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

定义组蛋白 H3K4 单甲基转移酶功能障碍在尿路上皮癌中的作用

• 批准号: 10522552
• 负责人: Yu Chen
• 金额: $ 60.23万
• 依托单位: SLOAN-KETTERING INST CAN RESEARCH
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-09-01 至 2027-08-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10522552
• 关键词: Ablation; Affect; Allografting; Archives; Benign; Binding; Bladder; Carcinogens; Cell Line; Chromatin; Clinical; Clonal Expansion; Complex; DNA; Data; Down-Regulation; EZH2 gene; Enhancers; Epigenetic Process; Event; Exhibits; Functional disorder; Gene Expression; Genes; Genetic; Genetic Engineering; Genetic Transcription; Genetically Engineered Mouse; Genomic Segment; Histologic; Histology; Histone H3; Histones; Human; KRASG12D; Kidney; Lesion; Location; Lysine; MLL gene; Malignant Neoplasms; Malignant neoplasm of ureter; Malignant neoplasm of urinary bladder; Maps; Mediating; Messenger RNA; Methyltransferase; Mitogen-Activated Protein Kinase Kinases; Modeling; Molecular; Morbidity - disease rate; Morphology; Mus; Muscle; Mutation; Neoplasm Metastasis; Nitrosamines; Oncogene Activation; Oncogenes; Oncogenic; Organoids; Output; Pathway interactions; Patients; Phosphotransferases; Predisposition; Proteins; RNA; Recurrence; Recurrent disease; Regulation; Risk; Role; Running; Sampling; Secondary to; Signal Transduction; Site; Techniques; Time; Transcriptional Regulation; Transitional Cell; Transitional Cell Carcinoma; Tumor Suppression; Tumor-infiltrating immune cells; Up-Regulation; Ureter; Urethra; Urothelial Cell; Urothelium; allotransplant; base; bladder transitional cell carcinoma; cancer invasiveness; combinatorial; fitness; genetic signature; histone demethylase; histone methylation; histone methyltransferase; kinase inhibitor; loss of function mutation; men; migration; mortality; mouse model; preclinical study; programs; promoter; protein complex; response; single-cell RNA sequencing; stemness; transcription factor; transcriptome; tumor; tumorigenesis

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.

项目概要/摘要 尿路上皮癌 (UC) 涉及膀胱、肾脏和输尿管内衬的尿路上皮细胞，是一种主要的癌症 是美国尤其是男性发病和死亡的原因。膀胱UC在临床上可分为 非肌肉侵入性（NMIBC）和肌肉侵入性（MIBC）。 MIBC 占转移的绝大多数 和死亡率，治愈率只有 50% 左右。 Patients with treated NMIBC are at risk of recurrence or 在先前或从头部位进展为 MIBC。超过一半的尿路上皮癌，无论起源于何处，都存在 组蛋白去甲基化酶 KDM6A (UTX) 和两种高度同源组蛋白的功能缺失突变 甲基转移酶 KMT2C (MLL3) 和 KMT2D (MLL4)。这些蛋白质形成 MLL3/4-COMPASS （与 Set1 相关的蛋白质复合物）类复合物，部分通过调节增强子功能 增强子处组蛋白的甲基化。增强子是调节谱系特异性的 DNA 区域 转录程序。最近的研究表明，在遥远的地方患有两种尿路上皮癌的患者 站点（输尿管和膀胱）具有相同的 COMPASS 样突变。进一步组织学测序 良性尿路上皮识别复合物随时间扩展的频繁突变。我们的假设是这些 尿路上皮“现场癌化”下的突变。我们的实验室培育出了一只基因工程小鼠 删除尿路上皮中 Kmt2c、Kmt2d 或组合的模型。这些小鼠的尿路上皮 未表现出组织学异常。然而，转录组分析显示尿路上皮表现增加 干性和功能研究表明它们表现出增强的类器官形成能力。当跨入 Pten条件缺失小鼠，在肿瘤发生中存在较强的协同性。本次活动的总体目标 建议是利用我们最近生成的尿路上皮小鼠模型，这种类似于指南针的复杂损失来 从机制上了解其在肿瘤尿路上皮抑制中的作用。具体来说，在目标 1 中，我们力求确定 携带突变的尿路上皮的干性、克隆动力学、癌基因和致癌物易感性 这种类似于 COMPASS 的复合体，使用谱系追踪、类器官培养和单细胞 RNA 测序。瞄准 2，我们试图确定 MLL3/4-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，我们将寻求确定尿路上皮细胞中 Kmt2c 和 Kmt2d 的缺失如何影响增强子和 启动子功能。活性增强子是具有转录因子结合的开放染色质的基因组区域， 增强子RNA的不同转录，并环向启动子。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 转录和启动子环化 使用最先进的表观遗传学技术。