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.

项目总结/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，是一种主要的恶性肿瘤。 是美国发病率和死亡率的主要原因，尤其是男性。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，只有约50%的治愈率。接受过NMIBC治疗的患者有复发或复发的风险 在既往或初治研究中心进展为MIBC。无论起源部位如何，超过一半的尿路上皮癌都含有 组蛋白去甲基化酶KDM6A（UTX）和两种高度同源的组蛋白 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）样复合物，部分通过 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，两个尿路上皮癌患者在遥远的地方， 位点（输尿管和膀胱）具有相同的COMPASS样突变。组织学上的进一步测序 良性泌尿系统肿瘤在随着时间的推移而扩展的复杂性中识别出频繁的突变。我们的假设是， 在尿道癌的"现场癌变"下的突变。我们的实验室培育出了一只基因工程老鼠 在泌尿系统中缺失Kmt2c、Kmt2d或其组合的模型。这些老鼠的泌尿系统 没有组织学异常然而，转录组分析显示， 干性和功能研究表明它们表现出增加的类器官形成能力。当交叉进入 pten条件性缺失小鼠，在肿瘤发生中具有强的协同作用。本报告的总体目标 我们的建议是利用我们最近产生的尿路上皮这种COMPASS样复合物丢失的小鼠模型， 了解其在肿瘤尿路上皮抑制中的作用。具体而言，在目标1中，我们寻求确定 尿结核携带突变的干性、克隆动力学、癌基因和致癌物易感性， 这种COMPASS样复合物，使用谱系追踪，类器官培养和单细胞RNA测序。在aim中 2.我们试图确定MLL3/4-COMPASS功能障碍与癌基因之间的功能相互作用， activation.在目的3中，我们将试图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的趋异转录，并与启动子成环。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转录和与启动子的成环 使用最先进的表观遗传学技术