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

项目 2：DNA 损伤反应在基因毒性应激后克隆竞争中的作用

• 批准号: 10332336
• 负责人: Daisuke Nakada
• 金额: $ 59.23万
• 依托单位: BAYLOR COLLEGE OF MEDICINE
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-04-08 至 2027-03-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10332336
• 关键词: Affect; Anti-Inflammatory Agents; Antioxidants; Apoptosis; Apoptosis Promoter; Ascorbic Acid; Biological Assay; Bone Marrow; CHEK2 gene; Carboplatin; Cardiovascular Diseases; Cell Cycle; Cells; Cisplatin; Clonal Expansion; Collaborations; Cysteine; DNA Damage; DNA Repair; DNMT3a; Data; Development; Elderly; Ensure; Environment; Etoposide; Exposure to; FLT3 gene; Genes; Genotoxic Stress; Goals; Hematologic Neoplasms; Hematology; Hematopoiesis; Hematopoietic stem cells; Human; Individual; Inflammation; Inflammatory; Inflammatory Response; Intervention; MLL-AF9; Malignant - descriptor; Malignant Neoplasms; Melphalan; Metformin; Methods; Modeling; Mosaicism; Mus; Mutant Strains Mice; Mutate; Mutation; Non-Steroidal Anti-Inflammatory Agents; Oncogenes; Oxidative Stress; PPM1D gene; Pathway interactions; Pharmaceutical Preparations; Process; Recurrence; Replication Error; Reproducibility; Resistance; Risk; Role; Smoking; Symptoms; TP53 gene; Testing; Transplantation; Variant; Work; cell transformation; cell type; chemotherapy; cigarette smoke; cytotoxic; data sharing; digital; experimental study; exposure to cigarette smoke; genome sequencing; genotoxicity; high risk; human data; innovation; insight; mortality; mouse model; mutant; novel; pressure; prevent; programs; reconstitution; response; single cell sequencing; stressor; whole genome

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.

项目摘要/摘要 项目2的总体目标是确定DNA损伤响应中的突变（DDR）途径如何相互作用 遗传毒性应激源引起克隆造血（CH）和恶性转化。 CH是相关的 〜20个复发基因，血液系统恶性肿瘤的风险增加了11倍。在 CH中通常突变的基因是PPM1D，TP53，ATM，CHEK2和SRCAP，所有这些都涉及 DDR。这一发现增加了DNA损伤施加选择性压力的可能性，允许DDR突变。 造血干细胞和祖细胞（HSPC）以克隆的膨胀，导致CH。确实，我们已经表明 携带CH相关PPM1D突变的鼠HSPC比野生型HSPC获得了竞争优势 顺铂治疗后。比较CH相关突变对促进克隆扩张的影响 遗传毒性应激源，我们创建了一种新型的CH小鼠模型，其中五个CH突变HSPC被共转移 用野生型HSPC和用顺铂治疗的受体小鼠（称为5倍马赛克模型）。我们发现PPM1D TRP53突变细胞胜过其他CH突变体，例如DNMT3A和TET2，支持了以下概念 遗传毒性应激源专门为DDR突变细胞选择。在项目2中，我们将检验以下假设 DDR中的突变赋予暴露于DNA损害剂的HSPC的选择性优势，例如 化学疗法或香烟烟雾，这与复制误差突变签名有关。在AIM 1中， 我们将在上述五个常见的DDR基因中使用5倍的马赛克策略。我们将 将这些小鼠暴露于化学疗法或香烟烟雾中，并确定特定的暴露是否有利 特定的突变HSPC。尽管在CH中经常发现DDR突变，但它们在恶性中不太常见 克隆，提出了一个问题，即DDR-突变的CH如何促进血液学恶性肿瘤。在这里，我们将使用我们的 新颖的5X模型，以检查DDR突变的HSPC是否促进细胞外部血液系统恶性肿瘤 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 是针对凋亡，炎症或氧化应激的干预措施，减轻CH的风险 转化为血液系统恶性肿瘤。该项目与其他项目高度融合，因为它将 通过将数据与项目进行比较，将遗传毒性应激源的影响与其他炎症原因区分开 1，它将与项目1共享技术创新。通过这些模型，我们将能够迭代验证 项目3中的人类中发现的压力源突变相互作用。项目2的结果将提供新颖的见解 DDR突变如何允许HSPC在暴露于遗传毒性胁迫化疗后扩展 和香烟烟。这些机械研究可能揭示了针对CH的潜在干预措施，重要的是 恶性转化。该项目将使用核心A进行单细胞测序实验和鼠的WGS 殖民地。核心B（行政）将通过协调程序的各个方面来支持该项目，以确保 严格和可重复性，并促进公众共享数据。