PROJECT SUMMARY/ABSTRACT Aging is associated with functional decline of the hematopoietic system and clonal hematopoiesis (CH), a poorly understood process by which long-lived hematopoietic stem cells (HSCs) and their progeny with certain somatic mutations undergo positive selection. Individuals with CH have increased risk of developing blood cancer, cardiovascular disease, type 2 diabetes, and all-cause mortality. Thus, CH is a healthspan-limiting condition. Understanding how and why CH occurs with aging, and defining effective interventions to extend healthspan, have strong potential to reduce the frequency or severity of CH-associated diseases in aged individuals. Our laboratory has recently made formative discoveries that the aging bone marrow microenvironment and impaired HSC mitochondrial function cooperate to cause HSC aging. These discoveries have inspired us to define the components of aging that contribute to positive selection of CH- mutant HSCs. The major goal of this proposal is to determine processes by which hematopoietic-intrinsic and -extrinsic factors cause age-associated CH. Using a mouse model of a common CH mutation in DNMT3A, our preliminary data demonstrate that both hematopoietic-intrinsic and -extrinsic adaptive mechanisms facilitate selective advantage of Dnmt3a-mutant HSCs in the context of aging. We hypothesize that Dnmt3a-mutant HSCs are adapted to survive and self-renew in an aging microenvironment through enhanced mitochondrial metabolism, and that they promote premature senescence of the microenvironment to further their selective advantage. In Aim 1, we will identify the specific mitochondrial alterations in Dnmt3a-mutant HSCs that functionally increase their competitive advantage in an aging microenvironment. In Aim 2, we will determine the mechanisms by which Dnmt3a-mutant HSCs induce senescence of mesenchymal stromal cells in the BM microenvironment. In Aim 3, we will determine the extent to which targeting mesenchymal stromal cell senescence will reduce the functional competitive advantage of Dnmt3a-mutant HSCs and progression to hematologic pathology. Successful completion of this project will determine the mechanisms by which hematopoietic-intrinsic and -extrinsic processes confer a competitive advantage to Dnmt3a-mutant HSCs during aging. We expect that understanding these mechanisms will allow prioritization of targets for therapeutic intervention to limit CH-associated pathologies including myeloid malignancies, cardiovascular disease, and type 2 diabetes.