Multiomic analyses reveal new targets of polycomb repressor complex 2 in Schwann lineage cells and malignant peripheral nerve sheath tumors

Abstract

Background: Malignant peripheral nerve sheath tumors (MPNSTs) can arise from atypical neurofibromas (ANF). Loss of the polycomb repressor complex 2 (PRC2) is a common event. Previous studies on PRC2-regulated genes in MPNST used genetic add-back experiments in highly aneuploid MPNST cell lines which may miss PRC2-regulated genes in NF1-mutant ANF-like precursor cells. A set of PRC2-regulated genes in human Schwann cells (SCs) has not been defined. We hypothesized that PRC2 loss has direct and indirect effects on gene expression resulting in MPNST, so we sought to identify PRC2-regulated genes in immortalized human Schwann cells (iHSCs).

Methods: We engineered NF1-deficient iHSCs with loss of function SUZ12 or EED mutations. RNA sequencing revealed 1327 differentially expressed genes to define PRC2-regulated genes. To investigate MPNST pathogenesis, we compared genes in iHSCs to consistent gene expression differences between ANF and MPNSTs. Chromatin immunoprecipitation sequencing was used to further define targets. Methylome and proteomic analyses were performed to further identify enriched pathways.

Results: We identified potential PRC2-regulated drivers of MPNST progression. Pathway analysis indicates many upregulated cancer-related pathways. We found transcriptional evidence for activated Notch and Sonic Hedgehog (SHH) signaling in PRC2-deficient iHSCs. Functional studies confirm that Notch signaling is active in MPNST cell lines, patient-derived xenografts, and transient cell models of PRC2 deficiency. A combination of MEK and γ-secretase inhibition shows synergy in MPNST cell lines.

Conclusions: We identified PRC2-regulated genes and potential drivers of MPNSTs. Our findings support the Notch pathway as a druggable target in MPNSTs. Our identification of PRC2-regulated genes and pathways could result in more novel therapeutic approaches.

Keywords: MPNST; NF1; PRC2; nirogacestat; notch signaling.

Figure 1.

Differential expression analyses reveal 1327 genes that are altered by PRC2 status in NF1-deficient human Schwann cell (SCs), are upregulated or downregulated during transformation, and are potential mediators of the MPNST phenotype. PCAs of engineered immortalized human Schwann cell (iHSC) lines using (A) gene expression data. (B) Overlap of differentially upregulated genes from MPNSTs, NF1 and PRC2 (SUZ12 or EED)-deficient human SCs leads to 145 upregulated differentially expressed genes; 1210 genes are upregulated when PRC2 is lost (shown in bolded overlap). (C) Heatmap of overlapping 1327 differentially expressed, PRC2-regulated genes across all engineered human SCs. Values are gene-wise Z scores. (D) Overlap of downregulated differentially expressed genes from MPNSTs with PRC2 restored from Zhang et al. and 1210 upregulated genes from iHSCs. Heatmaps of the (E) H3K27me3 and (F) H3K27ac status of 172 direct and indirect targets of PRC2. The downregulation Venn diagram is shown in Supplementary Figure S2C. All Western blot quantification can be found in Supplementary Figure S6.

Figure 2.

Genes derepressed by PRC2 inactivation do not show consistent changes in promoter methylation. Integrative analysis of DNA methylation and gene expression of engineered cell lines using a scatterplot of mean difference of beta values versus log2 expression fold change. Each point represents a gene-probe pair of the engineered lines. Adapted from Xu et al.

Figure 3.

NOTCH signaling is a significantly upregulated pathway when PRC2 is lost in engineered iHSCs. Gene set enrichment analysis using clusterProfiler of iHSCs with (A) NF1 and PRC2 loss. (B) Gene expression heatmap of NOTCH signaling pathway players across all engineered lines. (C) NOTCH signaling pathway rendered by Pathview compiling RNA seq (middle panel), methylome (left panel), and proteome (right panel) data representing the ratio in change of each signaling pathway player after PRC2 loss in iHSCs.

Figure 4.

Gene expression analysis of patient ANF and MPNSTs reveals activation of the Notch pathway. (A) Visual grading of various NOTCH pathway effectors from TMA data using immunohistochemistry. (B) Representative images of TMA staining. The scale bar is 70 µm. All shown MPNSTs are scored as intensity 3 and ANFs are 0-1. Other examples of staining intensity can be found in Supplementary Figure S5. (C) Gene expression heatmap of NOTCH signaling pathway players across ANF (n = 12) and MPNST (n = 8) patient samples. Notch pathway players were probed in multiple (D) MPNST and (E) PDX cell lines. (+) control refers to MDA-MB-231 cells for all marks except HES1 and NOTCH3 where MCF7 cells were used.

Figure 5.

NOTCH signaling modulates growth behaviors in transient PRC2 knockdown Schwann cell models and PRC2-deficient MPNSTs. (A) Western blots to confirm SUZ12 was knocked down in iPNF cells. (B) NOTCH1 and NOTCH3 were probed in a transient SUZ12 knockdown in iPNF cells. (+) control refers to CUTLL1 cells for NOTCH1 and MCF7 cells for NOTCH3. (C) RT-qPCR of upstream and downstream markers of NOTCH signaling on generated transient cell line models (pooled siRNA). (D, E) Western blots to confirm successful transfection of GOF vectors of Notch pathway players (NOTCH1, NOTCH3, and Luciferase control) in iPNF lines. (F) Average cell counts of GOF cell lines following 24-h transwell migration assay. (G) Proliferation of iPNF GOF lines in a 6-well plate was tracked over 5 days; 20 000 cells were plated on day 0. Two-way ANOVA of Luc vs NOTCH1 yields a P-value of .004 and Luc vs NOTCH3 yields a P-value of .0143. (H) Western blots to confirm siRNA knockdown of NOTCH receptors in MPNST cell lines. (I) Proliferation of siRNA knockdown MPNST cell lines with AlamarBlue in 96-well plate format. ***P ≤ .001, ****P ≤ .0001.