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

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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.