocampus than in the serum. The results showed that the Chaigui granule could alleviate the decrease of CYP19A1 and E2 levels in the hippocampus of rats induced by CUMS, demonstrating the regulatory effect of the Chaigui granule on sex hormone levels in the hippocampus. At the same time, the results showed that the positive drugs venlafaxine had no significant impact on the levels of sex hormones in the serum and hippocampus of CUMS depressed rats. However, some studies have shown that the levels of testosterone in the testes of venlafaxine-treated rats were significantly elevated (Fds et al., 2021). Many clinical studies showed that venlafaxine treatment led to the decline of patients’ sexual function. It is considered that low testosterone level is related to venlafaxine treatment (Clayton et al., 2009). Shuganjieyu also had no significant effect on the levels of sex hormones in the serum and hippocampus of CUMS depressed rats. Through literature research, it was found that shuganjieyu combined with paroxetine hydrochloride can increase the level of serum E2 in perimenopausal women (Xi et al., 2021). Still, no study has demonstrated a significant callback effect of shuganjieyu on sex hormone levels in depressed patients.
First, the mRNA and protein expression results of CYP19A1 can further prove that the Chaigui granule can promote the conversion of T to E2 by regulating CYP19A1 in the hippocampus. Second, Studies have found that E2 can specifically bind to different ERs, activating different cascade signaling pathways and exerting anti-depressant effects. Among them, the MAPK/ERKs signaling pathway is the main pathway for learning, memory, and emotional response in the brain (Yang et al., 2014). Chaigui granule had a regulatory effect on ERα mRNA but did not significantly improve ERα protein levels. mRNA is a template for protein synthesis, whereas transcription and translation are required for protein synthesis. However, the synthesis from gene to protein is a very complex process, and many steps are needed for the final synthesis of protein. Although there is a close relationship between gene expression and protein expression, an increase in gene expression level does not necessarily lead to an increase in protein level. But, the Chaigui granule had a significant effect on ERβ mRNA status and protein expression, which indicated that E2 in the hippocampus might specifically bind ERβ to exert its biological influence demonstrating that the Chaigui granule could improve the regulation of downstream pathways by E2. It also has been shown that injection of ERKs antagonists into the dorsal hippocampus of experimental rats revealed that rats showed significant depression-like behavior (Duman et al., 2007). By analyzing the mRNA levels of relevant molecules on the ERKs pathway, we found that the expression of ERK1/2 and p-ERK1/2 was differentially regulated by the Chaigui granule after the pharmacological intervention, which further demonstrated that the Chaigui granule could promote the specific binding of E2 to its receptor ERβ, which in turn activates the downstream ERK1/2 signaling pathway to exert antidepressant effects. However, T is also strongly associated with depression, and many studies support that testosterone can improve depressive mood (Osadshiy and Soldatkin, 2022). It has also been shown that depression-like behavior can be induced by decreasing serum and brain testosterone levels in rats (Li et al., 2022). Our previous results also showed a regulatory effect on T levels, for which we also measured the expression of AR mRNA. The results showed that the Chaigui granule could reverse the decrease of AR mRNA expression induced by depression. However, there was no modulation of AR protein in the administration groups. It suggests that Chaigui granule may exert antidepressant efficacy in multiple ways. Besides, Ven had a more substantial modulatory effect on ERβ than ERα, suggesting that venlafaxine could exert an anti-depressant influence by increasing the mRNA contents of ERβ, ERK1, and ERK2. Venlafaxine is an atypical bicyclic anti-depressant that effectively antagonizes the reuptake of 5-HT and NA. However, it was found that venlafaxine strongly regulated ER. After extensive literature research, the anatomical distribution of ER is consistent with the innervation area of 5-HT neurons, and estrogen has a particular regulatory effect on 5-HT. Therefore, venlafaxine may affect ER by regulating 5-HT (Bethea et al., 2002). This study clarified its anti-depressant mechanism by regulating sex hormone levels and thus ERKs signaling pathway, but there are still shortcomings. In the selection of experimental animals, it was initially thought that the sex hormone levels of male rats were relatively stable; therefore, male rats were used in this study. However, a follow-up study of female rats should be conducted comprehensively and in-depth to fully consider the effect of estrogen changes in female rats during different physiological cycles.
In this study, it was found that the therapeutic administration of Chaigui granule had significant antidepressant efficacy. Through further studies, it was found that Chaigui granule increased the CUMS-induced decrease in T and E2 contents in serum and hippocampus. After measuring the content of CYP19A1 in the hippocampus, the Chaigui granule was found to promote the conversion of T to E2 in the hippocampus. This may be an important reason why Chaigui granule exerts its antidepressant effect. To test our conjecture, estradiol receptors as well as downstream ERK signaling pathways, were investigated. From the results of RT-qPCR and WB, we concluded that the Chaigui granule could exert an anti-depression effect by promoting the conversion of T to E2 in the hippocampus and activating the ERK signaling pathway downstream of E2.
CUMS, A chronic unpredictable mild stress; ELISA, Enzyme-linked immunosorbent assay; RT-qPCR, reverse transcription-quantitative PCR; WB, Western blot; ERK, extracellular regulated protein kinases; T, testosterone; E2, estradiol; CREB, cAMP-response element binding protein; SPF, Sucrose preference test; OFT, Open field test; FST, Forced swimming test; AR, androgen receptors | true | true | true |
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PMC9637994 | Joseph S. Bowness,Tatyana B. Nesterova,Guifeng Wei,Lisa Rodermund,Mafalda Almeida,Heather Coker,Emma J. Carter,Artun Kadaster,Neil Brockdorff | Xist-mediated silencing requires additive functions of SPEN and Polycomb together with differentiation-dependent recruitment of SmcHD1 | 17-03-2022 | chromatin,epigenetics,X inactivation,Xist RNA,Polycomb,SMCHD1,SPEN | Summary X chromosome inactivation (XCI) is mediated by the non-coding RNA Xist, which directs chromatin modification and gene silencing in cis. The RNA binding protein SPEN and associated corepressors have a central role in Xist-mediated gene silencing. Other silencing factors, notably the Polycomb system, have been reported to function downstream of SPEN. In recent work, we found that SPEN has an additional role in correct localization of Xist RNA in cis, indicating that its contribution to chromatin-mediated gene silencing needs to be reappraised. Making use of a SPEN separation-of-function mutation, we show that SPEN and Polycomb pathways, in fact, function in parallel to establish gene silencing. We also find that differentiation-dependent recruitment of the chromosomal protein SmcHD1 is required for silencing many X-linked genes. Our results provide important insights into the mechanism of X inactivation and the coordination of chromatin-based gene regulation with cellular differentiation and development. | Xist-mediated silencing requires additive functions of SPEN and Polycomb together with differentiation-dependent recruitment of SmcHD1
X chromosome inactivation (XCI) is mediated by the non-coding RNA Xist, which directs chromatin modification and gene silencing in cis. The RNA binding protein SPEN and associated corepressors have a central role in Xist-mediated gene silencing. Other silencing factors, notably the Polycomb system, have been reported to function downstream of SPEN. In recent work, we found that SPEN has an additional role in correct localization of Xist RNA in cis, indicating that its contribution to chromatin-mediated gene silencing needs to be reappraised. Making use of a SPEN separation-of-function mutation, we show that SPEN and Polycomb pathways, in fact, function in parallel to establish gene silencing. We also find that differentiation-dependent recruitment of the chromosomal protein SmcHD1 is required for silencing many X-linked genes. Our results provide important insights into the mechanism of X inactivation and the coordination of chromatin-based gene regulation with cellular differentiation and development.
X chromosome inactivation (XCI) evolved in mammals to equalize the levels of X-linked gene expression in XX females relative to XY males (Lyon, 1961). The XCI process, which is developmentally regulated, is orchestrated by the X inactive specific transcript (Xist), a 17-kb non-coding RNA that accumulates in cis across the future inactive X (Xi) chromosome (Brockdorff et al., 1992; Brown et al., 1992; Lee and Jaenisch, 1997; Penny et al., 1996). Xist RNA recruits several factors that collectively modify chromatin/chromosome structure to silence X-linked genes (Boeren and Gribnau, 2021). Although XCI commences rapidly with the onset of Xist RNA expression, gene silencing is established progressively over a period of days (Borensztein et al., 2017; Lin et al., 2007; Marks et al., 2015; Sousa et al., 2019). Several X-linked genes exhibit partial or complete escape from X inactivation (Posynick and Brown, 2019). Gene silencing by Xist RNA is mediated principally by the A-repeat element, a tandem repeat located at the 5′ end of the transcript (Wutz et al., 2002). Recent studies have identified the RNA binding protein (RBP) SPEN as the critical silencing factor that recognizes this element (Lu et al., 2016; McHugh et al., 2015; Monfort et al., 2015). Thus, SPEN loss of function or deletion of the A-repeat result in strong abrogation of chromosome silencing (Chu et al., 2015; Dossin et al., 2020; McHugh et al., 2015; Moindrot et al., 2015; Monfort et al., 2015; Nesterova et al., 2019). SPEN interacts with the corepressor NCoR-HDAC3 through a C-terminal SPOC domain (Ariyoshi and Schwabe, 2003; Dossin et al., 2020), and this pathway plays an important role in Xist-mediated silencing (McHugh et al., 2015; Żylicz et al., 2019). Recent evidence indicates that the SPEN SPOC domain may also recruit other factors that contribute to Xi gene silencing (Dossin et al., 2020) and that SPEN has a SPOC-independent function in ensuring localization and accumulation of Xist RNA across the Xi chromosome (Rodermund et al., 2021). Accordingly, deletion/mutation of the SPEN SPOC domain alone does not fully recapitulate the silencing deficiency observed in SPEN-null cells or after deletion of the A-repeat (Dossin et al., 2020; Rodermund et al., 2021). Several factors in addition to SPEN have been implicated in Xist-mediated silencing (reviewed in Brockdorff et al., 2020). The most notable example is the Polycomb system, comprising the multiprotein Polycomb repressive complexes PRC1 and PRC2, which catalyze the histone modifications H2AK119ub1 and H3K27me3, respectively (de Napoles et al., 2004; Plath et al., 2003; Silva et al., 2003). Polycomb recruitment to Xi is initiated by the RBP hnRNPK, which also binds to a tandem repeat element in Xist RNA exon 1: the B/C-repeat (here PID region) (Bousard et al., 2019; Colognori et al., 2019; Nesterova et al., 2019; Pintacuda et al., 2017). hnRNPK interacts with PCGF3/5-PRC1 complexes to promote deposition of H2AK119ub1. This step triggers a positive feedback cascade leading to recruitment of other PRC1 complexes and PRC2 (Almeida et al., 2017). PCGF3/5 loss of function or deletion of the Xist PID region impairs Xi gene silencing, but not to the same degree as SPEN loss of function (Bousard et al., 2019; Colognori et al., 2019, 2020; Nesterova et al., 2019). Polycomb-mediated gene silencing in XCI has been attributed principally to PRC1 and H2AK119ub1 rather than PRC2/H3K27me3 (Almeida et al., 2017; Nesterova et al., 2019). Similar to SPEN, the Polycomb pathway has been reported to have a secondary function in Xist RNA localization (Colognori et al., 2019). Recruitment of SPEN and the Polycomb system to Xi occurs rapidly at the onset of Xist RNA expression, consistent with their role in early establishment of gene silencing (Dossin et al., 2020; Nesterova et al., 2019; Żylicz et al., 2019). Conversely, some other XCI-associated factors, notably the histone variant macroH2A and the chromosomal protein SmcHD1, are concentrated on Xi after several days of Xist RNA expression, as determined by analysis of differentiating XX mouse embryonic stem cells (mESCs) (Gendrel et al., 2012; Mermoud et al., 1999). SmcHD1 recruitment to Xi is dependent on establishment of PRC1-mediated H2AK119ub1 (Jansz et al., 2018a). Loss of SmcHD1 function results in female-specific embryo lethality attributable to failure of silencing in a subset of Xi genes (Blewitt et al., 2008; Gendrel et al., 2013; Mold et al., 2013). There is evidence that SmcHD1 facilitates long-term maintenance of X inactivation (Sakakibara et al., 2018; Wang et al., 2019), but a possible role in establishment of gene silencing has not been investigated in depth. SmcHD1 is important for DNA methylation of Xi CpG islands (Blewitt et al., 2008; Gendrel et al., 2012) and establishment of a unique chromosome architecture on Xi (Gdula et al., 2019; Jansz et al., 2018b; Wang et al., 2018). In this study, we analyze a series of loss-of-function mESC lines to elucidate the interplay of SPEN, Polycomb, and SmcHD1 in Xist-mediated X chromosome silencing.
In recent work, we derived SPENSPOCmut interspecific XX mESC lines with point mutations that abrogate SMRT/NCoR interaction with the SPEN-SPOC domain (Figure S1A; Ariyoshi and Schwabe, 2003; Oswald et al., 2016; Rodermund et al., 2021) and found that Xist-mediated silencing is only partly reduced, contrasting with reports using complete loss-of-function SPEN mutations (Dossin et al., 2020; Monfort et al., 2015; Nesterova et al., 2019). Equivalent conclusions were reached in an independent study that analyzed XX mESCs with deletion of the SPOC domain (Dossin et al., 2020). The degree of silencing observed after 1 day of Xist induction is highly similar in the two studies (Figure S2A), suggesting that the precise SPENSPOCmut point mutation has a biological effect equivalent to that seen with complete deletion of the SPOC domain. The silencing activity that is retained after mutation of the SPOC domain is referred to hereafter as SPOC-independent silencing. To further investigate SPOC-independent silencing, we extended our analysis across a more complete X inactivation time course. We optimized a protocol for deriving neuronal precursor cells (NPCs) from mESCs (Conti et al., 2005; Splinter et al., 2011), enabling analysis of the temporal trajectory of Xist-mediated silencing in a highly synchronous and homogeneous model. We assayed X-linked gene silencing by chromatin-associated RNA sequencing (ChrRNA-seq) over daily time points of NPC differentiation and found that Xist-mediated gene silencing is largely complete by around day 7 in wild-type (WT) interspecific XX lines with doxycycline-inducible Xist on the Mus musculus domesticus allele (iXist-ChrXDom) (Figure S2B). Accumulation of Xist RNA, as quantified from ChrRNA-seq data, increased progressively over the same time course (Figure S2B). We then analyzed Xist-mediated silencing during NPC differentiation in SPENSPOCmut mESCs, which were derived from the iXist-ChrXDom parental line. Consistent with our previous study (Rodermund et al., 2021), partial silencing is observed in SPENSPOCmut mESCs after 1 day of Xist induction (Figures 1A and S2C). SPOC-independent silencing persisted and, in fact, marginally increased at later time points up to day 6 of NPC differentiation (Figure 1A). These findings contrast with the near complete loss of silencing observed using the SPENΔRRM mutation (Nesterova et al., 2019) and A-repeat deletion (Coker et al., 2020), as shown in Figure S2C. Levels of Xist RNA were slightly reduced compared with WT iXist-ChrXDom (Figure 1B), but not to the dramatic extent observed in the SPENΔRRM or XistΔA lines (Figure S2C). Despite SPOC-independent silencing, from analysis of selected pluripotency (Nanog and Klf2) and neuronal (Nes and Vim) marker genes, we found tentative evidence of defective differentiation (Figure S2D), potentially indicating the mechanism by which two active X chromosomes in female mESCs antagonize exit from pluripotency (Schulz et al., 2014). Additionally, in later-stage differentiated cultures, we noticed X chromosome elimination in cell lines strongly deficient in X inactivation, reflecting selection against cells with two active X chromosomes (Colognori et al., 2020). We therefore limited our analysis to the first 6 days of differentiation, when over 90% of cells demonstrated clear Xist territories, and there were no subpopulations of XO cells apparent in WT or mutant lines (Figure S2E). To investigate the basis of SPOC-independent silencing, we defined a gene subset that shows greater silencing in SPENSPOCmut cells after 6 days of Xist induction (Figures 1C and S2F) and then compared their characteristics with those of other minimally silenced, strictly SPOC-dependent Xi genes. Using this approach, we found that SPOC-independent silencing is associated with lower expressed genes and with genes that are closer to the Xist locus (Figures 1D and 1E). We also observed an association with a local chromatin environment enriched in H3K27me3, as defined by the ChromHMM model (Figure S2G; Ernst and Kellis, 2017; Nesterova et al., 2019). These characteristics resemble those identified previously as being associated with Xi genes that are more affected by disruption of the Polycomb pathway (Nesterova et al., 2019; Pintacuda et al., 2017), indicating that the Polycomb system may underpin SPOC-independent silencing. Consistent with this idea, re-examination of H2AK119ub1 native chromatin immunoprecipitation sequencing (ChIP-seq) from SPENSPOCmut (Rodermund et al., 2021) revealed a greater enrichment of H2AK119ub1 over SPOC-independent genes compared with SPOC-dependent genes after 24 h of Xist induction in mESCs (Figure 1F).
To further investigate the link between SPOC-independent silencing and the Polycomb system, we examined the effect of depleting Polycomb function in SPENSPOCmut mESCs. We made use of the FKBP12F36V/dTAG-13 degron system (Nabet et al., 2018) to acutely deplete PCGF3/5-PRC1, the complex required to initiate Polycomb recruitment to Xi (Almeida et al., 2017). Using CRISPR-Cas9-facilitated homologous recombination, we introduced an FKBP12F36V degron to the N termini of PCGF3 and PCGF5 in iXist-ChrXDom mESCs (Figure S1B). Treatment of the tagged mESC line, iXist-ChrXDom FKBP12F36V-PCGF3/5, with the cell-permeable small molecule dTAG-13 resulted in rapid and complete degradation of both proteins within 15–30 min, with no detectable effect on protein levels of RING1B or SUZ12, core subunits of PRC1 and PRC2, respectively (Figure 2A). Consistent with a previous analysis of PCGF3/5 conditional knockout mESCs (Fursova et al., 2019), we observed an ∼30% global reduction in H2AK119ub1, determined by calibrated native ChIP-seq after 36 h of dTAG-13 treatment, attributable to reduced “blanket” coverage over intergenic or gene body regions rather than at canonical PRC1 target sites (Figure S3A). Genome-wide levels of H3K27me3 were broadly unchanged (Figure S3B). iXist-ChrXDom FKBP12F36V-PCGF3/5 was validated to confirm near-complete loss of Xi-specific accumulation of Polycomb-mediated H2AK119ub1 and H3K27me3, as determined by allelic ChIP-seq analysis (Figure 2B). ChrRNA-seq of Xist-mediated silencing in cells treated with dTAG-13 for 12 h prior to Xist induction demonstrated a moderate silencing deficiency (Figure 2C) equivalent to that reported in our previous work (Nesterova et al., 2019), with all X-linked genes somewhat deficient in silencing in the absence of PCGF3/5, although to varying degrees, on day 6 of NPC differentiation (Figure S3C). Levels of Xist RNA were broadly similar between untreated and dTAG-treated ChrRNA-seq samples (Figure S3D). We went on to engineer the SPEN SPOC mutation in iXist-ChrXDom FKBP12F36V-PCGF3/5 mESCs to analyze the effect of PCGF3/5 degradation on SPOC-independent silencing. Treatment of the combined mutants with dTAG-13 resulted in complete loss of Xist-mediated silencing over NPC differentiation in two independent clones (Figures 2D and S3E). As above, we only analyzed differentiation time points up to day 6 because of progressive selection for X chromosome elimination in differentiated cells with two active X chromosomes. Levels of Xist RNA were equivalent to SPENSPOCmut clones (Figure S3F; cf. Figure 1B). PCGF3/5-PRC1 complexes function globally in genome regulation, and it is conceivable that indirect effects of perturbing other pathways contribute to the silencing deficit we observed. To further validate our findings, we introduced the SPEN SPOC mutation into the iXist-ChrXDom XistΔPID XX mESC line, in which the Xist B/C-repeat region required for hnRNPK/Polycomb recruitment in X inactivation is deleted (Figure S1C; Nesterova et al., 2019). Allelic ChrRNA-seq analysis was carried out to assess Xist-mediated gene silencing after Xist induction and NPC differentiation. As shown in Figures 2E and S3G, SPOC-independent silencing was abolished in two independent clones. Levels of Xist RNA were similar to those seen in the parental XistΔPID line (Figure S3H). These results further support the conclusion that SPOC-independent silencing in XCI is attributable to activity of the Polycomb system. Abrogation of Xi Polycomb recruitment and the SPEN SPOC mutation have been reported to have effects on Xist RNA localization (Colognori et al., 2019; Markaki et al., 2021; Rodermund et al., 2021), and this could potentially contribute to the aberrant silencing observed when both pathways are depleted. Qualitative analysis of Xist RNA domains using RNA fluorescence in situ hybridization (FISH) and conventional microscopy indicates monoallelic Xist upregulation and cloud formation after 1 day of Xist induction in mESCs for all cell lines presented so far (Figures S4A–S4D). We did, however, observe a tendency toward larger Xist territories in FKBP12F36V-PCGF3/5 cells after dTAG-13 treatment (Figure S4B) and occasional Xist RNA dispersal in double mutant cells (Figures S4C and S4D). With this in mind, we went on to quantify Xist localization parameters using computational analysis of super-resolution 3D structured illumination microscopy (3D-SIM) of Xist RNA FISH in the iXist-ChrXDom FKBP12F36V-PCGF3/5 model. As summarized in Figures 3A and 3B, PCGF3/5 degradation alone and in combination with SPENSPOCmut resulted in expanded Xist RNA territories and an increased number of Xist RNA foci. A proportion of cells had partially or extensively dispersed Xist RNA, with the latter category being larger with the combined mutation (Figures 3C and 3D). We conclude that mislocalization of Xist RNA may make some contribution to the observed gene silencing deficiencies, but given that Xist expression and Xi domain formation are maintained in most cells, the deficit is unlikely to account for the complete abolition of silencing in the combined knockout. Our findings suggest that SPOC-independent silencing is, in large part, mediated by the Polycomb pathway, implying that Polycomb functions in parallel with rather than downstream of SPEN as the two principal pathways mediating establishment of Xist-mediated gene silencing.
The SPEN and Polycomb systems are recruited rapidly to Xi at the onset of Xist RNA expression, but silencing of individual genes proceeds over several hours and, in some cases, days (Borensztein et al., 2017; Lin et al., 2007; Loda et al., 2017; Marks et al., 2015; Nesterova et al., 2019; Sousa et al., 2019). There is evidence that cellular differentiation promotes Xist-mediated silencing, although this is based on analysis of only two X-linked genes (Loda et al., 2017). To further address this latter point, we assayed allelic silencing chromosome wide after long-term (10 days) continuous Xist RNA induction in undifferentiated mESCs compared with NPC differentiation conditions. For these experiments, we used iXist-ChrXDom and a reciprocal interspecific XX mESC line with the inducible promoter driving Xist RNA expression on the Mus musculus castaneus X chromosome (iXist-ChrXCast) (Figures 4A–4C, S5A, and S5B). Unlike iXist-ChrXDom, iXist-ChrXCast is informative across the entire X chromosome, enabling allelic expression analysis of a larger number of X-linked genes. As shown in Figures 4A and 4B, silencing is significantly reduced in undifferentiated mESCs compared with cells differentiated to NPCs. Principal-component analysis of autosomal gene expression (Figure 4C) and marker gene analysis (Figures S5A and S5B) confirm retention of mESC identity in these experiments. We went on to determine whether there is a relationship between dependence on cellular differentiation and silencing dynamics. We defined a silencing half-time (t1/2) for each X-linked gene by fitting the silencing trajectory with an exponential decay model, summarizing over multiple ChrRNA-seq time points and replicates from NPC differentiation time courses of iXist-ChrXDom and iXist-ChrXCast (Figure S5C). Half-times were strongly correlated between cell lines (Figure S5D; R = 0.82, Spearman’s rank correlation) and were used to classify genes into three equally sized groups showing fast, intermediate, and slow silencing kinetics (Figure S5E). Consistent with prior reports (Marks et al., 2015; Nesterova et al., 2019; Sousa et al., 2019), we found that proximity to the Xist locus and expression levels prior to Xist induction influence the dynamics of gene silencing, with higher-expressed genes and genes farther from Xist silencing more slowly (Figure S5F). We then examined the relationship between silencing dynamics and genes that fail to complete silencing after 10 days of Xist induction in undifferentiated mESCs and found a clear correspondence between X-linked genes showing differentiation dependence and intermediate/slowly silencing groups (Figure 4D).
A possible explanation for the link between Xi silencing and differentiation is involvement of a factor(s) not available in undifferentiated mESCs. A candidate for this function is the chromosomal protein SmcHD1, which is recruited to Xi dependent on PRC1 activity (Jansz et al., 2018a), but only after several days, based on analysis of XX mESCs undergoing embryoid body differentiation (Gendrel et al., 2012). Re-examination of the kinetics of SmcHD1 association with Xi by immunofluorescence (IF) in our NPC model with inducible Xist expression confirms that SmcHD1 recruitment is a late step, becoming detectable only after 3–4 days of Xist induction and NPC differentiation (Figures 5A and 5B). Differences in timing relative to prior analysis using embryoid body differentiation may reflect the lag in onset of Xist RNA expression from the native versus inducible promoter. Western blot analysis demonstrates that overall levels of SmcHD1 are equivalent in mESCs and throughout NPC differentiation (Figures 5C and S6A), indicating that SmcHD1 availability itself does not account for delayed Xi recruitment. SmcHD1 recruitment to Xi could not be detected in XX mESCs after 10 days of continuous induction of Xist RNA expression (Figures 5D and 5E). Differentiation-dependent recruitment of SmcHD1 was previously interpreted to support a role in maintenance rather than establishment of Xi gene silencing (Gendrel et al., 2012). However, silencing of genes showing intermediate/slow kinetics is incomplete at the time of SmcHD1 recruitment (Figure S6B), raising the possibility that SmcHD1 has a role in establishing silencing for these gene groups. With this in mind, we performed an analysis of the relationship between silencing dynamics and SmcHD1 dependency, with the latter being based on classifying genes as dependent (n = 56), partially dependent (n = 143), not dependent (n = 101), or escapees (genes also expressed from Xi in the WT, n = 18) using ChrRNA-seq data from XX mouse embryonic fibroblast (MEF) lines derived from SmcHD1-null embryos (Figure S6C; Gdula et al., 2019). As illustrated in Figure 5F, we observed a clear association between SmcHD1 dependence and silencing dynamics, with SmcHD1-dependent genes strongly overlapping with slowly silencing genes. We also found that SmcHD1 dependency is associated with genes showing incomplete silencing after long-term Xist expression in undifferentiated mESCs, as illustrated in Figure S6D for the iXist-ChrXCast cell line.
The aforementioned observations suggest that SmcHD1 could have a role in establishment of Xi silencing (in addition to its defined role in maintenance of Xi silencing), specifically in relation to genes that exhibit a relatively slow silencing trajectory. To test this idea directly, we used CRISPR-Cas9-mediated mutagenesis to generate SmcHD1 knockout (KO) cell lines in iXist-ChrXDom and iXist-ChrXCast backgrounds (Figures S1D and S7A). Generation of homozygous KO clones was confirmed by western blot analysis (Figure 6A). KO lines were then validated to confirm that enrichment of Polycomb-linked histone modifications over Xi is maintained (Figure S7B) and that the nuclear SmcHD1 signal and Xi enrichment are absent after 7 days of Xist induction under NPC differentiation conditions (Figures 6B and S7C). We went on to analyze Xi gene silencing in SmcHD1 KO mESCs using an extended time course of NPC differentiation. Data for a single iXist-ChrXDom-derived cell line and two iXist-ChrXCast-derived SmcHD1 KO cell lines alongside WT controls are presented in Figures 6C and S7D. At early time points, up to 5–7 days of differentiation, silencing in SmcHD1 KO cells proceeded with a trajectory similar to that seen in WT cells. However, at later time points, Xi gene silencing plateaued in SmcHD1 KO cells. To further investigate this observation, we analyzed silencing for the different SmcHD1 dependency groups as defined above. As shown in Figure 6D, silencing of SmcHD1 “not dependent” genes proceeded to completion, whereas SmcHD1-dependent and partially dependent genes retain some activity on Xi at all stages of NPC differentiation. Similar results were obtained for SmcHD1 KO iXist-ChrXCast mESCs (Figure S7E). Differences between the WT and KO for SmcHD1-dependent and partially dependent genes become apparent at day 5 of NPC differentiation (Figures 6D and S7E, bottom panels), which correlates with the time of SmcHD1 recruitment to the Xi chromosome (Figures 5A and 5B). These results demonstrate that SmcHD1 contributes to establishment of silencing at a specific subset of genes during Xist-mediated chromosome silencing in a differentiation-dependent manner and, accordingly, that completion of the X inactivation process occurs only in cells that have transitioned away from the pluripotent state.
Our findings reinforce the view that Xist-mediated gene silencing is largely attributable to chromatin modification by the SPEN and Polycomb pathways but also highlight that the two pathways function in parallel. It is important to stress that this is not due to regulation of mutually exclusive gene subsets but, rather, a varying contribution to silencing on a gene-by-gene basis. Genes more affected by disruption of the Polycomb system are generally expressed at a lower level in mESCs and are located in chromatin environments with higher initial H3K27me3 levels, indicative of proximity to sites where the Polycomb system is targeted independent of Xist RNA expression. In contrast, genes strongly dependent on the SPEN system tend to be more highly expressed in mESCs (Nesterova et al., 2019), which is in line with recent evidence that SPEN is rapidly recruited to active promoters and enhancers of X-linked genes upon Xist expression (Dossin et al., 2020). That SPEN and Polycomb function in parallel is supported by the observations that SPOC-independent silencing persists in mESCs differentiated into NPCs and that complete loss of silencing occurs in SPENSPOCmut mESCs after depletion of PCGF3/5 or deletion of the Xist B/C-repeat, the Polycomb complex subunits and Xist RNA region required for Xist-mediated Polycomb recruitment, respectively. A caveat when reaching these conclusions is that SPENSPOCmut and Polycomb mutations have minor but significant effects on Xist RNA behavior/localization and that this is increased in mESCs with both pathways abrogated. However, as noted above, these effects are unlikely to account for the complete loss of silencing we observed in the latter scenario. Recent work has shown that the catalytic activity of PRC1 complexes, specifically deposition of H2AK119ub1, is essential for maintenance of Polycomb target gene repression in mESCs (Blackledge et al., 2020; Tamburri et al., 2020). We have reported previously that the effects of Polycomb on Xist-mediated silencing are largely attributable to PRC1 activity (Nesterova et al., 2019), and it follows that this is likely linked to H2AK119ub1 deposition over Xi. However, exactly how H2AK119ub1 affects Xist-mediated silencing remains to be established. Possible mechanisms include direct effects on chromatin structure (for example, compaction or transcriptional inhibition) and indirect effects involving reader proteins. In X inactivation, recruitment of the chromosomal protein SmcHD1 is an example of the latter (Jansz et al., 2018a). However, a contribution of PRC1 in Xist-mediated silencing is seen prior to SmcHD1 recruitment, demonstrating that other mechanisms, direct or indirect, are also important. Further studies are required to determine whether this is linked to direct effects of H2AK119ub1 on chromatin structure/transcription or other unidentified H2AK119ub1 reader proteins. Recent work characterizing an analogous model cell line lacking Xist-mediated Polycomb recruitment (ΔΒ/MS2-Xist) reported reduced Xi compaction compared with WT Xist, as determined by DAPI staining and DNA paint measurements of chromosome volume (Markaki et al., 2021). Although potentially offering evidence of a direct role of Polycomb in chromosome compaction, these effects were most pronounced after 4 days of mESC differentiation, so a contribution of SmcHD1 to this compaction phenotype (see below) cannot be discounted. Although the SPEN and Polycomb pathways are sufficient for initial establishment of Xist-mediated silencing, repression of individual X-linked genes progresses with very different trajectories, ranging from hours to several days. In this study, we find that, for genes that are normally inactivated relatively slowly, cellular differentiation is required to complete the silencing process. Thus, dependence on cellular differentiation for completion of silencing is apparent for a significant proportion of X-linked genes. Formally, this could be attributable to a factor or factors whose presence is limited to differentiated cells or to different properties of mESCs relative to differentiated derivatives; for example, a more rapid cell cycle. Support for the former possibility is exemplified by the chromosomal protein SmcHD1, which is recruited to Xi only in differentiated mESC derivatives. Our analysis demonstrates a role of SmcHD1 in establishment of silencing, affecting genes with intermediate/slow silencing dynamics. The pathway that elicits Xi SmcHD1 recruitment in differentiated cells is unknown. We speculate that a factor present only in differentiated cells facilitates SmcHD1 recognition of H2AK119ub1. Previous work indicates that this is unlikely to be the loading factor LRIF1, which mediates SmcHD1 recruitment to H3K9me3-modified chromatin via interaction with HP1 proteins but is dispensable for SmcHD1 localization to Xi in MEFs (Brideau et al., 2015). Similarly, the mechanism of action of SmcHD1 in X inactivation is also poorly understood. Previous work has pointed to functions in chromosome compaction (Nozawa et al., 2013), DNA methylation of CpG islands (Blewitt et al., 2008; Gendrel et al., 2012), and eviction of cohesin/CTCF in the context of formation of Xi-specific higher-order chromosome structures (Gdula et al., 2019; Jansz et al., 2018b; Wang et al., 2018). Further studies are needed to address this issue in terms of SmcHD1 function in establishment and maintenance of Xist-mediated silencing. Our findings provide a comprehensive view of key steps underpinning the establishment of Xist-mediated silencing, as summarized in Figure 7. In a wider context, the role of SmcHD1 in reinforcing gene repression by the Polycomb system specifically in differentiated cells is likely to be relevant at other Polycomb target loci (for example, Hox gene clusters), and therefore provides an important paradigm for how epigenetic mechanisms contribute to locking gene expression states as cells transition from pluripotency to terminal differentiation.
Analysis of gene silencing in X inactivation as described here is performed in a cell culture model that may differ subtly from the physiological setting, early embryogenesis. Our analysis of differentiated cells is restricted to NPCs, and it is possible that other cell lineages would show some differences in the extent or rate of silencing of specific genes. Xist expression in our model is driven by an introduced inducible promoter, and this may mean that there are differences in the quantitative output of Xist RNA and its timing relative to that which occurs from the native Xist promoter. Use of SNPs ascribing the allelic origin of X-linked transcripts to the M. m. castaneus and M. m. domesticus genomes is informative for a good proportion of genes, but some genes are not evaluated because of absence of SNPs, very low gene expression levels, or the recombination event in the iXist-ChrXDom line. Finally, in experiments that examine the effect of combined loss of Polycomb and SPEN-SPOC-mediated silencing, we cannot precisely quantify the degree to which Xist RNA localization effects contribute to the observed loss of silencing.
Further information and requests for resources and reagents should be directed to and will be fulfilled by the lead contact, Neil Brockdorff ([email protected]).
All oligos, plasmids, and cell lines generated in this study are available from the Lead contact without restriction.
All female (XX) mouse embryonic stem cell (mESC) lines used for this study were originally derived from the parental F1 2-1 line (129/Sv-CAST/EiJ, a gift from J. Gribnau). mESCs were routinely maintained in Dulbecco’s Modified Eagle Medium (DMEM; Life Technologies) supplemented with 10% foetal calf serum (FCS; ThermoFisher), 2 mM L-glutamine, 0.1 mM non-essential amino acids, 50 μM β-mercaptoethanol, 100 U/mL penicillin/100 μg/mL streptomycin (all from Life Technologies) and 1000 U/mL LIF (made in-house). mESCs were grown on gelatin-coated plates under standard culture conditions (37°C, 5% CO2, humid) atop a ‘feeder’ layer of MitomycinC-inactivated (Merck Life Science) SNLP mouse fibroblasts and passaged upon ∼80% confluency every 2-3 days using TrypLE Express (ThermoFisher) at room temperature. Xist expression was driven by a TetOn promoter induced by addition of 1 μg/mL of doxycycline (Merck Life Science, D9891). Prior to experiments, cells were pre-plated for 30-40 min on gelatinized dishes to allow feeder cells to preferentially attach, with slower-attaching mESCs then taken from suspension and plated on feederless gelatinized dishes to be harvested for further protocols upon confluency (ie. 2-3 days later). For calibrated ChIP-seq experiments, Drosophila S2 (Sg4) cells were grown adhesively at 25°C in Schneider’s Drosophila Medium (Life Technologies) supplemented with 1 × Pen/Strep and 10% heat-inactivated FCS. Cell counting was performed with a Countess 3 Automated Cell Counter (ThermoFisher).
Single guide RNAs used for generating CRISPR-Cas9-mediated double-strand breaks at target loci were designed using the CRISPOR online tool (Concordet and Haeussler, 2018) and are listed in the Key resources table. Sequences in bold are those encoding the sgRNA sequences complementary to target sites in the genome. Cloning into sgRNA plasmid vectors was performed using reverse complement oligos and the single-step digestion-ligation Zhang lab protocol (Broad Institute) into the pX459 background (Ran et al., 2013; Addgene plasmid #62988). Homology vectors for CRISPR-assisted homologous recombination were cloned by Gibson Assembly using oligonucleotides synthesized from Invitrogen. Briefly, 300-500 bp homology fragments were amplified by PCR from iXist-ChrX genomic DNA using FastStart High Fidelity enzyme (Merck Life Science). N-terminal FKBP12F36V fragments were originally amplified from pLEX_305-N-dTAG (Addgene #91797) (Nabet et al., 2018). Gibson assembly ligation into a restriction-enzyme digested pCAG backbone plasmid was then performed using Gibson Assembly Master Mix (NEB) according to manufacturer’s guidance. Products from digestion-ligation or Gibson assembly reactions were transformed into XL10-Gold ultracompetent bacteria (Agilent). DNA was isolated from bacterial colonies using the Miniprep kit (Qiagen) and confirmed as containing the desired plasmid via Sanger sequencing.
Generation of the parental iXist-ChrX cell lines is described in our previous study (Nesterova et al., 2019) (iXist-ChrXDom = clone B2, iXist-ChrXCas = clone C7). SPENSPOCmut and FKBP12F36V-PCGF3/5 mutant cell lines were generated by CRISPR-assisted homologous recombination by co-transfection of cloned homology and Cas9-sgRNA plasmids at a molar ratio of 6:1 (2.5 μg of homology vector, ∼1 μg of sgRNA vector). SmcHD1 KO cells were transfected with 1 μg of each Cas9-sgRNA plasmids for CRISPR/Cas9-mediated mutagenesis. Transfections were performed as follows: 1–1.5 × 106 mESCs were plated into wells of a 6-well plate ∼24 hours prior to transfection. Pen/strep were removed from the growth media ∼3 hours prior to transfection of plasmid vectors using Lifofectamine2000 (ThermoFisher) according to the manufacturer’s protocol. The following day, each well was split into several 90 mm plates at low density and cells were subjected to puromycin selection (∼3 μg/mL) from 48 to 96 hours post-transfection. Following puromycin wash-out, cells were grown under regular mESCs conditions for a further 8-10 days until clonal colonies could be isolated in 96-well plates and positive clones validated and expanded. Summary details for all mESC lines used in this study, sgRNA sequences and plasmid vectors can be found in the Key resources table or Table S1. Oligonucleotides used for screening and PCR validation of cell lines are also listed in Table S1.
mESC to NPC differentiation protocols from the literature (Conti et al., 2005; Splinter et al., 2011) were adapted and optimized for iXist-ChrX lines as follows. mESCs were first extensively separated from feeder cells by pre-plating four times for 35-40 min each. 0.5 × 106 mESCs were then plated to gelatin-coated T25 flasks and grown in N2B27 media (50:50 DMEM/F-12:Neurobasal (Gibco) supplemented with 1 × N2 and 1 × B27 (ThermoFisher), 1 mM L-glutamine, 100 μM β-mercaptoethanol, 50 U/mL penicillin/50 μg/mL streptomycin (all from Life Technologies) with 1 μg/mL doxycycline for continuous Xist induction. On day 7, cells were detached from the base of the flask with Accutase (Merck Life Sciences), and 3 × 106 cells were plated to grow in suspension in 90 mm bacterial petri dishes containing N2B27 + Dox media supplemented with 10 ng/mL EGF and FGF (Peprotech). On day 10, embryoid-body-like cellular aggregates were collected by mild centrifugation (100 g for 2 min) and plated back onto gelatin-coated 90 mm dishes in N2B27 + Dox + FGF/EGF media. When NPC outgrowths reached ∼80% confluency, cells were detached by Accutase treatment and split at 1:3–1:4 ratio on gelatinized 90 mm petri dishes. For long term differentiated timepoints cells were maintained in N2B27 + Dox + FGF/EGF media and passaged as above. For dTAG13-treated FKBP12F36V-PCGF3/5 and combined FKBP12F36V-PCGF3/5 + SPENSPOCmut lines, 100 μM dTAG-13 was added 12 hours prior to initial pre-plating and maintained in the growth media throughout the protocol.
Nuclear extracts were made from cell pellets of confluent 90 mm dishes (3 × 107 cells, 1 × packed cell volume, PCV). Briefly, cell pellets were washed with PBS then resuspended in 10 × PCV buffer A (10 mM HEPES-KOH pH7.9, 1.5 mM MgCl2, 10 mM KCl, with 0.5 mM DTT, with freshly added 0.5 mM phenylmethylsulfonyl fluoride (PMSF) and complete protease inhibitors (Roche)). After 10 min on ice to allow cell swelling, cells were centrifuged (1500 g for 5 min at 4°C) and resuspended in 3 × PCV buffer A + 0.1% NP40 (Merck Life Science). After 10 further min on ice, nuclei were collected by centrifugation (400 g for 5 min at 4°C) then resuspended in 1 × PCV buffer C (250 mM NaCl, 5 mM HEPES-KOH pH 7.9, 26% glycerol, 1.5 mM MgCl2, 0.2 mM EDTA-NaOH pH8, with fresh 0.5 mM DTT and protease inhibitors). NaCl was then added dropwise up to a concentration of 350 mM, and the extract was incubated for 1 hour on ice with occasional agitation. After centrifugation (16000 g for 20 min at 4°C) the supernatant was taken as soluble nuclear extract. This was quantified by Bradford’s assay (Bio-Rad) and stored at −80°C until use.
Nuclear extracts were used for Western blot analysis of all proteins shown in this study. Samples were loaded onto home-made polyacrylamide gels and transferred to PVDF or nitrocellulose membranes using a Trans-blot Turbo (Bio-Rad). Membranes were then blocked for 1 hour at room temperature in 10 mL blocking buffer: 100 mM Tris pH 7.5, 0.9% NaCl, 0.1% Tween (TBST) and 5% Marvel milk powder. Blots were incubated overnight at 4°C with primary antibody (see Key resources table), washed four times with blocking buffer, then incubated with constant agitation at room temperature for 1 hour in secondary antibody of the relevant species conjugated to horseradish peroxidase. After washing twice more in blocking buffer, once in TBST, and once in PBS (10 min each), membranes were developed and visualized using Clarity Western ECL substrate (Bio-Rad).
Immunofluorescence was performed as described in our previous study (Nesterova et al., 2019). Cells were grown on gelatinized slides or 13 mm diameter coverslips (VWR) for at least a day prior to experimental fixation. Slides were washed with PBS and then fixed with 2% formaldehyde for 15 min followed by 5 min of permeabilization in 0.4% Triton X-100. All procedures were performed at room temperature (RT). Cells were briefly washed with PBS before blocking with a 0.2% PBS-based solution of fish gelatin for three washes of 5 min each. Primary antibody dilutions were prepared in fish gelatin solution with 5% normal goat serum and added to cells on slides for 2 hours of incubation in a humid chamber at room temperature. Slides were then washed three times in fish gelatin solution. Secondary antibodies were diluted 1:400 in fish gelatin solution and incubated with cells on slides for 1 hour in a humid chamber. After incubation, slides were washed twice with fish gelatin and one time with PBS before mounting using Vectashield mounting medium with DAPI. Excess mounting medium was removed and the coverslips were sealed to slides using nail varnish. Cells were analysed and scored for the presence of Xi domains on an inverted fluorescence Axio Observer Z.1 microscope using a PlanApo ×63/1.4 NA oil-immersion objective. Images were acquired using AxioVision software.
Cells for each sample were split to grow on gelatin-coated 22 mm coverslips in wells of 6-well plates and fixed at ∼70% confluency after ∼48 hours. Xist expression was induced for 1 day, and in the case of FKBP12F36V lines dTAG-13 was added 12 hours prior to doxycycline addition. At collection, cells on coverslips were washed once with PBS, fixed in the 6-well plate with 3% formaldehyde pH 7 for 10 min, then washed once with PBS, twice with PBST.5 (0.05% Tween20 in PBS), and transferred into a new 6-well dish for permeabilization in 0.2% Triton X-100 in PBS for 10 min at RT. After three further PBST.5 washes, cells on cover slips were subjected to ethanol dehydration by an initial incubation with 70% EtOH (for 30 min at RT), then progressive exchanges to 80%, 90% and finally 100% EtOH. Xist FISH probe was prepared, starting on the previous day, from an 18 kb cloned cDNA (pBS_Xist; Key resources table) spanning the whole Xist transcript using a nick translation kit (Abbott Molecular). The FISH hybridization mix consisted of: 3 μL Texas Red-labelled Xist probe (∼50 ng DNA), 1 μL 10 mg/mL Salmon Sperm DNA, 0.4 μL 3 M NaOAc and 3 volumes of 100% EtOH per sample. This was precipitated by centrifugation (20,000 g for 20 min at 4°C), washed with 70% EtOH, dried by air or speed vacuum, resuspended in 6 μL deionized formamide (Merck Life Science) per hybridization, then incubated in a shaker (1400 rpm) at 42°C for at least 30 min. 2× hybridization buffer (4× SSC, 20% dextran sulfate, 2 mg/mL BSA (NEB), 1/10 volume nuclease free water and 1/10 volume vanadyl-ribonucleoside complex (VRC; pre-warmed at 65°C for 5 min before use)) was denatured at 75°C for 5 min, placed back on ice to cool, then mixed with hybridization mix. Each coverslip was hybridized with 30 μL probe/hybridization mix in a humid box at 37°C overnight. The next day, coverslips were washed 3 times for 5 min at 42°C with pre-warmed 50% formamide/2× saline-sodium citrate buffer (1/10 20× SSC in PBST.5), then subjected to further washes (3 x 2XSSC, 1 × PBST.5, 1 × PBS, each for 5 min using a 42°C hot plate) before being mounted with VECTASHIELD with DAPI (Vector Labs) onto Superfrost Plus microscopy slides (VWR). Slides were dried and sealed using clear nail polish prior to imaging. 5-10 images (20-40 cells per image) were acquired with AxioVision software on an inverted fluorescence Axio Observer Z.1 microscope (Zeiss) using a PlanApo ×63/1.4 NA oil-immersion objective. Images for all lines were scored together, blinded, for the presence or absence of a noticeable Xist RNA domain.
Cells were grown on 18 × 18 mm No. 1.5H precision cover slips (Marienfield) for 3D-SIM microscopy and treated as described above. Coverslips were washed twice with PBS and fixed using 3% formaldehyde (pH 7) in PBS for 10 min at room temperature. A stepwise exchange of PBST.5 was carried out, before cells were permeabilized using 0.2% Triton X-100 PBS for 10 min at room temperature, then blocked for 30 min (2% BSA/0.5% fish skin gelatine/PBST.5, 2U/μL RNAsin Plus (Promega) at room temperature). Coverslips were washed twice in PBST.5, then once in 2× SSC before an overnight incubation at 37°C with FISH probe/hybridization buffer (prepared as above) in a humid chamber. The following day coverslips were washed with 2× SSC as detailed above, then stained with 2 μg/mL DAPI in PBST.5 for 10 min at room temperature. Coverslips were washed again with PBS, then milliQ water, before mounting as above but using Vectashield mounting medium (no DAPI), and imaged within a week using the DeltaVision OMX V3 Blaze system (GE Healthcare).
3D-SIM imaging was performed on a DeltaVision OMX V3 Blaze system (GE Healthcare) equipped with a 60×/1.42 NA Plan Apo oil immersion objective (Olympus), pco.edge 5.5 sCMOS cameras (PCO), and 405, 488, 593 and 640 nm lasers. Image stacks were acquired with a z-distance of 125 nm and with 15 raw images per plane (5 phases, 3 angles). Spherical aberration after reconstruction was reduced by using immersion oil of different refractive indices (RIs) matched to respective optical transfer functions (OTFs). Here, immersion oil with an RI of 1.514 was used for the sample acquisition and matched to OTFs generated using immersion oil of RI 1.512 for the blue, and 1.514 for the red channel. OTFs were acquired using 170 nm diameter blue emitting PS-Speck beads and 100 nm diameter green and red emitting FluoSphere beads (Thermo Fisher Scientific).
The raw data was computationally reconstructed with softWoRx 6.5.2 (GE Healthcare) using channel-specific OTFs and Wiener filter settings of 0.005. A lateral (x-y) resolution of approximately 120 nm and an axial (z) resolution of approximately 320 nm was achieved (Miron et al., 2020). All data underwent assessment via SIMcheck (Ball et al., 2015) to determine image quality via analysis of modulation contrast to noise ratio (MCNR), spherical aberration mismatch, reconstructed Fourier plot, and reconstructed intensity histogram values. Reconstructed 32-bit 3D-SIM datasets were thresholded to the stack modal intensity value and converted to 16-bit composite z-stacks to discard negative intensity values using SIMcheck’s “threshold and 16-bit conversion” utility and MCNR maps were generated using the “raw data modulation contrast” tool of SIMcheck. To eliminate false positive signals from reconstructed noise, we applied a modulation contrast filtering using an adapted in-house Fiji script (Rodermund et al., 2021; Schindelin et al., 2012). Here, all pixels in the reconstructed dataset where the corresponding MCNR values in the raw data map fall below an empirically chosen threshold of 4.0 are set to zero intensity. Thereafter, the resulting ‘masked’ reconstructed dataset is blurred with a Gaussian filter with 0.8 pixel radius (xy) to smoothen hard edges.
Color channels were registered in 3D with the open-source software Chromagnon 0.85 (Matsuda et al., 2018) determining alignment parameter (x,y,z-translation, x,y,z-magnification, and z-rotation) from a 3D-SIM dataset acquired on the date of image acquisition of multicolor-detected 5-ethenyl-2′-deoxyuridine (EdU) pulse replication labeled C127 mouse cells serving as biological 3D alignment calibration sample (Rodermund et al., 2021).
Reconstructed 3D-SIM image stacks were pre-processed and subjected to modulation contrast filtering as described above (Rodermund et al., 2021). Thereafter, lateral color channel alignment was performed as described above. The resulting images were used as representative images of whole nuclei. For further analysis however, the DAPI channel was discarded, and Xist territories were cropped manually using Fiji to exclude signal from neighboring cells. The cropped dimensions were later used to define Xist territory volume in all different cell types. The processed 3D-SIM image files were analyzed using an in-house adapted makefile script for masking of the signal and centroid determination by watershed algorithm (Rodermund et al., 2021). The output data was used to determine the number of Xist foci in all different cell types. Additionally, localization phenotypes observed in the different cell lines and conditions were scored by eye. Xist RNA territories were scored as either “localized”, “slightly dispersed” or “fully dispersed” based on the fully processed 3D-SIM images.
Between 5 × 106 (NPC) and 3 × 107 (mESC) cells were collected from confluent 90 mm dishes, washed once with PBS, then snap-frozen and stored at −80°C. Chromatin extraction was performed as follows: Cell pellets were lysed on ice for 5 min in RLB (10 mM Tris pH7.5, 10 mM KCl, 1.5 mM MgCl2, and 0.1% NP40). Nuclei were then purified by centrifugation through 24% sucrose/RLB (2800 g for 10 min at 4°C), resuspended in NUN1 (20 mM Tris pH7.5, 75 mM NaCl, 0.5 mM EDTA, 50% glycerol, 0.1 mM DTT), and then lysed by gradual addition of an equal volume NUN2 (20 mM HEPES pH 7.9, 300 mM NaCl, 7.5 mM MgCl2, 0.2 mM EDTA, 1 M Urea, 0.1 mM DTT). After 15 min incubation on ice with occasional vortexing, the chromatin fraction was isolated as the insoluble pellet after centrifugation (2800 g for 10 min at 4°C). Chromatin pellets were resuspended in 1 mL TRIzol (Invitrogen) and fully homogenenized and solubilized by eventually being passed through a 23-gauge needle 10 times. This was followed by isolation of chromatin-associated RNA through TRIzol/chloroform extraction with isopropanol precipitation. Precipitated RNA pellets were washed twice with 70% ethanol. Final ChrRNA samples were then resuspended in H2O, treated with TurboDNAse and measured by Nanodrop (both ThermoFisher). 500 ng–1 μg of RNA was used for library preparation using the Illumina TruSeq stranded total RNA kit (RS-122-2301).
Calibrated native ChIP-seq was performed largely as described in our previous studies (Nesterova et al., 2019; Rodermund et al., 2021) using buffers supplemented with 5 mM of the deubiquitinase inhibitor N-ethylmaleimide (Merck Life Science) for H2AK119ub1 ChIP. 4 × 107 mESCs and 1 × 107 Drosophila Sg4 Cells (20% cellular spike-in) were carefully counted using a Countess 3 Automated Cell Counter (ThermoFisher) and pooled. Cells were then lysed in RSB (10 mM Tris pH8, 10 mM NaCl, 3 mM MgCl2, 0.1% NP40) for 5 min on ice with gentle inversion before nuclei collection by centrifugation (1500 g for 5 min at 4°C). Nuclei were resuspended in 1 mL of RSB +0.25 M sucrose +3 mM CaCl2, treated with 200U of MNase (Fermentas) for 5 min at 37°C, quenched with 4 μL of 1M EDTA, then centrifuged at 2000 g for 5 min. The supernatant was transferred to a fresh tube as fraction S1. The remaining chromatin pellet was incubated for 1 hour in 300 μL of nucleosome release buffer (10 mM Tris pH7.5, 10 mM NaCl, 0.2 mM EDTA), carefully passed five times through a 27G needle, and then centrifuged at 2000 g for 5 min. The supernatant from this S2 fraction was combined with S1 to make the final soluble chromatin extract. For each ChIP reaction, 100 μL of chromatin was diluted in Native ChIP incubation buffer (10 mM Tris pH 7.5, 70 mM NaCl, 2 mM MgCl2, 2 mM EDTA, 0.1% Triton) to 1 mL and incubated with Ab (see Key resources table) overnight at 4°C. Samples were incubated for 1 hour with 40 μL protein A agarose beads pre-blocked in Native ChIP incubation buffer with 1 mg/mL BSA and 1 mg/mL yeast tRNA, then washed a total of four times with Native ChIP wash buffer (20 mM Tris pH 7.5, 2 mM EDTA, 125 mM NaCl, 0.1% Triton X-100) and once with TE pH 7.5. All washes were performed at 4°C. The DNA was eluted from beads by resuspension in elution buffer (1% SDS, 100 mM NaHCO3) and shaking at 1000 rpm for 30 min at 25°C, and was purified using the ChIP DNA Clean and Concentrator kit (Zymo Research). Enrichment of ChIP DNA at predicted sites for each chromatin modification was confirmed by qPCR using primers given in Table S1 and SensiMix SYBR (Bioline, UK). 25–100 ng of ChIP DNA was used for library prep using the NEBNext Ultra II DNA Library Prep Kit with NEBNext Single indices (E7645).
Next-generation sequencing DNA libraries were loaded on a Bioanalyzer 2100 (Agilent) with High Sensitivity DNA chips to verify fragment size distribution between 200-800 bp. Sample libraries were quantified using a Qubit fluorometer (Invitrogen) and, optionally, by qPCR with KAPA Library Quantification DNA standards (Roche) and SensiMix SYBR (Bioline) before being pooled together. 2 × 81-cycle paired-end sequencing was performed using an Illumina NextSeq500 (FC-404-2002).
The standard ChrRNA-seq data mapping pipeline is reported in our previous study (Nesterova et al., 2019). Briefly, raw fastq files of read pairs were first mapped to rRNA by bowtie2 (v2.3.2; Langmead and Salzberg 2012) and rRNA-mapping reads discarded (typically <2%). The remaining unmapped reads were aligned to an N-masked mm10 genome with STAR (v2.4.2a; Dobin et al., 2013) using parameters: “-outFilterMultimapNmax 1 -outFilterMismatchNmax 4 -alignEndsType EndToEnd”. Aligned reads were assigned to separate files for either the Cast or Dom/129S genomes by SNPsplit (v0.2.0; Krueger and Andrews, 2016) using the “- paired” parameter and a SNPfile containing the 23,005,850 SNPs between Cast and Dom/129S genomes (UCSC). Read fragments overlapping genes, for both the ‘unsplit’ and ‘allelic’ files of each sample, were counted by the program featureCounts (Liao et al., 2014) using an annotation file of all transcripts and lncRNAs from NCBI RefSeq and the parameters “-t transcript -g gene id -s 2”. Principle Component Analysis (PCA) of iXist-ChrX samples was performed using the DESeq R package (Anders and Huber, 2010). A variance stabilizing transformation (VST) was applied to a count matrix containing all samples and the top 500 differentially expressed autosomal genes were taken for calculation of principle components. Further allelic analysis was performed using R and RStudio on allelic count matrix output files from featureCounts. X-linked genes with at least 10 allelically-assigned fragments (i.e. containing reads that overlap SNPs) in >80% of WT samples were retained for gene silencing analysis. Gene silencing was assessed by calculating the allelic ratio of read counts, given by . An additional filter on the allelic ratio in uninduced mESCs (0.1 < allelic ratio < 0.9) was also applied, as strongly monoallelic genes are likely to be technical artifacts of singular mis-annotated SNPs. Kinetic modeling of gene silencing dynamics was performed using WT iXist-ChrX samples collected in NPC differentiation time course experiments. Exponential model curve fitting was performed using the “nlsLM” function from the “minpack.lm” R package (Elzhov et al., 2016) to a model of the form:(y = allelic ratio; t = time; = final allelic ratio; = initial allelic ratio) where is fixed for genes that undergo complete inactivation but is allowed as a parameter for escapees (defined as allelic ratio >0.1 in mature NPCs). Fitting was done first to the entire dataset in order generate initial parameter estimates. These were then used as inputs for linear regression to fit the model to the silencing trajectory of each gene individually. Model fitting was possible for the vast majority of allelic chrX genes analyzed. Silencing halftimes were calculated by the formula:where , and are parameters of the exponential model fit, and F = 0.5 (to calculate half of ). Halftimes were used to categories X-linked genes into equal classes of fast, intermediate, and slow kinetics of silencing. For instances where genes are directly categorized or compared by their ‘Initial Expression Level’, this was done using mRNA-seq data from iXist-ChrXDom mESCs (two replicates averaged together). This data, which contains very few intronic reads, allows for the calculation of a ‘Transcripts per kilobase Million (TPM)’ value for each gene in the count matrix, and hence categorisation of genes into equal groups of low, medium or high expressed. For instances where the relative expression of the same gene (or in the case of Xist, the number of chromatin-associated transcripts) was compared across ChrRNA-seq samples, a simpler RPM (aka CPM; Reads/Counts per Million) transformation of the count matrix was used. Summary tables characterizing X-linked genes by all the comparison metrics used in this study are provided in Table S2. Plots used to visualize ChrRNA-seq analysis in this study (eg. box, scatter, bar, violin, PCA) were primarily generated using ggplot2 and associated packages in R.
For ChIP-seq experiments quantitatively calibrated with Drosophila Sg4 cells, raw fastq reads were mapped with bowtie2 (v2.3.2; Langmead and Salzberg, 2012) to the N-masked mm10 genome concatenated with the dm6 Drosophila genome. Parameters “–very-sensitive –no-discordant –no-mixed -X 2000” were used and unmapped read pairs were removed. Alignment files were sorted using samtools (Li et al., 2009), PCR duplicates were marked and discarded by the picard-tools ”MarkDuplicates” programme, and reads were allelically-assigned using SNPsplit (Krueger and Andrews, 2016). For spike-in calibration, reads mapping to the mm10 and dm6 genomes, in both IP and matched input samples, were counted by samtools. Calibration factors were then calculated according to the derived formula for occupancy ratio (ORi) (Hu et al., 2015) and are provided in Table S3.
Meta-profiles from ChIP-seq datasets collected in iXist-ChrXDom-derived cell lines were generated from normalized/calibrated bigWig files using the “reference-point” mode of “computeMatrix” in the deeptools suite (Ramírez et al., 2014), followed by the “plotProfile” function. Profiles were centered either on TSSs from gene annotations (UCSC refGene), or published datasets of SUZ12 and RING1B peak locations in mESCs (Fursova et al., 2019).
Total and allele-specific alignment (BAM) ChIP and input files were processed into bedGraph format by bedtools “genomeCoverageBed” (Quinlan and Hall, 2010) and calibrated by factors given in Table S3. The custom Python script ExtractInfoFrombedGraph_AtBed.py (https://github.com/guifengwei) was then used to extract values of signal for 250 kb windows spanning the 103.5 Mb ‘chrX1’ region that can be allelically-analysed. These files were loaded into RStudio for further data processing. Briefly, IP files were first normalized to appropriate input files to calculate enrichment (IP/input) for each window across the chromosome. Line graphs of allelic enrichment were calculated for each sample by subtraction of Xa enrichment from Xi enrichment (Xi – Xa) and are thus ‘internally’ normalized to be more robust to technical variability (eg. in ChIP efficiency) between samples. Data points in boxplots represent allelic enrichment for each window calculated as the ratio of Xi enrichment compared to Xa enrichment (Xi/Xa). ‘Poor mappability’ regions were defined as windows with outlier signal in non-allelic input (+/− 2.5 median absolute deviation). ‘Low allelic’ regions were defined as windows ranking in the lowest 5% of signal in allelic input files. 79/414 windows classified as either ‘poor mappability’ or ‘low allelic’ windows are masked by shaded regions in line graphs and excluded from associated boxplots.
Statistical tests were performed in R using base or “ggpubr” packages. For instances where statistical significance is indicated in figures, the test employed is stated in the accompanying legend. The Wilcoxon rank-sum (aka Mann-Whitney U) test was favored for comparisons between two independent, unpaired samples. This test determines whether it is equally likely a randomly selected value from one sample will be less than or greater than a randomly chosen value from another. It is a non-parametric alternative to the unpaired t test which does not assume normal distributions about a mean, and so is appropriate for calculating significance for many comparisons in this study. Fisher’s exact test was used for comparing the categorical data collected of Xist RNA dispersal in Figure 3. Sample sizes are provided where applicable in either the main text or figure legends. In all cases, the quantity represented by n (eg. the number of genes, cells, or experimental replicates) is made clear. | true | true | true |
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PMC9638004 | Daniel J. Wilcock,Andrew P. Badrock,Chun W. Wong,Rhys Owen,Melissa Guerin,Andrew D. Southam,Hannah Johnston,Brian A. Telfer,Paul Fullwood,Joanne Watson,Harriet Ferguson,Jennifer Ferguson,Gavin R. Lloyd,Andris Jankevics,Warwick B. Dunn,Claudia Wellbrock,Paul Lorigan,Craig Ceol,Chiara Francavilla,Michael P. Smith,Adam F.L. Hurlstone | Oxidative stress from DGAT1 oncoprotein inhibition in melanoma suppresses tumor growth when ROS defenses are also breached | 21-06-2022 | melanoma,lipid droplets,DGAT1,fatty acids,reactive oxygen species,SOD1,oxidative stress | Summary Dysregulated cellular metabolism is a cancer hallmark for which few druggable oncoprotein targets have been identified. Increased fatty acid (FA) acquisition allows cancer cells to meet their heightened membrane biogenesis, bioenergy, and signaling needs. Excess FAs are toxic to non-transformed cells but surprisingly not to cancer cells. Molecules underlying this cancer adaptation may provide alternative drug targets. Here, we demonstrate that diacylglycerol O-acyltransferase 1 (DGAT1), an enzyme integral to triacylglyceride synthesis and lipid droplet formation, is frequently up-regulated in melanoma, allowing melanoma cells to tolerate excess FA. DGAT1 over-expression alone transforms p53-mutant zebrafish melanocytes and co-operates with oncogenic BRAF or NRAS for more rapid melanoma formation. Antagonism of DGAT1 induces oxidative stress in melanoma cells, which adapt by up-regulating cellular reactive oxygen species defenses. We show that inhibiting both DGAT1 and superoxide dismutase 1 profoundly suppress tumor growth through eliciting intolerable oxidative stress. | Oxidative stress from DGAT1 oncoprotein inhibition in melanoma suppresses tumor growth when ROS defenses are also breached
Dysregulated cellular metabolism is a cancer hallmark for which few druggable oncoprotein targets have been identified. Increased fatty acid (FA) acquisition allows cancer cells to meet their heightened membrane biogenesis, bioenergy, and signaling needs. Excess FAs are toxic to non-transformed cells but surprisingly not to cancer cells. Molecules underlying this cancer adaptation may provide alternative drug targets. Here, we demonstrate that diacylglycerol O-acyltransferase 1 (DGAT1), an enzyme integral to triacylglyceride synthesis and lipid droplet formation, is frequently up-regulated in melanoma, allowing melanoma cells to tolerate excess FA. DGAT1 over-expression alone transforms p53-mutant zebrafish melanocytes and co-operates with oncogenic BRAF or NRAS for more rapid melanoma formation. Antagonism of DGAT1 induces oxidative stress in melanoma cells, which adapt by up-regulating cellular reactive oxygen species defenses. We show that inhibiting both DGAT1 and superoxide dismutase 1 profoundly suppress tumor growth through eliciting intolerable oxidative stress.
The growth and survival of cancer cells is underpinned by dysregulated cellular metabolism (DeBerardinis and Chandel, 2016), which further promotes cancer progression by reprogramming stromal cells, mediating evasion of immune responses, and promoting metastasis (Andrejeva and Rathmell, 2017; Lee et al., 2018; Lyssiotis and Kimmelman, 2017). A central facet of cancer cell metabolism is a shift in glucose usage away from oxidative phosphorylation toward biosynthetic reactions. Without compensation by fatty acid oxidation (FAO) and glutaminolysis, this shift would result in deficiencies in citric acid cycle intermediates needed for ATP production (DeBerardinis and Chandel, 2016). Although catabolism of fatty acids (FA) maintains ATP production, increased lipogenesis (lipid generation) is simultaneously required for cell membrane synthesis, for which FA are principal building blocks (Koundouros and Poulogiannis, 2020). Furthermore, lipid signaling molecules such as phosphatidylinositides, diacylglycerides (DAG), lysophosphatidic acid, and prostaglandins, implicated in multiple cancer hallmarks (Chen and Huang, 2019), are also derived from FA. To satisfy these competing demands, cancer cells increase FA supply. This can be achieved by initiating de novo FA synthesis, normally a function of adipocytes and hepatocytes but an acquired characteristic of cancer cells driven by elevated FA synthase (FASN) expression (Koundouros and Poulogiannis, 2020). Alternatively, FA can be acquired from the diet, directly from the circulation or indirectly from adipose tissue. Cancer cells utilize secreted lipases like lipoprotein lipase (LPL) to hydrolyze FA from triglycerides, and FA transporter proteins (FATP) like CD36 and SLC27 family members and FA-binding proteins (FABP) to facilitate uptake (Lengyel et al., 2018). Enhanced FA uptake by cancer cells promotes tumor growth and dissemination (Nieman et al., 2011; Pascual et al., 2017; Watt et al., 2019). Intracellular lipases also liberate FA from intracellular lipid stores (Maan et al., 2018). One such lipase, monoacylglycerol lipase (MAGL or MGLL), is upregulated in aggressive cancers, wherein its suppression impedes tumor growth and metastasis (Nomura et al., 2010). Whereas adipocytes are adapted to store FA, non-adipose, non-transformed cells that become overloaded with FA suffer from lipotoxicity, characterized by reduced insulin signaling (insulin resistance) and increased cell death (Brookheart et al., 2009). Multiple processes underlie lipotoxicity, including increased ceramide synthesis, dysregulation of phospholipid production compromising mitochondrial and ER membrane integrity, impaired ATP generation, and induction of reactive oxygen species (ROS) (Brookheart et al., 2009). How dysregulated metabolism favoring FA acquisition is tolerated by cancer cells and how the toxic by-products of rampant FAO (principally ROS, including lipid peroxides) are suppressed or neutralized, are incompletely understood. Targeting the ability of cancer cells to manage potentially cytotoxic metabolites that arise from the rewiring of metabolic pathways is an intriguing therapeutic avenue warranting further exploration. Diacylglycerol O-acyltransferase 1 (DGAT1) is an ER-resident enzyme that catalyzes the final step in triacylglyceride (TAG) synthesis from DAG and FA. DGAT1 is required for lipid droplets (LD), cytosolic organelles comprising a core of neutral lipids (mainly triglycerides and sterol esters) delimited by a monolayer of phospholipids (Wilfling et al., 2013). The signficance of DGAT1 up-regulation and subsequent formation of LD to cancer has recently been demonstrated in glioblastoma models, in which DGAT1 inhibition led to tumor cell death through induction of lipotoxicity and oxidative stress (Cheng et al., 2020). Here, we demonstrate the oncogenic ability of DGAT1 in melanoma using zebrafish and find that DGAT1 again exerts this effect primarily through shielding melanoma cells from lipotoxicity. Consequently, DGAT1 inhibition results in oxidative stress in melanoma cells but this is countered by adaptive up-regulation of ROS-neutralizing enzymes such as superoxide dismutase 1 (SOD1). However, simultaneous DGAT1 and SOD1 inhibition leads to catastrophic levels of ROS accumulating in tumor cells, triggering their death.
Few oncoproteins directly dysregulating cellular metabolism have been identified. Point mutations in the isocitrate dehydrogenases IDH1 and IDH2 have been confirmed as oncogenic drivers in only 0.9%–3% of all cancers (Molenaar et al., 2018) (Figure S1A). The gene encoding FASN is frequently amplified in cancer (Koundouros and Poulogiannis, 2020) (Figure S1A); however, FASN inhibitors have failed to gain clinical approval (Flavin et al., 2010). LPL, CD36, FATP1 (SLC27A1), and FATP2 (SLC27A2), implicated in increased FA uptake into melanoma cells (Alicea et al., 2020; Henderson et al., 2019; Pascual et al., 2017; Zhang et al., 2018), are unaffected by point mutation in cancer and display gene amplification in 2%–8% of cases (Figure S1A). Given this dearth of therapeutic targets but also the significance of lipid metabolism for melanoma development (Fischer et al., 2018), we interrogated genes that we previously identified as accompanying progression of oncogenic RAS-driven melanoma in zebrafish (Henderson et al., 2019). Plotting expression fold change against association with patient survival, dgat1a/DGAT1 emerged as an outlier, being both highly up-regulated in zebrafish melanoma and significantly associated with reduced patient survival (Figures 1A and 1B). In contrast, expression of the gene encoding the functionally related, although structurally distinct, Dgat2/DGAT2 did not change significantly, nor was it associated with patient survival (Figures 1A and 1B). Furthermore, we confirmed elevated expression of human DGAT1 but not DGAT2 mRNA in melanoma tumors relative to both skin and nevi (Figure 1C) and elevated DGAT1 protein in human melanoma cell lines relative to primary melanocytes irrespective of NRAS or BRAF mutational status (Figure 1D). We next considered what might underlie DGAT1 up-regulation in melanoma. Visualization of structural alterations of the DGAT1 gene in the Cancer Genome Atlas firehose legacy cutaneous melanoma dataset using cBioPortal revealed significant focal amplification (as defined by the stringent GISTIC 2.0 algorithm) in up to 7% of melanoma cases with available copy number variation (CNV) data (Figure 1E), comparable to other recognized melanoma oncogenes (namely CCND1, KIT, CDK4, MITF, TERT, and MDM2). An extra copy of chromosome 8q, containing the DGAT1 locus but also other putative melanoma oncogenes ASAP1/DDEF, MYC, and GDF6 (Ehlers et al., 2005; Schlagbauer-Wadl et al., 1999; Venkatesan et al., 2018) (Figure 1F), has been observed in approximately 30% of melanoma cases (Bastian et al., 1998). Consistently, DGAT1, ASAP1, MYC, and GDF6 are co-amplified in melanoma and other cancers (Figures S1B and S1C). However, of these four, DGAT1 mRNA expression displayed the strongest association with reduced patient survival (Figures 1B and S1D). Furthermore, our previous comparative oncogenomic analysis (Venkatesan et al., 2018) uncovered amplification of dgat1a together with gdf6b (both on chromosome 19) in oncogenic-BRAF-driven zebrafish melanoma, concomitant with up-regulation of dgat1a and gdf6b mRNA (Figure 1G and Table S1). In contrast, neither dgat1b nor gdf6a on chromosome 16, nor any myc or asap1 paralogs (on chromosomes 2 and 24) were amplified or up-regulated (Figures 1G and S1E, and Table S1). Finally, we found DGAT1 to be frequently amplified in other human cancers, a feature of many well-characterized oncogenes, most notably in up to 26% of cases of ovarian cancer (Figures S1A and S1F). Strikingly, DGAT1 amplification was associated with significantly poorer progression-free survival across multiple cancer types (Figure 1H). Thus, from a cancer genomics perspective, DGAT1 exhibits the hallmarks of an oncogene.
In human melanoma, DGAT1 amplification was observed to co-occur with BRAF and NRAS mutation but also independently of either (Figure 2A). To elucidate further the oncogenic potential of DGAT1, we utilized a melanocyte rescue and lineage-restricted expression system as described previously (Ceol et al., 2011; Venkatesan et al., 2018). Remarkably, we found that forcing Dgat1a expression in zebrafish melanocytes lacking functional p53 was sufficient to induce melanoma (Figure 2B). Moreover, Dgat1a cooperated with both oncogenic BRAF and NRAS to accelerate the development of nodular tumors (Figures 2C–2E). Thus, in zebrafish, Dgat1 behaves as a melanoma oncoprotein. Significantly, effects of forcing Dgat2 expression were indistinguishable from the non-oncogenic EGFP control (Figure 2D), indicating that Dgat1 stimulation of tumorigenesis cannot be replicated by the functionally related Dgat2. In human melanoma models, DGAT1 also exhibited oncogenic characteristics. Forced expression of DGAT1 in the DGAT1Low melanoma cell line 888MEL led to an increase in cellular proliferation in all four clones tested (Figures 2F and S2A). Cancer cells can sustain their growth despite transient or limited nutrient availability in the tumor microenvironment (Ackerman and Simon, 2014). We therefore investigated the role of DGAT1 over-expression in allowing melanoma cells to tolerate nutrient and oxygen deprivation in culture. DGAT1-overexpressing melanoma cells exhibited a 100%–200% increase in cell number compared with parental DGAT1Low cells under conditions of greatest stress: 1% serum and hypoxia (1% O2) (Figures 2G, S2B, and S2C). A growth advantage conferred by DGAT1 over-expression was also observed in normal human melanocytes (NHM) (Figures 2H and S2D). Our zebrafish and human models demonstrate that in melanocytes DGAT1 has the hallmarks of an oncoprotein, conferring a growth advantage especially under stress conditions likely encountered in the tumor microenvironment.
Having uncovered DGAT1 as an oncoprotein, we next assessed the impact of DGAT1 antagonism in human melanoma cell lines. DGAT1 depletion using siRNA reduced cell proliferation and decreased the fractions of cells in S-phase (Figures 3A and S3A), in contrast to the increased proliferation and cell cycle progression observed with stable DGAT1 over-expression (Figures 2F and S2A). As DGAT1 has been mooted as a clinical target for combating obesity, several potent and selective small-molecule inhibitors are already available for repurposing (DeVita and Pinto, 2013). To determine whether DGAT1 depletion effects were due to the enzymatic function of DGAT1, we utilized four selective DGAT1 inhibitors (AZD3988, AZD7687, A922500, or T863). Similarly to siRNA-mediated depletion, pharmacological antagonism of DGAT1 suppressed growth of DGAT1High (A375, MM485, and SKMEL105) and DGAT1Amplified (LOXIMVI and SKMEL5) melanoma cells over 96 h (Figures 3B and S3B), accompanied by decreased cell cycle progression (Figure 3B). In contrast, no changes in cell growth or cell cycle progression were observed following DGAT2 depletion or inhibition (Figures S3C–S3E). Given that DGAT1 over-expression exhibited the largest relative effect on cell growth under conditions of cellular stress, we hypothesized that, conversely, the impact of DGAT1 inhibition would be exacerbated when cells were exposed to nutrient and hypoxic stress. Indeed, we found that DGAT1 antagonism significantly impaired proliferation when external lipid sources were restricted, with the greatest reduction in cell number observed at the lowest levels of serum (Figure 3C). Moreover, exposing cells to hypoxic conditions (1% O2) led to a further decrease in cell number upon DGAT1 inhibition (Figure 3D). Further highlighting the key role of DGAT1 up-regulation in melanoma cells, even under standard tissue culture conditions, siRNA knockdown or pharmacological inhibition of DGAT1 led to apoptosis, quantified through an increase in cleaved caspase-3 (Figures 3E and 3F). The significant impact of DGAT1 antagonism on melanoma cell proliferation and survival, exacerbated under conditions mimicking tumor microenvironmental stresses, highlights DGAT1 as a possible therapeutic target in melanoma.
To address the effect of DGAT1 on melanoma lipid metabolism, we performed ultra-high-performance liquid chromatography-mass spectrometry (UHPLC-MS) to identify and contrast lipid species extracted from NRASG12D Dgat1a-over-expressing or NRASG12D EGFP-expressing zebrafish tumors. This analysis revealed increased concentrations of almost all TAG species detected in tumors with forced Dgat1a expression, with longer polyunsaturated FA (PUFA) chains showing the greatest increases (Figures 4A and S4A and Table S2), although changes in individual species were not deemed statistically significant after correction for multiple testing. Several factors confounded the detection of lipid species changes in Dgat1a-over-expressing tumors. First, EGFP-expressing tumors also over-express Dgat1a and have ample LD (Henderson et al., 2019). Second, lipids originating from associated stromal cells dilute the lipids derived from tumor cells. In order to overcome the complexity of whole-tumor analysis, we turned to melanoma cell lines. Over-expression of DGAT1 in human melanoma cells resulted in a striking increase in LD (Figure 4B), consistent with cells adopting an engorged morphology (Figure 4B). Moreover, this was also observed in NHM upon DGAT1 over-expression (Figure S4B). In parallel, UHPLC-MS lipidomic analysis revealed an increase in almost all TAG species (Figure 4B and Tables S3 and S4), corroborating the trend in TAG seen in zebrafish tumors over-expressing Dgat1a (Figure 4A). Conversely, antagonism or depletion of DGAT1 in melanoma cell lines over-expressing endogenous DGAT1 led to a reduction in the amount of LD beginning between 12 and 24 h and continuing until 72 h (Figures 4C and S4C), as previously reported for glioblastoma cells (Cheng et al., 2020) and other cell types (Nguyen et al., 2017). In contrast, LD were not affected by DGAT2 depletion (Figure S4D). As expected, UHPLC-MS lipidomic analysis revealed a reduction of multiple TAG species already at 24 h that was maintained until 72 h, (Figures 4D and S4E and Tables S5 and S6), with TAG-containing PUFA particularly affected (Figure S4A). Alongside the anticipated changes in TAG levels following manipulation of DGAT1 activity, we also observed a decrease in several acylcarnitine (AcCa) species upon DGAT1 over-expression (Figure 4B and Tables S3 and S4) and a reciprocal increase in AcCa species following DGAT1 inhibition (Figures 4D, 4E, and S4E and Tables S5 and S6). AcCa, formed by esterification of FA (typically long chain) to L-carnitine, are transported into mitochondria to be processed into acetyl-CoA through β-oxidation. The production of AcCa determines the rate of FAO (Falomir-Lockhart et al., 2019). Moreover, excessive FAO can result in mitochondrial dysfunction (Koves et al., 2008). Indeed, we observed impaired ATP production following DGAT1 inhibition, implied by the increased phosphorylation of both AMPK and RAPTOR (Figure 4F). Furthermore, we observed reduced oxygen consumption in DGAT1-inhibited cells after 48 h (Figure 4G), implying decreased mitochondrial respiratory function despite levels of AcCa remaining elevated (Figures 4D and S5E). Additionally, post-24-h DGAT1 inhibition, we observed loss of mitochondrial membrane potential, decreased PINK1 cleavage, and increased levels of mitophagy factor PARKIN (Figure 4H). Moreover, by blocking AcCa synthesis using etomoxir (a carnitine palmitoyltransferase inhibitor), we could partially restore mitochondrial membrane potential and cell proliferation (Figures S4F and S4G). Taking these results together, we conclude that DGAT1 maintains mitochondrial function in melanoma cells by regulating the availability of FA for FAO.
To better understand the effects of DGAT1 inhibition on cellular signaling and metabolism, we performed MS-based whole-proteome analysis in SILAC-labeled A375 cells. Analysis of differentially expressed proteins highlighted three key areas: (1) FAO (consistent with increased AcCa availability), (2) peroxisome proliferator-activated receptor (PPAR) signaling, presumably indicating the increased availability of lipid regulators of PPAR transcription factors, and (3) nuclear regulatory factor 2 (NRF2) signaling, which is a well-established response to ROS production that dampens ROS-mediated cellular damage (Sajadimajd and Khazaei, 2018) (Figures 5A, S5A, and S5B). Quantitative gene expression analysis further corroborated the effects of DGAT1 inhibition on these three key biological processes (Figure 5B). To measure the impact of DGAT1 suppression on ROS generation, we stained cells with fluorescent probes whose emission is changed by ROS. We observed increasing ROS production over time upon DGAT1 inhibition in multiple melanoma cell lines (Figures 5C and S5C). We also observed an increase specifically in mitochondrial ROS upon both DGAT1 inhibition and depletion (Figures S5D and S5E), a predicted consequence of excessive FAO (Koves et al., 2008). Through a chain reaction, oxygen-centered ROS can yield highly reactive and cytotoxic lipid peroxides responsible for ferroptosis (Conrad and Pratt, 2019), a form of programmed cell death. Accordingly, DGAT1 inhibition led to an increase in lipid peroxidation from 24 h, with further increases by 48 h both in the cytoplasm (Figure 5D) and in the mitochondria specifically (Figure 5E). Additionally, depletion of DGAT1 resulted in increased protein conjugation with 4-hydroxynonenal (4HNE) (Figure 5F), a lipid peroxidation by-product (Dalleau et al., 2013). Conversely, we observed decreased 4HNE protein conjugation in DGAT1-over-expressing melanoma cells (Figure 5G) and in zebrafish tumors over-expressing Dgat1a (Figure 5H), indicating that lipid peroxidation and its suppression by DGAT1 occurs not only in cell lines but also within tumors. We next investigated whether ROS induction upon DGAT1 suppression affected melanoma cell survival. Introduction of the ROS-scavenging agents Tempol and Ebselen partially suppressed apoptosis induced by DGAT1 depletion (Figure 5I). Conversely, stable over-expression of DGAT1 was protective against ROS-mediated cell death triggered by chemical ROS inducers (Figure S5F). Moreover, ferrostatin-1, a potent ferroptosis inhibitor (Skouta et al., 2014), rescued the reduction in cell number observed upon DGAT1 inhibition, an effect also observed with Tempol and Ebselen. Combining ferrostatin-1 with either Tempol or Ebselen led to the greatest rescue of cell number and further suppressed apoptosis driven by DGAT1 inhibition (Figure S5G). Thus, DGAT1 promotes the survival of melanoma cells by suppressing the ROS production and lipid peroxidation that would otherwise occur as a result of their increased FA acquisition.
There are few pan-cancer-druggable metabolic oncoproteins whose suppression is well tolerated. We wished to establish whether pharmacological inhibition of DGAT1 would be a viable strategy to halt tumor growth in mice. We implanted DGAT1High A375 melanoma cells in mouse flanks and commenced daily oral treatment with the DGAT1 inhibitor A922500 (90 mg/kg/day) when tumors reached approximately 100 mm3. Surprisingly, A922500 treatment had no significant impact upon tumor growth in vivo (Figure 6A) despite an increase in tumor ROS levels revealed by increased 4HNE conjugation to protein (Figure S6A). We hypothesized that this might be due to more effective ROS neutralization in vivo, which would need to be antagonized in order to suppress tumor growth. We therefore investigated expression of the ROS-detoxifying enzymes SOD1 and -2, two known NRF2 target genes (Dreger et al., 2010; Ma et al., 2021; Park and Rho, 2002; Taylor et al., 2008). Indeed, inhibition of DGAT1 in melanoma cell lines led to increased SOD1 and -2 as well as SESTRIN2 (SESN2) (Figure S6B), a NRF2 activator induced upon mitochondrial respiratory malfunction (Bae et al., 2013; Garaeva et al., 2016), in agreement with our earlier MS-based whole-proteome and quantitative PCR analyses demonstrating activation of NRF2 signaling following DGAT1 inhibition (Figures 5A and 5B). To confirm the role of SESN2/NRF2 signaling in inducing SOD expression following DGAT1 inhibition, we depleted SESN2 using siRNA and in parallel treated cells with NRF2 inhibitor ML385 (Singh et al., 2016). These treatments prevented the induction of SOD1, SOD2 and an additional established NRF2 target, HMOX1, by DGAT1 inhibition (Figures S6C–S6F), consistent with SOD induction being driven through a SESN2/NRF2-signaling axis. Moreover, further analysis of the A375 tumors treated with A922500 revealed increased SESN2, SOD1, and SOD2 expression (Figure S6A), concomitant with increased lipid peroxidation, confirming that our A922500 administration had been effective in inducing ROS and NRF2 signaling in tumor cells in vivo. Assuming that ROS-neutralizing factors such as SOD enzymes were limiting the tumor inhibitory effects of DGAT1 antagonism, we next examined whether blocking the induction of SOD1 would augment the impact of DGAT1 inhibition on melanoma cell growth and survival. Indeed, we found that combining DGAT1 inhibition with SOD1 knockdown led to increased ROS generation concomitant with a dramatic reduction in cell number when compared with inhibition of DGAT1 alone (Figures S6G–S6I). In the clinic, tetrathiomolybdate (TTM) anions are used to chelate cupric cations and have been shown to potently inhibit SOD1, which uses cupric cations as a co-factor, leading to clinical trials of TTM as anti-cancer agents (Juarez et al., 2006; Lowndes et al., 2008; Redman et al., 2003). Again, compared with DGAT1 inhibitor treatment alone, the combination of DGAT1 inhibitor and TTM led to a significant increase in ROS generation and 4HNE protein conjugates (Figures 6B and S6J), concomitant with a marked decrease in cell growth and induction of apoptosis (Figures 6B and S6K). We next assessed the impact of combining inhibition of DGAT1 and SOD1 on tumor growth by using not only the BRAFV600E-mutant A375 xenograft model but also an NRASQ61R-mutant MM485 xenograft model. In both models, similarly to DGAT1 inhibition alone, SOD1 inhibitor treatment alone had no significant impact on tumor growth (Figures 6C and 6D). Strikingly, combined DGAT1 and SOD1 inhibition led to a profound reduction in tumor growth, with most animals demonstrating tumor regression in the MM485 xenograft model (Figures 6C and 6D). Western blotting of protein extracts from tumors revealed the expected increase in 4HNE protein conjugates in tumors treated with DGAT1 inhibitor or SOD1 inhibitor alone, and elevated SOD1 expression following treatment with DGAT1 inhibitor alone. Significantly, 4HNE protein conjugates were most elevated in tumors treated with a combination of the DGAT1 and SOD1 inhibitors (Figures 6E and 6F). Thus, combinatorial DGAT1 and SOD1 inhibition synergized to create a toxic overload of ROS in tumors, severely retarding the growth of melanoma cells in vivo.
The role of dysregulated lipid metabolism, encompassing more than just de novo FA synthesis, in promoting neoplasia is increasingly apparent (Guri et al., 2017; Nomura et al., 2010; Watt et al., 2019). Previously, we demonstrated that enhanced FA uptake accompanies neoplastic progression in melanocytes (Henderson et al., 2019), and this was shown to be a metabolic feature of tumor-initiating cells in particular (Pascual et al., 2017) and to drive melanoma tumor development, dissemination, and resistance to targeted therapy (Alicea et al., 2020; Zhang et al., 2018). However, the adaptations allowing melanoma cells to tolerate exposure to excess FA had been overlooked. We now demonstrate that DGAT1 is a bona fide metabolism oncoprotein that stimulates melanoma tumorigenesis by conferring protection against ROS, including lipid peroxidation, through inducing LD formation (see model; Figure 7). DGAT2, a functionally related albeit structurally distinct enzyme, appeared less important than DGAT1 for melanoma LD formation, cell growth, and survival, as was shown previously to be the case for glioblastoma cells (Cheng et al., 2020). This distinction in oncopotency may reflect the distinct roles performed by DGAT1 and DGAT2 in LD formation and adipocyte biology: DGAT1 is exclusively ER resident and responsible for initiating LD formation (Wilfling et al., 2013) and for protecting ER membranes in adipocytes against lipotoxicity (Chitraju et al., 2019); DGAT2 can relocate to LD from the ER and is responsible for further enlarging LD when FA are abundant (Wilfling et al., 2013). Moreover, whereas Dgat1-null mice are viable, Dgat2-null mice die at birth lacking >90% of TAG (Stone et al., 2004). We uncovered frequent DGAT1 amplification and up-regulation in melanoma but also in many other cancers, notably ovarian, breast, uterine, esophageal, liver, pancreatic, head and neck, prostate, stomach, and lung cancers. Abundant LD have been observed in a range of cancers, consistent with widespread up-regulation of DGAT1 (Cheng et al., 2020; Cruz et al., 2020; Petan et al., 2018), indicating that DGAT1 is likely to perform an oncogenic role in these other cancers too. The co-occurrence of DGAT1 amplification with FASN, CD36, or MAGL amplification in human cancers was significant, arguing for these co-aberrations being synergistic, in keeping with DGAT1 facilitating safe accumulation of FA in cancer cells. We propose that FA sequestration as TAG in LD allows melanoma cells to accumulate and utilize FA safely, thereby avoiding cell death. As previously described in non-transformed embryonic fibroblasts (Nguyen et al., 2017) and glioblastoma cells (Cheng et al., 2020), we observed that DGAT1 suppression resulted in overloading of mitochondria with AcCa, driving excessive FAO and ROS production. Thus, the pathophysiological role of DGAT1 is to regulate the supply of FA to mitochondria within tolerable limits. Evidently, cancer cells, having elevated FA, require more DGAT1 to achieve this. Lipid peroxides are especially cytotoxic and are generated by the action of oxygen-centered radicals on PUFA preferentially (Gaschler and Stockwell, 2017). As PUFA are concentrated in LD, where they are shielded from peroxidation (Jarc et al., 2018), enhanced LD levels mediated by DGAT1 up-regulation confer dual protection from cell death, both by moderating FAO-triggered ROS production and by simultaneously suppressing PUFA peroxidation. LD are known to protect non-transformed and transformed cells against a range of cellular stresses typically encountered in the tumor microenvironment, including nutrient deprivation and hypoxia (Cruz et al., 2020; Petan et al., 2018). Indeed, these conditions induce LD formation downstream of autophagy (Nguyen et al., 2017). Additionally, low pH up-regulates DGAT1 and consequently increases LD formation. In turn, LD are required for acidosis-induced metastasis (Corbet et al., 2020). Nutrient deprivation and hypoxia compromise de novo FA synthesis and desaturation. Unsaturated FA are essential for membrane fluidity and cell viability, and in the absence of desaturation reactions or the ability to scavenge unsaturated FA from the circulation, cancer cells must draw on LD for unsaturated FA in order to survive (Ackerman et al., 2018). The above roles for LD are consistent with our observation that the effect of DGAT1 suppression on cell growth was exacerbated by serum withdrawal and hypoxia and explains why conversely DGAT1 over-expression conferred its greatest growth advantage under these conditions. The ability of DGAT1 inhibitor alone to suppress tumor growth was recently shown in a glioblastoma xenograft model (Cheng et al., 2020). However, we did not observe efficacy of DGAT1 inhibition alone in our mouse melanoma xenograft models, most likely due to a striking upregulation of the anti-ROS response. In order to antagonize this adaptation, we employed TTM, a clinically relevant SOD1 inhibitor. Combinatorial DGAT1 and SOD1 inhibition led to a profound impairment of melanoma growth in vivo. DGAT1 is pan-cancer amplified, and it remains to be determined to what extent other cancers can mount the anti-ROS responses we observed in our melanoma xenograft models. Thus, future studies should focus on evaluating the efficacy of DGAT1 inhibition alone, or in combination with inhibition of anti-ROS mechanisms, to induce the most well-tolerated and beneficial outcomes for cancer patients.
Our study relied on established human melanoma cell lines to explore DGAT1 function, and these may have adapted to ex vivo culture in ways that make them less representative than primary cells. To antagonize DGAT1 in cell cultures, we mostly used 30 μM A922500 to ensure complete inhibition over the 24–72 h required to fully deplete LD and to suppress cell viability. Although in excess of doses required to inhibit transiently DGAT1 catalytic activity, this concentration is still beneath that required to suppress the activity of related acyltransferases including DGAT2 (King et al., 2009). Moreover, an even higher dose of A922500 (47 μM) was unable to suppress LD dependent on DGAT2 or to markedly suppress viability in glioblastoma cells over-expressing DGAT2 (Cheng et al., 2020), consistent with A922500 being a selective DGAT1 inhibitor at this concentration. That we can reproduce the effects of A922500 on LD depletion and cell viability with other DGAT1 inhibitors and with siRNA targeting DGAT1 and observe the converse upon over-expressing DGAT1 further corroborate that the effects of A922500 are DGAT1 dependent. To demonstrate the interaction between DGAT1 and SOD1 on tumor growth in vivo, we have again relied on pharmacological agents that may have generated off-target effects that contributed to tumor growth inhibition. Additionally, durability of tumor growth suppression was not established in our study, nor host tolerance to chronic drug treatment, albeit that treatment was well tolerated by mice over the time course it was evaluated. As a further limitation, xenograft models do not fully recapitulate features of spontaneous tumors in notably developing in the absence of adaptive immunity. Potentially, PDX models, autochthonous genetically engineered mouse melanoma models, or ex vivo tumor explant culture might have allowed us to maintain more features of melanoma in which to assess the impact of DGAT1 depletion or antagonism.
Further information and requests for resources and reagents should be directed to and will be fulfilled by the lead contact, Adam Hurlstone ([email protected]).
• Plasmids and cell lines generated in this study can be requested from the lead contact.
Regulated procedures involving zebrafish were ethically approved by The University of Manchester Animal Welfare and Ethical Review Body (AWERB), or by the UMMS Institution Animal Care and Use Committee (A-2016, A-2171), and carried out under a license issued by the appropriate national regulatory authority. Zebrafish were housed at ∼28°C under a 14 h light/10 h dark cycle. Transgenic zebrafish expressing BRAFV600E or NRASG12D have been previously described (Casar et al., 2018; Ceol et al., 2011) and were crossed onto a mitfaw2/w2 (mitfa−/−) background to suppress melanocyte development and further onto a tp53M214K/M214K background to promote tumorigenesis. Melanocyte restoration and simultaneous over-expression of Dgat1a, Dgat2 or EGFP was then achieved by injection of embryos with a mitfa-minigene containing plasmid as previously described (Ceol et al., 2011). Briefly, zebrafish dgat1a and dgat2 were amplified from cDNA of wild-type 48 h post-fertilization zebrafish embryos, and subcloned into the pDONR221 vector (see Table S7 for oligonucleotide sequences). The pDest-mitfa:dgat1a-pA and pDest-mitfa:dgat2-pA destination vectors were created using an LR clonase reaction consisting of p5E-mitfap, pME-dgat1a or pME-dgat2, p3E-pA and an empty destination vector. Expression plasmid was injected into zebrafish zygotes along with Tol2 mRNA. pCS2-TP plasmid for Tol2 mRNA generation was a kind gift from Dr Koichi Kawakami (National Institute of Genetics). Sufficient embryos for all experimental arms were generated simultaneously, pooled and then randomly assigned to a construct, although formal randomization techniques were not used. Zebrafish were group-housed according to the construct. Only zebrafish embryos with near complete melanocyte rescue at 5 days were retained for further analysis. Analysis of tumor formation was not performed blinded to the construct identity. Sample sizes were not predetermined based on statistical power calculations but were based on our experience with these assays. To assess the statistical significance of differences in overall survival, we used Mantel–Cox’s log-rank tests.
Mice were housed in the University of Manchester Biological Safety Unit. CD1® nude mice (female, 8 weeks of age) were injected subcutaneously into the left flank with 4 × 106 A375 cells or MM485 cells (in PBS). When animals had developed melanoma nodules of ∼100 mm3, animals were allocated randomly to a treatment group and drug administration was initiated (4–6 mice per group). Treatment was by oral gavage once daily with vehicle (1% Tween80 in PBS), DGAT1 inhibitor A922500 (90 mg/kg) or SOD1 inhibitor ammonium tetrathiomolybdate (5 mg/kg). After the indicated number of days, tumors were isolated and analyzed as described.
Human melanoma cell lines were cultured in High Glucose DMEM with 10% FBS, and penicillin–streptomycin (Sigma) at 37°C and 5% CO2. Normal human melanocytes were purchased from Cascade Biologics and cultured according to the manufacturer's guidelines. Lenti-X cells were cultured in High Glucose DMEM with 10 %v/v FBS, and penicillin–streptomycin (Sigma) at 37°C and 5% CO2. All cells tested negative for mycoplasma and cell lines were authenticated using STR profiling (See Table S8 for DGAT1 characteristics of cell lines used in this study.).
Compounds were used at the following concentrations unless otherwise noted: 50 μM AZD3988 (Tocris), 30 μM A922500 (Stratech), 50 μM AZD7687 (Stratech), 70 μM T863 (Sigma), 1 μM Oligomycin (Sigma), 0.5 μM FCCP (Sigma), 1 μM Antimycin-A (Sigma), 1 μM Rotenone (Sigma), 50 μM PF-06424439 (Sigma), 5 μM Ebselen (Tocris), 1 mM Tempol, 200 μM Paraquat (Sigma), Menadione (Sigma), 100 μM Etomoxir (Sigma), 2 μM Ferrostatin-1 (Sigma), 5 μM ML385 (Selleckchem), and 1 μM ATN-224 (Cayman Chemical). Antibodies against DGAT1 (ab54037) and 4-Hydroxynonenal (ab46545) were purchased from abcam. Antibodies against Vinculin (66305-1-Ig), Beta-Tubulin (10094-1-AP), PINK1 (23274-1-AP), Parkin (14060-1-AP), SOD1 (10269-1-AP), SOD2 (24127-1-AP), SESN2 (10795-1-AP) and GAPDH (60004-1-Ig) were purchased from Proteintech. Antibodies against phospho-AMPK (50081), phospho-RAPTOR (2083), Caspase-3 (9662), phospho-ERK (4370) and GFP (2956) were purchased from Cell Signalling. The antibody against gamma-tubulin (T5326) was purchased from Sigma. The antibody against ERK2 (C-14) was from Santa Cruz Biotechnology.
The following plasmid used was purchased from Addgene: pcDNA3.1-mMaroon1 (83840). The GFP and WPRE elements were excised from pCDH-MCS-T2A-copGFP (a kind gift from Andrew Gilmore, The University of Manchester) using BspEI and KpnI. mApple (BspEI and XhoI adapters) and WPRE (XhoI-KpnI adapters) were PCR amplified, digested and subcloned to create the pCDH-MCS-T2A-mApple vector. DGAT1 was further subcloned into the both the pCDNA3.1 vector and pCDH-MCS-T2A-mApple using the MCS. All plasmids were transfected using Lipofectamine (Invitrogen) following standard protocols. All siRNA was transfected using Lipofectamine RNAi Max (Invitrogen) following standard protocols.
Briefly, Lenti-X™ HEK293 cells (Takara Bio) were transfected with pMDLg/pRRE, pMD2.G, pRSV-Rev plasmids (all kind gifts from Angeliki Malliri, Cancer Research UK Manchester Institute) and pCDH-EF1α-DGAT1-T2A-mApple viral vectors using Fugene (Promega) following standard protocols. The viral containing supernatant was filtered using a 0.45 μm filter and frozen at −80°C prior to transduction of target cells. The supernatant containing the viral particles was added to target cells along with 10 ng/mL Polybrene (Millipore) for 24 h. Target cells were then grown and selected from single cell colonies.
Cells were washed with PBS and lysed with sample buffer (62.5 mM TRIS pH 6.8, 2 %w/v Sodium dodecyl sulfate (SDS), 10 %v/v glycerol, 0.01%w/v bromophenol blue, 3 %v/v 2-mercaptoethanol). Lysates were then sonicated and heated to 95°C for 10 minutes prior to being evenly loaded onto SDS-polyacrylamide gels using the Mini Trans-Bot electrophoresis system (Biorad), followed by transfer to PVDF using standard western blotting procedures.
Indicated cells were stained with 2 μM BODIPY 493/503 (ThermoFisher Scientific) and 5 ng/mL Hoecsht 3342 (Cell Signalling) for 30 minutes prior to fixing in 4 %w/v paraformaldehyde and imaging using a Leica microscope system. Images were processing using Fiji.
Indicated cells were fixed in 4 %w/v paraformaldehyde and stained with LipidTox Green (ThermoFisher Scientific) according to manufacturer’s instructions, and 5 ng/mL Hoecsht 3342 (Cell Signalling) for 15 minutes prior to imaging using a Leica microscope system. Images were processing using Fiji.
RNA from cell lines was isolated with TRIZOL® (Invitrogen). After chloroform extraction and centrifugation, 5 μg RNA was DNase treated using RNase-Free DNase Set (Qiagen). 1 μg of DNase treated RNA was then taken for cDNA synthesis using the Protoscript I first strand cDNA synthesis kit (New England Biolabs). Selected genes were amplified by quantitative real time PCR (RT-qPCR) using Sygreen (PCR Biosystems). Relative expression was calculated using the delta-delta CT methodology and beta-actin was used as reference housekeeping gene. Sequences for primers used can be found in the Table S7.
Indicated cell lines were trypzinized and pelleted by centrifugation at 500 g for 5 min, washed with PBS. For mitochondrial membrane potential cells were stained with 2 μM JC-1 (Life Technologies) for 30 minutes at 37°C. For positive control samples 0.5μM FCCP was added simultaneously with JC-1. Data was acquired by the BD BIOsciences Foretessa and quantified using the Flowjo software. A minimum of 10,000 cells were analyzed per condition.
Indicated cell lines were trypzinized and pelleted by centrifugation at 500 g for 5 min, followed by a PBS wash. For lipid peroxidation cells were stained with either 5 μM BODIPY™ 581/591 C11 (ThermoFisher Scientific) or MitoPerOx (Abcam) for 30 minutes at 37°C. Data was acquired by the BD BIOsciences Foretessa and quantified using the Flowjo software. A minimum of 10,000 cells were analyzed per condition.
Indicated cell lines were trypzinized and pelleted by centrifugation at 500 g for 5 min, followed by a PBS wash. For mitochondrial specific ROS detection, cells were stained with 2.5 μM Mitosox (ThermoFisher Scientific) for 30 minutes at 37°C. Data was acquired by the BD BIOsciences Foretessa and quantified using the Flowjo software. A minimum of 10,000 cells were analyzed per condition.
Indicated cells were stained and fixed with 0.5 %w/v crystal violet (Sigma) in 4 %w/v paraformaldehyde/PBS for 30 minutes. Fixed cells were then solubilized in 2 %w/v SDS/PBS and absorbance measured at 595 nm using Synergy H1 microplate reader (BioTek).
Indicated cells were labelled with 20 μM 5-ethynyl-2′-deoxyuridine (EdU) for 4 h and processed following the manufacturer’s protocol (Click-iT® EdU Alexa Fluor® 488 Imaging Kit, Thermo Fisher). Prior to imaging cells were then stained with 5 ng/mL Hoecsht 3342 for 15 minutes. Stained cells were analyzed using a using a Leica microscope system. Images were processing using Fiji.
Indicated cell lines were seeded into 24-well plates at a density of 15,000–20,000 cells per well, depending on growth rate and the design of the experiment. After 24 h drugs or siRNA were added, and cells were imaged every hour using the Incucyte ZOOM (Essen Bioscience) Phase-contrast images were analyzed to detect cell proliferation based on cell confluence. For cell apoptosis, caspase-3 and caspase-7 green apoptosis-assay reagent (Life Technologies) was added to the culture medium following manufacturer’s instructions. Cell apoptosis was analyzed based on green fluorescent staining of apoptotic cells.
Cells were stained with 5 μM Dihydroethidium for 20 minutes in the dark at 37°C. Fluorescence was measured at excitation 480 nm emission 570 nm using Synergy H1 micro plate reader (BioTek). Fluorescence values were normalized to cell number by staining the cells with crystal violet after fluorescence read.
We evaluated both point mutations and CNV in the TCGA SKCM firehose legacy, TCGA pan-cancer and Cancer Cell Line Encyclopedia datasets using the cBioPortal platform (Gao et al., 2013). The GISTIC2.0 algorithm was used to identify focal amplifications (Mermel et al., 2011). Gene Ontology analysis was carried out using both enrichR (Chen et al., 2013) and metascape software (Zhou et al., 2019). Association between mRNA expression in TCGA datasets and survival was evaluated using OncoLnc (Anaya, 2016). mRNA levels determined by microarray were accessed through the Oncomine platform (Rhodes et al., 2004).
Data was tested for normality using the Shapiro-Wilk test. Data was considered to be normally distributed if p > 0.05. Differences in the number of lipid droplets per cell, relative cell number and percentage EdU incorporation between DMSO and drug treated cells were assessed using an unpaired two-sided t-test, or Mann-Whitney test if data were not normally distributed. In comparing the differences in these same characteristics between cells transfected with either non-target or one of several siRNA oligonucleotides, a one-way ANOVA with Tukey’s multiple comparisons test (or Friedman with Dunn’s multiple comparisons test if data were not normally distributed) was used to measure significance. Differences were considered significant if p < 0.05. All data obtained were analyzed using Graphpad Prism 8.1.
For quantitative mass spectrometry, A375 cells were labelled in SILAC DMEM supplemented with 10 %v/v dialyzed fetal bovine serum (Sigma), 2 mM glutamine, 100 U/mL penicillin and 100 μg/mL streptomycin for 15 days to ensure complete incorporation of amino acids. Two cell populations were obtained: one labelled with natural variants of the amino acids (light label; Lys0, Arg0) and a second one with heavy variants of the amino acids (L-[13C6,15N4]Arg (+10) and L- [13C6,15N2]Lys (+8)) (Lys8,Arg10). The light amino acids were from Sigma, while their heavy variants were from Cambridge Isotope Labs.
Cells from the two SILAC conditions treated as indicated were lysed at 4°C in ice cold modified RIPA buffer (50 mM Tris, pH 7.5, 150 mM NaCl, 1 %v/v NP-40, 0.1 %w/v sodium deoxycholate, 1 mM EDTA, 5 mM β-glycerolphosphate, 5 mM sodium fluoride, 1 mM sodium orthovanadate, 1 complete inhibitor cocktail Tablet per 50 mL). Proteins were precipitated for two hours at −20°C in four-fold excess of ice-cold acetone. The acetone-precipitated proteins were solubilized in denaturation buffer (10 mM HEPES, pH 8.0, 6 M urea, 2 M thiourea) and the SILAC-labelled lysates were mixed 1:1 based on protein concentrations. Proteins were reduced with 1 mM dithiothreitol (DTT) for 60 min, alkylated with 5.5 mM chloroacetamide (CAA) for 60 min and digested first with endoproteinase Lys-C (Wako, Osaka, Japan) and then, after a five-fold dilution with 50 mM ammonium bicarbonate (ABC), with trypsin (modified sequencing grade, Sigma). The peptide mixture was desalted and concentrated on a C18-SepPak cartridge (Waters, USA) and eluted with 50 %v/v acetonitrile. Equal amounts of SILAC lysates were then mixed 1:1, reduced with DTT, and alkylated with CAA before being resolved on SDS-PAGE (8–12 %w/v, Invitrogen). Separated proteins were fixed in the gel and visualized with colloidal Coomassie staining (Invitrogen). Each gel lane was excised and separated into eight segments that were sliced, destained with 50 %v/v EtOH in 25 mM ABC and dehydrated with 100 %v/v EtOH. Proteins were digested with sequence-grade trypsin (Sigma) overnight. Trypsin activity was quenched by acidification with trifluoroacetic acid (TFA) and peptides were extracted from the gel sections with increasing concentrations of acetonitrile. Organic solvent was evaporated in a vacuum centrifuge, as described (Francavilla et al., 2016).
Enriched in-gel digested peptides were desalted and concentrated on STAGE-tips with two C18 filters and eluted using 40 %v/v acetonitrile, dried and reconstituted in 5 %v/v acetonitrile in 0.1 %v/v formic acid prior to analysis by LC-MS/MS using an UltiMate 3000 Rapid Separation LC (RSLC, Dionex Corporation, Sunnyvale, CA) coupled to a QE HF (Thermo Fisher Scientific, Waltham, MA) mass spectrometer. Mobile phase A was 0.1 %v/v formic acid in water and mobile phase B was 0.1 %v/v formic acid in acetonitrile and the analytical column utilized was a 75 mm × 250 μm inner diameter 1.7 μm CSH C18 (Waters). Samples were transferred to a 5 μL loop before loading on to the column at a flow of 300 nL/min for 5 minutes at 5 %v/v B. The loop was subsequently taken out of line and the peptides separated using a gradient that went from 5 %v/v to 7 %v/v B and from 300 nL/min to 200 nL/min in 1 min followed by a shallow gradient from 7 %v/v to 18 %v/v B in 64 min, then from 18 %v/v to 27 %v/v B in 8 min and finally from 27 %v/v B to 60 %v/v B in 1 min. The column was washed at 60 %v/v B for 3 min before re-equilibration to 5 %v/v B in 1 min. At 85 min, the flow was increased to 300 nL/min until the end of the run at 90 min. Mass spectrometry data was acquired in a data dependent manner for 90 min in positive mode. Peptides were selected for fragmentation automatically by data dependent analysis on a basis of the top 8 (phospho-proteome) or top 12 (proteome) peptides with m/z between 300 to 1750 Th and a charge state of 2, 3 or 4 with a dynamic exclusion set at 15 sec. The MS Resolution was set at 120,000 with an AGC target of 3e6 and a maximum fill time set at 20 ms. The MS2 Resolution was set to 30,000, with an AGC target of 2e5, a maximum fill time of 45 ms, isolation window of 1.3 Th and a collision energy of 28.
Raw data were analyzed with the MaxQuant software suite, version 1.5.6.5, with the integrated Andromeda search engine (Cox et al., 2011). Proteins were identified by searching the HCD-MS/MS peak lists against a target/decoy version of the human Uniprot database, which consisted of the complete proteome sets and isoforms (2016 release) supplemented with commonly observed contaminants such as porcine trypsin and bovine serum proteins. Tandem mass spectra were initially matched with a mass tolerance of 7 ppm on precursor masses and 0.02 Da or 20 ppm for fragment ions. Cysteine carbamidomethylation was searched as a fixed modification. Protein N-acetylation, oxidized methionine and either deamidation of asparagine and glutamine (proteome analysis) were searched as variable modifications. Labelled lysine and arginine were specified as fixed or variable modification, depending on prior knowledge about the parent ion (MaxQuant SILAC identification). False discovery rate was set to 0.01 for peptides, proteins and modification sites. Minimal peptide length was six amino acids. Only peptides with Andromeda score >40 were included. Potential contaminants, reverse sequenced peptides and phosphorylation sites with a localization probability of less than 0.75 (class I) (Olsen et al., 2006) were filtered from the dataset. The remaining data were filtered to remove sites or peptides without quantification in at least two of the three replicates for each time point. The median of the replicates was taken. Sites or peptides with a SILAC ratio of greater than 1.5 were considered up-regulated whilst those with a ratio less than 0.75 were considered down-regulated. Gene network visualization was performed using enrichR (Chen et al., 2013). For the proteome analysis a minimum of three to seven peptide identifications with at least two being uniquely assigned to the particular protein were required. Sequence coverage of the identified proteins was at least 5%. Gene Ontology analysis was carried out using enrichR (Chen et al., 2013) and metascape (Zhou et al., 2019).
The samples were maintained at 4°C and analyzed applying two Ultra-High-Performance Liquid Chromatography-Mass Spectrometry (UHPLC-MS) methods using a Dionex UltiMate 3000 Rapid Separation LC system (Thermo Fisher Scientific, MA, USA) coupled with a heated electrospray Q Exactive Focus mass spectrometer (Thermo Fisher Scientific, MA, USA). Lipid extracts were analyzed on a Hypersil GOLD column (100 × 2.1 mm, 1.9 μm; Thermo Fisher Scientific, MA, USA). Mobile phase A consisted of 10 mM ammonium formate and 0.1 %v/v formic acid in 60 %v/v acetonitrile/water and mobile phase B consisted of 10 mM ammonium formate and 0.1 %v/v formic acid in 90 %v/v propan-2-ol/water. Flow rate was set for 0.40 mL.min-1 with the following gradient: t = 0.0, 20 %v/v B; t = 0.5, 20 %v/v B, t = 8.5, 100 %v/v B; t = 9.5, 100 %v/v B; t = 11.5, 20 %v/v B; t = 14.0, 20 %v/v B, all changes were linear with curve = 5. The column temperature was set to 55°C and the injection volume was 2 μL. Data were acquired in positive and negative ionization mode separately within the mass range of 150–2000 m/z at resolution 70,000 (FWHM at m/z 200). Ion source parameters were set as follows: Sheath gas = 48 arbitrary units, Aux gas = 15 arbitrary units, Sweep gas = 0 arbitrary units, Spray Voltage = 3.2 kV (positive ion)/2.7 kV (negative ion), Capillary temp. = 380°C, Aux gas heater temp. = 450°C. Data dependent MS2 in ‘Discovery mode’ was used for the MS/MS spectra acquisition using following settings: resolution = 17,500 (FWHM at m/z 200); Isolation width = 3.0 m/z; stepped collision energies (stepped CE) = 20, 40, 100 [positive ion mode]/40, 60, 130 [negative ion mode]. Spectra were acquired in five different mass ranges: 150–510 m/z; 500–710 m/z; 700–860 m/z; 850–1010 m/z; 1000–2000 m/z. A Thermo ExactiveTune 2.8 SP1 build 2806 was used as instrument control software in both cases and data were acquired in profile mode. Quality control (QC) samples were analyzed as the first ten injections and then every seventh injection with two QC samples at the end of the analytical batch. Two blank samples were analyzed, the first as the sixth injection and then the second at the end of each batch.
Raw data acquired in each analytical batch were converted from the instrument-specific format to the mzML file format applying the open access ProteoWizard (version 3.0.11417) msconvert tool (Chambers et al., 2012). During this procedure, peak picking and centroiding, were achieved using vendor algorithms. Isotopologue Parameter Optimization (IPO - version 1.0.0, using XCMS - version 1.46.0) (Libiseller et al., 2015) was used to perform automatic optimization of XCMS (Smith et al., 2006) peak picking parameters. For centWave peak picking algorithm following parameters and ranges were used: min_peakwidth (from 2 to 10); max_peakwidth (from 20 to 60); ppm (from 5 to 15); mzdiff (−0.001 to 0.01); snthresh (10); noise (10,000); prefilter (3); value_of_prefilter (100); mzCenterFun (wMean); integrate (1); fitgauss (FALSE); verbose.columns (FALSE). Optimised XCMS parameters for raw data files deconvolution were: min_peakwidth (6); max_peakwidth (30); ppm (14); mzdiff (0.001); snthresh (10); noise (100); prefilter (3); value_of_prefilter (100); mzCenterFun (wMean); integrate (1); fitgauss (FALSE); verbose.columns (FALSE). For feature grouping method density was used with following: minfrac (0.5); minsamp (1); bw (0.25); mzwid (0.01); max (50); sleep (0). A data matrix of metabolite features (m/z-retention time pairs) versus samples was constructed with peak areas provided where the metabolite feature was detected for each sample.
The data for pooled QC samples were applied to perform QC filtering. The first five QCs for each batch were used to equilibrate the analytical system and therefore subsequently removed from the data before the data was processed and analyzed. The data from the pooled QC samples were used to apply QC filtering. For each metabolite feature detected QC samples 1–8 were removed (i.e. leaving a blank and 2 QCs at the start of each batch) and the relative standard deviation and percentage detection rate were calculated using the remaining QC samples. Blank samples at the start and end of a run were used to remove features from non-biological origins. Any feature with an average QC intensity less than 20 times the average intensity of the blanks was removed. Any samples with >50% missing values were excluded from further analysis. Metabolite features with a RSD > 30% and present in less than 90% of the QC samples were deleted from the dataset. Features with a <50% detection rate over all samples were also removed. Prior to statistical analysis, the data was normalized using probabilistic quotient normalization (PQN) (Dieterle et al., 2006). For multivariate analysis missing values were replaced by applying k nearest neighbor (kNN) missing value imputation (k = 5) followed by log transformation (Parsons et al., 2007).
LipidSearch (version 4.2, Thermo Fisher Scientific) was used to annotate peaks based on their MS/MS fragmentation patterns. For lipid annotation, all experimental LC-MS/MS spectra data were searched against a MS/MS lipid library in the LipidSearch software database using the following potential ion forms: positive ion = [M+H]+, [M+NH4]+, [M+Na]+, [M+K]+, [M+2H]2+; negative ion = [M-H]-, [M+HCOO]-, [M+CH3COO]-, [M+Cl]-, [M-2H]2-. The quality of the annotation was graded as A-D. This is defined as: Grade A = all fatty acyl chains and class were completely identified; Grade B = some fatty acyl chains and the class were identified; Grade C = either the lipid class or some fatty acyls were identified; Grade D = identification of less specific fragment ions. Only peaks with an MS/MS identification were discussed in this manuscript. All lipid annotations are reported at a confidence of level 3 according to the Metabolomics Standards Initiative (Sumner et al., 2007).
For univariate statistics the normalized data was used to avoid including imputed values in the calculations. Fold changes were computed between all pairs of groups. For 2-group comparisons a t-test was applied to determine features showing a significant different between groups. For comparisons exploring two factors a 2-way ANOVA with an interaction term was applied to determine features showing a significant difference between factor levels. For features found to be significant, Tukey’s Honest Significant Difference (HSD) was applied to determine between which levels the difference was significant (p < 0.05). A False Discovery Rate (FDR) correction (Benjamini-Hochburg) was applied to adjust for multiple testing and control the number of false positives (q < 0.05) for both t-test and ANOVA.
All peak matrix processing, univariate and multivariate analyses were performed in the R environment using STRUCT (STatistics in R Using Class Templates) and STRUCTToolbox packages, which make use of PMP and SBCMS packages. These packages are maintained by Phenome Centre Birmingham and available on GitHub (https://github.com/computational-metabolomics). | true | true | true |
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PMC9638016 | David Millrine,Thomas Cummings,Stephen P. Matthews,Joshua J. Peter,Helge M. Magnussen,Sven M. Lange,Thomas Macartney,Frederic Lamoliatte,Axel Knebel,Yogesh Kulathu | Human UFSP1 is an active protease that regulates UFM1 maturation and UFMylation | 03-08-2022 | UFM1,ubiquitin,cysteine protease,endoplasmic reticulum,ER,ribosome,ubiquitin-like modifier,membrane protein,UFC1,UBA5 | Summary An essential first step in the post-translational modification of proteins with UFM1, UFMylation, is the proteolytic cleavage of pro-UFM1 to expose a C-terminal glycine. Of the two UFM1-specific proteases (UFSPs) identified in humans, only UFSP2 is reported to be active, since the annotated sequence of UFSP1 lacks critical catalytic residues. Nonetheless, efficient UFM1 maturation occurs in cells lacking UFSP2, suggesting the presence of another active protease. We herein identify UFSP1 translated from a non-canonical start site to be this protease. Cells lacking both UFSPs show complete loss of UFMylation resulting from an absence of mature UFM1. While UFSP2, but not UFSP1, removes UFM1 from the ribosomal subunit RPL26, UFSP1 acts earlier in the pathway to mature UFM1 and cleave a potential autoinhibitory modification on UFC1, thereby controlling activation of UFMylation. In summary, our studies reveal important distinctions in substrate specificity and localization-dependent functions for the two proteases in regulating UFMylation. | Human UFSP1 is an active protease that regulates UFM1 maturation and UFMylation
An essential first step in the post-translational modification of proteins with UFM1, UFMylation, is the proteolytic cleavage of pro-UFM1 to expose a C-terminal glycine. Of the two UFM1-specific proteases (UFSPs) identified in humans, only UFSP2 is reported to be active, since the annotated sequence of UFSP1 lacks critical catalytic residues. Nonetheless, efficient UFM1 maturation occurs in cells lacking UFSP2, suggesting the presence of another active protease. We herein identify UFSP1 translated from a non-canonical start site to be this protease. Cells lacking both UFSPs show complete loss of UFMylation resulting from an absence of mature UFM1. While UFSP2, but not UFSP1, removes UFM1 from the ribosomal subunit RPL26, UFSP1 acts earlier in the pathway to mature UFM1 and cleave a potential autoinhibitory modification on UFC1, thereby controlling activation of UFMylation. In summary, our studies reveal important distinctions in substrate specificity and localization-dependent functions for the two proteases in regulating UFMylation.
The ubiquitin-like protein ubiquitin fold modifier 1 (UFM1) is emerging as a central regulator of protein homeostasis through its role in ribosome quality control (Gerakis et al., 2019; Banerjee et al., 2020; Witting and Mulder, 2021). The physiological importance of protein UFMylation is evidenced by mutations in UFM1 pathway components that result in neurodevelopmental pathophysiology including cerebellar ataxia, encephalopathy, epilepsy, peripheral neuropathy, and systemic skeletal abnormalities characterized by abnormal cartilage development (Colin et al., 2016; Duan et al., 2016; Egunsola et al., 2017; Di Rocco et al., 2018). In mice, knockout of UFM1 pathway components results in early-stage embryonic lethality that is attributed to defective hematopoiesis (Tatsumi et al., 2011). UFMylation is therefore essential for normal physiology, with evidence of involvement in tissue development. Recent studies linking UFM1 to ER stress responses and secretory pathways highlight a potential mechanism to explain these observations. Indeed, UFM1 is conjugated to ER membrane-associated ribosomal subunits (Walczak et al., 2019; Liang et al., 2020), which is induced by ribosomal stalling (Wang et al., 2020). Produced as an 85-amino-acid precursor, UFM1 must first be proteolytically activated through the removal of a serine-cysteine dipeptide at its C terminus (Komatsu et al., 2004). Like ubiquitin, UFM1 is conjugated to substrates through an enzymatic cascade of E1 (UFM1-activating enzyme 5; UBA5), E2 (UFM1-conjugating enzyme 1; UFC1), and E3 (UFM1 specific ligase 1; UFL1) enzymes, resulting in the formation of an isopeptide bond between the C-terminal glycine of UFM1 and the substrate lysine (Komatsu et al., 2004). These core enzymes are supported by accessory factors that include UFM1-binding protein 1 (UFBP1/DDRGK1) and CDK5 regulatory subunit-associated protein 3 (CDK5RAP3), whose function is poorly defined (Yang et al., 2019; Stephani et al., 2020). UFBP1 and UFL1 localize to the ER and they are thought to catalyze the UFMylation of Ribosomal Protein L26 (RPL26) (Walczak et al., 2019; Stephani et al., 2020). UFMylation of RPL26 in proximity to the Sec61 translocon and oligosaccharyl-transferase complex occurs after ribosome stalling and initiates specialized autophagy of the ER membrane to facilitate clearance of arrested nascent peptides and ribosomes through a lysosomal pathway (Walczak et al., 2019; Liang et al., 2020; Wang et al., 2020). Termed ER-phagy, this organelle-specific degradation pathway involves the wholesale targeting of regions of the rough ER for lysosomal degradation (Khaminets et al., 2015). In a pathway dependent on mitochondrial respiration, acute amino acid starvation stimulates ER-phagy via a pathway requiring UFM1 system components and modification of RPL26 (Liang et al., 2020). The UFM1 system may, therefore, control turnover of the translational apparatus in response to the cellular and metabolic state. An elaborate system of regulation appears to have evolved solely for this purpose, as RPL26 is the most compelling UFM1 substrate described to date (Walczak et al., 2019). While the precise function is unclear, it appears that proper functioning of the pathway requires an equilibrium of UFM1 conjugation that supports stalled ribosome clearance and ER turnover without damaging the cell’s capacity for protein biogenesis. Tight regulation is reflected in the specificity of the pathway which, unlike the highly redundant ubiquitin system, is coordinated by only a handful of enzymes (Komatsu et al., 2004; Gerakis et al., 2019; Walczak et al., 2019; Liang et al., 2020). At present only two enzymes, UFM1 specific protease 1 and 2 (UFSP1 and UFSP2), are known to cleave UFM1 conjugates, with UFSP1 reported to be catalytically inactive in humans (Kang et al., 2007; Ha et al., 2008). UFM1 requires the peptidase-linked cleavage of the C-terminal serine84-cysteine85 peptide to achieve its mature form, a prerequisite for the attachment of UFM1 onto substrates. However, in UFSP2−/− cell lines, UFM1 modification of the ribosomal subunit RPL26 is enhanced, not abolished (Muona et al., 2016; Walczak et al., 2019), leading us to challenge the notion that UFSP2 is the only UFM1-specifc protease in humans. Hence, we hypothesize that additional unreported mechanisms must exist to support UFM1 maturation in the absence of UFSP2. Indeed, the probable existence of additional UFM1-specific proteases has been noted by others (Walczak et al., 2019; Witting and Mulder, 2021). In the present study we sought to identify this unknown UFM1 peptidase. To our surprise, we isolated from cells UFSP1 that is larger than the presently annotated form and has activity toward the UFM1 precursor. Analysis of knockout cell lines identified overlapping and unique contributions of UFSP1 and UFSP2 to ribosome modification and processing of precursor UFM1. Furthermore, we identify a role for localization of UFSP2 at the ER via its interaction with the ER resident protein ODR4 for its ability to remove UFM1 from RPL26. In addition, UFSP1 is unable to reverse RPL26 UFMylation, highlighting the specificity in the system. Intriguingly, we observe a striking accumulation of UFMylated UFC1 in cells lacking UFSP1. Based on our observations, we propose dual roles for UFSP1 in activating UFMylation, first at the level of UFM1 maturation and second, by removing a potential autoinhibitory modification on UFC1. Thus, UFSP enzymes act at disparate points of the pathway to ensure appropriate UFMylation.
To confirm the existence of additional peptidases with activity toward precursor UFM1, we generated UFSP2−/− cell lines using CRISPR-Cas9 approaches. Consistent with previous studies, we observed an increase in both RPL26-(UFM1)1 and RPL26-(UFM1)2 species as a result of UFSP2 deficiency (Figures 1A and S1A–S1D) (Ishimura et al., 2017; Walczak et al., 2019; Liang et al., 2020; Wang et al., 2020; Kulsuptrakul et al., 2021). We next developed an experimental system to monitor UFM1 peptidase activity, whereby cell lysates were incubated with a reporter protein comprising pro-UFM11−85 fused at its carboxy terminus to GFP via a short peptide linker. Intriguingly, lysates derived from parental wild-type (WT) and UFSP2−/− HEK293 cells showed equivalent ability to cleave the UFM1-GFP fusion construct to liberate mature UFM1, suggesting the presence of an additional protease (Figures 1B and S1E). Cleavage of the fusion protein could be prevented by preincubation with broad-acting cysteine peptidase inhibitors iodoacetamide or N-ethylmaleimide, suggesting that the proteolytic activity observed in UFSP2−/− cells is due to a cysteine-based protease (Figures 1C and S1E). Taken together, these data reveal the existence of additional UFM1-targeting cysteine peptidases with the capacity to activate precursor UFM1.
We next employed biochemical approaches to identify the enzyme responsible. Lysates from HEK293 cells were fractionated sequentially over heparin and Source-Q columns, and ensuing fractions were screened for peptidase activity by monitoring cleavage of the UFM1-GFP reporter (Figure 2A). UFM1-specific peptidase activity was restricted to two sequential fractions and, strikingly, these fractions did not have detectable amounts of UFSP2 (Figures 2B and S2A). Importantly, this activity could be recapitulated in fractionations using UFSP2−/− cells (Figure 2C). We were therefore successful in enriching additional UFM1 peptidase activity, distinct from UFSP2. To identify the protease, we adopted an unbiased approach using liquid chromatography-mass spectrometry (LC-MS) profiling of the active fraction from UFSP2−/− cells. These data identified 974 proteins, including 21 proteins documented as having deubiquitinase or hydrolase activity (Figure S2B). Among these, ubiquitin carboxy-terminal hydrolase L1 (UCHL1) distributed in close alignment with the novel peptidase activity (Figure S2C), an attractive candidate given its historic association with the maturation of ubiquitin precursors (Grou et al., 2015). However, when tested, neither recombinant UCHL1 nor related family members (UCHL3, UCHL5, or BAP1) could cleave the UFM1-GFP reporter (Figure S2D). Hence, we depleted UCHL1 using CRISPR-Cas9 and repeated the fractionation and MS analyses (Figures S2E–S2G). To our surprise, these analyses revealed UFM1-specific peptidase-1 (UFSP1), characterized as an inactive homolog of UFSP2, among the top candidates in both active fractions (Figure 2D). Analysis by immunoblotting confirmed the restricted distribution of UFSP1 in the two active fractions (Figure 2E). Therefore, our cell-fractionation studies successfully captured an active form of UFSP1, a surprising observation considering the present consensus surrounding UFSP1 non-functionality in humans (HUGO Gene Nomenclature Committee [HGNC] annotation).
The UFSP1 activity we observe raises the possibility that the HGNC annotation is incorrect and the UFSP1 expressed in cells spans a longer stretch at the N terminus that contains the catalytic residues. Aggregate ribosome profiling data across multiple studies (Ribo-seq; GWIPS-viz; https://gwips.ucc.ie/) supported our hypothesis that regions upstream of the annotated UFSP1 locus are actively translated. A high number of protected reads was observed 5′ to the incorrect start site with coverage of the catalytic cysteine (C54) and a putative CTG start codon (Figure 3A). Reported previously, this non-canonical initiation site is a rare example of translation initiation from codons other than ATG and is proposed to be the only in-frame start codon capable of producing a functional UFSP1 cysteine protease (Ivanov et al., 2011). We therefore reanalyzed MS data from the two active fractions to search for peptides that correspond to regions upstream of the annotated start site. This analysis identified 12 matching peptides corresponding to the N terminus of UFSP1 (65% coverage) and included the catalytic cysteine (Figure 3B). Indeed, close inspection of curated isoforms of UFSP1 (Uniprot, Ensembl, and NCBI) showed that of the two human UFSP1 variants, one isoform (A0A5F9ZGY7) shares amino acid residues described as essential for the catalytic activity of murine UFSP2 (Figure 3C) (Kang et al., 2007; Ha et al., 2008). This conclusion is supported by cross-species bioinformatic analysis of UFSP1 transcripts that support the existence of the longer version (Figures S3A–S3C). Further confirmation is obtained in immunoblotting of endogenously expressed UFSP1, which migrates at a size consistent with the predicted molecular mass (∼24 kDa) upon translation of the correct transcript (Figure 2E). Importantly, this is larger than the incorrectly HGNC-annotated UFSP1 (15 kDa). These results imply that the long isoform is the primary UFSP1 protein expressed in human cells that is catalytically active. To test this experimentally, we expressed and purified recombinant UFSP1 corresponding to the 24 kDa form and the incorrectly annotated protein (Figure S3D). When incubated with the UFM1-GFP fusion protein, only the recombinant long-isoform UFSP1, but not its truncated form, showed cleavage activity in vitro (Figure 3D). Of note, UFSP1 is expressed at very low levels in cells as judged by RNA-sequencing data (GTEx) and quantitative proteomics data (Figures 3E–3G), possibly explaining why the correct UFSP1 may have escaped detection, further contributing to the acceptance of the misannotated form. Taken together, our analyses provide convincing evidence that the long-isoform UFSP1 we here identify is the correct endogenous UFSP1 that is catalytically competent via a cysteine-thiol-based mechanism, thus correcting the long-held misconception that human UFSP1 is an inactive protease.
Since nothing is known about the function of UFSP1 in cells, we next sought to identify the roles of UFSP1 in regulating UFMylation and to dissect the relative contributions of UFSP1 and UFSP2. First, a comparison of UFMylation in UFSP1−/− and UFSP2−/− cells revealed an increase of RPL26 UFMylation in UFSP2−/− cells, which was missing in the WT and UFSP1−/− cells. Instead, UFSP1−/− cells showed an increase in UFMylated UFC1 that could be confirmed in immunoprecipitations of UFM1 (Figure 4A). To confirm these observations and identify the lysine residue on UFC1 that is modified, we immunoprecipitated UFM1 from UFSP1−/− cell lines and analyzed them by LC-MS. Importantly the immunoblotting data were corroborated by detection of MS spectra corresponding to UFC1 peptides in which Lys122 was modified by Val-Gly (the C-terminal UFM1 dipeptide that remains on UFMylated residues after tryptic digest) (Figure S4A). Hence, in the absence of UFSP1, there is an accumulation of UFC1 UFMylated at K122. Interestingly, K122 is situated near the catalytic cysteine of UFC1 (Figure 4B). Immunoblot analysis of cell lysates revealed a basal level of UFMylated UFC1 across all cell lines tested (n = 15) (Figure S4B). While this observation was true of both human and murine cell lines, we note the presence of an unannotated short-isoform variant of UFC1 unique to mice (Figures S4B and S4C). While the earlier assays used a UFM1-GFP reporter to reveal peptidase activity in UFSP1, to confirm that UFSP1 has isopeptidase activity, we performed a series of in vitro assays with various in vitro generated substrates including UBA5 and UFC1 modified with UFM1 via an isopeptide bond. Furthermore, through the reconstitution of UFM1 pathway components, we were able to synthesize UFMylated products (Peter et al., 2022). These are a heterogeneous mixture of UFM1 conjugates including K69-linked polyUFM1 chains in addition to automodified UFC1 and UFL1(Peter et al., 2022). UFSP1 effectively cleaved UFM1 from these different substrates and catalyzed the disassembly of polyUFM1 chains in vitro (Figures 4C and 4D). Taken together, these data show that in addition to its peptidase function in targeting precursor UFM1, UFSP1 is an effective isopeptidase with activity toward diverse substrates.
To further explore the observation of enriched K122-modified UFC1 and establish that these observations are not cell line specific, we generated a series of UFSP1 and UFSP2 knockouts in three different cell lines, HEK293, U2OS, and HeLa, and multiple clones were confirmed by sequencing (Figure S5A). Consistent with our earlier observation (Figures S1C and S1D), we observe increased mono- and di-UFMylated RPL26 in UFSP2−/− but not UFSP1−/− cell lines (Figure 5A). Meanwhile, in all the different UFSP1−/− cell lines tested, we observed a size shift in UFC1 of approximately 10 kDa (Figure 5A). This size shift corresponded to modification of UFC1 with UFM1 and accounted for up to 50% of cellular UFC1 in UFSP1−/− cell lines (Figure 5A). These results suggest that UFC1 is constitutively modified with UFM1 in cells whose removal depends on UFSP1. By contrast, we observed no effect of loss of UFSP1 or UFSP2 on UFMylation of reported UFM1 substrates P53 and histone H4 (Figures S5B and S5C) (Qin et al., 2019; Liu et al., 2020). In the absence of either UFSP1 or UFSP2, cells appear able to generate sufficient mature UFM1. However, in cell lines lacking both UFSP enzymes (UFSP1−/−/UFSP2−/−), a complete loss-of-function phenotype is manifested by a total absence of detectable UFMylation (Figure 5B). This could be rescued through recombinant overexpression of UFSP1 (Figure S5D). These data are consistent with a requirement for either UFSP1 and/or UFSP2 in the generation of mature UFM1 and suggest that both enzymes contribute, in a partially redundant manner, to precursor UFM1 maturation. To confirm that the complete loss of UFMylation observed in double knockout cell lines stems from an absence of mature UFM1 in these cells, UFSP1−/−/UFSP2−/− cell lines in HEK293 and HeLa backgrounds were transiently transfected with constructs expressing either mature UFM11–83 or its precursor counterpart (UFM11−85). Overexpression of mature UFM1, but not proUFM1, successfully rescued mono- and di-UFMylated RPL26 (Figure 5B). These data are consistent with in vitro analysis of peptidase activity in cell lysates where cell lysates from UFSP1−/−, UFSP2−/−, and UFSP1−/−/UFSP2−/− HEK293 cell lines were incubated with the proUFM1-GFP probe. Here, cleavage activity was only completely abolished in the absence of both enzymes (Figure 5C). Furthermore, close inspection of immunoblot analyses reveals a size shift in UFM1 electrophoretic mobility consistent with an increase in molecular weight corresponding to proUFM1 in cell lines lacking both UFSP enzymes (Figure 5D). While these experiments clearly demonstrate complete loss of UFM1 maturation in cells lacking both UFSP1 and UFSP2, this does not preclude the existence of additional proteases in specific cell types.
We next explored whether differences in subcellular localization of UFSP1 and UFSP2 might contribute to the differences in substrate specificities observed in knockout cells. UFSP2 interacts with odorant response abnormal protein-4 (ODR4), a transmembrane protein that is localized to the ER membrane where it is thought to anchor UFSP2 in proximity to the ER-ribosome interface (Chen et al., 2014). In contrast, sequence analysis and structural predictions suggest that UFSP1 will not interact with ODR4 and is likely to instead reside in the cytosol. This makes it more possible for UFSP1 to be the protease mainly responsible for UFM1 maturation. To disrupt the ER localization of UFSP2, we generated ODR4 knockout cell lines and monitored UFMylation (Figures 6A and S6A). Intriguingly, UFSP2 protein levels are markedly reduced in ODR4−/− cell lines and vice versa, suggesting that UFSP2 and ODR4 are engaged in a mutually stabilizing relationship (Figure 6A). Moreover, UFSP2 and ODR4 knockout cell lines were an exact phenocopy in their role in restraining levels of RPL26 UFMylation (Figure 6A). In cell lines lacking both UFSP1 and ODR4, levels of di-UFMylated but not mono-UFMylated RPL26 were reduced. Interestingly, this is similar to our observation of a cell line heterozygously deficient for UFSP1 (UFSP1+/−/UFSP2−/−) where the second UFM1 modification on RPL26 was absent (Figures 6A and S6B). These data may indicate a preference for mono-UFMylated RPL26 in circumstances where mature UFM1 is limiting and aligns with the description of RPL26 modification as sequential with K132 UFMylation dependent on prior modification of K134 (Walczak et al., 2019). Finally, we investigated whether UFSP1 could contribute directly to the UFMylation pathway in ribosomal quality control. Upon induction of ribosome stalling following treatment with low-dose anisomycin, RPL26 UFMylation is induced only in the membrane fraction of WT cells (Figure 6B, top). In the absence of UFSP2, significant RPL26 UFMylation is observed, which does not increase further upon ribosome stalling (Figure 6B, bottom). In contrast, UFSP1 knockout cells remained competent for the induction of ribosome UFMylation upon ribosome stalling (Figure 6B). These data suggest that ribosome UFMylation is dynamic and is mainly regulated by UFSP2. UFSP1, on the other hand, indirectly regulates RPL26 UFMylation by facilitating UFM1 maturation and cleaving UFM1 from UFMylated UFC1. To address the biological significance of these findings, the proteomes of the different cell lines (UFSP1−/−, UFSP2−/−, and UFSP1−/−/UFSP2−/− HEK293) were analyzed by data independent acquisition (DIA) quantitative proteomics. A total of 577 proteins passed the fold change and significance thresholds in at least one phenotype (Benjamini-Hochberg adjusted p < 0.05; log2 fold change > 1) (Figures 6C and S6C). Heatmap analysis (k-means method) revealed that distinct subsets of proteins are altered in UFSP1 and UFSP2 knockout cells (Figures 6C, S6D, and S6E). Gene ontology overlaps calculated using a hypergeometric distribution tool provided by the molecular signatures database (msigdb; https://www.gsea-msigdb.org/) suggest contributions of UFSP1 to small-molecule metabolism (Figure 6D). By contrast, in UFSP2−/− HEK293 cells, processes including tissue development (VEGFA, FGFR1, BMP7, TIMP1, COL6A6) and lipid metabolism (LOX, ALOX5) were heavily represented, while UFSP1−/−/UFSP2−/− cells exhibited features of both UFSP1−/− and UFSP2−/− knockout cell lines (Figures 6C, 6D, and S6C–S6E). Proteins subject to the most extreme changes (COL6A6, CDKN2A, RPL22L1) were common to all genotypes (Figure 6E). Although ER-linked proteins were represented, we observed no effect on canonical ER stress or UPR-linked pathways (Figure S7A). Immunoblot analysis of cell fractions confirms that UFSP1 is cytosolic in localization and, together with bioinformatic interpretation of UFSP2-ODR4 interactions, supports our conclusion that cellular localization is key to understanding the unique functionality observed in proteomes of UFSP1−/− and UFSP2−/− knockout cell lines (Figures 7A–7C).
Considering that 99 deubiquitinating enzymes have been identified in the analogous ubiquitin system (Lange et al., 2021), it seemed remarkable that the UFM1 pathway in humans could be reliant on only one enzyme. Together with the confounding observation of active UFMylation in UFSP2−/− cell lines, it has been clear to many in the field that additional enzymes must exist to process precursor UFM1 into its mature counterpart (Witting and Mulder, 2021). Our study now reveals that the annotation of UFSP1 as inactive is mistaken, as we identify human UFSP1 to be translated from a non-canonical CTG start codon. We report that human UFSP1 is not only an active cysteine protease that is expressed in cells but also one with key functions in the UFM1 pathway. Overall, we define at least three contributions of UFSP enzymes. First, consistent with reports elsewhere, we find UFSP2 to be a key regulator of RPL26 modification. Second, UFSP1 acts to restrain levels of UFC1 modified with UFM1. Finally, in a partially redundant manner, both UFSP enzymes contribute to UFM1 precursor maturation and the maintenance of a cellular pool of mature UFM1 (Figure 7A). One of two mechanisms may contribute to the different substrate activities of UFSP enzymes. First, the cellular expression levels of UFSP1 are very low, possibly limiting its ability to counter RPL26 UFMylation. Second, subcellular localization: the cytosolic localization of UFSP1 compared with the ER localization of UFSP2 makes it more likely for UFSP1 to be the UFM1-maturing enzyme. The transmembrane domain of ODR4 provides a means for UFSP2 to associate with the ER membrane, bringing it into proximity of the ribosome and UFM1 ligase machinery (Chen et al., 2014). Structure prediction of ODR4 using AlphaFold reveals it to adopt an MPN fold (Jumper et al., 2021). Interestingly, the crystal structure of Ufsp from C. elegans reveals the presence of an MPN fold in addition to the catalytic domain (Kim et al., 2018). It remains to be investigated whether ODR4 only mediates ER localization of UFSP2 or has additional roles to allosterically modulate the activity of UFSP2. Superposition of an AlphaFold-predicted complex of UFSP2-ODR4 reveals that UFSP1 lacks domains required for interaction with ODR4 (Figure 7B). Hence, UFSP1 is localized to the cytosol where it is unable to interact with UFMylated RPL26 at the endoplasmic reticulum. We observe a striking accumulation of UFMylated UFC1 in UFSP1−/− cells but not UFSP2−/− cells. This suggests that UFC1 is constantly UFMylated in cells, and this modification is removed by UFSP1. Interestingly, previous studies have suggested regulation of E2 activity by covalent modification (Stewart et al., 2016). It may be that UFC1 UFMylation alters protein interactions with other UFM1-conjugating enzymes or putative substrates, either sterically or through the addition of a UFM1-interacting surface. For instance, the SUMO-specific E2 Ubc9 (UBE2I) is auto-SUMOylated, a modification that serves to attract proteins with SUMO-interacting motifs (Stewart et al., 2016). Of note, a UFM1 interacting motif has been described and may serve a similar purpose here (Padala et al., 2017; Kumar et al., 2021). Alternatively, auto-UFMylation may interfere with E2 catalytic activity. A recent case study of UBE2S automodification identified a lysine residue precisely five amino acids from the catalytic cysteine (K+5) (Liess et al., 2019). In thioester transfer assays, automodification at this site on UBE2S reduced the overall efficiency of ubiquitin transfer from the E1 by steric hindrance. Intriguingly, up to 25% of E2 enzymes possess a conserved lysine residue at +5 amino acids from the catalytic cysteine (Liess et al., 2019). Following a similar theme, monoubiquitylation of UBE2T at K86 (5 amino acids from the catalytic cysteine K91) is suggested to reduce E2 activity linked to the Fanconi anemia DNA repair pathway mediated by the E3 ubiquitin ligase FANCL (Machida et al., 2006). Our work identifies UFC1 to be modified on K122, a site located +6 residues from the catalytic C116. While further work is required, we speculate a similar inhibitory role for this UFC1 modification. These observations may reflect a common regulatory feature of E2 enzymes. If so, then UFSP1 may be the first documented instance of a protease acting to relieve E2 autoinhibition, eventually influencing the rate of overall UFMylation. Hence, UFSP1 may act at two levels to activate UFMylation, firstly in UFM1 maturation and secondly as part of a regulatory loop to remove the inhibitory UFC1 modification. Given that UFSP1 gene expression is remarkably low relative to other UFM1 pathway components, it is possible that under specific circumstances, UFSP1 gene induction may function as an inducible ON-OFF switch or accelerator for ribosome UFMylation. Further studies will be required to assess whether this is the case and to assess the precise kinetics of UFMylation in cellular models with UFSP1 overexpression or deletion. Moreover, studies examining whether UFSP1 contributes to other functions attributed to the UFM1 pathway, including regulation of the DNA damage response via histone H4 UFMylation or ER-phagy induced by metabolic stress, are likely to reveal novel insights (Qin et al., 2019; Liang et al., 2020). In particular, our proteomic analysis aligns with observations elsewhere that UFMylation may be integral to the development of extracellular matrix (Egunsola et al., 2017; Walczak et al., 2019). A serendipitous outcome of our study is the correction of the long-held misconception of UFSP1 as an inactive peptidase homolog. This misunderstanding stems from the annotation of UFSP1 as the “inactive UFM1-specific protease-1” (HGNC annotation) and is based on a hypothetical interpretation of an N-terminal truncated version of UFSP1 that lacks catalytic residues when compared with UFSP2. Perhaps early studies characterizing UFSP1, performed in murine systems, were reliant on annotations that predate entry of the long isoform described here (Kang et al., 2007; Ha et al., 2008). Clearly, the naming of UFSP1 as the “inactive UFM1-specific protease-1” will require revision. Our study corrects the view that human UFSP1 is catalytically inactive, and in doing so has laid foundations for future investigation into the unique contributions of UFSP family proteases to ER and cellular homeostasis.
A major substrate of UFSP1 appears to be UFC1, as UFMylated UFC1 modified at K122 accumulates in cells lacking UFSP1. Our work does not determine the cellular conditions that promote UFC1 modification at this potential autoinhibitory site. Furthermore, it will be important to establish the kinetics of this UFC1 modification in cells. Our investigation of UFSP1 and UFSP2 function in this study has employed constitutive knockout cell lines that may be subject to cellular adaptation. Future studies using acute depletion or inhibition of UFSPs will shed light on the dynamics and effect of rapid changes in UFMylation. Moreover, quantitative and temporal comparison of UFSP1 and UFSP2 activity on their respective substrates will inform the significance of this regulatory circuit.
Further information and requests for resources and reagents should be directed to and will be fulfilled by the lead contact, Yogesh Kulathu ([email protected]).
Plasmids and cell lines are available upon request to the study lead author listed above. Identifier codes for plasmids and cell lines are included in the key resources table. All cell lines used in this study are maintained in a dedicated cell bank and are traceable by Cell line name, Clone number, and CRISPR project ID. The authors declare no restriction on the use of materials detailed herein.
Cell lines were cultured in DMEM (GIBCO) supplemented with 10% v/v Fetal Bovine Serum (FBS), 50mg/ml Penicillin Streptomycin, and 2mM L-Glutamine. Cell cultures were maintained in a 5% CO2 incubator in a humidified environment and routinely checked for mycoplasma. Cell lines used in this study include U2OS, HeLa, HEK293, and commercially available Flp-In T-REx HEK293 cells (Invitrogen; R78007). Cell lines were sourced from a dedicated facility at MRC-PPU core services.
HEK293T cells (10 confluent plates) were collected in phosphate-buffered saline/PBS (Gibco; 14190-094) supplemented with 1mM EDTA and 1mM EGTA. Cells were washed once in PBS, resuspended in ice-cold cracking buffer (50mM Tris pH7.5, 1mM DTT, 0.1mM EDTA, 0.1mM EGTA), and incubated on ice for 15 minutes before lysis by mechanical stress (>20 sequential passes through 21-23-gauge needles). The lysate was cleared by centrifugation (17000 x g for 5 minutes), passed through a 25mm/45μm polyethersulfone filter (Sigma Aldrich; WHA68962504), and de-salted into a desalting buffer (30mM MOPS pH7.0, 5% glycerol, 1mM DTT, 0.015% Brij 35) on a Sephadex G25 column using an Akta Pure Fast Protein Liquid Chromatography (FPLC) device. The lysate was next applied to a 1mL Heparin HiTrap column (GE-Healthcare Life Sciences, now Cytiva) with elution on an increasing salt gradient into 1mL fractions (Greiner bio-one 96 well blocks; 780270). A salt gradient was introduced using a high salt buffer (30mM MOPS pH7.0, 1.2M NaCl, 5% glycerol, 1mM DTT, 0.015% Brij 35). Heparin column flow-through was next passed through a Source Q HR 5/5 column into 30mM Tris-HCl pH8.2, 5% Glycerol, 1mM DTT, and 0.015% Brij 35 with elution on a salt gradient (High salt buffer supplemented with 1.0M NaCl). SourceQ fractions (1mL volume) were eluted into a 96 well block at 1mL intervals. Heparin and SourceQ binding fractions were immediately snap-frozen in liquid nitrogen and stored at −80°C until use. For mass spectrometry, fractions were washed/buffer exchanged in an Amicon ultra centrifugal concentrator (Millipore; UFC500396) with 2mL 30mM Tris-HCl pH 7.5 containing 1mM TCEP, followed by 2mL 50mM triethylammonium bicarbonate buffer (Sigma Aldrich T7408-100mL) containing 1mM TCEP. Samples were then concentrated to approximately 100μL volume and alkylated with 40mM IAA (Sigma Aldrich; I1149-5G) for 3 hours in the dark at room temperature. Samples were next reduced by adding 2mM DTT and incubating at room temperature for a further 15 minutes (Formedium; DTT100). After overnight digestion with 10μg/ml mass-spectrometry grade Trypsin (Pierce; 90057), samples were submitted to the MRC-PPU mass-spectrometry facility for analysis.
To prepare samples a 3:4 dilution was made in NuPAGE LDS sample buffer (ThermoFisher; NP007) supplemented with 10% v/v β-mercaptoethanol. Samples were heated to 95°C for 5 minutes before gel loading. Gel electrophoresis was performed using an XCell SureLock electrophoresis tank (ThermoFisher; EI0001) with 4–12% Bis-Tris NuPAGE pre-cast 12, 15, and 26 well gels (Invitrogen; NP0322, NP0323, and WG1403BX10). Protein was transferred to a 45μm nitrocellulose membrane (Amersham; 10600002) at 90V for 90minutes in a Mini Trans-Blot Cell (Biorad;). Membranes were blocked for one hour in 5% Bovine Serum Albumin (Sigma Aldrich; A7906-100G)) dissolved in TBST. Primary antibodies were diluted 1:1000 in 5%BSA TBST and incubated overnight with shaking at 4°C. Membranes were washed three times for 10 minutes per wash in TBST. Membranes were next incubated with fluorescent secondary antibody (IR800) diluted 1:20,000 in TBS containing 5%BSA, 0.1% Tween for 30 minutes at RT with shaking. After washing in TBST for a further 30 minutes (3 × 10-minute washes) membranes were visualized on a Lycor Odyssey CLx.
To generate cell lysates for RPL26 immunoblots, one near-confluent 15cm plate of HEK293 cells was gently collected in 0.5mM EDTA/0.5mM EGTA and placed on ice. Cells were washed once in ice-cold PBS and resuspended in lysis buffer (1%NP40, 50mM Tris-HCl pH7.5, 150mM NaCl) supplemented with a protease inhibitor cocktail (1mM benzamidine, 1mM AEBSF, Protease inhibitor cocktail (Roche; 48679800)). Lysates were cleared by centrifugation (20,000 x g, 5 minutes) and mixed with LDS sample buffer as before. For enzymatic assays, cell lysates were generated in the absence of protease inhibitors as described in the cell fractionation procedure above. For chemical induction of UFMylation cells were treated with 200nM Anisomycin dissolved in DMSO for 20 minutes before harvesting.
HEK293 or HeLa cells were washed once in PBS, collected in ice-cold PBS, and pelleted by centrifugation at 1000 x g. Cell pellets (∼1 × 107) were resuspended in 1 mL of 0.02% w/v digitonin, 50 mM HEPES pH 7.5, 150 mM NaCl, 2 mM CaCl2, and 1 x protease inhibitor cocktail tablet EDTA-free. Lysates were incubated on ice for 10 min and centrifuged at 17000 x g for 10 min at 4°C. The supernatant was transferred to a new Eppendorf tube (cytoplasmic extract). The remaining pellet was washed with 1 mL PBS and resuspended in 1 mL of 1% Triton X- 100, 50 mM HEPES pH 7.5, 150 mM NaCl, and 1x protease inhibitor cocktail tablet EDTA-free. Lysates were incubated on ice for 10 minutes and centrifuged at 17000 × g for 10 min at 4°C. The supernatant was transferred to a new Eppendorf tube (membrane extract). The remaining pellet was washed with 1 mL PBS and sonicated in 1 mL of 1% SDS, 25 mM Tris pH 8, 150 mM NaCl, 2.5 mM EDTA, and 1x protease inhibitor cocktail tablet EDTA-free (nuclear extract). Equal volumes of the collected fractions were resolved by SDS-PAGE and subjected to immunoblot analysis.
Pro-UFM1 (NP_057701.1) was cloned in frame with an N-terminal GFP tag and intervening short peptide linker (GSGEGRG) into the pcDNA5/FRT/TO bacterial expression vector (Invitrogen; V652020). A C-terminal histidine tag (Hisx6) with a C3 protease site facilitated the generation of native protein after purification with Ni2+/NTA affinity beads. UFSP1 short and long variant isoforms were cloned into the pGEX6P1 vector for bacterial expression. UFSP2 (modified with a stabilizing R136A mutation) was cloned into the petDuet (His6-TEV-UFSP2; DU59927) bacterial expression vector in frame with an N-terminal 6xHis-tag. A full list of cDNA constructs is included in the key resources table.
Recombinant GST-3C-tagged UCH-L1, UCH-L3, UCHL-5, and BAP were obtained from MRC-PPU Reagents and Services (https://mrcppureagents.dundee.ac.uk/). UFSP1 short (Q6NVU6) and long (A0A5F9ZGY7) isoforms and UFC1 (DU47927, DU68653, and DU73281, respectively) were expressed with GST-3C tags and purified using Glutathione S-transferase (GST) affinity purification. Briefly, expression constructs were transformed into E.coli BL21(DE3) competent cells, and expression of the recombinant protein was induced with 0.25 mM IPTG overnight (∼16 hours) at 18°C. Cells were sedimented by centrifugation, resuspended in lysis buffer (50 mM Tris-HCl pH 8.0, 300 mM NaCl, 2 mM DTT), and supplemented with a protease inhibitor cocktail (1 mM benzamidine, 1 mM AEBSF, 1x protease inhibitor cocktail (Roche; 48679800)), and lysed by Ultra sonification. The lysate was cleared by ultracentrifugation at 30,000 x g for 30 minutes and mixed with Glutathione Sepharose 4B beads for approximately 1.5 hours at 4°C. Beads were washed with high salt wash buffer (50 mM Tris-HCl pH 8.0, 500 mM NaCl, 2mM DTT) and low salt wash buffer (50 mM Tris-HCl pH 8.0, 150mM NaCl, 2 mM DTT, 10% Glycerol). Protein was eluted by incubation with 0.1 mg 3C protease (MRC-PPU Reagents and Services) in 10 mL low salt wash buffer overnight at 4°C. The cleaved protein was further purified on a Superdex-75 gel filtration column. The purified protein was concentrated, aliquoted, snap-frozen in liquid nitrogen, and stored at −80°C until further use.
pET15b-6xHis-3C-UBA5 (DU32106) and pET15b-6xHis-TEV-UFM1 (DU73256) and pET15b-6xHis-3C-UFM1-GSGEGR-GFP (DU59553) were transformed into E.coli BL21(DE3) competent cells. Expression of the recombinant protein and cell lysis was performed as described above. The lysate was cleared by ultracentrifugation at 30,000 x g for 30 minutes, and then mixed with Ni2+ NTA beads in binding buffer (25 mM Tris pH 8.0, 300 mM NaCl, 10 mM imidazole, 1 mM DTT). Beads were then washed with 30-bed volumes of wash buffer (25 mM Tris pH 8.0, 300 mM NaCl, 20 mM imidazole, 2 mM DTT). UBA5 and UFM1 were eluted with elution buffer (50 mM Tris pH 8.0, 200 mM NaCl, 300 mM imidazole, 1 mM DTT). UFM1 and UFM1-GFP were eluted by incubation with TEV and 3C protease (MRC-PPU Reagents and Services), respectively, overnight at 4°C. Proteins were further purified on a Superdex-200 HiLoad ™ 16/600pg (UBA5, UFM1-GFP) or Superdex-75 HiLoad ™ 16/600pg (UFM1) gel filtration column. Peak fractions were concentrated to 2–16 mg/mL, snap-frozen in liquid nitrogen, and stored at −80°C until use.
UFMylated (UBA5-UFM1) was generated by incubating UBA5 (0.01 mM) with UFM1 (0.01 mM) in reaction buffer (50 mM Tris pH 8.0, 200 mM NaCl, 5 mM ATP, 5 mM MgAc) for 17 hours at 23°C. UFC1-UFM1 was generated by incubating UFC1 (0.05 mM) with UBA5 (2 μM) and UFM1 (0.05 mM) in reaction buffer for 4 h at 37°C. Reaction products were purified on Superdex-200 HiLoad ™ 16/600pg (UBA5-UFM1) and Superdex-75 HiLoad ™ 16/600pg (UFC1-UFM1). Peak fractions were concentrated, snap-frozen in liquid nitrogen and stored at −80°C until use.
Whole-cell lysates (WCL) were extracted from UFSP2−/− HEK293 cells by mechanical lysis (syringe), thiol proteases ‘activated’ by addition of 10mM Dithiothreitol (DTT) (Kulathu et al., 2013; Lee et al., 2013). and incubated with the UFM1-GFP fusion protein. Cell fractions and/or recombinant enzymes were pre-activated on ice in Activation Buffer (50mM Tris-HCl pH7.5, 50mM NaCl) supplemented with 10mM freshly prepared DTT. The activated enzyme was next incubated with 5ug (3μM) recombinant UFM1-GFP fusion protein for 3 hours at 37°C. Cleavage of the GFP tag was analyzed by Coomassie stain and/or immunoblot analysis. For assays involving chemical inhibitors, Iodoacetmide (Sigma-Aldrich; I1149-5G) or N-ethylmaleimide (Sigma-Aldrich; 04259-5G) were added to the enzyme for 1 hour at room temperature in the dark before mixing with recombinant UFM1-GFP. All enzymatic reactions were completed in ultra-pure distilled water (Millipore QPOD; ZMQSP0D01).
CRISPR guide RNAs were designed with support from T. MacCartney at MRCPPU Reagents and Services. CRISPR sense and anti-sense guides were cloned into pX335 (Addgene plasmid 42335; Feng Zhang lab; Massachusetts Institute of Technology) and pBABED puro U6 (DU48788) plasmids respectively. The pX335 construct contains a chicken β–actin promoter-driven expression cassette for Cas9. In a separate strategy, single guide RNAs were cloned into the px459 vector (Addgene; 48139). Full details of guide-RNAs, frameshift mutations, and relevant sequencing data are included in the supplementary figures and key resources table. Procedures are described elsewhere (Ran et al., 2013a, 2013b): briefly, 1–2 million cells were seeded into a 10cm dish in antibiotic-free Dulbecco’s Modified Eagle Medium (DMEM) and transfected with 1μg plasmid DNA using Lipofectamine 2000 (Invitrogen; 1168019) according to the manufacturer’s instructions. Cells were selected in 2μg/ml puromycin for 24 hours followed by a 24-hour recovery period in pre-conditioned media. Cells were plated at clonal dilution (0.7 cells/well) or submitted for single-cell sorting, expanded, and screened by sequencing and/or immunoblot analysis.
For UFSP1, UFSP2, and ODR4 knockout clones, a ∼1-1.5Kb fragment that included guide-RNA target sites was PCR amplified using Q5 High-Fidelity DNA Polymerase (NEB; M0491). Primers were designed using the NCBI Primer Blast tool and are documented in the key resources table. PCR products were purified by spin column (QIAGEN;28104) and cloned into a plasmid vector using the StrataClone blunt PCR cloning kit (Agilent; 240207). Colonies were selected and grown in 4mL 2xTY media supplemented with Ampicillin (10μg/ml). Plasmid DNA was extracted using the QIAprep Spin Miniprep kit (QIAGEN; 27104) and submitted for sequencing at the MRC PPU DNA sequencing and services division. Mutations were aligned to the Hg38 assembly (UCSC genome browser) using ClustalW (European Bioinformatics Institute; Muscle). Primers are detailed in the key resources table.
Western blots were processed in ImageStudio Lite (Licor) and arranged in Adobe illustrator. Original Graphics and cartoons were developed in Adobe Illustrator. Data filtering and analysis of public resources (MEROPS, GTEx) were completed in RStudio. Heatmaps were generated using the Complex Heatmap R-package (ComplexHeatmap) in R studio and clustered using default parameters (Euclidean method). Protein structures were visualized in ChimeraX. For the heatmap in Figure 6C, a k-means clustering approach (n = 3) was applied following statistical analysis using the Elbow method. Proteins within clusters are grouped by the Euclidean method.
Protein homology was analyzed using Consurf (Tel Aviv University; https://consurf.tau.ac.il/) and visualized in Pymol (Educational license V2) by Schrodinger (https://pymol.org/2/). Mass-spectrometry results were aligned with the MEROPS database (European bioinformatics Institute; https://www.ebi.ac.uk/merops/) to discover novel peptidases. Gene expression data was downloaded from the Genotype-Gene expression project (GTEx) (Broad Institute; https://www.gtexportal.org/home/). For protein copy number analysis across 32 human tissues, the dataset PXD016999 from ProteomeXchange (https://doi.org/10.1016/j.cell.2020.08.036) (Jiang et al., 2020) was reanalyzed using MaxQuant 2.0.3.1.6.
fasta files corresponding to the amino acid sequence of human UFSP1 (Q6NVU6; A0A5F9ZGY7) and UFSP2 (H0Y9B0; H0YA18; D6RA67; Q9NUQ7) protein-coding transcripts were downloaded from Uniprot with reference to Ensembl annotation. Analogous sequences from other species were obtained with reference to the NCBI Homologene resource. Sequence alignment was performed using the ClustalW algorithm in Muscle (European Bioinformatics Institute). The ClustalW output was visualized in Jalview and edited in Adobe Illustrator.
For each sample, a confluent 15cm plate of HEK293 cells was resuspended in ice-cold PBS (1mM EDTA/1mM EGTA), pelleted by centrifugation, and immediately lysed by addition of SDS-lysis buffer (5% SDS, 50mM TEAB pH8.5). Lysates were boiled for 5 minutes at 95°C followed by sonication using a Diagenode Biorupter at high energy for 10 cycles (30sec ON, 30sec OFF). Lysates were cleared by centrifugation at 20,000 x g for 20 minutes and quantified by BCA assay (Pierce; 23225). 200μg protein was prepared as follows; TCEP stock solution (100mM TCEP, 300mM TEABC) was added to a final concentration of 10mM TCEP (1:10), and samples were incubated at 60°C for 30 minutes. Samples were rested at room temperature and freshly prepared iodoacetamide (IAA) added to 40mM final concentration. After 30 minutes at room temperature shielded from light and with gentle agitation, samples were acidified by the addition of mass-spectrometry grade 12% phosphoric acid to a final concentration of 1.2% (1:10). Sample ‘clean-up’ was completed using S-trap micro-columns with overnight on-column digestion using 13μg trypsin per 200μg of protein input. Eluted peptides were lyophilized by speed-vacuum and submitted to the MRC-PPU core mass-spectrometry facility. For differential expression analysis data were processed using LIMMA. Data was analyzed in Dia-nn 1.8 (Demichev et al., 2020; Steger et al., 2021). Selected MSMS spectra of VG-modified peptides were annotated using IPSA.
Statistical details are included in the figure legends. All experiments shown are representative of at least three independent experiments. Observations of CRISPR knockout cell lines include multiple biological replicates (independently isolated clones with different mutations) as described in the figure legends. For proteomics data analysis, three technical replicates (three plates of the same CRISPR clone) were processed in parallel. For analysis of proteomics data, we considered a Benjamini & Hochberg adjusted p-value of <0.05 as significant. An arbitrary Log2 fold cut-off value of >1 was applied to focus the analysis on proteins with the most robust change. Gene Ontology enrichments were calculated using a hypergeometric tool (msigdb) with a p-value of less than 0.05 considered significant. For heatmap analysis, k-means clustering was performed using R base functions in Rstudio. To determine the appropriate number of clusters the elbow statistic was applied using the FactoExtra R-package. Visualization and euclidean clustering of proteins within k-means clusters was performed using the Complex heatmap package. R-packages are detailed in the key resources table. | true | true | true |
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PMC9638459 | Sonakshi Rastogi,Aditi Singh | Gut microbiome and human health: Exploring how the probiotic genus Lactobacillus modulate immune responses 10.3389/fphar.2022.1042189 | 24-10-2022 | lactobacillus,inflammation,gut microbiome,probiotics,gut-lung axis,gutbrain axis,gut-heart axis,gut-bone axis | The highest density of microbes resides in human gastrointestinal tract, known as “Gut microbiome”. Of note, the members of the genus Lactobacillus that belong to phyla Firmicutes are the most important probiotic bacteria of the gut microbiome. These gut-residing Lactobacillus species not only communicate with each other but also with the gut epithelial lining to balance the gut barrier integrity, mucosal barrier defence and ameliorate the host immune responses. The human body suffers from several inflammatory diseases affecting the gut, lungs, heart, bone or neural tissues. Mounting evidence supports the significant role of Lactobacillus spp. and their components (such as metabolites, peptidoglycans, and/or surface proteins) in modulatingimmune responses, primarily through exchange of immunological signals between gastrointestinal tract and distant organs. This bidirectional crosstalk which is mediated by Lactobacillus spp. promotes anti-inflammatory response, thereby supporting the improvement of symptoms pertaining to asthma, chronic obstructive pulmonary disease (COPD), neuroinflammatory diseases (such as multiple sclerosis, alzheimer’s disease, parkinson’s disease), cardiovascular diseases, inflammatory bowel disease (IBD) and chronic infections in patients. The metabolic disorders, obesity and diabetes are characterized by a low-grade inflammation. Genus Lactobacillus alleviates metabolic disorders by regulating the oxidative stress response and inflammatory pathways. Osteoporosis is also associated with bone inflammation and resorption. The Lactobacillus spp. and their metabolites act as powerful immune cell controllers and exhibit a regulatory role in bone resorption and formation, supporting bone health. Thus, this review demonstrated the mechanisms and summarized the evidence of the benefit of Lactobacillus spp. in alleviating inflammatory diseases pertaining to different organs from animal and clinical trials. The present narrative review explores in detail the complex interactions between the gut-dwelling Lactobacillus spp. and the immune components in distant organs to promote host’s health. | Gut microbiome and human health: Exploring how the probiotic genus Lactobacillus modulate immune responses 10.3389/fphar.2022.1042189
The highest density of microbes resides in human gastrointestinal tract, known as “Gut microbiome”. Of note, the members of the genus Lactobacillus that belong to phyla Firmicutes are the most important probiotic bacteria of the gut microbiome. These gut-residing Lactobacillus species not only communicate with each other but also with the gut epithelial lining to balance the gut barrier integrity, mucosal barrier defence and ameliorate the host immune responses. The human body suffers from several inflammatory diseases affecting the gut, lungs, heart, bone or neural tissues. Mounting evidence supports the significant role of Lactobacillus spp. and their components (such as metabolites, peptidoglycans, and/or surface proteins) in modulatingimmune responses, primarily through exchange of immunological signals between gastrointestinal tract and distant organs. This bidirectional crosstalk which is mediated by Lactobacillus spp. promotes anti-inflammatory response, thereby supporting the improvement of symptoms pertaining to asthma, chronic obstructive pulmonary disease (COPD), neuroinflammatory diseases (such as multiple sclerosis, alzheimer’s disease, parkinson’s disease), cardiovascular diseases, inflammatory bowel disease (IBD) and chronic infections in patients. The metabolic disorders, obesity and diabetes are characterized by a low-grade inflammation. Genus Lactobacillus alleviates metabolic disorders by regulating the oxidative stress response and inflammatory pathways. Osteoporosis is also associated with bone inflammation and resorption. The Lactobacillus spp. and their metabolites act as powerful immune cell controllers and exhibit a regulatory role in bone resorption and formation, supporting bone health. Thus, this review demonstrated the mechanisms and summarized the evidence of the benefit of Lactobacillus spp. in alleviating inflammatory diseases pertaining to different organs from animal and clinical trials. The present narrative review explores in detail the complex interactions between the gut-dwelling Lactobacillus spp. and the immune components in distant organs to promote host’s health.
The human body is inhabited by trillions of dynamic and diverse microbial communities that potentially regulate the physiology of the host, popularly known as the “Microbiome” (Malard et al., 2021). The human microbiome performs imperative biological functions such as immune system homeostasis, regulation of host metabolism, prevention of pathogens invasion and improvement of the epithelial barrier function (Malard et al., 2021). The highest density of microbes resides in human gastrointestinal tract, known as the “Gut microbiome” (Dwivedi et al., 2021. The predominant bacterial phyla in the gastrointestinal tract are Firmicutes and Bacteroidetes, followed by Actinobacteria and Proteobacteria (Rinninella et al., 2019). Of note, the members of the genus Lactobacillus that belong to phyla Firmicutes are the most important probiotic bacteria of the gut microbiome. These gut-residing Lactobacillus species not only communicate with each other but also with the gut epithelial lining to balance the gut barrier integrity, mucosal barrier defence and ameliorate the host immune responses (Martín et al., 2019). In addition, Lactobacillus species exhibit microbial roles by competitively excluding opportunistic pathogens from inhabiting functional niches in the gut, restraining attachment of pathogens on epithelium as well as directly killing pathogens by producing lactic acid, acetic acid, propionic acid, bacteriocins and reactive oxygen species (ROS) (Dempsey and Corr, 2022). Besides microbial roles, commensal Lactobacillus species regulate both adaptive and innate immune responses by inducing T cells, Natural Killer (NK) cells, macrophages differentiation, cytokine-production and stimulating toll-like receptors (TLRs). They also manifest immunomodulatory effect by increasing immunoglobulin-A (IgA) producing B cells expression in Peyer’s patches in the lamina propria, where they block pathogens adhesion to the intestinal epithelium (Cristofori et al., 2021). The human body suffers from several inflammatory diseases affecting the gut, lungs or neural tissues. Mounting evidence supports the significant role of Lactobacillus species in suppressing inflammatory responses by downregulating the expression of T-helper17 (Th17) inflammatory cells and their signature cytokines IL-17F, and tumor necrosis factor-alpha (TNF-α). Numerous works of literature is available that highlights the beneficial role of these commensals in ameliorating symptoms of cardiovascular-related diseases (CVD), osteoporosis, metabolic diseases such as diabetes and obesity (Zaiss et al., 2019; Archer et al., 2021; Companys et al., 2021). The genus is also reported to regulate cholesterol metabolism as well as gut-derived metabolites production such as trimethylamine-N-oxide (TMAO), short chain fatty acids (SCFAs), lipopolysaccharides (LPS), and bile acids (BA). Osteoporosis is associated with bone inflammation and resorption (Britton et al., 2014). The Lactobacillus spp. and their metabolites also act as powerful immune cell controllers and exhibit a regulatory role in bone resorption and formation, supporting bone health. (Zaiss et al., 2019). In view of these considerations, the significant role of Lactobacillus spp. in the alleviation and treatment of human diseases presents attractive therapeutic potential. Thus, the aim of this review is to analyze the current research works implicating the potent use of these microbes and their components in mediating immune responses that affect host health. PubMed was used to search for all of the studies published over the last 15 years using the key words “lactobacillus” or “Gut microbiota” and “inflammation”. More than 550 articles were found, and only those published in English and providing data on aspects related to human diseases were included in the evaluation.
Nowadays, the gut microbiome is a prominent area of scientific research as it holds imperative role in human health and pathology. Of note, the members of genus Lactobacillus are the most important probiotic bacteria. Lactobacillus spp. act by regulation of luminal pH, enhancement of barrier function by increasing mucus production, secretion of antimicrobial peptides, and by changing the gut microbial composition (Dempsey and Corr, 2022). Their cogent functional attributes include improving digestion, maintenance of gastrointestinal-barrier integrity, competition to opportunistic pathogens, neuromodulation, participation in maturation of the immune system in early life and preservation of immune homeostasis during entire life, production of metabolites, vitamins and other components (Dempsey and Corr, 2022). Detailed taxonomic profiling reveals that the genus Lactobacillus belongs to phylum Firmicutes, class Bacilli, order Lactobacillales and family Lactobacillaceae. They are Gram-positive, catalase-negative, non-spore forming, obligate saccharolytic rods or coccobacilli with low GC (guanine and cytosine) content of the genome (Zheng et al., 2020). Members of the genus Lactobacillus are well-adapted to the hostile environment persisting in oral, gastrointestinal and vaginal tract. In healthy adult’s feces, the concentration of different lactobacilli species accounts for upto 105–108 CFU/g. Among human gut-dwelling microbial species, Lactobacillus crispatus, Lactobacillus gasseri, Lactobacillus ultunensis, Ligilactobacillus ruminis, Limosilactobacillus reuteri, Lactobacillus kalixensis, Lactocaseibacillus casei, Limosilactobacillus gastricus, Limosilactobacillus antri, Lactobacillus rhamnosus, Ligilactobacillus salivarius, Limosilactobacillus fermentum, etc. are found to be permanent and form essential part (Zheng et al., 2020). Probiotic supplementation with lactobacilli species assists in altering the gut microbiota composition, thereby reducing dysbiosis and maintaining the microbial balance with a greater abundance of useful bacteria. A probiotic formulation comprising of L. rhamnosus GG, L. acidophilus, L. plantarum, L. paracasei, and L. delbrueckii when given orally tends to stimulate the other gut microbial species such as Prevotella and Oscillibacter that exhibited anti-inflammatory activity in rats (Zeng et al., 2021).
As probiotics, lactobacilli play significant roles through various GM-derived metabolites andpossess several health ameliorating attributes common among them are alleviation of chronic diseases, immune system stimulation, pathogen protection, and nutritional physiology.
When the gut barrier becomes dysfunctional, it leads to several inflammatory conditions, such as irritable bowel syndrome (IBS), inflammatory bowel disease (IBD) and obesity. Gut-dwelling Lactobacillus spp. were found to restore gastrointestinal barrier function directly or indirectlyin mice inflammation models (Martín et al., 2019). Indirect mechanisms through which they interact with the immune system involve modulating gut microbiota, regulating intestinal epithelial barrier integrity, viscoelastic mucin layer properties, antagonistic peptides/factors production and competitive exclusion of pathogens (Cristofori et al., 2021).
The gut epithelial barrier separates the internal intestinal milieu from the luminal environment, thereby ascertaining the permeability of nutrients and other molecules as well as exerting a protective role by arresting the entry of microbes and toxic compounds. Studies have reported that commensal Lactobacilli spp. maintain gut barrier integrity which depends upon the multi-protein complexes such as tight junctions, gap junctions, adherens and desmosomes (Kocot et al., 2022). These complexes are primarily made up of transmembrane proteins namely, claudin, occludin, and junctional adhesion molecules which interact with adjacent cells via zonula occludens (ZO) and actin fibers. In case of chronic inflammatory diseases or enteric infections, the intestinal barrier integrity is disrupted. Studies have reported increased expression of ZO-1, occludin and claudin proteins by L. rhamnosus CNCM-I in enterohemorrhagic E. coli O 157:H7 infected Caco-2 cell lines (Laval et al., 2015). Similarly, L. casei DN-114 001 up-regulates ZO-1 protein expression via activation of Toll-like receptor (TLR)-2 in Caco-2 cells. The TLR-2 binding leads to Protein kinase C (PKC) activation which causes tight junction proteins translocation, thereby enhancing gut barrier function (Kocot et al., 2022).
Gut epithelium is covered by high molecular weight glycoproteins, mainly mucins which are produced by goblet cells (Wuet al., 2020). The viscoelastic mucin layer provides protection against digestive enzymes, supports food passage and prevents translocation of microbes across lamina propria, thus imparting gut homeostasis (Wu et al., 2020). Studies have reported certain Lactobacillus strains to regulate mucin gene expression thereby changing the mucus layer attributes and indirectly affecting the gut immune responses. Of note, L. rhamnosus CNCM I-3690 regulated Muc2 and Muc3 gene expression in mucus-producing goblet cells in mice inflammation model, thus preventing gut barrier dysfunction and inflammation (Martín et al., 2019).
The vital function of lactobacilli strains lies in their ability to prevent pathogenic growth by synergistically inhibiting enteropathogens and stimulating the host immune system. The lactobacilli that showed in vitro antagonistic activity against periodontal and enteric pathogens are L. oris, L. paracasei, L. crispatus, L. gasseri, L. salivarius, L. plantarum, L. delbrueckii, L. rhamnosus, L. acidophilus, and L. fermentum, thereby improving gut homeostasis (NĘdzi-GÓra et al., 2020; Dempsey and Corr, 2022). Among the known factors that contribute to the antimicrobial ability of Lactobacillus spp. is their tendency to produce a wide range of metabolites such as organic acids, hydrogen peroxide, nitric oxide, SCFAs and bacteriocin that impede the growth of pathogens (Sulijaya et al., 2020). Organic acids, particularly lactic acid is a potent inhibiting factor as it creates acidic cytoplasmic pH as well as permeabilize the outer membrane of Gram-negative microorganisms (Sulijaya et al., 2020). For example, L. acidophilus 4,356 inhibited the growth of H. pylori by abundantly producing lactic acid (Modiri et al., 2021). Nitric oxide (NO) is another inhibitory microbial metabolite. Probiotic lactobacilli can effectively elevate NO synthesis or can stimulate host macrophages for NO production. For example, L. fermentum LF1 produces NO via the NO synthase pathway via oxidation of l-arginine to l-citrulline (Dempsey and Corr, 2022). Some may even ribosomally synthesize short peptides known as bacteriocins which possess broad-spectrum inhibitory activity against foodborne pathogens and spoilage bacteria. For example, L. plantarum LPL-1 produced 4,347 Da plantaricin LPL-1 (Wang et al., 2018) while curvacin A is from L. curvatus (Ahsan et al., 2022). In the genome of several other lactobacilli species, genes encoding for pediocin and plantaricin are found (Ahsan et al., 2022). The hydrogen peroxide producing lactobacilli also induce growth stagnation in pathogens. L. johnsonii UBLJ01 genome analysis revealed NADH oxidase, lactate oxidase and pyruvate oxidase genes involved in H2O2 synthesis while in vitro test reported their strong antagonistic potential (Ahire et al., 2021). In addition to the synthesis of inhibitory substances, Lactobacilli spp. can decrease the toxicity by degradation of microbial toxins, particularly by impeding toxin expression or by binding to the pathogen’s outer membrane. For example, L. rhamnosus JB3 reduced the infection of H. pylori in the gut by either forming lipid rafts or downregulating the expression of virulent genes (Do et al., 2021). Recently, literature is available that highlights the ability of lactobacillus spp. in detoxifying mycotoxins that are known to cause carcinogenesis and immune-suppression in the host. Of note, L. coryniformis BCH-4 and L. plantarum MiLAB 393 produced fungicide compounds cyclo (L-Leucyl-L-Prolyl) and 3-phenyllactic acid cyclo (L-Phe-L-Pro)respectively which significantly reduced the viability of aspergillus and candida species (Ström et al., 2002; Salman et al., 2022). Additionally, Sunmola et al., 2019 reported the anti-viral activity of L. plantarum and L. amylovorus AA099 against enteroviruses such as echovirus whose site of primary replication is in the gastrointestinal tract. Similarly, Kawahara et al., 2022 reported the antiviral activity of S-layer proteins from L. crispatus KT-11 strains by suppressing the amplification of rotavirus protein 6 (VP6) expression in human intestinal epithelial Caco-2 cells. Also, Mousavi et al., 2018 demonstrated that L. crispatus microcolonies formation on the cell surface tends to block the entry of herpes simplex virus-2 particles, thus help in inhibiting the primary infection step. The rotaviruses are among the ones that bring about severe recurrent diarrhea in infants while herpes simplex virus I cause oral herpes in adults (Kawahara et al., 2022). The understanding of the exact mechanisms is still limited but a few of them include the synthesis of sialic acid and bacteriocins, immune stimulation and obstruction in the binding of viruses (Mousavi et al., 2018; Kawahara et al., 2022).
Inflammations are physiological responses to tissue injury and/or infections, elicited by pro-inflammatory cytokines produced by monocytes, B cells, T cells, dendritic cells (DC), natural killer (NK) cells, and macrophages. Substantial evidence from colitis-induced murine model studies claims the anti-inflammatory and immunomodulatory action of Lactobacillus spp. in gut inflammation (Cristofori et al., 2021). These effects largely depend upon cytokine production and immune cells proliferation. Pro-inflammatory cytokines, such as IL-8 play important role in the recruitment of immune cells during an inflammatory response. Notably, L. acidophilus can suppress IL-8 production and enhance Toll-like receptor-2 (TLR-2) expression through the regulation of TLR-2 mediated mitogen activated protein kinase (MAPK) signalling pathways and Nuclear Factor kappa-light chain-activated B cells (NF-кB) in inflammatory epithelial cells of the intestine (Li et al., 2021). Likewise, several strains of lactobacillus (such as L. acidophilus CCFM137, L. fermentum CCFM381, L. plantarum CCFM634 and CCFM734) also displayed anti-inflammatory potential by upregulating the expression of TLR-2/TLR-6 heterodimer receptor which act as an inflammatory intracellular signalling network (Ren et al., 2016). Inflammatory bowel disease (IBD) is systemic disorder that significantly perturbs the intestinal epithelial layer of the gastrointestinal tract causing four pathological conditions, Crohn’s disease (CD), ulcerative colitis (UC), microscopic colitis and pouchitis. Compare et al., 2017 reported L. casei DG lower pro-inflammatory IL-6, IL-8, TLR-4 and IL-1a and increase IL-10 cytokinelevels in the colonic mucosa of post-infectious IBD subjects. Additionally, L. casei and L. bulgaricus significantly lowered the pro-inflammatory cytokine TNF-α in colonic mucosal samples from CD patients (De Conno et al., 2022). The Treg cells are another immunological player involved in immune-modulation and tolerance. L. casei M2S01 displayed anti-inflammatory action in diseases, such as CD and microscopic colitis, by enhancing Treg cell activation, IL-10 levels and restoring gut microbial flora (Liu Y et al., 2021). Necrotizing enterocolitis (NEC) is another serious gastrointestinal inflammatory condition affecting particularly premature newborns. An ex-vivo study carried out on human intestinal cells from the ileus of NEC infants when treated with L. rhamnosus HN001 displayed reduced NF-kB inflammatory pathway activation through inhibition of TLR-4 (Good et al., 2014). Furthermore, commensal lactobacillus spp. activates mucosal immunity by increasing IgA antibodies, resulting in the immobilization and agglutination of pathogens. The dose-dependent consumption of lactobacillus particularly, L. plantarum, L. acidophilus, L. casei, L. delbrueckii subsp. bulgaricus, L. rhamnosus accrued the number of IgA-producing immune cells connected with mucosa lamina propria as well as stimulates the immunoglobin receptors present on epithelial cells of the intestine (Chen et al., 2022). Adhering to the host ileal tract, lactobacilli modulate another important aspect, apoptosis and cancer, both of which are associated with mucositis. For example, L. rhamnosus GG actively induces antiapoptoticAkt/protein kinase B and inhibits pro-apoptotic factors via p36 MAPK pathway while cell wall components of an array of lactobacilli such as lipoteichoic acid tends to stimulate NO synthase, thereby initiating a cascade of events that bring about pathogen-infected cell death (Banna et al., 2017). Important immune events include activation of macrophages through TNF-α cytokine production and NO-mediated upregulation of two surface phagocytosis receptors (FcγRIII and TLR-2). Sun et al., 2020 reported LPS-induced inflammatory response in Caco-2 cells owing to upregulation of anti-inflammatory cytokines genes (IL10, IL4, transforming growth factor-β3 (TGF–b3 and IFN-y)and downregulation of pro-inflammatory cytokines (IL6, IL1B, IL8 and TNF-α) along with higher expression of TLR-2 and NOD-like receptor genes. Also, genome analysis unfolds the activation of phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt) signaling pathway in Caco-2 cells by L. gasseri JM1. Another in vivo study demonstrated induction of innate and adaptive immune responses by L. acidophilus NCFB 1748 and L. paracasei DC412 on BALB/c inbred mice and Fisher-344 inbred rats through polymorphonuclear (PMN) cell recruitment, TNF-α secretion and phagocytosis (Azad et al., 2018). Lactobacilli encode for certain motifs within their genome, such as unmethylated CpG motifs which are recognized by TLRs that are expressed particularly in B cells and macrophages (Xiao et al., 2022). The role of Lactobacillus spp. in modulating immune responses is illustrated in Figure 1. In recent research, studies involving immunomodulating role of lactobacillus spp. in pain management and treating allergic symptoms have gained momentum. To date, few researchers have demonstrated the prowess of Lactobacillus spp. in relieving inflammatory pain by regulating pro-inflammatory cytokines such as TNF-α, IL-6 and IL-1β as well as expression of COX II in the host intestinal epithelium (Santoni et al., 2021). Although the exact pathway and mechanisms involved in inflammatory pain relief are yet not investigated in detail. L. reuteri DSM17938 displayed antinociceptive activity causing relief in children with functional abdominal pain (Jadrešin et al., 2020). The probable mechanism involves the activation of submucosal immune cells that trigger sensitivity in nerve terminals and/or tight junctions. Another randomized, placebo-controlled, double-blind, multi-centric study by Martoni et al., 2020, reported improvement in abdominal pain and IBS symptoms in adult subjects after the administration of B. lactis UABla-12and L. acidophilus DDS-1 for over 6 weeks. Maixentet al., 2020 also reported the efficacy of the cocktail of two L. acidophilus strains in relieving pain in IBD patients.
Lactobacillus as whole bacteria or their components (such as peptidoglycans, metabolites and surface proteins) have shown to exert an immunomodulating role in treating patients with chronic respiratory disorders. The probable mechanisms by which Lactobacillus modulate lung immunity and promote respiratory health is via the “Gut-Lung axis” which is bidirectional (Du et al., 2022). In the intestinal mucosa, pattern recognition receptors (PRRs, such as NLRs or TLRs) present on immune cells recognize Lactobacillus species or their components resulting in the activation of innate immune cells which tend to reach pulmonary tissues through lymphatic circulation (Stavropoulou et al., 2021; Du et al., 2022). To elucidate, oral administration of L. paracasei CNCM I-1518 caused innate lymphoid group 3 cells (ILC3s) to migrate from the gut to the lungs where ILC3 provides resistance to pneumonia (Gray et al., 2017). Moreover, L. rhamnosus GG and L. murinus oral supplementation promotes the migration of Treg cell to the lungs, thereby augmenting pulmonary inflammation (Zhang et al., 2018; Han et al., 2021). The Treg cells not only have the potent anti-inflammatory ability but are also found to block Th2 type immune response in the host. In addition to this, Lactobacillus interaction with gastric mucosa results in the secretion of cytokines by immune cells, which through circulation reach lung tissues where they alter the immune response. Kollinget al., 2018 reported oral intake of L. rhamnosus CRL1505 results in higher TNF-α, IFN-β, IFN-α, and IFN-ɣ cytokines levels in the lungs leading to a significant reduction in respiratory inflammations. Of note, certain lactobacillus strains secrete metabolites, particularly short chain fatty acids (SCFAs) such as acetate, propionate, butyrate that can regulate host pulmonary immune responses. SCFAs affect immune response in two ways; one is when unmetabolized SCFAs directly migrate to the lungs through circulation and enhance G protein-coupled receptors (GPCRs) activation or histone deacetylase inhibition (Koh et al., 2016). The other is where SCFAs migrate to bone marrow through circulation where they increase the differentiation of macrophage and dendritic progenitor cells (MDPs) and convert them into Ly6C–monocytes, these in turn reach the lungs and differentiate into anti-inflammatory alternatively activated macrophages (AAMs). These AAMs reduce neutrophiles recruitment and stimulate Treg cells to produce anti-inflammatory cytokines (IL-10, TGF-β), thus lower lung injury and inflammation (Anand and Mande, 2018; Du et al., 2022). Animal studies have reported higher butyrate production leads to an increase in Tregs cells and IL-10 production mediated by GPR109A receptor activation as well as restoration of IL-10 in pulmonary tissues by inhibiting histone deacetylase (Vieira et al., 2019). Spacova et al., 2020 showed that L. rhamnosus GR-1 significantly prevented the severity of airway inflammation and hyperactivity by modulating Th2-mediated immune responses as well as shifting gut microbiome composition in the allergic asthma model, supporting the existence of gut-lung axis. Likewise, Li et al., 2019 investigated the efficacy of six lactobacillus spp. (L. fermentum, L. salivarius, L. rhamnosus, L. casei, L. reuteri and L. gasseri) on the gut microbiome and airway inflammation of house dust mite (HDM)-treated asthmatic mice models. Among these, strains of L. reuteri displayed improved airway inflammation with reduced total HDM-IgG1, IgE and Th2-associated pro-inflammatory cytokines with a shift in gut microbial flora. To summarize, members of Lactobacillus spp. or their metabolites have potent anti-inflammatory response, thereby supporting the alleviation of symptoms of asthma, respiratory tract infections and chronic obstructive pulmonary disease (COPD) in patients. The animal and clinical trials associated with Lactobacillus spp. in inflammatory disorders of various organ systems are summarized in Table 1.
The role of gut-resident Lactobacillus species in human brain development and functions has also been reported. The mechanism involves the exchange of neural, hormonal and immunological signals between the gastrointestinal tract and the central nervous system (CNS), primarily known as the“Gut-Brainaxis”. This bidirectional communication is mediated by tryptophan precursors and microbial metabolites such as gamma-aminobutyric acid (GABA), histamine, 5-hydroxytryptamine (5-HT), glutamine, LPS, branched-chain amino acid (BCAAs), bile acids, SCFAs, and catecholamines which regulate potent pathways that are implicated in neuroglial cell function, neurogenesis, myelination, blood–brain barrier function and synaptic pruning (Suganya and Koo, 2020). Of note, human intestinal isolates, lactobacilli were able to produce GABA in the enteric nervous system (ENS). Studies also reported the modulation of the gut microbiota on supplementation with L. rhamnosus JB-1 activated the expression of γ-aminobutyric acid (GABA) receptors thereby resulting in marked improvement in cognitive responses in mice via the vagus nerve (Breit et al., 2018). Several species of Lactobacillus have shown marked effects as neuromodulators and neurotransmitters (such as monoamines, serotonin, and brain-derived neurotrophic factor) (Suganya and Koo, 2020). The microbial metabolite, such as SCFAs produced primarily by lactobacilli in the gut directly affect brain neurological functions through vagal, endocrine, humoral and immune pathways by either entering circulation or crossing the blood-brain barrier (BBB). SCFAs tend to activate Treg cells, endocrine cells and neuronal cells directly in order to increase regulatory cytokines level that maintains brain functions (Silva et al., 2020). LPS induces neurodegenerative and neuroinflammatory disease through TLR stimulation, especially TLR4 in the microglial cells and astrocytes. Studies reported that LPS/TLR4 signaling on microglial cells affects the CNS, particularly by enhancing inflammatory cytokines levels in the gut or CNS of Autism spectrum disorder (ASD) patients. Liu et al., 2019 reported oral supplementation of L. plantarum PS128 for 1 month drastically ameliorated ASD-related symptoms as compared with the placebo group in a double-blind, randomized, placebo-controlled study. Existing evidence supports the significant role of Lactobacillus species and their beneficial metabolites in alleviating neuroinflammatory and neurodegenerative disorders in experimental models or clinical settings. Multiple sclerosis (MS) is a neuroinflammatory autoimmune disease that is characterized by myelin sheath degradation and axonal damage (Suganya and Koo, 2020). Kwon et al., 2013 reported that oral administration of L. casei, L. acidophilus, and L. reuteni to experimental mice resulted in delayed MS progression by enhancing Foxp3+ and IL10 + Tregs expression and reducing the pro-inflammatory Th1/Th17 polarization in the peripheral immune system and inflammation site. Another similar research showed suppressed MS symptoms on supplementation with L. paracasei DSM 13434 and L. plantarum DSM 15312, particularly mediated by IL-10 producing CD4+CD25+Tregs on mice (Lavasani et al., 2010). In addition to this, human gut isolates L. reuteri NK33, L. mucosae NK41, B. longum NK46, and B. adolescentis NK98 have been reported to reduce stress-induced anxiety/depression in mice. The probable mechanisms involve modulation of gut microbial composition and inflammatory immune responses by blocking the NF-κB pathway and reducing LPS, corticosterone, IL-6, and TNF-α levels in serum (Jang et al., 2019). Alzheimer’s Disease (AD) is a neurodegenerative disorder wherein considerable decline in memory, activities, cognitive ability and thinking is observed in older adults (Suganya and Koo, 2020). Studies showed intake of L. acidophilus, B. bifidum and B. longum in rats for 12 weeks significantly augmented their spatial learning and memory, long-term potentiation (LTP), paired-pulse facilitation (PPF) ratios, and lipid profiles (Rezaei Asl et al., 2019). Further, Castelli et al., 2020 supported the neuroprotective role of Lactobacillus spp. in human neuroblastoma cells, by activating pTrK, P13K/Akt, p-CREB, pERK5 pathways and restoring gut microflora. Parkinson’s disease (PD) is another common neurodegenerative disorder marked by mood deflection, cognitive disturbances, resting tremors, slowness of movement, autonomic dysfunctions, sensory and sleep alternations, involving both the central and peripheral nervous system (Suganya and Koo, 2020). Studies have supported the role of L. acidophilus (LA02) and L. salivarius (LS01) in reducing pro-inflammatory cytokines (TNF-α, IL-6, IL-17A) and reactive oxygen species (ROS) levels and increasing anti-inflammatory cytokines (IL-4, IL-10) in peripheral blood mononuclear cells (PBMCs) thereby relieving Parkinson’ disease (PD)-associated symptoms (Magistrelli et al., 2019). The intricate crosstalk between gut and distant organs is illustrated in Figure 2.
A plethora of literature is available that documented the role of gut-dwelling Lactobacillus species in alleviating cardiovascular diseases (CVD) that specifically include diseases of the heart and blood vessels such as congestive heart failure, myocardial infarction, atherosclerosis, coronary artery disease, angina, peripheral vascular disease and aneurysm (Companys et al., 2021). Hypertension and high serum cholesterol are one of the major predisposing factors for CVD. Intestinal Lactobacillus colonies exert protective effect on the heart by regulating atheroinflammatory response, cholesterol metabolism, oxidative stress response, and gut-derived functional metabolites production such as TMAO, SCFAs, LPS, and BA (Companys et al., 2021; Papadopoulos et al., 2022). Of note, researchers also support that lactobacillus interventions could significantly impart a cardioprotective role by regulating the abundance and diversity of selective gut microbial flora (Zhao et al., 2021). To exemplify, L. salivarius Ls-33 supplementation changed gut microbiota composition by escalating the Prevotella-Bacteroides–Porphyromonas group/Firmicutes ratio in obese heart patients (Zhao et al., 2021). In the human body, high cholesterol level in the blood is considered one of the major predisposing factors for the development of atherosclerotic plaques in the arteries. Lactobacillus in the intestine plays an important role in assimilating cholesterol and the metabolism of triglycerides and fatty acids. The proposed mechanism of action includes: 1) Bile salts degradation through the action of microbial bile salt hydrolase (BSH), making them relatively less soluble and thus reducing their reabsorption by the intestinal epithelial layer and increasing their excretion in feces as observed in L. gasseri LBM220 (Rastogi et al., 2021) and L. mucosae SRV5 and SRV10 (Rastogi et al., 2020) 2) Microbial conversion of cholesterol to relatively soluble form coprostanol which can readily assimilate and excreted out via feces 3) Incorporation of cholesterol by the microbial membrane as observed in strain L. acidophilus ATCC 43121 4) production of short chain fatty acids (SCFA). SCFA can directly impede hepatic cholesterol synthesis by inhibiting 3-hydroxymethyl-3-glutaryl-CoA reductase enzyme in the liver (Amiri et al., 2021). GM analysis revealed that lactobacillus supplementation in mice model, selectively promotes the SCFAs metabolizing commensals, such as Ruminococcus, Eubacterium and Roseburia, thereby facilitating higher levels of fermented SCFAs (Amiri et al., 2021). The study also showed L. fermentum 296 when given to high-fed rat model for 4 weeks, significantly increased HDL and reduced harmful LDL owing to their increased production of SCFA (Cavalcante et al., 2019). Further, certain lactobacillus strains are directly involved in hypercholesterolemia, such as L. plantarum 06CC2 which remarkably reduced serum low-density lipoprotein (LDL), triglycerides (TC), free fatty acid levels, apolipoprotein B and increase apolipoprotein A-I levels in HFD Balb/c mice. At the same time, this strain also targets hepatic tissues for lipid deposition by regulating the expression of enzymes involved in cholesterol metabolism (Yamasaki et al., 2020). Apart from BAs and SCFAs, increasing evidence demonstrates that gut-derived microbial metabolites, particularly TMAO and LPS are also involved in the progression of CVD. Trimethylamine (TMA) is a metabolic product that is metabolized by gut commensals from dietary choline and l-carnitine which when absorbed reaches the liver where it is oxidized to TMAO. The high TMAO level causes inhibition of cholesterol reverse transport, pro-inflammatory changes in arterial vessel walls, induction of high cholesterol accumulation in macrophages, platelet hyper-responsiveness and arterial thrombosis (Zhao et al., 2021). Studies have shown Lactobacillus species alleviate TMAO-associated CVD risk by restraining the growth of gut microbes that produce key enzymes involved in TMA production. This was evidenced by research work carried out by Qiu et al., 2018, wherein L. plantarum ZDY04 intervention leads to significantly lowered TMAO circulating levels and TMAO-induced atherosclerosis by altering the relative microflora population of the genus Mucispirillum and the families Erysipelotrichaceae, Lachnospiraceae, and Bacteroidaceae in the animal model. L. plantarum Dad-13 supplementation in obese adults leads to a higher population of Bacteroidetes and lower Firmicutes flora in the gut (Rahayu et al., 2021). Also, L. plantarum supplementation directly reduced serum TMAO levels as well as circulating inflammatory factors of IL8, IL-12, and leptin in CVD patients. Furthermore, LPS as a bacterial outer membrane component also induces CVD by activating macrophages to secrete pro-atherosclerotic inflammatory cytokines (IL-1, TNF-α, IL-12, IL-6, IL-8) that accelerated atherosclerosis and heart failure occurrence owing to down-regulation of mitochondrial fatty acids oxidation in cardiomyocytes (Zhao et al., 2021). Also, LPS increases platelet aggregation through TLR4-mediated leukocyte cathepsin G activation causing thrombosis (Zhao et al., 2021). The L. brevis alleviated CVD onset by blocking LPS-induced inflammatory cytokine expression in the host (Chang et al., 2016). Similarly, L. paracasei FZU103 was found to modulate the LPS-induced inflammatory axis and cholesterol metabolism in mice fed with HFD. Metagenomic studies also demonstrated shift in GM, with higher abundance of Alistipes, Ruminococcus, Helicobacter and Pseudoflavonifractor but lower population of Tannerella, Blautia, and Staphylococcos in gut (Lv et al., 2021). In L. plantarum LP91-fed LPS-induced mice, expression of atherosclerotic inflammatory factors, particularly TNF-α and IL-6, vascular cell adhesion molecule, E-selectin was found to be downregulated, thus showing improvement in CVD symptoms (Aparna Sudhakaran et al., 2013).
Many recent publications have reported the beneficial role of Lactobacillus spp. in assuaging the onset of metabolic disturbances, such as obesity and diabetes mellitus, which are characterized by a low-grade inflammation (Archer et al., 2021).
Obesity is a multifaceted metabolic disease associated with changes in adipose tissues (AT), which is a complex endocrine organ involved in energy homeostasis. Usually, AT are subsuming adipocytes, lymphocytes, macrophages, fibroblasts, endothelial cells that produce plasminogen activator inhibitor (PAI-1), adiponectin, leptin, vascular regulators angiotensin II and cytokines (Andersen et al., 2016). The onset of obesity leads to dysfunctional AT with physiological changes in vascularization, oxidative stress levels, secreted adipokines and inflammatory state of infiltrated lymphocytes (Jo et al., 2009). In obese patients, a rise in free fatty acids (FFAs) level causes activation of signalling pathways, particularly TLRs which contribute to the pro-inflammatory response by increasing the production of molecules such as TNF-α, IL-6, IL1β, leptin, resistin, and chemokines in cells from lamina propria of gut (Khan et al., 2021). In the gut, activation of TNF-α stimulates apoptosis signalling pathways, FFAs levels and downregulates the expression of GLUT-4 transporters. In the high-fed diet (HFD) mice group, infiltration of CD8+ T cells, TNF-α, IFN-ɣ, and CX3CR1int macrophages in adipose tissues in response to high glucose, FFAs and apoptosis leads to inflammation. Of note, one of the driver for inflammatory alterations and loss of gut epithelial integrity in obesity includes, leakage of bacterial endotoxin LPS (Khan et al., 2021). This leads to an inadequate distribution of lymphocytes, changes in cytokines levels, gut microbiota and immune response to dietary antigens. Much research has been centred on the importance of lactobacillus in treating obesity by alleviating lipid accumulation, oxidative damage, inflammation, and gut dysbiosis. A recent publication reported synergistic effects of L. curvatus HY7601 and L. plantarum KY1032 in lowering excess weight and fat accumulation in the HFD mice group with reduced inflammatory biomarkers (Park et al., 2013). Choi et al., 2020, reported weight loss in HFD-fed mice when treated with L. plantarum LMT1-48 with fewer lipids accumulation, immune cells infiltration in AT and adipocyte size. Notably, multiple strains of lactobacillus-particularly, L. casei IMVB-7280, L. paracasei HII0, L. paracasei CNCM I-4034, L. rhamnosus CGMCC1.3, L. rhamnosus LA68 and L. casei IBS041 have proved positive effects in reducing obesity symptoms, likely, reduced weight gain, lower cholesterol levels, lower adiposity and inflammation (Wiciński et al., 2020). Similarly, L. reuteri GMNL-263 ameliorated symptoms pertaining to obesity in HFD-fed rats by decreasing serum pro-inflammatory factors levels and remodeling white adipose tissue (WAT) energy metabolism (Chen et al., 2018). Besides, Joung et al., 2021 reported lower fat accumulation in AT in HFD-fed mice on L. plantarum administration, which was attributed to altered gut microbiota with reduced Firmicutes/Bacteroidetes ratio. Overall, Lactobacillus strains have achieved remarkable results in the treatment of obesity-related symptoms.
Diabetes mellitus, a chronic metabolic disease is characterized by a consistently high serum glycemic index. A study of the Global Burden of Disease 2015 has reported that diabetes is one of the major causes of mortality in urban populations. There has been a significant association between the inflammatory condition and metabolic disturbances among diabetic patients. The postulated mechanism includes raised levels of the pro-inflammatory cytokine, TNF-α which inactivates insulin receptor (IRS-I) by phosphorylating serine residue. While other cytokines IFN-γ, TNF-α, and IL-1β works in a synergistic manner by infiltrating the β-cells of the pancreas, subsequently inducing cellular apoptosis and damage. Thus, higher pro-inflammatory cytokines levels in muscle, liver and adipose tissues are major drivers of diabetes pathology as they inhibit insulin signalling causing insulin resistance (Tsalamandris et al., 2019; Bezirtzoglou et al., 2021). Interestingly, L. plantarum Y44 showed downregulation of pro-inflammatory cytokine genes in the liver, intestine and muscle tissues particularly by activating regulatory anti-inflammatory cytokine IL-10, highlighting their immunomodulatory role (Liu et al., 2020). Likewise, L. casei Shirota strain showed reduced pro-inflammatory cytokines IL-4, IL-6 and C-reactive protein (CRP) levels in Streptozotocin (STZ)-induced diabetic rat models (Zarfeshani et al., 2011). Archer et al. (2021) also reported L. fermentum MCC2759 exhibited a reduction in glucose profile and pro-inflammatory cytokines, IL-10 in the liver, MAT, muscle and intestinal tissues in STZ-induced diabetic rats. This study also proved enhanced insulin sensitivity (GLP-1, GLUT-4, adiponectin), intestinal barrier integrity (ZO-1) and TLR-4 receptor expression. In addition to this, L. casei is reported to reduce serum glucose contents owing to improvement in post-immune responses via suppression of IL-2 and IFN- γ production (Qu et al., 2018). Another study by Park et al. (2015) reported that the anti-hyperglycemic effect on male db/db mice on administration of L. rhamnosus GG (LGG) is associated with increased ER stress and suppressed macrophages, leading to increased insulin sensitivity. Also, alteration in the expression of some diabetes-associated genes plays a role. Probiotic L. rhamnosus NCDC17 has reported to have antidiabetic capacity owing to its ability to upregulate mRNA expression of glucose metabolism and insulin sensitivity related genes such as GLUT4 (glucose uptake related genes), pp-1 (glycogen synthesis related genes) and PPAR- γ (insulin sensitivity related genes) and downregulating G6PC (gluconeogenesis related genes) (Singh et al., 2017). Further, consumption of probiotics affects the gut microflora composition which in turn alleviates intestinal epithelium and suppresses the immune response by reducing the TLR4 signalling pathway, ultimately increasing insulin sensitivity (Li et al., 2021). Some other postulated mechanisms include enhanced glucagon-like peptide-1 (GLP-1) secretion from intestinal L-cells to inhibit postprandial hyperglycemia by elevating the level of insulin released from pancreatic beta cells and reducing glucotoxicity (Kesika et al., 2019). A study involving L. kafiranofaciens M and L. kefiri K administration was found to stimulate GLP-1 secretion with concomitant rise in glucose metabolism (Kocsis et al., 2020).
Metabolic bone disorder, osteoporosis is characterized by poor bone tissues, deteriorated bone mass and porosity which subsequently lead to weak and brittle bones that are susceptible to fractures (Akkawi and Zmerly, 2018). Notably, Lactobacillus spp. as whole bacteria or their metabolites and/or structural components (such as SCFAs, hydrogen sulfide (H2S), insulin-like growth factor- I (IGF-I), LPS and peptidoglycans, etc) act as powerful immune cells controller and exhibit regulatory role in bone resorption and formation (Zaiss et al., 2019). Thereby, confirming their role as a linker in the “Gut-Bone axis”. Factors namely, IGF-I produced predominately in hepatic cells in response to dietary intake and gut microbes directly mediate the gut-bone axis (Zaiss et al., 2019). The microbial metabolite, H2S acts as a gasotransmitter, is produced by gut-resident Lactobacilli spp. and stimulates bone formation and postnatal skeletal development by activating Wnt signaling (Grassi et al., 2016). In osteoblasts, wnt signaling activation mediates enhanced osteoblastogenesis and arrests osteoblast apoptosis. Moreover, SCFAs have gained tremendous attention for their capacity to diffuse bone tissues and regulate immune responses (Zaiss et al., 2019). SCFA, particularly butyrate and propionate induce the proliferation of mature Treg cells. The maturation of Tregs depends upon GPR109a and GPR43 receptors expressed on dendritic cells (DC). The Treg cells suppress the CD4+ T cells that are present on the endosteal surfaces of the bone by producing immunosuppressive cytokines IL-10 and TGF-β; resulting in improved osteoporosis and bone mass density (BMD) by directly increasing osteoblast differentiation and reducing osteoclastogenesis (Smith et al., 2013). Also, activated Tregs cells are required for calciotropic parathyroid hormone–stimulated (PTH-stimulated) bone formation as it induces bone anabolism via Treg/Wnt10b/Wnt signaling pathway (Yu et al., 2018). Dietary supplementation with L. rhamnosus LGG for 4 weeks directly increased circulating and intestinal butyrate levels, confirming its capacity to diffuse from the intestine to distant organs like bone. In bone tissues, the butyrate induces Tregs and improves bone health. This study by Tyagi et al., 2018 further proved that L. rhamnosus LGG also altered gut microbial composition, with a higher Clostridia population that is known to elicit the generation of SCFAs in the gut. Thus, L. rhamnosus LGG substantially mediates the pathway linking SCFAs, Tregs, and bone formation. To date, multiple studies have utilized animal models to determine lactobacillus efficacy in reducing both primary and secondary osteoporotic bone loss. Oral intake of L. reuteri ATCC 6475 by healthy male mice for 1 month resulted in improved bone mineral content, vertebral and femoral trabecular bone density, trabecular thickness and trabecular number when compared to untreated controls. Increased bone density leads to higher levels of bone formation rate as evidenced by the osteoblast marker osteocalcin. L. reuteri ATCC 6475 acts by systemically suppressing gene expression of pro-osteoclastogenic and pro-inflammatory cytokines in both the bone marrow and the intestine (Nilsson et al., 2018). These anti-inflammatory effects, which are also observed in other species of Lactobacillus tend to directly increase the calcium transport across the intestinal barrier. Immune cell activation largely depends on calcium levels. In case of hypocalcemia, treatment of rats with yogurt-enriched with L. casei, L. reuteri and L. gasseri improved calcium absorption in a PTH-dependent manner. Likewise, L. rhamnosus (HN001) also improved calcium and magnesium retention in rat models (Collins et al., 2017). Ohlsson et al., 2014 treated mice with either a single L. paracasei strain (DSM13434) or a mixture of three strains (L. plantarum DSM 15312, DSM 1531 and L. paracasei DSM13434) in the water for 14 days resulted in increased cortical bone mineral content and decreased levels of urinary fractional excretion of calcium and resorption marker C-terminal telopeptides as compared to control. Different Lactobacillus strains act via distinct and/or overlapping pathways, such as L. helveticus was found to not only increase bone density by elevating calcium uptake, but also secrete bioactive peptides valyl-prolyl-proline (VPP) and isoleucyl-prolyl-proline (IPP) (Parvaneh et al., 2018). Further, conversion of insoluble inorganic salts into soluble forms, protection of intestinal mineral absorption sites, triggering of the modulation of calcium-binding proteins, and minimization of the interaction of minerals with phytic acids are the main actions reported by different strains of Lactobacillus that supported bone health.
An exponential advancement in sequencing processing, genome assembly and annotation technologies, has resulted in thousands of publicly available genomes of Lactobacillus spp. Access to these data has revolutionized the molecular view of probiotic bacteria, significantly accelerating the research deciphering the complexity associated with interactions between resident microbiota and the mucosal immune system. Notably, advancements in genomic tools, particularly functional genomics, proteomics, transcriptomics, and secretomics have helped in understanding the intricate dynamic host-microbe crosstalk that extends far beyond gastrointestinal health. To exemplify, studies on the bi-directional crosstalk between the GIT and the brain (gut-brain axis) are revealing the neurochemical importance of gut homeostasis. Similarly, studies involving the GIT and the lungs crosstalk (gut-lung axis) highlight the significance of microbial metabolites in regulating host’s immune responses. In addition this, Lactobacillus spp. modulate mucosal immunity through the interaction of proteinacious microorganism-associated molecular patterns (MAMPs) with pattern recognition receptors (PRRs) on antigen-presenting cells (APCs), such as dendritic cells and macrophages. Recently, the proteomic and genomic profiles of several lactobacilli were bioinformatically screened to create a secretome database cataloging the various extracellular proteins. For example, a proteomic-based method was used to identify S-layer associated proteins (SLAPs) in L. acidophilus. After extraction, the SLAPs were identified through mass spectrometry and referenced to the secretome database. The mutational analysis of SLAPs showed immunomodulatory phenotype using in vitro bacterial-DC co-incubation assays (Huang et al., 2021). However, it is important to note that the transcriptional networks induced by each probiotic were unique to each strain studied and that they show distinct metabolic and immunogenic profiles in the host. Thus, there is a growing need for human clinical trials with experimental designs, reflecting the future progress that has been made in the field of probiotics and GIT microbiome research.
Taken together, the intestinal microbial species, particularly Lactobacilli spp., within gut are identified as potential and talented players in restoring gastrointestinal barrier function, immune stimulation and gut microbial flora. So far, we have highlighted the intricate crosstalk between gut-dwelling Lactobacilli spp., or their metabolites and the immune components in distant organs to promote host’s health. The healthy balance in the intestinal ecosystem is preserved by the circuitry of monitoring mechanisms between potentially pro-inflammatory cell [Th cells secreting IFN-y, Th17 cells that secrete interleukin IL-17, and IL-22], and anti-inflammatory Foxp3+ receptor Tcells. Certain strains of lactobacilli stimulate the anti-inflammatory fork of the adaptive immune system by controlling Treg maturation or by driving IL-10 and IL-12 production. Given the current epidemic of inflammatory disorders plaguing present society, a call is necessary for feasible, available, and safe treatments to prevent and fight against it. Even though inflammatory and metabolic disorders pathogenesis is multifactorial and highly complexed, yet recent literature suggests modulation of gut microbial flora and immune responses using probiotics as the primary therapeutic intervention. Consequently, gut microbiome modulation to preserve a stable, consistent metabolic environment may be helpful in preventing and as additional treatment in affected patients. In future, far more in-depth clinical studies will be required to substantiate the therapeutic approaches with lactobacillus in directly maintaining gut microbiota homeostasis and regulate functional metabolites (such as TMAO, SCFAs, BAs, and LPS) which further lower risks associated with immune inflammation, high lipid cholesterol and oxidative stress. From author’s viewpoint, those looking to ameliorate their overall health by improving their gastrointestinal microbial complexity might find it more beneficial to target on consuming a fermented diet (such as yoghurt, kimchi, miso, sauerkraut) rich in Lactobacillus spp. To conclude, therapy with Lactobacillus spp. still provides a potential frontier in the treatment and prevention of inflammatory diseases. | true | true | true |
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PMC9638682 | 36329597 | Abdulhakeem D Hussein,Ekhlas Aziz Bakr,Mohammed Hadi Ali Al-Jumaili | Association between ABO blood groups and the risk of infection with SARS-CoV-2 in Iraq | 03-11-2022 | COVID-19,blood group,clinical outcome,quantitative reverse transcription polymerase chain reaction,mortality,respiratory illness,vaccination | Objective The primary goals of this research were to analyze the relationship between ABO blood types and the severity of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection and investigate the effect of vaccination in Iraq. Methods Data and outcomes were gathered from the medical records of 200 patients. Patients were categorized by blood group and vaccination status in the analysis. Results In total, 200 hospitalized patients (125 men and 75 women) with confirmed SARS-CoV-2 infection and blood group (ABO) and clinical data were enrolled. Of the 200 patients, 155 (77.5%) were vaccinated against SARS-CoV-2. The results illustrated that 25 patients died, which might have been attributable to a lack of vaccination or older age. Our analysis revealed that blood group O individuals were much less likely to be infected by SARS-CoV-2 than non-O subjects, whereas blood group A individuals carried a higher risk of infection. Conclusions Our findings illustrated that immunization significantly reduces COVID-19 risk across all age groups, but there has been an increase in the number of cases because of decreased vaccine efficacy in older patients and persons with comorbidities. However, 45% vaccination coverage lowered the outbreak’s peak. | Association between ABO blood groups and the risk of infection with SARS-CoV-2 in Iraq
The primary goals of this research were to analyze the relationship between ABO blood types and the severity of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection and investigate the effect of vaccination in Iraq.
Data and outcomes were gathered from the medical records of 200 patients. Patients were categorized by blood group and vaccination status in the analysis.
In total, 200 hospitalized patients (125 men and 75 women) with confirmed SARS-CoV-2 infection and blood group (ABO) and clinical data were enrolled. Of the 200 patients, 155 (77.5%) were vaccinated against SARS-CoV-2. The results illustrated that 25 patients died, which might have been attributable to a lack of vaccination or older age. Our analysis revealed that blood group O individuals were much less likely to be infected by SARS-CoV-2 than non-O subjects, whereas blood group A individuals carried a higher risk of infection.
Our findings illustrated that immunization significantly reduces COVID-19 risk across all age groups, but there has been an increase in the number of cases because of decreased vaccine efficacy in older patients and persons with comorbidities. However, 45% vaccination coverage lowered the outbreak’s peak.
In December 2019, a pneumonia outbreak occurred in Wuhan, China and rapidly spread globally. The World Health Organization termed the illness coronavirus disease 2019 (COVID-19), which is caused by the beta-coronavirus severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The extensive spread of COVID-19 occurred through human-to-human transmission, which occurred among symptomatic patients as well as asymptomatic and pre-symptomatic infected people. Fever, cough, fatigue, anorexia, myalgia, and diarrhea are the most common symptoms of COVID-19 in humans. In the week following the onset of symptoms, the condition frequently worsens from mild to severe illness. Some epidemiological and clinical variables of a community have been demonstrated to influence the risk of COVID-19 and increase the risk of severe illness. Age, sex, and several chronic disorders including heart disease and diabetes are the main risk variables. Although elderly patients are predisposed to COVID-19, multiple risk factors have been identified. In addition, cases of severe infection have been reported in healthy people. However, the biological indicators predictive of the occurrence of COVID-19 or its progression have not been identified. Meanwhile, several elements (host, viral, and environmental factors) were proposed to explain the COVID-19 clinical phenotype. In light of these findings, investigations assessing the relationships of blood groups, SARS-CoV-1, Plasmodium falciparum, Helicobacter pylori, Norwalk virus, hepatitis B virus, and Neisseria gonorrhoeae with COVID-19 susceptibility and severity have been conducted. Carbohydrate epitopes on the surface of human cells comprise the ABO blood groups discovered by Landsteiner. The trisaccharide moieties GalNAc1–3-(Fuc1,2)-Gal- and Gal1–3-(Fuc1,2)-Gal- are determinants of antigenicity for the A and B blood groups, respectively, whereas Fuc1,2-Gal- is the antigenic determinant for blood group O. Although blood groups are inherited genetically, environmental variables can affect which blood groups present in a population are passed to the next generation. ABO blood groups have been linked to the susceptibility to viral infection. For example, the susceptibility to Norwalk virus and hepatitis B infection has been linked to the blood type. It was also discovered that people with blood group O were less likely to contract the SARS coronavirus. Therefore, the association of COVID-19 susceptibility with ABO blood groups was explored in this study.
The data of the clinical study were obtained from Al Karama Teaching Hospital (Baghdad, Iraq) to investigate the relationship between blood groups and COVID-19. Patients with a confirmed diagnosis of COVID-19 by quantitative reverse transcription polymerase chain reaction (RT-qPCR) using nasopharynx swab samples who had clinical and blood group (ABO and AB) data were eligible for enrollment. The study was approved by the ethics committee of Dijlah University College (approval date: February 2022), and the requirement for informed consent was waived. A blood test was used for both the clinical and laboratory investigations. The patients’ case report forms were used to collect information as well as blood group, sex, age, hospital ward, and disease outcome data. In this study, we followed the relevant EQUATOR guidelines for reporting observational studies, and all patient details were de-identified. The patients were divided into groups by blood group, age, vaccination status, and hospital ward to assess their relationships with the outcomes of COVID-19 (recovered or dead).
Data were collected from 200 patients, including 125 men and 75 women. Regarding the association between vaccine reactogenicity and ABO blood groups, it was observed that people with blood group A have a higher risk of COVID-19, whereas those with blood group O have a lower risk of contracting the virus. However, the study revealed no link of vaccine reactogenicity with ABO blood groups for either available vaccine. However, people with various ABO blood groups exhibit varying degrees of reactogenicity to immunization by RNA vaccines developed by Pfizer and Sinopharm. Individuals with blood group A experienced more severe adverse effects than those with blood group O, and the extent of reactogenicity appeared to be linked to the blood group, as presented in Table 1 and Figure 1. The frequencies of blood types in the vaccinated and unvaccinated groups are presented in Table 1 and Figure 2.
The results suggest that blood groups are linked to the clinical outcomes of COVID-19. Meanwhile, the results revealed no major relationship between sex and the outcome of COVID-19 results. The results identified a substantial relationship between age and the outcome of COVID-19, as the risk of death was higher among older patients (Figure 3). This discovery is perhaps unsurprising given that elderly people have higher rates of diabetes, heart disease, hypertension, and chronic pain, which are comorbidities of respiratory disease and disorders that are related to a weakened immune system. In addition, there was a strong link between the ward of hospitalization and disease outcome. Specifically, the probability of mortality was higher among patients admitted to the ICU, CCU, and emergency ward. In addition, we noticed that patients who did not have chronic diseases stayed in the hospital to receive treatment. Specifically, some patients were hospitalized for 1 to 3 days, whereas others stayed for 1 to 2 weeks. Meanwhile, patients who were hospitalized for 1 to 2 months were admitted to the ICU because they did not receive treatment in the first few days of infection. For patients with concomitant chronic diseases, the duration of hospitalization typically ranged from 1 to 2 months, although some patients who died had a shorter hospital stay (Table 1). As presented in Figure 3, 200 samples were obtained from COVID-19–infected patients.
The discovery of risk variables linked to SARS-CoV-2 infection and outcomes has become a research priority because of the severity of the disease, which is usually unanticipated. Following the earliest findings in the literature on the link between COVID-19 and ABO blood groups, a number of studies investigated whether ABO blood groups were linked to the risk of COVID-19, the severity of the disease, and the risk of disease-related mortality. At the beginning of the infection, fever, dry cough, dyspnea, lethargy, myalgia/arthralgia, and cold are the most common symptoms. Less common symptoms include earache, hemoptysis, headache, nausea or vomiting, chest discomfort, diarrhea, loss of taste, sputum production, and loss of smell. Compared with individuals without diabetes, patients with diabetes had higher white blood cell, neutrophil, and lymphocyte counts and higher C-reactive protein and blood urea nitrogen levels. They also had lower red blood cell counts and hemoglobin levels. Excluding pharyngeal exudate, which was most frequently observed among patients with blood group A, clinical symptoms were more frequently observed in blood group O patients than in those with blood group A or B. Only patients with blood group O experienced hemoptysis, jaundice, abnormal lung auscultation, and hepatomegaly, indicating that anti-A antibodies from patients with blood group O are more protective than those from patients with blood group B. The predominant immunoglobulin isotypes of anti-A antibodies are IgG in blood group O and IgM in blood group B. The results of our study are consistent with those of the study conducted by Gerard et al., who found that subjects with anti-A antibodies (blood groups O and B) were significantly underrepresented in COVID-19 compared with patients with blood groups A and AB (P < 0.001), A (P < 0.001), or AB (P = 0.032). They then examined whether there was a difference in anti-A antibodies between groups O and B. The prevalence of group O carriage was significantly lower among patients with COVID-19 (P < 0.001), whereas the prevalence of group B carriers was significantly higher (P < 0.001), indicating that anti-A antibody from blood group O carriers is more protective than anti-A antibody from blood group B carriers. Another study in Iraq revealed that when compared with patients with other blood groups, patients with blood group A had the highest rate of COVID-19, whereas the COVID-19 rate was lowest among blood group O carriers. Meanwhile, Ad’hiah and colleagues reported that blood group A might be associated with an increased risk of COVID-19, particularly among men. In addition, several studies found that people with blood group O have a lower risk of COVID-19, whereas people with non-O blood groups, particularly group A, have a higher risk. In a French study involving 998 samples collected from blood donors, the seroprevalence rate of SARS-CoV-2 neutralizing antibodies was lower among group O donors than among non-O donors. The results of other studies as reviewed by Sapha et al. indicated that individuals with blood group A have higher risks of SARS-CoV-2 infection and severe outcomes, whereas people with blood group O are protected against the infection to some extent. One main hypothesis to explain these findings is that people in blood groups O and B have naturally occurring anti-A antibodies that might act as a partial defense against SARS-CoV-2 virions because of differences in the nature of these anti-A antibodies. A useful application of this theory would be in convalescent plasma therapy, in which donors with higher titers of natural antibodies based on blood group could be selectively recruited to optimize treatment. Although there were no significant variations in age or sex among the blood groups, the majority of patients with blood type A were older than 60 years. Consequently, the rates of comorbidities were higher among blood group A patients than among non-A patients. Diabetes mellitus and hypertension were the most frequent comorbidities among blood group A patients. Furthermore, patients with blood group A who tested positive for COVID-19 had higher risks of chronic renal disease and diabetes mellitus. Cough, shortness of breath, and headache were substantially more common among COVID-19–positive individuals with blood group A than in their counterparts with blood type B. Patients with blood groups A and O more frequently experienced body aches than those with other blood groups. It should be noted that some prior studies contradicted our results. In a study by Zietz et al., non-O patients had a slightly higher prevalence of infection. When compared with group O, the risk of intubation was lower for group A and higher for groups AB and B, whereas the risk of death was higher for group AB and lower for groups A and B. Meanwhile, some studies revealed no link between ABO blood groups and the susceptibility to SARS-CoV-2 infection. The disparity among studies could be attributed to differences in sample sizes, ABO heterogeneity among populations or geographical areas, differences in genetic backgrounds, and differences in viral strain. Variation in blood group phenotypes across countries, as well as genetic differences, might influence the heterogeneity of COVID-19 clinical phenotypes. The findings of such studies could lead to individuals at a higher risk of severe infection being vaccinated earlier or being monitored and treated more closely. These findings suggest that blood groups can influence the susceptibility to SARS-CoV-2 infection and the clinical course of COVID-19. The prevalence of blood group phenotypes in both the recovered and deceased patients is presented in Figure 4 and Table 2.
The relationship among ABO blood groups and COVID-19 can be explained by a number of mechanisms. Anti-A antibodies, the generation of SARS-CoV-2 glycan antigens, the impact of genetics, coagulation system polymorphisms, and ABO gene mutations have all been hypothesized to influence the link between ABO blood groups and COVID-19 susceptibility. Glycosyltransferases A and B were demonstrated to affect glycosylation in a variety of respiratory epithelial cells. Anti-A antibodies, which are normally present in people with blood group O or B, might affect the interaction of the SARS-CoV-2 S protein with its membrane receptor angiotensin-converting enzyme 2 (ACE2) receptor. The present study found that blood group O decreased the risk of critical and severe outcomes compared with blood group A. Anti-A antibodies found in group O people inhibit binding to antigens similar to those found on the SARS-CoV-2 envelope. Anti-A antibodies in group O people bind to the SARS-CoV-2 S protein, limiting the interaction of the SARS-CoV-2 S protein with the ACE2 receptor, potentially preventing viral entry into the lung epithelium (Figure 5). Any protection against infection or disease severity provided by the blood group status is only partial. Furthermore, the mechanism of these protective effects has not been fully clarified.
In patients with COVID-19, blood group O is linked to a lower likelihood of severe outcomes. Blood group O individuals have higher von Willebrand and VIII factor levels, resulting in a lower risk of cardiovascular disease. COVID-19 coagulopathy and vasculopathy traits have been revealed to play a significant role in the onset of acute respiratory distress syndrome. Consequently, it is believed that the lower risk of illness development in patients with blood group O was attributable to this occurrence. Furthermore, blood group O carriers have reduced levels of ACE, which converts angiotensin I to angiotensin II. Given that angiotensin II can encourage inflammatory responses and increase blood pressure, the lower ACE levels in group O carriers could explain the lower risk of severe symptoms.
Our data illustrated people with blood group A are most vulnerable to COVID-19. Blood group A was linked to higher rates of mechanical ventilation and death than the other blood groups. By binding to A and/or B antigens expressed on the viral envelope, anti-A and/or anti-B antibodies operate as virus-neutralizing antibodies, preventing target cell contagion. Human anti-A antibody binds to the SARS-CoV-2 S protein, which can block the interaction of the virus with the ACE2 receptor, thereby blocking its access to the lung epithelium. The increase in ACE1 levels because of ABH gene polymorphisms found in people with non-O blood types increases the predisposition to cardiovascular problems, and such patients account for the majority of severe cases of COVID-19. The most common immunoglobulin isotype of anti-A antibodies in blood group O is IgG, whereas IgM, which is prevalent found in blood group B, plays an important role clinically because anti-A antibodies from individuals with blood groups O and B have different properties. A useful application of this approach might be applied to convalescent plasma therapy, in which donors with greater natural antibody titers based on the blood group might be preferentially selected to improve treatment outcomes.
According to the study results, the COVID-19 rate among patients in this study decreased in the order of A > O >B > AB. The study also found that patients with blood group A might be more susceptible to COVID-19. Conversely, the risk of COVID-19 was lower among patients with blood group O. However, mounting data suggest that ABO blood groups contribute to disease biology at the biochemical and physiological levels, and it has already been recognized to contribute to the severity of COVID-19. Furthermore, the association between vaccination reactogenicity and ABO blood groups was investigated in this study. This relationship remains uncertain, and more research is needed. Meanwhile, the proposed mechanisms are only hypotheses. The small sample size of this study represents a limitation, and further research with a large cohort is needed to validate the findings. The study found that the majority of patients who arrived at the hospital had already received the vaccine, and various age groups were represented. Our results indicated that vaccination resulted in a large reduction of COVID-19 risk across all age categories, but there has been an increase in the number of cases because of lower vaccine effectiveness in older patients and individuals with comorbidities. Conversely, immunization with 45% coverage reduced the peak of the outbreak and resulted in a daily incidence of less than 1% from the beginning of vaccination. | true | true | true |
PMC9638800 | Jian Zhou,Weiwei Qian,Cuiliu Huang,Cunjun Mai,Yimei Lai,Zhiqin Lin,Guie Lai | Combined targeting of KRT23 and NCCRP1 as a potential novel therapeutic approach for the treatment of triple-negative breast cancer | 01-10-2022 | Triple-negative breast cancers,Keratin23 (KRT23),non-specific cytotoxic cell receptor 1 (NCCRP1),tumor immune microenvironment | Background Breast cancers characterized by triple-negative status tend to be more malignant and have a poorer prognosis. A risk model for predicting breast cancer risk should be developed. Methods We obtained gene expression and clinical characteristics data using the Clinical Proteomic Tumor Analysis Consortium (CPTAC) and The Cancer Genome Atlas (TCGA) database. Differential gene screening between patients with triple-negative breast cancer (TNBC) and non-triple-negative breast cancers (NTNBC) was performed according to the “edgeR” filter criteria. Univariate and multivariate Cox regression analyses were used to construct a risk model and identify prognosis-related genes. XCELL, TIMER, EPIC, QUANTISEQ, MCPCOUNTER, EPIC, CIBERSORT-ABS, and CIBERSORT software programs were used to determine the extent of tumor immune cell infiltration. To evaluate the clinical responses to breast cancer treatment, the half maximal inhibitory concentration (IC50s) of common chemotherapeutics were calculated using “pRRophetic” and “ggplot2”. Cell proliferation was assayed using cell counting kit-8 (CCK8) and 5-Ethynyl-2′-deoxyuridine (EdU) Cell Proliferation Kit. A dual-luciferase reporter assay confirmed the gene regulatory relationship of sex determining region Y-box 10 (SOX10). Results An assessment model was established for Keratin23 (KRT23) and non-specific cytotoxic cell receptor 1 (NCCRP1) using the univariate and multivariate Cox regression analyses. In addition, high expression levels of KRT23 and NCCRP1 indicated high proliferation and poor prognosis. We also found that the gene expression patterns of multiple genes were significantly more predictive of risks and have a higher level of consistency when assessing risk. In vitro experiments showed that the expressions of KRT23 and NCCRP1 were increased in TNBCs and promoted cell proliferation. Mechanically, the dual-luciferase reporter assay confirmed that SOX10 regulated the expressions of KRT23 and NCCRP1. The risk score model revealed a close relationship between the expressions of KRT23 and NCCRP1, the tumor immune microenvironment, and chemotherapeutics. Conclusions In conclusion, we constructed a risk assessment model to predict the risk of TNBC patients, which acted as a potential predictor for chemosensitivity. | Combined targeting of KRT23 and NCCRP1 as a potential novel therapeutic approach for the treatment of triple-negative breast cancer
Breast cancers characterized by triple-negative status tend to be more malignant and have a poorer prognosis. A risk model for predicting breast cancer risk should be developed.
We obtained gene expression and clinical characteristics data using the Clinical Proteomic Tumor Analysis Consortium (CPTAC) and The Cancer Genome Atlas (TCGA) database. Differential gene screening between patients with triple-negative breast cancer (TNBC) and non-triple-negative breast cancers (NTNBC) was performed according to the “edgeR” filter criteria. Univariate and multivariate Cox regression analyses were used to construct a risk model and identify prognosis-related genes. XCELL, TIMER, EPIC, QUANTISEQ, MCPCOUNTER, EPIC, CIBERSORT-ABS, and CIBERSORT software programs were used to determine the extent of tumor immune cell infiltration. To evaluate the clinical responses to breast cancer treatment, the half maximal inhibitory concentration (IC50s) of common chemotherapeutics were calculated using “pRRophetic” and “ggplot2”. Cell proliferation was assayed using cell counting kit-8 (CCK8) and 5-Ethynyl-2′-deoxyuridine (EdU) Cell Proliferation Kit. A dual-luciferase reporter assay confirmed the gene regulatory relationship of sex determining region Y-box 10 (SOX10).
An assessment model was established for Keratin23 (KRT23) and non-specific cytotoxic cell receptor 1 (NCCRP1) using the univariate and multivariate Cox regression analyses. In addition, high expression levels of KRT23 and NCCRP1 indicated high proliferation and poor prognosis. We also found that the gene expression patterns of multiple genes were significantly more predictive of risks and have a higher level of consistency when assessing risk. In vitro experiments showed that the expressions of KRT23 and NCCRP1 were increased in TNBCs and promoted cell proliferation. Mechanically, the dual-luciferase reporter assay confirmed that SOX10 regulated the expressions of KRT23 and NCCRP1. The risk score model revealed a close relationship between the expressions of KRT23 and NCCRP1, the tumor immune microenvironment, and chemotherapeutics.
In conclusion, we constructed a risk assessment model to predict the risk of TNBC patients, which acted as a potential predictor for chemosensitivity.
Breast cancer with triple-negative receptors lacks the genes for the estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor 2 (HER2)/neu. It accounts for 15–20% of all breast cancers and has a poor prognosis (1,2). In the absence of ER, PR, and HER2/neu, optimizing the therapeutic management of patients is difficult (2,3). Despite the improvements in combined surgical and radiochemotherapy (4-6), there remains a pressing need for new therapeutic approaches and molecular targets. Keratin23 (KRT23) belongs to the acidic type I keratins; it is strongly expressed in colon adenocarcinomas but absent in normal colon mucosa. KRT23 knockdown decreases proliferation and affects the DNA damage response of colon cancer cells (7). Similarly, researchers have found that phosphoprotein KRT23 accumulates in microsatellite-stable (MSS) colon cancers in vivo and impacts the viability and proliferation in vitro (8). However, KRT23 has not been reported in triple-negative breast cancers (TNBC). Non-specific cytotoxic cell receptor 1 (NCCRP1) is a paralog of the F-box superfamily of proteins (ubiquitin ligases) that regulate the cell cycle (9,10). In particular, NCCRP1 can act as a prognostic signature in pancreatic cancer and squamous cell carcinoma (11,12). However, the role of NCCRP1 in TNBC is unknown. In this study, we first assessed the roles of KRT23 and NCCRP1 in predicting the diagnosis and prognosis of TNBC using bioinformatics analysis based on the Clinical Proteomic Tumor Analysis Consortium (CPTAC). We then validated the messenger RNA (mRNA) and protein expressions of KRT23 and NCCRP1 in breast cancer. In addition to elucidating the molecular mechanisms regulating the biological actions of KRT23 and NCCRP1 in a breast cancer cell line, the purpose of the study was to investigate the effects of KRT23 and NCCRP1 on the proliferation of breast cancer cells. We present the following article in accordance with the MDAR reporting checklist (available at https://gs.amegroups.com/article/view/10.21037/gs-22-486/rc).
We obtained data on gene expression and clinical characteristics using the Clinical Proteomic Tumor Analysis Consortium (CPTAC) (https://cptac-data-portal.georgetown.edu/cptac) and The Cancer Genome Atlas (TCGA) database (https://tcga-data.nci.nih.gov/tcga). Differential gene screening between TNBC and non-triple-negative breast cancers (NTNBC) was performed according to the “edgeR” filter criteria (log2|fold change| >0.5, false discovery rate (FDR) <0.05). We then analyzed the obtained differential genes (Gene Ontology) GO and (Kyoto Encyclopedia of Genes and Genomes) KEGG enrichment pathways. This study used univariate and multivariate Cox regression analyses to construct a risk model and identify prognosis-related genes. Our survival analysis was based on the Kaplan-Meier analysis (KM). XCELL (13), TIMER (14), EPIC (15), QUANTISEQ (16), MCPCOUNTER (17), CIBERSORT-ABS (18), and CIBERSORT (18) software were used to determine the extent of tumor immune cell infiltration. To evaluate the clinical responses to breast cancer treatment, the half maximal inhibitory concentration (IC50s) of common chemotherapeutics were calculated using “pRRophetic” and “ggplot2”.
Breast cancer tissues and corresponding adjacent normal tissues were collected from 29 patients who underwent surgical resection at the First Affiliated Hospital of Gannan Medical University, China. After surgical removal, the samples were immediately frozen in liquid nitrogen. This study was conducted according to the Declaration of Helsinki (as revised in 2013) and was approved by the Ethics Committee of the First Affiliated Hospital of Gannan Medical University (No. LLSC-2022033101). Informed consent was obtained from the patients or their guardians.
RNA was extracted using an RNA Extraction Kit (Qiagen, USA) according to the manufacturer’s instructions. Next, the RNA was reverse-transcribed using the TAKARA reverse transcription kit (TaKaRa, Dalian, China). TaKaRa SYBR Premix Ex TaqTM II Kit (TaKaRa, Dalian, China) was used to amplify the complementary DNA (cDNA)templates by qRT-PCR. The ABI 7500 Real-Time PCR System (Applied Biosystems, CA, USA) was used to perform qRT-PCR. The primer sequences are listed in Table S1.
Human breast cancer cells (MDA-MB-231 cell lines) were purchased from the Cell Bank of Type Culture Collection of the Chinese Academy of Sciences (Shanghai, China). The MDA-MB-231 cells were cultured in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% Foetal Bovine Serum (FBS), 100 U/mL penicillin, and 100 mU/mL streptomycin (Corning, USA). Overexpression of KRT23 and NCCRP1 (lv-KRT23 and lv-NCCRP1) were constructed and transfected in MDA-MB-231 cells using lentiviral overexpression plasmids Ubi-MCS-SV40-puromycin. Cells transfected with lv-negative control (NC) lentivirus served as a negative control. Next, we used the hU6-MCS-CMV-puromycin plasmid to knock down KRT23 and NCCRP1 in cells (sh-KRT23 and sh-NCCRP1) before transfection in MDA-MB-231 cells, with sh-NC cells used as a negative control. The culture was performed in 5% carbon dioxide (CO2) incubators (Thermos, USA). The interfering nucleotide sequence was designed according to the Invitrogen RNA interference sequence design website (https://rnaidesigner.thermofisher.com/). The RNA interference target sequences are presented in Table S2.
Immunohistochemistry was performed according to the manufacturer's instructions (Solarbio, Beijing, China). Sections were observed, and images were captured under a Leica DMRB microscope (Leica Microsystems, Germany). An extraction kit for total proteins (Keygen, Nanjing, China) was used to extract proteins from cells. Protein concentrations were determined using the BCA-100 Protein Quantitative Analysis Kit (Shanghai, China). WB was performed according to the standard protocols, and WB analysis was conducted using the antibodies listed in Table S3.
Briefly, MDA-MB-231 cells (1×103) were seeded into 96-well plates. At various time points (0, 1, 2, 3, and 4 days), we determined the value of absorbance (at 450 nm) for each cell according to the instruction manuals (Dojindo, Japan). To conduct the EdU assay, we followed the instructions provided with the EdU Cell Proliferation Assay Kit (RiboBio Co., Ltd., Guangzhou, Guangdong, China). Finally, the EdU-stained cells were examined under a confocal microscope (Carl Zeiss, Germany).
To explore the effect of SOX10 on the KRT23 and NCCRP1 promoter activity, the sequences of the KRT23 and NCCRP1 promoters were sub-cloned downstream of the luciferase reporter gene to create pGL3-KRT23 pro-Wild-type (Wt) and pGL3-NCCRP1 pro-Wild-type (Wt) plasmids. To test the binding specificity, a corresponding mutant was created with a changed region binding site to create pGL3-KRT23 pro-Mut-type (Mut) and pGL3-NCCRP1 pro-Mut-type (Mut) plasmids. The sequences of the wild-type KRT23 and NCCRP1 promoters were obtained from the UCSC genome browser (http://genome.ucsc.edu/). Luciferase activity was measured using a luciferase assay kit (K801–200, BioVision Inc., Milpitas, CA, USA). The relative luciferase activity was calculated based on firefly fluorescence versus Renilla fluorescence.
All statistical analyses were performed by using R software packages version 3.4.2. Experimental data analysis was performed using GraphPad Prism 8, with P<0.05 considered statistically significant.
We determined the differential expression of proteins between TNBC and NTNBC based on the CPTAC database (available at https://cdn.amegroups.cn/static/public/gs-22-486-1.xlsx). Next, we analyzed the differential proteins by GO and KEGG enrichment pathways. GO enrichment analysis revealed five significantly enriched GO terms (carboxylic acid biosynthetic process, gland development, gland morphogenesis, organic acid biosynthetic, and urogenital system development) (P<0.05) (Figure 1A). The KEGG enrichment analysis identified five significantly enriched KEGG pathways (arginine biosynthesis, estrogen signaling pathway, fatty acid metabolism, prostate cancer, and staphylococcus aureus infection) (P<0.05) (Figure 1B). All differential proteins were analyzed to identify prognosis-related genes in triple-negative breast cancer. The univariate and multivariate Cox regression analyses found that KRT23 and NCCRP1 could independently predict prognosis (Figure 2A,2B), with high KRT23 and NCCRP1 expression levels indicating poor prognosis in triple-negative breast cancer (Figure 2C,2D). Immunohistochemistry showed that KRT23 and NCCRP1 had a high expression in TNBC tissues (Figure 2E). Next, the risk score was calculated for each of the 105 patients based on the KRT23 and NCCRP1 expression levels from CPTAC, and the patients were then divided into high- and low-risk groups based on a cutoff value (the median risk score). An analysis of the Kaplan-Meier survival data revealed that the high-risk group had worse overall survival (OS), which suggests that the risks may have a prognostic value (Figure 2F).
To further explore the biological role of KRT23 and NCCRP1 in breast cancer cells, the expressions of KRT23 and NCCRP1 were knocked down or overexpressed in MDA-MB231 cells using lentivirus (Figure 3A,3B). The EdU and CCK-8 assays showed that silenced KRT23 or NCCRP1 restored cell proliferation (Figure 3C-3E). However, the overexpression of KRT23 or NCCRP1 promoted cell proliferation (Figure 3F-3H). These results suggested that KRT23 or NCCRP1 might be essential oncogenes in TNBC.
The Venn diagram analysis identified the differentially expressed transcription factors, which co-regulated KRT23 or NCCRP1 expression in TNBC. The results showed that KRT23 and NCCRP1 had 12 differentially expressed common transcription factors (Figure 4A). An overall correlation between the 14 features is displayed in a heatmap, and a significant correlation was found between SOX10 expression and KRT23 or NCCRP1 expression (Figure 4B). Additionally, we found that SOX10 was positively correlated with KRT23 and NCCRP1 in TNBC tissues (Figure 4C,4D). Meanwhile, KRT23 and NCCRP1 expression was promoted by SOX10 overexpression (Figure 4E,4F). Dual-luciferase reporter assays in MDA-MB231 cells were performed to validate the regulation of KRT23 and NCCRP1 expression by SOX10 (Figure 4G).
In this study, multiple software programs (XCEL, TIMER, QUANTISEQ, MCPCOUNTER, EPIC, CIBERSORT-ABS, CIBERSORT) were used to analyze the relationships between patients in the high- and low-risk groups and immune cells in the risk prediction model (Figure 5A). To determine whether the chemotherapeutic treatment efficacy was correlated with the risk scores, we examined the relationship between risk scores and IC50. In the present study, we sought to determine the association between the risk score and the efficacy of common chemotherapeutic agents in the treatment of breast cancer in TCGA database. Our results revealed that a high-risk score was related to a lower half maximal inhibitory concentration (IC50) of chemotherapeutics such as Sorafenib (Figure 5B) and Lapatinib (Figure 5C), while a low-risk score was related to a lower IC50 of chemotherapeutics such as Bleomycin (Figure 5D), Embelin (Figure 5E), Tipifarnib (Figure 5F), and Temsirolimus (Figure 5G). These results indicated that the model was a potential predictor of chemosensitivity.
Breast cancers characterized by triple-negative status tend to be more malignant and have a poorer prognosis (1,2,4-6,19,20). To decrease the mortality rate and improve the prognosis of breast cancer patients, a model for predicting breast cancer risk should be developed. In this study, a risk model was constructed and validated using the CPTAC databases. Using multivariate and univariate Cox regression analyses, an assessment model was established for KRT23 and NCCRP1. We found that high expression levels of KRT23 and NCCRP1 were indicative of poor prognosis and that the gene expression patterns of multiple genes are considerably more predictive of future risks and have a higher level of consistency when assessing risk. KRT23 is a newly discovered member of the keratin family. Various tumor tissues, including pancreatic cancer (21), colorectal carcinoma (7,22-24), and hepatocellular carcinoma (25,26), have exhibited aberrant expression of KRT23. Our findings support the same results, namely that the expression of KRT23 was increased in TNBC and promoted cell proliferation. In this study, we investigated the expression and functions of NCCRP1 in cells. Our findings also found the same; the expression of NCCRP1 was increased in TNBC and promoted cell proliferation. Therefore, we can design specific antibodies against KRT23 and NCCRP1 to block their effects on breast cancer proliferation. To identify the transcription factors regulating the joint regulation of KRT23 and NCCRP1, we found their transcripts through the UCSC database and performed transcription factor prediction on all transcripts. Venn diagram analysis identified 12 differentially expressed transcription factors, which co-regulated KRT23 or NCCRP1 expression in TNBC. We confirmed our hypothesis that SOX10 regulated KRT23 and NCCRP1. We also analyzed the risk scores associated with immune cell infiltration and chemotherapeutics. The XCEL database showed a strong association with the common lymphoid progenitor, M2 macrophages, and CD4+ Th1 T cells in high-risk group patients. The TIMER database showed a strong association with macrophages in high-risk group patients. The QUANTISEQ database demonstrated a strong association with uncharacterized cells in high-risk group patients. The EPIC database showed a strong association with cancer-associated fibroblasts in high-risk group patients. The CIBERSORT-ABS database showed a strong association with neutrophils in high-risk group patients. The CIBERSORT database showed a strong association with M2 macrophages and neutrophils in high-risk group patients. Based on these results, we found that high-risk patients were closely associated with M2 type macrophage infiltration. Therefore, in the treatment of triple negative breast cancer, it can be combined with M2 macrophage inhibitor to improve the efficacy of chemotherapy. Similarly, we can also predict the relationship between KRT23 and NCCRP1 and immune cells by analyzing the correlation between KRT23 and NCCRP1 and immune-related genes. The sensitivity to chemotherapy was different between the high- and low-risk groups. In conclusion, these results indicated that the model was a potential predictor of chemosensitivity.
The article’s supplementary files as 10.21037/gs-22-486 10.21037/gs-22-486 10.21037/gs-22-486 10.21037/gs-22-486 | true | true | true |
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PMC9638856 | 36161689 | Hui Li,Ning Wang,Yu Jiang,Haofei Wang,Zengfeng Xin,Huazhang An,Hao Pan,Wangqian Ma,Ting Zhang,Xiaojian Wang,Wenlong Lin | E3 ubiquitin ligase NEDD4L negatively regulates inflammation by promoting ubiquitination of MEKK2 | 26-09-2022 | IL‐17R signaling,inflammation,MEKK2,NEDD4L,ubiquitination,Immunology,Molecular Biology of Disease,Post-translational Modifications & Proteolysis | Abstract Aberrant activation of inflammation signaling triggered by tumor necrosis factor α (TNF‐α), interleukin‐1 (IL‐1), and interleukin‐17 (IL‐17) is associated with immunopathology. Here, we identify neural precursor cells expressed developmentally down‐regulated gene 4‐like (NEDD4L), a HECT type E3 ligase, as a common negative regulator of signaling induced by TNF‐α, IL‐1, and IL‐17. NEDD4L modulates the degradation of mitogen‐activated protein kinase kinase kinase 2 (MEKK2) via constitutively and directly binding to MEKK2 and promotes its poly‐ubiquitination. In interleukin‐17 receptor (IL‐17R) signaling, Nedd4l knockdown or deficiency enhances IL‐17‐induced p38 and NF‐κB activation and the production of proinflammatory cytokines and chemokines in a MEKK2‐dependent manner. We further show that IL‐17‐induced MEKK2 Ser520 phosphorylation is required not only for downstream p38 and NF‐κB activation but also for NEDD4L‐mediated MEKK2 degradation and the subsequent shutdown of IL‐17R signaling. Importantly, Nedd4l‐deficient mice show increased susceptibility to IL‐17‐induced inflammation and aggravated symptoms of experimental autoimmune encephalomyelitis (EAE) in IL‐17R signaling‐dependent manner. These data suggest that NEDD4L acts as an inhibitor of IL‐17R signaling, which ameliorates the pathogenesis of IL‐17‐mediated autoimmune diseases. | E3 ubiquitin ligase NEDD4L negatively regulates inflammation by promoting ubiquitination of MEKK2
Aberrant activation of inflammation signaling triggered by tumor necrosis factor α (TNF‐α), interleukin‐1 (IL‐1), and interleukin‐17 (IL‐17) is associated with immunopathology. Here, we identify neural precursor cells expressed developmentally down‐regulated gene 4‐like (NEDD4L), a HECT type E3 ligase, as a common negative regulator of signaling induced by TNF‐α, IL‐1, and IL‐17. NEDD4L modulates the degradation of mitogen‐activated protein kinase kinase kinase 2 (MEKK2) via constitutively and directly binding to MEKK2 and promotes its poly‐ubiquitination. In interleukin‐17 receptor (IL‐17R) signaling, Nedd4l knockdown or deficiency enhances IL‐17‐induced p38 and NF‐κB activation and the production of proinflammatory cytokines and chemokines in a MEKK2‐dependent manner. We further show that IL‐17‐induced MEKK2 Ser520 phosphorylation is required not only for downstream p38 and NF‐κB activation but also for NEDD4L‐mediated MEKK2 degradation and the subsequent shutdown of IL‐17R signaling. Importantly, Nedd4l‐deficient mice show increased susceptibility to IL‐17‐induced inflammation and aggravated symptoms of experimental autoimmune encephalomyelitis (EAE) in IL‐17R signaling‐dependent manner. These data suggest that NEDD4L acts as an inhibitor of IL‐17R signaling, which ameliorates the pathogenesis of IL‐17‐mediated autoimmune diseases.
Inflammation represents an adaptive and dynamic host response against pathogens, physical/chemical injuries, toxins, or allergens. The stimulant triggers leukocyte infiltration, cytokines, and chemokines expression, and eventual subsiding of proinflammatory signaling. Dysregulated or prolonged inflammation has been closely related to a variety of human diseases such as autoimmune diseases, cancer, cardiovascular disease, and metabolic disorders (Coussens & Werb, 2002; Hotamisligil, 2017; Liu et al, 2020). Thus, immune‐regulatory and negative feedback mechanisms are critically required to harness inflammation and prevent excessive immunopathology post‐inflammation (Medzhitov, 2008). Inflammatory cytokines such as tumor necrosis factor α (TNF‐α), interleukin‐1 (IL‐1), and interleukin‐17 (IL‐17) are key regulators in the inflammation process. By binding IL‐1R, IL‐1 activates the MyD88‐IRAK1‐TRAF6‐TAK1 cascade (Boraschi et al, 2018). TNF‐α exerts its biological effects by binding two different receptors, TNFR1 and TNFR2. Upon activation, TNFR1 recruits adaptor proteins including TRADD, TRAF2, cIAP1, cIAP2, and RIP1. TNFR2 recruits TRAF2 and also TRAF1, TRAF3, cIAP1, and cIAP2, through their binding to TRAF2 (Kalliolias & Ivashkiv, 2016). Although with different biological functions, both IL‐1 and TNF‐α activate downstream MAPK and NF‐κB to induce inflammatory responses. Among the six members of the IL‐17 family (i.e., IL‐17A to IL‐17F), IL‐17A, commonly called IL‐17, is the first identified and also the most intensively studied member (Iwakura et al, 2011). IL‐17F shares the strongest sequence homology with IL‐17A. IL‐17A and IL‐17F activate downstream signaling pathways through a heterodimeric receptor composed of the IL‐17RA and IL‐17RC subunits (Gaffen, 2009). Upon IL‐17 and IL‐17F ligation, IL‐17RA and IL‐17RC recruit TRAF6 through adaptor protein Act1, ultimately activating MAPK, NF‐κB, C/EBPδ, and C/EBPβ to induce proinflammatory cytokines and chemokines expression (Qu et al, 2012). TNF‐α‐, IL‐1‐, and IL‐17‐induced signaling pathway protectively responds to pathogen invasion, injuries, toxins, and allergens. However, prolonged activated TNF‐α‐, IL‐1‐, and IL‐17‐induced signaling will cause the development of autoimmune diseases such as experimental autoimmune encephalomyelitis (EAE) (Sutton et al, 2009; Rostami & Ciric, 2013; Wolf et al, 2017). As is well demonstrated, deficiency or destruction of the negative regulator of the inflammatory signaling is responsible for the development of autoimmune diseases. As a negative regulator involved in TNF‐α‐ and IL‐1‐induced inflammatory signaling pathway, TNF‐α‐induced protein 3 (TNFIP3, also known as A20) has been reported to be associated with many human inflammation‐related diseases (Catrysse et al, 2014). Neural precursor cell expressed developmentally down‐regulated gene 4‐like (NEDD4L) is a highly conserved HECT‐type E3 ligase. NEDD4L mediates ubiquitination of epithelial Na+ channel (ENaC) and inhibits cell surface expression of ENaC in epithelia (Goulet et al, 1998; Abriel et al, 1999). NEDD4L also limits TGF‐β signal transduction by mediating Smad2/3 poly‐ubiquitination and degradation (Gao et al, 2009). NEDD4L has been reported to be associated with nerve disease in human and mouse models (Charbonnier‐Beaupel et al, 2015; Jewett et al, 2016; Manning & Kumar, 2018). Our recent data demonstrate that NEDD4L promotes Lys‐29‐linked ubiquitination of TRAF3, and thus positively regulates antivirus innate response (Gao et al, 2021). We also showed in a previous study that NEDD4L negatively regulates keratinocyte hyperplasia by promoting Lys‐27‐linked ubiquitination of GP130 and proteasomal degradation in keratinocytes (Liu et al, 2021). However, the physiologic role of NEDD4L in autoimmune diseases remains largely unknown. In the present study, we identify NEDD4L as a common negative regulator of IL‐17‐, TNF‐α‐, and IL‐1α‐induced signaling. NEDD4L constitutively and directly binds MEKK2 and reduces MEKK2 expression by promoting K27‐linked poly‐ubiquitination of MEKK2. Mekk2 knockdown inhibits IL‐17‐induced p38 and NF‐κB activation, as well as IL‐17‐induced proinflammatory cytokine and chemokine production. NEDD4L inhibits IL‐17‐induced inflammation in a MEKK2‐dependent manner. Further study shows that IL‐17 induces MEKK2 Ser520 phosphorylation, which is not only crucial for the activation of p38 and NF‐κB pathways but also for NEDD4L‐mediated MEKK2 degradation to suppress IL‐17R signaling. Furthermore, Nedd4l‐deficient mice are more susceptible to IL‐17‐induced peritonitis and pneumonia, and IL‐17R‐related EAE. These results suggest that as a negative regulator of MEKK2‐dependent IL‐17R‐mediated inflammation, NEDD4L severs as a pivotal immune modulator for dysregulated inflammation‐mediated autoimmune diseases.
To explore the role of NEDD4L in inflammation, we determined the effect of Nedd4l knockdown on IL‐17‐induced proinflammatory cytokine and chemokine expression. Synthesized Nedd4l‐specific small‐interfering RNA (siNedd4l) was transfected into HeLa cells to inhibit endogenous NEDD4L expression (Fig EV1A). Nedd4l knockdown significantly increased IL‐17‐induced Il‐6, Cxcl2, Ccl20 mRNA expression, and IL‐6 and CXCL2 production (Fig 1A and B). We next investigated the role of NEDD4L in inflammatory responses in embryonic fibroblasts (MEFs), which is well established for the IL‐17R signaling study (Zhong et al, 2012). As shown in Fig 1C and D, IL‐17‐induced Il‐6, Cxcl2, and Ccl20 mRNA expression, and IL‐6 and CXCL2 production were significantly increased in MEFs with Nedd4l homozygous deficiency (Nedd4l −/−) compared to the wild‐type (Nedd4l +/+) MEFs. Since IL‐17F has similar functions as IL‐17, Nedd4l homozygous deficiency significantly increased IL‐17F‐induced Il‐6, Cxcl2, and Ccl20 mRNA expression in MEFs (Fig 1E). Such significantly increased IL‐17A‐ or IL‐17F‐induced Il‐6, Cxcl2, and Ccl20 mRNA expression were also observed in MEFs with Nedd4l heterozygous deficiency (Nedd4l +/−) (Fig EV1B–D). Lung epithelial cells and astrocytes have been also identified to play a crucial role in IL‐17‐induced inflammation and autoimmune diseases (Kang et al, 2010; Zhong et al, 2012). Nedd4l deficiency in these cells resulted in significantly increased IL‐17‐induced Il‐6, Cxcl2, and Ccl20 mRNA expression compared to the wild‐type control cells (Figs 1F and G, and EV1E and F). These results suggest that NEDD4L plays a crucial role in IL‐17‐induced inflammation in vitro. Interestingly, increased TNF‐α‐ or IL‐1α‐induced Il‐6, Cxcl2, and Ccl20 mRNA expression was also observed in HeLa cells with Nedd4l knockdown and in MEFs with Nedd4l deficiency (Fig EV2A–D). These data suggest that NEDD4L might be a common negative regulator of TNF‐α‐, IL‐1α‐, and IL‐17‐induced inflammation.
To explore the regulatory role of NEDD4L in IL‐17‐induced inflammation responses in vivo, Nedd4l +/+ and Nedd4l +/− mice were intraperitoneally injected (i.p.) with IL‐17 or PBS, and Il‐6, Cxcl2, and Ccl20 mRNA expression in peritoneal mesothelial cells were analyzed by real‐time PCR. As shown in Figs 2A and EV2E, Nedd4l deficiency significantly increased IL‐17‐induced Il‐6, Cxcl2, and Ccl20 mRNA expression in peritoneal mesothelial cells. IL‐17‐induced chemokines amplify inflammatory responses by recruiting neutrophils to local tissue, as shown in an inflammation model in which airway administration of IL‐17 caused considerable pulmonary inflammation (Bulek et al, 2011; Zhong et al, 2012). To determine whether NEDD4L affects IL‐17‐induced pulmonary inflammation, Nedd4l +/+ and Nedd4l +/− mice were intratracheally injected with IL‐17 or PBS, followed by the analysis of Il‐6, Cxcl2, and Ccl20 mRNA expression in lung tissues and production of CXCL2 in the bronchoalveolar lavage fluid (BALF). As shown in Fig 2B, Nedd4l deficiency significantly increased IL‐17‐induced Il‐6, Cxcl2, and Ccl20 mRNA expression in lung tissues. CXCL2 production in BALF from Nedd4l +/− mice was also significantly increased compared to that from Nedd4l +/+ mice (Fig 2C). Consistent with increased proinflammatory cytokine and chemokine production in Nedd4l +/− mice, more CD45+ cells were recruited into the BALF of Nedd4l +/− mice than that of Nedd4l +/+ mice (Fig 2D). In particular, neutrophils (Gr1+CD11b+) in the BALF of Nedd4l +/− mice were increased more than 10 folds compared with that in wild‐type mice (Fig 2E), indicating that Nedd4l deficiency increased IL‐17‐induced pulmonary neutrophil infiltration. Histological analysis of lung tissue showed enhanced immunopathological changes in the lungs of Nedd4l +/− mice (Fig 2F). These results suggest that NEDD4L plays an important role in limiting IL‐17‐induced inflammation in vivo.
IL‐17 induces proinflammatory cytokine and chemokine production through MAPK and NF‐κB pathways. In HeLa cells, Nedd4l knockdown increased IL‐17‐induced phosphorylation of p38 and NF‐κB p65 subunit without affecting IL‐17‐induced phosphorylation of ERK1/2 and JNK1/2 (Fig 3A). In MEFs, Nedd4l deficiency increased IL‐17‐induced p38 and NF‐κB p65 subunit phosphorylation but not ERK1/2 and JNK1/2 phosphorylation (Fig 3B). Accordingly, Nedd4l knockdown in HeLa cells or deficiency in MEFs remarkably increased TNF‐α‐ or IL‐1α‐induced p38 and NF‐κB p65 phosphorylation (Fig EV3A–D). Consistently, Nedd4l deficiency in primary lung epithelial cells and astrocytes significantly promoted IL‐17‐induced p38 and NF‐κB p65 subunit phosphorylation (Fig 3C and D). Overexpression of exogenous wild‐type NEDD4L, but not the E3 enzyme activity mutation NEDD4L‐C942A (NEDD4L‐CA) in HeLa cells, inhibited IL‐17‐induced p38 and NF‐κB p65 subunit phosphorylation compared to the control group (Fig 3E). Similar signal transduction effect mediated by overexpression of exogenous NEDD4L could be reconstituted in Nedd4l‐silenced HeLa cells, suggesting that NEDD4L specifically regulates this signaling (Fig EV3E). These results indicate that NEDD4L plays a negative regulatory role in IL‐17‐induced signaling. IL‐17RA and IL‐17RC activate MAPKs and NF‐κB pathways through TRAF family members. However, neither Nedd4l knockdown nor Nedd4l deficiency affected expressions of the TRAFs (Fig 3A and B). Given the important role of MEKK family members in MAPK and NF‐κB activation (Zhao & Lee, 1999; Blonska et al, 2004), we set out to investigate the effect of Nedd4l knockdown on MEKKs. Interestingly, Nedd4l knockdown significantly increased MEKK2 expression in HeLa cells (Fig 3A). In MEFs, primary lung epithelial cells, and astrocytes, Nedd4l deficiency also increased MEKK2 expression (Fig 3B–D). Overexpression or reconstitution of exogenous wild‐type NEDD4L, but not NEDD4L‐CA, can inhibit MEKK2 expression compared to the control group (Figs 3E and EV3E). We compared MEKK2 expression in Nedd4l +/−, Nedd4l −/−, and wild‐type MEFs. As shown in Fig 3F, Nedd4l heterozygous deficiency was sufficient to increase MEKK2 expression in MEFs. Notably, Nedd4l deficiency in MEFs strongly prolonged the half‐life of MEKK2 protein compared to that in wild‐type MEFs (Fig 3G and H). Furthermore, overexpression of NEDD4L reduced MEKK2 expression (Fig EV3F), providing further evidence that NEDD4L inhibits the stability of MEKK2 protein.
To determine the mechanism through which NEDD4L reduced MEKK2 expression, we investigated the interaction between NEDD4L and MEKK2 in HeLa cells by immunoprecipitation with NEDD4L‐ or MEKK2‐specific antibody. As shown in Figs 4A and EV4A, NEDD4L was co‐immunoprecipitated with MEKK2, but not MEKK3, in both IL‐17‐stimulated and unstimulated cells, suggesting that NEDD4L was constitutively associated with MEKK2 in HeLa cells. In the pull‐down assay, NEDD4L recombinant protein was precipitated with GST‐MEKK2 protein (Fig 4B), suggesting that NEDD4L bound MEKK2 in vitro. To map the domains required for NEDD4L to interact with MEKK2, we constructed a series of plasmids expressing wild‐type and mutant NEDD4L in which C2 (∆C2), WW (∆WW), or HECT (∆HECT) domain was deleted, respectively (Fig EV4B). As shown in Fig 4C, the deletion of the WW domain but not the C2 and HECT domain disrupted the interaction between NEDD4L and MEKK2, demonstrating that the WW domain was necessary for NEDD4L to bind to MEKK2. As an E3 ubiquitin ligase, NEDD4L might inhibit the stability of MEKK2 protein by mediating the ubiquitination of MEKK2. To investigate whether NEDD4L regulates MEKK2 expression through proteasome‐dependent or lysosomal‐dependent pathway, we treated Nedd4l −/− and wild‐type control MEFs with a proteasome inhibitor MG132 or a lysosomal degradation inhibitor bafilomycin A1 (Baf A1). Following MG132 treatment but not Baf A1 treatment, MEKK2 expression in wild‐type control cells was elevated to the level comparable with that in Nedd4l −/− MEFs (Fig 4D), suggesting that NEDD4L suppresses the stability of MEKK2 protein by mediating MEKK2 ubiquitination in a proteasome‐dependent manner. In further support of our hypothesis, in vitro ligase activity assay demonstrated that it was NEDD4L protein, but not TRAF6 protein, efficiently increased MEKK2 ubiquitination (Fig 4E), suggesting that MEKK2 is a potential substrate of NEDD4L. Furthermore, NEDD4L deficiency reduced total poly‐ubiquitination of MEKK2, without affecting K63‐ and K48‐linked poly‐ubiquitination of MEKK2 in MEFs (Fig 4F). We further determined the effects of wild‐type versus mutant NEDD4L on MEKK2 ubiquitination in HEK293T cells. NEDD4L‐∆WW completely lost the capability to promote MEKK2 ubiquitination. Furthermore, deletion of the HECT domain also largely impaired such capability of NEDD4L. In contrast, deletion of the C2 domain enhanced the ubiquitination of MEKK2, which could be due to the relief of autoinhibitory interaction between its C2 and HECT domains, consistent with the notion that the C2 domain of HECT family ligases, including smurf2, NEDD4, and NEDD4L, inhibits the function of HECT domain (Wang et al, 2010; Malonis et al, 2017; Lorenz, 2018; Fig 4G). To determine the nature of NEDD4L‐mediated MEKK2 ubiquitination, we transfected HEK293T cells with NEDD4L‐ and MEKK2‐expressing vectors in the presence of constructs expressing wild‐type ubiquitin (HA‐Ub) or its mutants. As shown in Fig 4H and I, NEDD4L mainly promoted Lys‐27(K27O)‐linked poly‐ubiquitination of MEKK2 (Fig 4H). Mutation of Lys‐27 (K27R) completely abrogated the poly‐ubiquitination effect of NEDD4L on MEKK2 (Fig 4I), demonstrating that NEDD4L mediates K27‐linked poly‐ubiquitination of MEKK2. It is well established that the ubiquitin chain specificity of the HECT‐type E3 ligases is determined by their C‐terminal amino acids. NEDD4 family ligases, including NEDD4L, exhibit strict specificity toward K63 linkages (Maspero et al, 2013). However, in our study, NEDD4L mediates K27‐linked but not K63‐linked poly‐ubiquitination of MEKK2 in a dosage‐dependent manner (Fig 4J), which could be a consequence of the unique structure of NEDD4L. Further studies will be needed to identify the detailed differences between NEDD4L and other HECT‐type E3 ligases.
MEKK2 has been reported to participate in TNF‐α‐ and IL‐1‐induced signaling (Chayama et al, 2001; Hammaker et al, 2004). However, it remains unknown whether MEKK2 is also involved in IL‐17R signaling. Thus, our further experiments were focused on the role of MEKK2 in NEDD4L‐mediated negative regulation of IL‐17R signaling. We firstly used synthesized Mekk2‐specific small‐interfering RNA (siMekk2) to inhibit endogenous MEKK2 expression in HeLa cells, and then stimulated the cells with IL‐17 and detected IL‐17‐induced MAPKs and NF‐κB activation. As shown in Fig 5A, Mekk2 siRNA inhibited endogenous MEKK2 expression and IL‐17‐induced p38 and NF‐κB activation. Consistently, Mekk2 siRNA inhibited IL‐17‐induced Il‐6, Cxcl2, and Ccl20 mRNA expression in HeLa cells (Fig 5C). Similarly, Mekk2 knockdown also inhibited IL‐17‐induced p38 and NF‐κB activation and Il‐6, Cxcl2, and Ccl20 mRNA expression in MEFs (Fig 5B–D). These results suggest that MEKK2 is required for IL‐17R signaling. To investigate the role of MEKK2 in NEDD4L‐mediated regulation of IL‐17R signaling, we compared the effects of Nedd4l siRNA on IL‐17‐induced p38 and NF‐κB activation in the presence and absence of Mekk2 siRNA. As shown in Fig 5E, transfection of Nedd4l siRNA alone enhanced IL‐17‐induced p38 and NF‐κB activation in HeLa cells. However, in the presence of Mekk2 siRNA, Nedd4l siRNA could not enhance IL‐17‐induced p38 and NF‐κB activation (Fig 5E). Similarly, Mekk2 siRNA eliminated the enhancement of IL‐17‐induced p38 and NF‐κB activation by Nedd4l deficiency in MEFs (Fig 5F). Mekk2 knockdown nearly completely reversed the increase in IL‐17‐induced Il‐6, Cxcl2, and Ccl20 mRNA expression by Nedd4l knockdown in HeLa cells or Nedd4l deficiency in MEFs (Fig 5G and H). It has been identified that Smurf1, another HECT family E3, promotes MEKK2 ubiquitination and degradation in macrophages under the TLR9 activation (Wen et al, 2015). However, no combined effects were observed in Smurf1 and Nedd4l combined silenced HeLa cells (Fig EV4C and D). Syk is known to be a target for NEDD4L in mast cells (Yip et al, 2016). Syk acts as an upstream signaling molecule in IL‐17‐induced Act1‐TRAF6 interaction in keratinocytes, and inhibition of Syk can attenuate CCL20 production (Wu et al, 2015). We, therefore, checked the potential influence of Syk on NEDD4L‐mediated IL‐17 activation by treating HeLa cells with a Syk‐specific inhibitor, BAY 61–3,606. As shown in Fig EV4E, specific inhibition of Syk attenuated IL‐17‐induced Il‐6, Cxcl2, and Ccl20 mRNA expression, but could not eliminate the enhancement of IL‐17‐induced Il‐6, Cxcl2, and Ccl20 mRNA expression by Nedd4l silencing in HeLa cells. These results demonstrate that NEDD4L negatively regulates IL‐17R signaling via MEKK2.
MEKK2 Ser520 was reported to be phosphorylated following LPS stimulation and important for MEKK2 activation (Zhang et al, 2006). We prepared a phosphorylated MEKK2 Ser520‐specific antibody to detect MEKK2 phosphorylation in IL‐17‐treated HeLa cells. As shown in Fig 6A, IL‐17 stimulation induced remarkable MEKK2 Ser520 phosphorylation. The antibody against MEKK Ser520 was validated by western blotting analysis of wild‐type MEKK2 and its Ser520 mutants, including MEKK2 Ser520 residue mutating into continuously activated forms asparagine (S520D) and glutamic acid (S520E), or inactivated form alanine (S520A; Figs EV4F and EV4G). As the Ser520 phosphorylation in MEKK2 is regulated by IL‐17R signaling, we, therefore, tested if this phosphorylation is crucial for IL‐17‐induced proinflammatory cytokine and chemokine expression by mutating MEKK2 Ser520 residue into alanine (S520A). As shown in Fig 6B, MEKK2 S520A significantly inhibited the IL‐17‐induced proinflammatory cytokine and chemokine expression in HeLa cells, as well as the phosphorylation of p38 and NF‐κB p65 (Fig 6C). Reconstitution of exogenous wild‐type MEKK2, but not MEKK2‐S520A, can restore the phosphorylation of p38 and NF‐κB p65 in Mekk2‐silenced HeLa cells compared to the control group (Fig EV4H). We next explored how MEKK2 was activated in IL‐17R signaling. IL‐17 activates MAPK and NF‐κB through the Act1‐TRAF6‐TAK1 cascade. We examined whether MEKK2 interacts with these molecules by immunoprecipitation. TAK1 was found to associate with MEKK2 following IL‐17 stimulation (Fig 6D). We constructed plasmids expressing truncated MEKK2 N351 (a.a.1–351) and MEKK2 C352 (a.a.352–619; Fig EV4I). As shown in Fig EV4J, wild‐type MEKK2, mutant MEKK2 S520A, and MEKK2 C352 could be co‐immunoprecipitated with TAK1. In contrast, MEKK2 N351 could not be co‐immunoprecipitated with TAK1, demonstrating that TAK1 interacted with the C‐terminal kinase domain of MEKK2. We used Tak1‐specific siRNA to inhibit endogenous TAK1 expression (Fig 6E). Tak1 knockdown nearly completely blocked IL‐17‐induced MEKK2 phosphorylation (Fig 6E). These results suggest that MEKK2 interacts with TAK1 and is activated by TAK1 in IL‐17R signaling. We further investigated the effects of MEKK2 Ser520 phosphorylation on NEDD4L‐mediated MEKK2 degradation. As shown in Fig 6F, NEDD4L was co‐immunoprecipitated with MEKK2 (S520A) as efficiently as with wild‐type MEKK2, indicating that MEKK2 Ser520 phosphorylation was not required for the association between NEDD4L and MEKK2. However, NEDD4L failed to promote MEKK2 (S520A) ubiquitination and degradation (Fig 6G and H), demonstrating that MEKK2 Ser520 phosphorylation is indispensable for NEDD4L‐mediated MEKK2 ubiquitination. Taken together, IL‐17 induces MEKK2 S520 phosphorylation, which is required for downstream p38 and NF‐κB pathway activation. NEDD4L negatively regulates IL‐17R signaling via mediating Ser520‐phosphorylated MEKK2 degradation.
It has been reported that IL‐17R signaling in astrocytes plays a crucial role in mouse EAE, a well‐known mouse multiple sclerosis (MS) model (Kang et al, 2010). We then investigated the effect of NEDD4L heterozygous deficiency on the MOG35‐55‐induced EAE model by subcutaneous immunization of Nedd4l +/+ or Nedd4l +/− mice with MOG35‐55 and then intravenously injecting them with pertussis toxin (PTX). As shown in Fig EV5A, Nedd4l +/− mice suffered from more severe EAE disease symptoms compared to Nedd4l +/+ mice and had higher EAE clinical scores than their counterparts. Moreover, Nedd4l +/− mice displayed more inflammatory cell infiltration and more serious demyelination compared to their counterparts (Fig EV5B), as demonstrated by hematoxylin–eosin (H&E) and Luxol fast blue (LFB) staining, respectively. Consistent with these results, flow cytometry analysis of mouse CNS tissues (brains and spinal cords) demonstrated that CNS‐infiltrating CD11bhighGr1+ neutrophils were significantly increased in Nedd4l +/− mice compared to Nedd4l +/+ mice (Fig EV5C–F). We subsequently examined the expression levels of several proinflammatory cytokines and chemokines in the CNS tissues of Nedd4l +/+ and Nedd4l +/− EAE mice. As shown in Fig EV5G, the gene expression levels of Il‐6, Cxcl2, and Ccl20 were significantly increased in Nedd4l +/− EAE mice. These data demonstrate that Nedd4l deficiency promotes the pathogenesis of EAE. We next investigated whether the increased EAE pathogenesis in Nedd4l +/− mice is dependent on IL‐17R‐mediated signaling and inflammation. An anti‐IL‐17‐specific blocking antibody was continually i.p. injected into Nedd4l +/+ and Nedd4l +/− mice during the induction of EAE. As shown in Fig 7A and B, the injection of anti‐IL‐17 antibody resulted in significant inhibition of EAE disease symptoms, including clinical score, inflammation, and demyelination, in Nedd4l +/+ and Nedd4l +/− mice compared to the anti‐isotype control antibody‐injected group. The Nedd4l +/− mice exhibited much more severe EAE disease symptoms compared to Nedd4l +/+, which was obliterated after the injection of IL‐17‐blocking antibody (Fig 7A and B). Flow cytometry and real‐time PCR analysis of mouse CNS tissues demonstrated that CNS‐infiltrating CD11bhighGr1+ neutrophils and IL‐17‐induced genes, such as Il‐6, Cxcl2, and Ccl20, were significantly reduced in IL‐17‐blocking antibody‐treated Nedd4l +/− mice compared to mice treated with anti‐isotype control antibody, which was obliterated after the injection of IL‐17‐blocking antibody (Fig 7C–E). However, continual i.p. injection of a Syk‐specific inhibitor during the induction of EAE, BAY 61‐3606, could not eliminate the EAE phenotype difference between Nedd4l +/+ and Nedd4l +/− mice, suggesting that Nedd4l deficiency promotes the pathogenesis of EAE independent of Syk‐mediated signaling (Fig 7F and G). Collectively, these data suggest that NEDD4L regulates EAE pathogenesis via IL‐17R‐mediated MEKK2‐dependent signaling.
In the present study, we show that endogenous NEDD4L inhibits IL‐17, IL‐1α, and TNF‐α‐induced proinflammatory cytokine and chemokine production. NEDD4L promotes MEKK2 ubiquitination and inhibits the protein stability of MEKK2, which is required for IL‐17‐, TNF‐α‐, and IL‐1α‐induced signaling. NEDD4L is a highly conserved HECT‐type E3 ligase. It has been reported to be involved with multi‐physiological functions in mice by targeting the degradation of many important signal transduction‐associated proteins (Manning & Kumar, 2018). Here, we identified NEDD4L as a negative regulator of IL‐17‐mediated inflammation by showing that Nedd4l knockdown and Nedd4l deficiency increased IL‐17‐induced proinflammatory cytokine and chemokine production. Even heterozygous deficiency of Nedd4l significantly increased IL‐17‐induced proinflammatory cytokine and chemokine production in vitro and in vivo, emphasizing the importance of NEDD4L in the negative regulation of IL‐17R signaling. IL‐17 functions mainly by activating NF‐κB and MAPK pathways to induce proinflammatory cytokine and chemokine production. Although NF‐κB and MAPK activations have been demonstrated to be signal events downstream of adaptor protein Act1 and TRAF family members in IL‐17R signaling, the detailed mechanisms by which NF‐κB and MAPK are activated in IL‐17R signaling remain largely unknown. MAP3K family members TAK1 and Tpl2 have been involved in IL‐17‐induced NF‐κB and MAPK activation (Xiao et al, 2014). MEKK2 is involved in signal transduction of TCR, BMP, TGF‐β, FGF‐2, IL‐1α, TNF‐α, and TLR9 agonist CpG (Hammaker et al, 2004; Kesavan et al, 2004; Yamashita et al, 2005; Chang et al, 2011; Wen et al, 2015; Wu et al, 2021). Our results demonstrate that MEKK2 is required for IL‐17‐induced p38 and NF‐κB activation in HeLa cells and MEFs. NEDD4L constitutively and directly binds MEKK2 and inhibits MEKK2 expression by promoting K27‐linked poly‐ubiquitination of MEKK2. Mekk2 knockdown completely reverses Nedd4l deficiency mediated increase in IL‐17‐induced p38 and NF‐κB activation, and also completely reverses the increase in IL‐17‐induced cytokine and chemokine production, clearly demonstrating that NEDD4L negatively regulates IL‐17R signaling by inhibiting MEKK2 expression. In this study, we also show that NEDD4L inhibits TNF‐α‐ and IL‐1α‐induced signaling, which has been reported to require MEKK2 to activate JNK and NF‐κB pathways (Zhao & Lee, 1999; Hammaker et al, 2004). IL‐17, as well as TNF‐α, are important for host immune defense against certain pathogens. However, excessive IL‐17 activities have a high potential to induce damage to inflamed tissues. Thus, it is not surprising that multiple mechanisms restrict IL‐17R signaling to avoid pathogenic conditions. Several IL‐17R signaling negative regulators have been reported, including TRAF3, USP25, A20, SCFβ‐TrCP, and GSK‐3β (Zhong et al, 2012; Garg et al, 2013) (Shen et al, 2009; Zhu et al, 2010; Shi et al, 2011). We show here that NEDD4L negatively regulates IL‐17R signaling via mediating MEKK2 ubiquitination. Upon IL‐17 stimulation, MEKK2 is phosphorylated at Ser520, which is crucial not only for the activation of p38 and NF‐κB pathways but also for NEDD4L‐mediated MEKK2 degradation that negatively regulates IL‐17R‐induced inflammation. Excessive IL‐17‐, IL‐1α‐, and TNF‐α‐triggered signaling have been linked to the development of many inflammatory responses or autoimmune diseases. Our data show that Nedd4l deficiency in mice significantly promotes IL‐17‐induced pneumonia and peritonitis, and also MOG35‐55‐induced EAE in IL‐17R‐signaling dependent manner. NEDD4L has also been identified to regulate phosphorylated‐Syk K48‐linked ubiquitination in mast cells (Yip et al, 2016), and Syk acts as an upstream signaling molecule in IL‐17‐induced Act1‐TRAF6 interaction in keratinocytes (Wu et al, 2015), suggesting that NEDD4L may regulate IL‐17R signaling dependent on Syk. However, inhibition of Syk activation in HeLa cells and during induction of the MOG35‐55‐induced EAE model did not eliminate the phenotype difference in vitro and in vivo, which means NEDD4L regulates IL‐17R‐mediated inflammation and EAE is not dependent on Syk in our experimental system. Taken together, these data confirm that NEDD4L plays a crucial role in IL‐17R‐mediated inflammation and EAE. Taken together, the present study identifies MEKK2 as a mediator of IL‐17R signaling, and NEDD4L as a common negative regulator of IL‐17‐, TNF‐α‐, and IL‐1α‐induced signaling by inhibiting MEKK2 expression, providing novel insights into the mechanism of IL‐17‐, TNF‐α, and IL‐1α‐induced signaling. IL‐17 stimulation induces the MEKK2 phosphorylation and degradation, which are required for the induction of proinflammatory cytokine and chemokine. Thus, based on our findings, NEDD4L serves as a functional protein in inflammation and autoimmune diseases. NEDD4L could be a target for the treatment of IL‐17R‐related inflammation and autoimmune disorders.
Nedd4l‐deficient homozygous (Nedd4l −/−) and control BALB/cByJ (Nedd4l +/+) mice were purchased from JAX® Mice, America. In this study, Nedd4l‐deficient homozygous Nedd4l +/− mice were used in vivo diseases research for Nedd4l −/− mice show kidney defects and some other problems (Manning & Kumar, 2018). Off‐springs of the mice at the age of 6–8 weeks were employed in experiments. All animal experiments were carried out under the National Institute of Health Guide for the Care and Use of Laboratory Animals, with the approval of the Scientific Investigation Board of Laboratory Animal Center of Zhejiang University.
Antibodies for phospho‐ERK1/2 (#4370S), phospho‐JNK (#9251S), phospho‐p38 (#4511S), phospho‐p65 (NF‐κB) (#3033 s), ERK1/2 (#4696), JNK (#9251 s), p38 (#8690), NF‐κB p65 (#8242), and NEDD4L (#4013) were from Cell Signaling Technology (Cell Signaling Technology, Beverly, MA). Antibodies for TRAF2 (#5525–1), MEKK3 (#1673–1), and MEKK5 (#1772–1) were from Epitomics (Epitomics, Burlingame, CA). Antibodies for TAK1 (SC‐7967), MEKK2 (SC‐1088), TRAF3 (SC‐949), and TRAF6 (SC‐7221) were obtained from Santa Cruz Biotechnology (Santa Cruz Biotechnology, Santa Cruz, CA). Phospho‐MEKK2 Ser520‐specific antibody was prepared in Abmart (Shanghai, China). Human or mouse IL‐17A/F (human IL‐17, #200–17; Murine IL‐17A, #210–17; human IL‐17F, #200–25; and murine IL‐17F, #210‐17F) were purchased from PeproTech (PeproTech, Rocky Hill, NJ). E1, UBCH5b, and ubiquitin recombinant proteins were gifts from Prof. Zongping Xia (Zhejiang University).
To construct a plasmid‐expressing Myc‐tagged NEDD4L, cDNA‐encoding NEDD4L was amplified by PCR using mRNA of mouse peritoneal macrophage as a template, and cloned in the pCMV‐Entry plasmid. Flag‐tag in the plasmid was deleted by PCR and the plasmid could express Myc‐tagged NEDD4L. MEKK2 cDNA was amplified by PCR using mRNA of HeLa cells as a template and cloned in pCMV‐HA or pcDNA3.1 plasmid. The vectors for NEDD4L and MEKK2 mutants were subsequently generated by PCR amplification.
Male age‐matched Nedd4l +/+ and Nedd4l +/− littermates were treated by intraperitoneal injection of IL‐17 (0.5 μg per mouse) or PBS. Twenty‐four hours later, intraperitoneal leukocytes were removed by washing the peritoneal cavity with 5 ml PBS. And then 5 ml of 0.25% trypsin was injected into the peritoneal cavity. Ten minutes later, the trypsin solution was collected, and then 5 ml DMEM containing 10% FBS was used to wash the peritoneal cavity and residual mesothelial cells were collected. The expression of chemokines in the cells was measured by real‐time PCR analysis (Zhong et al, 2012).
Male age‐matched Nedd4l +/+ and Nedd4l +/− littermates were treated by intratracheal injection of IL‐17 (2 μg per mouse, 30 μl) or PBS (30 μl). Eight hours later, BLAF fluid was collected in 1.2 ml ice‐cold PBS through the trachea and centrifuged. Chemokines and cytokines in the supernatants were measured using ELISA assays. Cells in the precipitates were resuspended using 0.1 ml staining buffer and counted as lung‐infiltrating cells. The cells were also analyzed using flow cytometry after staining with anti‐Gr‐1 and anti‐CD11b antibodies. The total RNA of the lung tissues was isolated with 1 ml ice‐cold TriZol for real‐time PCR analysis. Lung tissues were dissected, fixed in 4% paraformaldehyde, embedded in paraffin, sectioned, and then stained with hematoxylin and eosin (Bulek et al, 2011).
GST‐tagged MEKK2 (M0324) and Myc‐tagged NEDD4L recombinant proteins were purchased from Sigma and Origene (Origene, Rockville, MD), respectively. GST‐MEKK2 (0.1 μg) bound to glutathione‐agarose beads were mixed with 0.05 μg Myc‐tagged NEDD4L and incubated at 4°C for 4 h with gentle rotation. The beads were washed three times with cell lysis buffer. Then, bound proteins were extracted with loading buffer and analyzed by immunoblot (Xia et al, 2009).
For in vivo ubiquitination assay, HEK293T cells were transfected with pCDNA3.1‐HA ubiquitin along with other vectors as indicated and cultured for 36 h. After being treated with MG‐132 for 6 h, the cells were lysed in 1% SDS and then heated for 5 min. After centrifuging, the supernatants were 10‐fold diluted with lysis buffer, followed by immunoprecipitation with the appropriate antibodies. Immunoprecipitants were analyzed by immunoblot with anti‐ubiquitin or antibody specific for K63‐linked ubiquitin/K48‐linked ubiquitin. The in vitro MEKK2 ubiquitination assay was carried out in a reaction mixture containing 0.2 μM E1, 0.1 μM UBCH5b, 0.4 μM Myc‐tagged NEDD4L, and 12.5 μM ubiquitin in buffer B at 30°C for 60 min before termination with SDS‐sample buffer (Xia et al, 2009).
Acute EAE was induced and assessed as previously described (Kang et al, 2010). Briefly, acute EAE was induced by subcutaneous immunization with 300 μg of the MOG35‐55 peptide (Met‐Glu‐Val‐Gly‐Trp‐Tyr‐Arg‐Ser‐Pro‐Phe‐Ser‐Arg‐Val‐Val‐His‐Leu‐Tyr‐Arg‐Asn‐Gly‐Lys, Sangon Biotech Co.) in CFA containing 5 mg/ml heat‐killed H37Ra strain of Mycobacterium tuberculosis (Chondrex, Inc) in the back region and both sides of the vertebrae. And the immunized mice were i.v. injected with pertussis toxin (List Biological Laboratories, Inc.) at a dose of 250 ng per mouse in PBS on the day of immunization and once more 48 h after the first injection. The clinical score was performed in a double‐blinded manner. Mice were examined every 2–3 days for disease symptoms and were double‐blinded and scored for disease severity using the EAE scoring rulers: 0, no clinical signs; 1, limp tail; 2, paraparesis (weakness and incomplete paralysis of one or two hind limbs); 3, paraplegia (complete paralysis of two hind limbs); 4, paraplegia with fore limb weakness or paralysis; and 5, moribund state or death.
Central nervous system tissues, including spinal cords and brains, were homogenized in ice‐cold tissue grinders, filtered through a 70 μm cell strainer (Falcon), and the cells were collected by centrifugation at 160 g for 5 min at 4°C. Cells were re‐suspended in 10 ml of 35% Percoll (GE) and centrifuged onto a 5 ml 70% Percoll cushion in 15‐ml tubes at 280 g for 25 min with a low accelerating or breaking speed. Cells at the 35–70% interface were collected and subjected to flow cytometry. Fluorescence‐conjugated monoclonal antibodies to FVD, CD45, CD4, CD8, CD11b, F4/80, and Gr‐1 were stained together for the surface marker analysis.
Paraffin‐embedded sections (4 mm thick) were subjected to either hematoxylin and eosin (H&E) or Luxol fast blue (LFB) to evaluate inflammation and demyelination, respectively. H&E and LFB were performed by the Histomorphology Platform, Zhejiang University, with the standard protocol performed according to the manufacturer's instructions.
Total RNA was isolated using TRIzol (TAKARA, Ostushiga, JAPAN) and cDNA was synthesized with a reverse‐transcription kit (TAKARA, Ostushiga, JAPAN). The expression of genes was detected by a LightCycler 480 system with SYBR Premix Ex Tap (TAKARA, Ostushiga, JAPAN). The data were calculated by a standard curve method and normalized to the expression of the gene‐encoding gapdh (human) or β‐actin (mouse). The specific primers for individual genes are in Appendix Table S1. The concentration of cytokines and chemokines in the cell culture medium was detected by ELISA assay (ebioscience, MN, USA and R&D, CA, USA).
The statistical analysis was performed using a two‐tailed Mann–Whitney U test or a two‐tailed Student's t‐test. Differences were considered significant at a P value of < 0.05.
Ning Wang: Data curation; software; formal analysis; validation; methodology. Yu Jiang: Resources; funding acquisition; validation. Huazhang An: Conceptualization; resources; funding acquisition; validation; methodology; writing – original draft; writing – review and editing. Haofei Wang: Validation; methodology. Hui Li: Conceptualization; resources; data curation; software; formal analysis; funding acquisition; validation; investigation; methodology; writing – original draft; writing – review and editing. Wenlong Lin: Conceptualization; formal analysis; funding acquisition; investigation; writing – original draft; project administration; writing – review and editing. Wangqian Ma: Resources; funding acquisition. Zengfeng Xin: Resources; funding acquisition. Hao Pan: Resources; funding acquisition. Xiaojian Wang: Conceptualization; resources; formal analysis; supervision; writing – original draft; project administration; writing – review and editing. Ting Zhang: Resources; funding acquisition; project administration. In addition to the CRediT author contributions listed above, the contributions in detail are: HL, WL, NW, and HW performed experiments. HL and WL performed the statistical analysis. YJ, ZX, HP, WM, and TZ provided some reagents. HA, XW, WL, and HL designed the study. HL, XW, WL, and HA drafted the manuscript.
The authors declare that they have no conflict of interest.
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PMC9638899 | 36271789 | Rafael Molina,Ricardo Garcia-Martin,Blanca López-Méndez,Anne Louise Grøn Jensen,J Rafael Ciges-Tomas,Javier Marchena-Hurtado,Stefano Stella,Guillermo Montoya | Molecular basis of cyclic tetra-oligoadenylate processing by small standalone CRISPR-Cas ring nucleases | 22-10-2022 | Abstract Standalone ring nucleases are CRISPR ancillary proteins, which downregulate the immune response of Type III CRISPR-Cas systems by cleaving cyclic oligoadenylates (cA) second messengers. Two genes with this function have been found within the Sulfolobus islandicus (Sis) genome. They code for a long polypeptide composed by a CARF domain fused to an HTH domain and a short polypeptide constituted by a CARF domain with a 40 residue C-terminal insertion. Here, we determine the structure of the apo and substrate bound states of the Sis0455 enzyme, revealing an insertion at the C-terminal region of the CARF domain, which plays a key role closing the catalytic site upon substrate binding. Our analysis reveals the key residues of Sis0455 during cleavage and the coupling of the active site closing with their positioning to proceed with cA4 phosphodiester hydrolysis. A time course comparison of cA4 cleavage between the short, Sis0455, and long ring nucleases, Sis0811, shows the slower cleavage kinetics of the former, suggesting that the combination of these two types of enzymes with the same function in a genome could be an evolutionary strategy to regulate the levels of the second messenger in different infection scenarios. | Molecular basis of cyclic tetra-oligoadenylate processing by small standalone CRISPR-Cas ring nucleases
Standalone ring nucleases are CRISPR ancillary proteins, which downregulate the immune response of Type III CRISPR-Cas systems by cleaving cyclic oligoadenylates (cA) second messengers. Two genes with this function have been found within the Sulfolobus islandicus (Sis) genome. They code for a long polypeptide composed by a CARF domain fused to an HTH domain and a short polypeptide constituted by a CARF domain with a 40 residue C-terminal insertion. Here, we determine the structure of the apo and substrate bound states of the Sis0455 enzyme, revealing an insertion at the C-terminal region of the CARF domain, which plays a key role closing the catalytic site upon substrate binding. Our analysis reveals the key residues of Sis0455 during cleavage and the coupling of the active site closing with their positioning to proceed with cA4 phosphodiester hydrolysis. A time course comparison of cA4 cleavage between the short, Sis0455, and long ring nucleases, Sis0811, shows the slower cleavage kinetics of the former, suggesting that the combination of these two types of enzymes with the same function in a genome could be an evolutionary strategy to regulate the levels of the second messenger in different infection scenarios.
The discovery of an adaptive prokaryotic immune system called Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR), in which the repeats associate with Cas (CRISPR associated) proteins, has constituted a revolution in life sciences. Their discovery (1–3)- and straightforward development into versatile nucleases (4–6) by guide RNA exchange paved the way for modifications à la carte that can be employed in biomedicine (7) and biotechnology (8–10). CRISPR-Cas systems are ribonucleoprotein (RNP) complexes that are highly diverse because of their different evolutionary origins. The CRISPR immune response consists of three stages mediated by a distinct subset of Cas proteins involving adaptation, CRISPR (cr) RNA maturation and interference, concluding with the recognition and cleavage of the target DNA or RNA (10–14). The CRISPR-Cas systems are divided into two classes according to the Cas proteins composing the nuclease module (15). Class 1 interference modules consist of a multi-subunit protein complex, while Class 2 modules comprise a single multidomain protein. The two classes are further divided into six types and many subtypes depending on which other Cas proteins are present in other functional modules. The Class 1 Type III constitutes a complex CRISPR immune system of particular interest, as its members deploy an intricated response controlled by a multipronged regulatory pathway to degrade both the mRNA and DNA of the invader (16–18). The large Type III interference complexes are characterized by the presence of the multidomain Cas10 signature protein, which commonly harbours two active sites for ssDNA cleavage (18–21) and a cyclase domain for cyclic oligoadenylate (cA) synthesis (22–24). The recognition of a target RNA triggers the catalytic activities of Cas10, and the cyclase domain polymerises ATP into cA species ranging between 3- and 6-AMP subunits (cAn) (22–25). These molecules act as a second messenger promoting the activation of CRISPR ancillary nucleases (Csx1/Csm6, Can1/Can2 and NucC families), which are the peons of the Type III immune response (22,23,26–28), degrading both host and invading nucleic acids in the cell, resulting in viral clearance, cell dormancy or cell death (29). These enzymes are key components of cyclic oligonucleotide-based antiphage signalling systems (CBASS). They contain CRISPR Associated Rossmann Fold (CARF) domains, which are involved in ligand binding. The CARF superfamily includes numerous domain architectures, in most of which the CARF domain is fused to a nuclease (30). The control of cAn levels require a regulatory system that can modulate or stop the activity of the indiscriminate nucleases. Although the CARF domains of some Csm6 proteins have been shown to slowly degrade cA4 or cA6, thereby self-limiting their ribonuclease activity (31–34), a major part of Type III systems includes standalone cyclic oligoadenylate degrading enzymes, also termed CRISPR ring nucleases (Crn), to fulfil this function. The first two members of this family were described in Sulpholobus solfataricus (Sso), where the Sso1393 (UniProt ID: Q97YD2) and Sso2081 (UniProt ID: Q7LYJ6) proteins, were shown to bind and degrade cA4 using a metal-independent mechanism, thus returning cells to a basal uninfected state (31). The same function has been assigned to the Sulfolobus islandicus (Sis) Sis0811 (UniProt ID: F0NH89) and Sis0455 (UniProt ID: F0NGX6) enzymes (32,35). These cyclic oligoadenylate degrading enzymes are CARF-domain proteins of the Crn1 family. From the structural point of view, two types of enzymes can be observed in this family. One of them includes a HTH domain joined to a CARF domain that belongs to the CARF7 major clade (36) (Sis0811 and Sso1393, Crn1 Large Standalone Ring Nucleases, LSRN), while the second one displays a small insertion in the C-terminal region of the CARF domain and belongs to the CARF_m13 minor clade (Sis0455 and Sso2081, Crn1 Small Standalone Ring Nucleases, SSRN) (Supplementary Figure S1). We have recently deciphered the catalytic mechanism of Sis0811 (37). However, no structural or mechanistic details are available for Sis0455, thus precluding our understanding of the SSRN group. Here, we determine the structure of Sis0455 in its apo and substrate-bound form. The structure reveals the dimeric assembly of the enzyme and the unique structural feature of the insertion in the C-terminus of the polypeptide. The substrate-bound conformation unveils the molecular details of cA4 recognition by the standalone ring nuclease. We observe that cA4 binding induces a conformational change from an open to a closed state. The conformational change cages the second messenger and avoids its dissociation by closing the catalytic site with the insertion located at the C-terminal of the CARF domains. Our time course mass spectrometry and binding experiments show that Sis0455 possesses a very high affinity for the substrate and suggest a slower catalytic mechanism than Sis0811. Our study provides the first structural insight into the substrate recognition and processing of this type of standalone ring nucleases, which together with the other ancillary proteins, regulate the Type III CRISPR defence system.
Sis0455 wild type sequence from Sis REY15A was synthesized by the Integrated DNA Technology (IDT, USA). The gene was then cloned by In-Fusion HD Cloning Plus (Tanaka) into pET-21 with a C-terminal extension encoding a TEV (Tobacco Etch Virus) protease target site and a 6× His-tag (histidine tag). The different mutants employed for the biochemical characterization of the enzyme were derived from this plasmid by site-directed mutagenesis carried out by the genomics service company Genewiz.
His-tagged Sis0455 and all its variants were expressed and purified from Escherichia coli BL21 pRARE cells. Cells were grown in LB media containing ampicillin (1 mM) and chloramphenicol (34 μg/ml) at 37 °C until an OD at 600nm wavelength of 0.6 was reached. Expression was induced by adding 0.5 mM of isopropyl β-d-1-thiogalactopyranoside (IPTG) at 37°C during 3h. The cells were harvested and re-suspended in lysis buffer (50 mM HEPES pH 7.5, 2 M NaCl, 5 mM MgCl2) in a ratio of about 10 ml buffer/1g cells supplemented with 1 protease inhibitor tablet (Roche Diagnostics GmbH), lysozyme and 1 μl Benzonase. Cells were lysed by sonication for 8 min at 35% amplitude with 15 seconds on and 20 s off cycle and then cell debris and insoluble particles were removed by centrifugation at 11 000 rpm for 45 min at 4 °C (Thermo Fisher Scientific, Multifuge X Pro). The supernatant was separated from the pellet and diluted to 500 mM NaCl (Dilution buffer: 50 mM HEPES pH 7.5, 5 mM MgCl2). This sample was then loaded onto a 5 ml Crude HisTrap FF column (GE Healthcare) equilibrated in buffer A (50 mM HEPES pH 7.5, 5 mM MgCl2, 500 mM NaCl). Elution of the proteins was performed by a stepwise gradient of buffer B (50 mM HEPES pH 7.5, 5 mM MgCl2, 500 mM NaCl, 500 mM Imidazole). Enriched protein fractions were applied onto a 5 ml HiTrap Q HP column (GE Healthcare) equilibrated with buffer A2 (20 mM Tris–HCl pH 8.0, 50 mM NaCl). The protein was eluted with a linear gradient of 0–100% buffer B2 (20 mM Tris–HCl pH 8.0, 1M NaCl). Protein-rich fractions were loaded onto a HiLoad 16/600 75 Superdex column (GE Healthcare) equilibrated in gel filtration buffer GF (25 mM HEPES pH 8.0, 300 mM KCl). The protein fractions were concentrated to ∼12 mg/ml (using 10 kDa MWCO Centriprep Amicon Ultra devices) and aliquots were flash-frozen in liquid nitrogen and subsequently stored at -80°C. Following the expression and purification protocol described above but growing cells in a Selenomethionine-enriched media, Selenomethionine-labeled Sis0455 protein was also purified for further crystallization experiments. Samples purity were monitored by SDS-PAGE gels.
SEC-MALS experiments were performed using a Dionex (Thermo Scientific) HPLC system connected in-line to a UV detector (Thermo Scientific Dionex Ultimate 3000, MWD-3000), a Wyatt Dawn8 + Heleos 8-angle light-scattering detector and a Wyatt Optilab T-rEX refractive index detector. SEC was performed using a Superdex 200 Increase 10/300 GL column (GE Healthcare) at 20°C in a buffer containing 50 mM HEPES pH 8.0, 300 mM KCl. For the analysis, 50 μl of Sis0455 were injected at 5.0 mg/ml and 0.5 ml/min flow rate. ASTRA (version 8.0.2.5) software was used to collect the data from the UV, refractive index, and light scattering detectors. The weight average molecular masses, Mw, were determined across the elution profile from static LS measurements using ASTRA software and a Zimm model, which relates the amount of scattered light to the weight average molecular weight of the solute, the concentration of the sample, and the square of the refractive index increment (dn/dc) of the sample.
Initial crystallization screenings with Sis0455 sample were performed at 293 K using the sitting-drop vapor-diffusion method and testing a collection of commercially available crystallization screens. The initial drops consisted of 0.15 μl of protein solution (13.54 mg/ml in 25 mM HEPES pH 8.0, and 300 mM KCl) and 0.15 μl well solution, and were equilibrated against 70 μl of well solution. After 60 days, the extensive initial screening only rendered plate-like crystals in 25% PEG 4000 and 0.05 M tricine pH 8.0. These crystals were subsequently scaled up and optimized using a dragonfly (TTP) screen optimizer yielding plate-like crystals grown in 22% PEG 4000 and 0.05 M tricine pH 8.0. Crystals were cryo-protected by adding 20% (v/v) glycerol to the mother liquor before flash-freezing in liquid nitrogen. Since selenomethionine-modified crystals were not reproducible under the native conditions mentioned above, initial crystallization screenings were performed from the scratch using the selenomethionine sample previously incubated with cA4 substrate before setting up the crystallization trials. After optimizing initial hits found in reservoir conditions based on 40% PEG 400, best diffracting crystals were grown in 41% PEG 400, 0.1M sodium acetate pH 4.5, 0.1M Li2SO4. As crystals were grown in cryo-conditions, they were directly flash-freeze in liquid nitrogen.
All data were collected from frozen crystals at 100 K with EIGER and PILATUS detectors at beamlines PXI and PXIII (SLS, Villigen, Switzerland) and at BioMax (MAX-IV, Lund, Sweden). Data processing and scaling were accomplished using XDS (38), POINTLESS and AIMLESS (39) as implemented in autoPROC (40). Statistics for the crystallographic data and structure solution are summarized in Table 1.
Sis0455:cA4 complex structure was solved from selenomethionine-modified crystals by experimental phasing using single-wavelength anomalous diffraction (SAD) method, as implemented in the program CRANK2 (41). Then, using a monomer from the Sis0455:cA4 complex as a searching model, Sis0455 apo structure was solved by molecular replacement method, as implemented in the program PHASER (42). The quality of the electron density maps provided from this molecular replacement solution allowed us to manually retrace the full model of Sis0455 apo structure. Both models were subjected to iterative cycles of model building and refinement with COOT (43), PHENIX (44) and REFMAC (45) yielding the refinement and data collection statistics summarized in the Table 1. The apo and complex final models have a Rwork/Rfree of 18/25 and 21/25 with 0.00 and 0.27% of the residues in disallowed regions of the Ramachandran plot, respectively. Figures were generated using PyMOL (The PyMOL Molecular Graphics System, Version 2.0 Schrodinger, LLC) and ChimeraX (46,47).
Cyclic tetraadenylate (cA4; catalog number, C355) was acquired from BIOLOG Life Science Institute (Bremen, Germany). Prior to ITC experiments both the proteins (Sis0455 and its variants) and the cA4 were extensively dialyzed against ITC buffer (50 mM HEPES pH 8.0, 300 mM KCl). Protein concentrations were determined using a spectrophotometer by measuring the absorbance at 280 nm and applying values for the extinction coefficients computed from the corresponding sequences by the ProtParam program (http://web.expasy.org/protparam/). The cA4 concentration was determined as well by measuring the absorbance at 260 nm and using a extinction coefficient of 54000 M-1 cm-1. cA4 at approximately 50 or 100 μM concentration was loaded into the syringe and titrated into the calorimetric cell containing the Sis0455 proteins at ∼5 or 10 μM, respectively. The reference cell was filled with distilled water. All ITC experiments were performed on an Auto-iTC200 instrument (Microcal, Malvern Instruments Ltd) at 25°C. The titration sequence consisted of a single 0.4 μl injection followed by 19 injections, 2 μl each, with 150 s spacing between injections to ensure that the thermal power returns to the baseline before the next injection. The stirring speed was 750 rpm. Control experiments with the cyclic tetra-adenylate injected in the sample cell filled with buffer were carried out under the same experimental conditions. These control experiments showed heats of dilution negligible in all cases. The heats per injection normalized per mole of injectant versus the molar ratio [cA4]/[Sis0455 variants] were fitted to a single-site model. Data were analysed with MicroCal PEAQ-ITC (version 1.1.0.1262) analysis software (Malvern Instruments Ltd).
Reactions of 50 μl containing 40 μM cA4, 2 μM Sis0455 in reaction buffer (20 mM Tris pH 8.0, 100 mM NaCl, 1 mM EDTA) were set and incubated at 70°C for one hour in a thermocycler. As a control, a reaction without any enzyme was also set. After this time, the reactions were stopped by lowering the temperature up to 4°C. Then the protein from each of the reactions was removed by performing a phenol extraction. This step was achieved by adding 50 μl of phenol to each of the reactions and followed by vortexing until the solution turns cloudy. Then the organic and aqueous phases were separated by centrifugation (60 s at 7000 rpm) and the aqueous phase dried down in a SpeedVac. The pellet containing the cyclic oligoadenylate compounds was resuspended in 8.6 mM TEA pH 8.3 (LC-MS buffer A) for further analysis. For the time course experiments, an initial larger volume of reaction was used and the reaction stopped at different time points (0, 2, 5, 10, 20, 30, 40, 50 and 60 min) and processed as above. All experiments were done, at least, in duplicates. The solutions containing the cyclic oligoadenylate compounds were injected into a UPLC system (UltiMate 3000, Dionex) using a Kinetex® EVO C18 reverse phase column (Phenomenex, 2.1 mm × 100 mm, 5.0 m particle size) at 30°C. The elution of the column was then analysed with a microOTOF-Q II mass spectrometer (Bruker Daltonik GmhH) equipped with an electrospray ionization (ESI) source (capillary voltage 4500 V, end plate offset –500 V, nebulizer gas (nitrogen) pressure 2.0 bar, flow 9 l/min and drying gas temperature 200°C). The mobile phase of the chromatography was applied at a flow rate of 0.2 mL/min and consisted of a gradient of Buffer A (8.6 mM TEA pH 8.3) and B (8.6 mM TEA pH 8.3, 5% acetonitrile) as follows: 0–4 min 0% B, 4–6 min 100% B, 6–8 min 0% B. Mass data acquisition was performed in negative-ion mode with a mass resolving power of 10 000 and a scan range of 300–1500. Data acquisition was done under the control of the module Hystar 3.2-SR 2 from Bruker Compass 1.3 software that integrates both the LC chromatographic separation and MS methods. Data analysis was done with DataAnalysis Version 4.0 SP5 (Bruker Daltonik GmhH). For the time courses series, the areas under the respective peaks were integrated using QuantAnalysis Version 2.0 SP5 (Bruker Daltonik GmbH).
Sis0455 is composed of a CRISPR Associated Rossmann Fold (CARF) domain (residues 1–178), which contains a unique insertion fragment at the C-terminal (Ct) region (residues 130–170) (Figure 1A, Supplementary Figure S1). After optimising initial crystallization hits from high throughput commercial screenings, the isolated Sis0455 protein (Supplementary Figure S2) yielded high-quality crystals that diffracted up to 2.27 Å resolution (Materials and Methods, Table 1). Molecular replacement trials using Sis0811 or other CARF domains as models failed in solving the phase problem. Therefore, we prepared a selenomethionine-modified sample to solve the phase problem using the SAD method. Selenium derivative crystals were obtained only in the presence of cA4. These crystals diffracted to 1.67 Å resolution (Materials & Methods, Table 1) and provided a high anomalous signal allowing the determination of the structure. The resulting electron density map permitted the unambiguous building of the Sis0455:cA4 structure (PDB code: 7Z55) (Figure 1B, Supplementary Figure S3a, b). Finally, the Sis0455 apo structure was solved by molecular replacement using this model as a searching model with the native protein data set (PDB code: 7Z56) (Figure 1C, Supplementary Figure 3Sc, d). Two monomers of Sis0455 form a dimer within the asymmetric unit (Figure 1B, C, Supplementary Figure S3c, d), in agreement with the molar mass, observed in solution in SEC-MALS experiments (Supplementary Figure S2b, Materials and Methods). The CARF domains shape the catalytic pocket at the interface of the dimer assembly, displaying the Ct insertion fragments oppositely oriented along the 2-fold dimer axis (Figure 1B, C). Hence, the 2-fold axis bisects the cA4 catalytic pocket and the Ct insertion fragments. The dimeric assembly is stabilized by the interaction between the α4 helix of each monomer (Supplementary Figure S1), and interactions between the α3 and β6 segments. The core of the CARF domain consists of a 6-stranded Rossmann-like fold, as in other related proteins (15), but unlike in other CARFs the core β5 and β6 strands do not form a ß-hairpin, as the Ct insertion fragment is located between them, adding the α5, η1, α 6 and η2 helices (Supplementary Figure S1). In addition, two disulphide bridges (C55–C155 and C56–C164) interconnect the CARF domain with the Ct insertion stabilizing the folding of each monomer (Supplementary Figure S3e). Sequence and structural alignments with other standalone ring nucleases, especially with its ‘cousin’ Sis0811 (PDB code: 7PQ2), reveal the conservation of K106 and S11 in Sis0455 (Figure 1D, Supplementary Figure S1). These residues have been proposed to play key roles in the enzymatic activity of these nucleases (32,37). The equivalent K169 and S12 in Sis0811 display a similar configuration in the apo structure (Figure 1D, Supplementary Figure S3c, d). As previously observed in Sis0811 and Sso1393, the conserved lysine residues are present at the very bottom of the catalytic pocket, while the conserved serine residues are located on each side of the active site (Supplementary Figure S3c, d). However, in Sis0455 the cavity is more positively charged by the presence of R105, which is exclusive of this type of ring nucleases (Supplementary Figures S1 and S3d).
To understand cA4 binding and degradation mechanism, we determined and analysed the structure of Sis0455 in complex with its substrate cA4 (Figure 1C, Supplementary Figure S3a, b, Table 1). The quality of the 2FoFc and omit electron density maps unambiguously revealed the presence of the cyclic oligoadenylate inside the catalytic pocket in a non-processed state (Figure 2A–C, Supplementary Figure S3a, b). The structure revealed that the substrate is trapped in the catalytic pocket isolating the cA4 molecule from the solvent (Figures 1B and 2, Supplementary Figure S3a, b). The structure revealed a closed state conformation of the enzyme, which is stabilized by interactions between the α5 helix in the Ct region of each protomer through polar interactions between N138-N138’, Q130-Y133’/Q130’-Y133 and E41-R134’ (Figure 2D, Supplementary Figure S3a, b). The cA4 is strongly stabilized in the catalytic pocket via a large interaction network (Figure 2C, Supplementary Figure S4), which involves polar interactions of the phosphate groups with S11-S11’ and Y133-Y133’, and the 2’OH group of the riboses with the T10–T10’ in each protomer. In addition, the cyclic molecule is also associated with R105–R105’ via water-mediated interactions, and polar interactions of the bases with T37–T37’ and E17–E17’. Furthermore, the bases are also stabilized through hydrophobic interactions with V42–V42’ and I128–I128’. As observed in other ring nucleases (32,37), the strictly conserved lysine residue (K106–K106’) at the bottom of the catalytic pocket generates the electropositive environment to accommodate the phosphate groups of cA4. Noteworthy is the conformation of the D75–D75’ residues in the active site, which seem to position the R105–R105’ to favour their interaction with the phosphate groups of the cyclic molecule (Figure 2C, Supplementary Figures S3a, b and S4).
The binding of cA4 in the cataytic pocket induces a large conformational change in the dimeric assembly (Figure 3, Supplementary Video S1). The two monomers undergo a jaw-like movement confining the substrate in the catalytic pocket. The Ct region of each protomer forms the closure of the shared catalytic pocket, while the CARF domains fold inwards, trapping and isolating the substrate into the positively charged active site. The extent of this conformational movement is reflected in a RMSD of 4.42 Å for the 325 Cα between the apo and ligand bound structures, which buries a surface of 1450 Å2. The key residues in the catalytic pocket undergo a conformational change between the apo and cA4 bound-state (Supplementary Figure S3a–d). The K106 and K106’ in Sis0455 change their arrangement to accommodate the cyclic substrate molecule, while S11 and S11’ residues move their Cα ∼5 Å to interact with cA4. These strictly conserved residues are supposed to be key during catalysis by homology with Sis0811 and Sso1393 nucleases (Supplementary Figure S1). Based in this comparison, the lysines would play an important role in the reaction intermediate stabilization, while the serines would be involved in the stabilization of the reaction product (37). However, the Sis0455:cA4 complex structure revealed a different cA4 recognition pattern, suggesting aditional residues playing important roles in the cleavage reaction. For instance, R105 and R105’ establish strong water-mediated interactions with the 2′OH of the riboses, thus positioning the substrate for the catalytic reaction (Figure 2C, Supplementary Figure S3a, b). Interestingly, the rotamer of these arginine residues in that substrate-interacting conformation is arranged by a salt-bridge interaction with D75, suggesting an indirect role in catalysis of the aspartic acid, as in the apo structure no interaction is observed between D75 and R105. The presence of the substrate induces a conformational change where both aspartic residues drive the arginines to configurate the pre-catalytic complex (Figure 2C, Supplementary Figure S3a, b).
To understand the role of the amino acids involved in cA4 binding, we performed isothermal titration calorimetry (ITC) binding assays with the wild type and mutants. The Sis0455 ring nuclease binds cA4 with a very high affinity (KD of 2.2 nM), which is almost five-fold higher than Sis0811 (KD 9.1 nM) (37) (Figure 4A). Interestingly, unlike Sis0811, which displayed an exothermic binding of cA4, the association of the cyclic molecule by Sis0455 is endothermic, suggesting that the conformational jaw movement observed upon ligand binding contributes to a solvent entropy change (Figure 3). The extensive protein surface burial to trap the substrate should result in solvent release in the polar active site upon cA4 binding, thus contributing favourably to the association. Based in the structural data and the sequence conservation, we selected key residues to perform glycine substitutions to examine the effect of the absence of the side chains in cA4 binding. The S11G and K106G substitutions diminished cA4 binding. The S11G variant displayed a decrease in the binding affinity of ∼10 fold (KD of 19.5 nM), while the binding of the K106G mutant severely affected the association with the ligand (KD of 4.1 μM) (Figure 4a), thus highlighting the importance of the lysine residues in cA4 binding. Unlike Sis0455, the K106G mutant displayed an exothermic binding profile, in agreement with the reduced polarity in the active site due to the lack of the lysine amino groups. The R105G as well as the D75G mutations abolished cA4 binding (Figure 4A, Supplementary Figure S5a), thus supporting the proposed role of D75 positioning R105, and confirming the key role of the arginine in ligand binding (Supplementary Figure S4a). The T10G mutant, which affects the 2’OH stabilization of the ribose, displays a substantial reduction of the cA4 binding affinity (KD of 35.3 nM), but still associates with the ligand. Mutations in the residues interacting with the bases, such as E17G/T37G, do not affect binding substantially (KD of 3.5 nM) (Supplementary Figure S5a). We also evaluated the effect of mutations in the α5 interacting helices of the Ct insertion in the single N138G, and double E41G/R134G and Q130G/Y133G mutants. Both N138G and E41G/R134G do not affect cA4 binding substantially (KD of 10.5 nM and 1.9 nM); however, the double mutant Q130G/Y133G severely affected the affinity of Sis0455 for the cyclic compound (KD of 2.6 μM). This double mutation destabilizes the closed state conformation by disturbing the interactions observed in the Ct insertion in the ligand bound structure (Figure 2D, Supplementary Figures S4 and S5a).
Next, we examined the effect of the mutants in the enzyme cA4 cleavage activity. We performed activity assays incubating the mutants with cA4 as in (37). The reaction mixture was analysed by mass spectrometry revealing the degradation products (Figure 4B, Supplementary Figure S5b, Materials and Methods). Five different species were detected: the intact cA4, P1 (ApA > P), P2 (hydrolysis product of P1, ApAp), P3 (intermediate reaction product, ApApApA > p) and P4 (hydrolysis of P3, ApApApAp). The assay confirmed that besides its important role in binding the conserved K106is essential for catalysis. The K106G substitution abolished the cleavage activity and did not generate any reaction product (Figure 4b). This result is similar to that observed in Sis0811(36), thus supporting its possible role as the key residue in stabilizing the transition state of the cleavage rection in both types of standalone ring nucleases. In addition, the R105G mutant also abrogated the nuclease activity confirming its important role in the reaction (Figure 4b). Therefore, both K106 and R105 are fundamental for cA4 degradation, however, the latter is only conserved in the SSRN group. By contrast, the conserved S11, which is present in both groups, does not seem to be critical in binding and catalysis for Sis0455, as it displayed a similar mass spectrometry pattern compared to the wild type (Figure 4B). This behaviour is different from that observed in the conserved S12 and S11 in Sis0811 and Sso1393, the standalone ring nucleases lacking the Ct insertion (32,37). Collectively, these observations suggest that while the role of the lysine residues is conserved between the two types of standalone ring nucleases, the serine does not have the relevance in catalysis which has been observed in the LSRN type. In addition, the important function of the conserved R105 in Sis0455 and Sso2081 is exclusive of the standalone ring nucleases including the Ct insertion. We also checked the effect on the enzyme activity of those amino acids that interact with cA4, either through the 2’OH of ribose (T10) or the base (E17/E37). In both cases, the substitution by glycine did not affect catalysis, the product profile of the reaction mixture was similar than the wild type (Supplementary Figure S5b). Finally, glycine substitutions in residues involved in the closing of the catalytic pocket in the Ct insertion were tested. The single N138G and double Q130G/Y133G substitutions did not affect catalysis (Supplementary Figure S5b). However, the E41G/R134G double mutant, which disturbs the polar interaction between the Ct insertions (Figure 2D), showed cA4 as major specie, also presenting P1 and the P3 linear products in a minor extent. Collectively, these observations indicate that the E41/R134 interaction is not relevant for binding the ligand, as its affinity is identical to the wild type. However, they seem important for stabilizing the closed state of the enzyme-substrate complex facilitating the configuration of the active site for catalysis.
The closest structural homologue of Sis0455 found by the DALI server (38) is the TsCard1:cA4 (48) complex (PDB code: 6WXX) (RMSD 2.7 Å, aligning 124 Cα out of 373 residues of TsCard1). The homology lies within the CARF domains of Card1 (UniProt Entry: F2NWD3) and Sis0455. The main difference arises from the Ct insertion in Sis0455, which includes the two extra helices (α5-α6) and the connecting loops, thus shaping different substrate-binding pockets from its dimeric assembly (Figure 5A, B). This suggests why the TsCard1-CARF dimer requires the action of residues in the Restriction Endonuclease (REase) domains to trap cA4 (48), while in the case of Sis0455 the Ct insertion closes the catalytic pocket. The superimpositon of the dimeric structures of Sis0455 and Sis0811 CARFs in complex with 2A2 (PDB code: 7PQ3) (RMSD: 2.2 Å) also shows that the catalytic pocket is not closed upon binding of the second messenger in Sis0811 (Figure 5C, D). The comparison of the active site of the cA4-bound structures show the conservation of the previuosly reported key residues for cA4 degradation in Sis0811 (S12 and K169) (37) (Figure 5E, F), which are also conserved in TsCard1 (S11 and K102). Although these key residues keep the same structural configuration in Sis0455, Sis0811 and TsCard1-CARF (Figure 5C), Sis0455 and Sis0811 degrade cA4, while TsCard1 does not cleave the cyclic ligand, suggesting differences in the active site. Interestingly, our activity assays (Figure 4B) and previous cleavage assays in Sso2081(32) show that the conserved R105, which is not present in TsCard1, is involved in the reaction (Figures 2C and 5E, F, Supplementary Figure S1). However, while the presence of this conserved arginine could justify the differences with SSRNs and TsCard1, LSRNs do not conserve this residue. On the other hand, TsCard1 conserves key residues involved in the bases recognition such as T39 (T37 in Sis0455 and T98 in Sis0811) and Y122 (Y133 in Sis0455 and Y191 in Sis0811) (Supplementary Figure S1). Hence, the contribution of other residues has to be taken into account to fully understand the molecular basis of cA4 recognition or cleavage. Collectively, the comparison of the active sites and biochemical properties indicate that a set of catalytic residues are conserved between the two groups of standalone ring nucleases; however, those containing the Ct insertion display extra residues which reshape the properties of the cleavage reaction.
In order to compare the degradation of cA4 by both ring nucleases from S.islandicus, we performed a time course experiment monitoring the reaction products. The cleavage reaction was stopped at different times, and the comsumption of cA4 and the generation of the linear P3 and P1 dinucleotide products were quantified at different times (Figure 6). Sis0455 and Sis0811 fully degrade cA4 producing 100% P1 product in 50 min. The overall view of the reaction suggest that both enzymes cleave one bond to generate the linear product before the second phosphodiester is hydrolyzed to produce the P1 product. However, the kinetics followed by these enzymes to cleave the two phosphodiester bonds is different. While the hydrolysis of the first phosphodiester is faster for Sis0811, converting almost 100% of the cyclic molecule in the P3 linear product in 20 min, the Sis0455 enzyme takes double time. Then Sis0811 employs 30 min to cleave the second phosphodiester bond converting all the linear product in the P1 dinucleotide. The reaction follows an overall similar scheme in Sis0455; however, the clevage of both phosphodiesters is slower in order to acomplish the complete degradation of the cyclic molecule in the same time. Therefore, Sis0455 linearizes cA4 slowly and follows a more progressive generation of P1 compared to Sis0811. The functional outcome of these different processing kinetics is unknown so far.
Our structure-function study provides experimental evidence of the catalytic mechanism of a member of the SSRN family (Figure 7). We propose that the cleavage reaction in Sis0455 will be initated by the 2’OH of the ribose, which would initiate the nucleophilic attack on the phosphate. Then, K106, which is conserved in the LSRN and SSRN enzymes, would stabilize the pentacovalent phosphorous formed in the transition state. The 2’–3’cyclic phosphate could be stabilized either by the S11 or T10 hydroxyl groups. These stages of the reaction are similar between the two groups of standalone ring nucleases. The structure of the Sis0455:cA4 complex suggest that T10 will favour the positioning of the 2’OH of the ribose for the nucleophilic attack. The cyclic 2’,3’-cyclic phosphate can be destabilized and disrupted over time (49), as it has been observed in our assays where some P2 product (ApAp) can be detected (Figure 4B and Supplementary Figure S5b). In addition, our analysis of the reaction products suggest that the cleavage reaction of the phosphodiester bonds does not occur in a concerted manner, as in the case of Sis0811 (37). The linear P3 product (ApApApA > p) was detected during our time course experiments (Figure 6), and the presence of the linear intermediate was also observed in a minor extent in the case of R105G and the K106G mutants. In addition, the abundance of P3 was increased for the E41G/R134G mutant, suggesting that disrupting the proper caging the compound in the active site would disturb the processing of the second phosphodiester; therefore, affecting the conversion of the linear P3 product into the P1 dinucleotide. Collectively, the data indicate that overall, the catalytic mechanism in the LSRN and the SSRN enzymes in S. islandicus is very similar. However, the processing of the first phosphodiester hydrolysis is faster in Sis0811 than in Sis0455, thus quickly removing the cyclic compound. These differences between the two enzymes in the cleavage reaction could arise from the requirement of the D75 to position R105 properly for triggering cA4 degradation.
A major part of the characterized Type III CRISPR-Cas systems utilizes cA4 as second messenger to trigger their immune response, while some other systems use a cyclic hexa-oligoadenylate (cA6) instead (23,25). The CARF domain architecture of the auxiliary proteins of each system is designed to specifically recognize one of these signals (33,35,48). The most common CARF family proteins are the Csx1/Csm6 ribonucleases, which are activated by their association with cA4 or cA6. These indiscriminate nucleases cleave RNA using their Higher Eukaryotes and Prokaryotes Nucleotide-binding (HEPN) domain (22,23), unleashing an indiscriminate RNase activity, which has been shown to result in cell growth arrest. Cellular growth is reestablished when infection is cleared from cells (29). A possible transcriptional role of cA4 has been suggested, as it was shown to bind to the CARF family transcription regulator Csa3, upregulating CRISPR loci and cas gene expression (50,51). The regulation of the indiscriminate degradation of cellular DNA and RNA by the cA activated Csx1/Csm6 nucleases is a crucial issue for the cell, as the activity of the indiscriminate nucleases confers Type III CRISPR systems a potential for self-destruction. The strategy of host nucleic acid degradation induces cell dormancy to slow viral propagation, but it can seriously affect cell survival if it is not strictly regulated. To address this problem the cell can switch off the production of the second messenger, however, this strategy will not eliminate the previously synthesized cA, which will remain residing in the cytoplasm. Therefore, the degradation of the signalling molecule is needed to downregulate the levels of the second messenger to switch off the Csx1/Csm6 nucleases. The development of such a regulatory strategy led prokaryotes to develop enzymes, such as the standalone ring nucleases, that cleave the phosphodiester bonds of the cyclic compound. Noteworthy, phages have also developed countermeasures, such as the degradation of these cyclic molecules (52,53), as strategies of immune evasion in phage biology to defeat host defence systems. The standalone ring nucleases are found in the crenarchaea, and the Sulfolobales usually encode several standalone ring nucleases orthologues alongside Csx1/Csm6 (54). Sso2081 and Sso1393 were the first members of the LSRN and SSRN identified in cellular lysates (32). Protein sequence analyses suggested that these orthologues most likely originated from gene duplication events (55). The genome of S. islandicus REY15A encodes two standalone ring nucleases, Sis0811 and Sis0455, which degrade the cA4 second messenger made by its Type III-B CRISPR system. Our Sis0455 structures reveals the jaw-like conformational change in the enzyme to accommodate the cyclic tetra-oligoadenylate in the active site, resembling the cA4 trapping mechanism described for the anti-CRISPR viral ring nuclease AcrIII-1 (52). The structural data together with our binding and activity experiments show that Sis0455 follows a mechanism similar to Sis0811 to degrade cA4. The same reaction products were identified in our mass spectrometry assay. Furthermore, the binding assays reveal that Sis0455 displays 5-fold higher afinity for cA4 than Sis0811 (37). While both enzymes degrade the cyclic compound in a non-concerted manner, generating a linear intermediate before its conversion in dinucleotides, the reaction in Sis0455 proceeds in a more progressive way than in Sis0811 (Figure 6). Sis0455 follows a slower kinetics to cleave the first phosphodiester bond of the cyclic compound, while Sis0811 linearizes cA4 very fast. These results suggest that the catalysis of the small ring nuclease would allow a longer lifetime of the intact substrate and the intemediate linear product in the cytoplasm before they are converted to dinucleotides. By contrast, Sis0811 would ‘linearise’ the major part of cA4 much faster. Overall, this result suggests that the different kinetics of these enzymes could be an evolutionary response in S. islandicus to regulate the levels of cA4 in different cellular scenarios during infection. This difference in cA4 degradation have been also observed in the enzymes from S. solfataricus, where Sso2081, the homolog of Sis0455, processes cA4 10-fold faster than Sso1393, the homolog of Sis0811 (32). This may reflect the regulatory needs of a given Type III CRISPR system. Collectively, the data suggest that presence of the two orthologues is a regulatory strategy which will overlap temporally or synergistically the activity of both enzymes for the control of cA4 levels. We propose that Sis0455, which displays a remarkable high affinity for the second messenger (KD 2 nM) is performing a continuous surveillance of the cA4 levels in the cytoplasm, as it will bind and slowly degrade low levels of the compound which could be present in the cell. This mechanism could be kept resident in the cell, avoiding the deleterious effect of a very early activation of SisCsx1, whose affinity for cA4 is 10-fold lower (KD 20 nM) (35). However, large quantities of cOA could be synthesized even at low levels of infection (56), and in that situation Sis0455 could not be able to eliminate cA4, either by binding or degrading the ligand. In this scenario Sis0811, whose afinity (KD 9.9 nM) (37) is 2-fold higher than that of SisCsx1, would kick-in eliminating the cyclic compound faster to avoid an early activation of the indiscriminate RNase. Type III CRISPR-Cas systems have tailored a complex regulatory system to control their immune response, which could have deleterious implications for the cell. Evolution has combined auxiliary proteins with different binding affinities and degradation rates to adapt the intensity of the immune response in different infection scenarios. Further functional studies are needed to fully explore and confirm how the Type III CRISPR-Cas standalone nucleases coordinate their action with the effector complexes and indiscriminate RNases to modulate bacterial defence.
Atomic coordinates and structure factors have been deposited in the Protein Data Bank under the accession codes 7Z55 and 7Z56 accordingly. The rest of the data are available from the corresponding author upon reasonable request.
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PMC9638941 | 36243977 | Gi Eob Kim,So Yeon Lee,Nils Birkholz,Kotaro Kamata,Jae-Hee Jeong,Yeon-Gil Kim,Peter C Fineran,Hyun Ho Park | Molecular basis of dual anti-CRISPR and auto-regulatory functions of AcrIF24 | 16-10-2022 | Abstract CRISPR-Cas systems are adaptive immune systems in bacteria and archaea that provide resistance against phages and other mobile genetic elements. To fight against CRISPR-Cas systems, phages and archaeal viruses encode anti-CRISPR (Acr) proteins that inhibit CRISPR-Cas systems. The expression of acr genes is controlled by anti-CRISPR-associated (Aca) proteins encoded within acr-aca operons. AcrIF24 is a recently identified Acr that inhibits the type I-F CRISPR-Cas system. Interestingly, AcrIF24 was predicted to be a dual-function Acr and Aca. Here, we elucidated the crystal structure of AcrIF24 from Pseudomonas aeruginosa and identified its operator sequence within the regulated acr-aca operon promoter. The structure of AcrIF24 has a novel domain composition, with wing, head and body domains. The body domain is responsible for recognition of promoter DNA for Aca regulatory activity. We also revealed that AcrIF24 directly bound to type I-F Cascade, specifically to Cas7 via its head domain as part of its Acr mechanism. Our results provide new molecular insights into the mechanism of a dual functional Acr-Aca protein. | Molecular basis of dual anti-CRISPR and auto-regulatory functions of AcrIF24
CRISPR-Cas systems are adaptive immune systems in bacteria and archaea that provide resistance against phages and other mobile genetic elements. To fight against CRISPR-Cas systems, phages and archaeal viruses encode anti-CRISPR (Acr) proteins that inhibit CRISPR-Cas systems. The expression of acr genes is controlled by anti-CRISPR-associated (Aca) proteins encoded within acr-aca operons. AcrIF24 is a recently identified Acr that inhibits the type I-F CRISPR-Cas system. Interestingly, AcrIF24 was predicted to be a dual-function Acr and Aca. Here, we elucidated the crystal structure of AcrIF24 from Pseudomonas aeruginosa and identified its operator sequence within the regulated acr-aca operon promoter. The structure of AcrIF24 has a novel domain composition, with wing, head and body domains. The body domain is responsible for recognition of promoter DNA for Aca regulatory activity. We also revealed that AcrIF24 directly bound to type I-F Cascade, specifically to Cas7 via its head domain as part of its Acr mechanism. Our results provide new molecular insights into the mechanism of a dual functional Acr-Aca protein.
As a result of the battle between bacteria and their invaders, including bacteriophages (phages) and other mobile genetic elements (MGEs), bacteria have evolved diverse defense mechanisms (1). In response, phages have developed anti-defense systems that work to suppress bacterial defense (2). Clustered Regularly Interspaced Short Palindromic Repeats (CRISPRs) and CRISPR-associated proteins (Cas) form CRISPR-Cas systems that provide adaptive immunity against invading genetic material (3–6). CRISPR-Cas systems are adaptive since they ‘record’ memories of past infections within their CRISPR arrays to elicit a rapid immune response to subsequent infections (7). CRISPR-Cas systems function via three distinct stages: adaptation, expression and interference (8,9). In the adaptation stage, invading DNAs are processed and integrated as spacers into a CRISPR array within the bacterial genome. Next, during the expression stage, the host CRISPR array is transcribed and processed into small CRISPR RNAs (crRNAs). Finally, in the interference stage, crRNA-guided complexes recognize crRNA-complementary sequences in invaders and either act alone or recruit additional proteins to cleave DNA or RNA of the invader (9–11). Due to the ability of specific DNA cleavage by CRISPR-Cas systems, they have been used for gene editing and their application on disease treatments is being tested (12–14). The long evolutionary interaction between bacteria and phages has led to the diversification of CRISPR-Cas systems, which are currently grouped into two broad classes (class 1 and class 2) encompassing six types (type I to type VI) based on the CRISPR locus organization, the cas gene composition, and their mechanisms (15). The class 1 systems, including types I, III, and IV, employ multi-subunit Cas proteins for performing multiple functions, whereas class 2 systems, including types II, V, and VI, utilize a single multi-domain Cas protein containing all necessary activities (15). The type I CRISPR-Cas systems are the most abundant and widely distributed, and are classified into seven subtypes, I-A through I-G, according to their signature cas genes and composition of Cas components (15). Multiple Cas proteins in type I systems associate with crRNA to form a CRISPR-associated complex for antiviral defense (Cascade) that recognizes invader DNA and recruits Cas3, the nuclease responsible for destroying target DNA (11). To counteract these anti-phage immune systems, phages have evolved systems to aid in their evasion of these immune systems (2). One of the most well-characterized evasion strategies in phages is to encode anti-CRISPR proteins (Acrs) that can neutralize the host CRISPR-Cas system (16). Since the first Acrs were discovered in phages capable of blocking the type I-F CRISPR-Cas system of Pseudomonas aeruginosa (17), around one hundred Acrs have been discovered based on functional screening and bioinformatic analysis (18–20). Because Acr proteins frequently lack sequence homology and common structural motifs, they are classified based on the targeted CRISPR-Cas systems (16,18). The AcrIF family inhibits the type I-F CRISPR-Cas system. Since the first AcrIF proteins (AcrIF1–5) were identified (17), 19 additional AcrIF proteins (AcrIF6–24) were discovered (21–23). The AcrIF family has also been a major focus of efforts to elucidate the structures and mechanisms of Acr proteins. These studies showed that the AcrIF family blocks type I-F activity in three distinct ways. The most common strategy is to block target DNA recognition by Cascade by direct binding of the Acr to Cascade component proteins. AcrIF1 (24), AcrIF2 (25), AcrIF4 (26), AcrIF6 (27), AcrIF7 (26), AcrIF8 (27), AcrIF10 (28) and AcrIF14 (26) use this mechanism to inhibit the type I-F CRISPR-Cas system. The second strategy is to inhibit Cas3 by direct interaction with the Acr. Masking the active site of Cas3 by AcrIF3 inhibits Cas3 interactions to target DNA and Cas3 bound by AcrIF3 fails to cleave target DNA (29). The final characterized inhibition strategy of the AcrIF family is enzymatic activity, represented by AcrIF11. AcrIF11 has an ADP-ribosyltransferase activity which mediates the ADP-ribosylation of the Cascade complex to prevent target DNA binding (30). AcrIF24 is among the most recently identified members of the AcrIF family. Interestingly, genetic analysis suggested that AcrIF24 has dual function as an Acr and an anti-CRISPR-associated (Aca) protein (23). Aca proteins are transcriptional regulators which commonly inhibit the expression of acr genes by forming an acr-aca operon (31–34). To understand the molecular basis underlying the functional mechanisms of AcrIF24, we determined its crystal structure. Our structure complements and adds to an AcrIF24 structural study that was published while our manuscript was in preparation (35). Based on our structural, microbiological and biochemical studies, we demonstrate the working mechanism of AcrIF24 as both an Acr and an Aca protein.
Primer sequences used in this study are listed in Supplementary Table S1. The full-length acrIF24 gene (encoding residues 1–228) from a Pseudomonas aeruginosa prophage (GenBank accession: WP_043084540) (23) was synthesized by Bionics (Daejeon, Republic of Korea) and cloned into a pET21a plasmid vector (Novagen, Madison, WI, USA). The NdeI and XhoI restriction sites were used for cloning. The resulting recombinant construct was transformed into Escherichia coli BL21(DE3) competent cells that were further cultured at 37°C in 1 l of lysogeny broth (LB) containing 50 μg/ml kanamycin. When the optical density at 600 nm (OD600) reached around 0.8, the temperature was adjusted to 20°C, and 0.5 mM isopropyl β-d-1-thiogalactopyranoside (IPTG) was added for induction of acrIF24 expression. The induced cells were further cultured for 20 h in a shaking incubator at 20°C. The cultured cells were harvested by centrifugation at 2000 × g for 15 min at 4°C, resuspended in 25 ml lysis buffer (20 mM Tris–HCl pH 8.0 and 500 mM NaCl), and lysed by ultrasonication. The cell lysate and supernatant were separated by centrifugation at 10 000 × g for 30 min at 4°C. The collected supernatant was mixed with Ni-nitrilotriacetic acid (NTA) affinity resins for 3 h, and the mixture was loaded onto a gravity-flow column (Bio-Rad, Hercules, CA, USA). The resin in the column was washed with 50 ml of lysis buffer to wash out unbound proteins. After washing, 3 ml elution buffer (20 mM Tris–HCl pH 8.0, 500 mM NaCl and 250 mM imidazole) was added to the column to elute the Ni-NTA bound target protein from the resin. Eluted AcrIF24 was concentrated to 30 mg/ml and applied onto a Superdex 200 10/300 GL column (GE Healthcare, Waukesha, WI, USA) connected to an ÄKTA Explorer system (GE Healthcare), which had been pre-equilibrated with SEC buffer (20 mM Tris–HCl pH 8.0 and 150 mM NaCl) for polishing the protein sample by size-exclusion chromatography (SEC). The eluted peak fractions from SEC containing AcrIF24 were collected, pooled, and concentrated to 12 mg/ml for crystallization. The purity of the protein was visually assessed using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). For obtaining type I-F Cascade complex, the plasmids pCsy_complex (#89232) and pCRISPR_DMS3g24 (#89244) were purchased from Addgene (36,37) and co-transformed into E. coli BL21 (DE3) cells. The Cascade complex was purified using the same method used for AcrIF24 purification. To express and purify individual subunits of Cascade, the full-length cas5, cas6, cas7 and cas8 genes were obtained by PCR using the pCsy_Complex plasmid as template DNA. Each obtained gene was cloned into a pET21a plasmid vector using NdeI and XhoI restriction sites. The purification methods used for Cas5, Cas6, Cas7 and Cas8 were the same as for purification of AcrIF24. The sequence encoding the AcrIF24 head domain deletion mutant was synthesized by Bionics (Daejeon, Republic of Korea), cloned into a pET21a plasmid vector (Novagen) and purified using the same method as for AcrIF24. Cas2/3 protein was prepared by a previously described purification method using the Cas2/3 expression construct obtained from the laboratory of Yue Feng (35)
The hanging drop vapor diffusion method was used for the crystallization of AcrIF24. Crystal plates were incubated at 20°C. Initial crystals were obtained by equilibrating a mixture containing 1 μl of protein solution (12 mg/ml protein in SEC buffer) and 1 μl of a reservoir solution containing 24% (w/v) polyethylene glycol 3350 (PEG 3350) and 0.4 M ammonium chloride (NH4Cl) against 500 μl of reservoir solution. The crystallization conditions were further optimized and the best crystals were obtained by adding 50 mM sodium fluoride (NaF), after which crystals appeared within 38 days. A single crystal was selected and soaked in reservoir solution supplemented with 40% (v/v) glycerol for cryo-protection. X-ray diffraction data were collected at −178°C on the beamline BL-5C at the Pohang Accelerator Laboratory (Pohang, Korea). Data was processed using HKL2000 software (38).
The AcrIF24 structure was determined using the molecular replacement phasing method which was performed by the PHASER program in the PHENIX package (39). The predicted structural model generated by AlphaFold2 was used as a search model (40). The initial model was built automatically using AutoBuild from the PHENIX package, and further model building with refinement was performed using Coot (41) and phenix.refine (42). The structure quality and stereochemistry were validated using MolProbity (43). All structural figures were generated using PyMOL (44).
Site-directed mutagenesis was performed using a Quick-change kit (Stratagene, San Diego, CA, USA) according to the manufacturer's protocols. Mutations were then confirmed by sequencing. Primer sequences used for mutagenesis are listed in Supplementary Table S1. Mutant proteins were prepared using the same method described for wildtype protein purification above.
The absolute molecular weight of AcrIF24 in solution was measured using SEC-coupled multi-angle light scattering (SEC-MALS). The protein solution was loaded onto a Superdex 200 Increase 10/300 GL 24 ml column (GE Healthcare) pre-equilibrated with SEC buffer. The flow rate of the buffer was controlled to 0.4 ml/min, and SEC-MALS was performed at 20°C. A DAWN-TREOS MALS detector was connected to an ÄKTA Explorer system. The molecular weight of bovine serum albumin was measured as a reference value. Data were processed and assessed using ASTRA software.
The amino acid sequences of AcrIF24 across different species were analyzed using Clustal Omega (45).
Promoter elements upstream of acrIF23 (which forms an operon with acrIF24) were identified using BPROM (46) and by manual curation compared to established consensus sequences. Inverted repeat sequences were identified using the Repeat Finder plugin of Geneious Prime Version 2022 1.1 (https://www.geneious.com).
The gBlock PF5881 was used as a template for amplification of the acrIF23-acrIF24 promoter using primers PF138 and PF139, and of the acrIF24 gene using PF209 and PF210. Oligonucleotide sequences used in this study are listed in Supplementary Table S1. The promoter PCR product was cut with SpeI and NsiI and inserted into pPF1439, a template plasmid for eyfp reporter studies (47), digested with the same enzymes, yielding pPF2963. The gene PCR product was cut with SacI and SphI and inserted into pBAD30 digested with the same enzymes, yielding pPF2964. To generate expression constructs for AcrIF24 variants with single point mutations, PCRs of the entire pPF2964 plasmid were performed with the primer pairs PF6867 and PF6868 (W110K), PF6869 and PF6870 (K197W) or PF6871 and PF6872 (R207W). PCR products were gel-purified and digested with DpnI to degrade the template plasmid, then directly transformed into E. coli DH5α to generate the plasmids pPF3482, pPF3483 and pPF3484, respectively. To generate the construct for the AcrIF24 double-mutant (Y128K/Y217W), the gBlock PF6976 was inserted into pBAD30 via the SacI and SphI sites, yielding pPF3487. All new plasmid constructs were confirmed by Sanger sequencing.
Reporter assays were performed using the P. carotovorum derivative PCF425, which has a deletion of two restriction endonuclease genes (48). Strains containing different combinations of the reporter plasmid with the acrIF23-acrIF24 promoter (pPF2963) and the acrIF24 expression plasmid (pPF2964 for the wild type or pPF3482, pPF3483, pPF3484 or pPF3487 for the different mutants) or the corresponding empty vectors (pPF1439 and pBAD30, respectively) were grown in LB containing 100 μg/ml ampicillin, 25 μg/ml chloramphenicol, 0.1% (w/v) arabinose and 100 μM IPTG at 1,200 rpm at 25°C. After 18 h of growth, fluorescence of plasmid-encoded mCherry and eYFP was measured by flow cytometry using a BD LSRFortessa cell analyzer. Cells were first gated based on forward and side scatter and cells positive for mCherry fluorescence were detected using a 610/20-nm bandpass filter (detector gain 606 V) and further analysed for eYFP fluorescence using a 530/30-nm bandpass filter (detector gain 600 V). Results for a strain carrying two empty vectors (pPF1439 and pBAD30) were subtracted from all other results to account for background fluorescence.
Examination of the in vivo activity of AcrIF24 against a phage-targeting CRISPR-Cas system first required creating a resistant host with a phage-targeting spacer in a type I-F CRISPR array. To generate a vector to promote acquisition of spacers targeting phage ZF40 in the P. carotovorum derivative PCF425, a 2.4kb-fragment of an uncharacterized ZF40 gene (locus tag F396_gp65) was first amplified by PCR using the primers PF2910 and PF2911 with a ZF40 lysate as the template. The resulting product was inserted into pPF1123 (49) using the restriction enzymes KpnI and SacI, yielding the plasmid pPF1526. Next, the oligonucleotides PF2914 and PF2958 were annealed. The annealing product contains a protospacer targeted by the P. carotovorum RC5297 type I-F CRISPR–Cas system combined with a non-consensus protospacer-adjacent motif (5′-TG-3′) previously shown to elicit priming in a related strain (50). This annealing product, which has SphI and SpeI restriction overhangs, was inserted into pPF1526 cut with the same enzymes, resulting in the spacer acquisition plasmid pPF1527. For spacer acquisition via priming, strain PCF425 was transformed with pPF1527 by electroporation. Plasmid uptake was expected to trigger spacer acquisition due to the presence of the priming protospacer on pPF1527, with acquisition potentially occurring from the plasmid-born ZF40 gene fragment. Resulting transformants were grown for 24 h at 30°C in LB medium without antibiotic selection to allow plasmid loss and then plated on LB-agar plates containing 100 μM IPTG to allow selection against colonies producing mCherry, which is encoded on pPF1527. Of mCherry-negative colonies, arrays of the type I-F CRISPR-Cas system were screened for spacer acquisition using PF2969 and PF2970 (array 1) or PF2971 and PF2972 (array 2). Expanded arrays were sequenced to identify the origin of the newly acquired spacers. One of the resulting strains, named PCF835, which acquired one spacer targeting phage ZF40, was selected for further experiments.
To test the activity of AcrIF24 variants against the native type I-F CRISPR-Cas system of P. carotovorum RC5297, pBAD30 or a derived construct for production of an AcrIF24 variant (pPF2964 for the wild type or pPF3482, pPF3483, pPF3484 or pPF3487 for the different mutants) was electroporated into strain PCF425 (without CRISPR immunity against phage ZF40) or PCF835 (with type I-F CRISPR immunity against phage ZF40). Each strain was grown overnight in LB medium containing 0.2% (w/v) arabinose for induction. From these cultures, 100 μl were added to top agar and poured on LB-agar plates containing 0.2% (w/v) arabinose. After solidification, 5 μl spots of a tenfold serial dilution of a ZF40 variant with its native acrIF8–aca2 operon deleted were placed on the top agar. Plaque formation was examined after 16 h of incubation at 25°C.
Varying concentrations of purified wildtype or mutant AcrIF24 were pre-incubated with 20 ng of annealed long inverted repeat DNA (IR-L) or 800 ng of annealed short inverted repeat DNA (IR-S) in binding buffer (10 mM HEPES pH 7.5, 1 mM MgCl2 20 mM KCl, 1 mM tris(2-carboxyethyl)phosphine (TCEP), and 5% (v/v) glycerol in a final volume of 20 μl) for 30 min on ice. Prepared samples were then separated by gel electrophoresis at 100 V on a 10% native 0.5× TBE (Tris–borate–EDTA) polyacrylamide gel. After electrophoresis, gels were stained with SYBR Gold (Invitrogen, Waltham, MA, USA) and visualized according to the manufacturer's instructions. Annealed DNA oligos, IR-L and IR-S, were generated by mixing complementary oligonucleotides synthesized by Bionix (Seoul, Republic of Korea) in a 1:1 molar ratio in annealing buffer (10 mM Tris pH 7.5, 50 mM NaCl, and 1 mM EDTA), heating to 100°C for 3 min, and cooling to 25°C for 1 h.
Purified AcrIF24 (wildtype and various mutants) at a concentration of 20 μM were pre-incubated with 1.5 μg annealed oligo DNA (IR-L or IR-S) at 4°C for 60 min in a final volume of 20 μl SEC buffer. Agarose gels (6%) were prepared with agarose LE powder (Gold Biotechnology) using 0.5× TB buffer. Prepared agarose gel was run on a Mupid-2 plus electrophoresis kit (Advance, Japan) in 0.5× TB buffer for 30 min at 100 V.
Protein-DNA complex formation between AcrIF24 (wildtype or mutants) and annealed oligo DNA (IR-L and IR-S) was evaluated via native (non-denaturing) PAGE with 8∼25% acrylamide gels. Coomassie Brilliant Blue was used for staining and detection of shifted bands. DNA and protein were used at concentrations of 10 and 20 μM, respectively.
SEC was performed to analyze complex formation between AcrIF24 and type I-F Cascade. AcrIF24 was mixed with type I-F Cascade or each individual protein component of Cascade, incubated for 30 min at 25°C, and applied to a size-exclusion column (Superdex 200 HR 10/30, GE healthcare), which was pre-equilibrated with SEC buffer. The peak fractions were collected and subjected to SDS-PAGE. Coomassie Brilliant Blue was used for staining and analyzing the pattern of co-migrated bands.
To test the anti-CRISPR activity of wildtype AcrIF24 and its mutants (W110K, ΔHead, K197Y, R207W, and Y128K/Y217W), reactions were performed in a 10 μl buffer system containing 0.64 μM Cascade complex, 0.16 μM Cas2/3, 0.04 μM dsDNA, and 100–1000 nM AcrIF24 or its mutant proteins. First, we incubated AcrIF24 or its mutants with the type I-F Cascade complex at 37°C in reaction buffer (20 mM HEPES pH 7.5, 100 mM KCl, 5% (v/v) glycerol, and 1 mM DTT) for 30 min. Then we added target DNA to a final concentration 0.04 μM and incubated at 37°C for 30 min. Cas2/3 was further added along with reaction buffer (5 mM MgCl2, 75 μM NiSO4, 5 mM CaCl2 and 1 mM ATP) and the reaction was performed at 37°C for 1 h. We quenched the reaction by adding proteinase K and incubating for an additional 10 min at room temperature. The reaction products were separated by electrophoresis on 10% polyacrylamide gels and visualized by staining with SYBR GOLD.
To understand the molecular basis underlying AcrIF24 anti-CRISPR function, AcrIF24 was overexpressed in E. coli and purified using two-step chromatography, affinity chromatography and size-exclusion chromatography (SEC). During SEC, the protein eluted at bigger than ∼44 kDa from a Superdex 200 gel-filtration column, indicating that AcrIF24 (∼26 kDa for monomer) may exist as a dimer in solution (Figure 1A). The purified AcrIF24 protein sample was successfully crystallized and diffracted to 2.5 Å. Due to the absence of structural homologues in the PDB database, we were unable to solve the phasing problem by molecular replacement (MR) at the initial stage of structure determination. However, the phasing problem was solved by molecular replacement using a structural model predicted by alphafold2 (40). The final structural model of AcrIF24 was refined to Rwork = 21.66% and Rfree = 28.58%. The diffraction data and refinement statistics are summarized in Table 1. The crystal belongs to space group P6522 with one molecule present in the asymmetric unit. The final structural model contains the complete AcrIF24 sequence (residues M1 to S228). The overall shape of the AcrIF24 structure resembled a bird and was composed of three distinct domains. Based on the domain locations in the bird shape, we named them head, wing and body domain (Figure 1B). The wing domain was formed by five β-sheets (β1–β5) and one long α-helix (α1) at the N-terminal part of AcrIF24, while the body domain was composed of six α-helixes (α3-α8) forming a helical bundle fold at the C-terminus of AcrIF24 (Figure 1C). The head domain was formed by four β-sheets (β6–β9) and one α-helix (α2) was localized between the wing and body domains (Figure 1C). Because the electrostatic surface features are sometimes important for predicting the function of a protein, we analyzed the electrostatic surface features of AcrIF24. This analysis showed that AcrIF24 contained a highly positively charged cleft in the bottom part of the body domain, although negatively and positively charged surfaces were evenly dispersed in the AcrIF24 structure (Figure 1D). B-factor analysis showed that most of the head domain had relatively higher B-factors (average 88.25 Å2), indicating that the head domain might be flexible (Figure 1E). The α8 helix and connecting loop at the body domain also had relatively higher B-factors (average 62.37 Å2). Because flexible protein features can become rigid upon interaction with a specific binding partner, the flexible head domain might be critical for the protein interactions for the proper function of AcrIF24. To investigate the structural novelty of AcrIF24, structural homologues were searched using the DALI server (51). The closest related structure picked by this server was Aca1 (52), having a Z-score of 6.9 and 2.5 Å root mean square deviation (RMSD) when superimposing 68 amino acids among 73 total amino acids of Aca1 with 72 amino acids among 228 total amino acids of AcrIF24 (Table 2). The structure of Aca1 was only superposed with the body domain of AcrIF24 (Figure 1F). The sequence identity of Aca1 with the body domain of AcrIF24 was 22%. This search indicated that the structures of the wing and head domains of AcrIF24 are novel without significant similarity to previously described structures. Although the overall structure of the body domain of AcrIF24 is similar to the entire structure of Aca family, structural superposition indicated that the two structures are not identical, having a high RMSD value (2.5 Å).
Although various Acrs inhibit CRISPR-Cas activity in monomeric form, previous studies have shown that the dimeric form of Acrs is often critical for their activity (29,53,54). Similarly, some Aca proteins have been shown to function as dimers (31,33,55). Given the possible dimeric form of AcrIF24 as judged by our SEC experiment, we used multi-angle light scattering (MALS) to confirm the stoichiometry by determining the absolute molecular mass of AcrIF24 in solution. MALS showed that the experimental molecular mass was 55.76 kDa (1.86% fitting error) with 1.002 polydispersity (Figure 2A). Because the theoretically calculated molecular weight of monomeric AcrIF24 with the C-terminal histidine tag was 26.03 kDa, the molecular mass analyzed by MALS likely corresponds to dimeric AcrIF24. Based on these SEC and MALS data, we concluded that AcrIF24 forms a homo-dimer in solution. Crystallographic packing analysis showed that two types of putative dimers were detected: a MolA/Sym1 dimer or a MolA/Sym2 dimer. The MolA/Sym1 dimer was constructed via head and body domains from each molecule, while the MolA/Sym2 dimer was formed via the wing and body domains from one molecule and the head domain of another molecule (Figure 2B). To find a symmetric molecule that forms a dimer with monomeric molecule A found in the crystallographic asymmetric unit, the protein-protein interactions (PPI) in both the MolA/Sym1 dimer and the MolA/Sym2 dimer were further analyzed using the PDBePISA PPI-calculating server (Figure 2C) (56). PPI analysis showed that the complex formation significance score (CSS) of the MolA/Sym1 dimer was 0.2 (the score ranges from 0 to 1 as the relevance of the interface to complex formation increases), while that of the MolA/Sym2 dimer was 0, indicating that the MolA/Sym1 dimer might be the biologically relevant form. A total of 62 residues (31 from each molecule) were involved in the formation of PPI of the MolA/Sym1 dimer, whose total surface buried an area of 1078.3 Å2, representing 8.3% of the total surface area (Figure 2C and D). Meanwhile, 15 residues from MolA and 20 residues from Sym2 were involved in the formation of the MolA/Sym2 PPI, whose total surface buried an area of 571.1 Å2, representing 4.4% of the total surface (Figure 2C). The main forces used for the formation of the MolA/Sym1 dimer PPI were six hydrogen bonds (H-bonds) and two salt bridges, which were generated at two distinct regions, one in the body domains and another in the head domains. For maintaining the head domain-mediated PPI, salt bridges were formed in between S228 and R221 of each molecule and H-bonds were formed between residues A227 and R221 of each molecule (Figure 2D and E). For maintaining the body domain-mediated PPI, extensive H-bonds were generated by R88, Y128, T129, and N126 of each molecule (Figure 2D and F). In contrast to the MolA/Sym1 dimer, the MolA/Sym2 dimer was an asymmetric dimer that might be unlikely to form in the cellular environment. Therefore, we propose that AcrIF24 naturally occurs as a MolA/Sym1 dimer. To confirm our hypothesis that the MolA/Sym1 dimer might be a favoured dimer model, we generated three MolA/Sym1 PPI disruption mutants, R221W (head domain-mediated PPI disrupting mutant), T129W (wing domain-mediated PPI disrupting mutant), and a Y128K/Y217W double mutant (both PPI disrupting mutant) and performed SEC-MALS with those mutants. This experiment clearly showed that the Y128K/Y217W double mutant produced a peak at a delayed position during SEC compared with that of the wildtype. The Y128K/Y217W double mutant produced a smaller-sized particle, which is likely a monomer of AcrIF24, indicating that MolA/Sym1 represents the true dimeric state (Figure 2G and H).
The dimeric nature of AcrIF24, the presence of a predicted helix-turn-helix (HTH) DNA binding motif similar to some known Aca proteins (23), and the high structural similarity of the body domain of AcrIF24 with Aca1 analysed by DALI search followed by structural superimposition (Table 2 and Figure 1F), led us to hypothesize that AcrIF24 functions analogously to an Aca protein by binding DNA and altering gene expression. Analysis of AcrIF24 using ConSurf demonstrated that the base of the body domain was highly conserved and contained the putative HTH motif within the C-terminus of the protein (Figure 3A), spanning helices α6–α8 (Figure 3B). Although the amino acid sequences of the head and body domains were highly conserved, the N-terminal wing domain was not conserved. Because the putative HTH motif was found in the body domain, AcrIF24 might use the body domain to recognise the specific promoter sequence for the Aca activity. The dimeric nature of AcrIF24 suggested that it would likely recognise an inverted repeat (IR) sequence to control acr expression. In the P. aeruginosa prophage, acrIF24 is present as the second gene in an operon also including acrIF23. Therefore, we predicted that the entire acrIF23-acrIF24 operon would be regulated by AcrIF24. We examined the intergenic region upstream of acrIF23 for a predicted promoter using BPROM and manual curation, which revealed –35 and –10 regions consistent with a strong promoter (TTGCAT-N17-TATAGT) (Figure 3C). Positioned almost perfectly between the -35 and -10 elements of this promoter was an IR (TAGCTCGATTCGAGCTA) with two perfect 8 bp half-sites (underlined) separated by 1 bp (Figure 3C). To test whether AcrIF24 functions to regulate the acrIF23-acrIF24 operon, we made a transcriptional fusion of the acr operon promoter to eyfp. Expression of acrIF24 led to a reduction in eYFP fluorescence as assessed by flow cytometry, indicating that AcrIF24 functions as an Aca to repress acr operon expression (Figure 3D). To examine if AcrIF24 binds directly to the acrIF23-acrIF24 promoter region, we performed electrophoretic mobility shift assays (EMSA) with purified AcrIF24. AcrIF24 bound in a concentration-dependent manner to a long DNA fragment [IR-L] that contained the promoter and the IR sequence, whereas BSA did not bind this DNA (Figure 3E). The IR sequence was sufficient for AcrIF24 recognition, since the protein also bound to a minimal 17 bp dsDNA fragment [IR-S] that solely contained the IR (Figure 3E). Since the dimer-disrupted mutant of AcrIF24 (Y128K/Y217W double mutant) lost its ability to form a complex with IR-L, we concluded that dimerization of AcrIF24 was critical for the recognition of IR sequence in the promoter (Figure 3F). Indeed, this mutant was unable to repress the promoter in our reporter assay (Figure 3D). The complete IR was necessary for AcrIF24 binding, because mutation of one half of the IR in either the long [IR-L] or short [IR-S] DNA fragments abrogated interactions (Figure 3G). In summary, AcrIF24 contains a C-terminal HTH motif and recognizes and binds an IR to repress acrIF23-acrIF24 operon expression.
After establishing a regulatory function for AcrIF24, we next aimed to identify the protein regions involved in DNA binding. A helix-turn-helix (HTH) domain was previously predicted at the C-terminus of the protein (23) which in our structure forms the body domain. To obtain a more detailed view of the DNA-binding residues on AcrIF24, we first used the DNA-binding residues prediction server, DRNApred (57). According to this prediction server, residues S10, T12, R16, Y20 and S45 on the wing domain and residues T177, S180, T199, R196, S191, Y198, K197, S203 and R207 on the body domain were selected as tentative DNA-binding residues (Figure 4A). Electrostatic surface features of dimeric AcrIF24, showing a highly positively charged cleft in the bottom part of the wing and body domains (Figure 4B), supported the DRNApred predictions. Based on these observations and predictions, we speculated that AcrIF24 may use its body domain to recognize and bind to the specific IR sequence of DNA and that the wing domain may also be involved in DNA recognition. To test our hypothesis, we performed mutagenesis studies. Among the predicted tentative DNA binding residues, the highly conserved K197 and R207 on the body domain were mutated to tyrosine and tryptophan, respectively, producing K197Y and R207W mutants (Figure 3B). These two body domain mutants were hypothesized to disrupt DNA binding. For making wing domain mutants that might disrupt DNA binding, residues R16 and S45 were selected and mutated to tryptophan and tyrosine, respectively, generating R16W and S45Y mutants. These two residues were not conserved across different species (Figure 3B). All mutant proteins were purified and tested for DNA binding. While R16W and S45Y wing domain mutants were still able to bind to DNA, the K197Y and R207W body HTH motif mutations completely abrogated binding to long and short promoter sequences (IR-L and IR-S) (Figure 4C and Supplementary Figure S1). We validated these results using EMSA in agarose gel and native-PAGE by detecting shifted protein bands generated by DNA interaction. As expected, K197Y and R207W mutants were not able to produce shifted bands, while wildtype and S45Y produced distinct shifts (Figure 4D and Supplementary Figure S2). Because the IR-L/AcrIF24 complex sometimes stuck in the well at the high concentration of AcrIF24 provided for EMSA assay, we verified the specific interaction of DNA and AcrIF24 using the same EMSA assay in a protein concentration-dependent manner (Figure 4E). These experiments more clearly showed that K197Y and R207W mutants completely lost their IR-L binding ability and R16W mutants partially lost its DNA binding capability (Figure 4E). All these binding experiments indicated that residues K197 and R207 in the body domain are critical for DNA binding. Although R16W produced a shift, the amount shifted was reduced compared with wildtype AcrIF24, indicating that R16 on the wing domain influences DNA binding (Figure 4D and E). In agreement with these results, the K197W and R207W mutant proteins were unable to repress the acrIF23-acrIF24 promoter in reporter assays (Figure 3D). In conclusion, the HTH body domain of AcrIF24 is involved in recognition of the IR in the acrIF23-acrIF24 promoter and this interaction is essential for promoter repression.
To understand how AcrIF24 functions as an anti-CRISPR, we initially tested a direct interaction of AcrIF24 with the P. aeruginosa type I-F Cascade complex. This type I-F complex is composed of the Cas proteins Cas5f1, Cas6f, six copies of Cas7f1 and Cas8f1, and includes a single 60 nt crRNA (Figure 5A). First, SEC and SDS-PAGE were performed with Cascade in the absence of AcrIF24 to obtain a SEC profile and the location of each Cascade subunit on an SDS-PAGE gel. This analysis showed that the main peak fraction of the SEC profile, containing all the Cascade subunits, was produced at a position corresponding to an ∼400 kDa size particle eluted from the SEC column (Figure 5B and C and Supplementary Figure S3A and B). Since the typical mass of type-I-F Cascade is around 400 kDa, this SEC analysis indicated that the complex used in this study was successfully purified. In addition, each subunit of Cascade, Cas5f1 (36.2 kDa), Cas6f (21.4 kDa), Cas7f1 (39.7 kDa) and Cas8f1 (50.1 kDa), was detected at the expected position on the SDS-PAGE gel as well (Figure 5C and Supplementary Figure S3B). To analyze a direct interaction of AcrIF24 with type I-F Cascade, purified AcrIF24 was mixed with purified Cascade complex, incubated and loaded onto the SEC column. This SEC experiment showed that the main peak of AcrIF24-Cascade eluted 1 ml earlier than that of Cascade lacking AcrIF24, suggesting that AcrIF24 and Cascade were interacting to form a larger complex (Figure 5B). This elution volume corresponds to a mass >670 kDa, suggesting that AcrIF24 interacts with Cascade and that this interaction leads to the aggregation of more than one Cascade complex with AcrIF24. Indeed, elution fractions from this major peak contained all Cascade subunits in addition to co-eluting AcrIF24 when visualized by SDS-PAGE (Figure 5C and Supplementary Figure S4A and B). These observations clearly showed that AcrIF24 directly interacted with Cascade. To determine which region of AcrIF24 mediates binding to Cascade, we mutagenized AcrIF24. Because AcrIF24 was divided into the three distinct domains (wing, head, and body), we selected conserved and exposed surface residues from these domains and mutated them to residues that may disrupt the interaction with Cascade. G22Y and G189K mutants represented wing domain and body domain disruption mutants, respectively, while both D105K and W110K mutants represented head domain disruption mutants (Figure 5D). To test if the mutations affected interaction with Cascade, we performed SEC and SDS-PAGE. The main peak eluted at almost the same volume on the SEC profile for each mutant (Supplementary Figure S5A-E). However, co-migration of AcrIF24 W110K with Cascade was significantly reduced (Figure 5E and F, and Supplementary Figure S5D), indicating that this head domain disruption mutant has an impaired capacity for binding to Cascade. Based on this result, we concluded that the head domain of AcrIF24 is necessary for binding to Cascade. To confirm this conclusion, we purified a head domain deletion mutant (ΔHead) and analyzed the effect of the deletion on the interaction of AcrIF24 to Cascade. As expected, the ΔHead mutant could not bind to Cascade by failing to co-migrate with Cascade on SEC followed by SDS-PAGE (Figure 5E and F, and Supplementary Figure S5F). Based on these experiments, we confirmed that the head domain of AcrIF24 is necessary for binding to Cascade. Next, we wondered which Cascade subunit(s) were critical for the interaction with AcrIF24. To answer this question, we purified each subunit of Cascade separately (Supplementary Figure S6) and performed SEC with a mixture of each subunit with AcrIF24 (Figure 5G–I); note that an interaction with Cas8f could not be tested due to insolubility. Although an apparent peak shift by forming a complex was not detected on the SEC profiles, AcrIF24 co-migrated with Cas7f1 but not with Cas5f and Cas6f on SDS-PAGE, indicating that AcrIF24 bound specifically to Cas7f1(Figure 5I). This result is in good agreement with recent a structural study published by Yang et al (35). The cryo-EM study of the complex between AcrIF24 and Cascade showed that AcrIF24 specifically binds to Cas7f1 when AcrIF24 inhibits the Cascade activity by direct binding.
We wanted to determine how AcrIF24 inhibits type I-F Cascade activity. Since many AcrIF proteins inhibit I-F Cascade binding to complementary dsDNA targets, we first tested whether this mechanism applies here. I-F Cascade was purified with a crRNA complementary to a target dsDNA. Addition of I-F Cascade to this probe led to binding, as observed by a shift on the EMSA (Figure 6A). The addition of Cas2/3 alone, or in combination with Cascade, had no effect on these reactions. Importantly, the addition of increasing concentrations of AcrIF24 reduced the quantity of bound dsDNA (Figure 6A). Therefore, AcrIF24 inhibits the ability of I-F Cascade to bind to its specific complementary invader targets. Cas2/3 was added in these assays but no particular role was detected in our assay. Next, we asked which domains of AcrIF24 are important for its I-F Cascade inhibitory activity. We first tested the role of the head domain by examining the ability of the W110K and ΔHead AcrIF24 variants to inhibit Cascade. Although the W110K mutant still retained function, deletion of the entire head domain rendered AcrIF24 unable to inhibit Cascade function in dsDNA binding (Figure 6B). These findings are in agreement with our binding analysis of AcrIF24 with Cascade (Figure 5E and F), where ΔHead lost complete binding activity, while the W110K mutant still possessed some binding capability. Mutations that disrupted DNA binding and promoter repression by AcrIF24 (i.e. K197Y and R207W; Figure 3D and Figure 4) had no effect on inhibition of I-F Cascade (Figure 6C), suggesting that the DNA-binding (Aca) function of AcrIF24 may be independent from its Acr activity in vitro. Furthermore, AcrIF24 dimerization was required to inhibit I-F Cascade, since the Y128K/Y217W mutant failed to disrupt DNA binding by Cascade (Figure 6D). Finally, we examined whether these in vitro results are also valid in an in vivo phage infection model. For this, we used Pectobacterium carotovorum RC5297, which has a type I-F CRISPR-Cas system, and an acr-less variant of phage ZF40. This phage efficiently infected P. carotovorum (-CRISPR), but infectivity was drastically reduced in the presence of a ZF40-targeting spacer in the host CRISPR-Cas system (+CRISPR, Figure 6E). However, even in the presence of a targeting spacer, overexpression of acrIF24 from a plasmid allowed the phage to overcome CRISPR-Cas defense. In contrast, the W110K mutation completely abrogated the protective effect of AcrIF24 and therefore displayed a stronger phenotype in vivo than in vitro, highlighting the importance of this head domain residue for Acr activity (Figure 6E). The dimer-disruption mutations (Y128K/Y217W) also completely abrogated Acr activity, in agreement with our in vitro results, supporting that dimerization is important for inhibiting Cascade. Interestingly, the mutant proteins unable to repress the promoter (K197W and R207W) also slightly affected Acr function, albeit not as strongly as the W110K mutation in the head domain. This suggests that the Acr and Aca functions of AcrIF24 are not completely separable (Figure 3D). In summary, dimer formation and the head domain of AcrIF24 are critical for its Acr activity.
The dual function of AcrIF24 as both an Acr and Aca was initially suggested by Pinilla-Redondo and colleagues (23). The gene encoding P. aeruginosa AcrIF24 was shown to confer anti-CRISPR activity and was in an operon lacking any aca gene. Part of the C-terminus of AcrIF24 was predicted as a HTH motif, which is a major DNA binding domain, suggesting that AcrIF24 contains Aca function in addition to anti-CRISPR activity (23). In this study, we solved the crystal structure of AcrIF24, which contains a novel domain composition reminiscent of a bird and consisting of head, body and wing domains. AcrIF24 is dimeric and binds a short inverted repeat sequence in the acrIF23-acrIF24 operon promoter to repress acr expression. Through mutagenesis, we demonstrated that the body domain, which contains the HTH motif, was essential for DNA binding. We further show that AcrIF24 binds directly to the type I-F Cascade through interactions with the Cas7f1 subunit. Overall, our results uncover the structure and regulatory and anti-CRISPR activity of a dual function Acr-Aca protein. AcrIF24 is a dimer through interactions between the head and body domains. Many DNA binding proteins with HTH motifs function as dimers. Indeed, dimerization of Aca proteins is critical for the recognition of inverted repeat (IR) DNA sequences in the promoter region of acr operons (52,55,58). In agreement, AcrIF24 bound to a short IR sequence in the promoter region of the acr operon and repressed transcription. The mechanism of action is likely due to blocking RNAP binding. While our manuscript was about to be submitted, another study on the structure of AcrIF24 was published (35). Interestingly, they were unable to solve the crystal structure of the complete protein, but of a deletion mutant with a linker instead of the head domain. This mutant dimerized still, despite lacking the head, even though the head appears to make important contributions to the protein-protein interface in our crystal structure. To understand operator recognition, both studies examined the roles of different amino acids in DNA binding. In terms of the body domain, we uncover roles of K197 and R207 for DNA binding, and Yang et al. show that R196 is also important (35). Because we show that an R16W mutation on the wing domain partially affected DNA binding, this domain might also contribute to promoter binding. The location of R16 at the bottom of AcrIF24 in line with the HTH motif supports the involvement of the wing in DNA recognition. Yang et al. show that their head domain deletion mutant still binds DNA, indicating this portion of AcrIF24 is not required for Aca activity. Interestingly, some AcrIF24 homologues are truncated, with the N-terminal wing domain missing, such as in B. glumae (Figure 3B). It is possible that AcrIF24 homologues evolved with or without the wing domain and that it provides an accessory DNA binding activity. Therefore, there are potentially two different classes of AcrIF24, a three domain version and a two domain version. The structure of AcrIF24 in complex with the IR DNA binding site will be critical for a detailed understanding of the exact DNA recognition strategy. Diverse mechanisms of various anti-CRISPR proteins have been demonstrated (16,25,53,59). We showed that AcrIF24 directly binds to the type I-F Cascade complex with critical involvement of the head domain. Direct interactions of an Acr with Cascade can block recruitment of target DNA or the Cas3 nuclease and are the most common Acr strategies for inhibiting CRISPR-Cas systems (26,27). Among the twenty-four AcrIF family members identified, at least nine, including AcrIF1, AcrIF2, AcrIF4, AcrIF6, AcrIF7, AcrIF8, AcrIF9, AcrIF10 and AcrIF14, directly bind to Cascade for inhibition (26,27,60). Our results suggest that AcrIF24 also blocks the ability of I-F Cascade to bind target DNA to inhibit its CRISPR-Cas activity. This is in agreement with the recent cryoEM structure of dimeric AcrIF24 in complex with two Cascade complexes that was published while our manuscript was in preparation (35). Indeed, we showed that the mass of Cascade (∼400 kDa) was dramatically increased by complex formation with AcrIF24 (around 700–800 kDa). Furthermore, our in vitro and in vivo data show that dimerization is essential for AcrIF24 to inhibit Cascade. These results, together with the study by Yang et al. indicate that interaction of dimeric AcrIF24 with two Cascade complexes results in CRISPR-Cas inhibition. Consistent with our study,Yang et al., showed that AcrIF24 binds to Cas7f1 subunits (Cas7.2f-Cas7.6f) in Cascade. Although most of AcrIF24 contributed to the Cas7 interaction, the major domain involved was the head. This is consistent with our mutagenesis studies showing reduced binding with a head mutant, and with our in vivo data showing complete loss of Acr activity in the W110K head-domain mutant. Furthermore, binding was completely abrogated when the head domain was deleted. Due to their ability to only solve the structure of the head via cryoEM in the AcrIF24-Cascade complex, the authors suggest that the head is disordered until interaction with Cascade. However, our intact, fully folded crystal structure of AcrIF24 indicates that AcrIF24 can be in ordered conformation without binding to Cascade. Indeed, superimposition of our AcrIF24 structure with AcrIF24 in complex with Cascade revealed identical structures except several loops on the head domain that are important for Cas7f recognition (Supplementary Figure S7A and B). This indicates that interaction with Cascade causes certain loops on, but not the entire, head domain to change conformation. Given that Acrs are natural CRISPR-Cas inhibitors, their potential applications in bio-medical therapeutics, including antibacterial compounds, gene editing, and regulation of gene drives, have been suggested (2,16,61). Moreover, the engineering of Acr proteins for better usage has been intensively studied (62). In this context, the structural information of AcrIF24 and its dual function can contribute new information not only to the basic understanding of phage-host interactions and the field of CRISPR-Cas but also to potential bio-medical applications of Acrs.
The coordinate and structure factor have been deposited into the Research Collaboratory for Structural Bioinformatics (RCSB) Protein Data Bank (PDB) under the PDB code of 7XI1.
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PMC9639037 | 36226560 | Minsu Yun,Sun‐Hee Park,Dong Hee Kang,Ji Wook Kim,Ju Deok Kim,Siejeong Ryu,Jeongyeob Lee,Hye Min Jeong,Hye Ran Hwang,Kyoung Seob Song | Inhibition of Pseudomonas aeruginosa LPS‐Induced airway inflammation by RIPK3 in human airway | 13-10-2022 | histone,LPS,pro‐inflammatory cytokine,Pseudomonas aeruginosa,RIPK3,ubiquitination | Abstract Although the physiological function of receptor‐interacting protein kinase (RIPK) 3 has emerged as a critical mediator of programmed necrosis/necroptosis, the intracellular role it plays as an attenuator in human lungs and human bronchial epithelia remains unclear. Here, we show that the expression of RIPK3 dramatically decreased in the inflamed tissues of human lungs, and moved from the nucleus to the cytoplasm. The overexpression of RIPK3 dramatically increased F‐actin formation and decreased the expression of genes for pro‐inflammatory cytokines (IL‐6 and IL‐1β), but not siRNA‐RIPK3. Interestingly, whereas RIPK3 was bound to histone 1b without LPS stimulation, the interaction between them was disrupted after 15 min of LPS treatment. Histone methylation could not maintain the binding of RIPK3 and activated movement towards the cytoplasm. In the cytoplasm, overexpressed RIPK3 continuously attenuated pro‐inflammatory cytokine gene expression by inhibiting NF‐κB activation, preventing the progression of inflammation during Pseudomonas aeruginosa infection. Our data indicated that RIPK3 is critical for the regulation of the LPS‐induced inflammatory microenvironment. Therefore, we suggest that RIPK3 is a potential therapeutic candidate for bacterial infection‐induced pulmonary inflammation. | Inhibition of Pseudomonas aeruginosa LPS‐Induced airway inflammation by RIPK3 in human airway
Although the physiological function of receptor‐interacting protein kinase (RIPK) 3 has emerged as a critical mediator of programmed necrosis/necroptosis, the intracellular role it plays as an attenuator in human lungs and human bronchial epithelia remains unclear. Here, we show that the expression of RIPK3 dramatically decreased in the inflamed tissues of human lungs, and moved from the nucleus to the cytoplasm. The overexpression of RIPK3 dramatically increased F‐actin formation and decreased the expression of genes for pro‐inflammatory cytokines (IL‐6 and IL‐1β), but not siRNA‐RIPK3. Interestingly, whereas RIPK3 was bound to histone 1b without LPS stimulation, the interaction between them was disrupted after 15 min of LPS treatment. Histone methylation could not maintain the binding of RIPK3 and activated movement towards the cytoplasm. In the cytoplasm, overexpressed RIPK3 continuously attenuated pro‐inflammatory cytokine gene expression by inhibiting NF‐κB activation, preventing the progression of inflammation during Pseudomonas aeruginosa infection. Our data indicated that RIPK3 is critical for the regulation of the LPS‐induced inflammatory microenvironment. Therefore, we suggest that RIPK3 is a potential therapeutic candidate for bacterial infection‐induced pulmonary inflammation.
Pseudomonas aeruginosa is a Gram‐negative bacterial pathogen related to a wide range of infections including lung disease. , Because of its metabolic flexibility and fundamental resistance to antimicrobials, P. aeruginosa grows well in a wide variety of materials and environments, including in‐hospital facilities and patient devices. Although it rarely infects healthy individuals, it is a leading and well‐known opportunistic pathogen, especially in immunocompromised patients with defective immune defences. P. aeruginosa is known to colonize and infect the lungs of patients with cystic fibrosis (CF) and advanced stages of chronic obstructive pulmonary disease (COPD). , Most importantly, P. aeruginosa has multiple antibiotic resistance and tolerance that allow it to survive antibiotic treatment against infection. Moreover, bacterial coinfection is a mutual complication of several viral pulmonary tract infections, such as severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2), and significantly increases morbidity and mortality. Therefore, a new therapeutic strategy is needed for antibiotic‐dependent microbial treatment. Receptor‐interacting serine/threonine‐protein kinase (RIPK) 3 is a major protein of necroptosis that can be initiated by various signals including the activation of death receptors, Toll‐like receptors, interferon receptors and pathogen infection. , Necroptosis is a system of regulated cell death characterized by necrotic features including cell swelling and disrupted cell plasma membrane. Necroptosis is tightly regulated by the activation of RIPK1 and RIPK3. RIPK3 is regulated by the proteins related to post‐translational modification, such as protein phosphatase 1B, deubiquitylating enzyme A20, C terminus of Hsp‐70‐interacting protein (CHIP) and Pellino E3 ubiquitin protein ligase 1 (PELI1). Although RIPK3 is a pivotal protein in necroptosis, post‐translational processes that may control RIPK3 activity and stability during P. aeruginosa infection in the airway remain poorly understood. Disruption of cell‐to‐cell barriers is a major feature of tissue inflammation. An excessive increase in epithelial permeability has critically harmful effects on tissue homeostasis by increasing body exposure to detrimental environmental agents and inducing uncontrollable inflammation. , , Therefore, actin remodelling plays a very important role in maintaining cell homeostasis by suppressing pathogen infection and controlling inflammation. The biochemical and physiological mechanism by which actin remodelling and trans‐epithelial electrical resistance (TEER) may be affected by RIPK3 during P. aeruginosa infection in respiratory diseases remains unclear. This study aims to characterize whether the RIPK3 protein has an anti‐inflammatory effect against P. aeruginosa in the airway. Overexpression of RIPK3 could down‐regulate LPS‐induced inflammation, but not siRNA‐RIPK3. Interestingly, the RIPK3 protein can bind histone to inhibit translocalization into the cytoplasm. After the methylation of histone with LPS stimulation, RIPK3 translocates into the cytoplasm to decrease the production of pro‐inflammatory cytokines dramatically. Our data indicate that the RIPK3 protein is critical for the regulation of the LPS‐induced inflammatory microenvironment in respiratory diseases.
A human lung disease spectrum tissue array was purchased from US Biomax (Rockville). The tissue array consisted of 24 cases (48 cores), which included normal lung tissue, lung hyperplasia of stroma, and pulmonary fibrosis with chronic inflammation of bronchiole, lung lobar pneumonia, pulmonary atelectasis, collapsed lung, pulmonary tuberculosis, pulmonary emphysema, and inflammatory pseudotumor plus lung small cell carcinoma, lung adenocarcinomas, and lung squamous cell carcinomas. Of these, we used three different groups of tissues: normal, hyperplasia and inflammation. Lung sections were processed with either anti‐RIPK3 antibody, phosphor‐RIPK3 antibody (Ser277) or goat anti‐rabbit IgG (Alexa Fluor 546), stained with Deep‐Red, and images were taken for each core. Five separate images of each section were given a score , , , , , , , , , by three independent scientists.
The LPS was purchased from Merck. Purified cytokines and specific ELISA kits were purchased from R&D Systems. Normal human bronchial epithelial (BEAS‐2b) cells were purchased from ATCC, and cells were cultured in BEBM (Lonza) with a BEGM kit at 37°C in a humidified incubator with 5% CO2. The culture plates were pre‐coated with a mixture of 0.01 mg/ml fibronectin (Sigma‐Aldrich), 2 mg/ml gelatin (Sigma‐Aldrich) and 0.01 mg/ml bovine serum albumin (Gibco BRL) dissolved in BEBM.
Cells were treated with LPS. Real‐time PCR was performed using a BioRad iQ iCycler Detection System (BioRad Laboratories; Hercules) with iQ SYBR Green Supermix. Reactions were performed in a total volume of 20 μl which included 10 μl of 2x SYBR Green PCR Master Mix, 300 nM of each primer and 1 μl of previously reverse‐transcribed cDNA template. The following primers were used: RIPK3, forward ACTCCCGGCTTAGAAGGACT, and reverse GCCCTGCTCCTCTTGGTAAG; IL‐6, forward CCACACAGACAGCCACTCACC, and reverse CTACATTTGCCGAAGAGCCCTC; IL‐1β, forward ACTCCCGGCTTAGAAGGACT, and reverse GCCCTGCTCCTCTTGGTAAG, and β2‐microglobulin, used as a reference for normalization, forward CGCTCCGTGGCCTTAGC and reverse GAGTACGCTGGATAGCCTCCA. Real‐time RT‐PCR was performed on a MiniOption Real‐Time PCR Detection System (Bio‐Rad). The parameters were 95°C for 10 min, followed by 40 cycles of 95°C for 15 s, 60°C for 30 s and 72°C for 30 s. All reactions were performed in triplicate. The relative quantity of mRNA was obtained using the comparative cycle threshold method and was normalized using β2‐microglobulin as an endogenous control.
The plasmid expressing wild‐type RIPK3 was kindly gifted by Dr. You‐Sun Kim (Ajou Univ School of Medicine, Suwon, Korea) and siRNA‐RIPK3 [CUCUCCGCGAAAGGACCAA (dTdT)], and siRNA‐control [CCUACGCCACCAAUUUCGU(dTdT)] were synthesized to Bioneer (Daejeon, Korea). Cells were plated in 6‐well plates 1 day prior to transfection using FuGENE6 (Roche; Indianapolis, IN) according to the manufacturer's instruction. The cells were grown to confluence in 6‐well plates. After treatment with LPS, cells were lysed with 2x lysis buffer (250 mM Tris‐Cl [pH 6.5], 2% SDS, 4% β‐mercaptoethanol, 0.02% bromophenol blue and 10% glycerol). Equal amounts of whole cell lysates were resolved by 8%–10% SDS‐PAGE and transferred to specific membranes. Membranes were blocked with 5% skim milk in Tris‐buffered saline (50 mM Tris‐Cl [pH 7.5] and 150 mM NaCl) for 1 h at room temperature. Blots were then incubated overnight with anti‐phospho‐RIPK3 (Phospho S227; Abcam, ab209384), phospho‐Ser/Thr Phe antibody (Cell Signalling Technology #9631) or total RIPK3 antibody (Cell Signalling Technology #13526) in TTBS (0.5% Tween 20 in Tris‐buffered saline). After washing with TTBS, the blots were further incubated for 45 min at room temperature with anti‐rabbit or anti‐mouse antibody in TTBS and visualized using the ECL system.
F‐actin staining was performed using ActinRed 555 ReadyProbe reagent (Molecular Probes; R37112) following the manufacturer's instructions. The cells were incubated for 30 min, the stain solution removed, and the cells rinsed with PBS. Images were obtained using a Nikon Eclipse 80i microscope (Eclipse 80i) with a 540 nm excitation filter and a 565 nm emission filter.
Before evaluation, the electrodes were sterilized and corrected according to the manufacturer's instructions (Merck). The shorter tip was placed in the culture plate insert, and the longer tip was placed in the outer well. The unit area resistance (Ω × cm2) was calculated by multiplying the sample resistance (Ω) by the effective area of the membrane (4.2 cm2 for 6‐well Millicell inserts).
Nano‐LC–MS/MS analysis was performed with a nano HPLC system (Agilent, Wilmington, DE). A nano‐chip column (Agilent, 150 mm × 0.075 mm) was used for peptide separation. The mobile phase A for LC separation was 0.1% formic acid in deionized water, and the mobile phase B was 0.1% formic acid in acetonitrile. The chromatography gradient was designed for a linear increase from 3% B to 45% B in 30 min, 45% B to 95% B in 1 min, 95% B in 4 min and 3% B in 10 min. The flow rate was maintained at 300 nl/min. Product ion spectra were collected in the information‐dependent acquisition mode and were analysed by Agilent 6530 Accurate‐Mass Q‐TOF using continuous cycles of one full scan TOF MS from 300–2000 m/z (1.0 s) plus three product ion scans from 150–2000 m/z (1.5 s each). Precursor m/z values were selected starting with the most intense ion, using a selection quadrupole resolution of 3 Da. The rolling collision energy feature was used, which determines collision energy based on the precursor value and charge state. The dynamic exclusion time for precursor ion m/z values was 60 s.
The mascot algorithm (Matrixscience) was used to identify peptide sequences present in a protein sequence database. Database search criteria were as follows: taxonomy, Homo sapiens; fixed modification, carbamidomethylated at cysteine residues; variable modification, oxidized at methionine residues; maximum allowed missed cleavage, 2; MS tolerance, 100 ppm; and MS/MS tolerance, 0.1 Da. Only peptides resulting from trypsin digests were considered. The peptides were filtered with a significance threshold of p < 0.05.
The cells were harvested by scraping into lysis buffer [25 mM HEPES, 150 mM NaCl, 0.5% NP‐40, 1 mM EDTA, 1 mM EGTA, and protease inhibitor tablet (Complete Mini; Roche)], sonicated (4 times each for 5 s) and centrifuged at 12,000 g for 15 min. Supernatant lysates were pre‐cleared with Dynabeads Protein G (Life Technologies) for 30 min at 4°C. Following centrifugation, appropriate antisera were added to the pre‐cleared lysates, incubated for 30 min at 4°C and added Dynabeads Protein G. Immunoprecipitated protein complexes were recovered using DynaMag‐2.
The cells were fixed with 4% paraformaldehyde, permeabilized with 0.5% Triton X‐100 for 5 min, blocked with 5% BSA for 1 h, labelled with diluted (1:100) anti‐RIPK3 antibody and incubated at 4°C overnight. The secondary antibody, Alexa Fluor 546, was diluted 1:100 in PBS containing 5% BSA and incubated for 2 h at room temperature. Nuclei were stained with diluted Deep‐Red (1:300).
The data are presented as the mean ± SD of at least three independent experiments. Where appropriate, statistical differences were assessed by the Wilcoxon–Mann–Whitney test. p‐Values < 0.05 were considered statistically significant.
To examine whether RIPK3 expression is clinically related to inflammation in human lung tissue, the immunohistochemistry was performed with RIPK3‐specific antibody in human pathological lung tissue array (Figure 1A). Whereas RIPK3 expression was strongly observed in normal tissues, it was decreased dramatically in the inflammation group. Interestingly, RIPK3 expression was detectable in both nuclear and cytoplasm in the normal tissue group, but decreased RIPK3 expression was observed mostly in the cytoplasm of the inflammation group. Interestingly, there was no difference in the level of phospho‐RIPK3 in normal tissue or patient tissue (Figure 1C). Representative images are shown with disease quantification scores (Figure 1B,D). Lung pathologies suggest that a dramatic decrease in RIPK3 expression is closely associated with inflammatory disease processes.
To examine whether LPS could control RIPK3 gene expression at an early event, BEAS‐2B cells were stimulated with LPS in a time‐dependent manner (Figure 2A). RIPK3 gene expression was diminished at 4 h after LPS treatment. To detect whether LPS induces RIPK3 phosphorylation to activate its own physiological functions, phospho‐antibodies were used. RIPK3 had not been phosphorylated after the treatment with LPS for 2 ~ 4 h using phospho‐Ser277 antibody as well as phospho‐Ser/Thr pan antibody (Figure 2B), suggesting that RIPK3 plays an independent role by a different mechanism in normal human bronchial epithelial cells without phosphorylation by LPS. In addition, to know whether overexpressed RIPK3 expression could regulate the LPS‐induced inflammatory microenvironment and F‐actin formation in bronchial cells, we used specific siRNA‐RIPK3 (Figure 2C). Overexpressed RIPK3 could significantly decrease pro‐inflammatory cytokine gene expression (IL‐6 and IL‐1β); however, siRNA‐RIPK3 did not affect the expression of these genes. More interestingly, with LPS only, F‐actin was generated throughout the cell at 30–45 min. Whereas F‐actin was continuously and strongly generated throughout the cell for 120 min with RIPK3 overexpressed, it was not increased by LPS in the cells transfected with siRNA‐RIPK3, and, apical extension and protrusion were dramatically decreased in the cells transfected with siRNA‐RIPK3 (Figure 2D). In addition, we found that even trans‐epithelial electrical resistance (TEER) was reinstated by the overexpression of RIPK3, but not by siRNA‐RIPK3 (Figure 2E). These results suggest that overexpression of RIPK3 could change the inflammatory microenvironment after LPS treatment in human bronchial epithelial cells.
Since RIPK3 is a multifunctional signalling mediator, we thought it might combine with a protein complex to apply its potential roles. To investigate RIPK3‐binding partners in mammalian cells, we immunoprecipitated the RIPK3 complex from BEAS‐2B cells transfected with the RIPK3 construct (Figure 3A). Coomassie‐blue staining detected a unique protein band in the RIPK3‐immune complex. The RIPK3‐specific band was analysed by LC–MS and identified as histone H1.2 and Ubiquin‐40S S27a protein (Figure 3A). Immunoprecipitation of the lysates was used to examine the association between endogenous H1B and RIPK3 and showed that RIPK3 was bound to H1B. Surprisingly, this interaction was dissociated after stimulation with LPS for 15 min (Figure 3B, upper panel). Similarly, anti‐Histone antibody efficiently immunoprecipitated Histone from cell lysates and also coimmunoprecipitated RIPK3 (Figure 3B, lower panel). RIPK3 protein (upper panel) or Hib protein (lower panel) level itself as an input control was not decreased under LPS‐incubation at these time points. These results suggest that histone 1B was bound to RIPK3 in the nucleus to prevent translocation towards the cytoplasm before LPS stimulation. Thus, we performed immunocytochemistry and analysed RIPK3 expression levels in the nuclear and cytoplasmic regions of BEAS‐2B cells with/without LPS stimulation (Figure 3C). Interestingly, RIPK3 expression was observed mainly in the nuclear region without LPS treatment, whereas it strongly translocated to the cytoplasmic region after LPS treatment, but not siRNA‐RIPK3 (Figure 3C). We performed the Western blot analysis with nuclear and cytosol fractionation to confirm the results of immunocytochemical staining. CREB was used as a fractionation control. We found RIPK3 expression was decreased in nuclear fraction, whereas it was slightly increased in cytosolic fraction after LPS treatment (Figure S1). In addition, to know which physiological mechanism disintegrated the interaction between H1B and RIPK3, we used a histone deacetylase inhibitor (SAHA) or an inhibitor of histone methyltransferase EZH2 (DZNep). SAHA (2 μM) was treated for 12 h or DZNep (0.5 μM) was treated for 72 h before LPS stimulation. The gene expression of pro‐inflammatory cytokines was significantly increased after DZNep (Figure 3D), but not SAHA (data not shown), indicating because the histone was methylated, it could not no longer be combined with RIPK3, and RIPK3 was translocated to the cytoplasm in an anti‐inflammatory function. According to the results of the LC–MS/MS (Figure 3A), RIPK3 was bound to ubiquitin without LPS stimulation. To understand the physiological significance of this interaction between RIPK3 and ubiquitin, an immunoprecipitation assay was performed (Figure 3E). RIPK3 ubiquitination by LPS reached a peak at 60 min and was maintained at 120 min. In addition, the expression of RIPK3 was the same as the previous result (Figure 2A). Interestingly, RIPK3 ubiquitination by LPS reached a peak at 60 min and gradually declined at 120 min in the cells transfected with the wild‐type RIPK3 construct, and the expression of RIPK3 was quickly degraded at 30 min after LPS treatment (Figure 3E, upper panel). Conversely, when immunoprecipitation assay was performed, ubiquitin bound rapidly, and then, ubiquitination has been processed. After 60 min, the binding to RIPK3 seemed weak (Figure 3E, lower panel). We propose that the anti‐inflammatory signalling of RIPK3 inhibits the inflammation signalling by LPS over a short time and then degrades, the signal of the inflammatory response by LPS is transmitted, and the ubiquitination of endogenous RIPK3 occurs at a later time.
To determine which signal pathway is activated within the cells stimulated by LPS, we performed Western blot analysis using a phospho‐specific antibody (Figure 4A). Because NF‐κB signalling pathway is essential for LPS signalling, we checked whether LPS‐induced NF‐κB signalling was affected by RIPK3. IκB phosphorylation was maximally activated at 30 min, and this effect decreased at 45 min (Figure 4A). Interestingly, RIPK3 dramatically inhibited LPS‐induced iκB phosphorylation for 30 min, but not siRNA‐RIPK3 (Figure 4B). Furthermore, to determine whether RIPK3 influences LPS‐induced pro‐inflammatory cytokine gene expression through the NF‐κB pathway, we used a p65 overexpression construct, which is a signalling protein downstream of iκB. Overexpression of p65 increased the expression of pro‐inflammatory cytokine genes but cotransfection with both RIPK3 and p65 constructs dramatically decreased expression (Figure 4C), indicating that NF‐κB appears to be controlled by RIPK3.
P. aeruginosa is a dominant organism within the hospital environment, an increasingly multidrug‐resistant microorganism, and the most common Gram‐negative pathogen causing hospital‐acquired pneumonia. , In the lung, P. aeruginosa has been known to opportunistically colonize patients with CF and chronic obstructive pulmonary disease. Chronic P. aeruginosa infections result from the dynamic and complex interactions between pathogen and host, where bacteria continue without causing overwhelming host injury, and where host defences fail to eliminate the pathogen. Although various diseases in humans are caused by P. aeruginosa, treatment is limited due to drug resistance. This study is focused on the use of intracellular proteins to overcome drug resistance and on providing new therapeutic options to overwhelm P. aeruginosa. RIPK3 is a multifunctional regulator of cell death and inflammation that controls signalling downstream of the tumour necrosis factor receptor family, DNA‐dependent activator of IFN‐regulatory factors and toll‐like receptors (TLRs). Several reports have shown that RIPK3 was regulated by post‐translational modification and regulatory proteins, such as protein phosphatase 1B (Ppm1b), deubiquitylating enzyme A20, the C terminus of Hsp‐70‐interacting protein (CHIP) or Pellino E3 ubiquitin protein ligase (PELI) 1. Although it is very important to identify/characterize RIPK3‐binding proteins or the physiological mechanism in the human body, there is no study on the function of RIPK3 in the respiratory tract during P. aeruginosa infection. As mentioned above, because infection with P. aeruginosa is very severe and harmful in many diseases, we investigated the effect of RIPK3 on P. aeruginosa‐induced acute airway inflammation. Both the expression of RIPK3 and phosphorylated RIPK3 were remarkably decreased in the human inflamed pathological lung group, compared to the normal group (Figure 1). Although phosphorylated RIPK3 promotes the intracellular apoptotic pathway , and overexpressed RIPK3 induced MLKL phosphorylation‐mediated programmed necrosis, it is still unclear why RIPK3 expression, but not phospho‐RIPK3 was dramatically decreased in human pathological lungs, and cell death was not observed in the tissues (Figure 1C). The possibilities are that (1) RIPK3 may act as an anti‐inflammatory regulator in the lung. Why the RIPK3 protein, which is expressed strongly in normal lung tissues, could not be observed in inflammatory tissues could mean that RIPK3 may promote anti‐inflammatory phenomena. We will explain this possibility in more detail later (Figure 2). (2) There will be other unknown proteins that can bind to RIPK3 and affect its physiological function in the human lung. A common feature of the RIPK3‐binding proteins mentioned in the above paragraph is the cytoplasmic location. Interestingly, cytoplasmic proteins could affect post‐translational modifications, but there have been no studies of events occurring in the nucleus and the post‐transcriptional modification so far. We will also explain this possibility in more detail later (Figure 3). Together, the function of RIPK3 is still not known accurately, and unlike the lungs of normal tissue, RIPK3 expression can be dramatically reduced in the inflammatory pathological lung, and more interestingly, the expression of RIPK3 after LPS stimulation translocated towards the cytoplasm, not the nucleus. Taken together, RIPK3 expression was significantly decreased and translocated towards the cytoplasm in human pathological lungs in our system. To examine how RIPK3 translocated from the nucleus to cytoplasm in the pathological lung or after treatment with LPS (Figure 3), 1D LC–MS/MS analysis was performed. According to several studies, RIPK3 mainly binds to cytoplasmic proteins, but RIPK3 is also expressed in the nucleus. In our system, RIPK3 was bound to the ubiquitin protein (Figure 3A). The protein complex with H1b protein bound to RIPK3 was in the nucleus. However, on stimulation(s), it became an acetylation of H1b that was no longer bound to RIPK3 and dissociated, proving that RIPK3 finally translocated to the cytoplasm and played an independent function in airway cells. Many studies show that gene expression is controlled by acetylation. However, to our knowledge, there is no study on histone in signalling mechanism or physiological phenomena related to RIPK3, and this study is the first in the respiratory system. Therefore, further research on this should be conducted in‐depth. RIPK3 is known as a switch protein that determines necroptosis or inflammation. Interestingly, Moriwaki et al. demonstrated that RIPK3 protects against dextran sodium sulphate (DSS)‐induced colitis and that RIPK3 is required for tissue repair by inducing an axis of IL‐23, IL‐1β and IL‐22 downstream of DSS‐induced injury, independent of its role in cell death. In our system, RIPK3 inhibited the expression of IL‐1β and IL‐6 genes by inhibiting the activity of the iκB pathway, thus finally creating an anti‐inflamed microenvironment (Figure 4C). These differences are caused by differences in the cells, but the main cause is differences in the surrounding environment. Unlike other tissues, the lungs are always in contact with external air, and gas exchange occurs in blood vessels and the alveolar of surrounding tissues. Recently, Han et al. reported that BEAS‐2B cells have the specific features of mesenchymal stem cells. In that paper, they used α‐MEM complete media with 10% FBS to incubate BEAS‐2B cells. We found that the morphology of BEAS‐2B cells was dramatically changed when the cells were incubated in α‐MEM with 10% FBS (Figure S2). The morphological change seems to be caused by latent TGFβ1, which is already contained at a high level in foetal bovine serum, based on a recent report that TGFβ1 could induce epithelial to mesenchymal transition in BEAS‐2B cells. To culture BEAS‐2B cells in this research, we utilized the BEGM TM Bronchial Epithelial Cell Growth Medium Bullet Kit TM (Ronza, CC‐3170), containing hEGF instead of TGFβ1. In our culture circumstances, the observation of morphological change of the cells was not detected at all. This media has been known to be proper to incubate primary bronchial epithelial cells and maintain the properties of epithelial cells. We found that RIPK3 expression was decreased in human pathological lungs and had an inhibitory effect on LPS‐induced airway inflammation and TEER. In addition, H1B bound to RIPK3 is dissociated by methylation after LPS stimulation. Translocated RIPK3 inhibited iκB activation to suppress the expression of pro‐inflammatory cytokine genes. Thus, these results suggest that RIPK3 may be a potential therapeutic candidate during P. aeruginosa infection‐induced respiratory diseases.
Minsu Yun: Conceptualization (equal); data curation (equal); investigation (equal); writing – original draft (equal). Sun‐Hee Park: Conceptualization (lead); data curation (lead); formal analysis (lead). Dong Hee Kang: Data curation (equal); methodology (equal). Ji Wook Kim: Data curation (equal); formal analysis (equal); investigation (equal). Ju Deok Kim: Investigation (equal); resources (equal); validation (equal). Siejeong Ryu: Supervision (equal); validation (equal). Jeongyeob Lee: Formal analysis (equal); validation (equal). Hye Min Jeong: Investigation (equal); methodology (equal). Hye Ran Hwang: Investigation (equal); methodology (equal). Kyoung Seob Song: Conceptualization (equal); validation (equal); writing – original draft (equal).
This study was supported by a grant from the National Research Foundation of Korea (NRF) and funded by the Korean government (NRF‐2020R1I1A2075001 and NRF‐2021R1A4A1031380 to K.S.S.).
We declare that we do not have any conflicts of interest.
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PMC9639322 | Tsung-Ming Chang,Pei-Yi Chu,Hui-You Lin,Kuo-Wei Huang,Wen-Chun Hung,Yan-Shen Shan,Li-Tzong Chen,Hui-Jen Tsai | PTEN regulates invasiveness in pancreatic neuroendocrine tumors through DUSP19-mediated VEGFR3 dephosphorylation | 06-11-2022 | PTEN,VEGFR3,DUSP19,Pancreatic neuroendocrine tumor,Invasiveness | Background Phosphatase and tensin homolog (PTEN) is a tumor suppressor. Low PTEN expression has been observed in pancreatic neuroendocrine tumors (pNETs) and is associated with increased liver metastasis and poor survival. Vascular endothelial growth factor receptor 3 (VEGFR3) is a receptor tyrosine kinase and is usually activated by binding with vascular endothelial growth factor C (VEGFC). VEGFR3 has been demonstrated with lymphangiogenesis and cancer invasiveness. PTEN is also a phosphatase to dephosphorylate both lipid and protein substrates and VEGFR3 is hypothesized to be a substrate of PTEN. Dual-specificity phosphatase 19 (DUSP19) is an atypical DUSP and can interact with VEGFR3. In this study, we investigated the function of PTEN on regulation of pNET invasiveness and its association with VEGFR3 and DUSP19. Methods PTEN was knocked down or overexpressed in pNET cells to evaluate its effect on invasiveness and its association with VEGFR3 phosphorylation. In vitro phosphatase assay was performed to identify the regulatory molecule on the regulation of VEGFR3 phosphorylation. In addition, immunoprecipitation, and immunofluorescence staining were performed to evaluate the molecule with direct interaction on VEGFR3 phosphorylation. The animal study was performed to validate the results of the in vitro study. Results The invasion and migration capabilities of pNETs were enhanced by PTEN knockdown accompanied with increased VEGFR3 phosphorylation, ERK phosphorylation, and increased expression of epithelial–mesenchymal transition molecules in the cells. The enhanced invasion and migration abilities of pNET cells with PTEN knockdown were suppressed by addition of the VEGFR3 inhibitor MAZ51, but not by the VEGFR3-Fc chimeric protein to neutralize VEGFC. VEGFR3 phosphorylation is responsible for pNET cell invasiveness and is VEGFC-independent. However, an in vitro phosphatase assay failed to show VEGFR3 as a substrate of PTEN. In contrast, DUSP19 was transcriptionally upregulated by PTEN and was shown to dephosphorylate VEGFR3 via direct interaction with VEGFR3 by an in vitro phosphatase assay, immunoprecipitation, and immunofluorescence staining. Increased tumor invasion into peripheral tissues was validated in xenograft mouse model. Tumor invasion was suppressed by treatment with VEGFR3 or MEK inhibitors. Conclusions PTEN regulates pNET invasiveness via DUSP19-mediated VEGFR3 dephosphorylation. VEGFR3 and DUSP19 are potential therapeutic targets for pNET treatment. Supplementary Information The online version contains supplementary material available at 10.1186/s12929-022-00875-2. | PTEN regulates invasiveness in pancreatic neuroendocrine tumors through DUSP19-mediated VEGFR3 dephosphorylation
Phosphatase and tensin homolog (PTEN) is a tumor suppressor. Low PTEN expression has been observed in pancreatic neuroendocrine tumors (pNETs) and is associated with increased liver metastasis and poor survival. Vascular endothelial growth factor receptor 3 (VEGFR3) is a receptor tyrosine kinase and is usually activated by binding with vascular endothelial growth factor C (VEGFC). VEGFR3 has been demonstrated with lymphangiogenesis and cancer invasiveness. PTEN is also a phosphatase to dephosphorylate both lipid and protein substrates and VEGFR3 is hypothesized to be a substrate of PTEN. Dual-specificity phosphatase 19 (DUSP19) is an atypical DUSP and can interact with VEGFR3. In this study, we investigated the function of PTEN on regulation of pNET invasiveness and its association with VEGFR3 and DUSP19.
PTEN was knocked down or overexpressed in pNET cells to evaluate its effect on invasiveness and its association with VEGFR3 phosphorylation. In vitro phosphatase assay was performed to identify the regulatory molecule on the regulation of VEGFR3 phosphorylation. In addition, immunoprecipitation, and immunofluorescence staining were performed to evaluate the molecule with direct interaction on VEGFR3 phosphorylation. The animal study was performed to validate the results of the in vitro study.
The invasion and migration capabilities of pNETs were enhanced by PTEN knockdown accompanied with increased VEGFR3 phosphorylation, ERK phosphorylation, and increased expression of epithelial–mesenchymal transition molecules in the cells. The enhanced invasion and migration abilities of pNET cells with PTEN knockdown were suppressed by addition of the VEGFR3 inhibitor MAZ51, but not by the VEGFR3-Fc chimeric protein to neutralize VEGFC. VEGFR3 phosphorylation is responsible for pNET cell invasiveness and is VEGFC-independent. However, an in vitro phosphatase assay failed to show VEGFR3 as a substrate of PTEN. In contrast, DUSP19 was transcriptionally upregulated by PTEN and was shown to dephosphorylate VEGFR3 via direct interaction with VEGFR3 by an in vitro phosphatase assay, immunoprecipitation, and immunofluorescence staining. Increased tumor invasion into peripheral tissues was validated in xenograft mouse model. Tumor invasion was suppressed by treatment with VEGFR3 or MEK inhibitors.
PTEN regulates pNET invasiveness via DUSP19-mediated VEGFR3 dephosphorylation. VEGFR3 and DUSP19 are potential therapeutic targets for pNET treatment.
The online version contains supplementary material available at 10.1186/s12929-022-00875-2.
Pancreatic neuroendocrine tumors (pNETs) are neoplasms of pancreatic origin with significant neuroendocrine differentiation and expression of neuroendocrine markers [1]. pNETs account for approximately 3% of all pancreatic neoplasms in Taiwan, and the incidence of pNETs has significantly increased worldwide in recent decades [2–4]. The prognosis of pNETs is significantly associated with stage. The overall survival time of the patients with pNETs with regional and distant metastases is significantly shorter than that of the patients with pNETs at a localized stage [3]. Metastasis is a great challenge for the clinical management of the patients. It is well known that the metastatic ability of cancer cells is strongly associated with cell motility, such as cell migration and invasion [5]. Therefore, it is necessary to understand the molecular mechanisms of cell invasiveness in pNETs to prevent and manage metastatic disease. Phosphatase and tensin homolog (PTEN) is a phosphatidylinositol-3,4,5-trisphosphate 3-phosphatase that acts as a tumor suppressor by inhibiting the AKT signaling pathway and regulates cell behaviors, including cell growth, motility, and invasiveness [6]. Low PTEN expression is associated with a higher proportion of liver metastases and shorter disease-free survival/progression-free survival and overall survival times in the patients with pNET [7, 8]. We previously showed that loss of PTEN promotes the proliferation of pNETs in vitro via upregulation of the AKT/mTOR/c-Myc axis [9]. Whether PTEN affects cell invasiveness in pNETs is unclear. Vascular endothelial growth factor receptor 3 (VEGFR3) is a member of the class III receptor tyrosine kinase family that contains seven immunoglobulin-like loops [10]. VEGFR3, which is predominantly expressed in lymphatic vessels and has been identified as a marker for lymphatic endothelial cells. The binding of vascular endothelial growth factor (VEGF) C and VEGFD to VEGFR3, along with the activation of downstream signaling, is essential for lymphangiogenesis [11, 12]. VEGFR3 is also expressed in various tumor cells and plays a role in tumor growth and metastasis in many cancer types [13, 14]. However, the functional role of VEGFR3 in pancreatic neuroendocrine tumorigenesis has not been fully explored. In this study, we investigated the roles of PTEN and VEGFR3 in the invasiveness of pNETs, and delineated the molecular mechanisms by which PTEN regulates VEGFR3 phosphorylation in pNETs.
We purchased human QGP-1 cells from the Japanese Collection of Research Bioresources (JCRB; Tokyo, Japan), and murine NIT-1 cells were obtained from the Bioresource Collection and Research Center (BCRC; Hsinchu, Taiwan). QGP-1 cells were cultured in RPMI-1640 medium (HyClone, South Logan, UT, USA) containing 10% fetal bovine serum (FBS) and antibiotics. NIT-1 cells were cultured in F12-Kaighn’s medium (Gibco, Grand Island, NY, USA) containing 10% FBS and antibiotics. The cell lines were used in this study at or before passage 15. Human wild-type and mutant PTEN (C124S, G129E, and Y138L) [6], and human dual-specificity protein phosphatase 19 (DUSP19) expression plasmids were purchased from Addgene (Cambridge, MA, USA). shRNAs targeting Luc (shLuc), PTEN (shPTEN), VEGFR3 (shVEGFR3), and DUSP19 (shDUSP19) were obtained from the National RNAi Core Facility of Academic Sinica (Taipei, Taiwan). shLuc was used as a control for gene knockdown. Recombinant human VEGFR3-Fc chimeric protein and recombinant human PTEN protein were purchased from R&D Systems (Minneapolis, MN, USA). The VEGFR3 kinase inhibitor, MAZ51, was purchased from Calbiochem (San Diego, CA, USA). Trametinib (GSK1120212) was purchased from AdooQ Biosciences (Irvine, CA, USA). Biotinylated VEGFR3 phosphopeptide was custom-made by Mission Biotech (Taipei, Taiwan). Recombinant human DUSP19 protein was purchased from Origene (Rockville, MD, USA).
Knockdown of PTEN, VEGFR3, and DUSP19 by shRNA in the indicated cell lines was conducted using a lentiviral transduction system according to the instructions of the National RNAi Core Facility of Academic Sinica. Overexpression of human wild-type PTEN, mutant PTEN (C124S, G129E, and Y138L), and human DUSP19 was performed using the Lipofectamine 2000 transfection reagent (Invitrogen, Carlsbad, CA, USA).
Total RNA was isolated from control, PTEN knocked down or PTEN overexpressed QGP-1 cells by using a total RNA mini kit (Geneaid, New Taipei City, Taiwan). A high-capacity cDNA reverse transcription kit (Applied Biosystems Inc, Waltham, MA, USA) was used to perform reverse transcription according to the manufacturer’s protocol. DUSP19 expression was examined by using SYBR green PCR master mix, and GAPDH was used as an internal control to check the efficiency of cDNA synthesis and PCR amplification. The primers used were: DUSP19-forward, 5′-CCAGTGCTTTTTCTTTGGTG-3′; DUSP19-reverse, 5′-TTGCTTTCTTTGCCCTCTTG-3′; GAPDH-forward, 5′-ACGTGATGCAGAACCACCTACTG-3′; and GAPDH-reverse, 5′-ACGACGGCTGCAAAAGTGGCG-3′. The PCR products were separated on a 3% 0.5× Tris–acetate-EDTA agarose gel and visualized under a UVP Biospectrum image system (Upland, CA, USA).
Whole cell lysates were harvested using a lysis buffer. Equal amounts of protein were subjected to SDS-PAGE and transferred onto PVDF membranes. The membranes were incubated with different primary antibodies, including anti-VEGFR3, anti-PTEN, anti-GAPDH (Santa Cruz Biotechnology, Dallas, TX, USA), anti-phospho-VEGFR3 (Cell Applications, San Diego, CA, USA), anti-SLUG, anti-vimentin, anti-p38, anti-phospho-p38, anti-ERK, anti-phospho-ERK, anti-phospho-JNK, anti-AKT, anti-phospho-AKT (Cell Signaling Technology, Danvers, MA, USA), anti-E-cadherin (BD Biosciences, Franklin Lakes, NJ, USA), and anti-DUSP19 (GeneTex, Irvine, CA, USA). The immunocomplexes were detected by probing with anti-mouse or -rabbit IgG conjugated with horseradish peroxidase (Jackson ImmunoResearch, West Grove, PA, USA). Immunoreactive bands were detected on membranes by adding an enhanced chemiluminescence reagent (PerkinElmer Western Lightning Plus-ECL, PerkinElmer, Waltham, MA, USA). Bands were visualized using a UVP BioSpectrum imaging system (Upland, CA, USA). The details of western blot analysis are described in our previous paper [15].
The in vitro invasion assay was performed using 24-well Transwell units with polycarbonate filters (pore size, 8 μm) coated with Matrigel (BD Biosciences) on the upper surface. The lower compartment of the Transwell unit was filled with a medium containing 10% FBS. A total of 1 × 104 cells were seeded in the upper compartment of the Transwell unit, which allowed these cells to invade. Non-invading cells on the upper surface of the membrane were removed after 24 h using a cotton swab. The invading cells on the bottom surface of the membrane were fixed with formaldehyde and stained with the Giemsa solution. Cells were counted under a microscope. For the migration assay, the polycarbonate filters were not coated with matrigel. The other procedures were the same as those used for the invasion assay.
For immunoprecipitation, cell lysates containing 1000 μg of cellular protein were incubated with anti-VEGFR3 antibody (sc-514825, Santa Cruz Biotechnology), anti-DUSP19 (GeneTex) or mouse IgG (sc-2762, Santa Cruz Biotechnology) overnight at 4 °C, and the immunocomplexes were collected with immunoprecipitation reagents. The collected proteins were released by boiling in SDS-PAGE loading buffer and subjected to SDS-PAGE. Western blotting was performed by probing the membranes with anti-VEGFR3 anti-mouse and anti-DUSP19 (GeneTex) anti-rabbit antibodies to detect interactions between VEGFR3 and DUSP19. For immunofluorescence staining, cells were cultured on coverslips, fixed with 4% formaldehyde for 15 min at room temperature, and washed with phosphate-buffered saline (PBS). The cells were permeabilized with 0.1% Triton X-100 for 10 min and incubated with 1% bovine serum albumin to block non-specific binding. Anti-VEGFR3 and anti-DUSP19 antibodies were applied to coverslips and incubated overnight at 4 °C. After extensive washing with PBS, Alexa Fluor 488-conjugated anti-rabbit IgG and Alexa Fluor 594-conjugated anti-mouse IgG (Invitrogen) were added to the coverslips and incubated at room temperature for 1 h. The coverslips were washed twice with PBS and then added with DAPI working solution for 5 min incubation at room temperature with protection from light. The coverslips were then washed twice with PBS and placed in mounting solution. The locations of the fluorescence signals were observed using a fluorescence microscope (LEICA DMI4000 B, Leica Microsystems, Wetzlar, Germany). Immunofluorescence images were also obtained and reconstituted by LEICA DMi8 microscope and Leica Application Suite X (LAS X) software (Leica Microsystems) to produce the XY section and XZ section images.
Biotinylated VEGFR3 phosphopeptide was added to the streptavidin-coated 96-well plates and incubated for 2 h at room temperature. The excess (biotinylated) phosphopeptide was then removed using wash buffer (PBS containing 0.05% Tween 20). Recombinant PTEN or DUSP19 was added to each well and the plate was incubated for 1 h at 37 °C. Recombinant PTEN or DUSP19 was removed using wash buffer and the primary antibody (anti-phospho-VEGFR3) was added to each well of the plate and incubated for 1 h at room temperature. The primary antibody was removed with wash buffer and horseradish peroxidase (HRP)-conjugated secondary antibodies (Biolegend, San Diego, CA, USA) was added to each well and incubated for 1 h at room temperature. Finally, each well of the plate was washed three times with wash buffer and incubated with tetramethylbenzidine (TMB) substrate solution (Clinical Science Products, Mansfield, MA, USA) for 15 min at room temperature prior to measuring the absorbance of each well at 405 nm (Sunrise absorbance reader, TECAN, Männedorf, Switzerland).
Male NOD-SCID mice (6 to 8-week old) were obtained from LASCO (Taipei, Taiwan). Animals were housed under specific pathogen–free conditions according to the guidelines of the Animal Care Committee of the National Health Research Institutes (NHRI), Taiwan. The mice were provided with free access to a standard sterilized laboratory diet and water. QGP-1 cells transduced with shLuc or shPTEN plasmid (1.0 × 107 cells/mouse) were inoculated into the mice by injection into the subcutaneous space in the flank. After approximately 2 weeks, the tumors had grown to a volume of approximately 150–200 mm3. There were 10 mice in the shLuc control group (QGP-1/shLuc) and 30 in the shPTEN group. Thirty mice, inoculated with QGP-1/shPTEN cells, were randomly divided into three groups. Mice in one group (shPTEN/control) were orally administered with PBS (control group) (n = 10). Another 10 mice were treated with 1 mg/kg of trametinib (shPTEN + trametinib group). The remaining ten mice were treated intraperitoneally with 8 mg/kg MAZ51 (shPTEN + MAZ51 group). Tumor volumes were measured twice a week until sacrifice. Tumor volumes were calculated using the formula V = length (mm) × width2 (mm) × (π/6) [16]. The animals were sacrificed by CO2 exposure in their home cages, and the tumors were excised for further analysis.
The animal tissues were fixed in 10% neutral-buffered formalin and then underwent tissue processing, embedding in paraffin wax to make paraffin blocks. Sections (4 µm) sliced using a microtome from paraffin-embedded tissue blocks were in 10 mM Tris-buffered saline containing 0.5% Tween 20, pH 7.6, rehydrated through sequential dilutions of alcohol, and washed in PBS. The slides stained with anti-SLUG primary antibody (1:100; sc-166476, Santa Cruz Biotechnology) were performed on the BOND-MAX Automated Immunohistochemistry Vision Biosystem (Leica Microsystem) using onboard heat induced antigen retrieval and a Leica Bond Polymer Refine Detection system (Leica Biosystems, Wetzlar, Germany). Diaminobenzidine (DAB) was used as the chromogen (Leica Biosystems) in all the immunostainings. To prepare a negative control, IHC was performed without the primary antibody. The invasion status was scored by the extent of tumor invasion to the surrounding tissues. Confinement of the tumor to the original area was scored as 0, focal invasion into the peripheral fibrofatty tissue as 1, multifocal invasion into the peripheral fibrofatty tissue as 2, and invasion into the muscular bundles as 3. To analyze SLUG expression, a semiquantitative assessment of the percentage of positively stained carcinoma cells (from 0 to 100%) along with the staining intensity (graded as 0, no staining; 1, weak staining; 2, moderate staining; and 3, strong staining) was used. Finally, a board-certified pathologist blinded to the specimen information independently assessed the SLUG expression.
RNA sequencing data of 33 pNETs were collected from the GEO GSE118014 database (http://www.ncbi.nlm.nih.gov/geo/). Gene expression was examined by Illumina HiSeq 2000. Raw data were obtained to analyze correlation between PTEN and DUSP19 genes [17].
SAS statistical software (version 9.4, SAS Institute Inc., Cary, NC, USA) was used to perform statistical analyses. Differences in the relative migration rate, invasion rate, and phosphatase activity between pNET cells under the indicated conditions were analyzed using the Wilcoxon rank-sum test. The difference in tumor invasion between the inoculated tumors under the indicated conditions was analyzed using the Wilcoxon rank-sum test. Differences in SLUG expression between inoculated tumors under the indicated conditions were analyzed using Fisher’s exact test. Spearman’s rank-order correlation was calculated and correlation coefficiency (ρ) was expressed to correlate mRNA expression level of PTEN and DUSP19 in pNET tumors from RNA sequencing database. A two-sided P value of less than 0.05 indicated significance.
To understand whether PTEN expression correlates with the invasiveness of pNETs, we knocked down and overexpressed PTEN in a human pNET cell line, QGP-1, and evaluated the invasion and migration abilities of QGP-1 cells. Figure 1A shows that migration and invasion were significantly enhanced in QGP-1 cells with PTEN knockdown. In contrast, migration and invasion were significantly suppressed in QGP-1 cells overexpressing PTEN (Fig. 1B). We evaluated VEGFR3 expression in QGP-1 cells with and without PTEN knockdown or overexpression. Figure 1C shows that VEGFR3 phosphorylation significantly increased in QGP-1 cells upon PTEN knockdown. The molecules associated with epithelial–mesenchymal transition (EMT) were also evaluated. Decreased protein expression of E-cadherin and increased protein expression of vimentin and SLUG were noted in QGP-1 cells with PTEN knockdown (Fig. 1C). Figure 1D shows that VEGFR3 phosphorylation was significantly reduced in the QGP-1 cells overexpressing PTEN. The protein expression of E-cadherin increased, whereas that of vimentin and SLUG decreased in QGP-1 cells overexpressing PTEN. The enhancement of migration and invasion and the increase in VEGFR3 phosphorylation in pNET cells was also demonstrated in a murine pNET cell line, NIT-1, with PTEN knockdown accompanied by associated changes in EMT molecules (Additional file 1: Fig. S1). These data demonstrated that PTEN regulates cell invasiveness and VEGFR3 phosphorylation in pNETs.
To confirm whether PTEN-regulated VEGFR3 phosphorylation is associated with the invasiveness of pNETs, we added the VEGFR3 kinase inhibitor MAZ51 to the culture medium of QGP-1 cells with PTEN knockdown to measure the migration and invasion capabilities of the cells (Fig. 2A). The increase in migration and invasion capabilities of QGP-1 cells upon PTEN knockdown was inhibited by the addition of MAZ51. VEGFC is a major ligand for VEGFR3, which induces receptor phosphorylation and activation of its downstream signaling pathway in human tissues and cancers [11, 12]. We previously showed that PTEN loss induces upregulation of c-Myc and that c-Myc promotes VEGFC upregulation and lymphangiogenesis in pNET cells [9, 18]. To evaluate whether upregulation of VEGFR3 expression in QGP-1 cells with PTEN knockdown is associated with enhanced VEGFC binding, we added a VEGFR3-Fc chimeric protein, R3Fc, to the culture medium of QGP-1 cells with PTEN knockdown. No suppressive effect on cell migration or invasion was observed in culture with the addition of R3Fc (Fig. 2A). Figure 2B shows that the increase in VEGFR3 phosphorylation in QGP-1 cells with PTEN knockdown was reduced by the addition of MAZ51 but not R3Fc. A similar result was observed in the murine pNET cell line, NIT-1 (Additional file 2: Fig. S2). In addition, we knocked down VEGFR3 in QGP-1 cells with PTEN knockdown and found that the enhanced migration and invasion capabilities of QGP-1 cells with PTEN knockdown was significantly suppressed by VEGFR3 knockdown (Fig. 2C). We evaluated the downstream signals of VEGFR3 associated with invasion and migration, including p38, JNK, and ERK, in QGP-1 cells with PTEN knockdown or overexpression, respectively. Only ERK phosphorylation significantly increased in QGP-1 cells after PTEN knockdown (Fig. 3A). A similar result was observed in NIT-1 (Fig. 3B). In contrast, ERK phosphorylation was significantly reduced in QGP-1 cells overexpressing PTEN (Fig. 3C). We added a MEK inhibitor, trametinib, to the culture medium of QGP-1 cells with PTEN knockdown. Figure 4A shows that the enhanced migration and invasion capabilities of QGP-1 cells with PTEN knockdown were significantly suppressed by the addition of trametinib, accompanied by a decrease in the phosphorylated ERK level in cells with PTEN knockdown. A similar result was observed in NIT-1 (Fig. 4B). These results indicate that PTEN regulates VEGFR3 phosphorylation independent of VEGFC, and that PTEN-regulated VEGFR3 phosphorylation is responsible for the invasiveness of pNET cells. Blockade of VEGFR3 or ERK can reverse the increase in the invasiveness of pNET cells with PTEN loss.
We then attempted to delineate the molecular mechanism through which PTEN regulatesVEGFR3 phosphorylation. Although PTEN principally has lipid phosphatase activity, it also has a protein tyrosine phosphatase catalytic domain [6, 19]. To evaluate whether PTEN directly dephosphorylates VEGFR3, we performed an in vitro phosphatase assay by incubating recombinant human PTEN with biotinylated VEGFR3 phosphopeptide. The phospho-signal of the phospho-VEGFR3 peptide was unchanged by addition of recombinant PTEN (Fig. 5A). This result indicated that PTEN does not regulate VEGFR3 phosphorylation through direct interaction with VEGFR3. We also investigated other potential mediators of VEGFR3 phosphorylation. A global analysis of the receptor tyrosine kinase–protein phosphatase interactome showed that VEGFR3 interacts with DUSP19 [20]. This finding suggests that DUSP19 may be a critical mediator of VEGFR3 dephosphorylation by PTEN. DUSPs are heterogeneous protein phosphatases that can dephosphorylate tyrosine and serine/threonine residues on the same substrate. DUSP19 is classified as an atypical DUSP [21]. We evaluated the protein and mRNA expression level of DUSP19 in QGP-1 cells with PTEN knockdown or overexpression. Figure 5B shows that the protein (upper panel) and mRNA (lower panel) level of DUSP19 was significantly reduced in QGP-1 cells with PTEN knockdown. In contrast, the protein and mRNA level of DUSP19 was significantly increased in QGP-1 cells overexpressing PTEN. The protein level of DUSP19 was positively correlated with that of PTEN but negatively correlated with VEGFR3 phosphorylation. In addition, Fig. 5C shows that the protein level of DUSP19 increased in QGP-1 cells overexpressing wild-type, lipid phosphatase-deficient (G129E), protein phosphatase-deficient (Y138L), and both lipid and protein phosphatase-deficient (C124S) PTEN [6]. This result indicated that the mechanism by which PTEN upregulates DUSP19 expression is independent of phosphatase activity. We overexpressed DUSP19 in QGP-1 cells, and reduced the phosphorylation of VEGFR3 in QGP-1 cells was noted (Fig. 5D). Furthermore, we knocked down DUSP19 expression in the QGP-1 cells overexpressing PTEN. Knockdown of DUSP19 restored VEGFR3 phosphorylation in QGP-1 cells overexpressing PTEN, which led to dephosphorylation of VEGFR3 (Fig. 5D). This result demonstrated that VEGFR3 phosphorylation is regulated by DUSP19. These results supported our hypothesis that PTEN transcriptionally upregulates DUSP19 expression to dephosphorylate VEGFR3 in pNET.
To confirm the receptor tyrosine kinase–protein phosphatase interaction of VEGFR3 and DUSP19, an anti-VEGFR3 antibody was used to pull down VEGFR3-associated protein complexes in QGP-1 cells with or without knockdown of DUSP19. DUSP19 was present in the protein complex with VEGFR3 from QGP-1 cells with knockdown of vector control (shLuc) but not present in the protein complex with VEGFR3 from QGP-1 cells with DUSP19 knockdown (shDUSP19). The mouse IgG was used as a negative control for pull down assay (Fig. 6A, left panel). On the other hand, an anti-DUSP19 antibody was used to pull down DUSP19-associated protein complexes in QGP-1 cells with or without knockdown of VEGFR3. VEGFR3 was present in the protein complex with DUSP19 from QGP-1 cells with knockdown of vector control (shLuc) but not present in the protein complex with DUSP19 from QGP-1 cells with VEGFR3 knockdown (shVEGFR3) (Fig. 6A, right panel). Immunofluorescence staining of QGP-1 cells showed that VEGFR3 and DUSP19 were colocalized on the cell membrane (Fig. 6B). Furthermore, z stack image of confocal microscope also showed the co-localization of VEGFR3 and DUSP19 (white arrow) (Fig. 6C). In addition, we performed an in vitro phosphatase assay by incubating recombinant human DUSP19 with a biotinylated VEGFR3 phosphopeptide. The phospho-signal of the phospho-VEGFR3 peptide was reduced by addition of recombinant DUSP19 (Fig. 6D). Taken together, these results confirmed that DUSP19 directly binds to VEGFR3 and dephosphorylates VEGFR3 via its tyrosine phosphatase activity.
We performed an animal study to evaluate the effect of PTEN loss on pNET invasiveness in vivo. Tumor growth in QGP-1 xenograft-bearing mice was not significantly increased by PTEN loss (Fig. 7A). We evaluated the invasion status of the tumors by microscopic observation of hematoxylin and eosin (H&E) staining of the excised xenografts and their surrounding tissues. The invasion score was significantly higher in QGP-1 xenografts with PTEN loss than in control tumors (Wilcoxon rank-sum test, P = 0.017). Representative H&E-stained images of QGP-1 xenografts with and without PTEN loss are shown in Fig. 7B. We administered vehicle control (PBS), the VEGFR3 inhibitor MAZ51, or the MEK inhibitor trametinib to mice bearing QGP-1 xenografts with PTEN loss for 2 weeks (5 days of drug treatment and 2 days of rest) and evaluated tumor growth and invasiveness by H&E staining. Figure 7A shows that tumor growth was not significantly inhibited by MAZ51 or trametinib. However, tumor invasion in mice bearing QGP-1 xenografts with PTEN loss was significantly suppressed by MAZ51 treatment (Wilcoxon rank-sum test, P = 0.021) and tended to be suppressed by trametinib treatment (Wilcoxon rank-sum test, P = 0.096). Representative H&E-stained images are shown in Fig. 7B. We evaluated the expression of the EMT marker SLUG in the xenografts. The staining of SLUG was in the cytoplasm of the tumor cells. A SLUG staining score of 3+ in ≥ 30% of the tumors was defined as high SLUG expression. There were five, nine, three, and seven mice with high expression of SLUG in the control (shLuc), shPTEN, shPTEN + MAZ51, and shPTEN + trametinib groups, respectively. The expression of SLUG in xenografts with PTEN loss (shPTEN) tended to be higher than that in the control (shLuc) tumors (Fisher’s exact test, P = 0.141), although the difference was not statistically significant. The expression of SLUG in xenografts with PTEN loss was suppressed by MAZ51 treatment (Fisher’s exact test, P = 0.020) compared to that in untreated tumors. Figure 7C shows the representative IHC staining of SLUG in the tumors.
In this study, we demonstrated that PTEN loss promotes pNET invasion and migration through the upregulation of VEGFR3 phosphorylation, ERK phosphorylation, and the expression of the EMT molecules vimentin and SLUG in tumor cells. PTEN regulates VEGFR3 phosphorylation through positive regulation of DUSP19 to directly interact with VEGFR3 and dephosphorylate it in pNET cells. The VEGFR3 inhibitor MAZ51 and MEK inhibitor trametinib may reduce the enhanced invasion and migration abilities of pNET cells induced by PTEN loss. The results were validated in a xenograft mouse model. PTEN is well known for its function as a tumor suppressor and a potent inhibitor of the PI3K-AKT-mTOR pathway to regulate cell growth and survival. PTEN also plays a role in other functions, such as cellular metabolism, motility, polarity and senescence [6]. Aberrant PTEN expression has been shown in various cancer types and is associated with tumor proliferation, metastasis, and prognosis in these cancers [22–24]. Low expression of PTEN has also been shown in pNETs and is associated with shorter patient survival times [7]. Previously, we showed that loss of PTEN and/or LKB1 was associated with increased proliferation of pNET cells [9]. The role of PTEN in the invasive or metastatic capability of pNETs is unknown. In studies regarding the invasive and metastatic capabilities of pNETs, Yang et al. showed that autotaxin and neuropilin-1 are upregulated by STAT3 activation upon IL-6 stimulation and are associated with the metastatic capability of the pNET cell line BON1 [25, 26]. Hunter et al. showed that heparinase, an enzyme that degrades heparan sulfate proteoglycans, promotes peritumoral lymphangiogenesis and tumor invasion in a RIP1-Tag2 (RT2) PanNET transgenic mouse model [27]. Sennino et al. reported that concurrent inhibition of VEGF signaling in the tumor microenvironment and c-MET signaling in tumor cells suppresses pNET growth, invasion, and metastasis [28]. In the current study, we showed that the expression status of PTEN affects pNET invasion and migration. VEGFR3 has been well reported for its function in tumor invasion and metastasis in addition to lymphangiogenesis [11–14]. Most studies have reported that VEGFR3 signaling in tumor cells depends on the VEGFC/VEGFR3 axis in an autocrine manner and mainly contributes to tumor invasion and metastasis [14, 29, 30]. Matsuura et al. reported that the autocrine loop of VEGFC/FLT-4 (VEGFR3) in tumor cells promotes tumor proliferation and lymphangiogenesis [29]. Yeh et al. showed that VEGFC/VEGFR3 upregulates SLUG expression via the KRAS-MAPK-YAP1 pathway and enhances cancer migration, invasion and stemness [14]. Li et al. demonstrated that OCT4 increases VEGFC expression, which results in VEGFR3 activation and induction of EMT in esophageal cancer. In addition, OCT4/VEGFC/VEGFR3/EMT signaling promoted tumor growth and intraperitoneal metastasis in a xenograft mouse model [30]. Although we previously showed that PTEN loss can upregulate the mTOR/c-Myc axis in pNETs and that c-Myc activation may upregulate VEGFC to promote lymphangiogenesis in pNETs [9, 18]. Our current study showed that the activation of VEGFR3 signaling in pNETs was not abolished by the addition of a VEGFC-neutralizing chimeric protein, R3-Fc, but was blocked by the VEGFR3 inhibitor MAZ51. This result demonstrated that the regulation of VEGFR3 phosphorylation by PTEN is VEGFC-independent. PTEN is a phosphatase that dephosphorylates both lipid and polypeptide substrates [6]. Although the major biological function of PTEN is to dephosphorylate lipid substrates, it has also been reported to dephosphorylate protein substrates, including cAMP-responsive element-binding protein 1 (CREB1) [31] and insulin receptor substrate 1 (IRS1) [19] to perform its tumor-suppressive function. In our study, as PTEN-regulated VEGFR3 phosphorylation in pNETs was independent of VEGFC, we hypothesized that PTEN acts as a protein phosphatase for VEGFR3. However, we failed to demonstrate direct interaction between VEGFR3 and PTEN. Intriguingly, we found that PTEN transcriptionally upregulates DUSP19 to dephosphorylate VEGFR3 and DUSP19 directly interacts with VEGFR3. A weak positive correlation of PTEN and DUSP19 was noted in the RNA sequencing data of 33 pNET tumors although not statistically significant [Spearman’s ρ = 0.1667 (95% CI − 0.187 to 0.482, and P = 0.3539)] (Additional file 3: Fig. S3) [17]. Because the sample size was small, further study with larger sample size of pNETs is warranted to confirm the positive correlation of PTEN and DUSP19. DUSP19 is an atypical DUSP that localizes in the cytoplasm. DUSP19 appears to mediate the regulation of MAPK and JNK signaling [21]. Wang et al. showed that DUSP19 expression was reduced in osteoarthritis patients and that DUSP19 inhibited chondrocyte apoptosis via JNK dephosphorylation [32]. Yao et al. showed that DUSP19 is negatively regulated by IL-1β and that overexpression of DUSP19 suppresses the activation of JAK2/STAT3 and matrix metalloproteinase (MMP) expression in rat chondrocytes [33]. Although DUSP19 has been shown to function in the regulation of these signaling pathways, its mechanism of action and its substrate are still unknown. Yao et al. analyzed the receptor tyrosine kinase–protein phosphatase interactome and showed the interaction of DUSP19 with VEGFR3 [20]. In our study, we demonstrated that VEGFR3 is the substrate of DUSP19 and that DUSP19 is positively regulated by PTEN in transcriptional level. These results clarify the association between PTEN, DUSP19, and VEGFR3. In addition, we demonstrated the regulation of the EMT regulator SLUG [34], which has been proven to be a downstream target of VEGFR3 and induces cell invasion [14], by PTEN both in vitro and in vivo. Taken together, our results suggest a novel mechanism for the regulation of invasiveness in pNETs with aberrant PTEN expression (Fig. 8). In this study, we demonstrated that the activation of VEGFR3/ERK and EMT molecules in pNETs is induced by PTEN loss to promote tumor invasiveness. Therefore, VEGFR3 and ERK can be targeted to inhibit the invasion and metastasis of pNETs. We chose the potent selective VEGFR3 inhibitor MAZ51 and MEK inhibitor trametinib to validate this hypothesis. Our data showed that both MAZ51 and trametinib effectively inhibited PTEN loss-induced increase in pNET invasion and migration. ERK is a downstream effector of VEGFR3 [35, 36]. Trametinib is an MEK inhibitor that inhibits the MEK and ERK signaling pathways and has been approved for use in the treatment of BRAF-mutant cancers, such as melanoma and anaplastic thyroid cancer, in combination with the BRAF inhibitor dabrafenib [37, 38]. It has also been investigated for use in advanced solid tumors in combination with the anti-VEGFR inhibitor pazopanib or chemoradiation in phase I trials [39, 40]. In our study, trametinib inhibited the invasion and migration of pNET cells as effectively as MAZ51 in vitro but less effectively than MAZ51 in vivo via inhibition of the ERK pathway. Most VEGFR inhibitors are not VEGFR3 selective. However, new ERK inhibitors and highly selective VEGFR3 inhibitors have been developed and are currently under investigation [41, 42]. The novel agents may be candidate anti-invasive drugs for treating pNETs. Our study identified VEGFR3, MEK, and ERK as potential therapeutic targets for controlling pNET metastasis. However, the animal study did not show significant inhibition of tumor growth. This result suggests that the combination of an anti-invasive agent and an anti-proliferative agent, such as sunitinib, everolimus, or chemotherapy, is worthy of further investigation in the treatment of pNETs. On the other hand, we showed that PTEN regulates VEGFR3 and the downstream signaling pathway of VEGFR3 is mediated through the regulation of DUSP19. Targeting DUSP1 and DUSP6 genetically or with a DSUP inhibitor was shown to inhibit malignant peripheral nerve sheath tumor growth and promote cell death [43]. Novel DUSP inhibitors are under investigation [44, 45]. Therefore, DUSP19 is also a potential therapeutic target for pNETs.
We demonstrate a novel mechanism by which PTEN loss promotes pNET invasion and migration by downregulating DUSP19 to inhibit DUSP19-mediated VEGFR3 dephosphorylation. Loss of PTEN is commonly observed in pNETs and is associated with poor prognosis. Our results provide important mechanistic clarification of pNET invasiveness and metastasis, and identify VEGFR3, ERK, and DUSP19 as potential therapeutic targets to prevent and treat pNET metastasis.
Additional file 1: Figure S1. The migration and invasion abilities of NIT-1 cells are negatively regulated by PTEN. (A) The relative migration (P = 0.021, Wilcoxon rank-sum test) and invasion (P = 0.021, Wilcoxon rank-sum test) abilities of NIT-1 cells with and without knockdown of PTEN. (B) The protein levels of VEGFR3, phosphorylated VEGFR3, E-cadherin, vimentin and SLUG in NIT-1 cells with and without PTEN knockdown.Additional file 2: Figure S2. VEGFR3 phosphorylation is responsible for pNET cell invasiveness in a VEGFC-independent manner. (A) The relative migration and invasion abilities of NIT-1 cells with (shPTEN) and without (shLuc) knockdown of PTEN and treated with the VEGFR3-Fc chimera protein (shPTEN-R3Fc) or the VEGFR3 inhibitor MAZ51 (shPTEN-MAZ51). Migration: shLuc vs. shPTEN, P = 0.021; shPTEN vs. shPTEN-MAZ51, P = 0.030; Wilcoxon rank-sum test. Invasion: shLuc vs. shPTEN, P = 0.021; shPTEN vs. shPTEN-MAZ51, P = 0.030; Wilcoxon rank-sum test. (B) The protein level of phosphorylated VEGFR3 in NIT-1 cells with and without knockdown of PTEN and treated with the VEGFR3-Fc chimera protein R3-Fc or the VEGFR3 inhibitor MAZ51.Additional file 3: Figure S3. DUSP19 showed a weak positive correlation with PTEN in pNET tumors. The mRNA expression of PTEN and DUSP19 from the RNA sequencing data of 33 pancreatic neuroendocrine tumors, which were collected from the GEO GSE118014 database (http://www.ncbi.nlm.nih.gov/geo/). Spearman’s ρ = 0.1667 (95% CI − 0.187 to 0.482, and P = 0.3539). | true | true | true |
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PMC9639392 | Murong Xu,Yutong Li,Ying Tang,Xiaotong Zhao,Dandan Xie,Mingwei Chen | Increased Expression of miR-155 in Peripheral Blood and Wound Margin Tissue of Type 2 Diabetes Mellitus Patients Associated with Diabetic Foot Ulcer Xu et al | 03-11-2022 | miR-155,diabetic foot ulcer,type 2 diabetes mellitus,microRNAs,biomarker | Purpose To investigate the correlations of miR-155 expression in the peripheral blood and wound margin tissue of patients with diabetic foot ulcer (DFU) and explore the clinical value of miR-155 as a potential biomarker for the diagnosis and treatment outcomes of DFU. Methods Sixty newly diagnosed T2DM patients without DFU (T2DM group), 112 T2DM patients with DFU (DFU group), and 60 healthy controls (NC group) were included. MiR-155 levels in the peripheral blood and wound margin tissue were determined by quantitative real-time PCR, while clinical features and risk factors of DFU were explored. Multiple stepwise logistic regression analysis was used to determine whether miR-155 expression was an independent risk factor for DFU. The diagnostic effectiveness of miR-155 level on DFU was evaluated using ROC curve analysis. Results A significant decrease in the expression level of miR-155 was observed in T2DM group compared with NC group (P < 0.05), while a markedly increased miR-155 expression level was noted in DFU group compared with T2DM group (P < 0.01). Moreover, there was a negative correlation between the expression levels of miR-155 with healing rate of DFU. Kaplan-Meier survival curve analysis showed that the cumulative rate of unhealed DFU in miR-155 high expression group is higher than that in miR-155 low expression group, both in peripheral blood and wound margin tissue (log rank, P = 0.004, P < 0.001, respectively). The multivariate logistic regression analysis confirmed that a high expression of miR-155 was an independent risk factor for DFU. The ROC curve analysis indicated that the AUC of miR-155 for the diagnosis of DFU was 0.794, with the optimum sensitivity being 96.82% and the optimum specificity of 95.93%. Conclusion The increased expression of miR-155 in peripheral blood of T2DM patients is closely related to the occurrence of DFU. MiR-155 is a potentially valuable biomarker for diagnosis and prognosis of DFU. | Increased Expression of miR-155 in Peripheral Blood and Wound Margin Tissue of Type 2 Diabetes Mellitus Patients Associated with Diabetic Foot Ulcer Xu et al
To investigate the correlations of miR-155 expression in the peripheral blood and wound margin tissue of patients with diabetic foot ulcer (DFU) and explore the clinical value of miR-155 as a potential biomarker for the diagnosis and treatment outcomes of DFU.
Sixty newly diagnosed T2DM patients without DFU (T2DM group), 112 T2DM patients with DFU (DFU group), and 60 healthy controls (NC group) were included. MiR-155 levels in the peripheral blood and wound margin tissue were determined by quantitative real-time PCR, while clinical features and risk factors of DFU were explored. Multiple stepwise logistic regression analysis was used to determine whether miR-155 expression was an independent risk factor for DFU. The diagnostic effectiveness of miR-155 level on DFU was evaluated using ROC curve analysis.
A significant decrease in the expression level of miR-155 was observed in T2DM group compared with NC group (P < 0.05), while a markedly increased miR-155 expression level was noted in DFU group compared with T2DM group (P < 0.01). Moreover, there was a negative correlation between the expression levels of miR-155 with healing rate of DFU. Kaplan-Meier survival curve analysis showed that the cumulative rate of unhealed DFU in miR-155 high expression group is higher than that in miR-155 low expression group, both in peripheral blood and wound margin tissue (log rank, P = 0.004, P < 0.001, respectively). The multivariate logistic regression analysis confirmed that a high expression of miR-155 was an independent risk factor for DFU. The ROC curve analysis indicated that the AUC of miR-155 for the diagnosis of DFU was 0.794, with the optimum sensitivity being 96.82% and the optimum specificity of 95.93%.
The increased expression of miR-155 in peripheral blood of T2DM patients is closely related to the occurrence of DFU. MiR-155 is a potentially valuable biomarker for diagnosis and prognosis of DFU.
Diabetic foot is a serious chronic complication of diabetes. Diabetic foot ulcer (DFU) is the most common manifestation of the diabetic foot and the most common cause of non-traumatic lower limb amputation.1 The risk of a patient with diabetes developing a foot ulcer across their lifetime has been estimated to be 19–34%.2 The pathogenesis of DFU is complex and has not been fully clarified. Early diagnosis, scientific evaluation and timely standardized treatment can effectively improve the prognosis of DFU.3 In recent years, an increasing number of studies have shown that abnormal expression of microRNAs (miRNAs) is closely associated with the occurrence and prognosis of DFU.4 MiRNAs are a class of endogenous non-coding RNAs with a length of approximately 18–25 nucleotides. They can regulate the expression of a target gene by specifically binding to the 3′ untranslated region of downstream target mRNA to guide the silencing complex to degrade mRNA or inhibit protein translation.5,6 MiR-155 is widely involved in many biological processes, such as the development and differentiation of immune cells,7,8 the inflammatory response,9 and it has significant effects on the functions of keratinocytes, fibroblasts, dermal mesenchymal stem cells, and other cells involved in the process of wound healing.10,11 Inhibition of miR-155 expression in local wound tissue can promote skin wound healing in diabetic rats.12,13 Knockout of microRNA-155 can ameliorate the Th17/Th9 immune response in mice and accelerate wound healing.14 In addition, recent studies have shown that mesenchymal stem cell-derived exosomes loaded with miR-155 inhibitor can improve diabetic wound healing.15 However, the clinical research on the correlation between miR-155 and DFU has not been reported. Accordingly, our study aimed to investigate the changes in the expression level of miR-155 in the peripheral blood and wound tissue of DFU patients and its relationship with the pathogenesis and prognosis of DFU.
The participants for this study were selected from the subjects described in a previous study16 and included 112 patients with type 2 diabetes mellitus (T2DM) having DFU complications (DFU group) who were hospitalized in the Department of Endocrinology at the First Affiliated Hospital of Anhui Medical University from January 2018 to December 2019. In these 112 cases, the duration of foot ulcer was more than four weeks, the ulcer area was 2–20 cm2, the Wagner grade was 2–4, the ankle-brachial index (ABI) was 0.7–1.3. Sixty patients with newly diagnosed T2DM without DFU in the Department of Endocrinology at our hospital in the same period were selected as a diabetic group (T2DM group). These patients had a course of T2DM ranging from one week to five months with no lower extremity atherosclerosis disease, and no diabetic peripheral neuropathy. In addition, sixty healthy subjects who underwent physical examination in the Health Management Center of our hospital in the same period were selected as a normal control group (NC group). All the subjects in the NC group underwent a 75 g oral glucose tolerance test to confirm normal glucose tolerance. All subjects had no severe heart, liver, or renal insufficiency; no autoimmune diseases; no severe sepsis; and no cancerous ulcer wounds. This study was approved by the Medical Ethics Committee of the First Affiliated Hospital of Anhui Medical University, and we obtained the informed consent of the subjects.
All patients with DFU were given routine systemic treatment previously reported,16 including anti-infection, reducing blood pressure, reducing blood glucose, correcting hypoproteinemia, nourishing nerves, improving blood supply of lower limb wounds, etc. Wound debridement was performed to remove blackened necrotic soft tissue and bone tissue. The full-sickness skin tissue within 0.5 cm of the wound margin was cut by skilled surgeons using tissue scissors according to the sampling protocol and stored in a refrigerator at −80°C. According to the specific conditions of each DFU, decompression or continuous negative pressure wound therapy was given. The course of the DFU was monitored, and the diabetic foot multidisciplinary team decided whether amputation should be performed. All patients with DFU were followed up until the wound healed completely, and the healing time was recorded. Complete wound healing after 8 weeks was defined as spontaneous complete closure, ie, 100% reepithelization,17 and recorded after eight weeks of treatment.
Venous blood from the elbow was drawn from all subjects into anticoagulation tubes (the anticoagulant was sodium fluoride/EDTA/heparin, selected according to different examination items) or non-anticoagulant tubes at 8:00–8:30 a.m. after fasting for 10 hours. Serum albumin (ALB), blood glucose, blood lipids, glycosylated hemoglobin A1c (HbA1c), white blood cell (WBC) count, hemoglobin (Hb), C-reactive protein (CRP), erythrocyte sedimentation rate (ESR), and other indicators were measured. Blood glucose, blood lipid, and ALB were measured by an automatic biochemical analyzer (Module P800, Roche, Switzerland). Fasting plasma glucose (FPG) was determined using the glucose oxidase method. Total cholesterol (TCH), triglycerides (TG), high-density lipoprotein cholesterol (HDL-C), and low-density lipoprotein cholesterol (LDL-C) were assayed by the oxidase colorimetric method. HbA1c was detected by high-pressure liquid chromatography, CRP by latex enhanced immunoturbidimetry, and ESR by the Wechsler method. The area of skin ulcers was measured by digital photography combined with ImageJ (Image J-ij133-jdk15, National Institutes of Health, Bethesda, USA) medical image analysis software, ABI was measured by a Doppler blood flow detector (DPL-03, Hangzhou Yuanxiang Medical, China), and transcutaneous oxygen partial pressure (TcPO2) was measured by a transcutaneous oxygen partial pressure detector (TCM400, Ledu, Denmark).
The expressions of miR-155 in peripheral venous blood (P-miR-155) and the expressions of miR-155 in wound margin tissue (T-miR-155) were measured by real-time quantitative reverse transcription PCR (qRT-PCR). RNA was extracted from 2-mL EDTA anticoagulant blood samples or 50 mg of wound margin tissue according to the instructions of the miRcute miRNA extraction and separation kit (TIANGEN, Beijing, China). cDNA was then synthesized according to the instructions of miRcute miRNA cDNA synthesis kit (TIANGEN, Beijing, China). The primer sequences of miR-155 were as follows: forward primer 5′-CGGCGGTTAATGCTAATTGTGAT-3′, reverse primer 5′-GTGCAGGGTCCGAGGT-3′; The primer sequences of the endogenous control U6 were as follows: forward primer 5’-GCTTCGGCAGCACATATACTAAAA-3’, reverse primer 5’-CGCTTCACGAATTTGCCTGTCAT-3’. qRT-PCR was carried out according to the instructions of the miRcute miRNA fluorescence quantitative detection kit (TIANGEN, Beijing, China). The cycling conditions were pre-denaturation at 95°C for 5 min, denaturation at 95°C for 20s, annealing at 58°C for 15s, and extension at 72°C for 10s, for a total of 42 cycles. Using U6 as the internal reference, the relative expression of miR-155 was calculated by the 2−ΔΔCt method. Each sample was repeated three times, and the average value was taken as the result.
SPSS 19.0 software was used for statistical analysis. The measurement data are expressed by the mean ± standard deviation (±S), whereas the non-normal measurement data are expressed by the median (quartile interval) [M (P25, P75)]. X2 test or t-test was used for comparison between the two groups; analysis of variance test was used for comparison between multiple groups; and LSD-t test was used for further pairwise comparison. Spearman correlation analysis was used to evaluate the correlation between the expression of miR-155 in both peripheral blood and wound margin tissue and other clinical variables. Multiple stepwise logistic regression analysis was used to determine whether miR-155 in peripheral blood was an independent risk factor for DFU. Kaplan–Meier survival curve analysis was used to study the correlation between the expression of miR-155 in peripheral blood and wound margin tissue and the wound healing of DFU. ROC curve analysis was used to explore the possibility of miR-155 in peripheral blood as a potential biomarker for the diagnosis of DFU. The best sensitivity and specificity of ROC curve were determined by common methods.18 All tests were bilateral, P < 0.05 was considered to be a statistically significant difference.
There were no significant differences in gender composition, age, TCH, or LDL-C levels among the three groups (P > 0.05). The levels of FPG, HbA1c, and TG in the T2DM group and DFU group were higher than those of the NC group, whereas the level of HDL-C was lower than that of the NC group, and the differences were statistically significant (P < 0.05). The expression level of miR-155 in the peripheral blood of the T2DM group was lower than that of the NC group, and that of the DFU group was higher than that of the NC group and T2DM group, and the differences were statistically significant (P < 0.05). In addition, there were no significant differences in TcPO2, ABI, CRP, ESR, ALB, WBC, and Hb values between the NC group and the T2DM group (P > 0.05). Moreover, the duration of diabetes, FPG, HbA1c, CRP, ESR, WBC count, and miR-155 expression levels in peripheral blood in the DFU group were higher than those in the T2DM group, whereas TcPO2, ABI, ALB, and Hb were lower in the DFU group than those in the T2DM group, and the differences were statistically significant (P < 0.05). There was no significant difference in TG and HDL-C levels between the two groups (P > 0.05) (Table 1).
The 112 patients with DFU were divided into two subgroups according to the median level of miR-155 expression in peripheral blood and wound margin tissue, respectively. The patients with miR-155 expression lower than the median were classified as the low expression group and those with expression higher than or equal to the median were classified as the high expression group. The clinical characteristics of DFU between the high expression group and the low expression group were compared. As shown in Tables 2 and 3, the expression levels of miR-155 in both peripheral blood and wound margin tissue of patients with DFU were negatively correlated with the healing rate of foot ulcer after eight weeks (P = 0.037, P = 0.035, respectively), and positively correlated with the course of foot ulcer (P = 0.033, P = 0.034, respectively), and Wagner grade of foot ulcer (P = 0.010, P = 0.013, respectively). There was no correlation between miR-155 expression in both the peripheral blood and wound margin tissue and other clinical characteristics of foot ulcers. In order to further explore the effect of the change of miR-155 expression in peripheral blood and wound margin tissue on wound healing, we used Kaplan–Meier survival curve analysis method. The results showed that the estimated time median of wound healing in peripheral blood miR-155 high expression group and low expression group were 10.23 weeks and 9.51 weeks, respectively; Similarly, the estimated time median of wound healing in wound margin tissue miR-155 high expression group and low expression group were 10.12 weeks and 8.67 weeks, respectively. Whether in peripheral blood or wound margin tissue, the cumulative rate of unhealed DFU in miR-155 high expression group is higher than that in miR-155 low expression group (log rank, P = 0.004, P < 0.001, respectively). The wound healing time of high expression group is longer than that of low expression group (P < 0.05) (Figure 1A and B).
In the NC group, no significant correlation was observed between the expression of miR-155 in peripheral blood and other clinical parameters (P > 0.05). In the T2DM group, the expression of miR-155 in peripheral blood was negatively correlated with the levels of FPG and HbA1c (P < 0.05) and had no significant correlation with other indicators (P > 0.05). In the DFU group, the expression of miR-155 in both peripheral blood and wound margin tissue was positively correlated with the course of foot ulcer, Wagner grade, CRP, and WBC count (P < 0.05), and had no significant correlation with other indicators (P > 0.05). In addition, the expression of miR-155 in peripheral blood was positively correlated with that in wound margin tissue in the DFU group (Tables 4 and 5).
In diabetic patients, DFU was used as a dependent variable, and sex, age, and other variables with P < 0.1 obtained from univariate logistic regression analysis (including the course of diabetes, FPG, HbA1c, TG, LDL-C, HDL-C, ALB, Hb, TcPO2, ABI, CRP, WBC, ESR, P-miR-155) were used as independent variables for multivariate stepwise logistic regression analysis. The course of diabetes, HbA1c, CRP, low TcPO2, high expression of P-miR-155 were independent risk factors for DFU (Table 6).
To further explore the potential value of miR-155 in the diagnosis of DFU, the expression level of miR-155 in the peripheral blood was assessed on an independent group of 172 peripheral blood samples including 60 cases of T2DM and 112 cases of DFU. The ROC curve was determined to evaluate the sensitivity and specificity of peripheral blood miR-155 in diagnosing DFU. The results showed that the AUC of peripheral blood miR-155 for diagnosing DFU was 0.794 (95% CI, 0.726–0.863, P < 0.001), the best cut-off point of miR-155 was 1.01, the sensitivity was 96.82%, and the specificity was 95.93% (Figure 2).
Based on the above research, it is easy to find that the miR-155 expression level in peripheral blood of newly diagnosed T2DM patients is significantly lower than that of subjects with normal glucose tolerance. Conversely, the expression level of miR-155 in DFU patients was dramatically higher than that of patients without DFU. Multiple logistic regression analysis showed that high expression of peripheral blood miR-155 was an independent risk factor for DFU. Further analysis revealed that the expression levels of miR-155 in the peripheral blood and wound margin tissue of patients with DFU were closely related to the Wagner grade, the healing rate of foot ulcers. The patients with high expression of miR-155 in the peripheral blood and wound margin tissue had more serious DFU status, lower healing rate, and longer healing time, indicating that the high expression of miR-155 is not only a strong risk factor for the pathogenesis of DFU but also a potential biomarker for the evaluation, treatment, and prognosis of DFU. To our knowledge, this is the first study to investigate the relationship between changes in miR-155 expression and the onset and treatment outcome of DFU in patients with T2DM. In the present study, peripheral blood miR-155 levels were notably decreased in patients with T2DM compared with control individuals with normal glucose tolerance. Correlation analysis revealed that in patients with T2DM, the expression of miR-155 was negatively correlated with FPG and HbA1c. Animal studies find that overexpression of miR-155 transgenes can improve glucose tolerance and insulin sensitivity in mice, leading to hypoglycemia; in contrast, miR-155 deficiency in mice can impair islet function and lead to hyperglycemia, impaired glucose tolerance, and insulin resistance in the liver, muscle, and adipocytes.19,20 Clinical studies show that the expression level of miR-155 in the peripheral plasma and monocytes of T2DM patients is significantly lower than that of sex-age-matched normal glucose tolerance subjects.21,22 These findings support the results of the present study. Notably, according to the results of this study, we cannot explain the reason for the down-regulation of miR-155 expression in T2DM patients. However, a previous study demonstrated that high blood sugar potentially downregulates the expression of miR-24 by inducing the activation of c-Myc.23 As for whether hyperglycemia directly downregulates the expression of miR-155, it is still uncertain. Therefore, more studies are needed to clarify the mechanism of miR-155 expression change in high glucose environment. However, in the DFU group, there was no significant correlation between the expression level of miR-155 in peripheral blood and wound margin tissue and FPG or HbA1c. We speculate that the reason is that other factors in DFU patients may have more influence on the expression of miR-155 than FPG and HbA1c. In addition, our study also demonstrated that in DFU group, both the expression level of miR-155 in peripheral blood and wound margin tissue was positively correlated with the index of inflammatory state, including CRP, ESR, and WBC count, suggesting that the high expression of miR-155 in the peripheral blood and wound margin tissue of patients with DFU may be related to the state of infectious inflammation. Liu et al found that sepsis patients exhibited a significantly elevated miR-155 level, which was positively related to greater severity of sepsis.24 Chen et al revealed that serum miR-155 level was up-regulated in community-acquired pneumonia patients, and lipopolysaccharides could induce the up-regulation of miR-155 in RAW264.7 cells in vitro.25 These results strengthened our findings. Moreover, although there was a significant difference in the course of diabetes between the DFU group and the T2DM group, further analysis found that there was no correlation between miR155 and the course of diabetes in either the DFU group or the T2DM group. Therefore, the significant difference in the course of disease between the DFU group and the T2DM group might have little effect on the difference of miR-155 expression between the two groups. In this study, the course of foot ulcers in the DFU group was at least four weeks, which can be classified as a chronic refractory wound.26 The clinical features of the DFU group are a long course of diabetes, poor control of blood glucose for a long time, abnormal lipid metabolism, peripheral angiopathy, and infectious inflammation. Multivariate regression analysis showed that the course of diabetes, HbA1c, TcPO2, and CRP were independent factors affecting the occurrence of foot ulcers, which was consistent with the results of previous studies.27,28 Further analysis showed that the levels of miR-155 in the peripheral blood and wound margin tissue of the DFU group were significantly higher than that of the T2DM group; the expression level of miR-155 in both peripheral blood and wound margin tissue of the DFU group was positively correlated with the course of foot ulcer and Wagner grade, and negatively correlated with the healing rate of foot ulcer after 8 weeks, the higher the expression of miR-155, the more difficult and longer the time required for complete healing of DFU wound. Multiple regression analysis showed that the high expression of miR-155 in both peripheral blood and wound margin tissue was an independent risk factor for foot ulcer. These results suggested that miR-155 may be involved in the occurrence of diabetic foot ulcers and can be used as a biomarker for the severity and the prognosis of DFU. At present, the mechanism of miR-155 involved in wound healing has not been fully clarified. It is widely believed that the persistent and excessive inflammatory state in the wound and the functional impairment of a variety of cells in the epidermis involved in wound healing are important factors in the difficulty of DFU healing.3,29 Studies12 show that the expression of miR-155 is increased in skin wounds of diabetic rats. The up regulation of miR-155 not only plays a role in promoting inflammation but also can affect the migration and proliferation of keratinocytes and damage the re-epithelialization of wounds by down-regulating the expression of fibroblast growth factor-7. However, after the knockout of miR-155, M1-like macrophages in wound tissue of mice decrease, M2-like macrophages and type I collagen deposition significantly increase, wound inflammation is alleviated, and repairability is enhanced.30 Another study also found that knocking down miR-155 in mice can also decrease the expression of inflammatory cytokines Th17/Th9 in wound tissue and promote wound recovery.14 In addition, in the skin wound model of diabetic rats, after local injection of miR-155 inhibitor interferes with miR-155 expression, the number of inflammatory cells, such as T lymphocytes, neutrophils, and macrophages in wound tissue decrease, the levels of interleukin-1β and tumor necrosis factor-α decrease, the inflammatory reaction is alleviated, neovascularization significantly increases, the collagen content in granulation tissue increases and is arranged more regularly, and wound healing is accelerated.12,13 Furthermore, angiogenesis in wound tissue plays an important role in DFU wound healing and endothelial progenitor cells (EPC) migrate to the peripheral circulation and differentiate into mature cells to participate in angiogenesis.31 Studies show that miR-155 in M1-like macrophage-derived exosomes can reduce the angiogenesis of endothelial cells,32 and target PTCH1 to mediate EPC dysfunction caused by high glucose.33 It turned out that the expression level of exosomal miR-155 in peripheral blood could be used as a non-invasive biomarker for the diagnosis and progression of hepatic fibrosis34 as well as a potential biomarker for the detection of lung cancer.35 In the present study, according to the results of ROC curve analysis, we also found that the expression level of miR-155 in peripheral blood of T2DM patients could serve as a potential biomarker for the prediction DFU. Moreover, we also discovered that the expression level of miR-155 in both peripheral blood and wound margin tissue was positively correlated with the course of DFU, and negatively correlated with the healing rate of DFU after eight weeks and the complete healing time. Therefore, the abovementioned results suggested the functionality of the expression level of miR-155 in both peripheral blood and wound margin tissue for the diagnosis and prognosis of DFU. Nonetheless, further studies are needed to identify the reasons for the increased expression of miR-155 in both peripheral blood and wound margin tissue of patients with DFU. Significantly, in the present study, miR-155 in peripheral blood and in wound margin tissue exhibited a good consistency in terms of their expression and predictive value for wound healing. Sampling of peripheral blood carries a small risk of trauma, and the determination of miR-155 in peripheral blood is relatively simple and convenient. Therefore, based on our findings and previous studies,36 our research group recommends that the treatment outcome of DFU can be predicted by detecting the expression of miR-155 in peripheral blood.
This study finds that the increased level of miR-155 expression in peripheral blood of type 2 diabetes patients is closely related to the occurrence of DFU, and can be a biomarker for diagnosis of DFU (Figure 3). In addition, the increased level of miR-155 expression in peripheral blood and wound margin tissue is closely related to the poor prognosis of DFU. The shortcomings of this study include that it is a single-center study, the sample size is relatively small, and selection bias may exist. Therefore, further studies are needed to confirm these findings. In addition, this study also cannot clarify the causal relationship between miR-155 and DFU. In the future, we need to further explore the mechanism of action of miR-155 and evaluate whether miR-155 can become a new therapeutic target for DFU. | true | true | true |
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PMC9639483 | 36330810 | Long He,Boqian Wang,Xueyi Wang,Yuewen Liu,Xing Song,Yijian Zhang,Xin Li,Hongwei Yang | Uncover diagnostic immunity/hypoxia/ferroptosis/epithelial mesenchymal transformation-related CCR5, CD86, CD8A, ITGAM, and PTPRC in kidney transplantation patients with allograft rejection | 04-11-2022 | Kidney transplantation,allograft rejection,immunity,hypoxia,ferroptosis,epithelial mesenchymal transformation | Abstract The aim of this study was to identify predictive immunity/hypoxia/ferroptosis/epithelial mesenchymal transformation (EMT)-related biomarkers, pathways and new drugs in allograft rejection in kidney transplant patients. First, gene expression data were downloaded followed by identification of differentially expressed genes (DEGs), weighted gene co-expression network analysis (WGCNA) and protein–protein interaction (PPI) analysis. Second, diagnostic model was construction based on key genes, followed by correlation analysis between immune/hypoxia/ferroptosis/EMT and key diagnostic genes. Finally, drug prediction of diagnostic key genes was carried out. Five diagnostic genes were further identified, including CCR5, CD86, CD8A, ITGAM, and PTPRC, which were positively correlated with allograft rejection after the kidney transplant. Highly infiltrated immune cells, highly expression of hypoxia-related genes and activated status of EMT were significantly positively correlated with five diagnostic genes. Interestingly, suppressors of ferroptosis (SOFs) and drivers of ferroptosis (DOFs) showed a complex regulatory relationship between ferroptosis and five diagnostic genes. CD86, CCR5, and ITGAM were respectively drug target of ABATACEPT, MARAVIROC, and CLARITHROMYCIN. PTPRC was drug target of both PREDNISONE and EPOETIN BETA. In conclusion, the study could be useful in understanding changes in the microenvironment within transplantation, which may promote or sustain the development of allograft rejection after kidney transplantation. | Uncover diagnostic immunity/hypoxia/ferroptosis/epithelial mesenchymal transformation-related CCR5, CD86, CD8A, ITGAM, and PTPRC in kidney transplantation patients with allograft rejection
The aim of this study was to identify predictive immunity/hypoxia/ferroptosis/epithelial mesenchymal transformation (EMT)-related biomarkers, pathways and new drugs in allograft rejection in kidney transplant patients. First, gene expression data were downloaded followed by identification of differentially expressed genes (DEGs), weighted gene co-expression network analysis (WGCNA) and protein–protein interaction (PPI) analysis. Second, diagnostic model was construction based on key genes, followed by correlation analysis between immune/hypoxia/ferroptosis/EMT and key diagnostic genes. Finally, drug prediction of diagnostic key genes was carried out. Five diagnostic genes were further identified, including CCR5, CD86, CD8A, ITGAM, and PTPRC, which were positively correlated with allograft rejection after the kidney transplant. Highly infiltrated immune cells, highly expression of hypoxia-related genes and activated status of EMT were significantly positively correlated with five diagnostic genes. Interestingly, suppressors of ferroptosis (SOFs) and drivers of ferroptosis (DOFs) showed a complex regulatory relationship between ferroptosis and five diagnostic genes. CD86, CCR5, and ITGAM were respectively drug target of ABATACEPT, MARAVIROC, and CLARITHROMYCIN. PTPRC was drug target of both PREDNISONE and EPOETIN BETA. In conclusion, the study could be useful in understanding changes in the microenvironment within transplantation, which may promote or sustain the development of allograft rejection after kidney transplantation.
In recent years, kidney transplantation has been considered as the best therapeutic intervention for patients with end-stage organ failure [1]. However, kidney transplantation brings the risk of allograft rejection. If leaving unchecked, allograft rejection reaction can destroy the graft. With the use of immunosuppressive agents, the incidence of transplant rejection has reduced [2]. Although the annual survival rate of kidney transplant has reached more than 90%, there is a 4–5% loss of function of the kidney graft. The 5-year survival rate of kidney transplant is 70%, whereas the 10-year survival rate is only 50% [2]. Regular monitoring of serum creatinine is an insensitive predictor and only increases upon the deficiency in kidney function [3]. Thus, it is important to identify potential diagnostic and therapeutic markers that associated with different molecular mechanisms in the process of allograft rejection in kidney transplant patients. Activation of the immune system in recipients is majorly responsible for allograft rejection [4,5]. The severity of the allograft dysfunction process is positively correlated with the incidence of T cell-mediated acute rejection [6]. Hypoxia, an inevitable event accompanying kidney transplantation, is regarded as a common cause for delayed graft function [7–10]. In response to hypoxia, tubular epithelial cells can produce multiple pro-inflammatory factors and trigger tubule interstitial inflammation [11–13]. Ferroptosis, characterized by membrane damage, is an iron-dependent and regulated cell death [14]. Ferroptosis-related indicators, including iron and lipid peroxides are associated with renal fibrosis [15–20]. Epithelial–mesenchymal transition (EMT) is the indispensable process in embryonic development and organ fibrosis [21]. It is noted that the EMT is involved in the progression of interstitial fibrosis in kidney allograft with chronic kidney allograft dysfunction [22]. Maybe, there are complex regulatory mechanisms among immunity, hypoxia, ferroptosis, and EMT, which may be important factors in allograft rejection after the kidney transplant. In view of this, the aim of the present study is to explore predictive immunity/hypoxia/ferroptosis/EMT biomarkers, pathways, and new drugs in the process of graft rejection in kidney transplant patients, thus enabling more accurate and less invasive diagnosis.
Gene expression data were downloaded from the Gene Expression Omnibus (GEO) dataset. Keywords of ‘kidney transplant’ and ‘Homo sapiens’ were used to filter the gene expression profile data. The corresponding data set was then filtered using the following criteria. Inclusion criteria for dataset are as follows: (1) there are more than five cases; (2) there is rejection information. Exclusion criteria for dataset are as follows: (1) the study is conducted at the cell line or animal level; (2) there is a single case in the study; (3) repetitive or overlapping study. Finally, a total of four datasets (involving kidney transplant biopsy sample) were included in the study, including GSE36059 (involving 122 patients with allograft rejection and 281 patients without allograft rejection), GSE48581 (involving 78 patients with allograft rejection and 222 patients without allograft rejection), GSE129166 (involving 35 patients with allograft rejection and 60 patients without allograft rejection), and GSE124203 (involving 774 patients with allograft rejection and 905 patients without allograft rejection). Randomly, GSE36059, GSE48581, and GSE129166 datasets were considered as a training set. GSE124203 datasets were considered as a validation set. For the above four datasets, the gene expression matrix files were downloaded and annotated using annotation files of GPL platform. For datasets of GSE36059, GSE48581, and GSE129166, the combat function in ‘SVA’ in R package was utilized to remove batch effect. The combined dataset included 235 cases and 563 normal controls.
In the training set, the ‘llimma’ package was used to identify DEGs in kidney transplant patients with allograft rejection. The screening criteria of DEGs were false discovery rate (FDR) <0.05 and |log2 fold change (FC)| >0.5. The volcano map was used for visualization of DEGs. The ‘WGCNA’ in R package was utilized to analyze the co-expression network of all genes, followed by the construction of the scale-free gene co-expression network. Genes with similar expression patterns were gathered together. Modular signature genes (ME) were defined as the first major component in each module. To identify the key modules most associated with allograft rejection, the ME of each module was calculated using the ‘moduleEigengenes’ function. Pearson’s method was applied to analyze the correlation with allograft rejection. Modules with the highest positive and negative correlation with allograft rejection were chosen as hub modules.
First, in order to study the function of common genes in hub modules and DEGs in kidney transplant patients with allograft rejection, David database was used for Gene Ontology (GO) analysis. In addition, GSVA analysis was carried out to reveal differences in metabolic pathways. Significantly enriched GO terms and pathways were identified under the threshold value of FDR <0.05. Second, common genes in hub modules and DEGs were put into the STRING database to study the regulatory relationship between proteins encoded by these genes. The PPI network was constructed by Cytoscape software. CytoHubba is one of the plug-ins in Cytoscape software, which provides 11 topology analysis methods [23]. Finally, a total of seven topology analysis methods were adopted to screen central genes, including Degree, EPC, MNC, MCC, Closeness, Betweenness, and Stress. The first 20 node genes of each algorithm score were identified through the R package ‘UpSet’ to screen the multi-center intersection genes, which were considered as key genes involved in allograft rejection after the kidney transplant.
The receiver operating characteristic (ROC) curve was used to determine the accuracy of key genes in the diagnosis of allograft rejection after the kidney transplant. The area under curve (AUC) is an evaluation index of model performance. The AUC value ranges from 0 to 1, where 0.7 is acceptable performance and 0.9 is excellent performance. First, ROC curves of the combinations of key genes were plotted. Then, ROC curve of the single key gene was plotted separately in allograft rejection and non-rejection groups. Finally, the accuracy of the model was verified in the validation set.
First, to explore the influence of miRNA-gene regulatory relationship on the occurrence and development of allograft rejection after the kidney transplant, the miRNA-key gene regulatory network was constructed based on the interaction data of miRDB Database. Second, TRRUST Database was used to study the role of TFs in key gene regulation.
First, the single-sample gene set enrichment analysis (ssGSEA) algorithm was used to quantify the abundance of each cell infiltrate in the immune microenvironment (IME). Gene sets that mark each infiltrating immune cell type in IME were obtained from previous studies [24,25]. To observe the immune status of kidney transplant patients with allograft rejection, enrichment score was used to represent the relative abundance of each infiltrating cell in IME in each sample. Second, the status of hypoxia in the kidney transplant patients with allograft rejection was inferred from the hypoxia marker gene set in the MSigDB Database, which includes 200 hypoxia-related genes. Third, status of ferroptosis and EMT in the kidney transplant patients with allograft rejection was inferred from the literature [26,27]. Finally, the correlation between immune, hypoxia, ferroptosis, EMT and key genes was analyzed.
In order to provide a new perspective for disease diagnosis, treatment, and research for kidney transplant patients with allograft rejection, drugs related to key genes were screened out based on DGIdb Database (https://dgidb.org/).
Statistical analysis was performed using R version 3.5.3 (R Foundation for Statistical Computing, Vienna, Austria). The Limma package was used for differential expression analysis. Modules positively associated with allograft rejection were screened using the ‘WGCNA’ package. The function of the gene set was studied by using David database. The regulation relationship between the gene set was performed by using STRING database. ROC analysis was performed using the R package ‘pROC’ to calculate the AUC to assess the accuracy of genes in the diagnosis of allograft rejection. Wilcoxn.test was used to compare the differences of different immune cells in allograft rejection. Pearson’s correlation analysis was used to analyze the relationship between genes and immune cells.
After data preprocessing, 21,655 intersection genes were identified in the training set (GSE36059, GSE48581, and GSE129166) (Figure 1(A)). A total of 319 DEGs were identified in the kidney transplant patients with allograft rejection, including 313 up-regulated and six down-regulated genes. Volcano map and heat map of all DEGs are shown in Figure 1(B,C), respectively.
WGCNA was used to identify genes related to allograft rejection after the kidney transplant. First, samples were clustered and four abnormal samples were deleted. When the parameter value of the weight coefficient is 24, the scale-free topology is approximate (Figure 2(A)). After building the cluster tree, the minimum number of genes in modules was set to 100, which separate seven modules (gray modules were not included). The dynamic cutting tree method was utilized to merge the modules with the dissimilarity degree <25%. Finally, five modules were identified (Figure 2(B,C)). As shown in Figure 2(D), the red module had the highest positive correlation with allograft rejection after kidney transplant (Pearson’s r = 0.45; p = 3E–41). Some up-regulated genes in the red module were identified, such as C-C motif chemokine receptor 5 (CCR5), CD86 molecule (CD86), CD8a molecule (CD8A), integrin subunit alpha M (ITGAM), and protein tyrosine phosphatase receptor type C (PTPRC). The blue module had the highest negative correlation with allograft rejection after kidney transplant (Pearson’s r = −0.2; p = 1E–08). Some down-regulated genes in the blue module were identified, such as 4-hydroxyphenylpyruvate dioxygenase (HPD) and afamin (AFM). Therefore, red and blue modules were chosen as hub modules, which involved 1066 genes.
Totally, 270 common genes were identified in hub modules (involving 1066 genes) and DEGs (involving 319 genes) in the kidney transplant patients with allograft rejection. Based on GO analysis, immune response, external side of plasma membrane and identical protein binding were the most significantly enriched biological process, cytological component, and molecular function, respectively (Figure 3(A)). In the GSVA analysis, a total of 148 metabolic pathways were identified. Some metabolic pathways were more active in the allograft rejection group, such as graft versus host disease and type I diabetes mellitus (Figure 3(B)).
These 270 common genes were put into STRING database to study the regulatory relationship between proteins encoded by these genes in kidney transplant patients with allograft rejection (Figure 4(A)). Outermost 39 genes were derived from the union of the first 20 genes of seven topology analysis methods. After screening of the first 20 node genes of each algorithm score, a total of five key genes were identified (Figure 4(B)), including CCR5, CD86, CD8A, ITGAM, and PTPRC. The heat map of the above five key genes is shown in Figure 5(A). Moreover, the up-regulation of the above five key genes was verified in the validation set (Figure 5(B)).
The diagnostic model for kidney transplant patients with allograft rejection was constructed based on five key genes in the training set (Figure 6(A)). AUC value was 0.802. The diagnostic model was also verified in the validation set (Figure 6(B)). The AUC value in the validation set was 0.903. This suggested that the diagnostic model based on five key genes had an excellent diagnostic performance for kidney transplant patients with allograft rejection. Additionally, the diagnostic value of the single key gene was analyzed in the training set (Figure 7(A)) and the validation set (Figure 7(B)). The AUC value of above five key genes was more than 0.7, which suggested a potential diagnostic value of these genes for kidney transplant patients with allograft rejection.
Based on interaction data of miRDB database, the miRNA-key gene regulatory network was constructed in kidney transplant patients with allograft rejection (Figure 8(A)). There were respectively 121, 30, 67, 60, and 65 related miRNA with PTPRC, ITGAM, CD8A, CD86, and CCR5. Three miRNA-key gene regulatory pairs were identified, including hsa-miR-8485-ITGAM/CD86, hsa-miR-12123-PTPRC, and hsa-miR-664a-3p-CCR5/CD8A. According to the TRRUST database, the role of TFs in regulation of five key genes was investigated (Figure 8(B)). It is noted that TFs of nuclear factor kappa B subunit 1 (NFKB1) and RELA proto-oncogene, NF-kB subunit (RELA) regulated the expression of CCR5 and CD86.
The ssGSEA was used to evaluate the status of 23 types of immune cell infiltration in the training set in kidney transplant patients with allograft rejection (Figure 9(A)). Infiltration degree of 23 types of immune cells was high in the kidney transplant patients with allograft rejection. In the validation set (Figure 9(B)), apart from neutrophil and immature dendritic cells, the infiltration degree of the rest of 21 types of immune cells was elevated in kidney transplant patients with allograft rejection. Interestingly, all 23 types of immune cells were significantly positively correlated with five key genes (Figure 9(C)). For example, activated CD4 T cells, activated CD8 T cells, myeloid-derived suppressor cells (MDSCs), regulatory T cells, and T follicular helper cells were significantly positively correlated with PTPRC, CD8A, CD86, ITGAM, and CCR5, respectively.
There are 200 hypoxia-related genes in the MSigDB database. Moreover, these 200 genes are highly expressed in hypoxia state. A total of seven common genes were identified between 200 hypoxia-related genes and 319 DEGs, including caveolin 1 (CAV1), C-X-C motif chemokine receptor 4 (CXCR4), interferon stimulated exonuclease gene 20 (ISG20), placenta associated 8 (PLAC8), S100 calcium binding protein A4 (S100A4), transforming growth factor beta induced (TGFBI), and TNF alpha induced protein 3 (TNFAIP3). These seven genes were up-regulated in kidney transplant patients with allograft rejection in training set (Figure 10(A)) and validation set (Figure 10(B)). Moreover, all seven hypoxia-related genes were significantly positively correlated with five key genes (Figure 10(C)). It is noted that TNFAIP3, ISG20, PLAC8, TGFBI, and CXCR4 were significantly positively correlated with PTPRC, CD8A, CD86, ITGAM, and CCR5, respectively.
Ferroptosis status was predicted based on the suppressors of ferroptosis (SOFs) and drivers of ferroptosis (DOFs) in the literature. Some SOFs, such as CD44 molecule (CD44) and carbonic anhydrase 9 (CA9) were respectively significantly up-regulated and down-regulated in kidney transplant patients with allograft rejection in training set (Figure 11(A)) and validation set (Figure 11(B)). Some DOFs, such as ATM serine/threonine kinase (ATM) and phosphatidylethanolamine binding protein 1 (PEBP1) were respectively significantly up-regulated and down-regulated in kidney transplant patients with allograft rejection in training set (Figure 11(C)) and validation set (Figure 11(D)). Depending on the correlation analysis between SOFs and five key genes (Figure 11(E)), CD44 was significantly positively correlated with five key genes. CA9 was the most significantly negatively correlated with PTPRC. According to the correlation analysis between DOFs and five key genes (Figure 11(F)), ATM was the most significantly positively correlated with PTPRC. PEBP1 was the most significantly positively negatively with ITGAM.
Based on the evaluation of EMT status, EMT2 and EMT3 were higher in the kidney transplant patients with allograft rejection (Figure 12(A)). Similarly, EMT2 and EMT3 were higher in the kidney transplant patients with allograft rejection in the validation set (Figure 12(B)). It is worth mentioning that EMT2 and EMT3 were significantly positively correlated with ITGAM (Figure 12(C)).
Drugs associated with four key genes were screened based on DGIdb database (Figure 13). It is a pity that no related drugs were found for CD8A in the DGIdb database. CD86, CCR5, and ITGAM were respectively drug target of ABATACEPT, MARAVIROC, and CLARITHROMYCIN. In addition, PTPRC was drug target of both PREDNISONE and EPOETIN BETA.
CCR5, a chemokine receptor, is associated with the pathogenesis of a wide spectrum of health conditions, such as inflammatory diseases and autoimmune diseases. In a rat renal acute rejection model, CCR5 is significant up-regulated after allogeneic transplantation [28]. Interruption of the CCR5 is related to prolongation of allograft survival [29,30]. In addition, in kidney transplant recipients, those who are homozygous for CCR5 delta 32 have improved survival [31]. CD86, expressed on antigen-presenting cells, suppresses host immunity [32,33]. The numbers of circulating CD86+ after kidney transplant are significantly higher than those at pre-transplantation [34]. CD8A is significant up-regulated after kidney transplantation [28]. ITGAM, a member of the β2 integrin family of adhesion molecules, is expressed by cells of the myeloid lineage [35]. ITGAM is expressed by some kidney tubules. ITGAM plays essential roles in the adhesion of monocytes, macrophages, and the uptake of pathogens [36,37]. PTPRC is involved in regulating B cell and T cell receptor signaling. PTPRC is up-regulated in stable and acute kidney transplant patients [38,39]. In this study, CCR5, CD86, CD8A, ITGAM, and PTPRC were up-regulated and had the positive correlation with allograft rejection in kidney transplant patients. It is noted that a combination or single gene of the above five genes had a potential diagnostic value for kidney transplant patients with allograft rejection. Thus it can be seen that CCR5, CD86, CD8A, ITGAM, and PTPRC play crucial roles in the process of allograft rejection and can be considered as potential diagnostic markers for allograft rejection after the kidney transplant. Both innate and adaptive immune systems play critical roles in allograft rejection after the kidney transplant, among which T lymphocytes are the main cells for recognizing allografts [40]. According to function, T cells are divided into CD4+ T cells, CD8+ T cells and Treg cells [41,42]. Significantly higher RNA expression levels of CD4 are found in blood samples of patients with T-cell-mediated kidney transplant rejection [43]. Natural killer (NK) cells interact directly with CD4+ T lymphocytes and induce acute rejection mechanisms [44]. CD8+ T lymphocytes infiltrate the kidney during allograft rejection [45]. CD8+ senescent T cells are linked to a reduced possibility of allograft rejection after kidney transplantation [46,47]. Relatively few effector memory CD8+ T cells and effector CD8+ T cells are found in the peripheral blood of patients receiving immunosuppressive therapy after kidney transplantation [48]. In peripheral blood of kidney transplant patients, low regulatory T cells are related to allograft rejection and poor outcomes [49–55]. In addition, regulatory T cells can suppress memory CD8+ T cell and contribute to allograft survival [56]. T follicular helper cells induce differentiation of B lymphocyte and contribute to rejection [57–60]. Inhibition differentiation and function of T follicular helper cell can prevent the development of anti-donor antibody responses in transplantation [61–63]. In kidney transplantation, MDSCs reveal a strong immune suppressive ability [64]. In kidney transplant patients, MDSCs expand T regulatory cells [65]. In the present study, all 23 types of immune cells were significantly positively correlated with CCR5, CD86, CD8A, ITGAM, and PTPRC. Moreover, activated CD4 T cells, activated CD8 T cells, MDSCs, regulatory T cells, and T follicular helper cells were significantly positively correlated with PTPRC, CD8A, CD86, ITGAM, and CCR5, respectively. It is indicated that PTPRC, CD8A, CD86, ITGAM, and CCR5 may play key roles in the immune systems, which are associated with allograft rejection after the kidney transplant. In allografts, local over expression of vascular endothelial growth factor (VEGF) results in chronic rejection. Hypoxia is the major stimulating factor of VEGF expression [66,67]. Herein, seven hypoxia-related genes were up-regulated in kidney transplant patients with allograft rejection. It is noted that TNFAIP3, ISG20, PLAC8, TGFBI, and CXCR4 were significantly positively correlated with PTPRC, CD8A, CD86, ITGAM, and CCR5, respectively. In kidney transplantation, TNFAIP3 expression is linked to outcome prediction [68]. ISG20 is up-regulated in acute rejection after kidney transplant [69]. ISG20 could be a novel therapeutic target of renal fibrosis [70]. TGFBI can promote renal fibrosis [71]. The antagonist of CXCR4 effectively reduces the rejection intensity after transplantation [72,73]. The positive correlation between hypoxia-related genes and PTPRC, CD8A, CD86, ITGAM, and CCR5 may be associated with allograft rejection after the kidney transplant. Ferroptosis is considered to play key regulatory roles in acute kidney injury. However, the role of ferroptosis in immune rejection after kidney transplantation remains unclear [74]. In this study, two SOFs, CD44, and CA9 were respectively significantly up-regulated and down-regulated in kidney transplant patients with allograft rejection. Two DOFs, ATM, and PEBP1 were respectively significantly up-regulated and down-regulated in kidney transplant patients with allograft rejection. There is a prominent continuous expression of CD44 by the endothelial cells of kidney allograft in acute rejection [75]. CD44 absence leads to attenuated kidney injury following ischemia or reperfusion injury [76]. CA9, a membrane protein, regulates cell proliferation in response to hypoxia [77,78]. CA9 can serve as a potential target for renal cell carcinoma-specific immunotherapy [79]. Activation of ATM is found in renal ischemia or reperfusion injury [80]. PEBP1, plays roles in anti-inflammatory effects under homeostatic/basal conditions, is associated with kidney allograft rejection [81,82]. This suggested the association of ferroptosis and allograft rejection after kidney transplantation. In addition, CD44 was significantly positively correlated with PTPRC, CD8A, CD86, ITGAM, and CCR5. CA9 and ATM were respectively the most significantly negatively and positively correlated with PTPRC. PEBP1 was the most significantly positively negatively with ITGAM. These results indicated that SOFs (CD44 and CA9) and DOFs (ATM and PEBP1) showed a complex regulatory relationship between ferroptosis and PTPRC, CD8A, CD86, ITGAM, and CCR5. EMT plays key roles in the fibrosis process of renal grafts [83]. In the present study, EMT2 and EMT3 were higher in the kidney transplant patients with allograft rejection. Moreover, EMT2 and EMT3 were significantly positively associated with ITGAM. EMT2 and EMT3 were significantly linked to renal cell carcinoma [84]. Positively correlation between EMT2, EMT3, and ITGAM may be involved in the fibrosis process after kidney transplant. Based on regulatory networks between miRNAs and PTPRC, CD8A, CD86, ITGAM, and CCR5, three miRNA-key gene regulatory pairs were identified, including hsa-miR-8485-ITGAM/CD86, hsa-miR-12123-PTPRC, and hsa-miR-664a-3p-CCR5/CD8A. In addition, TFs of NFKB1 and RELA regulated the expression of CCR5 and CD86. NFKB1 is an inflammatory marker. After kidney transplantation, the NFKB1 promoter polymorphism (-94ins/delATTG) is related to susceptibility to cytomegalovirus infection [85]. Increased expression of RELA is associated with renal thrombotic microangiopathy [86]. Our result suggested that the regulation relationship between miRNA, TFs and PTPRC, CD8A, CD86, ITGAM, and CCR5 could be associated with inflammatory response in the development of allograft rejection after the kidney transplant. It is reported that existing immunosuppressive drugs are not sufficient to completely prevent allograft rejection in kidney transplant patients [87,88]. Therefore, it is needed to find potential drug targets for kidney transplant patients with allograft rejection. Based on DGIdb database, PTPRC was drug target of both PREDNISONE and EPOETIN BETA. In addition, CD86, CCR5, and ITGAM were respectively drug target of ABATACEPT, MARAVIROC, and CLARITHROMYCIN. PREDNISONE is an essential component of immunosuppression protocols during the first three decades of clinical kidney transplantation [89]. Anemia is a common complication of kidney transplantation. In kidney transplant recipients with moderate renal insufficiency, correction of anemia with EPOETIN BETA can slow the decline in glomerular filtration rate, reduce the incidence of end-stage renal disease, and improve quality of life without increasing the risk of cardiovascular events [90]. Treatment success of ABATACEPT has been found in post-kidney transplant patients [91]. MARAVIROC impairs lymphocyte chemotaxis with a theoretical reduction in organ transplant rejection [92]. CLARITHROMYCIN is utilized to prevent and treat infection in kidney transplant recipients [93]. Thus, it can be seen that PTPRC, CD86, CCR5, and ITGAM could be considered as potential targets of PREDNISONE, EPOETIN BETA, ABATACEPT, MARAVIROC, and CLARITHROMYCIN, which may provide novel treatment options for kidney transplant patients with allograft rejection. Beside above five diagnostic key genes positively correlated with allograft rejection, two genes negatively correlated with allograft rejection were found, including HPD and AFM. HPD is down-regulated in renal ischemia–reperfusion injury [94]. AFM is a biomarker of acute kidney transplant rejection [95]. Decrement in AFM is observed in early acute kidney allograft rejection [96]. It is reported that decreased expression of HPD and AFM may be associated with allograft rejection after the kidney transplant. In addition, based on GSVA analysis, some metabolic pathways were more active in the allograft rejection group, such as graft versus host disease and type I diabetes mellitus. Graft versus host disease is a rare complication after kidney transplantation [97]. New-onset diabetes after transplantation, another complication in kidney transplant recipients, can increase the risk of infections, allograft loss, and mortality [98,99]. In conclusion, five diagnostic genes were identified in kidney transplantation patients with allograft rejection, including CCR5, CD86, CD8A, ITGAM, and PTPRC. Highly infiltrated immune cells, highly expression of hypoxia-related genes and activated status of EMT were significantly positively related to these diagnostic genes. SOFs and DOFs showed a complex regulatory relationship between ferroptosis and five diagnostic genes. CD86, CCR5, and ITGAM were respectively drug target of ABATACEPT, MARAVIROC, and CLARITHROMYCIN. PTPRC was drug target of both PREDNISONE and EPOETIN BETA. Our study could be useful in understanding changes in the microenvironment within kidney transplantation. However, there are limitations to our study. First, the mRNA or protein expression validation analysis of CCR5, CD86, CD8A, ITGAM, and PTPRC is needed in kidney transplant biopsy sample from transplant recipients with graft rejection compared to who do not present dysfunction events. Second, the potential pathological mechanism of these genes should be investigated in cell lines or animal models. Third, the potential interaction mechanism between immune cell and CCR5, CD86, CD8A, ITGAM, and PTPRC are needed to investigate in the future study. | true | true | true |
PMC9639510 | Kaikai Zhang,Ming Cheng,Jingtao Xu,Lijian Chen,Jiahao Li,Qiangguo Li,Xiaoli Xie,Qi Wang | MiR-711 and miR-183-3p as potential markers for vital reaction of burned skin Forensic Sciences Research | 21-04-2020 | Forensic sciences,forensic pathology,vital reaction,skin burn,miR-711,miR-183-3p | Abstract In forensic practice, the identification of antemortem burns and postmortem burns is of the utmost importance. Reports from previous studies have shown that miRNAs, with lengths stretching over 18–25 nucleotides, are highly stable and resistant to degradation. However, there has been little research into the application of miRNAs in identifying antemortem and postmortem burns. This study compared the expression of miR-711 and miR-183-3p levels in mouse and postmortem human burned skins using RT-qPCR assay. RT-qPCR examination of burned mouse skins showed that increased miR-711 and miR-183-3p expression in comparison to intact skin tissues. The increased expressions of these two miRNAs were observed until 120 h after death in burned mouse skins, whereas no significant changes were found in postmortem burned skins. In human burned skins, the increased levels of these two miRNAs at 48 h following autopsy occurred in 19 of 26 subjects, which appeared to be related to the severity of the burn. These findings suggest that miR-711 and miR-183-3p may act as biomarkers for vital reaction of skin burn. Key points This study investigated miR-711 and miR-183-3p levels in mouse and postmortem human burned skins using RT-qPCR. Increased miR-711 and miR-183-3p levels were observed in burned mouse skins. The increased expressions of these two miRNAs were observed until 120 h after death in burned mouse skin. The increased levels of these two miRNAs were observed until 48 h after autopsy in 19 of 26 forensic cases, which appeared to be related to the severity of the burn. | MiR-711 and miR-183-3p as potential markers for vital reaction of burned skin Forensic Sciences Research
In forensic practice, the identification of antemortem burns and postmortem burns is of the utmost importance. Reports from previous studies have shown that miRNAs, with lengths stretching over 18–25 nucleotides, are highly stable and resistant to degradation. However, there has been little research into the application of miRNAs in identifying antemortem and postmortem burns. This study compared the expression of miR-711 and miR-183-3p levels in mouse and postmortem human burned skins using RT-qPCR assay. RT-qPCR examination of burned mouse skins showed that increased miR-711 and miR-183-3p expression in comparison to intact skin tissues. The increased expressions of these two miRNAs were observed until 120 h after death in burned mouse skins, whereas no significant changes were found in postmortem burned skins. In human burned skins, the increased levels of these two miRNAs at 48 h following autopsy occurred in 19 of 26 subjects, which appeared to be related to the severity of the burn. These findings suggest that miR-711 and miR-183-3p may act as biomarkers for vital reaction of skin burn. This study investigated miR-711 and miR-183-3p levels in mouse and postmortem human burned skins using RT-qPCR. Increased miR-711 and miR-183-3p levels were observed in burned mouse skins. The increased expressions of these two miRNAs were observed until 120 h after death in burned mouse skin. The increased levels of these two miRNAs were observed until 48 h after autopsy in 19 of 26 forensic cases, which appeared to be related to the severity of the burn.
Wound examination is an important step, especially during the assessment of a burned body [1, 2]. Traditional methods of distinguishing antemortem burns from postmortem burns are based on the external and internal vital reactions, such as increased carbon monoxide-haemoglobin in circulation, erythema and blisters, soot deposits in respiratory and/or digestive lumen [3]. Those which are quickly workable have their drawbacks when it comes to some complex conditions: postmortem burns and putrefaction can also cause the appearance of erythema and blisters, and soot deposits and elevated carbon monoxide-haemoglobin can be absent in open areas [4]. In recent years, some studies have been conducted to determine wound vitality, though most of them mainly focused on proteins and mRNAs [5–10]. MicroRNAs (miRNAs/miRs), whose lengths are about 18–25 nucleotides, control gene expression by binding to the 3′-UTR of relevant mRNAs [11, 12]. Because of their wide distribution, easy storage, high conservation, and participation in various life activities, miRNAs have become a hot topic in clinical research [13]. However, there has been little research into the application of miRNAs in identifying antemortem and postmortem injuries. The primary function of miR-711 is to modulate cell proliferation, apoptosis, as well as the cell cycle, and has become a probable biological target for the diagnosis and treatment of tumours [14, 15]. MiR-183 may be associated with the process of human non-small cell lung cancer and the regulation of the blood–brain barrier [16, 17]. Our previous study [1] used a miRNA microarray method to assess gene expression landscape of skin burn model. MiR-711 and miR-183-3p levels increased in antemortem burned skins. However, the application value of these miRNAs in forensic investigations has not been optimally exploited. Therefore, the levels of miR-711 and miR-183-3p in human and mouse burned skins were explored and the utility of these miRNAs as vitality indicators of burned skins was evaluated.
Male BALB/c mice (7–9 weeks old; (25 ± 3)g) were purchased from the Animal Centre of Southern Medical University (Guangzhou, China). A burned skin model was created as detailed in a prior study [1]. After the deep partial-thickness burn generated for 30 min, all mice were sacrificed with an overdose of anesthesia (60 mg/kg i.p.) and maintained at 24 °C. A 5 mm × 5 mm burned skin patch was cut at 0, 6, 12, 24, 48, 72, 96, and 120 h time-points (three mice per group). Skins obtained from unburnt mice served as the control (three mice per group). To obtain postmortem burn samples, after shaving the dorsal hairs, the mice were killed immediately (three mice). Dorsal skin burns were made 30 min after death with the same heated sheet of copper for 4 s. Then the postmortem burned skin specimen was harvested and stored in liquid nitrogen at −80 °C.
Twenty-six human burned skin specimen harvested from forensic autopsy cases, with eight from females and 18 from males. The subjects’ age ranged from 23 to 55 years old. Intact regions without burning or other kinds of injuries were harvested from shoulder or abdominal skins of the same subjects as the control samples. Information concerning all subjects is presented in Supplementary material 1. First-degree burns are often superficial and appear reddish without blisters. Second-degree burns display erythema and skin blistering. For third-degree burns, substantial necrosis is observed in the epidermis and dermis [2]. The period from the injury to the time of death was considered as the survival period. We defined warm time as the period from death to cold storage and postmortem interval as the period from death time to autopsy. Tissue histopathology was carried out as per the routine procedures. Each tissue sample was subcategorized into sections weighing 0.1 g each and stored in sterilized and 2.0 mL microtubes (BIO-BIK) at 24 °C for 0, 24 and 48 h. Thereafter, they were immediately frozen and stored.
The Agilent 2100 Bioanalyzer (Agilent Technologies, Palo Alto, CA, USA) was used to determine the RNA integrity number (RIN). Subsequently, cDNA was synthesized from the RNA using the PrimeScript RT reagent Kit (TaKaRa, Shiga, Japan). RT-qPCR was run on the Illumina Eco Real-Time PCR System (San Diego, CA, USA) under standard conditions. The mRNA level was calculated and normalized to the mRNA level of U6 using 2−△△Ct method. All reactions, protocols, and conditions are provided in Supplementary materials 2 and 3.
The gene expression products were assessed as triplicates and presented as mean ± SEM. Individual groups were compared using the non-parametric Mann–Whitney U test. Spearman’s rho method was employed for correlation analysis of paired parameters. All statistics were performed using GraphPad Prism version 5.01 (GrophPad Software, San Diego, CA, USA). P-value <0.05 was considered significant.
Data analysis revealed that RIN values were not significantly different between postmortem burn, control, and antemortem burn groups. However, a postmortem interval-dependent decrease in RIN was observed in the burned mice skin specimen (Figure 1). Higher levels of miR-711 and miR-183-3p were recorded in burned skins in comparison with the control and postmortem burned regions (Figure 2). We further examined whether postmortem intervals regulated miR-711 and miR-183-3p levels. Increased miR-711 and miR-183-3p levels in the burned regions were observed within 120 h after death (Figure 3).
Distinct differences were observed in RIN values among the groups but sex, age, postmortem interval, or warm time were not markedly different among the groups as evidenced by results of Spearman’s rho (P > 0.05). MiR-711 and miR-183-3p levels revealed considerable inter-individual variations in intact skin samples (Figure 4). Thus, fold changes of miR-711 and miR-183-3p levels in burned regions were compared with the intact skins from the same individuals. In burned regions, increased miR-711 and miR-183-3p levels were observed in 19 of 26 cases. For first-degree burn samples, miR-711 and miR-183-3p levels were increased in three of six cases (Figure 5). For second-degree burn samples, miR-711 and miR-183-3p levels were increased in 11 of 15 cases (Figure 6). For third-degree burn samples, these miRNAs were up-regulated in all five cases (Figure 7). The effects of postmortem intervals were also evaluated. RIN values showed a postmortem interval-dependent reduction in burned human skin tissues (Figure 8). Increased miR-711 and miR-183-3p levels were detected until 48 h after autopsy in all the above-mentioned 19 cases (Supplementary materials 4–6).
The identification of antemortem and postmortem burns is extremely important for the field of forensic pathology. When a corpse is found at a fire scene, a forensic pathologist needs to find out whether the person was alive upon sustaining the burns and determine whether the death occurred due to thermal injury. However, conventional findings to identify antemortem burns are usually unspecific, or even absent. Therefore, novel markers for burn vitality are needed. Previous studies, using the immunohistochemical method, reported that increased P-selectin, fibronectin, heat-shock protein 70, von Willebrand factor, and PECAM-1 were detected in the respiratory tract and lungs of fire victims, which support intravital reaction in fatal burns [5, 10, 18, 19]. Kubo et al. [4] reported that the increased mRNA expression of AQP3 was observed in antemortem burn skin. MiRNAs are very short nucleic acids which are resistant to extreme temperatures and pH. Some studies have reported that miRNAs are stable at room temperature and can be identified after long-term fixation [20]. Some miRNAs even remain detectable in storage at −20 °C for 10 years [21, 22]. In our previous study [1], an miRNA microarray technique was used to determine the miRNA expression profiles. A total of 24 differentially expressed miRNAs were observed in burned mice skins compared to that in unburned skins. Among these differentially expressed miRNAs, levels of miR-711 and miR-183-3p were significantly increased. This study reproduced the burned skin model, and evaluated the application of miR-711 and miR-183-3p as potential markers for vital reaction. In our animal experiment, we found that miR-711 and miR-183-3p levels were higher in the antemortem burned regions than those in the intact skin samples. In addition, postmortem burn did not induce changes in miR-711 and miR-183-3p levels in mouse skins, suggesting that these two miRNAs are potential biomarkers for differentiating antemortem from postmortem burns. In contrast to mice specimen, tissues obtained during autopsy are usually affected by postmortem intervals [23]. The effects of putrefaction and autolysis should, therefore, not be ignored. Therefore, the effects of postmortem intervals on these two miRNAs were also investigated. RIN is an indicator for assigning total RNA integrity [24]. In this study, RIN values tended to decrease in a postmortem interval-dependent manner in mice skin specimen, showing considerable degradation of RNA. However, the increased miR-711 and miR-183-3p levels can still be observed until 120 h after death. These results showed that RNA integrity was compromised by postmortem intervals, but miR-711 and miR-183-3p were considerably stable, at least for 120 h after death. Twenty-six human skin tissues were used to validate the mice model results. These two miRNAs levels were markedly higher in burned regions of 19 cases relative to intact regions. These significant changes can still be observed until 48 h after autopsy. Interestingly, the increased miRNAs seemed to be related to burn degree. The increased miR-711 and miR-183-3p levels were observed in three of six cases for first-degree burn samples, in 11 of 15 cases for second-degree burn samples, and in all five cases for third-degree burn samples. Further studies should therefore explore the underlying mechanisms. MiR-711 has been shown to be mainly involved in cell proliferation, apoptosis and regulation of the cell cycle. Its up-regulation suggests that it may participate in the repair of skin tissue after burns and promote the apoptosis of injured cells. MiR-183-3p has been reported to be involved in the opening of the blood–brain barrier. In this study, it may be related to the regulation of skin capillary permeability after burns and increase the biological functions, such as inflammatory factor exudation. These may be the possible reasons for the significant increase of these two miRNAs after burns, which still require further investigation. In conclusion, the present study using mouse and postmortem human burned skin tissues suggests that the detection of miR-711 and miR-183-3p levels might be useful for the determination of vital reaction of burned skin.
Kaikai Zhang and Ming Cheng carried out the genetic studies and participated in the miRNA microarray analysis; Jingtao Xu and Jiahao Li carried out the RT-qPCR and performed the statistical analysis; Lijian Chen and Qiangguo Li established the animal model and drafted the manuscript; Xiaoli Xie conceived of the study and participated in its design; Qi Wang participated in coordination and helped to draft the manuscript. All authors contributed to the final text and approved it. | true | true | true |
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PMC9640266 | Yunxin Zhang,Wentao Zhang,Wenlong Xia,Junwei Xia,Haishan Zhang | Downregulation of hsa-miR-135b-5p Inhibits Cell Proliferation, Migration, and Invasion in Colon Adenocarcinoma | 21-10-2022 | Colon cancer is the most common malignant tumor of the gastrointestinal tract, and approximately 80%–90% of colon cancers are colon adenocarcinomas (COADs). This study aimed to screen key microRNAs (miRNAs) associated with COAD. Differentially expressed (DE) miRNAs were screened between COAD and adjacent cancer samples based on the Gene Expression Omnibus (GEO) and the Cancer Genome Atlas obtained from datasets. The miRNAs of interest were validated using quantitative real-time polymerase chain reaction. Moreover, the effects of hsa-miR-135b-5p on the biological behavior of COAD cells were observed. To obtain the target genes of hsa-miR-135b-5p, transcriptome sequencing of the SW480 cells was performed, followed by protein-protein interaction (PPI) network and hsa-miR-135b-5p-target gene regulatory network construction and prognostic analysis. Downregulation of hsa-miR-135b-5p significantly inhibited SW480 cell proliferation, migration, and invasion and significantly facilitated apoptosis (P < 0.05). A total of 3384 DEmRNAs were screened, and enrichment analysis showed that the upregulated mRNAs were enriched in 25 Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways and 326 Gene Ontology Biological Processes (GO-BPs) while the downregulated mRNAs were enriched in 20 KEGG pathways and 276 GO-BPs. A PPI network was then constructed, and H2BC14, H2BC3, and H4C11 had a higher degree. In addition, a total of 352 hsa-miR-135b-5p-gene regulatory relationships were identified. Prognostic analysis showed that FOXN2, NSA2, MYCBP, DIRAS2, DESI1, and RAB33B had prognostic significance (P < 0.05). In addition, the validation analysis results showed that FOXN2, NSA2, and DESI1 were significantly expressed between the miR-135b-5p-inhibitor and negative control groups (P < 0.05). Therefore, downregulation of hsa-miR-135b-5p inhibits cell proliferation, migration, and invasion in COAD, and carcinogenesis may function by targeting FOXN2, NSA2, MYCBP, DIRAS2, DESI1, and RAB33B. | Downregulation of hsa-miR-135b-5p Inhibits Cell Proliferation, Migration, and Invasion in Colon Adenocarcinoma
Colon cancer is the most common malignant tumor of the gastrointestinal tract, and approximately 80%–90% of colon cancers are colon adenocarcinomas (COADs). This study aimed to screen key microRNAs (miRNAs) associated with COAD. Differentially expressed (DE) miRNAs were screened between COAD and adjacent cancer samples based on the Gene Expression Omnibus (GEO) and the Cancer Genome Atlas obtained from datasets. The miRNAs of interest were validated using quantitative real-time polymerase chain reaction. Moreover, the effects of hsa-miR-135b-5p on the biological behavior of COAD cells were observed. To obtain the target genes of hsa-miR-135b-5p, transcriptome sequencing of the SW480 cells was performed, followed by protein-protein interaction (PPI) network and hsa-miR-135b-5p-target gene regulatory network construction and prognostic analysis. Downregulation of hsa-miR-135b-5p significantly inhibited SW480 cell proliferation, migration, and invasion and significantly facilitated apoptosis (P < 0.05). A total of 3384 DEmRNAs were screened, and enrichment analysis showed that the upregulated mRNAs were enriched in 25 Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways and 326 Gene Ontology Biological Processes (GO-BPs) while the downregulated mRNAs were enriched in 20 KEGG pathways and 276 GO-BPs. A PPI network was then constructed, and H2BC14, H2BC3, and H4C11 had a higher degree. In addition, a total of 352 hsa-miR-135b-5p-gene regulatory relationships were identified. Prognostic analysis showed that FOXN2, NSA2, MYCBP, DIRAS2, DESI1, and RAB33B had prognostic significance (P < 0.05). In addition, the validation analysis results showed that FOXN2, NSA2, and DESI1 were significantly expressed between the miR-135b-5p-inhibitor and negative control groups (P < 0.05). Therefore, downregulation of hsa-miR-135b-5p inhibits cell proliferation, migration, and invasion in COAD, and carcinogenesis may function by targeting FOXN2, NSA2, MYCBP, DIRAS2, DESI1, and RAB33B.
Colon cancer is the most common malignant tumor in the gastrointestinal tract and ranks third and second in terms of morbidity and mortality, respectively, among all solid cancers [1–3]. Approximately 80–90% of colon cancers are colon adenocarcinomas (COADs) based on pathologic classification [4, 5]. In China, due to the increase in poor living and dietary habits, the incidence rate of COAD has also been increasing [6]. More than 83% of patients with COAD are at an advanced stage upon diagnosis, and nearly half of them are accompanied by metastasis from other sites and have a poor prognosis [7]. Surgical treatment is the most effective treatment for COAD; however, the effect of surgical resection is closely related to the preoperative staging of patients [8, 9]. Thus, further studies on the molecular mechanism of COAD occurrence and development and exploration of new key molecules may provide novel ideas for the treatment of COAD. MicroRNAs (miRNAs) are non-coding RNAs 20–24 nt long and regulate target gene expression by binding to the 3′ untranslated region of the target gene, which affects a series of physiological processes [10]. Studies have revealed that miRNAs are involved in almost all signaling pathway regulations in cancer and that there are differences in tumor diagnosis, staging, progression, prognosis, and chemotherapy [11–13]. Uncontrolled proliferation is a major feature of cancer and is the basis of its development [14]. As regulators, miRNAs affect tumor growth by targeting key members of the COAD-related proliferation signaling pathway [15]. The expression of many miRNAs is different between normal and COAD tissues. For instance, Mi et al. revealed that high miR-31-5p expression facilitated COAD progression by targeting TNS1 [16]. Zhao and Qin found that miRNA-708, which targets ZNF549, regulates COAD development through the PI3K/AKt pathway [17]. Liu and Di Wang revealed that miR-150-5p inhibits TP53 to facilitate the proliferation of COAD [18]. Therefore, screening for novel miRNAs in COAD is important. Therefore, this study was conducted to explore the key miRNAs correlated with the development of COAD as well as the molecular mechanisms involved. First, the common differentially expressed (DE) miRNAs were screened between COAD and adjacent cancer samples based on the Gene Expression Omnibus (GEO) and the Cancer Genome Atlas (TCGA) datasets. In addition, miRNAs of interest were verified using quantitative reverse transcription polymerase chain reaction (qRT-PCR), and hsa-miR-135b-5p was screened. Moreover, the effects of hsa-miR-135b-5p on the biological behavior of COAD cells were observed, and transcriptome sequencing was performed to identify target genes of hsa-miR-135b-5p. This study provides new clues for the treatment of COAD. A flowchart of the study is presented in Figure 1.
The processed miRNA expression profile data of the GSE125961 dataset (six COAD tissues and six adjacent cancer tissues) were downloaded from the GEO database (https://www.ncbi.nlm.nih.gov/geo/), after which the miRNA data were normalized. TCGA-COAD miRNA data (450 COAD samples and eight adjacent cancer samples) were also obtained from the University of California, Santa Cruz, database (https://xenabrowser.net/datapages/), and the miRNAID was converted to the mature miRNA ID of the V21 version.
After data preprocessing, DEmiRNAs were identified between COAD and adjacent cancer samples using the “limma” package (version 3.10.3) [19], with the threshold set at P < 0.05 and |log fold change (FC)| > 2. Moreover, overlapping DEmiRNAs screened from the GEO and TCGA datasets were obtained.
miRWalk3.0 [20] was used to predict the target genes of the overlapping DEmiRNAs in the miRTarBase, TargetScan, and miRDB databases. The target genes in all three databases were then obtained to build a miRNA-target gene regulatory network using Cytoscape (version 3.2.0) [21].
The 20 COAD and corresponding paracancerous tissues (including seven females and 13 males aged 46–78 years) were obtained from the China-Japan Union Hospital of Jilin University. This study was approved by the ethics committee of the China-Japan Union Hospital of Jilin University (N:2021081011). Informed consent was obtained from all subjects.
Human COAD cell lines (SW480, HT29, and HCT116) and a human colon epithelial cell line (NCM460) were purchased from Procell Life Science & Technology Co. NCM460 cells were cultured in 90% Dulbecco's Modified Eagle Medium: F12; SW480 cells were cultured in 90% L-15 base medium; and HT29 and HCT116 cells were cultured in 90%McCOY's 5 A base medium. The cells were supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin solution at 37°C in 95% air and 5% CO2, which was then replaced with a complete medium, and the cells were cultured for 24–48 h. The cells were then transfected with hsa-miR-135b-5p inhibitors or a negative control using Lipofectamine 2000 following the manufacturer's instructions (incubation at room temperature for 20 min).
Total RNA was extracted using TRIzol, and RNA concentration and quality were determined using a microplate reader (Infinite M100 PRO; TECAN, Switzerland). Total RNA was reverse-transcribed using a reverse transcription kit (MR101-01; Vazyme Biotech Co., Ltd., China), and the cDNA was used for qRT-PCR. snRNA U6 was used as an internal reference. The primer sequences are listed in Table 1.
After 0, 24, 48, or 72 h of incubation, cell viability was analyzed using CCK8 (C0037; Beyotime, China). The cells were first cultured in an incubator at 37°C and with 5% CO2 for 0, 24, 48, or 72 h, followed by treatment with CCK8 (20 μL per well) at 37°C for 2 h. OD450 was measured using a microplate reader (Infinite M100 PRO; TECAN). Each experiment was performed in triplicate.
After transfection with hsa-miR-135b-5p inhibitors or the negative control, the cells were collected in a flow tube, washed with phosphate-buffered saline (PBS), and then centrifuged. Cell apoptosis was assessed using an Annexin V/fluorescein isothiocyanate (FITC) and propidium iodide (PI) apoptosis detection kit (C1062L; Beyotime) according to the manufacturer's instructions. Annexin V-FITC, PI, and 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid buffer were mixed at a ratio of 1 : 2 : 5 to make a dye liquor, of which 100 μL was used to resuspend 1 × 106 cells. Cell apoptosis was analyzed using FlowJo software. Each experiment was performed in triplicate.
After transfection with hsa-miR-135b-5p inhibitors or the negative control, the cells were collected and centrifuged. A transwell assay was performed to detect cell migration and invasion, as described previously [22]. After incubation at 37°C for 16 h, the transwell chamber was washed with PBS and fixed in 95% ethanol for 5 min. The cells were stained with crystal violet for 10 min, washed with PBS, and analyzed under an optical microscope (IX73; Olympus, Japan) using ImageJ software. Each experiment was performed in triplicate.
Total RNA was obtained from hsa-miR-135b-5p inhibitor or negative control-transfected SW480 cells using TRIzol reagent. RNA integrity, purity, and concentration were determined using NanoDrop2000. Sequencing libraries were generated using NEBNext® Ultra™ RNA Library Prep Kit for Illumina® (E7530S; New England Biolabs, USA) according to the manufacturer's instructions, and index codes were added to attribute sequences to each sample. Sequencing was performed on an Illumina sequencing platform with the PE300 bp sequencing mode. After cluster generation, the library preparations were sequenced on an Illumina HiSeq platform, and paired-end reads were generated. Quality control of the reads was conducted using in-house written scripts. Raw reads in FASTQ format were processed using in-house Perl scripts. Transcriptome sequencing data were uploaded to the National Center for Biotechnology Information database using the BioProject ID PRJNA870261.
Raw counts were normalized using the median ratio method in the “DESeq2” package (version 1.18.1) [23], and differential expression analysis was performed to identify DEmRNAs between the hsa-miR-135b-5p inhibitor and negative control groups using the Wald test with cutoff values of P < 0.05 and |log2 FC| ≥ 1. In addition, enrichment analysis was performed on the identified up and downregulated mRNAs using the “clusterProfiler” package (version 3.2.11) [24] in R (version 3.4.4) with a threshold of P.adjust <0.05, and count ≥ 2. The Benjamini and Hochberg method was used to adjust the P value.
A PPI network of the top 50 upregulated and downregulated mRNAs was built using the STRING database (version 11) [25], and the parameters were set as follows: species, Homo sapiens; and PPI score > 0.4..
miRWalk3.0 [20] was used to identify the target genes of hsa-miR-135b-5p in miRWalk, miRtarbase, TargetScan, and miRDB databases. Target genes with a score ≥ 0.9 in more than two databases were acquired and intersected with the DEmRNAs, after which overlapping mRNAs were obtained. A regulatory network was then constructed using Cytoscape (version 3.6.1) [21].
The gene expression of RNA sequencing (log2(fpkm − uq + 1)) and clinical data (TCGA Colon and Rectal Adenocarcinoma (COADREAD) Phenotype) of Genomic Data Commons (GDC) TCGA (cohort: GDC Pan-Cancer) were obtained from the TCGA database [26]. Then, the matrix data of TCGA COADREAD mRNA and the clinical information of overall survival time in miRNA-target were acquired. The “survival” package (version 2.42–6) [27] in R (version 3.4.4) was used to perform prognostic analysis with a threshold of P < 0.05.
SPSS 22.0 software was used for statistical analysis. One-way analysis of variance and Newman–Keuls multiple comparison tests were used to compare the differences between groups. Statistical significance was set at P < 0.05.
According to the cutoff value of P < 0.05 and |log FC| > 2, 223 DEmiRNAs (170 upregulated and 53 downregulated) and 134 DEmiRNAs (60 upregulated and 74 downregulated) were identified from the GEO and TCGA datasets, respectively (Figures 2(a) and 2(b)). A total of 26 overlapping DEmiRNAs were obtained, including 17 upregulated and nine downregulated miRNAs (Figure 2(c)). In addition, a total of 194 miRNA-target gene regulatory relationships were acquired, including 17 miRNAs and 188 genes (Figure 3).
Of the 26 overlapping DEmiRNAs, six miRNAs that were not reported in COAD were selected, namely, hsa-miR-135b-5p, hsa-miR-19a-3p, hsa-miR-33a-5p, hsa-miR-328-3p, hsa-miR-139-5p, and hsa-miR-490-3p. qRT-PCR was performed on tissue and SW480 cells, and the results showed that only hsa-miR-135b-5p expression was significantly higher in the tumor groups (both in cell and tissue samples) than in the control groups (P < 0.05; Figure 4(a)).
Experiments were performed to determine whether hsa-miR-135b-5p influences the biological behavior of COAD cells. The expression of hsa-miR-135b-5p was significantly reduced in the hsa-miR-135b-5p inhibitor group compared with the inhibitor negative control and control groups (P < 0.05; Figure 4(b)). In addition, the CCK8 assay results showed that after reducing hsa-miR-135b-5p expression, cell growth was significantly reduced (P < 0.05; Figure 4(c)). Transwell assays showed that migration and invasion of COAD cells were significantly inhibited after reducing hsa-miR-135b-5p expression (P < 0.05; Figure 4(d)). Meanwhile, flow cytometric analysis revealed that cell apoptosis was markedly increased after reducing hsa-miR-135b-5p expression (P < 0.05; Figure 4(e)).
A total of 3384 DEmRNAs (2012 upregulated and 1372 downregulated) were identified between the hsa-miR-135b-5p inhibitor and negative control groups (Figure 5(a)). Enrichment analysis showed that the upregulated mRNAs were enriched in 25 Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways (e.g., ribosome, oxidative phosphorylation, and systemic lupus erythematosus) and 326 Gene Ontology Biological Processes (GO-BPs; e.g., signal recognition particle-dependent cotranslational protein targeting to membrane, cotranslational protein targeting to membrane, and protein targeting to endoplasmic reticulum) as shown in Figures 5(b) and 5(c), while the downregulated mRNAs were enriched in 20 KEGG pathways (e.g., extracellular matrix (ECM)-receptor interaction, protein digestion and absorption, and hematopoietic cell lineage) and 276 GO-BPs (e.g., cell-substrate adhesion, extracellular matrix organization, and extracellular structure organization) as shown in Figures 5(d) and 5(e).
A PPI network containing 45 nodes and 65 interaction pairs (Figure 6(a)) was constructed based on the identified DEmRNAs. H2BC14 (degree = 9), H2BC3 (degree = 9), and H4C11 (degree = 9) had higher degrees in the PPI network (Table 2).
A total of 352 regulatory relationships were identified, and 10 overlapping genes were obtained (Figure 6(b)), namely, NSA2, FOXN2, DIRAS2, DESI1, SV2C, RAB33B, MCTS1, CNIH4, SLCO5A1, and MYCBP (Figure 6(c)).
Prognostic analysis was conducted on the 10 overlapping genes, and the results showed that FOXN2 (P = 0.0085), NSA2 (P = 0.044), MYCBP (P = 0.0047), DIRAS2 (P = 0.0015), DESI1 (P = 0.022), and RAB33B (P = 0.037) had prognostic significance (P < 0.05; Figure 7). Of them, DIRAS2 was related to poor prognosis, while the other genes were related to better prognosis. Moreover, the six prognosis-related genes were validated, and the results showed that FOXN2 expression was significantly reduced while NSA2 and DESI1 expression was significantly increased in the miR-135b-5p-inhibitor group than in the negative control group (Figure 8). In contrast, MYCBP was not expressed in either group (P < 0.05).
Dysregulated miRNAs play crucial roles in tumorigenesis of human cancers [28, 29]. In this study, we found that downregulation of hsa-miR-135b-5p significantly inhibited SW480 cell proliferation, migration, and invasion and significantly facilitated apoptosis. In addition, a total of 3384 DEmRNAs were identified, and enrichment analysis showed that the upregulated mRNAs were enriched in 25 KEGG pathways and 326 GO-BPs and the downregulated mRNAs were enriched in 20 KEGG pathways and 276 GO-BPs. A PPI network was then constructed wherein H2BC14, H2BC3, and H4C11 had a higher degree. Furthermore, a total of 352 hsa-miR-135b-5p-gene regulatory relationships were identified. Prognostic analysis showed that FOXN2, NSA2, MYCBP, DIRAS2, DESI1, and RAB33B have prognostic significance. We first used miRNA expression profile data to screen the DEmiRNAs in COAD and adjacent cancer samples, and a total of 26 overlapping DEmiRNAs were obtained from the GEO and TCGA datasets. Six miRNAs of interest were selected among the 26 overlapping DEmiRNAs for validation via qRT-PCR, and the results showed that only hsa-miR-135b-5p was expressed at significantly higher levels in the tumor groups than in the control groups. Numerous studies have reported that hsa-miR-135b-5p is dysregulated in many human cancers and plays a crucial role in cancer progression. Naorem et al. demonstrated that hsa-miR-135b-5p is dysregulated in triple-negative breast cancer [30]. Lazzarini et al. showed that hsa-miR-135b-5p is differentially expressed in normal myometrium and leiomyomas [31]. Magalhães et al. found that in both diffuse and intestinal gastric cancer subtypes, APC is modulated by hsa-miR-135b-5p [32]. However, hsa-miR-135b-5p in COAD has not yet been reported. In this study, our in vitro experiments revealed that changes in hsa-miR-135b-5p expression influenced the biological behavior of COAD cells. Downregulation of hsa-miR-135b-5p resulted in significantly reduced growth, migration, and invasion and markedly increased apoptosis of COAD cells, which may provide novel insights into the treatment of COAD. To understand the exact mechanism underlying the effects of hsa-miR-135b-5p in COAD, transcriptome sequencing was performed. A total of 3384 DEmRNAs were screened, and enrichment analysis showed that the upregulated mRNAs were enriched in 25 KEGG pathways, and the downregulated mRNAs were involved in 20 KEGG pathways, including ribosome, oxidative phosphorylation, and ECM-receptor interaction. Ribosomes are essential for cellular growth, survival, and proliferation, and disruption of ribosome biogenesis can promote cell cycle arrest; thus, ribosome biogenesis is related to cancer [33]. Many studies have shown that oxidative phosphorylation can be upregulated in cancers and may be used as a target in cancer therapy [34–36]. The ECM is a non-cellular component of tissue, and previous studies have reported that ECM-receptor interactions play an important role in the development and metastasis of colorectal cancer [37–39]. Thus, we hypothesized that hsa-miR-135b-5p promotes COAD progression via the ribosome, oxidative phosphorylation, and ECM-receptor interaction pathways. Additionally, a PPI network was constructed, and H2BC14 (degree = 9), H2BC3 (degree = 9), and H4C11 (degree = 9) had a higher degree in the network. Valle et al. found that H2BC3, also known as HIST1H2BB, has growth-suppressing roles and can be used as a high-grade serous carcinoma precision medicine biomarkers [40]. Meanwhile, only a few studies on H2BC14 and H4C11 in cancer have been reported. The target genes of hsa-miR-135b-5p were searched and were intersected with the DEmRNAs, resulting in a total of 10 overlapping genes. Prognostic analysis showed that FOXN2, NSA2, MYCBP, DIRAS2, DESI1, and RAB33B had prognostic significance. In addition, the six prognosis-related genes were validated, and FOXN2, NSA2, and DESI1 were found to be significantly expressed between the miR-135b-5p-inhibitor and negative control groups. Ye and Duan found that FOXN2 is downregulated in breast cancer and modulates invasion, migration, and epithelial-mesenchymal transition via regulation of SLUG [41]. Liu et al. reported that FOXN2 can inhibit the invasion and proliferation of human hepatocellular carcinoma cells [42]. Jeong et al. revealed that HOXC6-mediated miR-188-5p expression induces cell migration by inhibiting the tumor suppressor FOXN2 [43]. Dai et al. found that the lncRNA WT1-AS inhibits cell aggressiveness via the miR-203a-5p/FOXN2 axis and is associated with the prognosis of cervical cancer [44]. NSA2, also known as TINP1, promotes tumor cell proliferation and significantly reduces p53 and p21 expression [45]. Wang et al. showed that NSA2 plays an important role in the development of ovarian cancer [46]. However, further in-depth studies are required to confirm this. Despite the findings, this study has some limitations. First, additional relevant experiments should be conducted to validate the six prognosis-related genes and pathways identified in this study. Second, further studies are required to analyze the specific mechanisms of hsa-miR-135b-5p in the progression of COAD. Third, the function of hsa-miR-135b-5p should be explored in vivo, and the clinical application of miR-135b-5p should be further analyzed.
In summary, downregulation of hsa-miR-135b-5p may target FOXN2, NSA2, and DESI1, thereby inhibiting cell proliferation, migration, and invasion in COAD. Therefore, hsa-miR-135b-5p can be used as a therapeutic target for COAD treatment. | true | true | true |
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PMC9640294 | 36093879 | Shang-Yeh Lu,Wei-Zhi Hong,Bruce Chi-Kang Tsai,Yu-Chun Chang,Chia-Hua Kuo,Thomas G. Mhone,Ray-Jade Chen,Wei-Wen Kuo,Chih-Yang Huang | Angiotensin II prompts heart cell apoptosis via AT1 receptor-augmented phosphatase and tensin homolog and miR-320-3p functions to enhance suppression of the IGF1R-PI3K-AKT survival pathway | 31-08-2022 | angiotensin II,heart damage,hypertension,mir-320,phosphatase and tensin homolog,renin–angiotensin–aldosterone system | Background: Hypertension is a severe public health risk factor worldwide. Elevated angiotensin II (Ang II) produced by the renin–angiotensin–aldosterone system can lead to hypertension and its complications. Method: In this study, we addressed the cardiac-injury effects of Ang II and investigated the signaling mechanism induced by Ang II. Both H9c2 cardiomyoblast cells and neonatal rat cardiomyocytes were exposed to Ang II to observe hypertension-related cardiac apoptosis. Results: The results of western blotting revealed that Ang II significantly attenuated the IGF1R-PI3K-AKT pathway via the Ang II-AT1 receptor axis and phosphatase and tensin homolog expression. Furthermore, real-time PCR showed that Ang II also activated miR-320-3p transcription to repress the PI3K-Akt pathway. In the heart tissue of spontaneously hypertensive rats, activation of the IGF1R survival pathway was also reduced compared with that in Wistar-Kyoto rats, especially in aged spontaneously hypertensive rats. Conclusion: Hence, we speculate that the Ang II-AT1 receptor axis induces both phosphatase and tensin homolog and miR-320-3p expression to downregulate the IGF1R-PI3K-AKT survival pathway and cause cell apoptosis in the heart. | Angiotensin II prompts heart cell apoptosis via AT1 receptor-augmented phosphatase and tensin homolog and miR-320-3p functions to enhance suppression of the IGF1R-PI3K-AKT survival pathway
Hypertension is a severe public health risk factor worldwide. Elevated angiotensin II (Ang II) produced by the renin–angiotensin–aldosterone system can lead to hypertension and its complications.
In this study, we addressed the cardiac-injury effects of Ang II and investigated the signaling mechanism induced by Ang II. Both H9c2 cardiomyoblast cells and neonatal rat cardiomyocytes were exposed to Ang II to observe hypertension-related cardiac apoptosis.
The results of western blotting revealed that Ang II significantly attenuated the IGF1R-PI3K-AKT pathway via the Ang II-AT1 receptor axis and phosphatase and tensin homolog expression. Furthermore, real-time PCR showed that Ang II also activated miR-320-3p transcription to repress the PI3K-Akt pathway. In the heart tissue of spontaneously hypertensive rats, activation of the IGF1R survival pathway was also reduced compared with that in Wistar-Kyoto rats, especially in aged spontaneously hypertensive rats.
Hence, we speculate that the Ang II-AT1 receptor axis induces both phosphatase and tensin homolog and miR-320-3p expression to downregulate the IGF1R-PI3K-AKT survival pathway and cause cell apoptosis in the heart.
Hypertension represents the largest health risk factor in modern society because it is a severe public health problem owing to its associated morbidity and mortality worldwide. Due to increases in the global population and population aging, the number of hypertensive patients has continued to increase in recent decades [1,2]. In the human body, blood pressure (BP) is regulated by the renin–angiotensin–aldosterone system (RAAS), which also maintains salt and water homeostasis through this hormone system in human body. However, RAAS also has an important role in the pathogenesis of hypertension [3,4], as it can control BP via the octapeptide angiotensin II (Ang II) mediated by angiotensin-converting enzyme hydrolysis. Ang II is a biologically active peptide at the center of the RAAS in BP regulation [4,5]. Ang II binds to the AT1 receptor (AT1R) to cause smooth muscle cell contraction, generate systemic vasoconstriction, release aldosterone, and reabsorb sodium to increase renovascular resistance, decrease renal BP, and increased BP. Ang II can also decrease BP by stimulating the AT2 receptor. Ang II–AT2 receptor interaction leads to vasodilation, natriuresis, and antiproliferative actions to decrease BP [3]. However, an abnormal high level of Ang II causes many heart diseases, including hypertrophic cardiomyopathy and heart failure. The AT1R is thought to mediate the major cardiovascular effects of Ang II. The Ang II-AT1R pathway induces cardiomyocyte apoptosis, cardiac hypertrophy, and heart remodeling [6,7]. It's determined Ang II as a critical factor in heart function. Another important factor for heart is insulin growth factor 1 (IGF1), which is involved in survival, oxidative stress, apoptosis, and proliferation [8]. Some studies have shown a synergistic effect between Ang II and IGF1. For example, the interaction between Ang II and insulin/IGF1 is found to be synergistic, rather than additive, in stimulating ERK1/2 activation in H295R cells [9]. In our previous studies, we present that IGF1 deficiency and/or IGF1 receptor (IGF-1R) resistance induces apoptosis in cardiomyoblast cells and that apoptosis is synergistically augmented by Ang II [10]. However, the mechanism underlying the synergy between Ang II and IGF1 is not clearly understood. MicroRNAs are a class of small, endogenous, single-stranded, non-coding RNAs that maintain multiple biological processes in the cell [11]. Previous research has shown that miR-320-3p is an important miRNA that controls cell apoptosis. For example, miR-320-3p overexpression can cause apoptosis in hypoxic pulmonary arterial smooth muscle cells [12]. It also reduces cardiomyocyte survival after ischemia/reperfusion injury via Akt3 inhibition [13]. Some studies have shown that miR-320-3p might be involved in the IGF1R signaling pathway and could be involved in apoptosis and angiogenesis [14,15]. Phosphatase and tensin homolog (PTEN), a tumor suppressor gene, has biological functions in several different cells, including vascular smooth muscle cells, endothelial cells, and cardiomyocytes [16]. PTEN inhibits the phosphoinositide 3-kinase (PI3K) signaling pathway to negatively regulate cell growth, metabolism, and survival [17]. In heart, PTEN also acts as a negative regulator of several cardiovascular diseases. The level of PTEN is increased in the hearts of mice postmyocardial infarction, but a reduction of PTEN stimulates the PI3K/Akt/VEGF pathway to improve cardiac function and vascular remodeling in such animals [18]. Similarly, the inhibition of PTEN in human umbilical vein endothelial cells decreases oxidized LDL-induced apoptosis [19]. PTEN degradation further augments Wharton's jelly-derived mesenchymal stem cell stability and protects cardiac function under hyperglycemic conditions [20]. Previous research has shown that miR-320 can cooperate with PTEN to repress tumor survival [21]. To date, however, there has been little discussion about the roles of both PTEN and miR-320 in the heart under hypertension stress. It is known that hypertension represents a pathological state of the heart. However, the mechanism underlying the interactions among Ang II, IGF1R, miR-320, and PTEN in cardiomyocytes during hypertension remains elusive. To understand the biological interaction among Ang II, IGF1R, miR-320, and PTEN in cardiomyocytes under hypertension stress, we hypothesized that these factors would have an additive or synergistic effect on the heart induced by hypertension-associated cardiac damage in this study. The aim of this study was thus to address this.
Angiotensin II (Cat#: A9525), losartan (Cat#: SML3317), SF1670 (Cat#: SML0684), and most chemo reagents were of analytical grade and purchased from Sigma-Aldrich (St. Louis, Missouri, USA) unless stated otherwise. The miR-320 mimic and inhibitor were purchased from Phalanx Biotech, Hsinchu, Taiwan.
Rat-derived H9c2 cardiomyoblasts were purchased from the Bioresource Collection and Research Center (BCRC, Hsinchu, Taiwan) and cultured in high-glucose DMEM (Hyclone, Logan, Utah, USA) supplemented with 10% fetal bovine serum (FBS) (Gibco, Grand Island, New York, USA), 2-mmol/l glutamine, 100-U/ml penicillin, 100-mg/ml streptomycin, and 1-mmol/l sodium pyruvate at 37 °C with 5% CO2[22]. The cells were seeded and grown to 70–80% confluency before treatment.
A neonatal Rat/Mouse cardiomyocyte isolation Kit (Cat#: nc-6031, Cellutron Life Technology, Baltimore, Maryland, USA) was used to isolate and culture neonatal rat cardiomyocytes, according to the manufacturer's guidelines. Whole hearts from 1- or 2-day-old Sprague–Dawley rats (BioLASCO, Taipei, Taiwan) were collected, and cardiomyocytes were isolated with digestion solution at 37 °C. Collected cells were seeded in 10 cm plates at 37 °C for 2 h to reduce contamination by cardiac fibroblasts. All unattached cells were cultured on SureCoat pre-coated plates with NS medium with serum (Cat#:m-8031, Cellutron Life Technology) containing 100 U/ml penicillin, and 100 μg/ml streptomycin in a 5% CO2 incubator for 24 h. After 24 h, all media were changed to NW medium (Cat#:m-8032, Cellutron Life Technology) and cardiomyocyte cultures were ready for use in further experiments.
The mimic and inhibitor of miR-320-3p were purchased from Phalanx Biotech (Hsinchu, Taiwan). H9c2 cardiomyoblasts or neonatal rat cardiomyocytes were transfected separately with the miR-320 mimic or miR-320 inhibitor using the jetPRIME transfection kit (Polyplus Transfection, Illkirch, France). After 6 h of transfection, Ang II was added to miR-transfected H9c2 cardiomyocytes or neonatal rat cardiomyocytes for 24 h.
The Animal Research Committee of China Medical University, Taichung, Taiwan approved all animal studies and all animal care (CMUIACUC-2019-258). The study followed the Guide for the Care and Use of Laboratory Animals (8th ed.) [23]. Male Wistar-Kyoto rats (WKY) (three 5-month-old rats, three 18-month-old rats, and three 24-month-old rats) and age-matched male spontaneously hypertensive rats (SHR) (three 5-month-old rats, three 18-month-old rats, and three 24-old-aged rats) were purchased from BioLASCO Taiwan Co., Ltd. All rats were bred in an animal room (22°C and 50% humidity with a 12-h light/12-h dark cycle) with drinking water and standard laboratory chow (Lab Diet 5001; PMI Nutrition International Inc., Brentwood, Missouri, USA). WKY and SHR rats were allocated to the following groups: young (5-month-old) group, middle-aged (8-month-old) group, and aged (24-month-old) control group. All rats were sacrificed in a prefilled CO2 chamber with 100% CO2. The left ventricles were separated from the heart and aseptically collected for subsequent studies.
The cell samples were prepared with RIPA buffer for 30 min on ice and centrifuged at 13 000 rpm for 60 min at 4 °C to collect the supernatants. The tissue extracts from each group were homogenized with RIPA buffer [100 mg tissue/1 ml radioimmunoprecipitation assay buffer (RIPA) buffer], placed on ice for 30 min, and centrifuged at 13 000 rpm for 40 min at 4 °C to collect the supernatants. The Bradford method (Bio-Rad Protein Assay; Bio-Rad, Hercules, California, USA) was used to determine the total protein concentration of each sample. The same amount (30 μg) of each protein sample was separated by SDS–PAGE at a constant voltage of 80 V. Electrophoresed proteins were transferred to a polyvinylidene difluoride (PVDF) membrane (Millipore, Bedford, Massachusetts, USA, 0.45 μm pore size) with a transfer apparatus (Bio-Rad). Tris-buffered saline with 0.1% Tween 20 detergent buffer (TBST) buffer with 5% skim milk was incubated with transferred PVDF membranes at room temperature for 1 h to avoid antibody non-specific binding. Primary antibodies, including those targeting β-actin, phospho-AktS473, Akt, caspase 3, glyceraldehyde 3-phosphate dehydrogenase (GAPDH), phospho-IGF1R, IGF1R, MAS, PI3K (Santa Cruz Biotechnology, Santa Cruz, California, USA), phospho-PI3K, PTEN (Cell Signaling Technology, Danvers, Massachusetts, USA), AT1R (Abcam, Cambridge, UK), and caspase 8 (Merck Millipore, Darmstadt, Germany) were diluted with antibody binding buffer and incubated with transferred PVDF membranes for approximately 12 h 4 °C. The transferred PVDF membranes were then washed three times in TBST buffer for 10 min after specific antibody binding; the membranes were incubated with secondary antibodies (GE Healthcare, Chicago, Illinois, USA) at room temperature for 1 h. The membranes were then washed three times for 10 min with TBST buffer. Protein expression was visualized using an enhanced chemiluminescence western blotting luminal reagent and quantified using a Fujifilm LAS-3000 chemiluminescence detection system (Tokyo, Japan) [24–26].
RNA purification and quantitative real-time PCR analysis were performed according to previously described methods with slight modifications. Total RNA was collected using Total RNA Isolation Reagent – TRIzol Reagent (Thermo Fisher Scientific, Waltham, Massachusetts, USA), and RNA samples (2 mg/ml) were converted to cDNA with reverse transcriptase using the Mir-X miRNA First-Strand Synthesis Kit (Takara Bio USA, Inc., San Jose, California, USA). cDNA (1 mg) was used with appropriate primers to perform real-time PCR using iQ SYBR Green Supermix (Bio-Rad Laboratories, Inc.). The primer sequence for mir-320-3p and that of the U6 primer, as an internal control, were provided with the Mir-X miRNA Synthesis Kit. To normalize with the GAPDH housekeeping gene, cDNA was prepared with the iScript cDNA Synthesis kit following the kit's protocol. Transcriptional levels of mir-320-3p, U6, and GAPDH expression were determined by SYBR green Connect Real-Time PCR Detection System (Bio-Rad). The expression levels of mir-320-3p were then normalized to U6 or GAPDH with a similar expression pattern. The inverse log of the DDCT was calculated [27–29].
The experimental methods of TUNEL assay were referred to the contents published by our laboratory with slight modification [30–32]. Cells were identified with a fluorescent microscope under an excitation wavelength in the 450–500 nm range and a detection wavelength in the 515–565 nm range (green). The positive signal was identified with an OLYMPUS BX53 microscope (Olympus Corporation, Shinjuku-ku, Tokyo, Japan).
When we sacrificed the animal and collected the heart, the hearts were fixed by formalin (neutral buffered, 10%). The sections were cut in 4 mm, deparaffinized by immersion in xylene, and rehydrated with gradient ethanol. The protocol of immunochemistry staining was referred to the contents published by our laboratory with slight modification [33,34]. Thereafter, the slides were identified with an OLYMPUS BX53 microscope (Olympus Corporation).
All experiments were performed at least three times. All results were quantified using ImageJ software and processed using Adobe Photoshop. Statistical analysis was conducted using SigmaPlot statistical software. Two groups were compared with Student's t test. Multiple comparisons of data were analyzed using a one-way analysis of variance, followed by Tukey's test. Statistical significance was set at P less than 0.05.
First, H9c2 cardiomyoblasts were utilized as a cell model to confirm that AT1R was upregulated by Ang II to repress the IGF1R signaling pathway. H9c2 cells were treated with Ang II, isoproterenol, and lipopolysaccharides, which are cardiotoxins, and only Ang II enhanced AT1R expression (Fig. 1a). The activation of IGF1R and Akt S473 phosphorylation was also reduced, but levels of the apoptosis markers cleaved caspase 8 and caspase 3 were increased (Fig. 1a). Next, different doses of Ang II and treatment periods were used to confirm that Ang II could repress the IGF1R pathway to induce heart cell apoptosis. As the concentration of Ang II was increased, AT1R expression also increased and IGF1R and Akt became less activated. The level of cleaved caspase 3 was also elevated following the increase in the Ang II concentration (Fig. 1b). The treatment periods of Ang II also affected AT1R and the downregulation of the IGF1R pathway. As time increased, Ang II also induced more AT1R to attenuate the phosphorylation of IGF1R and Akt S473 and upgraded cleaved caspase 8 and caspase 3 to activate cardiac cells apoptosis (Fig. 1c). These results suggest that AT1R expression can be enhanced by Ang II to suppress the IGF1R survival pathway and results in the apoptosis of H9c2 cardiomyoblasts. Further, Ang II-induced damage is dose-dependent and time-dependent.
Next, we focused on PTEN as it is a negative regulator of the IGF1R-PI3K-Akt signaling pathway [17]. Different heart-damaging compounds were used to evaluate their effect on PTEN expression in H9c2 cardiomyoblasts. Results showed that Ang II increased PTEN expression (Fig. 2a). Ang II-induced PTEN expression was dose-dependent and time-dependent. High doses of Ang II and longer treatments also enhanced PTEN expression (Fig. 2b and c). To investigate the mechanism underlying the modulation of PTEN and AT1R mediated by Ang II-induced injury, we incubated Ang II-exposed H9c2 cells with a PTEN inhibitor (SF1670) or an AT1R inhibitor (losartan). From these results, we inferred that Ang II lost its ability to upregulate PTEN when losartan was added and the activity of Akt was rescued, which showed that Ang II could not perform its negative function when AT1R was unable to transmit its signal. Treatment with SF1670 also restored the Akt signaling that was downregulated by Ang II. When SF1670 and losartan were combined to treat Ang II-exposed H9c2 cells, the level of phospho-Akt was nearly recovered (Fig. 2d). These results indicate that Ang II upregulates PTEN by binding to AT1R and inhibits Akt phosphorylation to induce apoptosis. The inhibition of AT1R and/or PTEN can upregulate the activation of Akt and reduce cell apoptosis. Interestingly, losartan treatment also reduced the expression of AT1R and inhibited Ang II-AT1R signaling (Fig. 3). It suggests that PTEN inhibition may also affect Ang II-AT1R function. Moreover, downstream IGF1R was increased, which further enhanced the activity of Akt and inhibited the expression of apoptotic markers, including caspase 8 and caspase 3 (Fig. 3). The results demonstrate that the inhibition of Ang II and/or PTEN activates the IGF1R survival pathway to suppress Ang II-related apoptosis in the heart.
Previous literature mentions that miR-320-3p also affects cardiomyocyte survival via Akt3 inhibition [13]. Hence, we next examined the expression level of miR-320-3p in Ang II-exposed H9c2 cells via real-time PCR. As expected, miR-320-3p expression was increased in Ang II-exposed H9c2 cells (Fig. 4a, Supplementary Fig. 1A). With an increase in time with Ang II, the increase in miR-320-3p was even more significant (Fig. 4b, Supplementary Fig. 1B). However, the increased level of miR-320-3p was reduced by losartan (an AT1R inhibitor) (Fig. 4c, Supplementary Fig. 1C). These results illustrate that Ang II binds to AT1R to stimulate the expression of miR-320-3p and suggest that miR-320-3p might play an important role in Ang II-induced effects on H9c2 cells. Next, a mimic and inhibitor of miR-320-3p were employed to investigate the main function of miR-320-3p in Ang II-treated H9c2 cells. As the concentration of the miR-320 mimic was increased, both the total expression and phosphorylation of Akt decreased, showing that miR-320-3p downregulated AKT signaling (Fig. 5a). Upon cotreatment miR-320 mimic with Ang II, the results significantly showed that the levels of the signal molecules associated with PI3K and Akt were all downregulated (Fig. 5b, Supplementary Fig. 2A). In contrast, when the miR-320 inhibitor was used, AKT activity increased (Fig. 5c). When the miR-320 inhibitor was cotreated with Ang II, it reversed the repression of the PI3K-AKT pathway caused by Ang II and also reduced the expression of cleaved caspase 3 (Fig. 5d, Supplementary Fig. 2B). We also used TUNEL assay to detect apoptotic cells in H9c2 cells. The image showed apoptosis was increased by Ang II and miR-320-3p mimic. On the contrary, miR-320-3p inhibitor significantly reversed the response of Ang II-induced apoptosis (Fig. 6). We also isolated neonatal rat cardiomyocytes to confirm the role of Ang II-induced miR-320-3p in the heart. Similar results were observed in neonatal rat cardiomyocytes. These results presented that the miR-320 mimic attenuated the phosphorylation of AKT, but the miR-320 inhibitor rescued the activity of AKT in Ang II-treated neonatal rat cardiomyocytes (Fig. 7). All results indicate that Ang II-AT1R signaling upregulates miR-320-3p expression in Ang II-exposed heart cells and that miR-320-3p can directly repress cell survival signals, which has a synergistic effect with Ang II-AT1R signaling, causing heart damage.
To confirm our previous results, three different aged rat groups were used in this study. As expected, the activation of IGF1R and AKT in the hearts of SHR rats was significantly lower than that in WKY rats. With increasing age, the IGF1R signaling pathway was associated with a greater reduction in activity in the hearts of hypertensive rats, especially in the oldest SHR rat group. Although all groups showed reduced expression of activated IGF1R and AKT with age, the IGF1R survival pathway in the WKY control group was always higher than that in the SHR group (Fig. 8a). The PTEN expression level was detected by immunohistochemistry staining, the similar result show SHR group was higher than WKY group. It could be support that PI3K-AKT pathway was reduced via PTEN in SHR group (Fig. 8b). These results provided evidence to confirm the reduction in the IGF1R survival pathway in the heart under hypertensive conditions. Interestingly, expression of the MAS1 receptor was also decreased in the hearts of SHR groups (Fig. 8a). Previous studies have shown that the MAS1 receptor can bind Ang1-7 and physiologically oppose the actions of Ang II [35]. Hence, fewer MAS1 receptors were expressed in hypertensive hearts and might also lead to abnormal heart function.
Hypertension has been well recognized as one of the major chronic diseases leading to morbidity and mortality in the 21st century, and the number of patients presenting with this risk factor will continue to increase to more than 30% of the general population in the future [1,2]. Ang II from RAAS is the main regulator controlling BP in the human body, and elevated levels of Ang II can result in hypertension and its related cardiac complications and chronic heart failure [36]. In a previous study, it was confirmed that activation of the Ang II-AT1R axis in the heart causes cardiac hypertrophy and other related heart damage [6]. When human AT1R is overexpressed in the hearts of mice, the cardiac morphology exhibits massive hypertrophy, and the structures of atria and ventricles also become significantly larger [6]. Intriguingly, Mathieu et al. [37] reported that Ang II-AT1R-induced heart damage is associated with sex-specific differences. Female AT1R overexpressed mice have diminished Ca2+ dynamics, more heart dysfunction, and increased mortality compared with male AT1R overexpressed mice. It implies that hypertension induced via Ang II-AT1R axis might has varies by sex. Activation of the IGF1R-PI3K-AKT signaling pathway is a cardiac-protective cascade. High-glucose-induced oxidative stress and HIF-1a transcriptional factor activation enhance IGFBP3 and inhibit the IGF1R-PI3K-AKT pathway to promote cardiomyocyte apoptosis [38]. However, the probiotic GMNL-263, oolong tea extract, or potato hydrolysate can trigger the IGF1R-PI3K-AKT survival pathway to protect heart functions [39–41]. In the current study, Ang II is not only found to bind AT1R to repress the IGF1R-PI3K-AKT survival pathway but also stimulated PTEN and miR-320-3p expression, resulting in a synergistic effect on suppression of the IGF1R-PI3K-AKT survival pathway. In the heart tissue of SHR rats, activation of the IGF1R survival pathway is also reduced compared with that in WKY rats, and the expression decreased with age. PTEN, a tumor suppressor protein, is a multifunctional lipid phosphatase that negatively regulates the PI3K signaling cascades [17]. Generally, activation of the PI3K-AKT pathway in vascular smooth muscle cells, endothelial cells, cardiomyocytes, and cardiac fibroblasts improves survival and reduces apoptosis. However, loss of PTEN function can alter heart function. For example, loss of PTEN function can decrease pathological hypertrophy and improve resistance to heart failure to biomechanical stress [16]. Ravi et al. [42] show that dysregulation of PTEN is an essential role in the progression of pulmonary hypertension. Hyperglycemic stress induces PTEN and causes heart damage, but a reduction in PTEN enhances cell survival and suppresses diabetic cardiac damage [20]. However, one brief report presents that overexpression of PTEN in mice can reduce Ang II-induced vascular fibrosis and remodeling [43]. Cardiomyocyte-specific inactivation of PTEN leads to hypertrophy and a reduction in cardiac contractility [44]. Therefore, PTEN can play two opposing roles in heart. In our study, we show that PTEN plays a negative factor to reduced IGF1R-PI3K-AKT survival pathway. The Ang II-AT1R axis is found to upregulate PTEN to cause apoptosis; however, the inhibition of Ang II and/or PTEN activated the IGF1R-PI3K-AKT survival pathway in response to Ang II-related apoptosis in the heart. Moreover, our results also presented that the level of miR-320-3p is also increased in Ang II-treated cardiomyocytes. Accumulating evidence has highlighted miR-320 as a negative regulator in the heart. Previous studies have shown that miR-320 affects fatty acid metabolism and causes lipotoxicity in the hearts of diabetic mice. The suppression of miR-320 also significantly limits diabetes-induced cardiac dysfunction [45]. Similarly, miR-320 downregulation decreases heart damage mediated by ischemia–reperfusion injury, whereas its overexpression has the opposite effect [46,47]. In patients with heart failure, miR-320 is slightly increased in heart tissues and plasma [48]. However, it could have different abilities in different cell types of the heart. For example, the overexpression of miR-320 in cardiomyocytes leads to cardiac dysfunction, whereas its overexpression in cardiac fibroblasts attenuates cardiac fibrosis and hypertrophy induced by transverse aortic constriction [48]. In the current study, we found that miR-320-3p overexpression reduced the activity of Akt, whereas inhibition had the opposite effect in both H9c2 cardiomyoblasts and neonatal rat cardiomyocytes under Ang II stimulation. These results suggest that the Ang II-AT1R axis induces the expression of PTEN and miR-320-3p to downregulate the IGF1R-PI3K-AKT survival pathway and cause cell apoptosis in the heart. In conclusion, collectively, Ang II is an important factor that causes hypertension and its related complications. In this study, the IGF1R-PI3K-AKT survival pathway in heart cells could be directly inhibited by the interaction between Ang II and AT1R to cause heart damage. Moreover, Ang II binding to AT1R was also found to activate PTEN function and miR-320-3p transcription to suppress the IGF1R-PI3K-AKT survival pathway in heart cells and induce apoptosis. In the heart tissues of SHR rats, activation of the IGF1R survival pathway was also reduced with age. Overall, as suggested by the current results in this study, the negative effect of Ang II on the heart is a synergistic effect, including AT1R, PTEN, and miR-320-3p (Fig. 9). The results of this study provide a more detailed biochemical mechanism to understand Ang II-induced cardiac injury.
The current study was supported by the China Medical University, Taichung, Taiwan, Grant/Award Number: CMU107-TC-03 and the China Medical University Hospital, Taichung, Taiwan, Grant/Award Number: DMR-110-014. Author contributions: S.-Y.L. and C.-Y.H. conceptualized and designed this study. S.-Y.L., W.-Z.H., and B.C.-K.T. collected and assembled the data. Y.-C.C. and C.-H.K. provided the materials for this study. S.-Y.L., W.-Z.H., and T.G.M. analyzed and interpreted the data. S.-Y.L., B.C.-K.T., and T.G.M. wrote a draft of the article. R.-J.C. and W.-W.K. provided reviewed and approved the final article. W.-W.K. provided administrative support. C.-Y.H. provided financial support. Consent for publication: The authors agree with the publication. Availability of data and material: The raw data used and/or analyzed during the current study are available from the corresponding author on reasonable request. The authors confirm that the data supporting the findings of this study are available within the article.
The authors declare that there are no conflicts of interest. | true | true | true |
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