Role of ATRX, a chromatin remodeler, in immunotherapy response

ATRX（染色质重塑剂）在免疫治疗反应中的作用

• 批准号: 10367734
• 负责人: Daniel SANGHOON Shin
• 金额: --
• 依托单位: VA GREATER LOS ANGELES HEALTHCARE SYSTEM
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-04-01 至 2026-03-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10367734
• 关键词: ATAC-seq; ATRX gene; Advanced Malignant Neoplasm; Atlases; Binding; Biological Assay; C57BL/6 Mouse; CRISPR/Cas technology; Cancer Model; Cancer cell line; Cell Line; Cells; ChIP-seq; Chromatin; Chromatin Structure; Clinical; Coculture Techniques; Colorectal Cancer; Complex; Coupled; Data; Development; Diagnosis; EZH2 gene; Elements; Embryo; Epigenetic Process; Exhibits; Feedback; Fibroblasts; Genes; Genetic Transcription; Genetically Engineered Mouse; Genome; Genomics; Granzyme; Health; Histone Deacetylase Inhibitor; Human; IRF1 gene; Immune; Immune Evasion; Immune system; Immunocompetent; Immunooncology; Immunotherapy; Institutes; Interferon Receptor; Interferon Type II; Interferons; JAK1 gene; JAK2 gene; Janus kinase; Kidney; Knock-out; Lead; Lentivirus Vector; MC38; Malignant Neoplasms; Malignant neoplasm of lung; Mediating; Melanoma Cell; Military Personnel; Modeling; Morbidity - disease rate; Mus; Mutation; Outcome; PD-1 blockade; Parents; Pathway interactions; Patients; Phenotype; Play; Polycomb; Process; Proteins; RNA Sequences; Reporting; Research; Research Design; Research Proposals; Resistance; Rest; Role; Signal Pathway; Signal Transduction; Signaling Molecule; Sleeping Beauty; Small Interfering RNA; Specificity; System; T-Lymphocyte; Testing; The Cancer Genome Atlas; Therapeutic; Transcript; Transposase; Treatment Efficacy; Untranslated RNA; Veterans; XCL1 gene; anti-PD1 antibodies; anti-PD1 therapy; antitumor effect; base; cBioPortal; cancer cell; cancer immunotherapy; chromatin remodeling; design; experimental study; immune checkpoint blockade; improved; in vivo; inhibitor; loss of function; loss of function mutation; lung cancer cell; melanoma; mouse model; mutant; patient subsets; perforin; polybromo; programmed cell death ligand 1; promoter; receptor; resistance mechanism; response; screening; side effect; tool; transcriptome; transcriptome sequencing; treatment response; trend; tumor; tumor growth; vector

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.

背景：对于许多晚期癌症患者而言，癌症免疫疗法是一个重大突破。 但是，收益仍然仅限于一部分患者，我们需要更好地了解 对提高治疗功效的反应和抗药性。我们已经确定了功能丧失（LOF）突变 与JAK1或JAK2（干扰素受体的立即下游信号分子）与 对PD-1封锁的阻力。我们还通过筛选PD-在人黑色素瘤细胞系中发现了这些突变 IFN-G处理（48个细胞系）的L1表达。 JAK1/2中的肿瘤港LOF，完全丢失了PD-L1 表达。有趣的是，其中一个人没有用主动信号通路的突变，但丢失了PD-L1 表达。我们探讨了为什么某些人类癌细胞即使完好无损地失去了自适应PD-L1表达 干扰素信号传导并假设表观遗传扰动正在介导这种表型。与此 方法，我们观察到使用ATRX siRNA降低了PD-L1的表达，该siRNA进一步测试了体内 鼠标模型。 ATRX KO MC38细胞的体内小鼠实验，抗PD-1抗体治疗产生 加速肿瘤生长或无效。当前的研究旨在了解 癌症免疫疗法中ATRX丧失介导的抗性。 客观/假设：ATRX，SWI/SNF样染色质重塑器正在调节可访问性 与免疫疗法反应相关的干扰素反应基因。 具体目的：我有两个目标。第一个目的是审问免疫机制 使用各种工具探测表观遗传状态的ATRX丢失。第二个目的是在体内建立 使用各种鼠类癌模型使用ATRX KO肿瘤生长。 研究设计：目标1。Subaim1）生成Atrx KO B16细胞，然后评估IFN-G的表观遗传学状态 使用芯片/ATAC-SEQ刺激（MC38和B16 ATRX KO克隆）。 Subaim 2）相关基因组 研究（CHIP/ATAC）与染色质相关的RNA序列（CHAR）。 Subaim 3）评估 ATRX KO克隆中IFN-G反应中的表观遗传修饰剂（使用各种表观遗传修饰剂，例如HDAC 抑制剂，脱甲基剂和EZH2抑制剂）。 Subaim 4）与鼠T细胞与ATRX的共培养测定 野生型母细胞和KO克隆。 AIM2。Subaim1）使用ATRX KO进行MC38和B16模型的体内实验。 Subaim2）Kras突变体 带有ATRX KO的鼠肺癌细胞系模型。 Subaim 3）肺癌和黑色素瘤中的ATRX KO 使用睡美人转座酶矢量系统进行基因工程的鼠标模型。 与军事健康相关：改善对许多类型的高级癌症的处理对于 退伍军人的健康。许多退伍军人被诊断出患有癌症时患有明显的发病率 这限制了他们的治疗选择。免疫疗法通常具有良好的耐受性，并且具有很大的潜力 但是，持久的反应，这种益处仅限于一部分患者（和退伍军人）。因此，是 必须通过了解反应的机制来提高免疫疗法的治疗功效 和阻力。拟议的研究旨在了解癌细胞如何逃避的机制 免疫系统通过调节染色质状态，重点关注ATRX的作用。