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

项目总结/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 与遗传毒性应激物一起引起克隆性造血（CH）和恶性转化。CH相关 约有20个基因反复突变，血液系统恶性肿瘤的风险增加了11倍。中 CH中通常突变的基因是PPM 1D、TP 53、ATM、CHEK 2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损伤施加了一种选择性压力，使DDR突变体能够在细胞内表达。 造血干细胞和祖细胞（HSPC）克隆扩增，导致CH。事实上，我们已经表明， 携带CH相关Ppm 1d突变的鼠HSPC获得了比野生型HSPC更大的竞争优势 顺铂治疗后。为了比较CH相关突变在促进克隆扩增中的作用， 基因毒性应激源，我们建立了一种新的CH小鼠模型，其中5个CH突变HSPC共移植 用野生型HSPC和用顺铂处理的受体小鼠（称为5x嵌合模型）。我们发现Ppm 1d Trp 53突变体细胞胜过其他CH突变体，如Dnmt 3a和Tet 2，支持以下概念： 遗传毒性应激物特异性地选择DDR突变细胞。在项目2中，我们将测试假设， DDR中的突变赋予暴露于DNA损伤剂的HSPC选择性优势， 化疗或吸烟，这与复制错误突变特征相关。在目标1中， 我们将使用上述5个常见DDR基因突变的5 ×嵌合策略。我们将 将这些小鼠暴露于化疗或香烟烟雾中， 特异性突变HSPCs。虽然DDR突变在CH中频繁发现，但在恶性肿瘤中较少见。 克隆，提出了一个问题，如何DDR突变CH促进血液恶性肿瘤。在这里，我们将使用我们的 一种新的5倍模型，用于检查DDR突变型HSPC是否促进细胞外源性恶性血液病 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是否能减轻CH及其风险， 恶性血液病该项目与其他项目高度融合，因为它将 通过与项目的数据比较，区分遗传毒性应激源与其他炎症原因的影响 1，并将与项目1分享技术创新。有了这些模型，我们将能够反复验证 项目3中在人类中发现的应激子-突变相互作用。项目2的结果将提供新的见解 DDR突变如何允许HSPC在暴露于遗传毒性应激源化疗后扩增 和香烟烟雾。这些机制研究可能揭示对CH的潜在干预措施，重要的是， 恶性转化本项目将使用Core A进行单细胞测序实验， 殖民地核心B（行政）将通过协调该计划的各个方面来支持该项目，确保 严谨性和可重复性，并促进公共共享数据。

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.