Project 2: The role of the DNA damage response in clonal competition following genotoxic stress

项目摘要/摘要 项目2的总体目标是确定DNA损伤反应(DDR)途径中的突变如何相互作用 基因毒性应激源导致克隆性造血(CH)和恶性转化。CH已关联 大约有20个基因反复突变，患恶性血液病的风险增加11倍。在这些人中 在CH中常见的突变基因有PPM1D、TP53、ATM、CHEK2和SRCAP，所有这些基因都参与了 DDR。这一发现提出了一种可能性，即DNA损伤施加了选择性压力，允许DDR突变 造血干细胞和造血祖细胞(HSPC)克隆性扩增，引起CH.事实上，我们已经证明了 携带CH相关PPM1D突变的小鼠HSPC获得相对于野生型HSPC的竞争优势 顺铂治疗后。比较CH相关突变在促进克隆扩张中的作用 基因毒性应激源，我们建立了一种新的CH小鼠模型，在该模型中，五个CH突变的HSPC被共同移植 用野生型HSPC和顺铂处理的受体小鼠(称为5x嵌合体模型)。我们发现PPM1D Trp53突变细胞胜过其他CH突变体，如DNMT3A和TET2，支持这一概念 基因毒性应激源专门为DDR突变细胞选择。在项目2中，我们将测试假设 DDR突变为暴露于DNA损伤剂的HSPC提供了选择性优势，如 化疗或吸烟，这与复制错误突变签名有关。在目标1中， 我们将使用5x马赛克策略，对上述五个常见的DDR基因进行突变。我们会 将这些小鼠暴露在化疗或香烟烟雾中，并确定特定的暴露是否有利于 特定突变型HSPC。虽然DDR突变在慢性肝炎中很常见，但在恶性疾病中较少见 克隆，提出了一个问题，即DDR突变CH是如何促进血液系统恶性肿瘤的。在这里，我们将使用我们的 检测DDR突变的HSPC是否促进细胞外源性恶性血液病的新型5x模型 方式，例如通过产生有利于转化细胞的环境。最后，在目标3中，我们将确定 针对细胞凋亡、炎症或氧化应激的干预措施是否可减轻慢性肝炎及其发病风险 向血液系统恶性转化。这个项目与其他项目高度集成，它将 通过与Project的数据比较，区分基因毒性应激源与其他炎症原因的影响 1，它将与项目1共享技术创新。有了这些模型，我们将能够迭代验证 项目3中在人类中发现的应激源-突变交互作用。项目2的结果将提供新的见解 DDR突变如何使HSPC在暴露于基因毒性应激源化疗后扩张 还有香烟味。这些机制研究可能揭示对CH的潜在干预，重要的是， 恶变。该项目将使用核心A进行单细胞测序实验和小鼠的WGS 殖民地。核心B(行政)将通过协调计划的所有方面来支持该项目，确保 严密性和可重复性，并促进数据的公开共享。

Project Summary/Abstract The overall goal of Project 2 is to determine how mutations in the DNA damage response (DDR) pathway interact with genotoxic stressors to cause clonal hematopoiesis (CH) and malignant transformation. CH is associated with ~20 recurrently-mutated genes and an 11-fold increase in risk of hematologic malignancies. Among the genes commonly mutated in CH are PPM1D, TP53, ATM, CHEK2, and SRCAP, all of which are involved in DDR. This finding raises the possibility that DNA damage exerts a selective pressure allowing DDR-mutant hematopoietic stem and progenitor cells (HSPCs) to clonally expand, causing CH. Indeed, we have shown that murine HSPCs carrying a CH-associated Ppm1d mutation gain competitive advantage over wild-type HSPCs after cisplatin treatment. To compare the effects of CH-associated mutations in promoting clonal expansion upon genotoxic stressors, we created a novel CH mouse model in which five CH mutant HSPCs were co-transplanted with wild-type HSPCs and treated recipient mice with cisplatin (termed 5x mosaic model). We found that Ppm1d and Trp53 mutant cells outcompeted other CH mutants, such as Dnmt3a and Tet2, supporting the concept that genotoxic stressors specifically select for DDR-mutant cells. In Project 2, we will test the hypothesis that mutations in the DDR confer a selective advantage to HSPCs exposed to DNA damaging agents, such as chemotherapies or cigarette smoke, which is associated with a replication error mutational signature. In Aim 1, we will use the 5x mosaic strategy with mutations in the five common DDR genes mentioned above. We will expose these mice to chemotherapies or cigarette smoke and determine whether particular exposures favor specific mutant HSPCs. Although DDR mutations are found frequently in CH, they are less common in malignant clones, raising a question as to how DDR-mutant CH promotes hematologic malignancies. Here, we will use our novel 5x model to examine whether DDR-mutant HSPCs promote hematologic malignancies in a cell extrinsic manner, such as by generating an environment favorable to transformed cells. Finally, in Aim 3 we will determine whether interventions targeting apoptosis, inflammation, or oxidative stress mitigate CH and its risk of transformation to hematologic malignancy. This Project is highly integrated with other Projects, as it will differentiate the effects of genotoxic stressors from other causes of inflammation by comparing data with Project 1, and it will share technical innovations with Project 1. With these models, we will be able to iteratively validate the stressor-mutation interactions found in humans in Project 3. Results from Project 2 will provide novel insights into how DDR mutations allow HSPCs to expand following exposure to the genotoxic stressors chemotherapy and cigarette smoke. These mechanistic studies may reveal potential interventions against CH and, importantly, malignant transformation. This Project will use Core A for single-cell sequencing experiments and WGS of murine colonies. Core B (administrative) will support this Project by coordinating all aspects of the Program, ensuring rigor and reproducibility, and facilitating public sharing of data.