Role of the Class IA PI3K in Hematopoietic Stem Cell Self-Renewal

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Proposal Summary/Abstract Adult hematopoietic stem cells (HSCs) are a rare and unique population of stem cells that reside in the bone marrow, where they undergo self-renewal and differentiation to maintain the blood system. To properly maintain the balance between self-renewal and differentiation, HSCs receive signals from growth factors and chemokines to activate the evolutionarily conserved phosphoinositide 3-kinase/Protein Kinase B (PI3K/AKT) signaling pathway. Pathologic activation of this pathway is frequently observed in cancers, including leukemia, making it a desirable target for cancer treatment. Several PI3K inhibitors are already used in the clinic and to better inform therapeutic targeting, it is crucial to understand the roles of PI3K in adult HSCs. Hematopoietic cells express three Class IA catalytic isoforms of PI3K (p110α, β, δ), all of which can transduce growth factor and cytokines signals. Out of these isoforms, p110β is unique, since in addition to transducing growth factor signals through receptor tyrosine kinases (RTK), it can directly interact with G-protein coupled receptor Gβɣ subunits to transduce chemokines, and also binds to RAC and to RAB5 GTPases. In mouse embryonic fibroblasts, the p110β-RAB5 interaction was shown to be important for the induction of autophagic cellular recycling process, which is essential for the maintenance of HSC metabolism and self- renewal. Individual Class 1A PI3K isoforms have unique functions in mature hematopoietic lineages, but they are dispensable for HSCs function. To study the redundant roles of Class 1A PI3K in HSCs, we have generated a triple knockout (TKO) mouse model with conditional deletion of p110α and p110β in hematopoietic cells, and germline deletion of p110δ. Analysis of these TKO mice reveals upon the loss of all three Class1A isoforms causes an increase in HSCs numbers, but decreased self-renewal and differentiation, with inefficient repopulation of all mature blood lineages. This phenotype is different from the phenotypes of any PI3K single isoform knockout mouse model, and even from p110α;δ double knockout animals, suggesting that p110β isoform plays an important compensatory role in HSCs. Moreover, my data suggests that loss of Class I PI3K causes a decrease in autophagy induction upon growth factor deprivation, though autophagy can still be induced with the mTOR inhibitor rapamycin. Thus, I hypothesize that loss of Class IA PI3K compromises autophagy induction, which causes altered HSC metabolism and impaired HSCs fitness. The proposed studies will use our PI3K TKO mouse model to elucidate in Aim 1 the cellular mechanism for defective self-renewal in HSCs. Aim 2 will establish the roles of autophagy in TKO HSC dysfunction. Lastly, Aim 3 will determine which binding interactions of p110β are the most important for its compensatory role in HSC function. In summary, this research will delineate the cellular and molecular mechanisms by which Class 1A PI3K supports HSC self-renewal and differentiation.