Project Summary/Abstract Clonal hematopoiesis (CH) is a common pre-malignant condition in which one or a few hematopoietic stem and progenitor cells (HSPCs) contribute disproportionately to peripheral blood. Heterozygous somatic mutations in about 20 genes are associated with CH, some of which are frequent in hematologic malignancies. Although evidence suggests individuals with CH are at high-risk for malignant transformation, the mechanisms by which CH arises and contributes to leukemogenesis remain poorly understood. Revealing these mechanisms is a priority for disease prevention, and an overall goal of our Program. In our preliminary studies, we showed that inflammation can drive expansion of Dnmt3a-mutant HSPCs. Further, we developed a novel mosaic mouse model by transplanting a mixed population of HSPCs from five different CH-associated mutant mouse lines as donor cells (5x mosaic mice). We then exposed these mice to modifiable external stressors and tracked expansion of HSPC clones harboring certain mutations. We observed stress-specific clonal selection of different mutant HSPCs. Building on these data and leveraging the team’s expertise, Project 1 will test the hypothesis that inflammatory stress leads to epigenetic and metabolic changes in HSPCs with CH-associated mutations to accelerate clonal expansion and malignant transformation. We will create 5x mosaic mice with mutations in five common CH-associated genes that play critical roles in epigenetics, splicing, and signaling responses: Dnmt3a, Tet2, Asxl1, Jak2, and Sf3b1. We will use this novel 5x mouse model to investigate the mechanisms through which infection (Aim 1) and high-fat diet-induced inflammation and metabolic changes (Aim 2) accelerate clonal selection, expansion, and malignant transformation of HSPCs. In both aims, we will further investigate whether intervening in inflammatory and metabolic pathways could attenuate clonal expansion and leukemia development. In Aim 3, we will explore the impact of inflammation on cooperative expansion of Dnmt3a- and Tet2-mutant HSPC clones. Our team has presented a large body of preliminary studies to strongly support the feasibility of our proposed innovative approaches and overall hypothesis. Our mouse models and the quantitative approaches used in Core A to delineate expansion and malignant transformation of individual HSPC clones in response to modifiable inflammatory stressors are technically innovative. This research is conceptually innovative, as it introduces novel insights into how modifiable risk factors selectively drive clonal expansion of HSPCs and leukemogenesis across an expanded range of genetic defects. This Project will interact extensively by sharing 5x-mosaic model and quantitative detection technologies as well as transcriptomic data with Project 2 (genotoxic stress and DDR genes), and by mechanistically testing causal connections between stressors, drugs, proteomic and epigenetic changes and CH from Project 3 (analys...