Novel SOX10 indel mutations drive schwannomas through impaired transactivation of myelination gene programs

Abstract

Background: Schwannomas are common peripheral nerve sheath tumors that can cause severe morbidity given their stereotypic intracranial and paraspinal locations. Similar to many solid tumors, schwannomas and other nerve sheath tumors are primarily thought to arise due to aberrant hyperactivation of the RAS growth factor signaling pathway. Here, we sought to further define the molecular pathogenesis of schwannomas.

Methods: We performed comprehensive genomic profiling on a cohort of 96 human schwannomas, as well as DNA methylation profiling on a subset. Functional studies including RNA sequencing, chromatin immunoprecipitation-DNA sequencing, electrophoretic mobility shift assay, and luciferase reporter assays were performed in a fetal glial cell model following transduction with wildtype and tumor-derived mutant isoforms of SOX10.

Results: We identified that nearly one-third of sporadic schwannomas lack alterations in known nerve sheath tumor genes and instead harbor novel recurrent in-frame insertion/deletion mutations in SOX10, which encodes a transcription factor responsible for controlling Schwann cell differentiation and myelination. SOX10 indel mutations were highly enriched in schwannomas arising from nonvestibular cranial nerves (eg facial, trigeminal, vagus) and were absent from vestibular nerve schwannomas driven by NF2 mutation. Functional studies revealed these SOX10 indel mutations have retained DNA binding capacity but impaired transactivation of glial differentiation and myelination gene programs.

Conclusions: We thus speculate that SOX10 indel mutations drive a unique subtype of schwannomas by impeding proper differentiation of immature Schwann cells.

Keywords: PMP2; SOX10; Schwann cell; myelination; schwannoma.

Graphical Abstract

Figure 1.

Recurrent SOX10 indel mutations in sporadic schwannomas lacking alterations in known nerve sheath tumor genes. (A) Genomic profiling on a discovery cohort of 39 schwannomas identified recurrent somatic mutations of the SOX10 gene in 8/30 (27%) sporadic/nonsyndromic schwannomas that lacked alterations in all other known nerve sheath tumor genes. See Supplementary Figure S1 for results of genomic profiling on an independent validation cohort of 57 additional schwannomas. (B) Diagram of the recurrent in-frame insertion and deletion mutations identified in SOX10, which all cluster at the C-terminus of the HMG-box DNA-binding domain of the encoded homeobox transcription factor. The two bolded mutations (p.Y173_Q174insKY [also annotated p.K172_Y173dup] and p.R176_R177insPQPR) were those used in functional studies. (C) Amino acid sequence of SOX10 in the region of the carboxy terminal end of the HMG domain (green shading) where the recurrent indel mutations in schwannomas are located demonstrating a high degree of conservation from humans through lower vertebrates including mouse and zebrafish. Uniprot ID for amino acid sequence: Homo sapiens, P56693-1; Pan troglodytes, A0A2J8LNY1; Loxodonta africana, G3U829; Mus musculus, Q04888; Rattus norvegicus, O55170; Gallus gallus, Q9W757; Danio rerio, Q90XD1. (D) Histopathology of the SOX10-mutant schwannoma arising from the right trigeminal nerve (cranial nerve V) in a 12-year-old male patient. Scale bar, 50 μm. (E) Unsupervised hierarchical clustering of genome-wide DNA methylation data for 22 schwannomas of varying genotypes along with 42 reference nerve sheath tumors. K-means clustering segregated these tumors into 4 epigenetic groups, which recapitulated the associated histopathologic diagnoses. Shown is a heatmap of the 20,000 most differentially methylated probes amongst the 64 tumors. See Supplementary Table S10 for sample manifest.

Figure 2.

Schwannomas with SOX10 indel mutation have a distinct epigenetic signature compared to those with NF2 mutation. (A) Unsupervised hierarchical clustering of DNA methylation data for 8 schwannomas with SOX10 indel mutations and 9 schwannomas with inactivating NF2 mutations. K-means clustering segregated these tumors into 2 epigenetic groups, which comprised those with SOX10 indel mutation and those with NF2 mutation. Shown is a heatmap of the 1,050 most differentially methylated probes amongst the 17 tumors. See Supplementary Table S11 for detailed annotations of these 1,050 differentially methylated CpG sites. B, Volcano plot of DNA methylation data for 8 schwannomas with SOX10 indel mutations and 9 schwannomas with inactivating NF2 mutations. The 100,000 CpG sites with the lowest adjusted p-values by DMP analysis are shown, and those CpG sites having adjusted p-values < 0.05 and greater than ± 2 log2 fold-change in mean β-values between the two molecular subgroups are colored pink. (C) Visualization of DNA methylation status at CpG sites within and immediately upstream of the MEIS1 gene locus on chromosome 2p14 in 8 schwannomas with SOX10 indel mutations and 9 schwannomas with inactivating NF2 mutations.

Figure 3.

Schwannomas arising from non-vestibular cranial nerves are highly enriched for SOX10 indel mutations. (A) Age distribution plot of 22 patients with SOX10-mutant schwannomas. (B) Anatomic location distribution plot of 95 schwannomas segregated by genotype reveals that non-vestibular cranial nerve tumors are highly enriched for SOX10 mutation. * P < 0.0001. (C) Imaging features of SOX10-mutant schwannomas. Shown are representative pre-operative magnetic resonance or computed tomography images from select patients including multiple with nonvestibular cranial nerve tumors. (D) Kaplan–Meier survival plot of 22 patients with SOX10-mutant schwannomas demonstrating a propensity for local recurrence/progression.

Figure 4.

SOX10 indel mutations impair transcriptional activation of Schwann cell differentiation and myelination genes. (A) Western blots of protein lysates extracted from SVG cells stably transduced with empty vector or FLAG-tagged wildtype SOX10, alongside lysate from a primary human schwannoma cell culture for comparison. (B) Gene Ontology analysis showing representative biologic processes that are enriched for genes whose expression is significantly higher in SVG cells transduced with wildtype SOX10 compared to empty vector. (C) Quantitative reverse transcription-PCR analysis of RNA extracted from SVG cells stably transduced with wildtype and mutant SOX10 isoforms. Relative gene expression of the indicated targets normalized to GAPDH expression is shown, with the mean of 3 replicates per experimental sample plotted with errors bars showing standard deviation. Source data in Supplementary Table S20. (D,E) Volcano plots of whole transcriptome RNA-sequencing gene expression data from SVG fetal glial cells following stable transduction with empty vector, SOX10 wildtype, or SOX10 R176_R177insPQPR mutant isoforms. Differentially expressed transcripts with P-values of < 1 × 10^-5 and log2 fold change > 1.2 derived from three sequencing replicates per experimental sample are highlighted in red. See Supplementary Figure S5 for Volcano plot of SOX10 K172_Y173dup mutant isoform. (F) Venn diagram of RNA-sequencing gene expression data from SVG fetal glial cells following stable transduction with empty vector, SOX10 wildtype, K172_Y173dup, or R176_R177insPQPR mutant isoforms showing intersection of differentially expressed transcripts. See Supplementary Table S19 for full gene lists. (D) Western blots of protein lysates extracted from SVG cells stably transduced with FLAG-tagged wildtype and mutant SOX10 isoforms, alongside lysates from parental unmodified SK-MEL-28 melanoma cells and U87MG glioma cells which both have robust MAP kinase signaling pathway activity for comparison.

Figure 5.

SOX10 indel mutant isoforms have retained DNA binding capacity at Schwann cell differentiation and myelination genes. (A) DNA sequence motif analysis of the 1,777 high-confidence chromatin binding peaks in SVG cells transduced with wildtype SOX10 revealed the known SOX10 binding motif ACAAAG. (B) Gene Ontology analysis showing representative biologic processes enriched in genes with chromatin binding peaks in the SOX10 wildtype samples. (C) Overlap of high-confidence chromatin binding peaks between the SOX10 wildtype samples and the two mutant isoforms. (D) SOX10 chromatin binding peak in the promoter region of the L1CAM gene from genome-wide ChIP-sequencing for SOX10 wildtype and two mutant isoforms. For each pair, the top alignment shows the immunoprecipitation sequencing whereas the bottom shows the input DNA.

Figure 6.

SOX10 indel mutations have retained DNA binding capacity but impaired transactivation at the Peripheral Myelin Protein 2 (PMP2) gene promoter. (A) Electrophoretic mobility shift assay (EMSA) of nuclear lysates from SVG cells stably transduced with empty vector, wildtype SOX10, or two tumor-derived SOX10 mutant isoforms using a biotinylated oligonucleotide probe corresponding to a segment of the PMP2 gene promoter containing a known SOX10 binding site. Binding reactions of the oligonucleotide probe with and without nuclear lysate and unlabeled oligonucleotide competitive inhibitor were resolved on a DNA retarding gel, and SOX10 bound DNA complexes were visualized by chemiluminescence using HRP-conjugated streptavidin. (B) Luciferase reporter assay of a PMP2 promoter reporter construct containing a known SOX10 binding site transfected into SVG cells stably transduced with empty vector, wildtype SOX10, or two SOX10 indel mutants. Plots show the mean of 3 replicates per experimental sample with errors bars showing standard deviation. Source data in Supplementary Table S22. (C) Western blots of protein lysates extracted from SVG cells stably transduced with SOX10 wildtype and K172_Y173dup mutant isoforms collected over time after the indicated number of serial passages. (D) Model of the role of wildtype SOX10 in Schwann cell differentiation and mutant SOX10 in driving schwannoma formation via impaired transcriptional activation of myelination and differentiation gene programs.