Abstract
Schwannomas are common peripheral nerve sheath tumors that can cause debilitating morbidities. We performed an integrative analysis to determine genomic aberrations common to sporadic schwannomas. Exome sequence analysis with validation by targeted DNA sequencing of 125 samples uncovered, in addition to expected NF2 disruption, recurrent mutations in ARID1A, ARID1B and DDR1. RNA sequencing identified a recurrent in-frame SH3PXD2A-HTRA1 fusion in 12/125 (10%) cases, and genomic analysis demonstrated the mechanism as resulting from a balanced 19-Mb chromosomal inversion on chromosome 10q. The fusion was associated with male gender predominance, occurring in one out of every six men with schwannoma. Methylation profiling identified distinct molecular subgroups of schwannomas that were associated with anatomical location. Expression of the SH3PXD2A-HTRA1 fusion resulted in elevated phosphorylated ERK, increased proliferation, increased invasion and in vivo tumorigenesis. Targeting of the MEK-ERK pathway was effective in fusion-positive Schwann cells, suggesting a possible therapeutic approach for this subset of tumors.
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Acknowledgements
This work was supported by the Canadian Institute of Health Research (CIHR) post-doctoral fellowship (S.A.). The contributions of T.J.P., K.D.A. and G.Z. to this project were supported by the Princess Margaret Cancer Foundation. We thank the staff of the Princess Margaret Genomics Centre (N. Winegarden, J. Tsao and N. Khuu) and Bioinformatics Services (C. Virtanen and Z. Lu) for their expertise in generating the sequencing data used in this study. G.Z. is supported by the Wilkins Family Chair in Brain Tumor Research, CIHR grants, and The Terry Fox Research Institute. K.D.A. is supported by funding from the MacFeeters-Hamilton Neuro-Oncology Research Program. The human immortalized Schwann cells were a gift from A. Hoke (The Johns Hopkins School of Medicine, Baltimore, Maryland, USA).
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Contributions
S.A., K.D.A. and G.Z. conceived the entire project, designed, performed and analyzed the majority of the experiments in this study, prepared figures and wrote the manuscript. M.R.W. and M.L. performed in vitro experiments and in vivo studies. S.J., M.D. and T.L. performed QC and targeted sequencing library preparations. T.T., G.K., A.M., O.K. and B.K. clinically annotated schwannomas samples. J.R.K., Y.M., N.L.-B., A.A., M.D., T.L., T.J.P., S.M.P., K.E.B. and P.D.T. provided technical assistance and data interpretation. A.D. and T.J.P. analyzed and interpreted fusion breakpoint, point mutations and indels from targeted re-sequencing. K.D.A. and G.Z. funded the study. J.P.B., P.K.A., W.L.B., I.F.D. and R.B. provided technical assistance and data interpretation. A.I. and S.M.P. provided computation expertise and data interpretation. M.G.F. and V.B. provided clinical expertise.
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Integrated supplementary information
Supplementary Figure 1 Recurrent mutations identified in schwannomas.
Distribution of mutations in NF2, ARID1B, ARID1A, DDR1, and CAST.
Supplementary Figure 2 Pathways enriched from mutations identified in schwannomas.
Mutations are enriched in several biological and signaling pathways using the Molecular Signatures Database (MSigDB v5.1, Broad Institute, Cambridge, MA, USA). All significant pathways are shown.
Supplementary Figure 3 Copy number variations in schwannomas.
(a) Control-FREEC (Control-FREE Copy number and allelic content caller) analysis to annotate chromosome 22 loss from initial discovery cohort of 26 patients with whole exome sequence data. 18/26 had 22q loss or NF2 loss. Representative 22 deletion and normal 22 images are shown. Green represents that exome sequencing-derived copy number data is comparable to matched blood, and blue indicates that exome copy number is less than 2n in tumor compared to blood. (b) NF2 gene expression (RNA-seq) is down-regulated in patient samples with 22q loss. (c) Representative copy number (CN) profile for a patient with chromosome 22q deletion (76/125) from both frozen and FFPE cohorts. (d) Representative CN profile for a non-22q deleted schwannomas patient (49/125 were normal).
Supplementary Figure 4 Unsupervised clustering of schwannomas based on DNA methylation.
(a,b) Change in Gini coefficient plot from k = 2 to k = 8 (a) and cumulative distribution function (CDF) plots generated from consensus k-means clustering (b) identifies strongest statistical support for the existence of two subgroups of schwannomas. (c) Non-negative matrix factorization (NMF) of the 1,500 most variable CpG probes supports two subgroups of schwannoma (cophenetic coefficient = 0.9997 at k = 2). (d) Cophenetic coefficient plot support two subgroups of schwannoma and stability of two clusters using different variable CpGs for clustering. (k denotes the number of clusters).
Supplementary Figure 5 Pathway analysis of genes based on differentially methylation probes in group 1 and 2 schwannomas.
(a,b) Gene set enrichment analysis (GSEA) on significantly enriched methylated gene probes in group 1 (predominantly vestibular schwannomas) (a) and group 2 schwannomas (predominantly spinal schwannomas) (b).
Supplementary Figure 6 SH3PXD2A-HTRA1 fusion expression in schwannomas.
(a) Quantitative real-time PCR using probes specific to the fusion breakpoint confirms the SH3PXD2A-HTRA1 fusion in 7/51 RNA samples from FFPE tissues (out of a total detection rate of 12/125 (10%) of our samples). (b) Alignment to an in silico reconstruction of the HTRA1-SH3PXD2A fusion, with discordant RNA reads mapping across the fusion junction. (c) Validation RT-PCR using primers specific to the HTRA1-SH3PXD2A fusion to confirm presence of the fusion identified from deFuse RNA-Seq analysis. (d) Sanger sequencing confirms the fusion sequence at the breakpoint. (e) RT-PCR for the SH3PXD2A-HTRA1 fusion in 20 vestibular schwannomas from NF2 patients. (f) The two positive cases (5 and 9) have the fusion in the tumor but not in blood.
Supplementary Figure 7 Characterization of SH3PXD2A-HTRA1 region on chromosome 10 from exome sequencing coverage.
(a) Zoomed-out view of chromosome 10. The dotted red vertical line on the left is chr10:105,452,000 (SH3PXD2A area) and the dotted red line on the right is chr10: 124,248,418 (HTRA1). Ample DNA exome coverage of intervening sequences in a fusion-positive sample and comparable exome coverage to matched blood and a fusion-negative patient suggests lack of large-scale genomic rearrangement to generate this fusion. (b,c) Zoomed-in image of aligned DNA reads around fusion breakpoint (vertical red line) for SH3PXD2A and HTRA1. No clear loss of read depth at exons involved at the fusion junction suggests that the rearrangement may occur in an intronic region or another genomic location.
Supplementary Figure 8 LC-MS analysis of fusion-positive samples.
(a,b) Peptide mapping of both HTRA1 and SH3PXD2A in fusion-positive cases. No C-term SH3PXD2A peptides or N-term HTRA1 peptides were identified, supporting evidence that these peptides came from only the fusion. (c) Co-outlier plot for SH3PXD2A and HTRA1 gene-expression from RNA-seq. All samples express wild-type SH3XD2A and HTRA1.
Supplementary Figure 9 HTRA1 and SH3PXD2A expression in fusion-positive and fusion-negative schwannomas.
(a,b) Sashimi plot demonstrates that fusion-positive and negative samples still have RNA-seq reads spanning the exon 1-2 junction. (c,d) Sashimi plot demonstrates that fusion-positive and negative samples still have RNA-seq reads spanning exon to exon junctions after exon 6. This supports that the fusion-positive patients still retains RNA expression of wild-type gene-partners in addition to the fusion.
Supplementary Figure 10 The SH3PXD2A-HTRA1 fusion, but not each of the wild-type gene partners, increase proliferation.
(a) Transient transfection of wild-type HTRA1, SH3PXD2A, SH3PXD2A-HTRA1 fusion and SH3PXD2A-HTRA1 S322A fusion in human Schwann cells. Error bars, s.e.m.; n = 5. (b) Trypan blue direct cell count using automated cell counter 96 h post-transfection. Error bars, s.e.m.; n = 5. (c) Anoikis assay for control and fusion-positive cells with viability measured 12 h post 48 h incubation in non-coated plates to prevent attachment. Error bars, s.e.m.; n = 3. (d) Immunoblot confirming HTRA1, SH3PXD2A single and dual knockdowns in human Schwann cells treated with control siRNA, pooled HTRA1, pooled SH3PXD2A or dual siRNA. (e) Direct cell count of human Schwann cells treated with siRNA from d. (f) Immuno-precipitation (IP) of wild-type and fusion protein. HEK-293 cells were transfected with myc-tagged HTRA1, HA-tagged SH3PXD2A-HTRA1 fusion or HA-tagged SH3PXD2A-HTRA1 S322A (a catalytic-dead protease). Following MYC and HA IPs, proteins were purified and eluted using HA or myc peptides. (g) Protease assay, 100 ng of Immuno-precipitated HTRA1, fusion, fusion dead protein or no protein was incubated with 10 μg of bovine serum albumin (BSA) overnight and run on a 10% gel. Samples were also incubated with protease inhibitors (Roche, Protease Inhibitor Cocktail) to inhibit protease activity. Cropped version of this blot is shown in Figure 5j.
Supplementary Figure 11 Immunofluorescence imaging of the SH3PXD2A-HTRA1 fusion.
(a-d) Immunofluorescence imaging of fusion-positive and fusion-negative HEI and immortalized Schwann cells using HA antibody, N-term HTRA1, SH3PXD2A and EEA1 (early endosome antigen 1) antibodies. Scale bar, 15 μm.
Supplementary Figure 12 Fusion-positive HEI-193 cells lose Schwann cell markers.
(a) Fusion-positive HEI-193 cells but not empty vector-expressing control cells form tumors in flank of NOD-SCID mice. (b) Immunoblotting of HEI-193 control and SH3PXD2A-HTRA1 fusion-positive cells for S100B and HA tag. (c) Immunoblotting of Schwann cells with empty vector control and SH3PXD2A-HTRA1 fusion-positive cells for S100B and HA tag. (d) H3K27me3 immunoblot confirms both fusion-positive and fusion-negative cells from patient samples have similar trimethylated histones, a functional readout of the PRC2 complex.
Supplementary Figure 13 Molecular subgroups of schwannoma based on RNA-seq.
(a) Similarity matrix based on consensus hierarchical clustering of gene expression data supports that the SH3PXD2A-HTRA1 fusions enrich into Group 1 (black cluster). (b) IL2/STAT5 GSEA plots from cell line data expressing the fusion and RNA-seq patient data from the fusion enriched cluster. This was the only enriched pathway identified between the two datasets.
Supplementary Figure 14 Fusion-positive cells are sensitive to trametinib.
(a) Immunoblot of HEI-193 and Schwann cells expressing empty vector controls, wild-type HTRA1 or SH3PXD2A. (b) Trametinib IC50 values for cells expressing empty vector, HTRA1 or SH3PXD2A. (c) Immunoblot of HEI-193 empty vector control or fusion expressing cells transfected with N-FLAG tagged NF2 full length cDNA. (d) Doubling time of control HEI-193 cells and cells expressing NF2, fusion or both. (e) Immunoblot Schwann cells expressing empty vector or the fusion expressing transfected with NF2 shRNA. (f) Doubling time from control Schwann cells, NF2 knockdown cells, fusion-positive cells and fusion-positive/NF2 knockdown cells.
Supplementary Figure 15 Immunohistochemistry of ARID1 protein in schwannoma.
(a) Immunohistochemistry (IHC) of ARID1A in wild-type, single and double ARID1 mutant schwannoma samples. (b) IHC of ARID1B in wild-type, single and double ARID1 mutant schwannoma samples.
Supplementary information
Supplementary Text and Figures
Supplementary Figures 1–15 and Supplementary Tables 1–3, 5, 6 and 10 (PDF 2874 kb)
Supplementary Table 4
Mutations identified in schwannoma patients (XLSX 80 kb)
Supplementary Table 7
RNA-seq fusion detection results (XLSX 54 kb)
Supplementary Table 8
SH3PXD2A-HTRA1 fusion peptides identified in patient samples (XLSX 13 kb)
Supplementary Table 9
Capture probes for targeted sequencing and fusion breakpoint identification (XLSX 69 kb)
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Agnihotri, S., Jalali, S., Wilson, M. et al. The genomic landscape of schwannoma. Nat Genet 48, 1339–1348 (2016). https://doi.org/10.1038/ng.3688
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DOI: https://doi.org/10.1038/ng.3688
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