PROJECT SUMMARY/ABSTRACT In recent years, large-scale sequencing revealed that the presence of clonally expanded hematopoietic stem/progenitor cells in the bone marrow and blood of healthy individuals is a widespread phenomenon in humans, generally referred to as clonal hematopoiesis (CH). Clonally expanded hematopoietic cells in many instances harbor mutations associated with myeloid malignancies and, importantly, their presence is associated with increased risk of developing myeloid malignancies (myelodysplastic syndrome, MDS and acute myeloid leukemia, AML). Leukemic progression is invariably associated with acquisition of additional mutations by the CH clone and further clonal expansions. CH is thus a premalignant condition that often constitutes the initiating event of leukemogenesis and thus offers a glimpse into the early events of malignant transformation. However, there is a relative scarcity of models to study early events of leukemogenesis, as mice do not develop CH and most human cancer models (immortalized cell lines, patient-derived xenografts) capture advanced cancers. My lab has pioneered the modeling of myeloid malignancies with human induced pluripotent stem cells (iPSCs). We recently developed a model of successive clonal evolution of AML. The overarching goal of this proposal is to investigate the molecular mechanisms underlying the oncogenic effects of CH mutations. In the first Aim we will characterize cell-intrinsic (transcriptome, chromatin accessibility, DNA methylation) and cell-extrinsic (cytokine secretion, differentiation propensity) effects of the 3 most common CH mutations – DNMT3A, TET2 and ASXL1 – using CRISPR-edited isogenic human iPSCs. In the second Aim we will explore the role of cell competition in the clonal advantage of cells harboring CH mutations before and after acquisition of additional mutations. In the third Aim, we will address the question of whether fully transformed AML cells maintain or lose dependency upon the initial CH mutation using engineered mutant alleles reversible via Cre-loxP recombination and testing the effects of correction of the CH mutation on the initiation and maintenance of AML in vitro and in vivo in xenografts. .