PROJECT SUMMARY Hematopoiesis has been considered an ordered process of blood cell formation from a strict hierarchy; however, it is now clear that intrinsic and extrinsic cellular forces influence a complex cellular ecosystem of differentiation. While recurrent mutations cause cell autonomous disruptions in pre-leukemic settings like clonal hematopoiesis of indeterminate potential (CHIP) and myelodysplastic syndrome (MDS), clonal selection is also dependent on extrinsic forces in the microenvironment like inflammation. In this context, understanding how perturbations in pre-malignant clones respond to pro-inflammatory factors becomes fundamental in therapeutic advancement. Our project seeks to establish a molecular and cellular framework for characterization of clonal and inflammatory hematopoiesis at single-cell resolution. We have found that alterations in mature innate immune cells contribute to a perturbed inflammatory environment, and more specifically factors that promote clonal expansion of CHIP mutant clones through differential activation of intracellular signlaing. Based on our preliminary data, we propose that this unique cytokine signaling network mediates cellular function and more so cellular metabolism specifically in mutant stem cells. The same activation in wild-type stem cells promotes differentiation and proliferation, with loss of stemness. Our research identified Signal Transducer And Activator Of Transcription 3 (STAT3) as one of the major intracellular signals mediating CHIP clonal function. In ongoing studies, we will to define STAT3 specific mechanisms of clonal cell maintenance. We propose to define how cytokine signaling pathways mediate clonal expansion (Aim 1). We will characterize the function and transcriptional characteristics of hematopoietic stem and progenitor cells using CHIP mutant pre-clinical models following induction and perturbation to define the molecular drivers for maintenance of transformed hematopoietic stem cells during inflammation (Aim 2). These experiments will define new regulatory networks by establishing and linking cytokine to active signaling and cellular metabolism. These results will provide the basis for studies to understand how inflammation during clonal hematopoiesis regulates mitochondrial metabolism for a selective advantage. These findings will allow us to exploit these interactions to impede pre- malignant clones by altering the marrow microenvironment and targeting signaling axis and cellular metabolism (Aim 3). Moving forward, these findings could lead to therapeutic interventions for improved cellular response mitigating CHIP and changing outcomes in hematological disease.