PI3K Signaling Is Essential for Leukemic Transformation of Tet2 Mutated Pre-Leukemic Hematopoietic Stem Cells

Abstract

Loss of Tet2 (Ten Eleven translocation 2) in mice results in increased self-renewal capacity of hematopoietic stem and progenitor cells (HSPCs) and increased myeloid malignancies associated with splenomegaly, monocytosis and extramedullary hematopoiesis following a long latency. Along with mutations in TET2, fms like tyrosine kinase 3 (FLT3) internal tandem duplications (ITD) are also frequently found in patients with AML and are associated with high blast percentages as well as poor prognosis. Competitive repopulation studies involving Tet2-/- LSK stem cells show increase in repopulating capacity as well as skewed differentiation towards monocytic/granulocytic lineage, leading to leukemia, with features similar to CMML. Thus, pointing to hematopoietic stem cell as the cell of origin for Tet2 loss mediated myeloid malignancies. Here, we show that gene expression data from AML patients showed increased expression of transcription factor c-Myc, which is associated with increase in AML blasts. In aged Tet2-/- mice, we found increased expression of c-Myc protein in HSPCs as well as increased expression of c-Myc mRNA in peripheral blood mononuclear cells. This increase in c-Myc expression in Tet2 deleted cells was associated with hgih PI3K activity. To study the role of PI3K signaling in Tet2-/- HSPCs, we generated Tet2:P85α (p85α encodes regulatory subunit of PI3K) double knockout mice. To obtain conditional deletion of P85α in hematopoietic cells, we crossed Mx-Cre mice with WT, Tet2, P85α, and Tet2:P85α mice, and deletion of P85α in HSPCs was achieved with poly-I:poly-C injection. In competitive transplantation assays, we found reduced cell engraftment of p85α deleted Tet2-/- HSPCs compared to Tet2-/- controls. Aged Tet2 depleted HSPCs showed an increase in neutrophils, monocytes, eosinophils and basophils, however a reduction in lymphocytes compared to wildtype mice. In contrast, complete correction of these features was observed in mice transplanted with HSPCs derived from PI3K ablated Tet2 knockout mice. In the original parental Tet2 depleted and P85 deficient mice and under conditions of transplantation, we observed a significant correction in multiple hematopoietic compartments including the LSK compartment, long term hematopoietic stem cell compartment (LT-HSCs), CMP, MEP and MPPs as well as correction in splenomegaly, and decrease in granulocytes and monocytes normally seen in aged Tet2 depleted mice. Erythroid cell development was analyzed by examining the expression of Ter119 and CD71 markers using flow cytometry. We found that inhibiting PI3K signaling via P85α deletion in Tet2 knockout mice completely corrected Tet2 loss mediated altered erythroid development. Importantly, PI3K signaling blockade rescued all of the features associated with Tet2 loss induced CMML like disease by reducing inflammatory cytokines production in aged Tet2-/- mice, while downstream of PI3K, reduced gene expression related to mTOR/p70S6K/eIF4, oxidative phosphorylation pathways together with mitochondrial genes was observed in Tet2-/- HSCs. Furthermore, we show that inhibiting PI3K signaling decreases the proliferation of Tet2-/- HSCs by modulating the expression of c-Myc. Further, we observed reduced proliferation of Tet2-/- HSCs treated with Myc inhibitor. Myc inhibition in Tet2-/- mice reduced WBC, lymphocyte, neutrophil, monocyte and RBC counts. Also, Myc inhibition caused decrease in the numbers of Tet2-/- HSCs. Inhibition of PI3Kinase in Tet2-/-Flt3/ITD mutant AML mice significantly decreased spleen infiltrated myeloid blast cells, myeloid progenitors and LSKs compared to vehicle group. Taken together, we show that PI3K signaling is absolutely required for the development of Tet2 driven CMML like disease party by regulating the levels of Myc in preleukemic HSCs.