PMC4823091_01
A novel HER3 somatic mutation in NSCLC. Clinical findings of a metastatic lung adenocarcinoma in a 14 year old male:
a. axial computed tomography (CT) image showing primary lesion in left bronchus of patient (arrow), b. H&E stained section of transbronchial biopsy specimen showing nests of poorly differentiated adenocarcinoma (arrow) infiltrating the bronchial mucosa (original magnification, x20), c. MRI scan of patient showing metastatic brain lesions in the white matter of both the left and right frontal lobes (arrows), d. H&E stained section showing metastatic lung adenocarcinoma in brain biopsy. Glial tissue is invaded by nests and sheets of tumor cells, growing in a cohesive pattern (arrow) (original magnification, x20) and e. Sanger sequencing chromatograms covering exon 21 of the HER3 gene revealing a double peak with a novel T-to-C base pair change resulting in a V855A mutation (arrows) in the pleural lung biopsy specimen of the patient (right panel). Patient's peripheral blood specimen (left panel) reveal the wild-type HER3 sequence only.
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As depicted in Fig. 1e, further examination of DNA extracted from the lung cancer specimen of the 14-year-old case-study revealed a double peak at nucleotide c.2564 located in exon 21 of the HER3 gene (NM_001982). The HER3-V855A mutation was detected in the tumor sample, but was not found in the patient's peripheral blood DNA (Fig. 1e), confirming that the mutation was of somatic origin. We also investigated the functional relevance of stable Ba/F3 transfectants co-expressing HER3-V855A and EGFR (Supplemental Fig. 1a). While Ba/F3 cells co-expressing HER3-V855A and EGFR exerted a robust growth response to TGFalpha treatment (Supplemental Fig. 1b), they were unable to form colonies with the same treatment (Supplemental Fig. 1c). TGFalpha (Supplemental Fig. 1c), but also demonstrated equal TGFalpha-induced growth response compared to the mutant HER3-V855A combination (Supplemental Fig. 1b). These data suggest that the pathogenic effects associated with the HER3-V855A mutation is not induced by heterodimerization with EGFR (upon TGFalpha stimulation).
PMC4823091_02
A novel HER3 somatic mutation in NSCLC. Clinical findings of a metastatic lung adenocarcinoma in a 14 year old male:
a. axial computed tomography (CT) image showing primary lesion in left bronchus of patient (arrow), b. H&E stained section of transbronchial biopsy specimen showing nests of poorly differentiated adenocarcinoma (arrow) infiltrating the bronchial mucosa (original magnification, x20), c. MRI scan of patient showing metastatic brain lesions in the white matter of both the left and right frontal lobes (arrows), d. H&E stained section showing metastatic lung adenocarcinoma in brain biopsy. Glial tissue is invaded by nests and sheets of tumor cells, growing in a cohesive pattern (arrow) (original magnification, x20) and e. Sanger sequencing chromatograms covering exon 21 of the HER3 gene revealing a double peak with a novel T-to-C base pair change resulting in a V855A mutation (arrows) in the pleural lung biopsy specimen of the patient (right panel). Patient's peripheral blood specimen (left panel) reveal the wild-type HER3 sequence only.
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F1
As depicted in Fig. 1e, further examination of DNA extracted from the lung cancer specimen of the 14-year-old case-study revealed a double peak at nucleotide c.2564 located in exon 21 of the HER3 gene (NM_001982). The HER3-V855A mutation was detected in the tumor sample, but was not found in the patient's peripheral blood DNA (Fig. 1e), confirming that the mutation was of somatic origin. We also investigated the functional relevance of stable Ba/F3 transfectants co-expressing HER3-V855A and EGFR (Supplemental Fig. 1a). While Ba/F3 cells co-expressing HER3-V855A and EGFR exerted a robust growth response to TGFalpha treatment (Supplemental Fig. 1b), they were unable to form colonies with the same treatment (Supplemental Fig. 1c). TGFalpha (Supplemental Fig. 1c), but also demonstrated equal TGFalpha-induced growth response compared to the mutant HER3-V855A combination (Supplemental Fig. 1b). These data suggest that the pathogenic effects associated with the HER3-V855A mutation is not induced by heterodimerization with EGFR (upon TGFalpha stimulation).
Protein structure visualization of the novel HER3-V855A mutation. a. Partial amino acid alignment of exon 21 sequence of the HER3 and EGFR tyrosine kinase domain. Also shown are the positions affected by the EGFR-L858R mutation and the novel HER3-V855A mutation (arrows). b. Partial amino acid alignment of the kinase domain of HER3 with other receptor tyrosine kinases adapted from the mutagrator kinase mutation interpretation tool reveals mutations (blue) at analogous residues. The HER3-V855A mutation is also analogous to the BRAF-L597V kinase mutation (red box). c. 3D structure of HER3 kinase domain (PDB: 3LMG) depicting the location of V855A mutation (red van der Waals radii).
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Although, the amino acid at position 855 in HER3 is not conserved relative to EGFR, the mutated amino acid remarkably maps at a position analogous to the location of the EGFR-L858R mutation (Fig. 2a). BRAF- L597V kinase mutation (Fig. 2b). BRAF-L597V is classified as an intermediate kinase active variant (approximately100 fold elevated BRAF activity compared to wild-type) that modestly increases ERK activation. The V855 residue is part of a conserved sequence motif (also includes L858 and L859) which stabilizes the inactive position of the alphaC helix (Fig. 2c), and we propose that the amino acid substitution may likely affect protein kinase activity. Of note, chronic treatment with NRG1beta neither induced HER3 or HER2 phosphorylation in Ba/F3 cells expressing HER3-V855A combined with a kinase-dead (KD) HER2 isoform (Supplemental Fig. 2b).
HER3-V855A combined with HER2 enhances neuregulin 1beta-induced activity in transformed Ba/F3 cells. a. Selected Ba/F3 transfectants were analyzed for cell surface protein expression by staining with HER-specific antibodies to confirm recombinant protein expression. HER2 and HER3 co-transfectants were labeled with PE-conjugated anti-HER2 or PE-conjugated anti-HER3 antibodies. b. Ba/F3 transfectants were cultured for 7 days in the absence or presence of the indicated stimulants. Cell growth was analyzed by the MTS assay. c. Ba/F3 co-transfectants were subjected to a methyl cellulose based colony formation assay in the presence of the indicated stimulants for 21 days. Magnification, 20X. d. Colonies were quantified using computerized photoshop CS6 analysis. e. Ba/F3 co-transfectants were cultured in the presence of NRGbeta for 5 days. Total cell lysates were analyzed by immunoblot analysis using the indicated antibodies. EV, empty vector. Also see supplemental Figure 1 and 2.
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F3
Stable cell populations were screened by fluorescence-activated cell sorting (FACS) and selected based on matched expression level (within two fold levels) of their relevant HER at the cell surface (Fig. 3a). As shown in Fig. 3b, Ba/F3 cells singly expressing wild-type HER3 or mutant HER3-V855A were unable to grow in IL-3 free culture conditions, even when supplemented with NRG1beta or TGFalpha ligands. The extent of NRG1beta-induced growth in the HER3-V855A/HER2 co-transfectant was even greater than levels observed in IL-3 treatment conditions (Fig. 3b). Furthermore, treatment with TGFalpha did not induce a growth response in both wild-type and mutant HER3 co-transfectants (Fig. 3b), further confirming that the presence of a HER3-specific cognate ligand is required to promote HER3/HER2 biological activity. As shown in Fig 3c & 3d, while NRG1beta treatment did not induce an increase in colony number between the wild-type and mutant HER3 co-transfectants, colony size was significantly greater inHER3-V855A compared to wild-type HER3 (p < 0.001). The colony size upon NRG1beta treatment was approximately 25-fold greater than under IL-3 treatment with the mutant HER3 co-transfectant (Fig. 3d). AKT and ERK 1/2 in the Ba/F3 co-transfectants. As shown in Figure 3e, a five-day chronic treatment with NRG1beta specifically increased levels of phosphorylated HER3 and AKT in HER3-V855A compared to wild-type HER3. HER2 and HER3 was observed in transformed H292 cells (Supplemental Fig 3b). H292 cells are NSCLC cells that display similar mucoepidermoid features as normal lung (non-cancerous) cells. Consistent with previous functional experiments (see Fig. 3), TGFalpha was unable to induce HER phosphorylation in transformed Ba/F3 cells (Fig. 4a). Of note, the cell surface protein expression of HER2 is much higher than HER3 in the transfected Ba/F3 cells (Fig. 3a).
HER3-V855A enhances neuregulin 1beta-induced activation of HER2 in transformed cells. a., b. Western blot analysis of Ba/F3 and HEK 293T transfectants treated for 16hrs in serum free conditions (SFM) and stimulated for 10 minutes with the indicated EGFR/HER cognate ligands. The cells were subsequently lysed and subjected to immunoblot analyses. Also see supplemental Figure 3.
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HER3-V855A enhances trans-phosphorylation of HER2, we performed immunoblot analysis on Ba/F3 and HEK 293Tlysates after 16hr incubation in serum-free conditions followed by10 minute acute ligand stimulation. HER3-V855A enhanced trans-phosphorylation of HER2 at Y1248 in both Ba/F3 and HEK 293T cells after NRG1beta stimulation (Fig. 4a and 4b). NRG1beta was essential for the induction of HER3 phosphorylation in wild-type and mutant HER3 co-transfectants, HER3 phosphorylation was absent in similar treatment conditions with corresponding single transfectants (Fig. 4a and supplemental Fig. 3a). NRG1beta-induced phosphorylation of mutant HER3 was strongly enhanced in HEK 293T cells, whereas the observed effect in Ba/F3 transfectants was more modest (Fig. 4a and 4b).
Enhanced interaction of HER2 with HER3-V855A Ba/F3 transfectants co-expressing HER2 and HER3 were treated in serum free conditions (SFM) for 1 hr followed by a 10-minute stimulation with NRG1beta. Total cell lysates were co-immunoprecipitated with either HER2 or HER3 antibodies, followed by western blot analysis. 20% input levels are indicated.
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Pull down of HER3 on HER2 immunoprecipitates revealed enhanced interaction of HER2 with HER3-V855A relative to wild-type HER3, specifically after incubation with NRG1beta (Fig 5a). Interestingly, reciprocal analysis with HER3 immunoprecipitates demonstrates that HER2 already interacts with the mutant HER3 complex in the absence of NRG1beta (with no observed effects with ligand stimulation), while NRG1beta stimulation is needed for interaction with the wild-type HER3 protein (Fig. 5b). This thus infers that a smaller fraction of the HER3 complex is pulled down in Fig. 5b, and may also result in differences in the amount of proteins stimulated by NRG1beta. The HER3-V855A co-transfectantformed significantly smaller colonies (7.2 +- 1.4) compared to the wild-type HER3 combination (78.9 +- 13.7) under erlotinib treatment (Fig. 5b and 5c). Pertuzumab (5 mug/mL) treatment induced less potent effects compared to afatinib, with significant reduction seen in mutant HER3 and wild-type HER2 phosphorylation, but not in active levels of AKT (Fig. 5d). Erlotinib and pertuzumab were less effective, yielding respectively a 20.4 +- 0.29% and 13.9+-0.6% increase in apoptotic cells compared to vehicle (Fig. 5e). All tested inhibitors were also less effective in suppressing HER-related activity and survival in the wild-type HER3/HER2 co-transfectant (Fig. 5d and 5e), indicating that tumors harboring HER3-V855A may predict response to targeted therapy.
Effectiveness of HER inhibitors on biological activity of HER3-V855A in transformed Ba/F3 cells. a. Dose-dependent growth inhibition of Ba/F3 co-transfectants treated with the indicated inhibitors and grown in the presence of NRG1beta for 7 days. Cell growth was assessed by the MTS assay. b. Ba/F3 co-transfectants were subjected to a methyl cellulose based colony formation assay in the presence of NRG1beta and treated with the indicated inhibitors or vehicle control (0.01% DMSO) for 21 days. c. Colonies were quantified using computerized photoshop CS6 analysis. Magnification, 20X (Mann Whitney U test: ***,p< 0.001. d. Following overnight serum starvation, Ba/F3 co-transfectants were treated with the indicated inhibitors or vehicle control (0.01% DMSO) for 2 hours and then stimulated with 100ng/mL of NRG1beta for 10 minutes. Immunoblot analysis was performed with the indicated antibodies. e. Ba/F3 co-transfectants treated with the indicated inhibitors (erlotinib (1muM), afatinib (1muM) and pertuzumab (20 mug/mL)) or vehicle control (0.1% DMSO) were grown in the presence of NRG1beta for 5 days and evaluated by the Annexin V-APC/7-AADbinding assay. f. Quantification of apoptosis with the sum of early and late apoptotic cell populations (only Annexin V stained) in the y-axis plot. Results are the averages from two replicate studies. Also see Table 1.
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Afatinib and pertuzumab each demonstrated high efficacy in inhibiting the growth of Ba/F3 cells co-expressing HER3-V855A and wild-type HER2 (Fig. 6a), with IC50 values of < 1 nmol/L and < 1mug/mL respectively (Table 1). Erlotinib, on the other hand, had a much weaker inhibitory effect on the mutant V855A combination (Fig. 6a) with a calculated IC50 value of 344+-4nmol/L (Table 1). As shown in Fig 6b, afatinib (100 nmol/L) and pertuzumab (5mug/mL) were effective in suppressing the formation of colonies in wild-type HER3 and mutant V855A co-transfectants. Afatinib (100nmol/L) potently inhibited NRG1beta-induced phosphorylation of HER3, wild type HER2 and AKT (Fig. 6d). Treatment with erlotinib (100nmol/L) also inhibited the HER pathway, albeit less potently than afatinib or pertuzumab treatment (Fig. 6d).
Prediction track depicting the impact of V855A on HER3 protein structure. a. SNPs from dbSNP (http://www.ncbi.nlm.nih.gov/SNP) were plotted with synonymous, non-synonymous and non-validated SNPs in green, red and gray respectively. b. Alignment generated conservation track shows the extracellular region (residues 20-643) in light green, the kinase domain (709-966) in red and the regulatory carboxyl-terminal end (967 - 1342) in gray. The cysteine rich furine-like repeats have been marked with numbers (1-5) along the track. The position of the valine to alanine substitution has been marked with an M. c. Server predicted wild-type and mutated models were energy minimized, refined and compared in means of root-mean-square deviation (RMSD) using a derivative of genetic algorithm. Regions altered by mutant V855A in the wild-type HER3 protein are represented as blue bars, colored according to conservation scores.
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Superimposition of predicted wild-type / mutated HER3 protein models reveals that the V855A mutation alters the kinase domain and the carboxyl-terminal end of the wild-type HER3 protein as shown by the blue bars along the prediction track depicted in Figure 7.