Acute myeloid leukemia (AML) affects 1 in 25,000 people per year, and is curable in only about 40% of cases. The mainstay of treatment is multi-agent chemotherapy and bone marrow transplantation, both of which have many complications and high mortality. This is largely attributed to the toxic effects that most antileukemic drugs have on hematopoietic stem cells (HSC), frequently leading to cytopenias. Therefore, it is crucial to determine which signaling pathways are important specifically in leukemic cells but not in HSCs, to enable the design of more specific and less toxic treatments for AML. The PI3 kinase (PI3K) pathway is pathologically activated in many human cancers, including AML. PI3K may also be important during hematopoiesis, as it is activated by hematopoietic growth factor receptors, such as erythropoietin receptor, c-kit receptor, and fms-like tyrosine kinase 3 (FLT3). In hematopoietic cells, receptor tyrosine kinases signal through the catalytic p110 subunit of PI3K, which has 3 isoforms (, , ), to activate the phosphorylation of the target kinase AKT. However, the roles of PI3K and its specific subunits in normal and leukemic stem cell function are poorly understood. We hypothesize that targeting individual subunits of PI3K may achieve the most selective anti-leukemic effect with minimal potential adverse affects. This proposal will utilize two novel genetic tools to investigate the roles of the PI3K subunits p110 and p110, individually and together, in leukemic cells and in normal HSCs: (1) mice with conditional deletion of p110 in hematopoietic cells and (2) double mutant mice with germline deletion of p110 and conditional deletion of p110. Preliminary data reveal that conditional deletion of p110 in HSCs results in impaired erythropoiesis, and slightly reduced HSC repopulating ability. As AKT signaling is maintained in p110-deleted bone marrow, we hypothesize that p110, a subunit expressed exclusively in hematopoietic cells, can complement for p110 in HSCs. In Specific Aim 1, we will analyze the effects of compound loss of p110 and p110 on HSC function and on AKT signaling in hematopoietic cells. Preliminary data suggests that deletion of p110 is insufficient to halt disease progression in at least some murine models of leukemia. Based on the failure of p110 excision to abrogate AKT signaling in hematopoietic cells, we hypothesize that it may be necessary to eliminate both p110 and p110 to inhibit the growth of leukemic cells. Specific Aim 2 will examine leukemia initiation and progression in mice after loss of p110, p110, or both using the bone marrow transplant system with retrovirally introduced mutated tyrosine kinases, including BCR-ABL, FLT3- ITD, and JAK2V617F. Specific Aim 3 will examine the roles of p110 and p110 in leukemic stem cell function in MLL-AF9-induced leukemia, which has been particularly difficult to target therapeutically. Together, these studies will provide important insights into the role and mechanism of PI3K signal transduction in HSCs, and the best strategies for therapeutic targeting of this pathway in leukemia.