Role of ATRX, a chromatin remodeler, in immunotherapy response

背景：癌症免疫治疗是许多晚期癌症患者的重大突破。 然而，受益仍然局限于一部分患者，我们需要更好地了解 反应和抗性，以提高治疗效果。我们已经确定了功能丧失（LoF）突变， JAK 1或JAK 2（干扰素受体的直接下游信号分子）与 对PD-1阻断的抵抗。我们还发现这些突变在人类黑色素瘤细胞系通过筛选PD- 用IFN-γ处理的L1表达（48个细胞系）。肿瘤在JAK 1/2中携带LoF，完全丢失PD-L1 表情有趣的是，其中一个没有突变，但失去了PD-L1， 表情我们探索了为什么一些人类癌细胞即使在完整的PD-L1表达的情况下也会失去适应性PD-L1表达， 干扰素信号传导，并假设表观遗传扰动介导了这种表型。与此 通过这种方法，我们观察到ATRX siRNA降低了PD-L1表达，并进一步在体内进行了测试。 小鼠模型。使用ATRX KO MC 38细胞的体内小鼠实验，产生抗PD-1抗体疗法 要么加速肿瘤生长要么无效。目前的研究旨在了解 癌症免疫治疗中ATRX缺失介导的耐药性。 目的/假设：ATRX是一种SWI/SNF样染色质重塑剂，可调节 干扰素应答基因与免疫治疗应答相关。 具体目标：我的研究有两个目标。第一个目的是询问免疫的机制， 逃避与损失ATRX使用各种工具来探测表观遗传状态。第二个目的是在体内建立 肿瘤生长与ATRX KO使用各种鼠癌症模型。 研究设计：目的1。Subaim 1）生成ATRX KO B16细胞，然后用IFN-g评估表观遗传学状态 使用ChIP/ATAC-seq. Subaim 2）关联基因组 使用染色质相关RNA序列（ChAR）的研究（ChIP/ATAC）。Subaim 3）评估 ATRX KO克隆中IFN-g应答的表观遗传修饰剂（使用各种表观遗传修饰剂，如HDAC 抑制剂、去甲基化剂和EZH 2抑制剂）。Subaim 4）鼠T细胞与ATRX的共培养测定 野生型亲本细胞和KO克隆。 目标2. Subaim 1）使用ATRX KO的MC 38和B16模型的体内实验。Subaim 2）Kras突变体 具有ATRX KO的鼠肺癌细胞系模型。Subaim 3）肺癌和黑色素瘤中的ATRX KO 使用睡美人转座酶载体系统的基因工程小鼠模型。 与军事卫生相关：改善许多类型晚期癌症的治疗对于 退伍军人的健康。许多退伍军人患有显着的发病率时，他们被诊断患有癌症 限制了他们的治疗选择免疫疗法通常耐受性良好，并且具有显著的潜力， 然而，持久的反应，受益仅限于一部分患者（和退伍军人）。因此有 必须通过了解反应机制来提高免疫治疗的疗效 和抵抗这项拟议中的研究旨在了解癌细胞如何逃避 免疫系统通过调节染色质状态，重点是ATRX的作用。

Background: Cancer immunotherapy is a major breakthrough for many patients with advanced cancer. However, benefits are still limited to a subset of patients, and we need to better understand the mechanisms of response and resistance to improve therapeutic efficacy. We have identified loss of function (LoF) mutations in JAK1 or JAK2 (immediate downstream signaling molecule of interferon receptor) that are associated with resistance to PD-1 blockade. We also found these mutations in human melanoma cell lines by screening PD- L1 expression with IFN-g treatment (48 cell lines). Tumors harbor LoF in JAK1/2, completely lost PD-L1 expression. Interestingly, one of them harbors no mutation with an active signaling pathway, yet lost PD-L1 expression. We explored why some human cancer cells lost adaptive PD-L1 expression even with intact interferon signaling and hypothesized that the epigenetic perturbation is mediating this phenotype. With this approach, we observed reduced PD-L1 expression with ATRX siRNA which was further tested with in vivo mouse models. In vivo mouse experiments with ATRX KO MC38 cells, anti-PD-1 antibody therapy produced either accelerated tumor growth or no effect. The current study is designed to understand the mechanism of resistance mediated by loss of ATRX in cancer immunotherapy. Objective/hypothesis: ATRX, a SWI/SNF-like chromatin remodeler is modulating the accessibility of interferon responsive genes that are associated with immunotherapy response. Specific aims: I have two aims for this study. The first aim is to interrogate the mechanisms of immune evasion with loss of ATRX using various tools to probe epigenetic state. The second aim is to establish in vivo tumor growth with ATRX KO using various murine cancer models. Study design: Aim 1. Subaim1) Generate ATRX KO B16 cells followed by Assess epigenetics state with IFN-g stimulation (both MC38 and B16 ATRX KO clones) using ChIP/ATAC-seq. Subaim 2) Correlate genomic studies (ChIP/ATAC) with Chromatin-Associated RNA-sequence (ChAR). Subaim 3) Assess the impact of epigenetic modifiers in IFN-g response in ATRX KO clones (using various epigenetic modifiers, such as HDAC inhibitor, demethylating agents and EZH2 inhibitor). Subaim 4) Coculture assay with murine T cells with ATRX wild-type parent cells and KO clones. Aim 2. Subaim 1) In vivo experiments with MC38 and B16 models with ATRX KO. Subaim2) Kras mutant murine lung cancer cell line models with ATRX KO. Subaim 3) ATRX KO in lung cancer and melanoma genetically engineered mouse models using sleeping beauty transposase vector system. Relevant to Military health: Improving treatment of many types of advanced cancers is critically important to the health of Veterans. Many Veterans suffer from significant morbidity when they are diagnosed with cancer that limits their therapeutic options. Immunotherapy is generally well tolerated and has a significant potential for durable response, however, the benefit is limited to a subset of patients (and Veterans). Therefore, it is imperative to improve therapeutic efficacy of immunotherapy by understanding the mechanisms of response and resistance. The proposed research is designed to understand the mechanisms of how cancer cells evade the immune system by modulating the chromatin state, focusing on the role of ATRX.