Project 1: Role of the H3K27 demethylase KDM6A in bladder cancer pathogenesis

项目摘要/摘要： 几乎所有的膀胱癌都含有与染色质状态调节有关的基因突变。 KDM6A是尿路上皮癌中这些染色质修饰基因中最常见的突变基因之一， 到目前为止，KDM6A改变在尿路上皮癌中比在任何其他人类癌症类型中都要常见得多。这里, 我们试图进一步阐明KDM6A失活在膀胱癌发病机制中的生物学作用。 为KDM6A突变尿路上皮患者制定合理治疗策略的前奏 癌症。基于KDM6A等基因尿路上皮癌细胞研究的初步数据 由CRISPR/Cas9靶向失活生成，我们假设KDM6A失活是早期事件 在膀胱癌发病机制中，通过解除对发挥关键作用的基因的调控来促进肿瘤的形成 调节RAS/MAP激酶信号。这项提议将寻求利用下一代技术的最新进展 测序、克隆分析、CRISPR基因编辑以及患者衍生的有机化合物和基因工程 小鼠(GEMM)建立KDM6A缺陷性膀胱癌细胞模型和小鼠模型 更准确地反映了人类疾病的基因组概况、组织学和生物学特性。这些 模型和一个独特的临床注释的人类膀胱癌队列和我们的合作专家- 该计划项目的调查人员随后将被用来对生物和 KDM6A在膀胱癌发病机制中的作用具体地说，我们将定义KDM6A 膀胱癌发生改变(目标1)；探索KDM6A缺失促进的生物学机制 转化的表型及其对药物反应的影响(目标2)；并开发癌基因-和 致癌物诱导Kdm6a功能丧失小鼠膀胱癌模型的研究 哪种KDM6A失活促进体内肿瘤的启动和/或进展(目标3)。一个长期的 这项工作的翻译目标将是使用从KDM6A突变肿瘤和细胞研究中获得的见解 线，有机和动物模型，以指导新的治疗方法的发展。 整合：拟议的研究与项目2具有很强的互补性，项目2的重点是 在膀胱癌中经常与KDM6A共改变的染色质重塑基因ARID1A，以及与KDM6A共同改变的 项目3，研究新患者来源的癌症进化、异质性和药物反应 有机体模型，其中几个模型含有KDM6A突变。这个项目的成功需要密切合作 与所有三个核心进行协作。具体来说，核心A将提供处理和分析方面的协助 目的1和帮助膀胱肿瘤的组织学和分子特征 在AIM 3的GEMM中；核心B将为AIM 2提供KDM6A突变和野生型人类器官细胞系 通过靶向删除目标3和核心C的尿路上皮中的Kdm6a，协助产生GEMM； 将为所有AIMS提供关键的生物信息和统计支持，并协助项目整合。

Project Summary/Abstract: Almost all bladder cancers harbor mutations in genes involved in the regulation of chromatin state. KDM6A is among the most commonly mutated of these chromatin-modifying genes in urothelial cancers, and KDM6A alterations are by far more common in urothelial cancer than in any other human cancer type. Here, we seek to further elucidate the biologic role of KDM6A inactivation in bladder cancer pathogenesis as a prelude to the development of rational therapeutic strategies for patients with KDM6A mutant urothelial cancers. On the basis of preliminary data derived from studies of KDM6A isogenic urothelial cancer cells generated by CRISPR/Cas9-targeted inactivation, we hypothesize that KDM6A inactivation is an early event in bladder cancer pathogenesis that promotes tumor formation by deregulating genes that play key roles in modulating RAS/MAP kinase signaling. This proposal will seek to leverage recent advances in next-generation sequencing, clonality analysis, CRISPR gene editing, and patient-derived organoid and genetically-engineered mouse (GEMM) modeling to generate cellular and murine models of KDM6A-deficient bladder cancer that more accurately mirror the genomic profile, histology, and biologic properties of the human disease. These models and a unique cohort of clinically annotated human bladder cancers and the expertise of our co- investigators on this Program Project will then be used to perform in-depth analyses of the biology and functions of KDM6A in bladder cancer pathogenesis. Specifically, we will define the timing at which KDM6A alterations arise in bladder cancer (Aim 1); explore the biologic mechanisms whereby KDM6A loss promotes the transformed phenotype and their implications for drug response (Aim 2); and develop oncogene- and carcinogen-induced mouse models of bladder cancer with Kdm6a loss-of-function to study the mechanisms by which KDM6A inactivation promotes tumor initiation and/or progression in vivo (Aim 3). A long-term translational goal of this work will be to use the insights gained from studies of KDM6A mutant tumors and cell line, organoid and animal models to guide the development of novel therapeutic approaches. Integration: The proposed studies are highly complementary with Project 2, which is focused on the chromatin remodeling gene ARID1A, which is often co-altered in bladder cancers with KDM6A, and with Project 3, which is studying cancer evolution, heterogeneity, and drug response in novel patient-derived organoid models, several of which harbor KDM6A mutations. The success of this project will require close collaboration with all three Cores. Specifically, Core A will provide assistance with the processing and analysis of human tumors for Aim 1 and aid in the histologic and molecular characterization of bladder tumors that arise in GEMMs in Aim 3; Core B will provide KDM6A mutant and wildtype human organoid cell lines for Aim 2 and assist with the generation of GEMMs with targeted deletion of Kdm6a in the urothelium for Aim 3; and Core C will provide critical bioinformatic and statistical support for all Aims, and assist with program integration.