PROJECT SUMMARY/ABSTRACT Clonal hematopoiesis of indeterminate potential (CHIP) is a common but poorly understood condition that predisposes asymptomatic individuals to leukemia and cardiovascular disease (CVD) and is associated with increased mortality. A recently-discovered condition, CHIP is characterized by the acquisition of age- associated somatic mutations that promote the expansion of specific blood cell populations, or clones, in a process called clonal hematopoiesis (CH). While CHIP-associated mutations, such as mutations in the epigenetic regulator Tet methylcytosine dioxygenase 2 (TET2), are frequently observed in leukemia, individuals with CHIP are at an even greater risk of CVD, including coronary heart disease and early-onset myocardial infarction, and the mechanisms underlying these cardiovascular manifestations are not well understood. While loss of Tet2 in hematopoietic cells accelerates atherosclerosis via the secretion of interleukin-1 (IL-1β) from pro-inflammatory macrophages, inhibition of IL-1 signaling only partially prevents atherosclerotic plaque formation, suggesting that additional inflammatory pathways contribute to this pathology. Indeed, our group has demonstrated that hyperactivation of toll-like receptor (TLR) signaling and downstream pro-inflammatory transcription factors promote CH and that a feedforward loop involving these pathways in hematopoietic stem and progenitor cells (HSPCs) propagates CH. However, it is not yet know if these pathways contribute to CHIP-associated CVD and if HSPCs play a direct role in this context. We hypothesize that the complex inflammatory response in Tet2-deficient CHIP-associated CVD relies on a pro-inflammatory feedforward loop between HSPCs and multiple mature hematopoietic lineages mediated by IL-1β, IL-6, and MYD88 signaling and that suppression of established CVD requires downstream inhibition of these pathways. To investigate this hypothesis, we propose the following specific aims: (1) to interrogate the inflammatory pathways underlying CHIP-associated CVD and (2) to investigate whether HSPCs and mature myeloid lineages contribute to a pro-inflammatory feedforward loop in CHIP-associated CVD. The objective of this fellowship is to determine the inflammatory signaling pathways and hematopoietic lineages underlying CHIP-associated CVD and to investigate their feasibility as therapeutic targets. To address these aims, genetic and pharmacologic approaches will be used to dissect the contributions of these pathways and cell types. Innovative techniques, including single cell-RNA sequencing, flow cytometry, and ultrasound, will be employed to elucidate the cell-type heterogeneity of CHIP and to monitor translationally-relevant indicators of disease progression and therapeutic response. The significance of this proposal is the identification of potential therapies for established CHIP-associated CVD. As patients with CHIP often present with established CVD, a better understanding o...