Red Blood Cell Production Research Articles

In vitro studies of erythropoiesis. II. The effect of anoxia on heme synthesis.

SINCE the original hypothesis in 1893 by Miescher, 1 it has been generally accepted that the stimulus for the production of red blood cells by the bone marrow is anoxia. That anoxic anoxia stimulates erythropoiesis is well known from the observations of polycythemia and marrow hyperplasia at high altitudes and in various pathologic conditions producing decreased arterial oxygen tensions. Polycythemia produced by anemic anoxia and by cobalt have been explained by assuming local bone marrow anoxia and interference with bone marrow respiratory enzymes, respectively. However, direct studies of bone marrow oxygen tension have failed to show marrow anoxia after slow bleeding sufficient to produce an excellent erythropoietic response 2 or in chronic anemia. 3

A Scalable Suspension Platform for Generating High-Density Cultures of Universal Red Blood Cells from Human Induced Pluripotent Stem Cells.

SummaryUniversal red blood cells (RBCs) differentiated from O-negative human induced pluripotent stem cells (hiPSCs) could find applications in transfusion medicine. Given that each transfusion unit of blood requires 2 trillion RBCs, efficient bioprocesses need to be developed for large-scale in vitro generation of RBCs.We have developed a scalable suspension agitation culture platform for differentiating hiPSC-microcarrier aggregates into functional RBCs and have demonstrated scalability of the process starting with 6 well plates and finally demonstrating in 500 mL spinner flasks. Differentiation of the best-performing hiPSCs generated 0.85 billion erythroblasts in 50 mL cultures with cell densities approaching 1.7 × 107 cells/mL. Functional (oxygen binding, hemoglobin characterization, membrane integrity, and fluctuations) and transcriptomics evaluations showed minimal differences between hiPSC-derived and adult-derived RBCs.The scalable agitation suspension culture differentiation process we describe here could find applications in future large-scale production of RBCs in controlled bioreactors.

Vitamin A supplements for preventing illness in children aged under 5: Mathematical and systematic approach at flooding disaster in Samarinda

Background: Heavy rains poured for several hours on sunday morning, causing the water level to rise at Benanga dam at the upstream of Karang Mumus river, the confluence of the Mahakam river. The Samarinda administration has applied an emergency protocol for fourteen days to assist 10,300 people affected by the flooding. Health City Office decided to distribute vitamin A to children under 5, as prevention strategy to avoid illness. Vitamin A refers to a subclass of retinoic acids long understood to help regulate immune function and to reduce morbidity of infectious diseases. Vitamin A is required for normal functioning of the visual system, maintenance of cell function for growth, epithelial integrity, production of red blood cells, immunity, and reproduction. Methods and materials: We evaluated the effect of prophylactic synthetic oral vitamin A supplementation compared with no treatment. We planned to conduct three subgroup analyses:1.Location: by flood affected area and buffer zone2.Age: 6–12 months v 1–5 years3.Sex: boys v girls. 1.684 children under 5 in the flood affected area, were given vitamin A, while children under 5 in the buffer area maintained with no treatment. The health status of these children were monitored for 3 months and reported directly to Health City Office Surveillance Department. Results: The morbidity of children under 5, in the buffer area were higher than in the flood affected area by 27%. 81% of cases of children under 5, in the buffer area within 3 months were Upper Respiratory Tract Infection, 12% were unspecified diarrhea. Only 27 children of 1.684 Children given Vitamin A were illness. No side affect or hypervitaminosis of vitamin A reported. Conclusion: Vitamin A supplementation is associated with reductions in illness problems in a range of settings compared to no treatment area in the buffer zone. Health Surveillance is maintained for approximately 3 months. However, there is a need for further studies comparing different doses and delivery mechanisms (for example, fortification). Until other sources are available, vitamin A supplements should be given to all children at risk of disaster, particularly where health access is limited.

Erythropoietin treatment and the risk of hip fractures in hemodialysis patients.

ABSTRACTErythropoietin (EPO) is the primary regulator of bone marrow erythropoiesis. Mouse models have provided evidence that EPO also promotes bone remodeling and that EPO‐stimulated erythropoiesis is accompanied by bone loss independent of increased red blood cell production. EPO has been used clinically for three decades to treat anemia in end‐stage renal disease, and notably, although the incidence of hip fractures decreased in the United States generally after 1990, it rose among hemodialysis patients coincident with the introduction and subsequent dose escalation of EPO treatment. Given this clinical paradox and findings from studies in mice that elevated EPO affects bone health, we examined EPO treatment as a risk factor for fractures in hemodialysis patients. Relationships between EPO treatment and hip fractures were analyzed using United States Renal Data System (USRDS) datasets from 1997 to 2013 and Consolidated Renal Operations in a Web‐enabled Network (CROWNWeb) datasets for 2013. Fracture risks for patients treated with <50 units of EPO/kg/week were compared to those receiving higher doses by multivariable Cox regression. Hip fracture rates for 747,832 patients in USRDS datasets (1997–2013) increased from 12.0 per 1000 patient years in 1997 to 18.9 in 2004, then decreased to 13.1 by 2013. Concomitantly, average EPO doses increased from 11,900 units/week in 1997 to 18,300 in 2004, then decreased to 8,800 by 2013. During this time, adjusted hazard ratios for hip fractures with EPO doses of 50–149, 150–299, and ≥ 300 units/kg/week compared to <50 units/kg/week were 1.08 (95% confidence interval [CI], 1.01–1.15), 1.22 (95% CI, 1.14–1.31), and 1.41 (95% CI, 1.31–1.52), respectively. Multivariable analyses of 128,941 patients in CROWNWeb datasets (2013) replicated these findings. This study implicates EPO treatment as an independent risk factor for hip fractures in hemodialysis patients and supports the conclusion that EPO treatment may have contributed to changing trends in fracture incidence for these patients during recent decades. Published 2021. This article is a U.S. Government work and is in the public domain in the USA. Journal of Bone and Mineral Research published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research (ASBMR).

A Review of Clinical Guidelines on the Management of Iron Deficiency and Iron-Deficiency Anemia in Women with Heavy Menstrual Bleeding.

IntroductionUp to one-third of women of reproductive age experience heavy menstrual bleeding (HMB). HMB can give rise to iron deficiency (ID) and, in severe cases, iron-deficiency anemia (IDA).AimTo review current guidelines for the management of HMB, with regards to screening for anemia, measuring iron levels, and treating ID/IDA with iron replacement therapy and non-iron-based treatments.MethodsThe literature was searched for English-language guidelines relating to HMB published between 2010 and 2020, using the PubMed database, web searching, and retrieval of clinical guidelines from professional societies.ResultsOverall, 55 guidelines mostly originating from North America and Europe were identified and screened. Twenty-two were included in this review, with the majority (16/22) focusing on guidance to screen women with HMB for anemia. The guidance varied with respect to identifying symptoms, the criteria for testing, and diagnostic hemoglobin levels for ID/IDA. There was inconsistency concerning screening for ID, with 11/22 guidelines providing no recommendations for measurement of iron levels and four contrasting guidelines explicitly advising against initial assessment of iron levels. In terms of treatment, 8/22 guidelines provided guidance on iron therapy, with oral iron administration generally recommended as first-line treatment for ID and/or IDA. Four guidelines recommended intravenous iron administration for severe anemia, in non-responders, or before surgery. Three guidelines provided hemoglobin thresholds for choosing between oral or intravenous iron treatment. Four guidelines discussed the use of transfusion for severe IDA.ConclusionMany of the guidelines for managing HMB recognize the importance of treating anemia, but there is a lack of consensus in relation to screening for ID and use of iron therapy. Consequently, ID/IDA associated with HMB is likely to be underdiagnosed and undertreated. A consensus guidance, covering all aspects of screening and management of ID/IDA in women with HMB, is needed to optimize health outcomes in these patients.

Effects of Erythropoietin in White Adipose Tissue and Bone Microenvironment.

Erythropoietin (EPO) is expressed primarily in fetal liver and adult kidney to stimulate red blood cell production. Erythropoietin receptor expression is not restricted to erythroid progenitor cells, and non-erythroid EPO activity includes immune response and bone remodeling. In bone fracture models, EPO administration promotes bone formation and accelerates bone healing. In contrast, in healthy adult mice, exogenous EPO-stimulated erythropoiesis has been concomitant with bone loss, particularly at high EPO, that may be accompanied by increased osteoclast activation. Other EPO-associated responses include reduced inflammation and loss of fat mass with high-fat diet feeding, especially in male mice. While EPO exhibited a sex-dimorphic response in regulation of fat mass and inflammation in obese mice, EPO-stimulated erythropoiesis as well as EPO-associated bone loss was comparable in males and females. EPO administration in young mice and in obese mice resulted in bone loss without increasing osteoclasts, suggesting an osteoclast-independent mechanism, while loss of endogenous EPO decreased bone development and maintenance. Ossicle formation of bone marrow stromal cell transplants showed that EPO directly regulates the balance between osteogenesis and adipogenesis. Therefore, during development, endogenous EPO contributes to normal bone development and in maintaining the balance between osteogenesis and adipogenesis in bone marrow stromal cells, while EPO treatment in mice increased erythropoiesis, promoted bone loss, decreased bone marrow adipogenesis, and increased osteoclast activity. These observations in mouse models suggest that the most prevalent use of EPO to treat anemia associated with chronic kidney disease may compromise bone health and increase fracture risk, especially at a high dose.

Genome-Wide Epigenetic Signatures of Adaptive Developmental Plasticity in the Andes.

High-altitude adaptation is a classic example of natural selection operating on the human genome. Physiological and genetic adaptations have been documented in populations with a history of living at high altitude. However, the role of epigenetic gene regulation, including DNA methylation, in high-altitude adaptation is not well understood. We performed an epigenome-wide DNA methylation association study based on whole blood from 113 Peruvian Quechua with differential lifetime exposures to high altitude (>2,500) and recruited based on a migrant study design. We identified two significant differentially methylated positions (DMPs) and 62 differentially methylated regions (DMRs) associated with high-altitude developmental and lifelong exposure statuses. DMPs and DMRs were found in genes associated with hypoxia-inducible factor pathway, red blood cell production, blood pressure, and others. DMPs and DMRs associated with fractional exhaled nitric oxide also were identified. We found a significant association between EPAS1 methylation and EPAS1 SNP genotypes, suggesting that local genetic variation influences patterns of methylation. Our findings demonstrate that DNA methylation is associated with early developmental and lifelong high-altitude exposures among Peruvian Quechua as well as altitude-adaptive phenotypes. Together these findings suggest that epigenetic mechanisms might be involved in adaptive developmental plasticity to high altitude. Moreover, we show that local genetic variation is associated with DNA methylation levels, suggesting that methylation associated SNPs could be a potential avenue for research on genetic adaptation to hypoxia in Andeans.

Article of Significant Interest in This Issue

Erythropoietin (EPO) is a plasma glycoprotein that stimulates red blood cell production. Though the transcriptional regulation of EPO has been well studied, the regulation of intracellular EPO trafficking remains poorly understood. Lin et al. (e00180-20) developed a genome-wide CRISPR knockout screen that identified SURF4 as the endoplasmic reticulum cargo receptor that promotes EPO secretion. SURF4 deletion results in intracellular accumulation and extracellular depletion of EPO, while SURF4 overexpression leads to enhanced EPO secretion. Collectively these studies point to a “cargo capture” model of EPO secretion. This work suggests that modulating the expression/function of SURF4 might treat certain erythroid disorders and also highlights a potential strategy for enhanced production of recombinant EPO.

Real-World Treatments and Thrombotic Events in Polycythemia Vera Patients: A Retrospective Analysis between 2018-2019 in US Population

BACKGROUND Polycythemia Vera (PV) is a rare myeloproliferative neoplasm associated with an increased production of red blood cells, white blood cells, and platelets. Most frequent treatment includes phlebotomy, hydroxyurea, interferon, and ruxolitinib. Current NCCN guideline recommends managing HCT levels to below 45%. The objective of this study was to determine real-world standards of care and patient characteristics, and to observe how treatment decisions vary by HCT level and thrombosis risk. METHODOLOGY We conducted a retrospective study using Symphony Health's longitudinal transactional healthcare claims database that includes prescription, medical and hospital claims across &amp;gt; 4,900 US payers representing 86% of US lives. Eligible patients had at least one ICD-10 diagnosis code for PV and at least one of the treatments including phlebotomy, hydroxyurea, busulfan, interferon, and ruxolitinib between Jan 1, 2018 and Dec 31, 2019 (index period). For eligible patients, all prior treatment history initiated as far back as January 2010 was used to report therapy changes. Patients were also required to have at least one PV diagnosis within a year of treatment initiation and at least 2 HCT lab results during the index period. PV treatment changes and characteristics were studied. RESULTS Out of 28,306 patients with PV, 4,264 patients had HCT lab data for 2 years (index period). Median duration of follow-up was 854 days (range 98-3,373days). Patient therapy duration was from 1 to 9 years. Median patient age was 65 (range 11-94), with 1,451 (34%) patients aged less than 60, 2,813 (66%) 60 years or older, and a substantial male predominance (62% vs 38%). 1,247 (29%) patients were classified as Low Risk (age&amp;lt; 60 with no TE history) and 3,017 (71%) patients as High Risk. Within the High-Risk group, 2,224 (52%) were age&amp;gt;60 without prior TE, 204 (5%) were age&amp;lt;60 with prior TE and 589 (14%) were age&amp;gt;60 with prior TE. For Low Risk patients' initial treatment was phlebotomy alone (85%) and a total of 73% of all Low Risk patients remained on phlebotomy alone. For High Risk patients' initial treatment was phlebotomy alone (60%) and 43% all of High-Risk patients remained on phlebotomy alone (Figure 1). The median HCT prior to treatment initiation was 52.9% and 48% during treatment. 936 (22%) patients achieved NCCN treatment guidelines with HCT levels always remaining under 45%, and 1,226 (29%) patients had HCT levels controlled between 45% and 50%. However, 2,102 (49%) patients had some or all HCT levels&amp;gt; 50% (Figure 2). With the most recent lab test, 2,180 (51%) of patients still had HCTs above 45% and 804 (19%) were still above 50%. In a sub-cohort of 653 High Risk patients with a prior TE and up to 5 years of follow up, 236 (36%) had at least one other TE; for the 1,774 High Risk patients who did not have the history of thrombosis, 161(9%) had at least one TE (Table 2). The most common TE since treatment began in patients with prior TE were deep vein thrombosis (n= 92 patients, 14%) and stroke (n= 95 patients, 15%). Among High Risk patients (n=397) who had another thrombotic event, 180 (45%) were treated by phlebotomy only and never switched to any other therapies. CONCLUSIONS Despite currently available treatments in US, patients' HCT level after treatment were higher than recommended as per guidelines. Failure to maintain HCT less than 45% increases the risk of future thrombotic events as shown by 36% of patients with prior TE experiencing another TE within the next 5 years. Disclosures Verstovsek: Sierra Oncology: Consultancy, Research Funding; ItalPharma: Research Funding; Blueprint Medicines Corp: Research Funding; NS Pharma: Research Funding; Promedior: Research Funding; Incyte Corporation: Consultancy, Research Funding; Protagonist Therapeutics: Research Funding; Novartis: Consultancy, Research Funding; Roche: Research Funding; AstraZeneca: Research Funding; PharmaEssentia: Research Funding; Genentech: Research Funding; Celgene: Consultancy, Research Funding; Gilead: Research Funding; CTI Biopharma Corp: Research Funding. Han:Protagonist Therapeutics: Consultancy. Chun Hayes:Protagonist: Consultancy. Woody:Protagonist: Current Employment. Valone:Protagonist: Current Employment. Gupta:Protagonist: Current Employment.

Momelotinib's Spleen, Symptom and Anemia Efficacy Is Maintained in Intermediate/High Risk Myelofibrosis Patients with Thrombocytopenia

Momelotinib's Spleen, Symptom and Anemia Efficacy Is Maintained in Intermediate/High Risk Myelofibrosis Patients with Thrombocytopenia

Ker-050, a Novel Inhibitor of Tgfβ Superfamily Signaling, Induces Red Blood Cell Production By Promoting Multiple Stages of Erythroid Differentiation

Diseases such as myelodysplastic syndrome (MDS) and myelofibrosis (MF) are characterized by ineffective hematopoiesis resulting in one or multiple cytopenias. Disease-causing defects occur across multiple cell lineages and stages of hematopoiesis, making development of an effective treatment for all patients challenging. Current treatment options to address anemia in these diseases target discreet stages in erythropoiesis, whereas defects leading to ineffective hematopoiesis can occur throughout the pathway. Therefore, a treatment that more globally modulates hematopoiesis has the potential to treat broad patient groups. The TGFβ superfamily plays a key role in regulating hematopoiesis; as signaling via SMAD2/3 activation results in cell quiescence, inhibiting precursors from progressing through later stages of hematopoiesis. KER-050, a modified ActRIIA ligand trap, promotes hematopoiesis through inhibition of ligands that signal though SMAD2/3, including activins and GDFs. In a Phase 1 clinical study, administration of KER-050 to healthy volunteers led to robust, rapid and sustained increases in red blood cells (RBCs), hemoglobin (HGB) and platelets, supporting an effect on the multiple stages of hematopoiesis. Here, we characterize the time course of KER-050-mediated effects on RBC production and changes in erythroid precursor cell populations in mice to characterize the mechanism of action of KER-050 on erythropoiesis. Mice treated with a single dose of a research form of KER-050 (RKER-050, 10mg/kg) had increased RBCs, HGB and hematocrit (HCT) (+8%, +9%, +7%, respectively) 12 hours after administration compared to vehicle-treated mice (VEH), and this effect was further increased on Day 7. There was also a reduction in the number of enucleated erythroid cells in the bone marrow and a parallel increase in the percent of immature reticulocytes (RET) in peripheral blood, suggesting an increased outflux of RET into circulation. This observation is consistent with RKER-050 promoting the maturation of late stage erythroid precursors leading to increases in RBCs, HGB and HCT as early as 12 hours post treatment and remaining 14 days post a single dose. In studies evaluating the effect of RKER-050 on bone marrow erythroid progenitors, a 2-fold increase in late orthochromatic erythroblasts/RETs (EryC) at Days 2 and 7 post-dose was observed. These data are consistent with RKER-050 promoting maturation and release of late-stage erythroid precursors into circulation. RKER-050 also elicited effects on early progenitors. Day 2 post-dose, there was a 2-fold increase in CFU-Es, with a 46% decrease in poly-erythrochromatic/early orthochromatic erythroid precursors (EryB) at Day 4, as compared to VEH. Day 7 post treatment, both CFU-Es and the EryB population returned to VEH levels. The increase in early progenitors appears to replenish the polychromatic erythroblasts (as shown by the return to VEH level of EryB precursors at Day 7), allowing for continued supply of maturing RETs. Consistent with this hypothesis, RKER-050-mediated changes in erythroid precursors continued to Day 14 with significantly increased early progenitor population (BFU-E +24%) and increased late stage erythroid precursors (EryB +40%, EryC 7-fold) while maintaining increased circulating RETs and RBCs. These data demonstrate that the RKER-050 treatment increases early progenitor cells which continue to mature and contribute to the overall upregulation of erythropoietic tone. Surprisingly, RKER-050 treatment resulted in a greater than 2-fold increase in serum levels of erythropoietin (Epo) at Days 4, 7 and 14. These counterintuitive results demonstrate that RKER-050 promotes erythropoiesis while at the same time increasing Epo. This effect may result in a feed-forward effect on the system and result in a sustained upregulation of erythropoietic tone. Overall, these data demonstrate that KER-050 stimulates terminal maturation of late-stage erythroid precursors, expands the early stage precursor population and progresses precursors through erythropoiesis. Additionally, KER-050 increases Epo within the milieu of elevated RBCs. The ability of KER-050 to target multiple stages along the erythropoiesis cascade makes it an appealing therapeutic candidate for diseases that cause anemia due to ineffective erythropoiesis, including MDS and MF, where defects can arise throughout the erythropoietic pathway. Disclosures Feigenson: Keros Therapeutics: Current Employment. Nathan:Keros Therapeutics: Current Employment. Materna:Keros Therapeutics: Current Employment. Gudelsky:Keros Therapeutics: Current Employment. Lema:Keros Therapeutics: Current Employment, Current equity holder in publicly-traded company. Tseng:Mitobridge: Current equity holder in private company; Keros Therapeutics: Current Employment, Current equity holder in publicly-traded company, Patents &amp; Royalties. Fisher:Keros Therapeutics: Current Employment, Current equity holder in publicly-traded company. Seehra:Keros Therapeutics: Current Employment, Current equity holder in publicly-traded company. Lachey:Keros Therapeutics: Current Employment, Current equity holder in publicly-traded company.

Elevated P21 (CDKN1a) Mediates Apoptosis of Beta-Thalassemic Erythroid Cells in Mice but Its Ablation Doesn't Improve Erythroid Maturation

Beta-thalassemias are caused by mutations in the β-globin gene leading to anemia. In β-thalassemia, excessive accumulation of unpaired α globin chains in erythroblasts, triggers redox-mediated reactions, which is associated with increased production of immature erythroid precursors that fail to mature. This impaired maturation is in part due to increased apoptosis of late maturing erythroblasts in β-thalassemic patients that aggravates anemia despite enhanced erythropoiesis leading to what is called ineffective erythropoiesis and ultimately resulting in extramedullary expansion of hematopoiesis. The mechanism of apoptosis in beta-thalassemia remains poorly understood. To investigate this, we examined the status of mediators of stress response during erythroid cell maturation ofHbbth3/+ (th3/+) mice, a model that mimics the beta-thalassemia intermedia phenotype in humans. We found that both Foxo3 and p53 were prematurely activated in th3/+ beta-thalassemic erythroblasts as compared to wild type controls. We crossed Hbbth3/+ (th3/+) and Foxo3-/- mice and found that red blood cell (RBC) count and hemoglobin content were improved (by 1g/L, n=10), and erythroblast apoptosis was decreased to similar levels as in the WT during erythroblast maturation of double mutant mice. However, loss of Foxo3 did not ameliorate the splenomegaly of th3/+mice. We also found that p53 direct target, p21 the cyclin-dependent kinase inhibitor was greatly upregulated in th3/+erythroblasts as well as in beta-thalassemic patients' erythroblasts. To address the contribution of p21, we crossed p21-/- and Th3/+. It showed a significant decrease of apoptosis in CD45- TER119+ erythroblasts both in the bone marrow and spleen of double mutant mice (30% and 23% reduction respectively, n=6 mice each genotype). Although, as in beta-thalassemic patients, serum erythropoietin (Epo) was elevated in the peripheral blood of th3/+mice, the double mutant mice had significantly lower level of Epo than th3/+ (45% reduction, n=3 mice per genotype).In p21-/-th3/+, CD45- TER119+ cells also showed lesser ROS accumulation(12% less, n=3 per genotypes). However, to our surprise, the deletion of p21 on beta-thalassemic background did not have any effect on splenomegaly (n=6 mice each genotype), complete blood count, hemoglobin, RBC production or bone marrow erythroid cell maturation (n=12 mice each genotype). To further examine the underlying mechanism, we analyzed cell cycle in double mutant p21-/-th3/+ erythroblast at distinct stages of maturation identified by CD45, TER119, CD44 and cell size (n=3 mice per genotype) using ki67 staining at distinct stages of maturation. We found p21-/-th3/+erythroblasts proliferate much less than their th3/+ counterparts (basophilic erythroblasts G2 14% less, polychromatic erythroblasts 20% less, p&amp;lt;0.05 n=3 mice per genotype). This may partially explain lack of improvement of RBC production and anemia despite enhanced erythroblast survival. ROS levels were also reduced in double mutant p21-/-th3/+ erythroblasts as compared to controls. Next we investigated the status of p53 and Foxo3 in double mutant p21-/-th3/+ erythroblasts as compared to controls. We confirmed as we had observed earlier that nuclear p53 and Foxo3 expression were greater in th3/+ primitive erythroid (TER119-/low, c-KIT+, CD71Hi) cells than in wild type (n=3 mice per genotype) controls. Strikingly, the double mutant p21-/-th3/+ erythroblasts exhibited the greatest nuclear Foxo3 in all four groups, while nuclear p53 was dramatically reduced by over 80% (n=2 mice each genotype. Each mouse taking &amp;gt;=30 cells to calculate nuclear MFI) even as compared to wild type. These combined studies suggest that ameliorating apoptosis may not improve anemia in beta-thalassemia. Disclosures Liang: Hemogenyx Pharmaceuticals LLC: Current Employment.

The Samd14-Capping Protein Complex Controls Cell Signaling in the Erythropoietic Stress Response

In anemia, restoring homeostatic levels of erythrocytes requires an erythropoietic regenerative response to accelerate red blood cell (RBC) production. Elucidating mechanisms that drive the process of erythropoiesis in the context of regeneration or "stress erythropoiesis" can reveal new strategies for targeting ineffective erythropoiesis. An important component of stress erythropoiesis involves stress-dependent activation of genes/proteins through transcriptional enhancers. We discovered an enhancer in intron 1 of the Sterile Alpha Motif (SAM) Domain 14 (Samd14) gene elevates Samd14 expression, facilitates SCF/c-Kit signaling, and is needed for survival in a hemolytic anemia model. However, it is dispensable for erythropoietic development. Our prior work demonstrated that the SAM domain of Samd14 promotes c-Kit-mediated cellular signaling to regulate progenitor function, and this SAM domain has functional attributes unique from those of structurally related SAM domains. Using immunoprecipitation-mass spectrometry, we determined that Samd14 interacts with the Capzβ protein. Capzβ is a component of the actin capping protein (CP) complex, which interact as α- and β heterodimers during actin filament assembly/disassembly. CP exerts diverse functions in cell motility, vesicular transport, cell signaling and cytokinesis. Using a series of Samd14 deletion constructs, we tested whether the Samd14-Capzβ interaction is important for Samd14 promotion of c-Kit signaling in stress erythroid progenitors. Our findings determined that the region of Samd14 required for binding to Capzβ (amino acids 38-54) were required to restore c-Kit signaling. Our ongoing studies are examining whether Samd14-Capzβ is similarly required for colony formation and cell survival of stress erythroid progenitors, and whether additional SAM domain-containing proteins have a similar role in regulating stress erythropoiesis. Understanding the fundamental drivers of regenerative erythropoiesis can lead to new therapeutic strategies and prognostic/diagnostic markers. Disclosures No relevant conflicts of interest to declare.

Safety and Efficacy of Idasanutlin in Patients (pts) with Hydroxyurea (HU)-Resistant/Intolerant Polycythemia Vera (PV): Results of an International Phase II Study

Safety and Efficacy of Idasanutlin in Patients (pts) with Hydroxyurea (HU)-Resistant/Intolerant Polycythemia Vera (PV): Results of an International Phase II Study

Metformin Upregulates Mir-26a to Improve Erythropoiesis in Preclinical Models of Diamond Blackfan Anemia through Suppression of Nlk Expression

Nemo-like Kinase (NLK) is an atypical member of the Mitogen-activated protein (MAP) kinase family and shares a highly conserved kinase domain with other MAP kinases. For this reason it is challenging to design small molecules that specifically inhibit NLK. In hematopoiesis, NLK expression is suppressed in all lineages except erythroid progenitors. In non-erythroid progenitors, micro-ribonucleic acid (miR)-181 is upregulated and binds to the 3' untranslated region (UTR) of NLK transcripts, resulting in dramatic degradation of NLK transcripts. NLK is not activated or required for healthy erythropoiesis, but in Diamond Blackfan Anemia (DBA), NLK is chronically hyper-activated, which contributes to failed erythroid expansion and disease pathogenesis. Given the sensitivity of NLK transcripts to 3'UTR binding-mediated degradation, we hypothesized that targeting NLK degradation by upregulating miRNAs that bind the NLK 3'-UTR could provide an alternative approach to directly inhibiting the kinase to suppress aberrant NLK activity in bone marrow progenitors from DBA patients. One miRNA binding site that is present in the NLK-3'UTR is miR-26a. We determined that miR-26a is upregulated 2.4-fold (p=0.009) in CD235+ erythrocytes by the Type 2 diabetes drug metformin. Metformin suppressed NLK expression 62.4% (p=0.006) and improved erythroid expansion 4-fold (p=0.001) in human and 6-fold (p=0.042) in zebrafish models of DBA. This effect required an intact miR-26a binding site within the NLK 3'UTR. However, metformin failed to induce miR-26a in murine models of DBA, however the exogenous expression of miR-26a suppressed NLK 36.4% (p=0.028) and improved erythropoiesis 2-fold (p=0.017). In summary, targeted suppression of NLK through metformin-mediated upregulation of NLK transcript-binding miRNAs effectively improved red blood cell production from 5.6% up to 33.3% of controls in a preclinical model of DBA. These results suggest that metformin is a potential drug for treatment of DBA. Disclosures Glader: Agios Pharmaceuticals, Inc.: Consultancy. OffLabel Disclosure: metformin. This drug is given to differentiating HSPCs in vitro to examine effect on specific kinases.

A Diamond-Blackfan Anemia Patient's Response to Eltrombopag and Genomic Analysis in Different Lineages

Diamond-Blackfan Anemia (DBA) is a rare, inherited bone marrow (BM) failure syndrome characterized by pure red cell aplasia generally presenting within the first year of life. The majority of cases are due to heterozygous mutations in ribosomal genes, either autosomal dominant inherited or sporadicde novoevents. Despite the recognition of the importance of ribosomal biology, the underlying mechanisms are still poorly understood. The only curative treatment is hematopoietic stem cell transplantation, otherwise steroids or transfusions will cause significant long-term morbidity. There is an unmet need for new therapies. We describe the unique clinical course and molecular analyses of a 32-year-old Hispanic woman, provide insights into disease pathophysiology as well as hope for an effective new treatment. She was diagnosed with DBA at the age of one month with isolated severe normochromic anemia. She failed various therapies, including corticosteroids, danazol, growth factors, and additional immunosuppression and developed significant iron overload despite desferal chelation. She was enrolled in a phase II clinical trial investigating the safety and efficiency of the thrombopoietin receptor agonist eltrombopag (EPAG) in patients with moderate aplastic anemia or unilineage cytopenias (NCT01328587). EPAG was administered in an escalating manner reaching 300mg daily. She achieved transfusion-independence by 16 weeks. This dose was continued for an additional 6 months and discontinued for protocol-defined robust response, ie Hb &amp;gt;10g/dL. After discontinuation, her hemoglobin steadily declined over a period of 8 months. EPAG was reinitiated, her Hb again rapidly improved and the EPAG dose was slowly tapered. Seven years since enrollment, the patient has remained on drug, currently at a dose of 25mg daily with normal blood cell counts and no EPAG related toxicity. BM cytogenetics and morphology have remained normal throughout follow up, as has a myeloid somatic mutation panel. Next generation sequencing has revealed a novel, pathogenic mutation in intron 4(c.356+3&amp;gt;C) of theRPS19gene affecting a splice donor site previously implicated in DBA. Testing of both parents was negative, documenting this to be ade novomutation. With digital droplet PCR (ddPCR), we have monitored the variant allele frequency (VAF) in fibroblasts (germline) and different hematopoietic populations (Figure 1). Fibroblasts showed a VAF 50% (heterozygous), while her whole BM and whole blood show a lower VAF between 25% and 37%, both before and on EPAG treatment. Across lineages were analyzed when she was on EPAG, lymphocytes showed the highest VAF (42% to 45%), followed by HSPCs and granulocytes at 24% to 32%. Of note, the erythrocytes progenitors (CD45-CD71+) had the lowest VAF of 8% to 14%. Sequencing of myeloid and erythroid colony forming units (CFU) with both pre and on EPAG BM samples confirmed only heterozygous mutations or wild-type (WT) CFUs in percentages comparable to their respective VAF detected by bulk ddPCR. These results suggest the emergence of WT clones within the BM either through mosaicism or a reversion event. The discrepancy in VAF between peripheral and BM samples is consistent with a proliferative advantage for WT clones, especially in the erythroid and myeloid lineages. Yet despite the presence of WT HSPCs, profound transfusion-dependent anemia persisted in this patient long-term. An imbalance between globin chain and heme production has been documented to result from ribosomal dysfunction in DBA, leading to increased iron-mediated cellular stress and death of erythroid progenitors. We hypothesize the residual mutant cells suppress the growth of WT cells, given the close proximity of large numbers of erythroid cells aggregated by macrophages in erythroid islands, potentially via passage of free iron. These observations have implications for the development of gene addition or gene correction therapies for DBA, suggesting that correcting a fraction of HSPCs will not revert the phenotype. Given the potent intracellular chelation activity of EPAG, and the lack of the EPAG target MPL receptor on erythroid progenitors, we hypothesize that iron chelation is a potential mechanism for improvement of red blood cell production by EPAG in DBA. The results in our patient have encouraged the opening of a prospective clinical trial (NCT04269889) to further explore the potential of EPAG for DBA patients. Figure Disclosures Young: Pharos I&amp;BT Co., Ltd.:Research Funding.Pakbaz:Amgen:Membership on an entity's Board of Directors or advisory committees;Dova:Membership on an entity's Board of Directors or advisory committees;Novartis:Membership on an entity's Board of Directors or advisory committees, Speakers Bureau;Terumo BCT:Membership on an entity's Board of Directors or advisory committees, Speakers Bureau;Global Blood Therapeutics:Speakers Bureau.Dunbar:Novartis:Research Funding.

Selective Inhibition of ALK2 Signaling Suppresses Serum Hepcidin and Increases Serum Iron

Hepcidin is an endocrine regulator of iron metabolism that, when elevated, can decrease levels of iron available for erythropoiesis and, as a result, decrease red blood cell production. Signaling though activin-like kinase-2 (ALK2), a TGFβ type 1 receptor, has been implicated in regulation of hepcidin-mediated iron regulation and mobilization; however, to date, ALK2's specific degree of involvement has not been convincingly elucidated, mainly due to the redundant effects of the type 1 receptors ALK3 and ALK5. Activation of type 1 receptors including ALK2, ALK3, and ALK5 via ligand BMPs and co-receptor hemojuvelin (m-HJV), results in downstream SMAD phosphorylation, increased hepcidin, and decreased serum iron while suppression of the receptor signaling would have the opposite effects. In order to assess the specific effect of ALK2 inhibition on hepcidin and iron mobilization, we utilized multiple modalities of inhibition and tested inhibitors in both naive and diseased animals. KTI-2338, a small molecule ALK2 kinase inhibitor, has been characterized to inhibit ALK2 signaling potently and selectively in in-vitro assays. To further assess the specific contribution of ALK2 to hepcidin expression and iron mobilization, we evaluated the effect of the novel neutralizing antibody KTI-A2.0MAb. This fully human antibody is targeted against the extracellular domain of ALK2 with no affinity for the other type 1 receptors and provides a unique tool for understanding ALK2 involvement in this system. In wild-type animals, targeting ALK2 signaling with either a small molecule or biologic therapeutic leads to decreased serum hepcidin and increased serum iron. To assess the efficacy of ALK2 inhibition in a disease state, we utilized an siRNA-based model of Iron Refractory Iron Deficiency Anemia (IRIDA). In IRIDA, patients exhibit a loss of functional TMPRSS6, a gene that encodes the transmembrane type II serine protease Matriptase-2 (MT-2). MT-2 suppresses hepcidin secretion by cleaving m-HJV, interrupting ALK2 signaling and downstream SMAD activation. Failure to cleave m-HJV allows continued activation of BMPRs, increased hepcidin, and decreased serum iron. Phenocopying what is observed in IRIDA patients, intravenous dosing of TMPRSS6 targeted siRNA results in suppressed TMPRSS6 expression and functional MT-2, increases in serum hepcidin, and decreases in serum iron. Therapeutic dosing of either a small molecule or biologic ALK2 inhibitor in the siRNA based IRIDA model resulted in rescue of hemoglobin, hematocrit, serum hepcidin, and serum iron in the disease state. Following treatment, hemoglobin, hematocrit, and serum iron were increased and serum hepcidin was decreased in treated groups compared to control cohorts receiving vehicle. Herein, we have evaluated multiple modalities of ALK2 inhibition in both healthy and disease states. We have characterized that inhibition of ALK2 signaling via either modality in both naïve and anemic mice contributes to a decrease in serum hepcidin and increase in serum iron levels. Though the use of a selective ALK2 targeted biologic does not completely preclude involvement of other BMP receptors such as ALK3, these data support our assertion that ALK2 signaling is an integral part of hepcidin-mediated iron mobilization, and illustrate the potential therapeutic benefit of ALK2 inhibition (with a small molecule inhibitor or a neutralizing monoclonal antibody) in anemia of high hepcidin including IRIDA and anemia of inflammation. Disclosures Backus: Keros Therapeutics: Current Employment, Current equity holder in publicly-traded company. Medeiros:Keros Therapeutics: Current Employment, Current equity holder in publicly-traded company. Lema:Keros Therapeutics: Current Employment, Current equity holder in publicly-traded company. Fisher:Keros Therapeutics: Current Employment, Current equity holder in publicly-traded company. Seehra:Keros Therapeutics: Current Employment, Current equity holder in publicly-traded company. Lachey:Keros Therapeutics: Current Employment, Current equity holder in publicly-traded company.

SATB1 Regulates GATA1 Protein Expression in Early Hematopoiesis and Is Deregulated in Diamond Blackfan Anemia

The induction of the master erythroid transcription factor, GATA1 during early erythropoiesis is critical for efficient red blood cell production. However, GATA1 is expressed at low levels in hematopoietic stem cells (HSCs) and is moderately induced at both the common myeloid progenitor (CMP) and megakaryocyte/erythroid progenitor (MEP) stages prior to lineage commitment. Diamond Blackfan Anemia is a rare disease, usually associated with ribosomal gene mutations, leading to significant decrease in GATA1 expression and block in early committed erythroid differentiation. Mild defects in other myeloid lineages are also observed, with limited clinical relevance. The importance of GATA1 downregulation in disease pathogenesis is manifested by rare patients with DBA carrying GATA1 mutations. To understand signaling pathways that contribute to the pathogenesis DBA, we perform RNA-seq with mRNA from human CD34+ fetal liver cells and found that the chromatin organizer, Special AT-rich sequence binding protein 1 (SATB1) was prematurely downregulated. Our results further demonstrated that sustained SATB1 expression is critical to maintain required levels of GATA1 protein at both the CMP and MEP stages of differentiation, but not in committed erythroid progenitors. In mice, SATB1 is modestly expressed in HSCs and upregulated during lymphopoiesis. SATB1 is downregulated during myeloid and erythroid differentiation and antagonizes myeloid and erythroid expansion. However, in human hematopoietic stem and progenitor cells (HSPCs), SATB1 is required for efficient expansion of these lineages. SATB1 maintains 78% expression in human MEPs, but is undetectable in early committed erythroid progenitors. In RPS19-insufficient human HSPCs, SATB1 was downregulated to 22% in MEPs (p=0.02). Re-expression of SATB1 corrected a significant subset of deregulated mRNAs, including GATA1 regulators. In the absence of SATB1, one such GATA1 regulator, heat shock protein 70 (HSP70), failed to be induced in ribosome-competent human MEPs, reducing GATA1 protein expression by 35.7% (p= 0.026). Concurrently, MEP expansion was inhibited by 64.5% (p=0.023), reducing erythroid and megakaryocyte expansion by 18.2% (p=0.024) and 20.4% (p=0.183) respectively. SATB1 facilitated the formation of chromatin loops linking together an enhancer element with HSP70 promoters required for HSP70 induction in early differentiation. Although GATA1 is significantly upregulated in committed erythroid progenitors, RPS19-insufficient human CD235+ erythrocytes express GATA1 28.4% of controls (p= 0.011). SATB1 re-expression increased GATA1 expression to 31.4% (p=0.089). Similarly, SATB1 re-expression increased CD235+ expansion from 13.9% to 39.5% (p=0.02) compared to controls. Our data indicate that premature SATB1 downregulation contributes to erythroid failure in DBA by reducing MEP expansion, but aberrant GATA1 expression observed in more mature erythrocytes is predominantly SATB1-independent. However, SATB1-re-expression improved CD11b+ myeloid expansion from 81.2% to 90.4% (p=0.045) and CD41a+ megakaryocyte expansion from 76.7% to 214.7% (p=0.038) respectively. Our results demonstrate that SATB1 plays an important role in human hematopoiesis and is an important regulator of GATA1. Disclosures Glader: Agios Pharmaceuticals, Inc.: Consultancy.

Administration of KER-047, a Novel ALK2 Inhibitor, Elicited Robust and Sustained Increases in Serum Iron in Healthy Participants

Background: Hepcidin is the master regulator of iron homeostasis. Its expression is controlled, in part, by signaling through TGF-β receptors including activin receptor-like kinase-2 (ALK2). As part of a negative feedback loop, high hepcidin levels increase expression of matriptase-2, which cleaves the ALK2 co-receptor hemojuvelin from the cell surface and suppresses ALK2 signaling. Dysregulation of the negative feedback system results in high hepcidin, which mobilizes iron from storage tissues and leads to insufficient iron for red blood cell production in the bone marrow, resulting in anemia. KER-047 is a selective ALK2 inhibitor that has the potential to normalize ALK2 signaling and the downstream sequalae that results in anemia as a result of elevated hepcidin. Aims: The objective of this Phase 1 study was to evaluate the safety, tolerability, pharmacokinetics, and pharmacodynamic effects of single and multiple ascending dose levels of KER-047 in healthy participants. Methods: In Part 1, two formulations of KER-047 were evaluated in single ascending oral doses ranging from 1 mg to 300 mg of a capsule formulation, and 30 mg to 450 mg of a liquid formulation or placebo. In Part 2, the liquid formulation was evaluated in multiple ascending doses (50 mg, 100 mg, 200 mg and 350 mg) of KER-047 or placebo, administered daily for up to 7 days. Endpoints included adverse events (AEs) and pharmacokinetic and pharmacodynamic parameters. Informed consent was obtained from all participants prior to enrollment in the study. Results: Part 1 enrolled 80 participants; 60 received a single dose of KER-047 and 20 received placebo. Part 2 enrolled 41 participants; 32 received KER-047 and 9 received placebo daily for up to 7 days. There were no serious adverse events in either part of the study. In Part 1, 3 (9.4%) participants discontinued the study; none discontinued due to AEs. In Part 2, 3 (9.4%) participants administered KER-047 and 1 (11.1%) placebo discontinued the study; of these, 2 participants discontinued due to an AE (one in the 200 mg group and one on placebo). In Part 2, 1 of 8 (12.5%) administered 200 mg and 4 of 8 (50%) participants administered 350 mg discontinued study drug due to AEs. The majority of AEs observed in participants administered KER-047 were mild or moderate in severity; severe AEs were reported only at the 350 mg multiple dose in 1 of 8 (12.5%) participants. AEs reported in ≥2 of participants administered KER-047 and higher than placebo were: headache, nausea, vomiting, diarrhea, gastroenteritis, chills, pyrexia, myalgia, decreased appetite, lymphopenia, neutropenia, and liver enzyme increases. Decreases in lymphocyte and neutrophil counts were observed at 200 mg and 350 mg. Mean KER-047 AUC and Cmax increased linearly, with greater than dose-proportional increases observed across multiple doses from 50-200 mg. Half-life values ranged from approximately 10 to 15 hours. Administration of KER-047 elicited rapid, robust and sustained dose-related increases in serum iron and transferrin saturation. Increases in serum iron and transferrin saturation were observed beginning on Day 2 after single doses and were sustained after multiple doses. Decreases in ferritin were also observed. Decreases in serum hepcidin post-dose were also observed in all the multiple dose cohort in which changes were evaluated. Reticulocyte hemoglobin content increased starting on Day 3 post-dosing indicating increased iron availability in the bone marrow. Summary: In healthy participants, administration of KER-047 elicited rapid, robust and sustained dose-related increases in serum iron and transferrin saturation that were associated with decreases in ferritin and hepcidin. The observed decrease in ferritin and hepcidin coupled with increases in reticulocyte hemoglobin content are indicative of increased iron mobilization, resulting in increased iron incorporation into hemoglobin. The tolerability profile of KER-047 in healthy participants has been characterized in this Phase 1 study. KER-047's unique pharmacologic effect on hepcidin and iron mobilization has the potential to treat anemia that results from elevated hepcidin. Table Disclosures Ordonez: Keros Therapeutics: Current Employment, Current equity holder in publicly-traded company; Ackea Therapeutics: Ended employment in the past 24 months. Ben:Nucleus Network: Current Employment. Barger:Keros Therapeutics: Current Employment, Current equity holder in publicly-traded company. Serino:BioBridges: Ended employment in the past 24 months; Keros Therapeutics: Current Employment. Tseng:Mitobridge: Current equity holder in private company; Keros Therapeutics: Current Employment, Current equity holder in publicly-traded company, Patents &amp; Royalties. Rovaldi:Keros Therapeutics: Consultancy. Lachey:Keros Therapeutics: Current Employment, Current equity holder in publicly-traded company. Seehra:Keros Therapeutics: Current Employment, Current equity holder in publicly-traded company.

Clasificación de las enfermedades del hematíe. Síndrome anémico y poliglobúlico. Concepto y clasificación

Anemia is a decrease in a person's normal red cell mass, making it insufficient for the optimal transportation of oxygen to tissues. Anemic syndrome is the set of signs and symptoms arising from this phenomenon and the compensatory mechanisms according to the severity and duration of the hypoxia. Polycythemia refers to an increase in the hemoglobin and/or hematocrit concentration in peripheral blood. The diagnosis of the specific cause of polycythemia is important for proper management of the patient. Anemia is the result of one or more combinations of three basic mechanisms: blood loss, a decrease in red blood cell production, or an increase in red blood cell destruction (hemolysis). According to the etiopathogenic classification, anemias are regenerative («peripheral») or non-regenerative («central»). A diagnosis of anemia is made in clinical practice with a hemoglobin figure below the reference values established by the WHO and other red blood cell parameters available on a complete blood count. A complete medical record with a comprehensive anamnesis and physical examination along with a complete blood count and laboratory tests, such as a peripheral blood and reticulocyte smear, Coombs test, and pattern of iron, among others, will allow us to establish an initial differential diagnosis of the anemia based on morphological (microcytic, normocytic, or macrocytic) and etiopathogenic (regenerative or non-regenerative) criteria.