The p110alpha and p110delta Isoforms of PI3 Kinase Are Dispensable for Hematopoietic Stem Cell Self-Renewal but Have Redundant Roles in B Cell Differentiation.

Abstract 2322

PI3 kinase (PI3K) is a key transducer of signals by hematopoietic growth factors, which control hematopoietic stem cell (HSC) proliferation, differentiation, self-renewal, and survival. Therefore, it can be expected that pharmacologic targeting of the PI3K pathway in malignancy could affect HSC function, and potentially result in myelotoxicity. The class IA PI3Ks are heterodimers, consisting of a catalytic 110-kd subunit termed p110, which has 3 isoforms in hematopoietic cells (α, β, and δ), and a regulatory subunit, which has 5 isoforms (p85α, p55α, p50α, p85β, or p55γ). Germline deletion of p85α and p85β causes a reduction in the number of fetal liver progenitors and impaired competitive repopulating ability of fetal liver HSCs (Haneline et al, Blood, 2006. 107(4):1375–82). This suggests that Class IA PI3K is an important regulator of HSC function. However it is unclear whether PI3K signaling is essential for adult hematopoiesis, and the roles of the individual PI3K isoforms in HSCs are unknown. In fibroblasts, the deletion of p110α impairs growth factor signaling and transformation by oncogenic signals. P110α^flox/flox;Mx1-Cre mice have anemia, but HSC number and function are unchanged (Gritsman et al, Blood, ASH Annual Meeting Abstracts, Nov 2009; 114: 3620). While germline deletion of p110δ affects B and T cell development and function, p110δ−/− offspring have normal blood counts, suggesting a minimal role for p110δ in HSCs (Jou et al, MCB, 2002. 22(24):8580–91).

To determine whether there is redundancy between p110α and p110δ in HSCs, we generated mice with the genotype p110δ−/−;p110α^flox/flox;Mx1-Cre (“PDFPA;Mx1-Cre”). After PolyI-PolyC (PIPC) injection to delete p110α in HSCs, PDFPA;Mx1-Cre mice develop leukopenia and anemia, decreased bone marrow (BM) cell counts and spleen weight, but survive for at least 8 months with no signs of disease. The B cell lineage is the most severely affected, with a 4-fold decrease in B220+ B cells in the BM of PDFPA;Mx1-Cre mice compared with WT controls, and a 3-fold decrease compared with p110δ−/−;p110α^flox/flox (PDFPA) controls. Multiparameter flow cytometry on the BM revealed a decrease in the frequency of LSK cells (Lin⁻c-kit⁺Sca1⁺ cells) in PDFPA;Mx1-Cre BM compared with WT;Mx1-Cre BM. While there was no effect on the frequency of long-term HSCs (CD150+CD48- LSK), there was a 5-fold decrease in the frequency of short-term HSCs (CD150-CD48-Flk2- LSK), multipotential progenitors (CD150-CD48-Flk2+ LSK), and common lymphoid progenitors (CLPs, Lin-IL7R+ckit^loSca1^loFlk2+), and a 2-fold decrease in the frequency of common myeloid progenitors (Lin-ckit+Sca1-FCRg^midCD34^hi). We observed a similar trend in p110δ−/− BM.

To determine whether p110δ and p110α are required for HSC self-renewal, we performed competitive and non-competitive repopulation assays. PDFPA;Mx1-Cre cells are capable of long-term multilineage reconstitution in a non-competitive setting. In the competitive setting (1:1 donor:competitor ratio), p110δ−/− BM cells were capable of normal trilineage long-term reconstitution, compared with WT BM. However, in recipients of PDFPA;Mx1-Cre BM, we observed a significant decrease in donor chimerism in the peripheral blood starting at 4 weeks after PIPC, with a complete lack of B cell donor reconstitution, compared with PDFPA controls. In contrast, myeloid and T cell reconstitution was detectable up to 20 weeks. There was a significant decrease in CLP donor chimerism in recipients of PDFPA;Mx1-Cre BM, while LT-HSC chimerism was unchanged. This suggests that p110α and p110δ have important redundant roles in HSC proliferation and differentiation along the lymphoid lineage, in addition to their known redundant roles in B cell development and BCR signaling (Ramadani etal, Science Signaling 2010. 3(134):p.1–10). Phospho-flow cytometry reveals significantly decreased Akt signaling in PDFPA;Mx1-Cre LSK cells in response to SCF.

In summary, we have uncovered important and partially redundant roles for p110α and p110δ in HSC proliferation and lymphoid differentiation, which most significantly impact early B cell development. Remarkably, despite being required for growth factor-induced Akt signaling in HSCs, these PI3K isoforms are not essential for HSC self-renewal in vivo. This work will have important implications for targeting the PI3K pathway in cancer treatment, and particularly in B cell malignancies.