Role of ATRX, a chromatin remodeler, in immunotherapy response

背景：肿瘤免疫治疗是许多晚期癌症患者的重大突破。 然而，益处仍然局限于一小部分患者，我们需要更好地了解 提高疗效的应答性和抵抗力。我们已经确定了功能丧失(LoF)突变 JAK1或JAK2(干扰素受体的直接下游信号分子)与 抵抗PD-1受体阻滞剂。我们还通过筛选PD-1在人类黑色素瘤细胞系中发现了这些突变。 经干扰素-g处理的48个细胞株的L1表达。JAK1/2肿瘤中含有LoF，PD-L1基因完全缺失 表情。有趣的是，其中一个没有突变，具有活跃的信号通路，但丢失了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情况下也失去了适应性的PD-L1表达 干扰素信号，并假设表观遗传扰动是该表型的中介。有了这个 方法：我们观察到ATRX siRNA降低了PD-L1的表达，并进一步在体内进行了测试 老鼠模型。用ATRX KO MC38细胞进行小鼠体内实验，产生抗PD-1抗体治疗 要么加速肿瘤生长，要么不起作用。目前的研究旨在了解心力衰竭的机制。 肿瘤免疫治疗中ATRX缺失介导的耐药性。 目的/假设：ATRX，一种类似SWI/SNF的染色质重构体，正在调节 与免疫治疗反应相关的干扰素反应基因。 具体目标：我对这项研究有两个目标。第一个目标是询问免疫的机制。 使用各种工具探测表观遗传状态以逃避ATRX的丢失。第二个目标是在体内建立 使用ATRX KO使用不同的小鼠癌症模型进行肿瘤生长。 研究设计：目的1)建立ATRX KO B16细胞，然后用干扰素-g评估表观遗传学状态 使用CHIP/ATAC-seq刺激(MC38和B16 ATRX KO克隆)。Subaim 2)相关基因组 用染色质相关RNA序列(CHAR)研究(CHIP/ATAC)。苏巴伊姆3)评估 ATRX KO克隆中干扰素-g反应的表观遗传修饰物(使用各种表观遗传修饰物，如HDAC 抑制剂、脱甲基剂和EZH2抑制剂)。小鼠T细胞与ATRX的共培养试验 野生型亲本细胞和KO克隆。 目的2.Subaim 1)用ATRX KO对MC38和B16模型进行体内实验。Subaim2)Kras突变体 用ATRX KO建立小鼠肺癌细胞模型。肺癌和黑色素瘤中的ATRX KO 利用睡美人转座酶载体系统构建基因工程小鼠模型。 与军事健康相关：改善对多种晚期癌症的治疗对 退伍军人的健康。许多退伍军人在被诊断为癌症时有很大的发病率 这限制了他们的治疗选择。免疫疗法通常耐受性良好，并有很大的潜力 持久的反应，然而，好处仅限于部分患者(和退伍军人)。因此，它是 了解免疫应答机制，提高免疫治疗疗效势在必行 和抵抗。这项拟议的研究旨在了解癌细胞如何逃避的机制。 通过调节免疫系统的染色质状态，重点发挥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.