Defining the role of the EGFR in MHC expression and anti-tumor immune responses.

描述(由申请人提供)： 目的：本研究的主要目的是：(1)明确致癌的EGFR/MAPK信号转导通路如何影响MHC分子的表达；(2)研究针对EGFR和MAPK通路中的酶的治疗对抗肿瘤免疫应答和免疫治疗应答的影响。研究计划：在目标1中，我们将使用特征良好的细胞系来模拟由EGFR过度表达、致癌RAS突变和致癌BRAF突变驱动的人类癌症。这些细胞系将被用来确定致癌的EGFR/MAPK信号转导和为对抗这一信号而开发的药物如何影响MHC-I类表达的转录和表观遗传调节。在目标2中，我们将这些药物与相关的小鼠肿瘤模型相结合，以确定这些药物如何影响体内对肿瘤挑战的免疫反应、相关免疫系统基因的表达以及对免疫治疗(如免疫检查点阻断和治疗性疫苗接种)的反应。方法：利用上述模型，我们将结合EGFR、BRAFV600E和MEK1/2抑制剂处理与染色质免疫沉淀实验，以确定EGFR/MAPK信号对转录因子募集和在MHCI启动子上放置表观遗传标记的影响。使用报告分析，我们将确定MHC I类启动子中响应EGFR/MAPK信号的区域。为了探索更复杂的调控机制，我们将研究EGFR/MAPK信号如何影响已知调节MHC表达的增强子阻断DNA元件的功能。我们还将确定EGFR/MAPK信号如何影响CTCF的招募/结合，CTCF是一种通过减轻复杂的染色质相互作用来调节MHC表达的蛋白质，它是一种通过减轻MHC内的位点而调节MHC表达的蛋白质。在我们的体内研究中，我们将使用治疗反应性和非反应性肿瘤模型来确定EGFR/MAPK抑制剂如何影响肿瘤生长、肿瘤反应性和抗原特异性CD8+T细胞的产生，以及使用流式细胞术、功能性细胞毒性T淋巴细胞(CTL)分析、RNA分析和其他方法对肿瘤微环境的影响。我们将探索一种新的假设，即这些药物在宿主抗肿瘤免疫中具有临床相关的肿瘤依赖和肿瘤非依赖效应，可用于增强免疫治疗的反应。临床意义：EGFR和MAPKs的抑制剂已经用于治疗具有不同反应率的晚期癌症退伍军人。为了帮助将这些药物与基于免疫的新疗法相结合，对这些激酶抑制剂如何影响体内免疫事件有一个复杂的了解将是至关重要的。事实上，尽管这些抑制剂已经在晚期癌症患者的临床试验中与免疫疗法相结合，但它们对免疫系统的影响在很大程度上是未知的。通过表征这些药物如何影响免疫反应，我们可以开发一个合理的框架，以优化这些药物的单独使用和与基于免疫的治疗相结合。此外，通过确定控制EGFR/MAPK介导的MHC抑制的机制，可以开发新的方法来破坏MHCI下调调节和导致的肿瘤细胞免疫逃逸，这种免疫逃逸通常发生在癌症中。

DESCRIPTION (provided by applicant): Objectives: The main objectives of this proposal are to: (1) define how oncogenic EGFR/MAPK signal transduction influences the expression of MHC molecules and, (2) characterize the impact of therapies that target the EGFR and enzymes in the MAPK pathway on anti-tumor immune responses and the response to immunotherapy. Research Plan: In Aim 1, we will use well-characterized cell lines to model human cancers driven by EGFR over-expression, oncogenic RAS mutations, and oncogenic BRAF mutations. These cell lines will be used to determine how oncogenic EGFR/MAPK signal transduction and the medications developed to combat this signaling, impact the transcriptional and epigenetic regulation of MHC class I expression. In Aim 2, we will couple these same medications with relevant murine tumor models to determine how these medications impact in vivo immune responses to tumor challenge, the expression of relevant immune system genes, and the response to immunotherapies such as immune checkpoint blockade and therapeutic vaccination. Methods: Using the above models, we will combine EGFR, BRAFV600E, and MEK1/2 inhibitor treatment with chromatin immunoprecipitation experiments to define the impact of EGFR/MAPK signaling on the recruitment of transcription factors and the placement of epigenetic marks at the MHCI promoter. Using reporter assays, we will define regions within MHC class I promoters that are responsive to EGFR/MAPK signaling. To explore more complex regulatory mechanisms, we will investigate how EGFR/MAPK signaling impacts the function of an enhancer-blocking DNA element known to regulate MHC expression. We will also determine how EGFR/MAPK signaling impacts the recruitment/binding of CTCF, a protein that regulates MHC expression by mitigating complex chromatin interactions, to sites within the MHC. For our in vivo studies, we will use therapy- responsive and non-responsive tumor models to determine how EGFR/MAPK inhibitors impact tumor growth, the generation of tumor-reactive and antigen-specific CD8+ T cells, and the tumor microenvironment using flow cytometry, functional cytotoxic T lymphocyte (CTL) assays, RNA analysis and other approaches. We will explore the novel hypothesis that these medications have clinically relevant tumor-dependent and tumor- independent effects on host anti-tumor immunity that can be used to enhance the response to immunotherapy. Clinical Relevance: Inhibitors of the EGFR and MAPKs are already used to treat Veterans with advanced cancer with variable response rates. To help integrate these medications with new and developing therapies that are immune based, it will be crucial to have a sophisticated understanding of how these kinase inhibitors influence immune events in vivo. Indeed, despite the fact these inhibitors are already being combined with immunotherapies in clinical trials for those with advanced cancer, their impact on the immune system is largely undefined. By characterizing how these medications influence immune responses, we can develop a rational framework to optimize the use of these medications alone and in combination with immune-based therapy. In addition, by defining the mechanisms that govern EGFR/MAPK-mediated MHC repression, new approaches can be developed to disrupt the MHCI down regulation and resulting tumor cell immune escape that occurs commonly in cancer.