The Class IA PI3 Kinase Isoforms Have Distinct but Overlapping Roles in Adult Hematopoiesis

The PI3 kinase (PI3K) signaling pathway is activated by most hematopoietic growth factors and chemokines that orchestrate normal adult hematopoiesis. Hematopoietic cells express four different catalytic isoforms of PI3K (p110α, β, δ, and γ), each encoded by a different gene (pik3ca, pik3cb, pik3cd and pik3cg, respectively). The Class IA (p110 α, β, δ) and Class IB (p110γ) enzymes use different regulatory subunits and are activated by different sets of upstream signals. While pik3ca and pik3cb are expressed ubiquitously, pik3cd is enriched in leukocytes. The roles of PI3K in adult hematopoiesis have been difficult to investigate using gene targeting in mice, since two of the isoforms (p110α and p110β) are required during embryogenesis. However, mice with germline deletion of pik3cd are viable and fertile with normal blood counts (Jou et al, MCB, 2002. 22(24):8580-91). Using the Mx1-Cre system to conditionally delete pik3ca in adult hematopoietic stem cells (HSCs), we previously showed that p110α is dispensable for adult HSC function (Gritsman et al, J Clin Invest 2014;124(4):1794-1809). We now report that conditional deletion of pik3cb using Mx1-Credoes not affect blood counts, bone marrow cellularity, survival, bone marrow HSC or progenitor numbers, or hematopoietic reconstitution in the transplantation setting. Therefore, none of the individual Class IA PI3K isoforms have critical roles in HSCs. However, it is still unclear whether these isoforms can substitute for each other in adult HSCs, or whether they are truly dispensable for HSC function.

To distinguish between these two possibilities and to uncover potentially redundant roles for PI3K isoforms in hematopoiesis, we have generated compound conditional knockout mice with the genotypes: pik3ca-lox/lox;pik3cd-/-;Mx1-Cre ("p110α-/-;p110δ-/-") and pik3ca-lox/lox;pikcb-lox/lox;Mx1-Cre ("p110α-/-;p110β-/-"). Interestingly, compound deletion of p110α and p110δ in HSCs results in significantly reduced white cell counts and anemia (Gritsman et al, Blood (ASH Annual Meeting Abstracts), Nov 2012; 120: 2322). In addition, p110α-/-;p110δ-/- mice have reduced numbers of bone marrow multipotential progenitors (MPPs), common myeloid progenitors (CMPs), and common lymphoid progenitors (CLPs), while HSC numbers are preserved. Furthermore, deletion of both p110α and p110δ leads to reduced B cell repopulation in competitive repopulation assays. This suggests that p110α and δ have redundant roles in HSC differentiation and B cell specification.

In contrast, we found that compound deletion of p110α and p110β has no effect on long-term competitive repopulation of any lineages for up to 20 weeks. We also observed normal donor chimerism in the HSC, MPP, and progenitor populations in transplant recipients of p110α-/-;p110β-/- bone marrow. After non-competitive transplantation, recipients of p110α-/-;p110β-/- bone marrow maintained normal blood counts over time. This indicates that, while p110δ is sufficient to support hematopoietic reconstitution of all lineages in the transplantation setting, p110β cannot support normal HSC differentiation by itself, particularly into the B cell lineage. However, p110α-/-;p110δ-/- mice do not develop pancytopenia, and repopulation of the myeloid and T cell lineages is still largely preserved, suggesting that long-term HSC function is intact. To determine whether HSC self-renewal and myeloid reconstitution after transplantation are possible in the absence of all three Class IA PI3Ks, we have generated triple knockout mice with the genotype pik3ca-lox/lox;pikcb-lox/lox;pik3cd-/-;Mx1-Cre ("TKO"). Analysis of the hematopoietic phenotype of TKO mice will be presented. These results will be valuable in predicting future hematologic toxicity in ongoing clinical trials with isoform-selective PI3K inhibitors or pan-PI3K inhibitors.