Defining epigenetic signaling to reshape pancreatic tumor microenvironment

项目摘要/摘要 胰腺癌患者的5年生存率保持在10%，主要是由于肿瘤对 标准的化疗和免疫疗法。深层免疫抑制肿瘤微环境的贡献 到治疗抗药性。值得注意的是，接受新辅助治疗的胰腺癌中有82%伴有功能丧失。 组蛋白修饰酶KMT2D的突变。表观遗传学已知会影响癌细胞的行为。 然而，肿瘤细胞内在的表观遗传学改变如何调节胰腺肿瘤的微环境 仍然难以捉摸。长期目标是开发新的治疗方法，将胰腺癌转化为 免疫热肿瘤，提高免疫治疗的疗效和患者的存活率。整体而言 本应用的目的是：1)确定KMT2D信号对肿瘤的影响 2)KMT2D调控激活素A表达的机制。这个 中心假说是胰腺癌KMT2D缺失后激活素A的上调重新编程癌症- 相关的成纤维细胞和免疫细胞促进亲肿瘤免疫抑制微环境。这个 这个项目的基本原理是KMT2D调节肿瘤的机制的知识 微环境将为未来临床前和临床治疗新策略的发展铺平道路 胰腺癌。中心假设将通过追求三个具体目标来检验：1)阐明 KMT2D信号对肿瘤微环境免疫成分的影响；2)定义 KMT2D信号对异质肿瘤相关成纤维细胞的调控；以及3)决定 KMT2D调控胰腺癌激活素A的分子机制在第一个和第二个下面 AIMS，我们将使用我们已建立的胰腺特异失活的胰腺癌基因小鼠模型 以确定KMT2D信号对癌症分化和激活的影响- 肿瘤中相关的成纤维细胞、巨噬细胞和T细胞以及配体-受体的相互作用 微环境及其内在机制。单细胞RNA测序技术，质量细胞仪， 并将采用多重荧光免疫组织化学方法。对于第三个目标，我们将描述 KMT2D与辅因子的相互作用及其调节激活素A的机制 用CHIP-SEQ、BruUV-SEQ和功能荧光素酶报告分析转录和增强子活性。这个 本申请中提出的研究具有创新性，因为它关注的是表观遗传学的一个新概念 来自肿瘤细胞的信号重塑肿瘤微环境，促进癌症进展和 使用最先进的动物模型和方法。这项拟议的研究具有重要意义，因为它将 KMT2D对胰腺癌转录调控机制及作用的研究进展 胰腺肿瘤微环境的信号转导将为未来的临床前和临床奠定基础 开发新的肿瘤微环境靶向治疗策略。

PROJECT SUMMARY/ABSTRACT The 5-year survival of pancreatic cancer patients remains at 10% primarily due to the tumor resistance to standard chemo and immunotherapies. The profound immunosuppressive tumor microenvironment contributes to treatment resistance. Notably, 82% of the neoadjuvant-treated pancreatic cancers carry loss of function mutations for KMT2D, a histone modification enzyme. Epigenetics is known to impact cancer cell behavior. However, how tumor cell-intrinsic epigenetic alterations modulate the pancreatic tumor microenvironment remains elusive. The long-term goal is to develop new treatments to convert pancreatic cancer to an immunologically hot tumor and improve the efficacy of immunotherapy and patient survival. The overall objectives in this application are to 1) determine the impact of KMT2D signaling on the tumor microenvironment and 2) characterize the mechanisms by which KMT2D regulates activin A expression. The central hypothesis is that upregulation of activin A upon KMT2D loss in pancreatic cancer reprograms cancer- associated fibroblasts and immune cells to promote a pro-tumoral immunosuppressive microenvironment. The rationale for this project is that the knowledge of the mechanisms by which KMT2D regulates the tumor microenvironment will pave the way for future preclinical and clinical development of new strategies to treat pancreatic cancer. The central hypothesis will be tested by pursuing three specific aims: 1) Elucidate the impact of KMT2D signaling on the immune composition in the tumor microenvironment; 2) Define the regulation of heterogeneous cancer-associated fibroblasts by KMT2D signaling; and 3) Determine the molecular mechanisms of activin A regulation by KMT2D in pancreatic cancer. Under the first and second aims, we will use our established pancreatic cancer genetic mouse models with pancreas-specific inactivation of KMT2D to determine the impact of KMT2D signaling on the differentiation and activation of cancer- associated fibroblasts, macrophages, and T cells, and ligand-receptor interactions in the tumor microenvironment and the underlying mechanisms. Single-cell RNA sequencing technology, mass cytometry, and multiplex fluorescent immunohistochemistry will be used. For the third aim, we will characterize the interactions between KMT2D and co-factors and the mechanisms of KMT2D-mediated regulation of activin A transcription and enhancer activity using ChIP-seq, BruUV-seq, and functional luciferase reporter assays. The research proposed in this application is innovative because it focuses on a novel concept that epigenetic signaling from tumor cells remodels the tumor microenvironment and contributes to cancer progression and uses state-of-the-art animal models and approaches. The proposed research is significant because it will shed light on the mechanism of transcriptional regulation by KMT2D in pancreatic cancer and the effect of KMT2D signaling on pancreatic tumor microenvironment, which will set the stage for future preclinical and clinical development of new tumor microenvironment-targeting treatment strategies.