Human EpoR Research Articles

Expression of Jak2 R1063H Leads to Altered Epor/Tpor Signaling, Changes in HSC Fitness and Represents Increased Risk of Thrombosis

Non-canonical weakly activating germline JAK2 variants may predispose progenitor cells to acquire JAK2 V617F during the evolution of myeloproliferative neoplasms (MPN) (Lanikova et al. Blood 2016;128(18):2266) and represent risk factors for MPN transformation into AML (Benton et al. Cancer 2019;125:1855). We have previously described and functionally characterized the JAK2 R1063H germline variant, cooperating with the JAK2 E846D variant in a case of hereditary MPN with erythrocytosis and megakaryocytic atypia (Kapralova et al. Blood 2016;128:1418) or with the acquired JAK2 V617F causing increased JAK2 signaling and higher disease severity in patients with essential thrombocythemia (Mambet et al. Blood 2018;132:2695). The mouse model bearing Jak2 R1063H mutation was created using microinjection of synthesized ssODNs containing the R1063H mutation, sgRNA mRNA and Cas9 protein into C57BL6/N-derived zygotes. In order to intensify a possible effect of the mutation, we bred and analyzed the knock-in Jak2 R1063H homozygous mice. The mice are born in normal (expected) frequency and display increased platelets (PLT) counts when compared to wild-type (wt) mice during life-span. Despite increased PLT counts, the plasma thrombopoietin level is normal whereas erythropoietin levels are increased without marked erythrocytosis. The preliminary co-immunoprecipitation experiments with human erythropoietin and thrombopoietin receptors (EPOR/TPOR) and JAK2 variants suggest increased TPOR and decreased EPOR binding with JAK2 R1063H kinase, possibly explaining the hematological phenotype. Also experiments with humanized mouse model of EPOR (Divoky et al. Proc Natl Acad Sci USA 2001;98:986) crossed with Jak2 R1063H mice supported these findings, where anemia due to hypoactivation of wt human EPOR signaling is more severe in Jak2 R1063H mice than in a control group and mild thrombocytosis is still present in the double mutant mice. The Jak2 R1063H model displays increased number of long-term hematopoietic stem cells (HSC) suggesting increased cycling in this population at young age (3 months) and shift to increased short-term HSC and multipotent progenitors with aging (12 months). The immunophenotypically-defined myeloid progenitor cells (LK, Lin-/Sca1-/cKithigh) are statistically increased in Jak2 R1063H mice due to disproportionately increased megakaryocytic/erythroid progenitors (MEP) over other myeloid progenitors at 3 months of age. The LK compartment is compromised in old Jak2 R1063H mice due to substantial myeloid bias by disproportionately increased CMP cells and marked decreased MEP cells, explaining the more pronounced anemia of aging in these mice. Some of these features are reminiscent of aging-associated myeloid and megakaryocytic bias, increased LKs, and relative exhaustion of progenitors with an erythroid lineage-priming program in aged bone marrow (BM) of a mouse model of primary familial and congenital polycythemia (PFCP) due to gain-of-function EPOR (Kralova et al, Am J Hematol 2022; doi: 10.1002/ajh.26658). However, the gain-of-function EPOR-introduced changes into the BM microenvironment are not associated with augmented hypoxia signaling and systemic inflammation and do not represent permanent challenges that would contribute to an increased incidence of myeloid malignancies in PFCP. In this regard, it will be interesting to compare these characteristics with the possible predisposing potential of Jak2 R1063H; further analysis of proliferative history of Jak2 R1063H HSC populations and expression profiling are ongoing. In the cohort of Jak2 R1063H mice, we also observed a relatively high frequency of sudden death in mice with median overall survival of 215 days (5/15) in comparison with the control group (0/15). We therefore measured the levels of D-dimers as a marker of thrombosis and found it statistically increased in the Jak2 R1063H group. Interestingly, in a WES study of patients with venous thromboembolism, the JAK2 R1063H was identified in one case, being considered a probable disease-causing variant (Lee at el. Blood Adv 2017;1:1224). In conclusion, our results confirm a direct effect of germline Jak2 R1063H mutation on EpoR/TpoR signaling but also on HSC biology, suggesting that the variant may represent a risk factor for BM remodeling in the process of myeloid cell transformation. Grant support: Czech Health Research Council NU21/03/00338.

Characterization of Iron Metabolism in Mouse Model of Congenital Polycythemia with Human Gain-of-Function Erythropoietin Receptor

Hepcidin (encoded by HAMP), the peptide hormone regulating systemic iron homeostasis, controls iron flux to plasma and thus ensures adequate iron supply for erythropoiesis. Increased erythropoietic activity negatively regulates hepcidin synthesis by the hormone erythroferrone (ERFE) produced by erythroid precursors in response to erythropoietin (EPO). Here we evaluated iron metabolism in a mouse model of polycythemia with augmented erythropoiesis and low EPO levels bearing the gain-of-function polycythemia-causing human EPO receptor gene (mtHEPOR) (Divoky PNAS 2002). The mtHEPOR embryos developed polycythemia around embryonic day (ED) 17.5, followed by unexpected but transient correction of polycythemia in perinatal life (postnatal day 7 - PD7) and reappearance of a polycythemic phenotype at 3-6 weeks of age. (Human patients with the gain-of-function polycythemia-causing human EPOR mutations are also born non-polycythemic and then develop polycythemia within a few weeks of neonatal life). The mtHEPOR mice had reduced Epo levels, augmented EPOR signaling and prolonged activation of Stat5 in erythroblasts when compared to mEpoR mice (Divoky JMolMed 2016). To assess whether developmental changes of erythron in this mouse model correlate with changes in iron metabolism, we determined temporal changes of Erfe and Hamp expression and body iron stores.We found that mtHEPOR mice have increased expression of Erfe at ED 17.5 (7.5-times) and PD7 (4-times) compared to mEpoR controls when normalized to a housekeeping gene beta-actin and independently to erythroid marker glycophorin A. Therefore Erfe in mtHEPOR mice would be increased both because of a higher number of erythroid precursors and because each precursor is producing more Erfe by increased transcription. This corresponds to reduced expression of Hamp in the liver of ED17.5 (4-times) and PD7 mtHEPOR mice (15-times) compared to mEpoR littermates. However, perinatally (PD1), Erfe expression dramatically decreased in mtHEPOR mice (7-times) concomitantly with increased Hamp expression (45-times), and both these factors reached expression levels detected in mEpoR at PD1. Expression of Erfe and Hamp between mtHEPOR and mEpoR mice was also not different in older mice (7 months of age), even though mtHEPOR mice were markedly polycythemic with persistent erythropoietic activity, markedly increased splenic erythropoiesis (the weight of spleens of control animals was 100-180 mg compared to 200-350 mg of mtHEPOR mice), and augmented EPOR-Stat5 signaling. However, mtHEPOR and mEpoR mice differed in transferrin saturation (TSAT) and tissue iron stores. The spleens of mtHEPOR mice had markedly increased iron deposits compared to mEpoR controls and stainable iron could only be detected in the livers of mtHEPOR mice but not in the livers of mEpoR controls. TSAT was elevated in mtHEPOR mice (77±3.2%) when compared to mEpoR (54.7±8.0%), consistent with the iron overload in mtHEPOR mice. Nevertheless, Hamp expression was comparable between mtHEPOR and mEpoR mice. This shows that Hamp expression was unexpectedly normal in respect to the presence of iron overload in mtHEPOR mice at 7 months of age.With further aging (mice 14 months and older), Erfe continued to increase and Hamp continued to decrease in mEpoR mice. In contrast, there was a trend towards decreased Erfe expression while Hamp expression became significantly elevated (2-times) in mtHEPOR mice. These changes were accompanied by a 2.4-fold decline in TSAT in both genotypes, which suggests that aged mEpoR mice develop iron deficiency while mtHEPOR mice remain iron-replete likely due to mobilization of iron from high iron stores.We show that the polycythemic phenotype, as well as Erfe and Hamp expression, in mtHEPOR mice undergoes dynamic changes during ontogenesis. We conclude that further research will be needed to understand the development of iron overload and changes in Erfe and Hamp expression in mtHEPOR mice characterized by erythroid hyperplasia and low Epo levels.Acknowledgment: BK, VD, and MH were supported by the Czech Science Foundation (projects GA15-13732S and GA17-05988S), KONTAKTII project LH15223, and in part by project LTAUSA17142. BK and JS contributed equally to this work. JTP and MH act as equivalent co-senior authors. DisclosuresNo relevant conflicts of interest to declare.

Selective Proliferation of Gene-Modified Human Erythroid Cells with Truncated Erythropoietin Receptors

Hematopoietic stem cell gene therapy for hemoglobin disorders, such as sickle cell disease, has stringent requirements for high-level gene marking with robust β-globin expression in erythroid cells for widespread successful clinical application. In vivo selection of gene-modified cells could allow for enhanced therapeutic effects. Erythropoietin (Epo) is a key cytokine for erythroid cell proliferation and differentiation. Various mutations in a C-terminal negative regulatory region of Epo receptor (EpoR) were reported in familial polycythemia. We hypothesized that coexpression of EpoR activated by C-terminal deletions could confer a selective advantage among gene-modified erythroid cells, thus raising the curative potential of therapeutic globin vectors.We generated a selectable truncated human EpoR (thEpoR) by deleting a 40 amino acid C-terminus, and prepared lentiviral vectors encoding both thEpoR and GFP (thEpoR/GFP) connected by internal ribosome entry site (IRES) controlled by murine stem cell virus promoter (Mp). K562 cells, Epo-dependent immortalized erythrocyte HUDEP-2 cells, and human CD34+ cell-derived erythroid cells (Epo-based differentiation) were transduced with thEpoR/GFP vector, and GFP-positive percentages (%GFP) were evaluated for 1-2 weeks. We observed increasing %GFP over 1 week for thEpoR/GFP-transduced HUDEP-2 cells at various Epo doses (3, 1, 0.3, and 0.1U/ml, p<0.01 in all doses) but not in the GFP only control (Figure). Additionally, there was no increase of %GFP for both thEpoR/GFP and GFP only vectors in Epo-independent K562 cells. Two weeks after erythroid differentiation of transduced CD34+ cells (2U/ml Epo), we observed higher glycophorin A-positive erythroid cell percentages (%GPA; 81±0 vs 50±3%, p<0.01), higher GFP and GPA-double positive percentages (%GFP+GPA+; 63±0 vs 44±3%, p<0.01), and larger cell expansion (2.8±0.6 vs 1.6±1.0 fold, p<0.01) in thEpoR/GFP transduction, as compared to GFP only control. These data demonstrate that thEpoR enhances erythroid cell expansion under Epo exposure in human erythroid cells. Accelerated erythroid differentiation can be induced by thEpoR in human CD34+ cells.Since Epo-based negative feedback among erythroid cells is required to maintain normal hematopoiesis, we switched from the constitutive Mp to the erythroid specific Ankyrin-1 (ANKp) or EpoR promoter with or without a WPRE enhancer. After erythroid differentiation of transduced CD34+ cells with the erythroid specific thEpoR/GFP vectors, we observed higher %GPA (81-85% vs 69±4%, p<0.01) in all thEpoR/GFP vectors, and higher %GFP+GPA+ (32±3 and 45±0 vs 22±1%, p<0.01) in thEpoR/GFP vectors with Mp and ANKp-WPRE, as compared to GFP only (Mp). Interestingly, higher γ-globin production in thEpoR/GFP transduction (12±0 vs 8±0%, p<0.01) was detected by HPLC, as compared to GFP only and no transduction controls. These data demonstrate that a thEpoR-based selective advantage in human erythroid cells can be controlled by erythroid specific promoters, and possibly induce γ-globin production.We then evaluated the ANKp-WPRE-derived thEpoR/GFP utilizing either an IRES or a 2A sequence to coexpress a GFP reporter (thEpoR-IRES-GFP, thEpoR-2A-GFP, and GFP-2A-thEpoR), since lower GFP intensity was observed in all thEpoR-IRES-GFP vectors (17-24 vs 95±5, p<0.01) than GFP only vector (Mp). In transduced CD34+ cell-derived erythroid cells, we observed higher %GFP+GPA+ in the GFP-2A-thEpoR vector (50±2 vs 37±3%, p<0.01), and greater cell expansion in thEpoR-IRES-GFP and GFP-2A-thEpoR vectors (100±7 and 88±6 vs 62±5 fold, p<0.05), as compared to GFP only vector with ANKp-WPRE. Lower GFP intensity was observed in all thEpoR/GFP vectors (14-34 vs 106±5, p<0.01) than the GFP only control, while the highest GFP intensity was obtained in GFP-2A-thEpoR (34±0). These data suggest that ANKp-WPRE with GFP-2A-thEpoR is optimal for erythroid specific expression, which should allow for both selection and detection of GFP signals in gene-modified erythroid cells in vitro and in vivo animal models.In summary, we developed erythroid specific thEpoR-expressing vectors, allowing for larger expansion and accelerated differentiation in human erythroid cells. While farther in vivo studies are desirable, this system should be beneficial for providing a selective advantage for gene-modified erythroid cells in gene therapy strategies for hemoglobin disorders. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Identification, Isolation and Transcriptome Analyses of Mouse, Rat and Man Erythroblastic Island Central Macrophages

Erythroblastic island (EBI), composed of a central macrophage and surrounding erythroid cells, is the first hematopoietic niche discovered for erythropoiesis. Yet, the identity of the central macrophage has so far remained elusive. Based on the previous findings that F4/80, VCAM1 and CD169 are potential mouse central macrophage markers, we first calculated the number of F4/80+VCAM1+CD169+ mouse macrophages in the mouse bone marrow and compared it to the number of Ter119+ erythroblasts. We found that the ratio of F4/80+VCAM1+CD169+ macrophage and erythroblasts is about 1:2. Given the fact that one central macrophage is surrounded by multiple erythroblasts, the above finding suggests that it is unlikely that all the F4/80+VCAM1+CD169+ macrophages are central macrophages.Erythropoietin (Epo) is essential for erythropoiesis. It has been reported that the Epo receptor (Epor) is expressed in peritoneal macrophages. These findings promoted us to speculate that EBI central macrophages may express Epor so that Epo acts on both erythroid cells and the central macrophages simultaneously in the niche to ensure efficient and optimal red cell production. To test this notion, we first examined whether mouse bone marrow and fetal liver macrophages express Epor using the Epor-GFPcre knockin mouse model. We found that ~5% of bone marrow F4/80+ macrophages and ~35% of fetal liver F4/80+ macrophages express Epor-GFP. As negative control, no Epor-GFP macrophages are noted in wild type F4/80+ macrophages. Importantly, ImageStream analyses revealed the native EBIs in bone marrow and fetal liver are formed by Epor+ but not Epor- macrophages. Bioinformatics analyses of RNA-seq data on the sorted Epor+ and Epor- macrophage populations revealed that molecules involved in central macrophage-erythroblast association such as VCAM1, CD169, and molecules known to be important for central macrophage function such as Dnase2a, ferroportin, are highly expressed in Epor+ macrophages. In marked contrast, highly expressed pathways in Epor- macrophages are associated with immune responses including antigen process and presentation. Intriguingly, the immune related pathways are dramatically downregulated in the Epor+ macrophages, suggesting that the Epor+ macrophages in bone marrow and fetal liver have evolved a specialized function in supporting erythropoiesis.To examine whether expression of Epor in EBI central macrophages is a conserved feature across species, we generated Epor-GFPcre knockin rat using the CRISP/Cas9 technology. Using CD163 as rat macrophage marker, we found that a subpopulation of rat bone marrow CD163+ macrophages expresses Epor-GFP. As a negative control, no Epor-GFP macrophages are noted in wild type CD163+ macrophages. To examine whether EPOR is expressed in human EBI central macrophages, antibody specificity for human EPOR is critical. To this end, we employed CRISP/Cas9 approach to knock out EPOR in K562 and Hela cell lines and validated the specificity of a commercially available anti-human EPOR antibody. Using CD163, CD169 as human macrophage markers, we found that EPOR is also expressed in a subpopulation of human macrophages. Moreover, in vitro EBI formation assay revealed that human EPOR+ but not EPOR- macrophages form EBIs with erythroid cells and that the EBI formation is enhanced by EPO.In summary, we for the first time, after discovery of the EBIs 60 years ago, have identified Epor+ macrophages in mouse bone marrow and fetal liver as EBI central macrophages. Our findings provide solid foundation for studying the mechanisms by which erythropoieis is supported EBI central macrophages. A better understanding of such mechanisms will provide extensive new knowledge on basic biology of erythropoiesis. It is also important to understand the pathology of erythropoietic disorders as well as to improve ex vivo erythrocyte production. DisclosuresNo relevant conflicts of interest to declare.

Erythropoietin Signaling Increases Choroidal Macrophages and Cytokine Expression, and Exacerbates Choroidal Neovascularization

Erythropoietin (EPO) is recognized for neuroprotective and angiogenic effects and has been associated with aging and neovascular age-related macular degeneration (AMD). We hypothesized that systemic EPO facilitates the development of choroidal neovascularization (CNV). Wild type mice expressed murine EPOR (mWtEPOR) in RPE/choroids at baseline and had significantly increased serum EPO after laser treatment. To test the role of EPO signaling, we used human EPOR knock-in mice with the mWtEPOR gene replaced by either the human EPOR gene (hWtEPOR) or a mutated human EPOR gene (hMtEPOR) in a laser-induced choroidal neovascularization (LCNV) model. Loss-of-function hWtEPOR mice have reduced downstream activation, whereas gain-of-function hMtEPOR mice have increased EPOR signaling. Compared to littermate controls (mWtEPOR), hMtEPOR with increased EPOR signaling developed larger CNV lesions. At baseline, hMtEPOR mice had increased numbers of macrophages, greater expression of macrophage markers F4/80 and CD206, and following laser injury, had greater expression of cytokines CCL2, CXCL10, CCL22, IL-6, and IL-10 than mWtEPOR controls. These data support a hypothesis that injury from age- and AMD-related changes in the RPE/choroid leads to choroidal neovascularization through EPOR-mediated cytokine production.

Effects of erythropoietin receptor activity on angiogenesis, tubular injury, and fibrosis in acute kidney injury: a "U-shaped" relationship.

The erythropoietin receptor (EpoR) is widely expressed but its renoprotective action is unexplored. To examine the role of EpoR in vivo in the kidney, we induced acute kidney injury (AKI) by ischemia-reperfusion in mice with different EpoR bioactivities in the kidney. EpoR bioactivity was reduced by knockin of wild-type human EpoR, which is hypofunctional relative to murine EpoR, and a renal tubule-specific EpoR knockout. These mice had lower EPO/EpoR activity and lower autophagy flux in renal tubules. Upon AKI induction, they exhibited worse renal function and structural damage, more apoptosis at the acute stage (<7 days), and slower recovery with more tubulointerstitial fibrosis at the subacute stage (14 days). In contrast, mice with hyperactive EpoR signaling from knockin of a constitutively active human EpoR had higher autophagic flux, milder kidney damage, and better renal function at the acute stage but, surprisingly, worse tubulointerstitial fibrosis and renal function at the subacute stage. Either excess or deficient EpoR activity in the kidney was associated with abnormal peritubular capillaries and tubular hypoxia, creating a "U-shaped" relationship. The direct effects of EpoR on tubular cells were confirmed in vitro by a hydrogen peroxide model using primary cultured proximal tubule cells with different EpoR activities. In summary, normal erythropoietin (EPO)/EpoR signaling in renal tubules provides defense against renal tubular injury maintains the autophagy-apoptosis balance and peritubular capillary integrity. High and low EPO/EpoR bioactivities both lead to vascular defect, and high EpoR activity overides the tubular protective effects in AKI recovery.

Solution structure of the transmembrane domain of the mouse erythropoietin receptor in detergent micelles.

Erythropoiesis is regulated by the erythropoietin receptor (EpoR) binding to its ligand. The transmembrane domain (TMD) and the juxtamembrane (JM) regions of the EpoR are important for signal transduction across the cell membrane. We report a solution NMR study of the mouse erythropoietin receptor (mEpoR) comprising the TMD and the JM regions reconstituted in dodecylphosphocholine (DPC) micelles. The TMD and the C-terminal JM region of the mEpoR are mainly α-helical, adopting a similar structure to those of the human EpoR. Residues from S216 to T219 in mEpoR form a short helix. Relaxation study demonstrates that the TMD of the mEpoR is rigid whilst the N-terminal region preceding the TMD is flexible. Fluorescence spectroscopy and sequence analysis indicate that the C-terminal JM region is exposed to the solvent. Helix wheel result shows that there is hydrophilic patch in the TMD of the mEpoR formed by residues S231, S238 and T242, and these residues might be important for the receptor dimerization.

Structural Insight into the Transmembrane Domain and the Juxtamembrane Region of the Erythropoietin Receptor in Micelles

Erythropoietin receptor (EpoR) dimerization is an important step in erythrocyte formation. Its transmembrane domain (TMD) and juxtamembrane (JM) region are essential for signal transduction across the membrane. A construct compassing residues S212–P259 and containing the TMD and JM region of the human EpoR was purified and reconstituted in detergent micelles. The solution structure of the construct was determined in dodecylphosphocholine (DPC) micelles by solution NMR spectroscopy. Structural and dynamic studies demonstrated that the TMD and JM region are an α-helix in DPC micelles, whereas residues S212–D224 at the N-terminus of the construct are not structured. The JM region is a helix that contains a hydrophobic patch formed by conserved hydrophobic residues (L253, I257, and W258). Nuclear Overhauser effect analysis, fluorescence spectroscopy, and paramagnetic relaxation enhancement experiments suggested that the JM region is exposed to the solvent. The structures of the TMD and JM region of the mouse EpoR were similar to those of the human EpoR.

Epo Receptors Are Not Detectable in Primary Human Tumor Tissue Samples

Erythropoietin (Epo) is a cytokine that binds and activates an Epo receptor (EpoR) expressed on the surface of erythroid progenitor cells to promote erythropoiesis. While early studies suggested EpoR transcripts were expressed exclusively in the erythroid compartment, low-level EpoR transcripts were detected in nonhematopoietic tissues and tumor cell lines using sensitive RT-PCR methods. However due to the widespread use of nonspecific anti-EpoR antibodies there are conflicting data on EpoR protein expression. In tumor cell lines and normal human tissues examined with a specific and sensitive monoclonal antibody to human EpoR (A82), little/no EpoR protein was detected and it was not functional. In contrast, EpoR protein was reportedly detectable in a breast tumor cell line (MCF-7) and breast cancer tissues with an anti-EpoR polyclonal antibody (M-20), and functional responses to rHuEpo were reported with MCF-7 cells. In another study, a functional response was reported with the lung tumor cell line (NCI-H838) at physiological levels of rHuEpo. However, the specificity of M-20 is in question and the absence of appropriate negative controls raise questions about possible false-positive effects. Here we show that with A82, no EpoR protein was detectable in normal human and matching cancer tissues from breast, lung, colon, ovary and skin with little/no EpoR in MCF-7 and most other breast and lung tumor cell lines. We show further that M-20 provides false positive staining with tissues and it binds to a non-EpoR protein that migrates at the same size as EpoR with MCF-7 lysates. EpoR protein was detectable with NCI-H838 cells, but no rHuEpo-induced phosphorylation of AKT, STAT3, pS6RP or STAT5 was observed suggesting the EpoR was not functional. Taken together these results raise questions about the hypothesis that most tumors express high levels of functional EpoR protein.

Absence of Functional Epo Receptors In Normal and Cancerous Human Tissues and Cell Lines,

Abstract 3169Erythropoietin (Epo) is a late acting growth factor, named because early studies suggested it had a singular effect on stimulation of red blood cell formation (erythropoiesis). Epo functions by activation of Epo receptors (EpoR) and while early studies suggested EpoR transcripts were expressed exclusively in the erythroid compartment, transcripts were later detected in other tissues and cell types including tumor cells with more sensitive RT-PCR strategies. This suggested that activation of EpoR by ESAs on tumor cells may explain results from some clinical studies where cancer patients in the ESAs treatment arm had decreased locoregional control and/or decreased survival. While EpoR transcripts could be detected, it was not clear that they were translated into EpoR protein or that the EpoR protein was functional. Attempts to examine EpoR protein expression in tumors were inhibited by nonspecificity and low sensitivity of available anti-EpoR antibodies.Recently specific and sensitive monoclonal antibodies were identified to human EpoR enabling experiments to explore EpoR protein expression in cells and tumors by western: CD34+ cells differentiated into erythroid progenitors and Epo-dependent UT-7/Epo cells served as positive controls and 769-P cells as a negative control. The latter expressed minimal EpoR mRNA and undetectable EpoR protein according to [125I]rHuEpo binding and western analysis. The limit of detection of EpoR by the most sensitive antibody, A82 in westerns was estimated to be less than 100 total (intracellular plus extracellular) EpoR/cell. By comparison, positive control cell types expressed over 10,000 EpoR/cell. In positive controls a 59 kDa protein, shown to be full-length EpoR, was readily detected with A82 with smaller EpoR proteins also present, but no proteins were detected in 769-P cells. The smaller proteins detected by A82 in the positive controls were shown by Mass-Spec analysis to be EpoR fragments. In contrast to the positive controls, EpoR proteins were not detected in either normal human breast or cancerous breast tissue (both Her2+ and Her2−) and there was little/no EpoR detected in a breast cancer lines (N=17) including MCF-7, though a band similar to EpoR in size was detected with the Santa Cruz anti-EpoR polyclonal, anti-peptide antibody, M-20. We therefore attempted to characterize this band. The band was not detected in MCF-7 cells with another specific anti-EpoR antibody (ab10653) nor was it observed in any immunoprecipitation:immunoblotting (IP:IB) experiments with combinations of 5 different anti-EpoR antibodies, though EpoR was readily detected in positive control. In contrast, the band was detected in IP:IB experiments with M-20 suggesting the protein is likely a non-EpoR protein that cross-reacted solely with that polyclonal antibody. Similar to breast tissues, EpoR was not detected with A82 in normal human primary keritinocytes, melanocytes and normal skin tissue. It was also not detected in squamous skin carcinoma and melanoma tissue or in cell lines derived from skin tumors (N=5). Matched normal and cancerous tissues from lung, colon, larynx, tongue and ovary were also negative for EpoR with A82. In contrast to tissues, low-level EpoR protein was detected in several of 21 cell lines derived from lung tumors. The EpoR protein in the one with the highest level (H838) was estimated to be ∼1,000–2,000 EpoR/cell, 10-fold lower than in erythroid progenitor cells and 40-fold lower than in UT-7/Epo cells. We therefore examined EpoR signaling in these cells. No phosphorylation of AKT or STAT5 (pAKT, pSTAT5) was detected in H838 cells with rHuEpo treatment. In control experiments increased pAKT was observed in H838 with a growth factor cocktail (EGF/ HGF/ IGF-1) and in UT-7/Epo cells treated with rHuEpo. These results suggested the EpoR in H838 cells was not functional. A factor dependent (IL-3) cell line (32D) was also engineered to express similar levels of EpoR as H838: these cells grew with IL-3 (10 ng/ml) but not with medium lacking growth factors, nor in medium containing 10 U/ml rHuEpo. In contrast, 32D cells with 20-fold higher levels of EpoR grew immediately with rHuEpo and at a rate similar to that with IL-3 but did not grow in absence of stimulation. These results suggest that EpoR expression level is important for Epo dependent growth of 32D cells, and that insufficient levels of EpoR in H838 cells may be one explanation for non-response to rHuEpo. Disclosures:Elliott:Amgen Inc: Employment, Equity Ownership. Swift:Amgen Inc: Employment, Equity Ownership. Busse:Amgen, Inc: Employment, Equity Ownership, Stock and Options. Rossi:Amgen: Employment, Equity Ownership. McCaffery:Amgen Inc: Employment, Equity Ownership.

Lineage Specific Erythropoietin Receptor Expression Regulation

AbstractAbstract 4779Erythropoietin receptor (EpoR) is a member of the cytokine receptor superfamily. During erythroid differentiation of hematopoietic stem cells, Epo acts through binding of EpoR on the surface of early erythroid progenitor cells to promote cell survival, proliferation and differentiation down the erythroid lineage. The extent of Epo response is dependent on the level of EpoR expression. The EpoR proximal promoter contains an inverted GATA-1 binding site (TTATCT) located at position -179 from the first codon of the human EpoR (hEpoR) gene. Three E boxes sites (CAGCTG) are also present in the 5′ untranslated transcribed region (UTR) of the EpoR gene and appear to be evolutionarily conserved in mammals. The expression of EpoR is not restricted to the erythroid lineage and can be found in several non-hematopoietic cells including endothelial, neural, muscle, cardiovascular and renal tissues. We previously found that EpoR is also expressed in primary satellite cells from skeletal muscle and in myoblast C2C12 cells but not in terminally differentiated myotubes. Epo stimulates proliferative and/or anti-apoptotic activities in myoblasts. Here we observed that erythroid MEL cells and C2C12 cells in the undifferentiated state express EpoR at a similar order of magnitude, but while EpoR expression increases with MEL cell differentiation, expression decreases with C2C12 differentiation. To gain insight on Epo activity during lineage specific differentiation, we compared EpoR expression during erythroid differentiation of MEL cells with myoblast differentiation of C2C12 cells. The conserved E-box region acts as a binding site for tissue specific basic-helix-loop-helix transcription factors, TAL1 in erythroid cells and the MyoD transcription factor family member in myoblasts. Deletion or mutation of the E-box motifs resulted in down regulated transcriptional activity of the EPO-R proximal promoter in erythroid and myoblast cells. These data suggest that the E-box region contributes to high activation of EPO-R transcription in both cell types. We also found that EPO-R expression is also dependent on the GATA-motif in the proximal promoter in myoblasts. While GATA-1, required for high level EpoR expression in erythroid cells is not expressed in myoblasts, we determined other GATA proteins in C2C12 cells associate with the GATA-1 binding site to provide transcription activity. However, unlike erythroid cells that exhibit high level induction of GATA-1 with erythroid differentiation and thus, high level EpoR expression, GATA proteins are down regulated with myoblast differentiation and EpoR expression is down regulated. Overall, during differentiation we observed an increase in histone acetylation and H3-K4 dimethylation in chromatin associated with the EpoR promoter in erythroid cells in contrast to a decrease in differentiating C2C12 cells, suggesting that the chromatin structure of EpoR in myoblast is less accessible than in erythroid cells and reflects the lower EpoR expression in non-hematopoietic cells. While DNA binding motifs for GATA and basic-helix-loop-helix transcription factors regulate EpoR expression in both erythroid and myoblast cells, important differences during lineage specific differentiation in EpoR chromatin accessibility and induction of the corresponding transcription factors that regulate EpoR expression explain in part the high induction of EpoR during erythroid differentiation in comparison to the low level expression in non-hematopoietic tissues. Disclosures:No relevant conflicts of interest to declare.

Erythropoietin-Induced Activation of the JAK2/STAT5, PI3K/Akt, and Ras/ERK Pathways Promotes Malignant Cell Behavior in a Modified Breast Cancer Cell Line

Erythropoietin (Epo), the major regulator of erythropoiesis, and its cognate receptor (EpoR) are also expressed in nonerythroid tissues, including tumors. Clinical studies have highlighted the potential adverse effects of erythropoiesis-stimulating agents when used to treat cancer-related anemia. We assessed the ability of EpoR to enhance tumor growth and invasiveness following Epo stimulation. A benign noninvasive rat mammary cell line, Rama 37, was used as a model system. Cell signaling and malignant cell behavior were compared between parental Rama 37 cells, which express few or no endogenous EpoRs, and a modified cell line stably transfected with human EpoR (Rama 37-28). The incubation of Rama 37-28 cells with pharmacologic levels of Epo led to the rapid and sustained increases in phosphorylation of signal transducers and activators of transcription 5, Akt, and extracellular signal-regulated kinase. The activation of these signaling pathways significantly increased invasion, migration, adhesion, and colony formation. The Epo-induced invasion capacity of Rama 37-28 cells was reduced by the small interfering RNA-mediated knockdown of EpoR mRNA levels and by inhibitors of the phosphoinositide 3-kinase/Akt and Ras/extracellular signal-regulated kinase signaling pathways with adhesion also reduced by Janus-activated kinase 2/signal transducers and activators of transcription 5 inhibition. These data show that Epo induces phenotypic changes in the behavior of breast cancer cell lines and establishes links between individual cell signaling pathways and the potential for cancer spread.

Erythropoietin receptor signaling regulates both erythropoiesis and megakaryopoiesis in vivo

Transgenic expression of a gain-of-function truncated mouse erythropoietin receptor gene ( EpoR) leads to expansion of the HSC pool in response to human erythropoietin (Epo). We have re-examined this observation using a knock-in mouse model, wherein the mouse EpoR gene was replaced in its proper genetic locus by a single copy of either a wild-type human or a polycythemia-inducing truncated human EPOR gene. Bone marrow cells obtained from knock-in mice were transplanted together with competitor bone marrow cells in a model that allows tracking of erythroid, platelet, and leukocyte contributions by each genotype. Secondary transplants were also performed. Stem/progenitor cells were identified phenotypically and isolated for colony-forming assays to evaluate cytokine responsiveness by cells with the wild-type human or truncated human EPOR gene. Augmented Epo signaling increased erythroid repopulation post-transplant as expected, but had no effect on short-term or long-term leukocyte repopulation. However, the wild-type human EPOR knock-in mouse showed decreases in both erythroid and platelet repopulation compared to marrow cells from the mutant human EPOR knock-in mouse or normal B6 animals. These results provide evidence supporting a role for Epo signaling in megakaryopoiesis in vivo and suggest a role for Epo signaling early in hematopoietic development.

Enrichment of Sca1+ hematopoietic progenitors in polycythemic mice inhibits leukemogenesis

Polycythemia vera (PV) is a myeloproliferative disorder characterized by a pronounced increase in the number of erythroid cells. However, despite this aberrant proliferation, the incidence of erythroleukemia is paradoxically rare in PV patients. In this study, we show that the progression of Friend virus-induced erythroleukemia is delayed in a mouse model of primary familial congenital polycythemia in which the wild-type Epo-receptor (EpoR) gene is replaced with a truncated human EPOR gene. Herein, we show that these mice exhibit enrichment of Sca1(+)/cKit(-) progenitors and several mature immune cells, such as dendritic cells and macrophages. In cotransplantation experiments, Sca1(+)/cKit(-) progenitors inhibit the tumorigenicity of Sca1(-)/cKit(+) erythroleukemic cells. A cell line established from Sca1(+)/cKit(-) progenitors is also capable of inhibiting leukemic proliferation in culture and in mice. This phenomenon of leukemic inhibition, also detected in the serum of PV patients, is partially attributed to increased nitric oxide secretion. In addition, the administration of erythropoietin into leukemic mice induces a polycythemia-like state associated with the expansion of Sca1(+)/cKit(-) progenitors and derivative immune cells, thereby inhibiting leukemia progression. This study indicates that a combination therapy incorporating the enrichment of Sca1(+)/cKit(-) progenitors may serve as a novel approach for the treatment of leukemia.

RNA Interference-Mediated Inhibition of Erythropoietin Receptor Expression Suppresses Tumor Growth and Invasiveness in A2780 Human Ovarian Carcinoma Cells

Although recombinant human erythropoietin (rHuEpo) has revolutionized the treatment of anemia, recent clinical trials suggested that rHuEpo use may be associated with decreased survival in cancer patients. Although the expression of erythropoietin (Epo) receptor (EpoR) has been demonstrated in various human cancers, the effect of exogenous Epo on the growth and therapy resistance of EpoR-bearing tumor cells is unclear at present. In the current study, we examined the hypothesis that EpoR may contribute to tumor growth independent of Epo in A2780 human ovarian carcinoma cells. A2780 human ovarian carcinoma cells showed high levels of EpoR expression, but lacked expression of Epo mRNA and biologically active Epo protein under both normoxic and hypoxic conditions. Exogenous Epo did not stimulate EpoR-mediated signaling, proliferation, invasiveness, or resistance to cytotoxic drugs in A2780 cells. In contrast, specific inhibition of EpoR expression using a short hairpin RNA (shRNA) expression plasmid resulted in markedly reduced proliferation and invasiveness in vitro. In addition, inhibition of EpoR expression led to abrogated in vivo ovarian cancer cell growth in a tumor xenograft system and resulted in decreased EpoR signaling. Our findings suggest that EpoR may be constitutively active in some cancer cells in the absence of Epo and provide the first evidence for a potential role of an Epo-independent, EpoR-mediated pathway in the growth of some human cancers.

Predictive testing for erythropoietin induced tumor progression in head and neck cancer

11007 Background: Although anemia is an independent poor prognostic factor in cancer, its correction using erythropoietin (Epo) may promote cancer progression and reduce survival, as suggested by several recent Phase III trials. We tested whether measuring Epo receptor (EpoR) mRNA levels in head and neck cancer could identify patients susceptible to Epo induced tumor growth. We also examined whether endogenous Epo might stimulate tumor growth. Methods: We measured EpoR mRNA levels in tumors from a previously reported Phase III trial in which 351 patients irradiated for head and neck cancer were randomized to Epo or placebo (Henke et al., Lancet 2003). 154 of these tumors had been evaluated previously with a polyclonal antibody raised against the human EpoR (C20) (Henke et al., J Clin Oncol 2006), but which also cross reacts with non-EpoR proteins (Elliott et al., Blood 2006). EpoR mRNA levels were measured in 106 tumors. We examined the relationship between EpoR mRNA level and locoregional progression free survival (LPFS), the primary study endpoint, in subjects assigned to Epo versus placebo. For patients enrolled in the placebo arm we also compared baseline serum Epo levels, C20 status, EpoR mRNA levels, and LPFS. Results: Epo-treated patients with unresected tumors that expressed above-median EpoR mRNA levels experienced a reduction in LPFS compared to the placebo group (p=0.02, n=s effect was not observed in patients with unresected tumors that expressed below-median EpoR mRNA levels (p=0.80, n=14). EpoR mRNA levels had no effect in patients with completely resected or partially resected tumors. In placebo patients with unresected or incompletely resected tumors, increased Epo levels were associated with impaired LPFS if their tumors were C20 positive (p=0.02, n=22), and improved LPFS if their tumors were C20 negative (p=0.09, n=15). C20 status and EpoR mRNA levels did not correlate (r=−0.11). Conclusion: Adverse effects of Epo on tumor progression may depend on tumor EpoR expression. Whether a relationship exists between other C20 targets and Epo induced tumor progression deserves further exploration. Our findings present clear hypotheses that should be tested in archival tumors from other Phase III trials. Author Disclosure Employment or Leadership Consultant or Advisory Role Stock Ownership Honoraria Research Expert Testimony Other Remuneration Hoffman-La Roche

Lack of functional erythropoietin receptors of cancer cell lines

Erythropoietin (Epo) therapy reduces red cell transfusion requirements and improves the quality of life of anemic cancer patients receiving chemotherapy. However, there is concern that Epo may promote tumor growth. We investigated by real-time RT-PCR, immunofluorescence microscopy, Western blotting and cell growth analysis whether human cancer cell lines (SH-SY5Y, MCF7, HepG2, U2-OS, HeLa, HEK293T, RCC4, HCT116, 7860wt and SW480) possess functional Epo receptors (EpoR). We detected EpoR mRNA in all cell lines. Neither hypoxia nor Epo treatment altered the level of EpoR mRNA expression. Four commonly used commercial antibodies proved to be unsuitable for immunoblot procedures because they cross-reacted with several proteins unrelated with EpoR. Depending on the antibody used, EpoR was localized to the plasma membrane, the cytoplasm or the nucleus. Experiments with small interfering RNA showed that EpoR protein was not expressed by the tumor cells except by UT7/Epo leukemia cells, which served as an EpoR positive control line, and by cells transfected with the human EpoR gene. Apart from UT7/Epo, none of the tumor cell lines responded to Epo treatment with phosphorylation of signaling molecules or with cell proliferation.

Truncated Human Erythropoietin Receptor Causes Fetal Polycythemia and Is Associated with a Prolonged Primitive Erythropoiesis and Hyperactive Definitive Erythropoiesis.

Gain-of-function mutations that cause truncations of the cytoplasmic domain of the human erythropoietin receptor (EpoR) result in the dominantly inherited disorder, primary familial congenital polycythemia. The EpoR truncation causes polycythemia, which is not always seen at birth, and in vitro hypersensitivity of erythroid precursors to erythropoietin (Epo). We have replaced the murine EpoR (MEpoR) gene with either a wild-type human EPOR (wthEPOR) gene or a mutant human EPOR gene (mthEPOR). This mutation, initially identified in a family with polycythemia, produces a truncated EpoR with a deletion of the cytoplasmic C-terminal domain after the first tyrosine residue. To investigate the effects of wthEPOR and mthEPOR genes on fetal erythropoiesis, we examined mouse fetuses homozygous for either wthEPOR or mthEPOR from E12.5 up to 5 weeks postnatally (PN). The mthEPOR fetuses were polycythemic and the wthEPOR fetuses were anemic during the early embryonic stage (E12.5∼E16.5) and at the adult stage (3 weeks and older). However, during the perinatal period (E18.5∼PN2 weeks), both of the phenotypes temporarily disappeared. To study the pathophysiology of the phenotypes, we analyzed erythropoiesis using differential expression of CD71 and TER119 surface antigens by FACS in the peripheral blood (PB) and fetal liver (FL). PB from wthEPOR and mthEPOR had a significantly higher population of earlier progenitors (proerythroblasts and early basophilic erythroblasts) from E12.5 to E16.5 compared to MEpoR. From E18.5 on, however, all genotypes showed a similar pattern. At E14.5, FL of wthEPOR and mthEPOR homozygotes were similar to MEpoR FLs at E12.5. Since a dynamic switch of primitive to definitive erythropoiesis occurs around E14.5, we evaluated nucleated erythroid cell populations in PB from E12.5∼E15.5 using the DNA dye DRAQ5 followed by FACS analysis. From E13.5 to E14.5, mthEPOR fetuses contained more nucleated cells than MEpoR fetuses (43 to 55% vs 37 to 29%; mthEPOR vs MEpoR), whereas wthEPOR fetuses had comparable numbers to MEpoR fetuses (36 to 25% vs 37 to 29%; wthEPOR vs MEpoR). Interestingly, all genotypes from E15.5 and thereafter contained over 90% enucleated cells with no significant difference in their PB. To understand the molecular mechanism of these features, we have analyzed Stat5 phosphorylation after Epo stimulation in FL from E14.5 and E18.5. At both stages, the mthEPOR FL had a markedly increased and sustained Epo-sensitive Stat5 phosphorylation compared to MEpoR FL. In contrast, in wthEPOR FL Stat5 phosphorylation was significantly lower even at the highest concentration of Epo. The membrane-localized EpoR levels were measured by fluorescence labeled anti-EpoR antibody. All genotypes showed comparable levels of EpoR on the membrane. We demonstrate here that with the exception of the perinatal period, mthEPOR causes polycythemia whereas wthEPOR causes anemia, even in fetal life. We propose that the polycythemia of mthEPOR during early embryogenesis (E12.5∼E14.5) is due to a delayed switch from primitive to fetal erythropoiesis, followed by hyperactive definitive erythropoiesis. These characteristics (the prolonged primitive erythropoiesis and hyperactive definitive erythropoiesis) could be explained, at least in part, by hyperactive and sustained Stat5 activation.

Endogenous Erythropoietin Signaling Is Required for Normal Neural Progenitor Cell Proliferation

Erythropoietin (Epo) and its receptor (EpoR), critical for erythropoiesis, are expressed in the nervous system. Prior to death in utero because of severe anemia EpoR-null mice have fewer neural progenitor cells, and differentiated neurons are markedly sensitive to hypoxia, suggesting that during development Epo stimulates neural cell proliferation and prevents neuron apoptosis by promoting oxygen delivery to brain or by direct interaction with neural cells. Here we present evidence that neural progenitor cells express EpoR at higher levels compared with mature neurons; that Epo stimulates proliferation of embryonic neural progenitor cells; and that endogenous Epo contributes to neural progenitor cell proliferation and maintenance. EpoR-null mice were rescued with selective EpoR expression driven by the endogenous EpoR promoter in hematopoietic tissue but not in brain. Although these mice exhibited normal hematopoiesis and erythrocyte production and survived to adulthood, neural cell proliferation and viability were affected. Embryonic brain exhibited increased neural cell apoptosis, and neural cell proliferation was reduced in the adult hippocampus and subventricular zone. Neural cells from these animals were more sensitive to hypoxia/glutamate neurotoxicity than normal neurons in culture and in vivo. These observations demonstrate that endogenous Epo/EpoR signaling promotes cell survival in embryonic brain and contributes to neural cell proliferation in adult brain in regions associated with neurogenesis. Therefore, Epo exerts extra-hematopoietic function and contributes directly to brain development, maintenance, and repair by promoting cell survival and proliferation independent of insult, injury, or ischemia.

The Wilms' Tumor Suppressor Wt1 Activates Transcription of the Erythropoietin-Receptor in Hematopoietic Progenitor Cells.

The Wilms' tumor suppressor, Wt1, has been implicated in the stage-specific quiescence and differentiation of hematopoietic progenitor cells. Since Wt1 deficiency compromises the proliferation and differentiation of erythroid blast-forming units (BFU-E) and colony-forming units (CFU-E), we analyzed the potential role of the transcriptionally active Wt1 isoform, Wt1(-KTS), in regulating the expression of the erythropoietin receptor (EpoR). CD117-positive hematopoietic progenitor cells isolated from the liver of Wt1-deficient murine embryos (Wt1−/−) at 11.5 dpc exhibited a 10-fold lower proliferative responsiveness to recombinant erythropoietin than CD117-positive cells from wild-type embryos (Wt1+/+), or embryos with heterozygous Wt1-deletion (Wt1+/−). Benzidine staining revealed a reduced fraction of hemoglobin-containing cells in Wt1-deficient cells in comparison to Wt1+/+ cells after 48 hrs of culture of CD117-positive cells in the presence of 6 U rhEpo/ml medium. Consistently, expression of the EpoR was significantly reduced in hematopoietic progenitor cells that lack Wt1 compared to Wt1+/+ cells. The decrease of EpoR mRNA was not due to a general down-regulation of gene expression in Wt1−/− CD117-positive cells, since levels of c-kit and c-mpl expression were normal. In the heart of wild-type and Wt1−/− embryos, which was analyzed as a non-hematopoietic tissue expressing both EpoR und Wt1, no difference in Epo-R expression was found. Transient transfection of Wt1(-KTS) into human hepatoma-derived HepG2 cells increased EpoR transcripts approximately 2-fold. A luciferase reporter plasmid carrying 309 bp of the proximal human EpoR promoter was stimulated 8-fold by co-transfection with Wt1(-KTS). The responsible cis-element in the EpoR promoter was identified by mutation analysis, electrophoretic mobility shift assay, and chromatin immunoprecipitation assay. In summary, we identified the EpoR gene to be the first downstream target of Wt1 within the hematopoietic cytokine receptor superfamily. Our data indicate that activation of the EpoR gene by Wt1 is a critical mechanism in normal murine hematopoiesis. However, our data do not support a role for Wt1 in EpoR expression in non-erythoid tissues, such as the heart.