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

摘要 癌细胞获得了基因和表观遗传改变，这些改变增加了健康，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 与黑色素瘤的进展有关。聚焦于表观遗传调控因子的活体RNAi筛选 KMT2D，一种H3K4甲基转移酶，在黑色素瘤中作为肿瘤抑制因子。KMT2D含有体细胞突变 在各种实体和血液系统的恶性肿瘤中，包括黑色素瘤(~15%)。一贯地，KMT2D在 携带活化BRAFV600E和PTEN失活基因的黑色素瘤基因工程小鼠模型 急剧加速了黑色素瘤的发生。显示KMT2D缺失的人小鼠黑色素瘤 与野生型肿瘤相比，增强子(H3K4me1和H3K27Ac)发生剧烈变化，表现较差 生死存亡。这些增强剂针对肿瘤细胞中的免疫调节基因，并一致地KMT2D KO 肿瘤表现为肿瘤微环境改变。这些数据表明增强子在黑色素瘤中起作用 由于KMT2D的突变可能促进黑色素瘤细胞的高度致瘤行为。我们假设 KMT2D丢失对黑色素瘤中的增强剂重新编程，并通过以下方式赋予癌细胞侵袭性 免疫微环境调节失调。这笔赠款的总体目标是了解 KMT2D在调节肿瘤微环境、检测增强子丢失功能和确定分子方面的作用 KMT2D在该系统中选择性调控增强子的机制。 目的1：探讨KMT2D改变免疫微环境的机制及其作用机制。 黑色素瘤中的突变。 在这个目标中，我们将利用iBIP；KMT2D小鼠模型和可用的人类肿瘤数据来确定变化 在KMT2D缺乏条件下的免疫微环境中。 目的2：确定KMT2D缺陷细胞免疫增强子缺失的功能。 为此，我们将对IFNG受体和IL11基因上增强子丢失的功能进行直接测试 基于CRISPR-Cas9的增强子缺失和表观遗传编辑实验。 目的：探讨KMT2D介导免疫增强剂调节的分子机制。 在这里，我们将进行机械实验，以确定KMT2D是如何优先被招募到免疫系统的 增强剂。 总之，我们的研究将确定肿瘤中的增强子重新编程，特别是那些由KMT2D引起的肿瘤 Lost重塑肿瘤微环境，为新的治疗策略提供洞察 黑色素瘤。

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.