Erythropoietin Stimulation Research Articles

Erythropoietin stimulation of human adipose tissue for therapeutic refilling releases protective cytokines

Apoptosis and inflammatory processes may be at the basis of reducing graft survival. Erythropoietin is a tissue-protective hormone with pleiotropic potential, and it interferes with the activities of pro-inflammatory cytokines and stimulates healing following injury, preventing destruction of tissue surrounding the injury site. It may represent a useful tool to increase the autograft integration. Through the use of multipanel kit cytokine analysis we have detected the cytokines secreted by human tissue adipose mass seeded in culture following withdrawal by Coleman’s modified technique in three groups: control, after lipopolysaccharides stimulation and after erythropoietin stimulation. In the control group, we have observed expression of factors that may have a role in protecting the tissue homeostatic mechanism. But the same factors were secreted following stimulation with lipopolysaccharides combined with others factors that delineated the inflammatory state. Instead through erythropoietin stimulation, the factors known to exert tissue-protective action were secreted. Therefore, the use of a trophic factors such as erythropoietin may help to inhibit the potential inflammatory process development and stimulate the activation of reparative/regenerative process in the tissue graft.

Factors Affecting Hemodialysis Adequacy in Cohort of Iranian Patient with End Stage Renal Disease.

Background:There are many factors that can affect dialysis adequacy; such as the type of vascular access, filter type, device used, and the dose, and rout of erythropoietin stimulation agents (ESA) used. The aim of this study was investigating factors affecting Hemodialysis adequacy in cohort of Iranian patient with end stage renal disease (ESRD).Methods:This is a cross-sectional study conducted on 133 Hemodialysis patients referred to two dialysis units in Sistan-Baluchistan province in the cities of Zabol and Iranshahr, Iran. We have looked at, (the effects of the type of vascular access, the filter type, the device used, and the dose, route of delivery, and the type of ESA used) on Hemodialysis adequacy. Dialysis adequacy was calculated using kt/v formula, two-part information questionnaire including demographic data which also including access type, filter type, device used for hemodialysis (HD), type of Eprex injection, route of administration, blood groups and hemoglobin response to ESA were utilized. The data was analyzed using the SPSS v16 statistical software. Descriptive statistical methods, Mann-Whitney statistical test, and multiple regressions were used when applicable.Results:The range of calculated dialysis adequacy is 0.28 to 2.39 (units of adequacy of dialysis). 76.7% of patients are being dialyzed via AVF and 23.3% of patients used central venous catheters (CVC). There was no statistical significant difference between dialysis adequacy, vascular access type, device used for HD (Fresenius and B. Braun), and the filter used for HD (p> 0.05). However, a significant difference was observed between the adequacy of dialysis and Eprex injection and patients’ time of dialysis (p <0.05).Conclusion:Subcutaneous ESA (Eprex) injection and dialysis shift (being dialyzed in the morning) can have positive impact on dialysis adequacy. Patients should be educated on the facts that the type of device used for HD and the vascular access used has no significant effects on dialysis adequacy.

Dissection of the Expression Patterns of Hydroxyurea Transporters during Erythropoiesis and Myelopoiesis

Background: Hydroxyurea (HU) is the only FDA approved drug for the treatment of moderate to severe sickle cell disease (SCD). It is becoming a standard of care for all SCD patients; clinical efficacy is attributed largely to enhanced expression of the γ-globin gene in erythroid progenitors. Dose-limiting toxicity of the drug in myeloid cells is believed to hinder its maximum effectiveness and utilization. Hitherto, the precise mechanisms that influence the on-target effects of HU in eyrthroid cells and the off-target cytotoxic effects in myeloid cells have not been defined. Cell surface transporters regulating the intracellular accumulation of compounds exert a major influence on drug action. For instance, differential expression of transporters in specific cell types elicits variable responses. We have previously shown that the organic cation/carnitine transporter 1 (OCTN1) and urea transporter B (UTB) modulate intracellular accumulation of HU. Both transporters are highly expressed in the bone marrow however their role in the on- and off-target effects of HU has never been examined. In this study, we reasoned that variation in the expression of UTB and OCTN1 on hematopoietic progenitor cells (HPCs) may influence how individual lineages of HPCs respond to the drug. Objective: In order to begin dissecting the role of OCTN1 and UTB transporters in modulating HU activity, the objective of the present study was to define lineage-specific expression patterns of UTB and OCTN1 in erythroid and myeloid progenitor cells during hematopoiesis. Methods: Human CD34+ HPCs isolated from peripheral blood were cultured in media containing erythropoietin or granulocyte-colony stimulating factor to induce erythropoiesis or myelopoiesis, respectively. Erythroid and myeloid differentiation was verified by flow cytometry and morphology. Specific populations of cells were isolated at defined time-points during differentiation, and gene expression for OCTN1 and UTB(encoded by SLC22A4 and SLC14A1 respectively) was determined by quantitative RT-PCR. Results: OCTN1 expression pattern was similar in erythroid and myeloid lineages but was variable during the course of differentiation. As cells differentiated from the common HPCs, OCTN1 expression was greatest in basophilic and polychromatic erythroblasts and promyelocytes. Expression increased 1.7- and 1.5-fold in erythroid and myeloid cells, respectively, compared to the common HPC. OCTN1 expression in late-stage precursor cells dropped to levels similar to those of common HPCs. Interestingly, UTB expression was 20-fold greater in othrochromatic erythroblasts and reticulocytes compared to common HPCs. However, the expression of this transporter decreased progressively in differentiating myeloid cells declining to a mean value 10-fold lower in late-stage myeloid band cells compared to the common HPCs. To examine whether HU treatment influences transporter expression, erythroblasts were treated with 100μM HU when OCTN1 and UTB expression was increased 10- and 20-fold, respectively (p&lt;0.001). After 72 hours of HU exposure, γ-globin expression increased 2.5-fold (p&lt;0.001) while OCTN1 expression increased 2-fold (p=0.01) compared to PBS-treated controls. UTB gene expression was significantly up regulated 1.5-fold (p&lt;0.02) at 24 and 48 hours after HU treatment. Taken together these data demonstrate differential expression patterns of OCTN1 and UTB in HPCs that are altered by HU. Conclusions: These studies are the first to document HU transporter expression during erythroid and myeloid differentiation. We have shown that HU is a substrate for both OCTN1 and UTB, and we now demonstrate that their expression patterns are consistent with experimental evidence of HU response. Optimal γ-globin induction is observed when early- to mid-stage progenitor cells are treated with HU and this coincides with significant increases in transporter expression. Preferential expression of UTB in erythroid progenitors suggests it may be a primary modulator of HU-mediated γ-globin induction with a lesser role in myeloid cytotoxicity. A detailed understanding of the expression patterns of OCTN1 and UTB in HPCs may help to refine the use of HU to improve its overall efficacy and utilization in SCD. Disclosures No relevant conflicts of interest to declare.

Erythroferrone (ERFE) Is Selectively Expressed in Human CD71+ Erythroprogenitor Cells and Deregulated Overexpression Is Associated with a Favorable Outcome in Low Risk Myelodysplastic Syndrome (MDS)

IntroductionRecently, Erythroferrone (ERFE) was discovered as a new regulator of hepcidin in the context of hematopoietic stress and erythropoietin (EPO) stimulation (Kautz et al., Nature Genetics 2014). ERFEhas been shown to be expressed by erythroprogenitor cells of the bone marrow in response to increased erythroid activity induced by phlebotomy, EPO treatment or simulation of infectious situations in mice. It induces increased iron availability by downregulation of hepcidin in the liver and therefore represents an important new factor in iron homeostasis to be explored as a potential diagnostic or therapeutic target in the context of anemia and iron overload. Myelodysplastic Syndromes (MDS) are a group of heterogeneous malignant hematologic diseases characterized by inefficient hematopoiesis, severe anemia and deregulated iron homeostasis. In order to determine the specific role of ERFE in MDS, we analyzed the gene expression of ERFE in different hematopoietic compartments of MDS patients and healthy controls and correlated the differential expression data with clinical parameters and survival.MethodsCD71+ erythroprogenitor cells (n=198 samples) were immunomagnetically purified from mononuclear bone marrow (BM) cells of a total of n=148 MDS and n=18 sAML patients. Chronological samples were available in n=21 cases. For controls, CD71+ BM cells were analyzed from n=35 healthy donors. In addition to CD71+ cells, CD61+, CD15+ , CD34+, selected from BM, as well as CD3+ selected peripheral blood (PB) cells were immunomagnetically collected from three MDS patients as well as two healthy young and two healthy old volunteers. After total RNA extraction using the AllPrep DNA/RNA Mini kit (Qiagen), cDNA was transcribed from RNA via Quantitect cDNA synthesis kit (Qiagen). Subsequently, ERFE expression was quantified from cDNA by quantitative PCR.ResultsIn comparative expression analyses of different hematopoietic BM progenitor fractions (CD34+, CD15+, CD61+ and CD71+), ERFE was almost exclusively expressed in the erythropoietic CD71+ compartment. ERFE expression profiles in the CD71+ subset revealed a highly significant overexpression of this gene in MDS IPSS-low/int-1-risk (fold change (FC)=4.3, p<0.0001), IPSS-int-2/high-risk (FC=6.23, p<0.0001) and sAML (FC=6.69, p<0.0001) relative to healthy controls. ERFE expression profiles in MDS and sAML did not correlate with clinical laboratory parameters such as hemoglobin, EPO levels, ferritin, cobalamine, folic acid, transferrin, transferrin saturation, soluble transferrin receptor, reticulocytes, zinc protoporphyrin and lactate dehydrogenase. A negative correlation was observable for c-reactive protein levels (p=0.0053, Spearman r=-0.29) suggesting a possible link between an inflammatory environment and ERFE regulation. In exemplary chronological time course samples, ERFE expression was upregulated subsequent to clinical therapies such as 5-Azacytidine or Lenalidomide. Interestingly, in the total cohort of MDS patients with survival data follow up (n=90), low ERFE expression was associated with a significantly worse survival than high ERFE expression (median survival 2.1 years versus not reached, HR: 4.4, p=0.0007). This observation was even more pronounced in the subgroup analysis of MDS IPSS low/int-1 risk patients (n=54, median survival 2.1 years versus not reached, HR: 22, p<0.0001).ConclusionThe observed highly aberrant overexpression of ERFE in CD71+ erythropoietic progenitor cells suggests an important role for this gene in the dysfunctional erythropoiesis of MDS. The observation of a correlation between ERFE expression and survival, especially in low risk MDS patients with no apparent coherence to other established clinical markers warrants further pursuit of ERFE expression profiles in CD71+ BM cells of MDS patients as a possible independent prognostic marker. Moreover, aberrant levels of ERFE could provide a promising target for novel therapeutic avenues that mechanistically address dysfunctional erythropoiesis in MDS. DisclosuresNo relevant conflicts of interest to declare.

Are erythropoietin stimulating proteins safe and efficacious in heart failure? Why we need an adequately powered randomised outcome trial (vol 9, pg 110, 2007)

Are erythropoietin stimulating proteins safe and efficacious in heart failure? Why we need an adequately powered randomised outcome trial (vol 9, pg 110, 2007)

Cooperative Effect of Erythropoietin and TGF-β Inhibition on Erythroid Development in Human Pluripotent Stem Cells.

Patient-specific human induced-pluripotent stem cells (hiPSCs) represent important cell sources to treat patients with acquired blood disorders. To realize the therapeutic potential of hiPSCs, it is crucial to understand signals that direct hiPSC differentiation to a hematopoietic lineage fate. Our previous study demonstrated that CD34(+)CD31(+) cells derived from human pluripotent stem cells (hPSCs) contain hemato-endothelial progenitors (HEPs) that give rise to hematopoietic cells and endothelial cells. Here, we established a serum-free and feeder-free system to induce the differentiation of hPSC-derived CD34(+)CD31(+) progenitor cells to erythroid cells. We show that extracellular matrix (ECM) proteins promote the differentiation of CD34(+)CD31(+) progenitor cells into CD235a(+) erythroid cells through CD41(+)CD235a(+) megakaryocyte-erythroid progenitors (MEP). Erythropoietin (EPO) is a predominant factor for CD34(+)CD31(+) progenitor differentiation to erythroid cells, whereas transforming growth factor beta (TGF-β) inhibits the development of CD34(+)CD31(+) progenitor cells. Apoptosis of progenitor cells is induced by TGF-β in early erythroid differentiation. Suppression of TGF-β signaling by SB431542 at early stage of CD34(+)CD31(+) progenitor differentiation induces the erythroid cell generation. Together, these findings suggest that TGF-β suppression and EPO stimulation promote erythropoiesis of CD34(+)CD31(+) progenitor cells derived from hPSCs.

Comparison of Efficacy of Continuous Erythropoietin Receptor Activator ( CERA ) in Chronic Kidney Disease Patients versus Patients on Hemodialysis : Single Center Experience

Background : prior to the availability of recombinant human erythropoietin (r-EPO, epoetin), patients on dialysis frequently demanded blood transfusions and excessive iron therapy, exposing them to the risks of iron overload, transmission of viral hepatitis, and sensitization, which reduced the chances of successful transplantation . Recombinant human erythropoietin has been used for more than 20 years for the treatment of renal anemia, Epoetin-Alfa and -beta representing the common traditional preparations. By the modification of the molecule's carbohydrate moiety or structure a longer duration of erythropoietin receptor stimulation was achieved. The continuous erythropoietin receptor activator C.E.R.A. once or twice a month was found sufficient to achieve serum hemoglobin target levels. This study was aimed to identify the efficacy of C.E.R.A (Methoxy polyethylene glycol-epoetin beta in achieving and maintenance of hemoglobin level in patients with chronic kidney disease and patients on regular Hemodialysis therapy. Patients and methods: 145 patients with either CKD stage 4 & 5 (e GFR < 45 ml/min) on follow up in nephrology outpatient department or on regular Hemodialysis in nephrology unit Dubai hospital. In this center, the CERA approved by the local pharmacy authority was used for treatment of anemia in chronic kidney disease monthly dose of (100 mg for 70 kg patient) and adjusted according to monthly HB level during the trial period for reaching and maintaining target HB level of 11-12.5 g/dl. Results : Patients on HD 78 patients (53.8%) and CKD patients 67 patients the mean HB level at the end of study period showed statistically significant rise in whole patient cohort with HB START (8.882 ±1.272 g/dl) and at the end of 24month (11.119±1017 g/dl) With P-value (< 0.001). HB at start was (9.013±1.298 g/dl) and (8.769±1.246) g/dl for CKD and HD respectively slightly higher in CKD group however without statistical difference (P-value 0.250). HB level at the 24 month period (end of trial period) was found statically higher in HD group (11.305±1.197g/dl) than CKD group (10.903±1.116g/dl) again without statistical significance P-value (0.038). Conclusion: It could be concluded that the long acting erythropoietin stimulating therapy using continuous erythrpoeitin receptor activator ( CERA ) as once monthly dose is effective and safe in maintaining target HB level in both CKD patients and patients on maintenance hemodialysis and there was no recorded side effects of its use either by intravenous or subcutaneous use.

Human rhabdomyosarcoma cells express functional erythropoietin receptor: Potential therapeutic implications.

The erythropoietin receptor (EpoR) is expressed by cells from the erythroid lineage; however, evidence has accumulated that it is also expressed by some solid tumors. This is an important observation, because recombinant erythropoietin (EPO) is employed in cancer patients to treat anemia related to chemo/radiotherapy. In our studies we employed eight rhabdomyosarcoma (RMS) cell lines (three alveolar-type RMS cell lines and five embrional-type RMS cell lines), and mRNA samples obtained from positive, PAX7-FOXO1-positive, and fusion-negative RMS patient samples. Expression of EpoR was evaluated by RT-PCR, gene array and FACS. The functionality of EpoR in RMS cell lines was evaluated by chemotaxis, adhesion, and direct cell proliferation assays. In some of the experiments, RMS cells were exposed to vincristine (VCR) in the presence or absence of EPO to test whether EPO may impair the therapeutic effect of VCR. We report for a first time that functional EpoR is expressed in human RMS cell lines as well as by primary tumors from RMS patients. Furthermore, EpoR is detectably expressed in both embryonal and alveolar RMS subtypes. At the functional level, several human RMS cell lines responded to EPO stimulation by enhanced proliferation, chemotaxis, cell adhesion, and phosphorylation of MAPKp42/44 and AKT. Moreover, RMS cells became more resistant to VCR treatment in the presence of EPO. Our findings have important potential clinical implications, indicating that EPO supplementation in RMS patients may have the unwanted side effect of tumor progression.

Abstract 2425: An in vitro platform to dissect drug responsiveness in refractory anemia with ringed sideroblasts (RARS)

Abstract Background: The pathophysiology of low-risk myelodysplastic syndrome (LR-MDS) remains unknown and therapeutic options are limited. In a phase-II trial, with oral rigosertib, a small molecule inhibitor of PI3K and PLK pathways, was shown to have a 39% response rate, in patients with LR-MDS. Rigosertib caused an increase in hemoglobin, a decrease in transfusion requirements, and, in some patients who were previously refractory, “re-sensitization” to erythropoietin (EPO) therapy. Thus far, the mechanism of this responsiveness remains unknown. Whole exome sequencing revealed broad spectrum mutations in these patients, including mutations in SF3B1, SRSF2 and TET2. However, there was no correlation between mutational spectrum and responsiveness. Methods: CD34+ cells were isolated from bone marrow (BM) mono nuclear cells derived from BM aspirates. Bone marrow stromal cells (BMSC) were isolated from bone biopsies of patients with MDS. Normal CD34+ cells and BMSC were obtained commercially. CD34+ cells were co-cultured with stromal cells plated a day before and stimulated with concentrations of EPO and rigosertib. Differentiation was assessed by staining cells with various markers of erythroid differentiation and analyzed by FACS. We show that co-culture of CD34+ stem cells from the bone marrow of patients with their own BMSC recapitulates key features of the MDS phenotype and rigosertib responsiveness. Results: Normal (non-MDS) CD34+ cells co-cultured with normal BMSC showed striking responsiveness to EPO stimulation, as evidenced by the increased production of CD45- low/GPA+/ band 3+/Integrin-Alpha4+ erythroid cells (2.47 fold increase compared to no EPO). In contrast, RARS CD34+ cells co-cultured with MDS stromal cells showed no erythroid differentiation with EPO stimulation (1.09 fold change compared to EPO). Notably, LR-MDS co-cultures showed increased erythroid differentiation with the addition of 20 nm and 100 nm rigosertib in the presence of EPO (1.28 and 1.4 fold increase, respectively, compared to EPO alone). Moreover, co-cultures obtained from 3/3 patients that showed responsiveness in vivo were responsive to combined EPO/rigosertib stimulation in vitro, while 2/2 non-responsive patients were not responsive in vitro. Conclusions: We have created an in vitro platform to dissect the mechanism of rigosertib responsiveness in RARS patients. This is a novel co-culture-based system that recapitulates key features of the RARS phenotype. Without reliable animal models for this disease, this platform may offer a viable method to characterize drug responsiveness, dissect mechanisms, and offer patient-specific drug responsiveness information, since individual CD34+ cells are co-cultured with a patient's own BMSC. We wish to use this in vitro co- culture test to prospectively predict responsiveness to experimental drugs in patients, thereby focusing the drug on only selected patients. Citation Format: Daniela Georgieva, Sheherzad Preisler, Michael Churchill, Abdullah Mahmood Ali, Azra Raza, Siddhartha Mukherjee. An in vitro platform to dissect drug responsiveness in refractory anemia with ringed sideroblasts (RARS). [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2425. doi:10.1158/1538-7445.AM2015-2425

Abstract 2040: Mutations in SF3B1 lead to aberrant splicing through cryptic 3′ splice site selection and impair hematopoietic cell differentiation

Abstract Heterozygous mutations in SF3B1, a component of the U2 complex involved in the recognition of 3′ splice sites (ss), have been reported with high frequency in refractory anemia with ring sideroblasts (RARS, a subtype of myelodysplastic syndrome, MDS) and have also been observed in chronic lymphocytic leukemia (CLL) and several solid tumors. To study the impact of SF3B1 mutations on splicing, RNAseq data obtained from breast cancer, melanoma, CLL and MDS samples with mutant (SF3B1MUT) or wild-type SF3B1 (SF3B1WT) were compared. The majority of aberrant junctions identified in the samples with mutant SF3B1 utilized an alternative 3′ss, suggesting its neomorphic function. Motif analysis of the sequences used by SF3B1MUT revealed the usage of a cryptic AG with a shorter and weaker polypyrimidine tract. Minigenes with modifications of these sites revealed the importance of both intronic and exonic sequence features for the recognition of the cryptic AG by SF3B1MUT. Of the aberrant junctions identified, several were common across all hotspot mutations and diseases; however, a unique aberrant splicing profile was found for each disease suggesting lineage and disease specific effects. The majority of splicing defects introduced a premature termination codon downstream of the cryptic AG leading to nonsense mediated decay (NMD) of aberrant transcripts and downregulation of gene expression, such as ABCB7. Gene-set enrichment analysis of aberrantly spliced and differentially expressed genes in SF3B1MUT MDS samples identified genes involved in cell differentiation and epigenetic pathways which are known to be deregulated in MDS. The impact on erythroid differentiation by SF3B1MUT was studied in transduced TF-1 cells following erythropoietin (EPO) stimulation. As expected, TF-1 SF3B1WT cells were able to differentiate normally after EPO treatment; however, expression of SF3B1K700E (the most common hotspot mutation found in RARS and CLL) induced a block in erythoid differentiation. This differentiation block was not observed with the expression of SF3B1G742D, a mutation found in CLL but not RARS, suggesting a context dependent role for SF3B1 mutations. Interestingly, the differentiation block observed in SF3B1K700E was associated with cytokine independent growth. Initial mining of RNAseq data from SF3B1MUT TF-1 cells highlighted several aberrantly spliced and NMD-downregulated genes previously implicated in MDS. Finally, a xenograft model was developed by subcutaneous implantation of transduced TF-1 cells. After several passages, an enrichment of TF-1 SF3B1K700E cells was observed, suggesting a growth advantage for SF3B1MUT cells over SF3B1WT cells. This data suggests that the K700E SF3B1 mutation can lead to a block in differentiation and competitive advantage as observed in human RARS. Citation Format: Silvia Buonamici, Samantha Perino, Kian Huat Lim, Jacob Feala, Rachel Darman, Esther Obeng, Richard R. Furman, Suzanna Bailey, Gregg Keaney, Pavan Kumar, Yoshiharu Mizui, Eunice Park, John Wang, Markus Warmuth, Lihua Yu, Ping Zhu, Benjamin L. Ebert, Peter Smith. Mutations in SF3B1 lead to aberrant splicing through cryptic 3′ splice site selection and impair hematopoietic cell differentiation. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2040. doi:10.1158/1538-7445.AM2015-2040

Abstract 4369: Rearrangements of the erythropoietin receptor are recurrent in Ph-like acute lymphoblastic leukemia and are sensitive to Jak2 inhibition

Abstract Introduction: Ph-like acute lymphoblastic leukemia (ALL) is characterized by a diverse range of genetic alterations that activate cytokine receptor and kinase signaling. We recently identified (Roberts KG, NEJM 2014) rearrangements of the erythropoietin receptor (EPOR) with immunoglobulin heavy (IGH) or kappa (IGK) chain loci in 154 Ph-like ALL. Here, we aimed to investigate their spectrum and recurrence in an extended cohort, as well as their pathogenic role and therapeutic potential in childhood Ph-like ALL. Methods: Whole transcriptome sequencing, real-time quantitative PCR (qPCR) and Sanger sequencing were performed to detect and map the EPOR rearrangements in 307 Ph-like ALL. Wild-type or EPOR rearranged alleles were expressed in IL-3-dependent mouse hematopoietic Ba/F3 cells and IL-7-dependent pre-B cells harboring alterations of Arf and/or the dominant negative IKZF1 allele IK6. Proliferation and downstream EPOR signaling were examined in absence or presence of erythropoietin (EPO). Leukemic growth was monitored in C57BL/6 mice transplanted with Arf-/- pre-B cells expressing rearranged EPOR. Luciferase-marked xenografts were established in NOD-SCID-IL2R gamma (NSG) null mice. Signaling, EPO-dependent proliferation and sensitivity to the JAK inhibitors were assessed ex vivo and in vivo. Results: EPOR rearrangements were identified in 8.3% of Ph-like ALL as the result of: i) inversion and cryptic insertion under the promoter region of IGH/IGK; ii) reciprocal translocation; or iii) fusion to 5′ LAIR1. All rearrangements resulted in ectopic overexpression of a C-terminal truncated receptor, confirmed by qPCR, that retained the phosphorylation site required for STAT5 activation, but lacked multiple tyrosine residues required for normal negative regulation. The rearranged alleles were expressed at higher and more prolonged levels than wild-type EPOR in Ba/F3 and Arf-/- mouse pre-B cells, and sustained cell proliferation with increased STAT5 phosphorylation following exogenous EPO stimulation. In vivo, the truncated receptor promoted leukemia development in C57BL/6 mice. Xenografted EPOR-rearranged leukemic cells exhibited high levels of truncated EPOR, STAT5 phosphorylation and ex vivo sensitivity to JAK2 inhibition using ruxolitinib, fedratinib, momelotinib, or pacritinib alone or in combination with daunrobucin, vincristine or dexamethasone (synergistic effect). The efficacy of Jak2 inhibition and the synergistic effect of combining ruxolitinib and dexamethasone were subsequently demonstrated in vivo. Conclusions: EPOR rearrangements are recurrent in Ph-like ALL and result in ectopic expression of truncated EPOR and activation of JAK2-STAT5 signaling pathway, sustaining leukemia development in vivo. Xenografted EPOR-rearranged leukemia cells exhibit ex vivo and in vivo sensitivity to JAK2 inhibitors alone or in combination with chemotherapy. Citation Format: Ilaria Iacobucci, Kathryn G. Roberts, Yongjin Li, Jinghui Zhang, Richard C. Harvey, Debbie Payne-Turner, Marcus Valentine, Kelly McCastlain, John Easton, I-Ming Chen, Michael Rusch, Steven M. Kornblau, Marina Konopleva, Elisabeth Paietta, Jacob M. Rowe, Ching-Hon Pui, Julie M. Gastier-Foster, Shalini Reshmi, Mignon L. Loh, Cheryl Willman, James R. Downing, Stephen P. Hunger, Charles G. Mullighan. Rearrangements of the erythropoietin receptor are recurrent in Ph-like acute lymphoblastic leukemia and are sensitive to Jak2 inhibition. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4369. doi:10.1158/1538-7445.AM2015-4369

SP759REVACLEAR IMPACT ON ERYTHROPOIETIN STIMULATING AGENT DOSES: A BUDGET IMPACT ANALYSIS FOR DIALYSIS PROVIDERS

SP759REVACLEAR IMPACT ON ERYTHROPOIETIN STIMULATING AGENT DOSES: A BUDGET IMPACT ANALYSIS FOR DIALYSIS PROVIDERS

Gene Expression of the Erythroid Regulator Erythroferrone (ERFE) is Highly Deregulated in CD71+ Erythroprogenitor Cells of Patients with Myelodysplastic Syndromes and Demonstrates Prognostic Relevance

IntroductionMyelodysplastic Syndromes (MDS) are clonal hematologic diseases that are characterized by inefficient hematopoiesis, severe anemia and resulting deregulated iron homeostasis. Apart from supportive therapy with red blood cell transfusions some MDS patients with anemia respond to treatment with erythropoiesis stimulating agents such as Erythropoietin (EPO). However, the majority of these patients also become refractory to EPO treatment during the course of disease, suggesting a dysfunctional regulation of erythropoiesis downstream of EPO signaling in MDS. Most recently, a crucial erythroid regulator of iron metabolism named Erythroferrone (ERFE) was newly discovered, which is selectively produced by bone marrow (BM) erythroprogenitor cells during hematopoietic stress and EPO stimulation (Kautz et al. ASH plenary session 2013 and Kautz et al., Nature Genetics 2014). Aberrant expression of ERFE has been shown to directly result in critical impairment of erythropoiesis. We therefore sought to examine the role of ERFEexpression in CD71+ erythroprogenitor cells derived from patients with MDS and secondary acute myeloid leukemia (sAML).MethodsCD71+ erythroprogenitor cells were immunomagnetically isolated from ficollized mononuclear BM cells of patients suffering from MDS (n=86, IPSS-low/int-1-risk n=69, IPSS-int-2/high-risk n=17), sAML (n=18) and age-matched healthy donors (n=17). In addition to CD71+ cells, CD34+, CD61+, CD15+ selected BM as well as CD3+ selected peripheral blood (PB) cells were immunomagnetically collected from three MDS patients as well as two healthy young and two healthy old donors. After total RNA was extracted using the AllPrep DNA/RNA Mini kit (Qiagen), cDNA was transcribed from RNA via Quantitect cDNA synthesis kit (Qiagen). Subsequently, ERFE expression was quantified from cDNA by quantitative PCR and normalized to corresponding GPIhousekeeping gene expression levels. Patient follow up (FU) data was available for n=55 MDS and n=14 sAML samples.ResultsAnalysis of ERFE expression in CD34+, CD15+, CD61+ and CD71+ BM as well as CD3+ and unselected mononuclear PB cells from MDS patients and healthy donors revealed almost exclusive expression of ERFE in CD71+ erythroprogenitor cells irrespective of disease state. Our analysis of ERFE expression profiles in this specific cell subset revealed a highly significant overexpression of this gene in MDS IPSS-low/int-1-risk (fold change (FC)=4.1, p<0.0001), IPSS-int-2/high-risk (FC=4.6, p=0.0003) and sAML (FC=6.5, p<0.0001) relative to age-matched healthy controls. Despite this marked profile of aberrantly regulated ERFE we identified a distinct fraction of patients with expression levels similar or even lower than those measured in healthy donors in 20% (11/55) and 36% (5/14) of analyzed MDS and sAML cases with FU. Univariate analysis revealed that low abundance of CD71+ ERFE transcripts was significantly associated with inferior overall survival (OS) in MDS patients (median survival 1.7 years vs. not reached, p=0.0066) and also sAML (median survival 0.1 vs. 0.8 years, p=0.031).ConclusionThe recent identification of the novel key regulatory gene ERFE in mouse models has greatly improved the understanding of the dynamic regulation of erythropoiesis. Our observation of almost exclusive ERFE expression in human BM erythroprogenitor cells further underlines its important role in human erythropoietic regulation both in healthy and myelodysplastic hematopoiesis. Moreover, strong upregulation of erythropoiesis stimulating ERFE in a large proportion of MDS patients usually suffering from anemia likely indicates its involvement in perturbed mechanisms of feedback signaling in MDS erythropoiesis. Pending integration with further clinical data, the current observation of significantly inferior survival probability for MDS and sAML patients with low ERFE expression levels indicates the potentially important biologic and clinical relevance of this novel regulatory gene in the pathogenesis of MDS. Consequently, aberrant levels of the erythroid hormone ERFEin MDS erythroprogenitor cells might provide a promising target for novel therapeutic avenues that mechanistically address dysfunctional erythropoiesis in MDS. DisclosuresNolte:Celgene Corp., Novartis Pharma: Honoraria, Research Funding.

PI3K and MEK1/2 molecular pathways are involved in the erythropoietin-mediated regulation of the central respiratory command

Erythropoietin stimulation modulates the central respiratory command in newborn mice. Specifically, the central respiratory depression induced by hypoxia is attenuated by acute (1h) or abolished by chronic erythropoietin stimulation. However, the underlying mechanisms remain unknown. As MEK and PI3K pathways are commonly involved in Epo-mediated effects of neuroprotection and erythropoiesis, we investigated here the implication of PI3K and MEK1/2 in the Epo-mediated regulation of the central respiratory command. To this end, in vitro brainstem–spinal cord preparations from 3 days old transgenic (Tg21; constitutively overexpressing erythropoietin in the brain specifically) and control mice were used. Our results show that blockade of PI3K or MEK1/2 stimulates normoxic bursts frequency in Tg21 preparations and abolish hypoxia-induced frequency depression in control preparations. These results show that MEK1/2 and PI3K pathways are involved in the Epo-mediated regulation of the central respiratory command. Moreover, this is the first demonstration that MEK1/2 and PI3K are involved in the brainstem central respiratory command.

Dissection of vertebrate hematopoiesis using zebrafish thrombopoietin

AbstractIn nonmammalian vertebrates, the functional units of hemostasis are thrombocytes. Thrombocytes are thought to arise from bipotent thrombocytic/erythroid progenitors (TEPs). TEPs have been experimentally demonstrated in avian models of hematopoiesis, and mammals possess functional equivalents known as megakaryocyte/erythroid progenitors (MEPs). However, the presence of TEPs in teleosts has only been speculated. To identify and prospectively isolate TEPs, we identified, cloned, and generated recombinant zebrafish thrombopoietin (Tpo). Tpo mRNA expanded itga2b:GFP+ (cd41:GFP+) thrombocytes as well as hematopoietic stem and progenitor cells (HSPCs) in the zebrafish embryo. Utilizing Tpo in clonal methylcellulose assays, we describe for the first time the prospective isolation and characterization of TEPs from transgenic zebrafish. Combinatorial use of zebrafish Tpo, erythropoietin, and granulocyte colony stimulating factor (Gcsf) allowed the investigation of HSPCs responsible for erythro-, myelo-, and thrombo-poietic differentiation. Utilizing these assays allowed the visualization and differentiation of hematopoietic progenitors ex vivo in real-time with time-lapse and high-throughput microscopy, allowing analyses of their clonogenic and proliferative capacity. These studies indicate that the functional role of Tpo in the differentiation of thrombocytes from HSPCs is well conserved among vertebrate organisms, positing the zebrafish as an excellent model to investigate diseases caused by dysregulated erythro- and thrombo-poietic differentiation.

Post-natal hypoxic activity of the central respiratory command is improved in transgenic mice overexpressing Epo in the brain

Previous studies indicated that erythropoietin modulates central respiratory command in mice. Specifically, a one-hour incubation of the brainstems with erythropoietin attenuates hypoxia-induced central respiratory depression. Here, using transgenic mice constitutively overexpressing erythropoietin specifically in the brain (Tg21), we investigated the effect of chronic erythropoietin stimulation on central respiratory command activity during post-natal development. In vitro brainstem-spinal cord preparations from mice at 0 (P0) or 3 days of age (P3) were used to record the fictive inspiratory activity from the C4 ventral root. Our results show that erythropoietin already stimulates the hypoxic burst frequency at P0, and at P3, erythropoietin effectively stimulates the hypoxic burst frequency and amplitude. Because the maturation of the central respiratory command in mice is characterized by a decrease in the burst frequency with age, our results also suggest that erythropoietin accelerates the maturation of the newborn respiratory network and its response to hypoxia.

Engineered Bovine Antibodies in the Development of Novel Therapeutics, Immunomodulators and Vaccines

Some bovine antibodies across all classes are unique, such as the CDR3 of the variable heavy-domain (VH CDR3), which is exceptionally long (up to 66 amino acids), unlike most conventional antibodies where the VH CDR3 loops range from 10 to 25 amino acids. The exceptionally long VH CDR3 is encoded by unusually long germline IGHD genes together with insertion of novel “a” nucleotide rich conserved short nucleotide sequence (CSNS) specifically at the IGH V-D junction. Such an exceptionally long VH CDR3 confers unique “knob and stalk” structural architecture where the knob, formed by intra-VH CDR3 disulfide bridges, is separated by 20 Å solvent exposed stalk composed of anti-parallel beta strands. The substitution of the knob with cytokines, such as, erythropoietin and granulocyte colony stimulating factor 3 (granulocyte colony stimulating factor), results in expression of functional fusion proteins with enhanced pharmacokinetics. The beta stranded stalk can be substituted with other rigid structures, for example, repeat alpha helices to form coiled-coil that mimics the beta-stranded stalk and, thus, opens opportunities for insertion of this structure in the CDRs of antibodies across species. Given the versatility of such a structural platform in bovine antibody VH CDR3, it provides the opportunity for the development of new generation of diagnostics, therapeutics, vaccines and immunomodulating drugs.

Nitric oxide and hypoxia stimulate erythropoietin receptor via MAPK kinase in endothelial cells

Erythropoietin receptor (EPOR) expression level determines the extent of erythropoietin (EPO) response. Previously we showed that EPOR expression in endothelial cells is increased at low oxygen tension and that EPO stimulation of endothelial cells during hypoxia can increase endothelial nitric oxide (NO) synthase (eNOS) expression and activation as well as NO production. We now observe that while EPO can stimulate NO production, NO in turn can regulate EPOR expression. Human umbilical vein endothelial cells (HUVEC) treated with 10–50μM of NO donor diethylenetriamine NONOate (DETANO) for 24h showed significant induction of EPOR gene expression at 5% and 2% of oxygen. Also human bone marrow microvascular endothelial cell line (TrHBMEC) cultured at 21 and 2% oxygen with 50μM DETANO demonstrated a time and oxygen dependent induction of EPOR mRNA expression after 24 and 48h, particularly at low oxygen tension. EPOR protein was also induced by DETANO at 2% oxygen in TrHBMEC and HUVEC. The activation of signaling pathways by NO donor stimulation appeared to be distinct from EPO stimulation. In reporter gene assays, DETANO treatment of HeLa cells at 2% oxygen increased EPOR promoter activity indicated by a 48% increase in luciferase activity with a 2kb EPOR promoter fragment and a 71% increase in activity with a minimal EPOR promoter fragment containing 0.2kb 5′. We found that DETANO activated MAPK kinase in TrHBMEC both in normoxia and hypoxia, while MAPK kinase inhibition showed significant reduction of EPOR mRNA gene expression at low oxygen tension, suggesting MAPK involvement in NO mediated induction of EPOR. Furthermore, DETANO stimulated Akt anti-apoptotic activity after 30min in normoxia, whereas it inhibited Akt phosphorylation in hypoxia. In contrast, EPO did not significantly increase MAPK activity while EPO stimulated Akt phosphorylation in TrHBMEC in normoxia and hypoxia. These observations provide a new effect of NO on EPOR expression to enhance EPO response in endothelial cells, particularly at low oxygen tensions.

Long-term hematological response in a patient with 5q- syndrome after suspension of lenalidomide therapy and further improvement with deferasirox therapy.

Long-term hematological response in a patient with 5q- syndrome after suspension of lenalidomide therapy and further improvement with deferasirox therapy.

Epo-Induced Erythroid Maturation Is Dependent On Plcγ1 Signalling

Erythropoiesis is a multi-step process in which the development of red blood cells occurs through expansion and differentiation of hematopoietic stem cells (HSCs) into more committed progenitors and finally into erythrocytes. Erythropoietin (Epo) is strictly required for erythropoiesis as it promotes survival and late maturation. In vivo and in vitro studies have pointed out the major role of erythropoietin receptor (EpoR) signalling through JAK2 tyrosine-kinase and STAT5a/b as a central regulator of erythropoiesis. STAT5a/b is essential in regulating early erythroblast survival, however, with regard to differentiation of erythroid progenitors current data are not definitive in establishing a critical, non-redundant role. Phospholipase C gamma 1 (PLCγ1) is known to act as key mediator of calcium-signalling that can substitute for PI3K/AKT in oncogenic models. Interestingly, genetic deletion of murine PLCγ1 in embryonic development using a conventional knockout mouse model resulted in lethality at E9.0 due to generalized growth failure and there was absence of erythrogenesis and vasculogenesis.Here, we revisited the role of Plcγ1 and investigated its function in signalling, differentiation and transcriptomic/epigenetic regulation of erythropoiesis: Upon Epo stimulation, we were able to demonstrate that Plcγ1 is a downstream target of EpoR/Jak2 signalling in lymphoid (Ba/F3) and myeloid (32D) progenitor cell lines (both transfected with EpoR and Jak2-WT) and in a erythroid progenitor (I/11) cell line. In order to specifically assess its role in erythroid development downstream of the EpoR-Jak2 axis, we focused on the murine pro-erythroblast cell line I/11 which is able to differentiate upon dexamethasone-/stem cell factor-withdrawal combined with erythropoietin stimulation. Interestingly, knockdown of Plcγ1 led to a dramatic delay (scr CD44high 21% vs. Plcγ1 shRNA CD44high 64%, p=0.02) in erythroid differentiation and accumulation of immature erythroid progenitors as assessed by flow cytometry technology. Knockdown of Plcγ1 did alter neither proliferation of cells nor the cell cycle distribution and activation of other EpoR downstream molecules as Stat5, Mek and Akt was not impaired. In addition, we analysed the colony-forming potential of Plcγ1-deficient I/11 and fetal liver cells (FLC) compared to controls. Colony formation was dramatically impaired in both - I/11 (scr 138 vs. Plcγ1 shRNA 32, p=0.03) and primary FLC (scr 107 vs. Plcγ1 shRNA 28, p<0.001) - when compared to control cells. Flow cytometry analysis of the colonies revealed a higher amount of immature populations (CD44high, KIT+) in PLCγ1-deficient cells as compared to controls whereas the content of TER119+ cells, reflecting more mature erythroid cells, was higher in controls.To elucidate on the mechanism of Plcγ1-mediated regulation of erythroid development, we performed global gene expression analysis in I/11 cells at various time points of differentiation after knockdown of Plcγ1. Several of the genes that change expression in the absence of Plcγ1 can be classified as transcription/co-transcription factors, epigenetic regulators, metabolic factors or adaptor molecules involved in intracellular signaling. Thus, Plcγ1-deficient cells showed up-regulation of the transcription factor RUNX1 and the adaptor molecule GRAP2 over time compared to controls whereas the epigenetic regulator H2AFY2 was significantly decreased. Stimulated by our observation that profound changes in global gene expression also included the epigenetic machinery (H2afy2), we speculated whether Plcγ1 signalling also modifies the global epigenetic landscape of I/11 pro-erythroblasts. Therefore, we performed genome-wide DNA methylome analysis in I/11 cells upon Plcγ1 knockdown using MCIP-seq (methyl-CpG immunoprecipitation combined with next-generation sequencing). The observed methylation changes were by far dominated by an apparent hypomethylation of differentially methylated regions (DMRs) in Plcγ1 knockdown cells as compared to control cells. In line with this, gene ontology analysis of DMRs revealed a highly significant enrichment of biological terms associated with developmental processes and cell differentiation.Taken together, our findings provide evidence for an essential role of Plcγ1 in regulating erythroid differentiation through alteration of the transcriptomic and epigenetic landscape. Disclosures:No relevant conflicts of interest to declare.