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

基本信息

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
项目类别：
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

项目摘要

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.

项目概要/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与基因毒性应激源一起导致克隆造血（CH）和恶性转化。 CH 关联约有 20 个反复突变的基因，血液系统恶性肿瘤的风险增加 11 倍。其中 CH 中常见的突变基因有 PPM1D、TP53、ATM、CHEK2 和 SRCAP，所有这些基因都与 DDR。这一发现提出了 DNA 损伤施加选择性压力的可能性，从而允许 DDR 突变造血干细胞和祖细胞 (HSPC) 克隆扩增，导致 CH。事实上，我们已经证明了携带 CH 相关 Ppm1d 突变的鼠 HSPC 比野生型 HSPC 获得竞争优势顺铂治疗后。比较 CH 相关突变对促进克隆扩增的影响基因毒性应激源，我们创建了一种新型 CH 小鼠模型，其中共移植了 5 个 CH 突变型 HSPC 使用野生型 HSPC 并用顺铂治疗受体小鼠（称为 5x 镶嵌模型）。我们发现 Ppm1d Trp53 突变细胞在竞争中胜过了其他 CH 突变细胞，例如 Dnmt3a 和 Tet2，这支持了以下观点：基因毒性应激源专门选择 DDR 突变细胞。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 损伤剂的 HSPC 选择性优势，例如化疗或吸烟，这与复制错误突变特征有关。在目标 1 中，我们将使用 5x 镶嵌策略，对上述 5 个常见 DDR 基因进行突变。我们将让这些小鼠接受化疗或吸烟，并确定特定的暴露是否有利于特定突变型 HSPC。尽管 DDR 突变在 CH 中常见，但在恶性肿瘤中较少见。克隆，提出了一个关于 DDR 突变体 CH 如何促进血液恶性肿瘤的问题。在这里，我们将使用我们的新型 5x 模型用于检查 DDR 突变 HSPC 是否会促进细胞外源性血液恶性肿瘤方式，例如通过产生有利于转化细胞的环境。最后，在目标 3 中我们将确定针对细胞凋亡、炎症或氧化应激的干预措施是否可以减轻 CH 及其风险转化为血液系统恶性肿瘤。该项目与其他项目高度集成，因为它将通过将数据与项目进行比较，区分遗传毒性应激源的影响与其他炎症原因的影响 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 中在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研究 DDR 突变如何使 HSPC 在暴露于基因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针对 CH 的潜在干预措施，重要的是，恶变。本项目将使用Core A进行小鼠的单细胞测序实验和WGS 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，并促进数据的公共共享。