Erythropoietin Receptor Research Articles

Comparison of Patient Survival According to Erythropoiesis-Stimulating Agent Type of Treatment in Maintenance Hemodialysis Patients.

This study aimed to evaluate the difference in patient survival according to the type of erythropoiesis-stimulating agent (ESA) treatment used in the Korean hemodialysis (HD) population. This retrospective study analyzed the laboratory data from a national HD quality assessment program and the claims of Korea. Included participants were divided into three groups according to the type of ESA used during the 6 months of each assessment period as follows: the EP group (n = 38,043, epoetin-α or epoetin-β), the DP group (n = 10,054, darbepoetin-α), and the MR group (2253, continuous erythropoietin receptor activator). The ESA doses in the EP, DP, and MR groups were 6451 ± 3586, 5959 ± 3857, and 3877 ± 2275 unit/week, respectively. The erythropoiesis resistance indexes (ERIs) in the three groups were 10.7 ± 6.7, 9.9 ± 7.6, and 6.3 ± 4.1 IU/kg/g/dL, respectively. Kaplan-Meier curves revealed similar rates of patient survival among the three groups (p = 0.530). A multivariate Cox regression analysis showed that the hazard ratios in the DP group and MR group were 1.00 (p = 0.853) and 0.87 (p < 0.001), respectively, compared to that of the EP group. The hazard ratio in the MR group was 0.87 (p = 0.001) compared to that of the DP group. Our study shows that the MR group had comparable or better patient survival than the EP and DP groups in the multivariate analysis. However, the ESA doses and ERI were considerably different among the three groups. It was difficult to determine whether the better patient survival in the MR group originated from the ESA type, ESA dose, ERI, or other hidden factors.

Indoxyl sulfate impairs erythropoiesis at BFU-E stage in chronic kidney disease

Chronic kidney disease (CKD) is a global health condition characterized by a progressive deterioration of kidney function. It is associated with high serum levels of uremic toxins (UT), such as Indoxyl Sulfate (IS), which may participate in the genesis of several uremic complications. Anemia is one of the major complications in CKD patients that contribute to cardiovascular disease, increase morbi-mortality, and is associated with a deterioration of kidney failure in these patients. Our study aimed to characterize the impact of IS on CKD-related erythropoiesis. Using cellular and pre-clinical models, we studied cellular and molecular effects of IS on the growth and differentiation of erythroid cells. First, we examined the effect of clinically relevant concentrations of IS (up to 250 μM) in the UT7/EPO cell line. IS at 250 μM increased apoptosis of UT7/EPO cells at 48 h compared to the control condition. We confirmed this apoptotic effect of IS in erythropoiesis in human primary CD34+ cells during the later stages of erythropoiesis. Then, in IS-treated human primary CD34+ cells and in a (5/6 Nx) mice model, a blockage at the burst-forming unit-erythroid (BFU-E) stage of erythropoiesis was also observed. Finally, IS deregulates a number of erythropoietic related genes such as GATA-1, Erythropoietin-Receptor (EPO-R), and β-globin. Our findings suggest that IS could affect cell viability and differentiation of erythroid progenitors by altering erythropoiesis and contributing to the development of anemia in CKD.

Subcutaneous C.E.R.A. for the Maintenance Treatment of Anemia in Pediatric Patients With CKD: A Phase 2, Open-Label, Single-Arm, Multicenter Study

The optimum starting dose of intravenous continuous erythropoietin receptor activator (C.E.R.A.) has been previously determined; this study ascertains the optimum starting dose of subcutaneous C.E.R.A. administration in pediatric patients. Phase 2, open-label, single-arm, multicenter study. Patients aged 3 months to 17 years with renal anemia and chronic kidney disease (CKD; including those treated with maintenance dialysis and those not treated with dialysis) who were receiving maintenance treatment with erythropoiesis-stimulating agents (ESAs). Subcutaneous C.E.R.A. administration every 4 weeks (starting dose was based on defined conversion factors). The primary outcome was the change in hemoglobin concentration between the baseline and evaluation period for each patient. Secondary efficacy measures and safety were also evaluated. Forty patients aged 0.4-17.7 years were enrolled. The study achieved its primary outcome: the mean change in hemoglobin concentration was an increase of 0.48g/dL; the 95% confidence interval (0.15-0.82) and standard deviation (±1.03) were within the prespecified boundaries (-1 to 1g/dL and<1.5g/dL, respectively). Mean hemoglobin concentrations were maintained within the target 10-12g/dL range in 24 of 38 patients and within±1g/dL of the baseline in 19 of 38 patients, and the median C.E.R.A. subcutaneous dose decreased over time. Efficacy in key subgroups (age group,dialysis type, prior ESA treatment) was consistent with the primary outcome. Thirty-eight patients completed the core period; 25 chose toenter the safety extension period. Safety wasconsistent with prior studies, with no new signals. Single-arm and open-label study; small sample size. Pediatric patients with anemia secondary to CKD who were on, or not on, dialysis could be safely and effectively switched from maintenance ESAs to subcutaneous C.E.R.A. administered every 4 weeks, using defined dose-conversion factors to determine the optimum starting dose. F. Hoffmann-La Roche Ltd. The SKIPPER trial registered at ClinicalTrials.gov with study number NCT03552393. Anemia, a complication of chronic kidney disease, is associated with poor quality of life and an increased risk of hospitalization and mortality. The current treatments for anemia include iron therapy and erythropoiesis-stimulating agents (ESAs); however, the relatively short half-lives of the ESAs epoetin alfa/beta or darbepoetin alfa may require more frequent dosing and hospital visits compared with the ESA known as continuous erythropoietin receptor activator (C.E.R.A.). A previous study demonstrated that children aged 5 years or more with anemia associated with chronic kidney disease who were on hemodialysis could be switched to intravenous C.E.R.A. from their existing epoetin alfa/beta or darbepoetin alfa treatment. This study provides evidence that subcutaneous C.E.R.A. can safely and effectively treat anemia in children, including those aged<5 years and regardless of whether they were on dialysis or the type of dialysis they received (peritoneal dialysis or hemodialysis).

The effect of erythroferrone suppression by transfusion on the erythropoietin-erythroferrone-hepcidin axis in transfusion-dependent thalassaemia: A pre-post cohort study.

To track post-transfusion changes on the erythropoietin (EPO)-erythroferrone (ERFE)-hepcidin axis, we collected blood samples from 82 regularly transfused patients with β-thalassaemia major (β-TM) immediately before and 4-6 days after transfusion. The post-transfusion haemoglobin, hepcidin, and ferritin levels were increased, while the EPO, ERFE, and soluble transferrin receptor were suppressed. In addition, hepcidin change was inversely associated with erythropoietic change, which was confirmed by an increase in the hepcidin-to-ERFE ratio after transfusion. Age was the main predictor of serum ERFE, followed by EPO, transfusion frequencies, and ferritin. We found ERFE to be a highly sensitive indicator of erythroid activity in β-TM and that the hepcidin-to-ERFE ratio after transfusion may be used as an appropriateness index of serum hepcidin regulation relative to the degree of erythropoiesis.

The Latest Evidence of Erythropoietin in the Treatment of Glaucoma.

Erythropoietin (EPO) is a circulating hormone conventionally considered to be responsible for erythropoiesis. In addition to facilitating red blood cell production, EPO has pluripotent potential, such as for cognition improvement, neurogenesis, and anti-fibrotic, anti-apoptotic, anti-oxidative, and anti-inflammatory effects. In human retinal tissues, EPO receptors (EPORs) are expressed in the photoreceptor cells, retinal pigment epithelium, and retinal ganglion cell layer. Studies have suggested its potential therapeutic effects in many neurodegenerative diseases, including glaucoma. In this review, we discuss the correlation between glaucoma and EPO, physiology and potential neuroprotective function of the EPO/EPOR system, and latest evidence for the treatment of glaucoma with EPO.

Cytokine Receptor-Like Factor 3 Negatively Regulates Antiviral Immunity by Promoting the Degradation of TBK1 in Teleost Fish.

The cytokine receptor-like factor 3 (Crlf3) belongs to the orphan class I cytokine receptors and is identified as a neuroprotective erythropoietin receptor. In previous studies of Crlf3, few focused on its role in innate immunity. Therefore, this study explored the regulatory role of Crlf3 in innate immunity. TANK-binding kinase 1 (TBK1) is a vital adaptor protein for the activation of the RLRs-MVAS-IRF3 antiviral signaling axis; thus, its expression and activity must be tightly regulated to maintain immune homeostasis and avoid undesirable effects. Here, we report that Crlf3 is a negative regulator of type I interferon production. The expression of Crlf3 is induced by poly(I·C) or Siniperca chuatsi rhabdovirus (SCRV) treatment. Silencing of Crlf3 enhanced poly(I·C)- and SCRV-induced type I interferon production, whereas overexpression of Crlf3 suppressed type I interferon production. Mechanistically, Crlf3 interacted with TBK1 via its N domain and then inhibited type I interferon production by promoting TBK1 proteasomal degradation through K48-linked polyubiquitination. Our study shows that Crlf3 is a key factor for viral escape from innate antiviral immunity in fish and provides a new perspective on mammalian resistance to viral invasion. IMPORTANCE The expression of Crlf3 was upregulated with SCRV invasion, which proved that Crlf3 was involved in the regulation of the antiviral immune response. In this study, we found that the existence of Crlf3 promoted the replication of SCRV. Therefore, it is reasonable to believe that SCRV evades innate immune attack with the assistance of Crlf3. In addition, we report that Crlf3 negatively regulates interferon (IFN) induction by promoting the degradation of TBK1 in fish. We showed that Crlf3 is evenly distributed in the cytoplasm and interacts with TBK1. Further studies showed that Crlf3 specifically mediates K48-linked ubiquitination of TBK1 and promotes TBK1 degradation, resulting in a marked inhibition of retinoic acid-inducible gene I (RIG-I) downstream signaling.

Effect of pentavalent inorganic arsenic salt on erythropoietin production and autophagy induction

Arsenic is abundant in the environment and takes the form of trivalent and pentavalent arsenic compounds. Arsenite has been reported to both promote and suppress erythropoietin (EPO) production and autophagy induction. EPO production is involved in hematopoiesis, and autophagy induction is involved in cytoprotection, both of which are thought to be cellular responses to arsenic stress. While there are reports that show the effects of EPO on autophagy induction, the relationship between EPO production and autophagy induction is unclear. Therefore, this study analyzed the effect of the pentavalent inorganic arsenic salt arsenate on EPO production in vitro and in vivo and EPO-induced autophagy in HepG2 cells. Exposure of HepG2 cells to low-concentration arsenate was observed to increase EPO production and induced autophagy. Moreover, a ROS scavenger suppressed the arsenate-induced increase in autophagy and EPO mRNA levels. Both EPO production and autophagy induction contributed to protection from arsenate-induced cytotoxic stress. HepG2 cells expressed the EPO receptor and production of EPO by HepG2 cells acted in an autoregulatory manner to suppress autophagy induction. In vivo administration of low-concentration arsenate to rats increased EPO mRNA levels in the liver and kidney. These results suggested that low-concentration arsenate promotes EPO production and autophagy induction in HepG2 cells, and the resultant EPO production contributes to cytoprotection of cultured cells via EPO receptor activation.

The indispensable role of the RNA helicase DDX5 in tumorigenesis induced by the myeloproliferative neoplasm-associated JAK2V617F mutant

A point mutation (V617F) in the Janus kinase 2 (JAK2) gene results in the production of disorderly activated tyrosine kinase, which causes myeloproliferative neoplasms (MPN). We herein demonstrated that the RNA helicase DDX5 was highly expressed at the mRNA and protein levels through the activation of signal transducer and activator of transcription 5 (STAT5) in Ba/F3 cells expressing a JAK2V617F mutant and erythropoietin receptor (V617F/EpoR cells) and MPN patient-derived HEL cells. A treatment with the JAK1/2 inhibitor, ruxolitinib and STAT5 inhibitor, pimozide significantly inhibited DDX5 mRNA expression and enhanced the degradation of DDX5 in these cells, suggesting that the JAK2V617F mutant positively regulates DDX5 mRNA expression and DDX5 protein stability by activating STAT5. The knockdown of DDX5 specifically inhibited the activation of mechanistic target of rapamycin (mTOR) in V617F/EpoR cells and HEL cells and significantly suppressed the proliferation of these cells. Furthermore, the knockdown of DDX5 markedly suppressed tumorigenesis, splenomegaly, and liver hypertrophy caused by an inoculation of V617F/EpoR cells in nude mice. Collectively, these results revealed that JAK2V617F exhibits transforming activity by inducing the expression of DDX5 in a STAT5-dependent manner, indicating the potential of the JAK2V617F/STAT5/DDX5 axis as a therapeutic target in the treatment of MPN.

Differential Effects of Monoferric Forms of Transferrin on Mouse Models of Anemia of Inflammation and Congenital Anemia

Transferrin (TF) is a bilobed 80kD glycoprotein with N- and C-lobe iron binding sites. TF circulates as four forms: unbound to iron (apo-TF), iron bound to the N-lobe, the C-lobe or to both lobes (diferric-TF). Most circulating TF under physiological conditions is monoferric (Dirusso et al., 1985). The TF forms interact with TF receptor-1 (TFR1), which is ubiquitously expressed and serves as the main mechanism for cellular iron delivery. TF also interacts with TF receptor-2 (TFR2) which is expressed on hepatocytes, erythroblasts, and other cells of the bone marrow and is thought to primarily influence cellular signaling events regulating hepcidin expression and erythropoiesis. In erythrocytes, loss of TFR2 results in abnormally high red blood cell production under conditions of iron-deficiency (Artuso et al., 2018), suggesting it functions as an iron sensor. Additional evidence suggests TFR2 modulates erythropoiesis via the erythropoietin (EPO) receptor (EPOR) (Forejtnikovà et al., 2010). To understand the role of monoferric TF forms in vivo, we previously generated N-lobe blocked (TfN-bl) or C-lobe blocked (TfC-bl) TF mutant mice. Characterization of these mice demonstrated dramatic differences in RBC levels, hepcidin expression relative to iron status, and EPO sensitivity (Parrow et al., 2019). More recently, we have become interested in characterizing the immune compartment of these mice. Recent studies point to iron being an important regulator of immune responses, which has important implications worldwide in understanding how to treat iron-deficient patients without exacerbating latent infections. Cells belonging to the immune branch have also been shown to express TFR2 and EPOR (Cantarelli et al., 2019; Roetto et al., 2011; Kawabata et al., 2001), however their function is largely unexplored. Therefore, in this work, we investigate the role of the two monoferric forms of TF on ineffective erythropoiesis (IE) using a mouse model of β-thalassemia (BT) intermedia (Hbbth3/+) and chronic anemia of inflammation using heat-killed Brucella Abortus (HKBA). Based on observations in BT mice treated with exogenous TF (Li et al., 2010), which led to reduced erythroid iron intake, reduced EPO requirement (as in TfC-bl mutant mice) and improved anemia, we hypothesized that Hbbth3/+ mice crossed with TfC-bl would demonstrate improved erythropoietic and iron parameters compared with Hbbth3/+TfN-bl. We measured effects on hematologic parameters, IE, and expression of relevant iron regulatory serum proteins. Hbbth3/+TfC-bl mice demonstrated significantly increased RBC counts, elevated hemoglobin, improved erythrocyte morphology, decreased splenomegaly, fewer bone marrow erythroblasts, and improvement of IE. Additionally, serum erythroferrone (ERFE) was significantly reduced and hepcidin levels were increased in Hbbth3/+TfC-bl relative to Hbbth3/+Tf+/+ controls. However, hematological parameters from Hbbth3/+TfN-bl mice did not show these improvements. In our studies examining the immune compartments of TfC-bl and TfN-bl mice we found striking differences in their expression of TFR1 and response to immunological challenge by HKBA. Our preliminary data shows that expression levels of TFR1 were elevated in both TF mutant mice relative to control at baseline on lymphocyte, monocyte and granulocyte populations of the bone marrow. When challenged with a single intraperitoneal injection of HKBA, TfC-bl-HKBA mice suffered a more severe anemic phenotype with a 30% probability of survival following HKBA treatment. On the contrary, TfN-bl-HKBA mice displayed a blunted response to the immunological challenge, with faster recovery and no death. Taken together, our work provides additional evidence that TF is not only delivering iron cargo to cells, but also functioning as a signaling molecule via the lobe that is bound to iron. Despite each mouse only possessing a TF capable of binding one atom of iron, the two mouse models show profound differences in the pathophysiology of BT and AI. We hypothesize that the monoferric TF forms exert their influence via TFR2. We are currently characterizing a new loxp flanked TFR2-FLAG mouse model generated by our lab, which has been crossed to the TF monoferric mutant mice to test this hypothesis. Additionally, we are also exploring the translation potential of the two TF forms to treat anemia in BT and AI.

Congenital Erythrocytosis Due to Novel CMTR1K769L Mutation Has Augmented Hypoxia Signaling and Features of Both Primary and Secondary Erythrocytosis

Primary erythrocytoses (PE) include germline gain of function mutations of erythropoietin (EPO) receptor (EPOR) gene and polycythemia vera (PV), a clonal disease due to acquired somatic JAK2 mutations. Both these PEs have intrinsically abnormal early erythroid progenitors (BFU-E) characterized by their in vitro EPO hypersensitive growth (EPORmutations), or even EPO independent growth (the hallmark of PV); both have low plasma EPO (Chapter 58, Williams Hematology, 10e). Secondary erythrocytoses (SE) have intrinsically normal BFU-Es but are caused by increased circulating erythropoiesis stimulators, chiefly EPO. In contrast, congenital Chuvash erythrocytosis is characterized by augmentation of hypoxia inducible factors (HIFs) causing high EPO and hypersensitive BFU-E's EPO responses; thus, it has features of both PE and SE. We report a 7-year-old normoxic girl with history of symptomatic erythrocytosis since birth of unknown etiology, with hemoglobin of 22 g/dL at birth, consistently elevated EPO at 50-75 mU/mL, and normal hemoglobin oxygen affinity. Her BFU-E grew without EPO yet no somatic PV defining mutations were found. A targeted analysis of previously described erythrocytoses genes was negative. Whole genome sequencing, performed under the auspices of Undiagnosed Disease Network, identified a heterozygous missense variant (p. Lys769Leu) of CMTR1(Cap Methyltransferase 1) gene, not listed in the Genome Aggregation Database (gnomAD). Cap methylation on mRNA protects mRNA from being degraded and promotes RNA translation by recruiting translation initiation factors (PMID:30312682). The variant was present in the proband but not in her asymptomatic mother (father was not available for the analysis). This variant was found in leukocytes and in nail DNA, indicating germline origin. Transcriptional X-chromosome inactivation assay (PMID:18641369) revealed polyclonal granulocytes and platelets, confirming that the erythrocytosis is caused by germline mutation, not by somatic mutation-driven clonal hematopoiesis. Because of her high EPO, we hypothesized that CMTR1K769L variant might cause high HIF activity. HIF-mediated gene regulation is tissue- and stage of differentiation-specific. Expression of HIF target genes SLC29A1, VEGFA, EDN1, and LDHA was upregulated in granulocytes while in platelets all target genes except SLC29A1 were upregulated. To test if CMTR1 regulates HIF transcriptional activity, 1) we correlated CMTR1 transcript levels with HIF target gene expression levels in granulocytes and platelets. CMTR1 transcript levels were positively correlated with expression levels of HIF target genes. 2) we tested if CMTR1 overexpression and knockdown alters HIF target gene expression. In erythroid cell lines (K562 and HEL), overexpressed CMTR1 increased SLC2A1 and VEGFA (HIF target genes transcripts) while knockdown of CMTR1 downregulated their expression. This demonstrates that CMTR1 positively regulates HIF transcriptional activity, suggesting that CMTR1K769L accounts for increased plasma EPO and for EPO mediated feature of SE. As erythroid progenitors express endogenous erythropoietin (PMID 9573013) which contributes to their differentiation and proliferation (PMID 10903342), we evaluated if CMTR1K769L also promotes endogenous erythroid EPO accounting for EPO independent growth of CMTR1K769L mutated BFU-Es and thus provides potential mechanism as a feature of PEfor the erythrocytosis. The proband's endogenous EPO transcript levels in in vitro expanded erythroid cells at day 4, when most of cells are erythroid progenitors, were 10 times higher than the mother's see Figure. We present a new cause of erythrocytosis associated with the novel germline mutation of principal epigenetic modifier of RNA, CMTR1 (PMID:33922187), associated with high EPO (SE). Her BFU-E grew without extrinsic EPO (PE) likely due to previously unobserved stimulation of EPO production in erythroid progenitors. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

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.

A First-in-Human Phase 1 Study of the Safety, Tolerability, and Pharmacokinetics of Polyethylene Glycol-Erythropoietin Injection, RD01, in Healthy Volunteers

Background: Polyethylene glycol-erythropoietin injection, also called RD01, is pegylated recombinant human epoetin alfa (rHuEPOα) which stimulates erythropoietin receptors on erythroid progenitor cells to induce the formation of new red blood cells. rHuEPO (both α and ꞵ, which are almost identical to endogenous erythropoietin in the sequence of amino acids, but are distinct in their glycosylation pattern) have short half-life in 6 to 24 hours after intravenous (IV) or subcutaneous (SC) injection (Halstenson 1991; Salmonson 1990), Darbepoetin alfa, which is more highly glycosylated epoetin and has a longer half-life in 24 to 72 hours (Macdougall 2007; Padhi 2006) and CERA is a pegylated rHuEPO ꞵ with a comparable half-life of about 130 hours (Macdougall 2006). However the pharmacokinetics of pegylated rHuEPOα has not been reported. Here we reported the results of the first-in-human study of the safety, tolerability, and pharmacokinetics of RD01 in healthy volunteers. Methods: In this double-blinded, placebo-controlled, dose escalation study, healthy volunteers were randomly assigned (10:2) to receive single dose of RD01 (0.2, 0.4, 0.8, 1.6, 2.4, 3.6, 4.8 μg/kg) or placebo via subcutaneous injection. There were 7 cohorts, and each cohorts consisted of 12 subjects. Study consisted of screening period (up to 14 days), treatment and assessment period (35 days), and an end-of-study visit (at day 35). RD01 pharmacokinetics parameters were estimated using non-compartmental analysis. Safety, tolerability, and immunogenicity were assessed throughout the study. Results: All adverse events (AEs) were of mild to moderate intensity. There were no deaths, serious AEs, or withdrawal from study due to AEs. The most frequent (＞5%) drug-related treatment emergent adverse events (TEAE) were serum iron decreased, serum ferritin decreased, unsaturated iron binding capacity increased, leukocyte count decreased, and diarrhea . No clinically significant findings in vital signs or electrocardiogram were observed. Positive ADA responses to RD01 were observed in 7 of 70 subjects. The exposure (AUC0-∞) and maximum concentration (Cmax) of RD01 were approximately linear in the range of 0.2-4.8 ug/kg dose. The median Tmax was 48~96 hours, while mean t1/2 as 82.4~160.6 hours (Table 1 The PK parameters of RD01). While RD01 of 0.2- 4.8 μg/kg dose-dependently elevated hemoglobin, reticulocyte and hematocrit. In volunteers who received RD01 above 0.8 μg/kg, the mean changes from baseline of hemoglobin concentration increased over 10 g/L within 7 days after dosing. Compared with placebo, RD01 above 0.8 μg/kg also produced greater mean maximum effect (Emax) in hemoglobin, reticulocyte and hematocrit. Conclusion: RD01 (0.2-4.8 μg/kg) was well tolerated by healthy Chinese subjects with a much longer t1/2, that supported further clinical development of RD01 (above 0.8 μg/kg) for patients with anemia. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Novel Immediate Direct Targets of EPO Signalling in Human Erythropoiesis

Introduction: Erythropoietin (EPO) regulates expression of genes that drive proliferation, survival and differentiation of erythroid progenitor cells into mature erythrocytes. The EPO receptor signals via JAK2 and STAT, PI3K and MAPK pathways. Only a few direct target genes of these pathways have been identified to date. Aim: To determine the repertoire of EPO-induced target genes in human erythroid cells Methods: We employed a well characterized conditionally immortalized EPO-dependent human erythroid progenitor model (HUDEP-2 cells). The cells require stem cell factor and EPO for growth, but can be starved of both and then re-challenged with either growth factor. A multi-omics approach was employed to find direct target genes of EPO stimulation. We performed ChIP-seq for pSTAT5, ATAC-seq, and a new method of primary RNA-seq based on SLAM-seq but using full length cDNA libraries. Results: Three hours of starvation followed EPO (5U/ml) stimulation for 1 hour resulted in rapid phosphorylation of STAT5, but not STAT1 or STAT3. ChIP-seq or pSTAT5 identified 3128 EPO-induced binding sites. The majority of peaks contain a palindromic 'GAS' motif (TTCYXRGAA), and are located at intronic (50%), distal (29%) and intergenic (15%) enhancers; only 3% are located at promoters. De novo motif discovery identified significant enrichment of DNA-binding motifs for GATA and KLF transcription factors (TFs), suggesting co-operativity between EPO signalling and the essential basal erythroid TFs, GATA1 and KLF1. We show GATA1 and KLF1 bind most of these by ChIP-seq. We found 14,535 new ATAC-seq peaks after EPO stimulation, at both enhancers and promoters. Only ~8% overlap with pSTAT5 ChIP-seq peaks, suggesting EPO-mediated phosphorylation and DNA-binding of undiscovered TFs. By searching for enriched motifs within these regions, we identified binding sites for TFs of the NFY, EGR, and NRF1 families and others, suggesting a hidden complexity of transcription factors that mediate responses to EPO. ChIP-seq for EGR1 is in progress and will be reported. We developed a novel metabolic labelling technique and bio-informatic pipeline, called BodySLAM-seq, to determine the immediate transcriptional targets of EPO. We used this to find ~100 differentially transcribed genes (DTGs) as well as differentially expressed genes (DEGs) in immediate response to EPO (45 min labelling period). Some are direct pSTAT5 target genes such as BCL2L1, PIM1, and CISH; others are novel targets involved in transcription regulation, erythropoiesis and signalling. Some are known immediate early genes such as EGR1. The RNAs for these are rapidly degraded in the absence of EPO and rapidly induced. Other genes are not degraded in the absence of EPO but are nevertheless rapidly induced. These were only detectable by BodySLAM-seq. Conclusions: We have discovered new pSTAT5 direct target genes, and also new DNA motifs that mediate responses to EPO, in human erythropoiesis. We have discovered a set of immediate early genes (IEGs) that are rapidly degraded in the absence of an EPO stimulus and rapidly induced again by EPO. These are involved in negative regulation of JAK-STAT signalling, cell proliferation, cell survival, and transcription. They are not involved in erythroid cell differentiation specifically. This suggests the main function of EPO is to induce cell proliferation and survival rather than differentiation.

Erythropoietin Receptor (EPOR) Signaling in the Osteoclast Lineage Contributes to EPO-Induced Bone Loss in Mice.

Erythropoietin (EPO) is a pleiotropic cytokine that classically drives erythropoiesis but can also induce bone loss by decreasing bone formation and increasing resorption. Deletion of the EPO receptor (EPOR) on osteoblasts or B cells partially mitigates the skeletal effects of EPO, thereby implicating a contribution by EPOR on other cell lineages. This study was designed to define the role of monocyte EPOR in EPO-mediated bone loss, by using two mouse lines with conditional deletion of EPOR in the monocytic lineage. Low-dose EPO attenuated the reduction in bone volume (BV/TV) in Cx3cr1Cre EPORf/f female mice (27.05%) compared to controls (39.26%), but the difference was not statistically significant. To validate these findings, we increased the EPO dose in LysMCre model mice, a model more commonly used to target preosteoclasts. There was a significant reduction in both the increase in the proportion of bone marrow preosteoclasts (CD115+) observed following high-dose EPO administration and the resulting bone loss in LysMCre EPORf/f female mice (44.46% reduction in BV/TV) as compared to controls (77.28%), without interference with the erythropoietic activity. Our data suggest that EPOR in the monocytic lineage is at least partially responsible for driving the effect of EPO on bone mass.

Erythropoietin signaling in peripheral macrophages is required for systemic β-amyloid clearance.

Impaired clearance of beta-amyloid (Aβ) is a primary cause of sporadic Alzheimer's disease (AD). Aβ clearance in the periphery contributes to reducing brain Aβ levels and preventing Alzheimer's disease pathogenesis. We show here that erythropoietin (EPO) increases phagocytic activity, levels of Aβ-degrading enzymes, and Aβ clearance in peripheral macrophages via PPARγ. Erythropoietin is also shown to suppress Aβ-induced inflammatory responses. Deletion of EPO receptor in peripheral macrophages leads to increased peripheral and brain Aβ levels and exacerbates Alzheimer's-associated brain pathologies and behavioral deficits in AD-model mice. Moreover, erythropoietin signaling is impaired in peripheral macrophages of old AD-model mice. Exogenous erythropoietin normalizes impaired EPO signaling and dysregulated functions of peripheral macrophages in old AD-model mice, promotes systemic Aβ clearance, and alleviates disease progression. Erythropoietin treatment may represent a potential therapeutic approach for Alzheimer's disease.

Epo-IGF1R cross talk expands stress-specific progenitors in regenerative erythropoiesis and myeloproliferative neoplasm

AbstractWe found that in regenerative erythropoiesis, the erythroid progenitor landscape is reshaped, and a previously undescribed progenitor population with colony-forming unit-erythroid (CFU-E) activity (stress CFU-E [sCFU-E]) is expanded markedly to restore the erythron. sCFU-E cells are targets of erythropoietin (Epo), and sCFU-E expansion requires signaling from the Epo receptor (EpoR) cytoplasmic tyrosines. Molecularly, Epo promotes sCFU-E expansion via JAK2- and STAT5-dependent expression of IRS2, thus engaging the progrowth signaling from the IGF1 receptor (IGF1R). Inhibition of IGF1R and IRS2 signaling impairs sCFU-E cell growth, whereas exogenous IRS2 expression rescues cell growth in sCFU-E expressing truncated EpoR-lacking cytoplasmic tyrosines. This sCFU-E pathway is the major pathway involved in erythrocytosis driven by the oncogenic JAK2 mutant JAK2(V617F) in myeloproliferative neoplasm. Inability to expand sCFU-E cells by truncated EpoR protects against JAK2(V617F)-driven erythrocytosis. In samples from patients with myeloproliferative neoplasm, the number of sCFU-E-like cells increases, and inhibition of IGR1R and IRS2 signaling blocks Epo-hypersensitive erythroid cell colony formation. In summary, we identified a new stress-specific erythroid progenitor cell population that links regenerative erythropoiesis to pathogenic erythrocytosis.

Effect of Erythropoietin on Mononuclear Cells of the Bone Marrow and Spleen.

We studied the effect of preconditioning of human bone marrow mononuclear cells with erythropoietin on the immunophenotype of immunocompetent cells and paracrine activity of mouse splenocytes. The expression of erythropoietin receptors on immunocompetent human bone marrow cells was shown to change after a short-term (60 min) exposure to erythropoietin. The number of T helpers carrying erythropoietin receptors decreased and the number of T suppressors, B lymphocytes, and monocytes carrying erythropoietin receptors increased. The presence of 30% conditioned medium from human bone marrow mononuclear cells or 33.4 U/ml of erythropoietin reduced apoptosis/necrosis, increased intracellular activity of NADPH-dependent oxidoreductases of splenocytes, and did not affect oxidative phosphorylation (did not enhance lactate production and glucose uptake by cells).

Erythropoietin-driven dynamic proteome adaptations during erythropoiesis prevent iron overload in the developing embryo.

Erythropoietin (Epo) ensures survival and proliferation of colony-forming unit erythroid (CFU-E) progenitor cells and their differentiation to hemoglobin-containing mature erythrocytes. A lack of Epo-induced responses causes embryonic lethality, but mechanisms regulating the dynamic communication of cellular alterations to the organismal level remain unresolved. By time-resolved transcriptomics and proteomics, we show that Epo induces in CFU-E cells a gradual transition from proliferation signature proteins to proteins indicative for differentiation, including heme-synthesis enzymes. In the absence of the Epo receptor (EpoR) in embryos, we observe a lack of hemoglobin in CFU-E cells and massive iron overload of the fetal liver pointing to a miscommunication between liver and placenta. A reduction of iron-sulfur cluster-containing proteins involved in oxidative phosphorylation in these embryos leads to a metabolic shift toward glycolysis. This link connecting erythropoiesis with the regulation of iron homeostasis and metabolic reprogramming suggests that balancing these interactions is crucial for protection from iron intoxication and for survival.

The Conundrum of Managing Anemia in Chronic Kidney Disease Patients on Dialysis with Associated Hemoglobinopathies- Perspective from a Single Nephrology Centre in North –Eastern India

Anemia is a common complication in chronic kidney disease (CKD) and has been associated with a reduced quality of life [1] as well as worse survival [2] and increased morbidity and mortality [3]. The prevalence of anemia is more severe as the estimated glomerular filtration rate (eGFR) declines and is 8.4% at stage 1 to 53.4% at stage 5. Similar data is observed in a more recent paper by the CKD Prognosis Consortium which also observed an increased prevalence of anemia among diabetic patients, independent of eGFR and albuminuria [4]. In CKD patients, EPO deficiency starts early in the course of CKD and appears initially, when eGFR falls below 30ml/min/1.73m2, this deficiency becomes more severe [5]. This absolute EPO deficiency can be caused by a decrease EPO production and /or errors in EPO sensing. CKD produces an alteration in oxygen delivery to the kidneys and results in adaptation of renal tissue to consume less oxygen and subsequent maintenance of normal tissue oxygen gradient. As a consequence, prolyl-hydroxylase domain (PHD) enzymes which regulates HIF activity remain active, the HIF heterodimer is not formed and the EPO gene is not activated [6]. Some CKD patients may also present with a functional EPO deficiency or EPO resistance, where normal range. EPO levels co-exist with low hemoglobin levels indicating a blunted bone marrow response to endogenous and exogenous EPO. Mechanisms hypothesized for EPO resistance include presence of pro-inflammatory cytokines thought to induce apoptosis and down regulation of expression of EPO receptor.

Association between Anti-Erythropoietin Receptor Antibodies and Cardiac Function in Patients on Hemodialysis: A Multicenter Cross-Sectional Study.

Cardiac dysfunction is an important prognostic predictor of cardiovascular mortality in patients on hemodialysis (HD). Erythropoietin (EPO) has been reported to improve cardiac function by binding to the EPO receptor (EPOR) on cardiomyocytes. This study investigated whether anti-EPOR antibodies were associated with left ventricular cardiac function in patients undergoing HD. This multicenter, cross-sectional observational study included 377 patients (median age, 70 years; 267 (70.8%) males) with chronic kidney disease (CKD) undergoing stable maintenance HD. Serum levels of anti-EPOR antibodies were measured, and echocardiography was used to assess the left ventricular mass index (LVMI) and left ventricular ejection fraction (LVEF). Anti-EPOR antibodies were found in 17 patients (4.5%). LVMI was greater (median of 135 g/m2 vs. 115 g/m2, p = 0.042), and the prevalence of LVEF < 50% was higher (35.3% vs. 15.6%, p = 0.032) in patients with anti-EPOR antibodies than in those without. Multivariable linear regression and logistic regression analysis (after adjusting for known risk factors of heart failure) revealed that anti-EPOR antibodies were independently associated with LVMI (coefficient 16.2%; 95% confidence interval (CI) 1.0–35.0%, p = 0.043) and LVEF <50% (odds ratio 3.20; 95% CI 1.05–9.73, p = 0.041). Thus, anti-EPOR antibody positivity was associated with left ventricular dysfunction in patients undergoing HD.