Identifying the Role of Tumor Cell Intrinsic DNMT1 in Anti-Tumor Immunity in Pancreatic Ductal Adenocarcinoma

项目摘要 胰腺导管腺癌（PDAC）是目前第三大癌症相关死亡原因 在美国不到9%的五年存活率主要是由于 早期发现和缺乏有效的治疗PDAC。新的免疫疗法，如免疫 已经彻底改变了其他癌症治疗的检查点阻断未能在 PDAC。这被认为是由于PDAC的免疫抑制微环境，其限制了PDAC的免疫抑制。 有效免疫治疗所必需的效应T细胞浸润和活化。了解如何提高 尽管存在免疫抑制微环境，但T细胞活化和浸润对于增加免疫应答是必不可少的。 免疫疗法在PDAC中的疗效。 使用胰腺癌的基因工程小鼠模型（“KPCY”），我们的实验室已经证明， 来源于原发性KPCY肿瘤的克隆可分为“T细胞炎症”或“非T细胞炎症”， 表型。虽然T细胞发炎的肿瘤对联合免疫疗法有反应，但非T细胞发炎的肿瘤对联合免疫疗法有反应。 肿瘤有抵抗力。对非T细胞炎症细胞的CRISPR筛选发现，DNMT1，一种DNA， 甲基转移酶通过DNA复制参与维持甲基化标记，对肿瘤的发生和发展具有重要意义。 PDAC的增长。在非T细胞炎症肿瘤细胞系中敲除DNMT 1，发现T细胞显著增加。 细胞浸润并减少体内肿瘤生长。然而，T细胞耗竭被发现可以拯救野生型 表型。基于最近对非特异性DNMT抑制剂氮杂胞苷的研究，DNMT 1的丢失是 假设去抑制基因组中的内源性逆转录病毒，导致dsRNA诱导， 激活“病毒模仿”免疫机制。本建议的目标是：（1）确定如何损失 DNMT 1促进T细胞依赖性抗肿瘤免疫，和（2）评估DNMT 1的治疗潜力。 靶向DNMT1与免疫疗法结合。 探讨肿瘤细胞内源性DNMT 1抑制T细胞依赖性抑制的机制 在肿瘤生长的过程中，我将首先评估内源性逆转录病毒和基因的DNA甲基化变化， 与DNMT1缺失引起的病毒模拟途径有关。然后，我将确定DNMT1是否丢失 病毒模拟途径的激活，以及抑制该途径是否拯救了野生型 表型（Aim 1）。为了确定DNMT 1作为靶点的翻译潜力，我将首先鉴定抗肿瘤的 由DNMT1缺失引起的T细胞和肿瘤细胞的变化。我将用DNMT 1试验小鼠免疫疗法 敲除肿瘤以确定对免疫疗法的敏感性的变化（Aim 2）。最终， 本研究的结果将使我们更好地了解DNMT 1在调节PDAC中的作用 免疫微环境及其作为治疗靶点潜力。

Project Summary Pancreatic ductal adenocarcinoma (PDAC) is currently the third leading cause of cancer-related death in the United States. The five-year survival rate of less than nine percent is attributed mainly to a difficulty in early detection and a lack of effective treatments for PDAC. Novel immunotherapies such as immune checkpoint blockade which have revolutionized treatment of other cancers have failed to achieve efficacy in PDAC. This is thought to be due to the immunosuppressive microenvironment of PDAC which limits the effector T cell infiltration and activation necessary for effective immunotherapy. Understanding how to increase T cell activation and infiltration despite an immunosuppressive microenvironment is essential to increasing the efficacy of immunotherapies in PDAC. Using a genetically engineered mouse model (“KPCY”) of pancreatic cancer, our lab has demonstrated that clones derived from primary KPCY tumors can be divided into “T-cell-inflamed” or “non-T-cell-inflamed” phenotypes. While T-cell-inflamed tumors are responsive to combination immunotherapy, non-T-cell-inflamed tumors are resistant. A CRISPR screen of non-T-cell-inflamed cells found that DNMT1, a DNA methyltransferase involved in maintaining methylation marks through DNA replication, is important for tumor growth in PDAC. Knockout of DNMT1 in a non-T-cell-inflamed tumor line was found to significantly increase T cell infiltration and decrease tumor growth in vivo. T cell depletion, however, was found to rescue the wild type phenotype. Based on recent studies of the non-specific DNMT inhibitor azacytidine, loss of DNMT1 is hypothesized to de-repress endogenous retroviruses in the genome, leading to dsRNA induction which activates a “viral mimicry” immune mechanism. The goals of this proposal are: (1) to determine how loss of DNMT1 promotes T-cell dependent anti-tumor immunity, and (2) to assess the therapeutic potential of targeting DNMT1 in conjunction with immunotherapy. To examine the mechanism of tumor cell intrinsic DNMT1 in inhibition of T-cell dependent suppression of tumor growth, I will first assess the changes in DNA methylation at endogenous retroviruses and genes related to the viral mimicry pathway provoked by loss of DNMT1. I will then determine if loss of DNMT1 leads to activation of the viral mimicry pathway, and whether inhibition of this pathway rescues the wild type phenotype (Aim 1). To determine the translational potential of DNMT1 as a target, I will first identify anti-tumor changes in T cells and tumor cells provoked by loss of DNMT1. I will trial immunotherapy in mice with DNMT1 knockout tumors to determine changes in sensitivity to immunotherapy (Aim 2). Ultimately, the insights gained from this study will provide us with a better understanding of the role of DNMT1 in regulating the PDAC immune microenvironment and its potential as a therapeutic target.