PI3Kinase Inactivation through Combined Loss of Catalytic Subunits p110α, p110β and p110δ Derails Hematopoietic Homeostasis

PI3Kinase is a lipid kinase that is involved in diverse cellular processes including cell growth, survival, differentiation and cell cycle. Deregulation of PI3K pathway has been associated with wide variety of cancers including several hematological disorders such as acute myeloid leukemia (AML). Hematopoietic growth factors such as Fms Related Tyrosine Kinase 3 (FLT3) ligand, stem cell factor (SCF), erythropoietin (EPO), and thrombopoietin (TPO) activate PI3K signaling pathway via tyrosine kinase receptors, growth factor receptors and G-protein coupled receptors. In mouse models of abrogation of PI3K signaling, involving AKT deletion, impairs the self-renewal potential of hematopoietic stem cells (HSCs). In contrast, mice models with constitutive activation of PI3K signaling, involving deletion of PTEN phosphatase, causes increased cycling of HSCs. PI3K regulatory subunits, P85α and P85β, are critical for fetal liver hematopoiesis. However, the role of PI3K in adult hematopoiesis and contribution of catalytic subunits of PI3K to hematopoietic homeostasis is not well studied. Given that HSCs express all three catalytic subunits of PI3K, we developed a mouse model for ablated PI3K pathway by depleting PI3K catalytic subunits p110α, p110β and p110δ. p110α and p110β subunits were conditionally deleted via MX-Cre system upon Poly-IC injection, and p110δ inactivation was achieved through p110δ inactivating knock-in mutation. Mouse tissues were harvested upon 5 weeks post poly-IC, at which time point the mice appeared moribund and were therefore analyzed for various hematopoietic lineages. These mice were anemic and peripheral blood analysis showed leukopenia, neutropenia and anemia with significantly reduced WBC, neutrophils, RBC, hemoglobin, hematocrits and platelet counts. Mice depleted of p110α, β and δ subunits-p110 triple knock out mice (p110-TKO) - demonstrated significantly reduced spleen size, reduced bone marrow cellularity with anemic bones relative to wild type mice. Hematopoietic stem cell compartment in p110-TKO mice showed increased frequency in Lineage^-veSca1^+veKit^+ve (LSK) population as well as an increase in more primitive HSCs defined as SLAM cells (CD150^+veCD48^-ve). In contrast, there was reduction in total number of common lymphoid progenitors (CLP), common myeloid progenitors (CMP), granulocyte-macrophage progenitors (GMP) and megakaryocyte-erythroid progenitors (MEP) in the bone marrow. These mice also showed dramatically reduced total B-cells and significantly reduced T-cells. However, these mice show increased frequency of GR1-Mac1 positive granulocytes in all hematopoietic compartments. Erythroid compartment was analyzed for various stages of red blood cell development using Ter119 and CD71 markers. Hampered RBC development was observed in p110-TKO mice with significantly increased immature erythroid cells (Ter119^lowCD71^low) in the bone marrow, spleen and peripheral blood compartments, with significantly reduced terminally differentiated cells (Ter119^+ve only). HSC self-renewal studies involving competitive repopulation assay showed that recipient mice have significantly reduced p110-TKO donor chimerism. Earlier studies from others have reported that p110α and p110δ subunits are dispensable for adult HSC function, however p110α is required for terminal erythroblast maturation and p110δ is required for leukocyte development. Our studies revealed that lack of all three catalytic subunits of PI3Kinase significantly impairs hematopoietic homeostasis with hampered stem cell development, erythroid cell development and a complete shut down in B-cell production.