Constitutive Activation of STAT5 and Bcl-XL Overexpression Can Induce Endogenous Erythroid Colony Formation in Human Primary Cells.

In contrast with secondary erythrocytosis, progenitor cells from polycythemia vera (PV) patients can undergo in vitro erythroid differentiation despite absence of erythropoietin (EPO) and presence of such endogenous erythroid colonies (EEC) is routinely used as a diagnostic assay. Recent focus on the JAK2 mutation V617F in PV patients argue for a direct implication of JAK2 dependent signaling pathways in EEC formation. Because STAT5 is the principal target of JAK2 in erythroid cells, we investigated whether EEC formation was only dependent on STAT5 activation or required other signaling pathways that would be activated by JAK2. For this purpose, we transduced a retroviral vector coding for a constitutively active form of STAT5 (MIGR-STAT5CA) in UT7 cells, a leukemic cell line with erythroid properties. We observed in cells transduced with the MIGR-STAT5CA vector a spontaneous induction of erythroid differentiation in comparison with cells infected with the empty vector MIGR, as assessed by GPA staining. We next investigated effects of STAT5CA on erythroid differentiation of human primary progenitors. Purified CD34⁺ cells obtained from peripheral blood (PB) of patients treated with G-CSF were transduced with the STA5CA vector, the CD36⁺/GPA⁻ erythroid progenitor cells were sorted and cultured in presence of SCF alone. When expressing STAT5CA, they both proliferate and undergo erythroid terminal differentiation despite the absence of EPO. We concluded that a phosphorylated form of STAT5 was sufficient to support in vitro erythroid differentiation of human primary cells. Because STAT5 has been shown to play a crucial role in erythropoiesis via induction of the antiapoptotic protein Bcl-xL, we next investigated whether effects of STAT5CA on erythroid maturation was dependent on Bcl-xL induction. Tansduction of human CD36⁺/GPA⁻ cells with a retrovirus containing the coding sequence of human Bcl-xL progenitors allowed survival, proliferation and GPA acquisition despite the absence of EPO. We next investigated whether STAT5CA or Bcl-xL overexpression in normal primary cells could reproduce the malignant phenotype observed in PV patients, i.e. induction of EEC formation. CD36⁺/GPA⁻ transduced with either the STAT5 CA or the Bcl-XL vectors were plated in methylcellulose in the absence of EPO. Bcl-xL as well as STAT5CA vectors could both induce endogenous erythroid colony formation. Regardless to these results, we hypothesized that the EEC formation observed in myeloproliferative disorders could be at least partially due to the JAK2 dependent activation of the STAT5/Bcl-XL pathway. Thus, both constitutive activation of STAT5 and Bcl-xL overexpression could substitute to EPO to induce terminal differentiation of human primary erythroid progenitors.