ABSTRACT Clonal hematopoiesis (CH) is a condition in which individual hematopoietic stem cells (HSCs) contribute disproportionately to peripheral blood production. Individuals with CH are at significantly greater risk of developing hematologic malignancies compared to those without CH. Analysis of CH-associated leukemias has recently revealed recurrent mutations in a number of cancer-associated genes, including PPM1D, which was previously not associated with leukemia development. In addition, PPM1D mutations have been observed in patients developing secondary leukemias after having received chemotherapy for prior non-hematopoietic solid tumors. PPM1D encodes the Wildtype p53-Induced Phosphatase 1 (WIP1), which is upregulated by p53 during DNA damage and acts homeostatically to dephosphorylate and downregulate DNA damage response proteins. PPM1D mutations are generally C-terminal truncating mutations that cause dramatic increases in WIP1 protein levels. It has been hypothesized that PPM1D mutations enhance fitness of hematopoietic stem and progenitor cells subjected to chemotherapeutic stress and may further contribute to malignant progression of HSC-derived cells in the context of other driver mutations. The precise mechanisms by which PPM1D mutations enhance hematopoietic cell fitness remain poorly understood. In this application we propose cellular, animal, and human patient studies to better understand how PPM1D mutations may drive clonal hematopoiesis and leukemogenesis with a long-term goal of testing therapeutic approaches that might target patients with malignancies that display such mutations. We propose four specific aims that include (1) the comparison of long term hematopoiesis and cancers in genetically engineered mice with a germline truncating mutation in Ppm1d; (2) the use of competitive HSC transplant experiments to analyze the fitness landscapes that impact the evolution and outcome of pre- leukemic hematopoiesis; (3) the use of genomic screens and cellular assays to identify pathways and elucidate functional mechanisms associated with PPM1D mutations in normal hematopoietic cells and leukemic cells; and (4) investigation of PPM1D-associated mutation signatures and clonal architecture of secondary leukemia and myelodysplastic syndrome cells arising in human patients exposed to previous chemotherapy for a primary tumor. These comprehensive studies on this newly discovered leukemia-associated oncogene may guide choice of therapeutic agents and elucidate mechanisms underlying the evolutionary selection of mutations that drive the progression from clonal dominance to malignancy.