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

项目总结/摘要： 多梳阻遏复合物2（PRC 2）复合物建立并维持多梳阻遏复合物2的二甲基化和三甲基化。 组蛋白H3位于基因组中的赖氨酸27（H3 K27 me 2/3），并调节染色质结构、转录、细胞 干性和分化。PRC 2是一种环境依赖性肿瘤抑制因子，其核心成分（例如， EZH 2、EED或SUZ 12）在各种癌症类型中失活。其中，高度恶性外周 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SUZ 12双等位基因失活的患病率（≥80%），导致PRC 2功能完全丧失。 癌症中PRC 2的缺失导致发育沉默的主调节因子的异常转录激活， 这导致增强的细胞可塑性和多种信号传导途径的异常激活。我们最近 发现PRC 2在癌症中的缺失导致免疫沙漠肿瘤微环境和对肿瘤的抵抗。 免疫检查点阻断。尽管如此，我们观察到，在PRC 2缺失的小鼠模型中，MPNST和MPNST的表达增加。 对于乳腺肿瘤，免疫治疗的反应可以通过免疫原性病毒感染来增强， 激活双链RNA（dsRNA）信号传导反应。此外，我们还确定并验证了 在MPNST中PRC 2丢失和DNA甲基转移酶1（DNMT 1）敲低之间的致死相互作用。体外和 用去甲基化剂（地西他滨）或选择性DNMT 1抑制剂（GSK 862、GSK 032）的体内治疗导致 与PRC 2野生型（wt）MPNST模型相比，在PRC 2损失中增强的细胞毒性和抗肿瘤作用。 从机制上讲，PRC 2缺失背景下的DNMT抑制剂（DNMTi）处理放大了PRC 2缺失的表达。 内源性逆转录转座子，随后导致先天免疫反应的激活，这可能是 通过反转录转座子形成dsRNA并通过PKR依赖性dsRNA传感触发细胞毒性来解释。 我们假设癌症中PRC 2的缺失可能为药物（例如，DNMTi，合成 dsRNA，免疫原性病毒），其激活肿瘤细胞中的dsRNA信号传导途径以诱导细胞毒性 并增强免疫原性。在这里，我们提出了综合多组学分析（例如，转录组， 表观基因组、单细胞[sc]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），以评估新的治疗策略及其机制， 在相关PRC 2缺失癌症中激活dsRNA应答和抗肿瘤作用，专注于特定目标： (1)剖析在不同PRC 2-损失中对DNMTi选择性敏感性的分子机制 （2）评估外源性dsRNA作为在PRC 2缺失肿瘤中诱导细胞毒性的策略;和（3） 评估在相关癌症模型中激活dsRNA应答的新治疗策略。这些研究 将产生利用dsRNA传感和激活先天免疫反应作为治疗的见解 这些研究为PRC 2缺失癌症的治疗策略提供了新的思路，并为生物标志物驱动的临床试验提供了关键的临床前数据。

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.