Loss of Dnmt3a Dysregulates Hematopoietic Homeostasis and Myeloid Skewing Via the PI3Kinase Pathway: PI3Kα/β Inhibition Corrects Extramedullary Hematopoiesis and Myeloid Skewing Due to Loss of Dnmt3a

Abstract

Mutations in DNA methyltransferase type 3A (DNMT3A) are frequently associated with myeloid malignancies including myelodysplastic syndromes (MDS), acute myeloid leukemia (AML), and myeloproliferative neoplasms (MPN) including systemic mastocytosis (SM). Presence of this mutation is associated with poor prognosis and reduced overall patient survival. Earlier studies have shown that hematopoietic specific loss of Dnmt3a in mice results in lethal, fully penetrant MPN with myelodysplasia (MDS/MPN) characterized by extramedullary hematopoiesis, peripheral cytopenias, splenomegaly and hepatomegaly. However, it's unclear how DNMT3A mutations contribute to myeloid skewing, and induce myeloid malignancies. Further, the mechanism(s) by which loss of DNMT3A impairs various mast cell functions leading to mastocytosis are also not defined. We show that loss of Dnmt3a in vitro results in accelerated and enhanced differentiation of mast cells from its bone marrow (BM) precursors and in vivo results in increased number of mast cells. Genome wide transcriptome analysis revealed elevated expression of the transcription factor MITF and a profound repression in the expression of C/EBPα in BM precursors derived from Dnmt3a-/- mice. Importantly, restoring the expression of Dnmt3a in Dnmt3a deficient BM cells, completely restored enhanced differentiation, along with correction in levels of MITF and C/EBPα, suggesting that early loss of C/EBPα in Dnmt3a-/- cells contributes to enhanced maturation of mast cells from its precursors. Furthermore, biochemical analysis revealed greater PI3Kinase activation in Dnmt3a-/- cells, and these findings were supported by genome wide transcriptome analysis. Importantly, mast cells derived from Dnmt3a-/- mice lacking the expression of p85α regulatory subunit of PI3Kinase or pharmacologic inhibition of PI3Kinase completely corrected cytokine induced hyperproliferation in these cells to near normal levels. Insights from mast cell studies led us to hypothesize that the aberrant hematopoietic regulation in Dnmt3a-/- mice described above may be due to hyperactivation of the PI3Kinase signaling in hematopoietic stem and progenitors (HSCPs). We investigated the role of PI3Kinase signaling in Dnmt3a loss induced myeloid malignancy using pharmacological inhibitor, GDC0941 (PI3Kα/δ inhibitor) and BAY1082439 (PI3Kα/β inhibitor). PI3Kα/β inhibitor treatment of Dnmt3a-/- mice markedly reduced monocytosis, neutrophilia, enhanced WBC counts, and improved RBCs, hematocrits and platelet numbers compared to control mice. In contrast, PI3Kα/δ inhibition moderately corrected the monocytosis and WBC aberrancy, however correction in erythroid dysregulation was not observed. Inhibiting PI3Kα/β signaling dramatically reduced splenomegaly and hepatomegaly in Dnmt3a-/- mice. In contrast, PI3Kα/δ inhibition moderately reduced the spleen size, however the correction in hepatomegaly was insignificant. Importantly, clonal hematopoiesis due to loss of Dnmt3a in a competitive transplant setting was rescued by inhibiting the PI3Kinase pathway. In the BM, PI3Kα/β inhibition moderately decreased LSK cells, however a marked decrease in GMP, and significant increase in MEP, and CLP was observed compared to controls. Flow cytometry analysis revealed that PI3Kα/β inhibition reduced Dnmt3a-/- BM cell migration to liver, and completely corrected the extramedullary hematopoiesis in the spleen compared to controls. Further, Dnmt3a loss induced alteration in erythroid development in the BM, spleen and PB was corrected upon PI3Kα/β blockade compared to controls. Genome wide transcriptome analysis revealed that PI3Kα/β inhibition markedly reduced the expression of GMP related genes, fetal liver hematopoiesis transcription program, and expression levels of inflammatory cytokines, growth factors, and chemokines in Dnmt3a depleted malignant mice. However PI3Kα/β inhibition increased the expression of genes involved in erythroid development in Dnmt3a-/- mice compared to controls. These results demonstrate that Dnmt3a ablation induces liver specific expansion of hematopoietic cells, and extramedullary hematopoiesis in spleen via aberrant activation of PI3Kinase signaling in HSCPs. Consistent with this notion, PI3K inhibitor treatment of malignant Dnmt3a-/- bearing mice showed significantly improved overall survival compared to controls. Disclosures No relevant conflicts of interest to declare.