Role of KMT2D and aberrant enhancers in modulating tumor microenvironment in melanoma

总结 癌细胞获得遗传和表观遗传改变，增加健康并通过 肿瘤进化的多个步骤。我们对功能性表观遗传事件的了解有限， 与黑色素瘤进展相关。一种专注于鉴定的表观遗传调节因子的体内RNAi筛选 KMT2D，一种H3K4甲基转移酶，作为黑色素瘤的肿瘤抑制因子。KMT2D携带体细胞突变 在各种实体和血液恶性肿瘤中，包括黑色素瘤（约15%）。因此，KMT2D在 携带激活的BRAFV600 E和PTEN失活的黑色素瘤的基因工程小鼠模型 大大加速了黑色素瘤的形成显示KMT2D缺失的人小鼠黑素瘤肿瘤 与野生型肿瘤相比，增强子（H3K4me1和H3K27Ac）发生了剧烈变化， 生存这些增强子靶向肿瘤细胞中的免疫调节基因，并且一致地KMT2D KO 肿瘤显示肿瘤微环境改变。这些数据表明黑色素瘤中增强子功能障碍 由于KMT2D突变可能促进黑素瘤细胞的高度致瘤性行为。我们假设 KMT2D缺失重编程黑色素瘤中的增强子并通过以下方式赋予癌细胞侵袭性 免疫微环境的失调。本补助金的总体目标是了解 KMT2D在调节肿瘤微环境中的作用，测试增强子损失的功能并确定分子 KMT2D在该系统中的优先增强子调节机制。 目的1：探讨KMT2D对免疫微环境的影响及其机制。 黑色素瘤的突变体 在这个目标中，我们将利用iBIP; KMT 2D小鼠模型和可用的人类肿瘤数据来确定改变 在KMT2D缺陷条件下的免疫微环境中。 目的2：确定KMT2D缺陷细胞中免疫增强子损失的功能性。 为此，我们将通过以下方法对IFNg受体和IL 11基因上增强子损失的功能性进行定向测试： 基于CRISPR-Cas9的增强子缺失和表观遗传编辑实验。 目的3：探讨KMT2D介导免疫增强剂调节的分子机制。 在这里，我们将进行机制实验，以确定如何KMT2D优先招募免疫 增强剂。 总之，我们的研究将确定肿瘤中的增强子重编程，特别是那些由于KMT2D 损失重塑肿瘤微环境，这可以为新的治疗策略提供见解， 黑素瘤

SUMMARY Cancer cells acquire genetic and epigenetic alterations that increase fitness and drive progression through multiple steps of tumor evolution. We have limited understanding of functional epigenetic events that are associated with melanoma progression. An in vivo RNAi screen focused on epigenetic regulators identified KMT2D, an H3K4 methyltransferase, as a tumor suppressor in melanoma. KMT2D harbors somatic mutations in various solid and hematological malignancies including in melanoma (~15%). Consistently, loss of KMT2D in a genetically engineered mouse model of melanoma bearing activated BRAFV600E and PTEN inactivation drastically accelerated melanomagenesis. Human mouse melanoma tumors with KMT2D loss displayed drastic alterations in enhancers (H3K4me1 and H3K27Ac) compared to wild type tumors and displayed poor survival. These enhancers targeted immune modulatory genes in the tumor cells and consistently KMT2D KO tumors showed altered tumor microenvironment. These data suggest that enhancer malfunction in melanomas due to mutations in KMT2D may promote highly tumorigenic behavior of melanoma cells. We hypothesize that KMT2D loss reprograms enhancers in melanoma and imparts aggressive properties to cancer cells by misregulation of immune microenvironment. Overall objectives of this grant to understand the define roles of KMT2D in modulating tumor microenvironment, test functionality of enhancer losses and determine molecular mechanism of preferential enhancer regulation by KMT2D in this system. Aim 1: To determine the mechanism of immune microenvironment alteration by KMT2D and its mutants in melanoma. In this aim, we will utilize iBIP;KMT2D mouse model and available human tumor data to determine alterations in immune microenvironment in KMT2D deficient conditions. Aim 2: To determine the functionality of immune enhancer losses in KMT2D deficient cells. In this aim, we will perform directed tests at functionality of enhancer loss on IFNg receptors and IL11 genes by CRISPR-Cas9 based enhancer deletion and epigenetic editing experiments. Aim 3: To determine the molecular mechanism of KMT2D mediated regulation of immune enhancers. Here we will perform mechanistic experiments to determine how KMT2D is recruited preferentially to immune enhancers. Together, our study will determine how enhancer reprogramming in tumors specifically those due to KMT2D loss reshape the tumor microenvironment which could provide insight into new therapeutic strategies in melanoma.