EPCR Raft Signaling Controls Activity of Hematopoietic Progenitor and Stem Cells

Aims: Hematopoietic stem cells (HSC) reversibly switch between dormancy and stress-induced proliferation, resulting in the maturation of hematopoietic progenitor cells (HPC) replenishing all differentiated blood cells. Our recent findings showed an important role for the EPCR (endothelial protein C receptor) in regulating HSC retention via integrin α₄β₁ (VLA4) and the small GTPase Cdc42 in the bone marrow (BM). To better understand the roles of EPCR signaling versus regulation of the anticoagulant pathway, we here studied EPCR C/S mice carrying a signaling point mutation abolishing intracellular palmitoylation implicated in raft localization. Experiments with isolated smooth muscle cells from these mice confirmed normal protein expression and anticoagulant function of the mutated receptor, providing a model to delineate the contribution of EPCR signaling to cellular activity and proliferation of hematopoietic progenitor and stem cells (HPSCs) in normal hematopoiesis and myeloid leukemia.

Methods: We analyzed HSPC frequencies in the bone marrow by multicolor flow cytometry at steady-state and after granulocyte colony stimulating factor (G-CSF) mobilization. Leukemia development was studied in the MLL-AF9-induced acute myeloid leukemia (AML) model.

Results: At steady state, we find increased frequencies of circulating HSC in the blood of EPCR C/S mice compared with controls, indicating that EPCR signaling mediates retention in the bone marrow niche. EPCR⁺ HSC from EPCR C/S mice displayed lower ESAM expression, marking the most primitive HSC subset, without demonstrating increased cell cycle activity relative to strain-matched controls. Cell cycle activity in myeloid progenitor cells was increased at steady-state and G-CSF-induced myelopoiesis and mobilization resulted in an increased release of HSPC, neutrophils and monocytes into the peripheral blood of EPCR C/S mice compared with controls. Consistent with increased proliferation of myeloid progenitors, MLL-AF9 AML induced in EPCR C/S bone marrow cells resulted in a more aggressive disease with shortened survival times compared with strain-matched control mice. The more aggressive AML was also demonstrated by higher leukemic load in the peripheral blood and increased frequencies of Ki67⁺ proliferative blasts in the bone marrow of MLL-AF9⁺ EPCR C/S recipients.

Conclusion: This novel mouse model revealed a critical role for EPCR signaling in regulating HSC dormancy, retention and HSPC proliferations. EPCR signaling is not only important for normal HSPC function, but also for leukemia progression raising new questions on interference of anticoagulants with normal and malignant myeloid hematopoiesis.

No conflict of interest has been declared by the author(s).