PROJECT SUMMARY / ABSTRACT The objective of this proposal is to identify new approaches for targeting clonal hematopoiesis (CH). CH is characterized by selective expansion of hematopoietic stem and progenitor cell (HSPC) clones harboring mutations in genes such as TET2 and DNMT3A. These HSPC in turn produce pathogenic mutant myeloid cells that can contribute to several aging-related co-morbidities including cardiovascular disease (CVD) and all-cause mortality. An expanded mutant HSPC pool may also contribute to increased risk of hematological malignancies. CH prevalence is significantly elevated in the elderly and individuals with prior genotoxic exposures, smoking history, and/or chronic inflammatory disease. These conditions are associated with chronically perturbed physiological homeostasis, characterized by hyper-inflammation. Understanding the mechanism(s) promoting the selective expansion of mutant HSPC is crucial for prioritizing therapeutic targets that can suppress CH. A central premise of our application is that that CH arises from a targetable interplay between inflammatory signals and altered metabolic programming that supports the energetic needs and thereby the preferential expansion of CH HSPC. Using the mouse as a model representing key features of human CH, our preliminary data show that CH HSPC exhibit increased levels of the transcription factor Hif-1 aberrant glycolytic metabolism and increased ATP production relative to wild-type HSPC. We find that inflammatory cytokines, particularly IL-1 strongly potentiates CH HSPC expansion, glycolytic metabolism and Hif-1 activity. Strikingly, treatment of CH mice with OLT-1177, an NLRP3 inflammasome inhibitor that prevents cleavage and activation of IL-1 potently suppresses CH. We hypothesize that CH is the result of an interdependent mechanism in which NLRP3-mediated IL-1 production potentiates Hif-1 and downstream glycolytic activity to support CH HSPC expansion. We propose that NLRP3 inhibition disrupts this circuit, limiting expansion of CH HSPC. To address the mechanism, we propose two Specific Aims: 1) we will characterize the metabolic features of Tet2/, Tet2+/ and Dnmt3aR878H/+ CH HSPC using in vivo mass spectrometry- and flow cytometry-based analyses of metabolism and glucose flux. We will also identify the extent to which CH HSPC rely upon Hif-1 and glycolysis for their energetic needs; 2) we will evaluate the requirement for NLRP3 in promoting aberrant glycolytic activity and/or preferential expansion of CH HSPC. We will use molecular genetics approaches to assess the role and mechanism of Hif-1 and NLRP3 in regulating CH HSPC metabolism and promoting their expansion in vivo. Using our non-conditioned adoptive BM transfer mouse model of CH, we will validate our mechanism using pharmacological inhibition of NLRP3 with OLT-1177 and establish whether NLRP3 blockade suppresses HSPC expansion, aberrant metabolic activity and accumulation of inflammatory immune cells ...