A Regulatory Network Between Notch and AKT Signaling Pathways Differentially Controls Megakaryocyte Development From Hematopoietic Stem or Committed Progenitor Cells.

Abstract 384

The Notch signaling pathway is implicated in a broad range of developmental processes, including cell fate decisions. However, the molecular basis for its role at the different steps of stem cell lineage commitment to a specific lineage is unclear. During hematopoiesis, the Notch signaling pathway is known to play an important role in T cell lineage development. Recently, we demonstrated that the Notch signaling pathway is also a positive regulator of megakaryocyte lineage specification from hematopoietic stem cells (HSC). The importance of a tight regulation of this latter role is highlighted by the aberrant activation of the canonical Notch pathway transcription factor RBPJ by OTT-MAL, a fusion oncogene specifically associated with infant acute megakaryoblastic leukemia (AMKL).

Here, we report a crosstalk between the Notch and PI3K/AKT pathways that provides new insights into the mechanism through which Notch signaling pathway regulates HSC differentiation into the erythro-megakaryocytic lineages. First, we observed that cells expressing a constitutively active Notch mutant had an increased level of phosphorylation of AKT compared to controls, indicating an association between Notch and AKT pathway activation. Using a Notch-GFP reporter mouse line, we confirmed that phosphorylation of AKT was increased in wild-type bone marrow cells upon physiological Notch stimulation (i.e. GFP+ cells) compared to control cells (i.e. GFP- cells) in vivo. Next, we assessed whether PI3K/AKT activation could replace or mimic Notch signaling during megakaryocyte development by transducing Lineage-Sca-1+cKit+ (LSK) cells or committed common myeloid progenitors (CMP) with a constitutively activated myristoylated AKT (myrAKT) mutant, followed by plating with or without Notch pathway stimulation on OP9-DL1 stroma or OP9 control stroma, respectively. MyrAKT-expressing LSK cells did not efficiently give rise to CD41⁺ megakaryocytic cells in the absence of Notch pathway stimulation, whereas myrAKT-expressing CMP showed partial rescue of development of megakaryocytes. Conversely, expression of a kinase-dead AKT mutant resulted in a pronounced reduction in megakaryocyte development from CMP, but had only a modest effect on LSK differentiation. Similar results were obtained with a chemical inhibitor of the AKT pathway. These results indicate that PI3K/AKT activation acts as an essential effector of the Notch pathway and can mimic Notch stimulation in CMP, whereas Notch-induced megakaryopoiesis from LSK cells is largely independent of the status of the PI3K/AKT pathway.

To investigate the role of PI3K-AKT pathway on megakaryocyte development in vivo, we used FoxO1/3/4-deficient and PTEN-deficient mice, and observed that both mouse lines had significantly increased megakaryopoiesis compared to control animals both in vivo and ex vivo after culture on OP9-DL1 stroma. Importantly, FoxO1/3/4-deficient progenitors had upregulation of Nrarp and Hes1, two Notch pathway targets, and chromatin immunoprecipitation assays revealed the presence of FoxO factors at the Hes1 promoter, indicating a feedback control of the PI3K/AKT pathway on Notch pathway activation. Taken together, these data demonstrate a complex regulatory network between the Notch and PI3K/AKT pathways during megakaryopoiesis. In addition, our results annotate developmental mechanisms in the hematopoietic system that enable a decision to be made either at the hematopoietic stem cell or the committed progenitor level to commit to the megakaryocyte lineage, supporting the existence of two distinct developmental pathways.