EpoR In Cells Research Articles

ETV6-RUNX1 (TEL-AML1) Deregulates the Erythropoietin Receptor (EPOR) and Promotes Cell Survival.

AbstractAbstract 3641ETV6-RUNX1 (TEL-AML1) fusion is usually an initiating and pre-natal event in childhood acute lymphoblastic leukaemia (ALL). Transformation results in the generation of a persistent pre-leukemic clone, which post-natally converts to ALL following the acquisition of necessary secondary genetic alterations. The erythropoietin receptor gene (EPOR) is consistently highly expressed ectopically in TEL-AML1+ ALL but the presence of a functional receptor on the cell surface and its role, if any, in leukemogenesis driven by TEL-AML1 remains to be confirmed. Similarly, there is much debate as to the role played by the cytokine erythropoietin (EPO) and signaling through EPOR in relation to non-erythroid cell survival. Here, we show by using biotinylated erythropoietin (EPO) and flow analysis that the pre-B ALL TEL-AML1+ cell line (REH) appeared to have higher levels of EPO receptor than other non-TEL-AML1 cell lines. Furthermore, when we “blind screened” CD19+ peripheral blood or bone marrow cells from 10 patients with pre-B ALL, we identified 5 patients that showed high expression of ligand-binding EPO receptor on the cell surface, 4 of which were subsequently shown to be TEL-AML1+. We show that the inducible expression of TEL-AML1 in lymphoid BaF3 cells or its constitutive expression in either a murine transgenic model or in normal human cord blood cells is also sufficient to increase expression of EPOR. In order to further assess the direct regulation of EPOR by TEL-AML1 we next performed EMSA and ChIP experiments to confirm occupancy of AML1 consensus binding sites within the EPOR promoter and luciferase reporter assays to confirm up-regulation of EPOR promoter activity in the presence of TEL-AML1. Given the proposed pro-survival properties of EPO on non-erythroid cells, we next asked if the observed increase in expression of the EPOR in cells expressing TEL-AML1 could correlate with an increased cell survival in the presence of EPO. Cell survival experiments including growth curves, Annexin V staining and analysis of anti-apoptotic gene markers revealed that IL3-dependent cells expressing TEL-AML1 showed a prolonged survival in the presence of EPO alone and the consequent activation of anti-apoptotic pathways. The subsequent removal of TEL-AML1 expression in these cells resulted in cell death even in the presence of EPO, further suggesting that this effect is a consequence solely of fusion gene expression. Moreover, the observed survival in the presence of TEL-AML1 and EPO was enhanced by the co-addition of stem cell factor (SCF). Interestingly, SCF associated intracellular tyrosines have been shown to play an essential role in signaling through EPOR-SCFR interactions. In the presence of EPO, signaling via EPOR was confirmed by phosophorylation of JAK2, STAT5 and AKT and the subsequent up-regulation of BCL-XL. Similarly, EPOR functionality was demonstrated in TEL-AML1 positive patient cells by analysis of JAK2 pathway signaling after EPO induction. We propose that such activation lead to the increase in cell survival also observed in patient cells in the presence of EPO - again suggesting a role for enhanced cell survival through the EPO-EPOR axis. In our final model of pre-leukemia, CD34+ cord blood cells were transduced in vitro with a lentivirus capable of expressing both TEL-AML1 and GFP and ‘primed’ for pre-B lineage commitment. GFP (TEL-AML1) positive cord blood cells also showed increased levels of cell surface EPO receptor when compared to empty vector controls. Collectively, these data support the contention that TEL-AML1 directly activates ectopic expression of a functional EPO receptor and provides cell survival signals that may contribute critically to persistence of the pre-malignant clone in patients. Disclosures:No relevant conflicts of interest to declare.

Identification of a sensitive anti-erythropoietin receptor monoclonal antibody allows detection of low levels of EpoR in cells

Erythropoietin (Epo) binds and activates the Epo receptor (EpoR) on the surface of erythroid progenitor cells resulting in formation of erythrocytes. Recently, EpoR was reported to be expressed on non-erythroid cells suggesting a role for Epo outside of erythropoiesis. However those studies employed antibodies with questionable specificity and the significance of the observations are controversial. In order to accurately determine the expression of EpoR proteins in cells, we have generated a panel of novel anti-human EpoR monoclonal antibodies. One of these antibodies (A82) was particularly sensitive and it detected the EpoR protein on intact cells by flow cytometry and by western blot analysis with cell lysates. Both methods were optimized and using them, EpoR protein was detected by western immunoblotting with lysates from fewer than 200 EpoR positive control cells and the positive signals were proportional to EpoR protein expression level with a minimal signal in EpoR negative cells. The proteins detected by western blot analysis using A82 included full-length EpoR (∼ 59 kDa) as well as smaller EpoR fragments derived from the EPOR gene. These results indicate that A82 can be used to examine low level EpoR expression in cells by western and flow cytometry allowing an improved understanding of EpoR expression and metabolism.