1 Clonal hematopoiesis (CH), a highly prevalent condition in the elderly, arises from somatic mutations that endow 2 a proliferative advantage to a subset of hematopoietic cells. CH increases the risk of myocardial infarction and stroke 3 independent of traditional risk factors. Relative to other common genetic variants giving rise to CH, the JAK2V617F (JAK2VF) 4 mutation that increases JAK/STAT signaling occurs at a younger age and imparts a substantial risk of premature coronary 5 heart disease (CHD). In CH mutated cells make up only a small fraction of blood cells, however, patients maintain an 6 elevated risk of CHD, suggesting mutant cells may have an outsized impact on their environment. Using murine models of 7 Jak2VF CH we found that IL-1 specifically promotes cell intrinsic proliferation of Jak2VF macrophages, but not wild type 8 (WT) macrophages even within the same atherosclerotic lesions. Administration of antibodies to IL-1 reduced Jak2VF 9 macrophage proliferation and improved features of plaque stability. scRNA-Seq analysis of lesion from mice with Jak2VF 10 expression in all bone marrow cells identified a prominent inflammatory myeloid population that was enriched for genes 11 associated with IL-1 signaling. We found that inflammatory myeloid cell abundance was decreased in the absence of the 12 pyroptosis executioner Gasdermin D (Gsdmd), potentially due to suppressed IL-1 secretion. Therefore, Specific Aim 1 13 will investigate the ability of Jak2VF cells to promote inflammatory phenotypes in WT cells within the same lesion through 14 IL-1 signaling. Mechanistically we have shown that the AIM2 inflammasome is activated in lesions from Jak2VF mice. 15 Deletion of Gsdmd from Jak2VF cells leads to increased DNA double strand breaks in lesions. Bone marrow derived 16 macrophages expressing Jak2VF, but lacking Caspase 1/11 have increased reactive oxygen species, suggesting DNA damage 17 occur upstream of inflammasome activation. Thus, Specific Aim 2 will determine if increased oxidized DNA in Jak2VF 18 macrophages promotes inflammasome activation and inflammation in lesions. Specific Aim 3 will then investigate if 19 truncation mutations in the CH variant protein phosphatase Mg2+/Mn2+ dependent 1D (PPM1D) that leads to inactivation of 20 DNA repair proteins, promotes atherosclerosis. The research proposed here will be accomplished by expanding my 21 conceptual knowledge of DNA damage in macrophages and atherosclerosis and will be complemented by training in single 22 cell sequencing technology to understand how immune cell populations change in lesions based on CH variant status. This 23 training will be supported by rigorous mentoring and collaboration with my scientific advisors during the K99 phase. 24 Together the proposed research and training will elucidate mechanism by which DNA damage and inflammasome activation 25 drive accelerated atherosclerosis in CH which will provide me the knowledge and skills to successfully...