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

项目概要/摘要： 几乎所有的膀胱癌都含有参与染色质状态调节的基因突变。 KDM 6A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， KDM 6A的改变在尿路上皮癌中比在任何其他人类癌症类型中更常见。在这里， 我们试图进一步阐明KDM 6A失活在膀胱癌发病机制中的生物学作用， 为KDM 6A突变型尿路上皮患者制定合理治疗策略的前奏 癌的基于来自KDM 6A等基因尿路上皮癌细胞研究的初步数据， 通过CRISPR/Cas9靶向失活产生，我们假设KDM 6A失活是一个早期事件， 在膀胱癌发病机制中，通过失调在膀胱癌中起关键作用的基因来促进肿瘤形成。 调节RAS/MAP激酶信号传导。该提案将寻求利用下一代技术的最新进展， 测序、克隆性分析、CRISPR基因编辑以及患者来源的类器官和基因工程 小鼠（GEMM）建模以产生KDM 6A缺陷型膀胱癌的细胞和鼠模型， 更准确地反映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，以及我们的合作伙伴的专业知识， 该计划项目的研究人员将被用于对生物学进行深入分析， KDM 6A在膀胱癌发病机制中的作用具体来说，我们将定义KDM 6A 改变出现在膀胱癌（目的1）;探索生物学机制，KDM 6A的损失促进 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 致癌物诱导的Kdm 6a功能丧失的膀胱癌小鼠模型，以研究其机制， 其中KDM 6A失活促进体内肿瘤起始和/或进展（目的3）。长期 这项工作的翻译目标将是利用从KDM 6A突变肿瘤和细胞的研究中获得的见解， 线，类器官和动物模型，以指导新的治疗方法的发展。 整合：拟议的研究与项目2高度互补，项目2的重点是 染色质重塑基因ARID 1A在膀胱癌中通常与KDM 6A共同改变， 项目3，研究癌症演变，异质性和药物反应，在新的患者源性 类器官模型，其中一些含有KDM 6A突变。该项目的成功将需要密切 与三个核心的合作。具体而言，核心A将协助处理和分析 目的1，并有助于膀胱肿瘤的组织学和分子特征， 核心B将为目标2提供KDM 6A突变体和野生型人类器官细胞系， 有助于产生靶向缺失Aim 3的尿路上皮中Kdm 6a的GEMM;和 将为所有目标提供关键的生物信息学和统计支持，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.