Multiomic Analyses Reveal New Targets of Polycomb Repressor Complex 2 in Schwann Lineage Cells and Malignant Peripheral Nerve Sheath Tumors - ChIPseq

Background Malignant peripheral nerve sheath tumors (MPNST) 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 addback 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 has not been defined. We hypothesized PRC2 loss has direct and indirect effects on gene expression resulting in MPNST, so we sought 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 1,327 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 signaling in PRC2-deficient iHSCs. Functional studies confirm 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 report 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. In order to model the progression from atypical neurofibromas to malignant peripheral nerve sheath tumors NF1, SUZ12, and EED were knocked out in immortalized human Schwann cells. CHIP-seq was generated histone modifications for H3K27me3 and H3K27ac for cell lines for all combinations of knockouts for NF1, SUZ12, and EED except that EED and SUZ12 knockouts were mutually exclusive.

背景 恶性周围神经鞘膜瘤（Malignant peripheral nerve sheath tumors, MPNST）可由非典型神经纤维瘤（atypical neurofibromas, ANF）进展而来。多梳蛋白抑制复合体2（polycomb repressor complex 2, PRC2）功能缺失是其常见分子事件。既往针对MPNST中PRC2调控基因的研究，多在高度非整倍体MPNST细胞系中开展基因回补实验，可能遗漏了NF1突变型ANF样前体细胞中受PRC2调控的基因。目前尚未明确人施万细胞中受PRC2调控的完整基因谱。本研究假设PRC2功能缺失可通过直接与间接途径调控基因表达，进而驱动MPNST发生，因此旨在永生化人施万细胞（immortalized human Schwann cells, iHSCs）中筛选受PRC2调控的基因。

方法 本研究构建了携带功能缺失型SUZ12或EED突变的NF1缺陷型永生化人施万细胞。通过RNA测序筛选得到1327个差异表达基因，以此定义PRC2调控基因集。为探究MPNST的发病机制，本研究将iHSCs中的差异表达基因与ANF和MPNST之间的一致性基因表达差异进行了比对分析。进一步采用染色质免疫共沉淀测序（chromatin immunoprecipitation sequencing, ChIP-seq）明确靶基因，并通过甲基化组学与蛋白质组学分析富集相关通路。

结果 本研究鉴定出可能参与MPNST进展的PRC2调控驱动基因。通路分析显示多条癌症相关通路呈上调状态。本研究发现，在PRC2功能缺失的iHSCs中存在Notch与音猬因子（Sonic Hedgehog）信号通路激活的转录组学证据。功能实验证实，Notch信号通路在MPNST细胞系、患者来源异种移植瘤以及PRC2功能缺失的瞬时细胞模型中均处于激活状态。MEK与γ-分泌酶抑制剂联合使用在MPNST细胞系中展现出协同抗肿瘤效应。

结论 本研究报道了MPNST中受PRC2调控的基因集及潜在驱动因子。本研究结果支持Notch信号通路可作为MPNST的可靶向治疗靶点。本研究鉴定的PRC2调控基因与通路可为开发新型治疗策略提供理论依据。为模拟从非典型神经纤维瘤到NF1相关恶性周围神经鞘膜瘤的进展过程，本研究在永生化人施万细胞中敲除了NF1、SUZ12与EED基因（注：EED与SUZ12敲除互为互斥事件，无法同时实现）。针对所有NF1、SUZ12、EED敲除组合的细胞系，本研究均开展了H3K27me3与H3K27ac两种组蛋白修饰的染色质免疫共沉淀测序检测。