Defining the role of histone H3K4 mono-methyltransferase dysfunction in urothelial carcinoma

项目摘要/摘要 尿路上皮癌(UC)涉及膀胱、肾脏和输尿管的尿路上皮细胞，是一种主要的 美国发病率和死亡率的原因，尤其是男性。膀胱型UC在临床上可分为 非肌肉侵袭性(NMIBC)和肌肉侵袭性(MIBC)。MIBC是绝大多数转移的原因 和死亡率，治愈率只有50%左右。接受治疗的NMIBC患者有复发或 在以前的或新的地点发展到MIBC。超过一半的尿路上皮癌，无论起源于何处，是否有港湾 组蛋白去甲基酶KDM6A(UTX)和两个高度同源的组蛋白功能突变丢失 甲基转移酶KMT2C(MLL3)和KMT2D(MLL4)。这些蛋白质形成MLI3/4-COMPASS (与Set1相关的蛋白质复合体)-样复合体，调节增强子功能，部分通过 组蛋白在增强剂上的甲基化。增强子是DNA中调节谱系特异性的区域 转录程序。最近的研究表明，患有两种尿路上皮癌的患者远在 输尿管和膀胱部位(输尿管和膀胱)存在相同的指南针状突变。组织学的进一步测序 良性尿路上皮在随着时间的推移扩张时发现复合体中的频繁突变。我们的假设是，这些 尿路上皮“野癌变”下的突变。我们的实验室已经培育出一只基因工程小鼠 尿路上皮中Kmt2c、Kmt2d或其组合缺失的模型。这些小鼠的尿路上皮 未见组织学异常。然而，转录组分析显示尿路上皮细胞数量增加。 茎和功能研究表明，它们表现出更强的类有机物形成能力。当进入到 PTEN条件性缺失小鼠，在肿瘤发生中有很强的协同性。这样做的总体目标是 建议利用我们最近产生的尿路上皮这种指南针状复杂丢失的小鼠模型来 从机制上理解其在肿瘤尿路上皮抑制中的作用。具体地说，在目标1中，我们试图确定 尿路上皮细胞突变株的茎性、克隆动态、癌基因和致癌物易感性 这个罗盘状的复合体，使用谱系追踪、器官培养和单细胞RNA测序。在AIM 2，我们试图确定MLI3/4-COMPASS功能障碍和癌基因之间的功能相互作用 激活。在目标3中，我们将试图确定Kmt2c和Kmt2d在尿路上皮细胞中的丢失如何影响增强子和 启动子功能。活性增强子是与转录因子结合的开放染色质的基因组区域， 增强子RNA的发散转录，并循环到启动子。我们将通过全局映射来研究每一步 组蛋白标记、染色质可及性、相关基因的mRNA转录和启动子的环路 使用最先进的表观遗传学技术。

Project Summary/Abstract Urothelial carcinoma (UC) involves the urothelial cells that line the bladder, kidney and ureters and is a major cause of morbidity and mortality in the US, especially in men. Bladder UC can be clinically separated into nonmuscle invasive (NMIBC) and muscle invasive (MIBC). MIBC accounts for the vast majority of metastasis and mortality, having only a ~50% cure rate. Patients with treated NMIBC are at risk of recurrence or progression to MIBC at prior or de novo sites. Over half of urothelial cancer, regardless of site of origin, harbor loss of function mutations in the histone demethylase KDM6A (UTX) and in two highly homologous histone methyltransferases KMT2C (MLL3) and KMT2D (MLL4). These proteins form the MLL3/4-COMPASS (COMplex of Proteins ASsociated with Set1)-like complex that regulate enhancer function, partly through methylation of histones at enhancers. Enhancers are regions of DNA that regulate lineage specific transcriptional programs. Recent studies have shown that patients with two urothelial carcinomas in far away sites (ureter and bladder) harbor the same COMPASS-like mutation. Further sequencing of histologically benign urothelium identify frequent mutations in the complex at expand over time. Our hypothesis is that these mutations under “field-cancerization” of the urothelium. Our lab has generated a genetically engineered mouse model with deletion of Kmt2c, Kmt2d, or the combination in the urothelium. The urothelium of these mice exhibit no histologic abnormalities. However, transcriptome analysis shows the urothelium exhibit increased stemness and functional studies show they exhibit increased organoid forming abilities. When crossed into the Pten conditional deletion mouse, there was robust cooperativity in tumorigenesis. The overall objective of this proposal is to utilize our recently generated mouse models of urothelial this COMPASS-like complex loss to mechanistically understand its role in tumor urothelial suppression. Specifically, in Aim 1, we seek to determine the stemness, clonal dynamics, oncogene and carcinogen susceptibility of urothelium harboring mutations in this COMPASS-like complex, using lineage tracing, organoid culture, and single-cell RNA-sequencing. In Aim 2, we seek to determine the functional interplay between MLL3/4-COMPASS dysfunction and oncogene activation. In Aim 3, we will seek to define how loss of Kmt2c and Kmt2d in urothelial cells affect enhancer and promoter function. Active enhancers are genomic regions of open chromatin with transcription factor binding, divergent transcription of enhancer RNA, and looping to promoters. We will study each step by global mapping of histone marks, chromatin accessibility, mRNA transcription of associated gene and looping to promoters using state-of the art epigenetics techniques.