Harnessing double stranded-RNA (dsRNA)-response and anti-tumor effect in PRC2-inactivated cancer

项目摘要/摘要： 多梳抑制复合体2(PRC2)复合体建立和维持 组蛋白H3位于基因组中的27位赖氨酸(H3K27me2/3)，调节染色质结构、转录、细胞 茎和分化。PRC2是依赖于上下文的肿瘤抑制因子，其核心成分(例如， EZH2、EED或SUZ12)在各种癌症类型中都是失活的。其中，高度恶性外周组织 神经鞘瘤(MPNST)是一种侵袭性软组织肉瘤，目前尚无有效的治疗方法。 EED或SuZ12双等位基因失活的患病率(≥80%)，导致PRC2功能完全丧失。 癌症中PrC2的缺失导致发育沉默的主调控因子的转录异常激活， 这导致细胞可塑性增强和多个信号通路的异常激活。我们最近 发现癌症中PRC2的缺失导致免疫沙漠肿瘤微环境和对 免疫检查站封锁。然而，我们观察到，在PRC2缺失的小鼠模型中，MPNST和 对于乳腺肿瘤，免疫治疗的反应可以通过感染免疫原性病毒来增强，这种病毒 激活双链RNA(DsRNA)信号反应。此外，我们已经确定并验证了一个 MPNST中PRC2缺失与DNA甲基转移酶1(DNMT1)基因敲除之间的致死性相互作用在体外和 体内用去甲基化药物(地西他滨)或选择性DNMT1抑制剂(GSK862、GSK032)治疗导致 与野生型(Wt)MPNST模型相比，在PRC2-Lost模型中增强了细胞毒性和抗肿瘤效应。 在机制上，DNMT抑制剂(DNMTi)在PRC2缺失的背景下处理放大了 内源性反转录转座子，并随后导致激活先天免疫反应，这可能是 解释为反转录转座子形成dsRNA并通过PKR依赖的dsRNA传感触发细胞毒性。 我们假设，癌症中PRC2的缺失可能为药物(例如，DNMTi、合成药物)创造治疗机会 DsRNA，免疫原性病毒)，激活肿瘤细胞中的dsRNA信号通路，诱导细胞毒 增强免疫原性。在这里，我们提出了整合的多组学分析(例如，转录组， 表观基因组、单细胞rna-seq和scatac-seq)和创新方法(例如，新的免疫原性 病毒，一种新的谱系跟踪系统)来评估新的治疗策略及其机制 在相关的PRC2缺失癌症中激活dsRNA反应和抗肿瘤效应，专注于特定的目的： (1)分析了不同PRC2缺失对DNMTi选择性敏感性的分子机制 肿瘤；(2)评估外源dsRNAs作为一种策略在PRC2缺失肿瘤中诱导细胞毒作用；以及(3) 评估在相关癌症模型中激活dsRNA反应的新治疗策略。这些研究 将对利用dsRNA传感和激活先天免疫反应作为治疗手段产生洞察力 在PRC2缺失癌症中的策略，并为生物标记物驱动的临床试验提供关键的临床前数据。

PROJECT SUMMARY/ABSTRACT: The polycomb repressive complex 2 (PRC2) complex establishes and maintains di- and tri-methylation of the histone H3 at lysine 27 (H3K27me2/3) in the genome and regulates chromatin structure, transcription, cellular stemness and differentiation. PRC2 is a context-dependent tumor suppressor whose core components (e.g., EZH2, EED, or SUZ12) are inactivated in various cancer types. Among these, high-grade malignant peripheral nerve sheath tumor (MPNST), an aggressive soft tissue sarcoma with no effective therapies, has a high prevalence (≥80%) of biallelic inactivation of EED or SUZ12, leading to complete loss of the PRC2 function. PRC2 loss in cancer results in aberrant transcriptional activation of developmentally silenced master regulators, which leads to enhanced cellular plasticity and aberrant activation of multiple signaling pathways. We recently uncovered that PRC2 loss in cancer leads to an immune-desert tumor microenvironment and resistance to immune checkpoint blockade. Nevertheless, we observed that, in murine models of PRC2-loss MPNST and mammary tumors, response to immunotherapy can be enhanced by infection with immunogenic virus, which activates double-stranded RNA (dsRNA) signaling responses. Moreover, we have identified and validated a lethal interaction between PRC2 loss and DNA methyltransferase 1 (DNMT1) knockdown in MPNST. In vitro and in vivo treatment with a demethylating agent (decitabine) or selective DNMT1 inhibitors (GSK862, GSK032) led to enhanced cytotoxicity and antitumor effects in PRC2-loss compared to PRC2 wild-type (wt) MPNST models. Mechanistically, DNMT inhibitor (DNMTi) treatment in the PRC2-loss context amplified the expression of endogenous retrotransposons and subsequently led to activation of innate immune responses, which could be explained by retrotransposons forming dsRNA and triggering cytotoxicity via PKR-dependent dsRNA sensing. we hypothesize that PRC2 loss in cancer may create therapeutic opportunities for agents (e.g., DNMTi, synthetic dsRNA, immunogenic viruses) that activate the dsRNA signaling pathways in tumor cells to induce cytotoxicity and enhance immunogenicity. Here, we propose integrative multi-omics analysis (e.g., transcriptome, epigenome, single-cell [sc]RNA-seq and scATAC-seq) and innovative approaches (e.g., novel immunogenic viruses, a novel lineage-tracking system) to evaluate novel therapeutic strategies and their mechanisms of activating dsRNA responses and anti-tumor effects in relevant PRC2-loss cancers, focusing on specific aims: (1) dissect the molecular mechanisms that underlie the selective sensitivity to DNMTi in different PRC2-loss cancers; (2) evaluate exogenous dsRNAs as a strategy to induce cytotoxicity in PRC2-loss tumors; and (3) evaluate novel therapeutic strategies of activating dsRNA-responses in relevant cancer models. These studies will generate insights into exploiting dsRNA-sensing and activation of innate immune responses as therapeutic strategies in PRC2-loss cancers and provide the pivotal preclinical data for biomarker-driven clinical trials.