PROJECT SUMMARY Clonal hematopoiesis (CH), as defined by the disproportionate representation of hematopoietic clones with somatic mutations, is highly prevalent in the elderly. CH confers a risk of progression to malignancy and has been associated with increased risk of cardiovascular disease and all-cause mortality. We are interested in studying CH specifically in the context of allogeneic hematopoietic cell transplantation (HCT). Due to the increasing number of aging recipients and related donors, CH in donor samples is becoming increasingly likely. Previous studies have implicated donor CH in donor cell leukemias and an increased risk of unexplained cytopenias and chronic graft-versus-host disease, and have shown preferential expansion of CH clones post-transplant. Paradoxically, donor CH has also been associated with reduced risk of relapse. Post- transplant, donor CH clones are under particular stress due to their rapid self-renewal and the inflammatory bone marrow microenvironment. However, little is known about the clonal dynamics and evolution that occurs in the bone marrow post-transplant and how they relate to the reported outcomes. Additionally, it is not clear how CH clones represented in the hematopoietic stem cell (HSC) populations compared to differentiated cells. In this project we propose to define the clonal dynamics and evolution post-allogeneic HCT at the single cell level. Our goal is to understand the interactions between the donor and recipient clones, how they develop and distribute across hematopoietic lineages, and how donor CH clones affect these processes. To achieve this goal, we will utilize longitudinal bone marrow samples from HCT recipients collected in the first two years post- transplant. We will apply a new high-throughput single cell system that allows for simultaneous genotypic and phenotypic information for each cell. Using this information, we will be able to track the evolution, expansion, and lineage distribution of each clone, and examine the role of pre-existing donor CH clones on this process. Additionally, we will compare the clonal architectures of the bone marrow HSC population with that of total peripheral blood to identify clones that are preferentially found in HSCs and track their emergence in mature blood cells. Overall, this project will provide a greater understanding of the risks in donor CH and what drives these risks. The insights we gain will also be valuable in understanding stem cell subclonal dynamics and competition under stress conditions and leukemic progression in general.