Abstract
Glioblastoma is one of the most lethal forms of adult cancer with a median survival of around 15 months. A potential treatment strategy involves targeting glioblastoma stem-like cells (GSC), which constitute a cell autonomous reservoir of aberrant cells able to initiate, maintain, and repopulate the tumor mass. Here, we report that the expression of the paracaspase mucosa-associated lymphoid tissue l (MALT1), a protease previously linked to antigen receptor-mediated NF-κB activation and B-cell lymphoma survival, inversely correlates with patient probability of survival. The knockdown of MALT1 largely impaired the expansion of patient-derived stem-like cells in vitro, and this could be recapitulated with pharmacological inhibitors, in vitro and in vivo. Blocking MALT1 protease activity increases the endo-lysosome abundance, impaired autophagic flux, and culminates in lysosomalmediated death, concomitantly with mTOR inactivation and dispersion from lysosomes.
Abstract
These findings place MALT1 as a new druggable target involved in glioblastoma and unveil ways to modulate the homeostasis of endo-lysosomes.
Introduction
Glioblastoma Multiforme (GBM) represents the most lethal adult primary brain tumors, with a median survival time of 15 months following diagnosis 1, 2 . The current standard of care for the treatment of GBM includes a surgical resection of the tumor followed by treatment with alkylating agent temozolomide and radiation. While these standardized strategies have proved beneficial, they remain essentially palliative 1, 3, 4 . Within these highly heterogeneous tumors exists a subpopulation of tumor cells named Glioblastoma Stem-like Cells (GSCs).
Introduction
Although the molecular and functional definition of GSCs is still a matter of debate, there is compelling evidence that these cells can promote resistance to conventional therapies, invasion into normal brain, and angiogenesis 5, 6, 7, 8, 9 . As such, they are suspected to play a role in tumor initiation and progression, as well as recurrence and therapeutic resistance. Owing to their quiescent nature, GSCs resist to both chemotherapy and radiation, which target highly proliferative cancer cells 6, 7 . Hence, there is a clear need to identify novel therapeutic targets, designed to eradicate GSCs, in order to improve patient outcome.
Introduction
GSCs constantly integrate external maintenance cues from their microenvironment, and as such represent the most adaptive and resilient proportion of cells within the tumor mass 5, 10 .
Introduction
Niches provide exclusive habitat where stem cells propagate continuously in an undifferentiated state through self-renewal 5 . GSCs are dispersed within tumors and methodically enriched in perivascular and hypoxic zones 11, 12, 13 . GSCs essentially received positive signals from endothelial cells and pericytes, such as ligand/receptor triggers of stemness pathways and adhesion components of the extracellular matrix 13, 14, 15, 16, 17 . They are also protected in rather unfavorable conditions where their stemness traits resist hypoxic stress, acidification, and nutrient deprivation 11, 12, 18 . Recently, it has been suggested that this latter capacity is linked to the function of the RNA binding protein QKI in the down-regulation of endocytosis, receptor trafficking and endo-lysosome-mediated degradation 18 .
Introduction
GSCs therefore down-regulate lysosomes as one adaptive mechanism to cope with the hostile tumor environment 18 .
Introduction
Lysosomes operate as central hubs for macromolecule trafficking, degradation, and metabolism 19 . Cancer cells usually show significant changes in lysosome morphology and composition, with reported enhancement in volume, protease activity, and membrane leakiness 20 . These modifications can paradoxically serve tumor progression and drug resistance, while providing an opportunity for cancer therapies. The destabilization of the integrity of these organelles might indeed ignite a less common form of cell death, named as lysosomal membrane permeabilization (LMP). LMP occurs when lysosomal proteases leak into the cytosol and induce features of necrosis or apoptosis, depending on the degree of permeabilization 19 . Recent reports also highlighted that lysosomal homeostasis is essential in cancer stem cell survival 18, 21 . Additionally, it has been shown that targeting the autophagic machinery is an effective treatment against apoptosis-resistant GBM 22, 23 . The autophagic flux inhibitor chloroquine can decrease cell viability and acts as an adjuvant for TMZ treatment in GBM. However, this treatment might cause neural degeneration at the high doses required for GBM treatment 24 . Therefore, it is preferable to find alternative drugs that elicit anti-tumor responses without the harmful effects on healthy brain cells.
Introduction
Here, we repurpose several members of a family of drugs, phenothiazines, to disrupt GSC lysosomal homeostasis, induce autophagic features, and subsequently reduce cell survival.
Introduction
Phenothiazines compose a class of neuroleptic and antihistaminic drugs, among which is Mepazine, which has been shown to block the MALT1 cysteine protease 25 . We further established that MALT1 sequesters QKI and maintains low levels of lysosomes, while its inhibition unleashes QKI and hazardously increases endo-lysosomes, which subsequently impairs autophagic flux. This leads to cell death concomitant with mTOR inhibition and dispersion from lysosomes. Disrupting lysosomal homeostasis therefore represents an interesting therapeutic strategy against GSCs.
Results

MALT1 Expression sustains Glioblastoma Cell Growth. Glioblastoma Stem-like Cells
(GSCs) are suspected to be able to survive outside the protective vascular niche, in a nonfavorable environment, under limited access to growth factors and nutrients 11, 13 . Because the transcription factor NF-κB is instrumental in many cancers and because it centralizes the paracrine action of cytokines 26, 27, 28, 29 , we revisited The Cancer Genome Atlas (TCGA) for known mediators of the NF-κB pathway that could be aberrantly engaged in GSC expansion.
MALT1 Expression sustains Glioblastoma Cell Growth. Glioblastoma Stem-like Cells
We found that the paracaspase mucosa-associated lymphoid tissue l (MALT1) expression was more significantly correlated with survival than other genes of the pathway (Fig. 1a ). This arginine-specific protease is crucial for antigen receptor-mediated NF-κB activation and Bcell lymphoma survival 30, 31 . In addition, when GBM patients were grouped by low or high MALT1 expression levels, there was a significant survival advantage for patients with lower MALT1 expression ( Fig. 1b) . Moreover, levels of MALT1 RNA are elevated in GBM (Grade IV) when compared with lower grade brain tumors (Grades II and III) or non-tumor samples ( Fig. 1c-d) . At the protein level, MALT1 expression was rather homogenously detectable in four GBM patient-derived cells with stem-like properties (GSCs) from various origins, 2 males (#1 and #12) and 2 females (#4 and #9), mesenchymal (#1 and #4), classical (#9), and neural (#12), and varied in ages from 59 (#12) to 68 (#1 and #9) and 76 years old (#4). MALT1 binding partner BCL10 was also expressed in GSCs, although its level of expression was not correlated with patient probability of survival ( Fig. 1a, 1e ). Of note, GSCs did not show clear signs of NF-κB activation, as visualized by phosphorylation and degradation of the NF-B inhibitor IB, as opposed to a treatment with TNFα ( Supplementary Fig. 1a ). In addition to its scaffold function in the modulation of the NF-κB pathway, MALT1 also acts as a protease for a limited number of substrates 32 . One of MALT1 known substrate, the deubiquitinating enzyme CYLD 33 , was constitutively cleaved in GSCs. This was not further increased upon stimulation with PMA/ionomycin, in contrast to Jurkat T cells, most likely due to a failure to co-opt canonical signaling in this cellular context ( Supplementary Fig. 1a ).
MALT1 Expression sustains Glioblastoma Cell Growth. Glioblastoma Stem-like Cells
This processing of CYLD was reduced upon treatment with the MALT1 enzymatic inhibitor Mepazine (MPZ) 25 , the competitive inhibitor zVRPR.fmk, or MALT1 siRNA treatment ( Supplementary Fig. 1b-d ), reinforcing the hypothesis that a fraction of MALT1 is most likely active in growing GSCs, outside its canonical role in antigen receptor signaling and immune cancer cells.
MALT1 Expression sustains Glioblastoma Cell Growth. Glioblastoma Stem-like Cells
To explore whether MALT1 was engaged in GSCs, the functional impact of MALT1 knockdown was evaluated on proliferation and stemness in vitro ( Fig. 1f -j). To this end, two individual short hairpin RNA sequences targeting MALT1 (shMALT1) cloned in a lentiviral bi-cistronic GFP expressing plasmid were delivered into GSC#1 and GSC#9 cells. We observed a reduced fraction of GFP-positive cells over time, while cells expressing nonsilencing RNA plasmids (shc) maintained a steady proportion of GFP-positive cells, indicating that MALT1-silenced cells were proliferating at a lower rate ( Fig. 1f ). Likewise, cells transfected with siMALT1 had a lower percentage of EdU-positive cells as compared to non-silenced control cells ( Fig. 1h -i). Additionally, GSCs either expressing shMALT1 or transfected with siMALT1 had less stem properties, as evaluated by limited dilution assay and tumorsphere formation ( Fig. 1g, 1j ). Taken together, these results indicate that MALT1 expression may be important for glioblastoma cell growth.
MALT1 Expression sustains Glioblastoma Cell Growth. Glioblastoma Stem-like Cells
Pharmacological Inhibition of MALT1 is Lethal to Glioblastoma Cells. Next, to evaluate the potential of targeting MALT1 pharmacologically, we treated GSCs #1, #4, #9, and #12 with MALT1 inhibitor Mepazine (MPZ) 25 at a dose of 20 µM. All four GSCs showed a marked reduction in stemness by both limited dilution and tumorsphere assays ( Fig. 2a-c ).
MALT1 Expression sustains Glioblastoma Cell Growth. Glioblastoma Stem-like Cells
This was accompanied by a marked reduction in the abundance of SOX2 and NESTIN stemness markers ( Fig. 2d ). Alongside the in vitro self-renewal impairment, GSC viability was largely annihilated ( Fig. 2e ). In contrast, MPZ treatment had no significant effect on viability of brain-originated human cells (endothelial cells, astrocytes, and neurons), ruling out a non-selectively toxic effect ( Fig. 2e ). In addition, GSC#9 showed an increase in propidium iodide (PI) uptake after MPZ treatment, demonstrating that reduced cell number was due at least partially to cell death ( Fig. 2f ). Differentiated sister GSCs (DGCs) also showed reduced viability in response to MPZ, indicating that targeting MALT1 may have a pervasive effect on different GBM tumor cells ( Fig. 2g ). Of note, when CYLD and BCL10 were knocked down through RNA interference, cell viability remained low upon MPZ treatment. This discards an instrumental action of these two MALT1 substrates downstream of MALT1 in MPZ-dependent cell demise ( Supplementary Fig. 1e-f ).
MALT1 Expression sustains Glioblastoma Cell Growth. Glioblastoma Stem-like Cells
MPZ is a drug, belonging to the phenothiazine family, and was formerly used in the treatment of schizophrenia 34 . We next evaluated whether other clinically relevant phenothiazines could block MALT1 protease activity and affect GSC viability ( Supplementary Fig. 2 ). Indeed, five of the seven tested phenothiazines reduced MALT1 protease activity upon antigen receptor activation in Jurkat T cells ( Supplementary Fig. 2 ). Furthermore, the effect on MALT1 inhibition was reflected in cell viability, with the best MALT1 protease activity inhibitors (Chlorpromazine and Fluphenazine) having robust effects on cell viability ( Fig. 2h ). Because MPZ has been shown to efficiently and safely obliterate MALT1 activity in experimental models 30, 35 , ectopically implanted GSC#9 mice were challenged with MPZ. Daily MPZ treatment reduced tumor volume in established xenografts, as well as number of NESTIN-positive cells (Figure 2i -j). This effect was prolonged for the week of measurement following treatment withdrawal. Together, these data demonstrate that targeting MALT1 pharmacologically is selectively toxic to GBM cells in vitro and in vivo.
MALT1 Inhibition alters Endo-lysosome Homeostasis.
To evaluate cell death modality triggered by MALT1 inhibition, caspases activity was blocked with the broad caspase inhibitor Q-VD-OPh (QVD) and increasing doses of MPZ were administered ( Supplementary   Fig. 3a ). QVD treatment did not rescue cell viability upon MPZ treatment, suggesting that cells were not dying through apoptosis, but rather another means. To gain insight into cell death mechanisms, transmission electron microscopy (TEM) was deployed to visualize morphological changes upon MPZ treatment. TEM images showed increased vacuoles and lysosomes compared to control cells (Fig. 3a ). The increase in lysosomes could be recapitulated upon siMALT1 treatment ( Supplementary Fig. 3b ) In fact, endo-lysosomal protein abundance was amplified upon MALT1 inhibition with MPZ, in a time-dependent manner ( Fig. 3b , Supplementary Fig. 3c ). Additionally, treatment with the MALT1 competitive inhibitor zVRPR.fmk and other phenothiazines, or MALT1 knockdown resulted in increased endo-lysosome abundance ( Fig. 3c -f, Supplementary Fig. 2c ), therefore disqualifying any drug-related action or deleterious accumulation in lysosomes. Conversely, other cellular organelles (early endosomes, mitochondria, golgi, and peroxisomes) remained unchanged upon MPZ treatment ( Fig. 3a , Supplementary Fig. 3c-d ). These endo-lysosomes appeared to be at least partially functional, as evidenced by pH-based Lysotracker staining, DQ-Ovalbumin and transferrin uptake ( Fig. 3d -e, Supplementary Fig. 3e-f ). Of note, at a later time point (16 hours) in MPZ treated cells, DQ-Ovalbumin staining was dimmer as compared to early time points (4 hours), which might signify lysosomal membrane permeabilization ( Supplementary Fig. 3e ). Moreover, ectopic tumors, excised from mice, challenged with a MPZ 2 weeks-regime showed a marked gain in LAMP2 staining and protein amount, as compared to vehicle treated tumors ( Fig. 3g ). Our data demonstrated that MALT1 knockdown and pharmacological inhibition provoke a meaningful endo-lysosomal increase.
MALT1 Inhibition alters Endo-lysosome Homeostasis.
Because autophagy is fueled by endo-lysosome activity, we also explored the impact of MALT1 inhibition on autophagy in GSCs, as estimated by LC3B modifications. The turnover of LC3B and the degradation of the autophagy substrate P62 also reflect autophagic flux 36 .
MALT1 Inhibition alters Endo-lysosome Homeostasis.
Upon MPZ treatment, there was an accumulation of lipidated LC3B (LC3B-II) and P62 protein amount over time, suggesting impaired autophagic flux ( Fig. 3h ). Treatment with MPZ also led to a significant increase in LC3B puncta at later time points (16 hours), subsequent to lysosomal increase (4 hours) ( Fig. 3i ). Super-resolution microscopy using structured illumination microscopy (SIM) further revealed that these LC3 structures were covered with LAMP2-positive staining (Fig. 3j ). This was concomitant with a reduced LC3B turnover, as evaluated via luciferase assay ( Fig. 3k ). Our data suggest that lysosomal increase upon MALT1 inhibition precedes autophagic flux impairment. At the functional level, there was increased CTSD release by GSCs treated with MPZ, implying lysosomal membrane permeabilization ( Fig. 3l ). Accordingly, treatment with lysosomal inhibitors partially rescued cells from MPZ-induced cell death ( Fig. 3m ). Thus, the protease activity of MALT1 appears to be required to maintain innocuous endo-lysosomes in GSCs.
MALT1 modulates the Lysosomal mTOR Signaling Pathway.
In order to further characterize the mode of action of MALT1 inhibition in GSCs, we performed RNAsequencing analysis on GSCs treated with MPZ for 4 hours, prior to any functional sign of death. Our results showed 7474 differentially expressed genes, among which no obvious endo-lysosomal protein encoding genes were identified, which was further confirmed by qPCR ( Fig. 4a -e, Supplementary Table 1 ). Of note, VGF, recently shown to promote GSC/DGC survival, was down-regulated upon MPZ treatment 37 ( Fig. 4e , Supplementary   Table 1 ). In line with a non-transcriptional regulation of lysosome biogenesis, knockdown of the master regulator of lysosomal transcription TFEB 38 failed to reduce autophagy signature and lysosomal protein up-regulation upon MPZ treatment ( Fig. 4f ). We thus hypothesized that the observed endo-lysosomal increase was due to modulation in their translation and/or RNA metabolism. When translation was blocked with cycloheximide, MPZ failed to increase endolysosomal protein amounts ( Supplementary Fig. 4a ). Likewise, RNAseq analysis unveiled putative changes in translation (peptide chain elongation, ribosome, cotranslational protein targeting, 3'-UTR mediated translational regulation), RNA biology (influenza viral RNA, nonsense mediated decay), metabolism (respiratory electron transport, ATP synthesis, oxidative phosphorylation, respiratory electron transport), and an mTOR signature (Bilanges serum and rapamycin sensitive genes) ( Fig. 4a-d ). Because mTOR sustains GSC expansion ( Supplementary Fig. 4b ) and its activation is linked to lysosomal biogenesis 14, 39, 40 , we further explored this possibility. Notably, MALT1 activity has been shown to participate in mTOR activation upon antigen receptor engagement, although the mechanism of action remains poorly understood 41,42 . In fact, MPZ and phenothiazine pharmacological challenge, as well as MALT1 siRNA blunted mTOR activation in GSCs, as evaluated through the activation and phosphorylation of AKT, p70S6K and S6 ribosomal protein ( Fig. 4g -j). MPZ treatment also reduced inhibitory phosphorylation of autophagy regulator ULK1 at serine 757 ( Fig. 4g ), which may partially account for increased autophagic features upon MPZ treatment.
MALT1 modulates the Lysosomal mTOR Signaling Pathway.
Furthermore, as phosphorylation of 4EBP1 increases protein translation by releasing it from EIF4E 43 , and as it can be resistant to mTOR inhibition 44 , we evaluated 4EBP1 phosphorylation levels over time in response to MPZ ( Supplementary Fig. 4c ). Although reduced shortly upon MPZ addition, phosphorylation returned at later time points, which may allow for the observed translational effect despite mTOR inhibition. As mTOR signaling is intimately linked to lysosomes 45 , we explored the impact of MPZ treatment on mTOR positioning. MPZ challenge induced a striking dispersion of mTOR foci (Fig. 4k ). In fact, mTOR staining divorced LAMP2-positive structures ( Fig. 4l , Supplementary Fig. 4d ).
MALT1 modulates the Lysosomal mTOR Signaling Pathway.
Interestingly, TFEB silencing influenced neither lysosome protein intensification nor mTOR recruitment at lysosomes ( Fig. 4f, Supplementary Fig. 4d ). Conversely, mTOR staining was dispersed from endo-lysosomes upon MPZ, zVRPR.fmk, or phenothiazines treatment, which likely accounts for decreased mTOR activity. These results suggest that MALT1 affects lysosomal homeostasis post-transcriptionally, and that this increase coincides with weak mTOR signaling, which may be due to displacement of mTOR from its lysosomal signaling hub.
MALT1 is Negatively linked to the Endo-lysosomal Regulator QKI. Shinghu et al. 18
recently demonstrated that the RNA binding protein Quaking (QKI) regulates endo-lysosomal levels in GBM. They showed that GBM-initiating cells maintain low levels of endolysosomal trafficking in order to reduce receptor recycling. QKI was suggested to regulate RNA homeostasis of endo-lysosome elements, independently of the TFEB-driven endolysosome biogenesis 18 . As our data suggest a counterbalancing role of MALT1 in lysosomal biogenesis, we revisited the TCGA and compared the expression of MALT1 with that of QKI in GBM patients. Interestingly, there was a negative correlation between the expression of the two genes ( Fig. 5a ). In addition, QKI and MALT1 were both linked to the expression of 7 common lysosomal lumen genes ( Fig. 5a ). This prompted us to examine QKI pattern in GBM. First, QKI was indeed expressed in a panel of GSCs, as well as in ectopic xenografts ( Supplementary Fig. 4e ). As expected, QKI displayed cytosolic and nuclear forms 46 , as evidenced by cellular fractionation and immunofluorescence ( Supplementary Fig. 4f-g) .
MALT1 is Negatively linked to the Endo-lysosomal Regulator QKI. Shinghu et al. 18
Similarly, human GBM samples from 2 patients showed pervasive QKI staining ( Supplementary Fig. 4h ). Given these findings, we decided to explore the possible link between MALT1 and QKI in GSCs. Co-immunoprecipitation experiments were thus deployed using QKI and the MALT1 binding partner BCL10 as baits. This showed that MALT1 was pulled down with QKI in GSC#1 and GSC#9, and vice versa (Fig. 5b ). Binding was, however, reduced in cells exposed to MPZ for 1 hour (Fig. 5c ). This suggests that active MALT1 tethered QKI in GSCs, while blocking MALT1 unleashed a fraction of QKI from the BCL10/MALT1 complex.
MALT1 is Negatively linked to the Endo-lysosomal Regulator QKI. Shinghu et al. 18
To next challenge the function of this putative neutralizing interaction of MALT1 on QKI, QKI expression was manipulated to alter QKI/MALT1 stoichiometry in GSCs. Strikingly, transient overexpression of QKI phenocopied the effect of MALT1 inhibition on endolysosomes. Reinforcing pioneer findings of QKI action on endo-lysosome components in transformed neural progenitors 18 , ectopically expressed QKI was sufficient to increase LAMP2 protein amount and lipidated LC3B ( Fig. 5d-e ). Accordingly, the augmented lysosome staining synchronized with mTOR displacement (Fig. 5f ). Corroborating the surge of lysosomes, the fraction of cells overexpressing QKI was drastically reduced over time, while fraction of cells expressing an empty vector remained stable, suggesting that exacerbated QKI expression hampered cell proliferation ( Fig. 5g ). Conversely, cells knocked down for QKI did not show the same MPZ-driven increase in LAMP2 and lipidated LC3B (LC3B-II), suggesting that QKI knockdown can partially rescue cells from endo-lysosomal increase ( Fig. 5h -i). Reinforcing this idea, mTOR staining was no longer dissipated from lysosomes upon MPZ treatment without QKI ( Fig. 5i ). Thus, QKI silencing rescued endolysosomes equilibrium in MPZ-treated cells, further indicating that MALT1 is negatively linked to the endo-lysosomal regulator QKI.
Discussion
Here we provide evidence that the paracaspase MALT1 activity is decisive for growth and survival in GBM. Our data indicate that MALT1 inhibition causes indiscipline of endolysosomal and autophagic proteins, which appears to occur in conjunction with a deficit in mTOR activity. In addition to the known MALT1 inhibitor Mepazine 25 , we show that several other clinically relevant phenothiazines can potently suppress MALT1 enzymatic activity, and have similar effects to MPZ on endo-lysosomes and cell death in GSCs. Since these drugs efficiently cross the blood-brain barrier in humans 47 and since they are currently used in the clinic, they represent an exciting opportunity for drug repurposing.
Discussion
The disruption of endo-lysosomal homeostasis appears to be the cause of death upon MALT1 inhibition in GSCs. As CSTD release is accelerated upon MALT1 blockade, and as inhibitors of lysosomal cathepsins (cathepsin inhibitor 1 and pepstatin A), but not pan-caspase blockade (QVD), can partially rescue cell viability, we hypothesize that cells may be dying from a form of caspase-independent lysosomal cell death (LCD) 19 . During this form of death, which may also be initiated by cathepins, lysosomal membrane permeabilization (LMP) allows cathepsins to act as downstream mediators of cell death upon leakage into the cytosol 19 . Additional studies will determine how exactly MALT1 inhibition drives lysosomal death in GSCs. Nevertheless, we found that inhibition of cathepsins provides only partial protection to cells treated with MPZ ( Fig. 3m ). Autophagic features may also play a part in cell death. Induction of autophagy likely occurs due to reduced inhibition of ULK1 ( Fig. 4g) as a consequence of mTOR dispersion from lysosomes 39,40 ( Fig. 4l ). Whether inducing or blocking autophagy are preferable therapeutic strategies in treating GBM remains hotly debated, with some groups reporting beneficial effects of blocking autophagy, and others preferring its activation as a therapeutic strategy 48, 23 . Here we show that the observed increased autophagic features are associated with reduced autophagic flux. Impairment in autophagic flux reduces a cell's ability for bulk degradation 36 . Others have shown that lysosomal dysfunction, such as LMP, can impede upon autophagic flux and eventually lead to cell death 49, 50 . Because of this, we infer that reduced autophagic flux is a downstream consequence of LMP, and ultimately contributes to LCD in our cells.
Discussion
MALT1 has previously been linked to mTOR activity 41,42 . For instance, MALT1 was reported to be necessary for glutamine uptake and mTOR activation after T cell receptor engagement 41 . As a consequence, the inhibition of MALT1 with zVRPR.fmk causes a reduction in the phosphorylation of S6 and p70S6K 42 . Our data now extend these findings to GSCs, however, the exact mechanism by which this occurs remains to be explored in both cellular backgrounds. Immunofluorescence analysis of mTOR positioning after MPZ treatment suggests that inhibition of mTOR is linked to its dispersion from the lysosomes, concurrent with lysosomal increase. However, we and others speculate that there may exist unidentified substrates of MALT1, which link its protease activity directly to mTOR activation 32 . This may also rationalize the need for constitutive MALT1 activity in GSCs, as mTOR is constantly active in these cells 14 . One hypothesis is that mTOR inhibition and/or dissociation from lysosomes originate from lack of processing of unknown MALT1 substrates and is then exacerbated once homeostasis is disrupted.
Discussion
How is QKI involved? Based on our data, we hypothesize that MALT1 sequesters QKI to prevent it from carrying out its RNA-binding function. Interestingly, MALT1 is already known to regulate other RNA-binding proteins Regnase-1/ZC3H12A, Roquin-1/RC3H1 and Roquin-2/RC3H2 51, 52 . We propose that upon MALT1 inhibition QKI is released and free to bind its RNA binding partners. QKI has already been shown to bind directly to lysosomal RNAs in progenitor cells 18 . It is thus tempting to speculate that QKI-dependent stabilization of lysosomal RNAs would preference the system towards more translation of these genes upon MALT1 inhibition. Indeed our RNA sequencing data suggests changes in translation and RNA biology upon MPZ treatment, however further study is needed to validate whether there is increased QKI binding to lysosomal RNAs upon MALT1 inhibition. Notably, QKI dependent lysosomal increase appears to be a post-transcriptional effect, independent of TFEB. As such, we propose a method of dual lysosomal control in GSCs whereby transcriptional biogenesis is tightly checked by known mTOR/TFEB pathway, and MALT1 acts on post-transcriptional regulation by isolating QKI from RNAs ( Supplementary Fig. 4i ).
Discussion
These findings place MALT1 as a new druggable target operating in non-immune cancer cells and involved in endo-lysosome homeostasis. Lysosomal homeostasis appears vital for glioblastoma cell survival and thus presents an intriguing axis for new therapeutic strategies in GBM. The Cancer Genome Atlas (TCGA) Analysis. The Cancer Genome Atlas (TCGA) was explored via the Gliovis platform (http://gliovis.bioinfo.cnio.es/) 53 . RNAseq databases (155 patients) were used to interrogate data related to MALT1 expression (levels of RNA, probability of survival, correlation with QKI expression). Optimal cut-offs were set. All subtypes were included and histology was the only selective criteria.
Materials and Methods
Cell Culture, siRNA and DNA transfection, lentiviral transduction. GBM patient-derived cells with stem-like properties (GSCs) were isolated as previously described 15, 54 .
Materials and Methods
Mesenchymal GSC#1 and #4, Classical GSC#9, and Pro-Neural GSC#12 were cultured as spheroids in NS34 medium (DMEM-F12, with N2, G5 and B27 supplements, glutamax and antibiotics, Life Technologies). In order to induce differentiation, GSCs were grown in TCTTCTGCAACTTCATCCA) or non-silencing short hairpin control (shc) were purchased from Open Biosystems. Lentiviral particles were obtained from pSPAX2 and pVSVg co-transfected HEK-293T cells and infected as previously described 55 . pFRT/FLAG/HA-DEST QKI was a gift from Thomas Tuschl (Addgene plasmid #19891) 56 , pRluc-LC3wt and pRluc-LC3BG120A were a gift from Marja Jaattela (Addgene plasmid #105002 and #105003) 57 .
Materials and Methods
They were introduced in GSCs using Neon electroporation system (MPK5000) according to manufacturer's instructions (Life Technologies). Tumorsphere Formation. To analyze tumorsphere formation, GSCs (100/µL) were seeded in triplicate in NS34 media as previously described 15 . Cells were dissociated manually each day to reduce aggregation influence, and maintained at 37°C 5% CO 2 until day 5 (day 4 for siRNA). Tumorspheres per field of view (fov) were calculated by counting the total number of tumorspheres in 5 random fov for each well. The mean of each condition was obtained from the triplicates of 3 independent experiments.
Antibodies and Reagents. Cathepsin
Limiting Dilution Assays. In order to evaluate the self-renewal of GSCs, limited dilution assays (LDA) were performed as previously described 58 . GSCs were plated in a 96-well plate using serial dilution ranging from 2000-1 cell/well with 8 replicates per dilution and treated as indicated. After 14 days, each well was binary evaluated for tumoursphere formation.
Antibodies and Reagents. Cathepsin
Stemness frequency was then calculated using ELDA software 59 . The mean stemness frequency for each treatment was calculated by averaging across 2 independent experiments. Animal Procedures. Tumor inoculation was performed on female Balb/C nude mice (Janvier) aged 6-7 weeks, as described previously 10 . Animals were randomly assigned to each group and group housed in specific pathogen free (SPF) conditions at 24ºC on a 12-hour daynight cycle. At all times, animals were allowed access to standard rodent pellets and water ad libitum. Mice were subcutaneously injected in each flank with 10 6 GSC#9 in 100 µL of PBS and growth factor-free matrigel (Corning). Once tumors were palpable, mice were injected intraperitoneally daily with MPZ (8 mg/kg) or vehicle (DMSO) for two consecutive weeks.
Antibodies and Reagents. Cathepsin
Tumor size was measured daily during treatment and for one week following treatment withdrawal, with calipers and tumor volume calculated using the following equation
Antibodies and Reagents. Cathepsin
Luciferase assays. Rluc-LC3B Luciferase assay was performed as previously described 57 .
Antibodies and Reagents. Cathepsin
Briefly, GSC#9 was transfected with 1 μg plasmid using a Neon Transfection System (Life Technologies 44. Qin, X., Jiang, B., and Zhang, Y. 4E-BP1, a multifactor regulated multifunctional protein. All data are representative of n=3, unless specified. Statistics were performed using a twotailed t-test with a 95% confidence interval for all experiments with p-values stated. 
Figure Legends


{'paper_id': '109292116', 'title': 'Control of the Endo-Lysosome Homeostasis by the Paracaspase MALT1 regulates Glioma Cell Survival', 's2_url': 'https://api.semanticscholar.org/CorpusID:109292116', 'source': 'S2ORC'}