Number Of Erythroid Progenitors Research Articles

Intracerebral Hemorrhage Leads to Anemia By Disrupting Erythropoiesis and Erythromyeloblastic Islands

Intracerebral hemorrhage (ICH), caused by the rupture of a cerebral vessel and subsequent entry of blood into the brain parenchyma, is a major form of acquired brain injury and contributes a remarkable proportion of stroke-related morbidity and mortality. Due to the few proven effective emergency and preventive treatments, patients who survived suffer varying degrees of poor prognosis that affect their quality of life. There is an urgent need to explore the cellular and molecular basis of the differences in the severity and prognosis of ICH. Anemia is a common manifestation of ICH. However, the mechanisms underlying ICH-induced anemia as we as the correlation between anemia and ICH progression/prognosis remain unclear. In this study, we found that approximately 35.1% (39 of 111) of ICH patients exhibited anemia and that decreased hemoglobin levels were positively associated with higher scores on the National Institutes of Health Stroke Scale, which quantifies the degree of neurological deficit, at admission. Furthermore, patients with decreased hemoglobin levels at admission showed higher modified Rankin scale scores at 3 months, which reflect the global disability of daily living at this time, suggesting that anemia was associated with a more severe progression and poor patient prognosis. To investigate the effects of ICH on bone marrow (BM) erythropoiesis, we conducted experiments in 12-month-old mice with collagenase-induced striatal ICH. Our results showed that ICH caused apparent anemia on day 3, associated with larger hematomas in the striatum and more severe neurologic deficits. We first evaluated the phenotype of hematopoietic progenitors and found that mice suffering ICH exhibited increased granulocyte monocyte progenitors but a significant decrease in common myeloid progenitors and megakaryocyte erythrocyte progenitors compared to control mice, suggesting a reduced source of erythroid progenitors. Further analysis revealed that the colony-forming ability of erythroid progenitors and subsequent terminal differentiation was significantly suppressed after ICH. To assess whether ICH-induced suppression of erythropoiesis is associated with erythromyeloblastic islands (EMBIs), the niche for erythropoiesis/myelopoiesis, we performed ICH using 12-month-old EpoR-tdTomato-Cre mice and found that the number of BM EMBIs and EpoR + macrophages decreased significantly. Moreover, the number and proportion of developing erythroid cells surrounding EMBIs decreased while LY6G + cells representing granulocytes increased prominently, indicating an altered microenvironment, with EMBI favoring granulopoiesis over erythropoiesis after ICH. Studies have shown that inflammation plays a crucial role in secondary brain injury after ICH. We hypothesize that inflammatory cytokines may contribute to the suppression of erythropoiesis observed in ICH mouse models. To test this, we measured inflammatory factors in both peripheral blood and BM fluid using Quantibody Mouse Inflammation Array. In particular, IL-6 concentrations in peripheral blood and BM fluid in ICH mice were significantly higher than controls. Treatment with MR16-1, an IL-6 antagonist, alleviated ICH-induced anemia and improved BM erythropoiesis in ICH mice, as evidenced by increased peripheral red blood cells and hemoglobin levels, elevated numbers of erythroid progenitors and terminal erythroblast. MR16-1 treatment also improved BM EMBI formation and increased EpoR + macrophages. In addition, MR16-1 treatment effectively improved neurologic deficits and reduced brain hematoma after ICH. Administration of recombinant IL-6 to wild-type mice resulted in decreased levels of red blood cells and hemoglobin along with suppression of erythropoiesis in the BM. Collectively, these results suggest that ICH modulates erythropoiesis and erythromyeloblastic islands function to ultimately lead to anemia and reveal a possible therapeutic opportunity to reverse ICH-induced anemia and improve ICH prognosis by targeting IL-6.

Isolation and evaluation of erythroid progenitors in the livers of larval, froglet, and adult Xenopus tropicalis.

Xenopus liver maintains erythropoietic activity from the larval to the adult stage. During metamorphosis, thyroid hormone mediates apoptosis of larval-type erythroid progenitors and proliferation of adult-type erythroid progenitors, and a globin switch occurs during this time. In addition, the whole-body mass and the liver also change; however, whether there is a change in the absolute number of erythroid progenitors is unclear. To isolate and evaluate erythroid progenitors in the Xenopus liver, we developed monoclonal ER9 antibodies against the erythropoietin receptor (EPOR) of Xenopus. ER9 recognized erythrocytes, but not white blood cells or thrombocytes. The specificity of ER9 for EPOR manifested as its inhibitory effect on the proliferation of a Xenopus EPOR-expressing cell line. Furthermore, ER9 recognition was consistent with epor gene expression. ER9 staining with Acridine orange (AO) allowed erythrocyte fractionation through fluorescence-activated cell sorting. The ER9+ and AO-red (AOr)high fractions were highly enriched in erythroid progenitors and primarily localized to the liver. The method developed using ER9 and AO was also applied to larvae and froglets with different progenitor populations from adult frogs. The liver to body weight and the number of ER9+ AOrhigh cells per unit body weight were significantly higher in adults than in larvae and froglets, and the number of ER9+ AOrhigh cells per unit liver weight was the highest in froglets. Collectively, our results show increased erythropoiesis in the froglet liver and demonstrate growth-dependent changes in erythropoiesis patterns in specific organs of Xenopus.

Administration of α-Klotho Does Not Rescue Renal Anemia in Mice.

Renal anemia is a common complication in chronic kidney disease (CKD), associated with decreased production of erythropoietin (EPO) due to loss of kidney function, and subsequent decreased red blood cell (RBC) production. However, many other factors play a critical role in the development of renal anemia, such as iron deficiency, inflammation, and elevated fibroblast growth factor 23 (FGF23) levels. We previously reported that inhibition of FGF23 signaling rescues anemia in mice with CKD. In the present study we sought to investigate whether α-Klotho deficiency present in CKD also contributes to the development of renal anemia. To address this, we administered α-Klotho to mice with CKD induced by an adenine-rich diet. Mice were sacrificed 24 h after α-Klotho injection, and blood and organs were collected immediately post-mortem. Our data show that α-Klotho administration had no beneficial effect in mice with CKD-associated anemia as it did not increase RBC numbers and hemoglobin levels, and it did not stimulate EPO secretion. Moreover, α-Klotho did not improve iron deficiency and inflammation in CKD as it had no effect on iron levels or inflammatory markers. Interestingly, Klotho supplementation significantly reduced the number of erythroid progenitors in the bone marrow and downregulated renal Epo and Hif2α mRNA in mice fed control diet resulting in reduced circulating EPO levels in these mice. In addition, Klotho significantly decreased intestinal absorption of iron in control mice leading to reduced serum iron and transferrin saturation levels. Our findings demonstrate that α-Klotho does not have a direct role in renal anemia and that FGF23 suppresses erythropoiesis in CKD via a Klotho-independent mechanism. However, in physiological conditions α-Klotho appears to have an inhibitory effect on erythropoiesis and iron regulation.

HIF-Mediated and Non-HIF-Mediated Differential Gene Expressions in Sickle Cell Reticulocyte and Their Impact on Clinical Manifestations

Reticulocytosis in sickle cell disease (SCD) is driven by tissue hypoxia from hemolytic anemia and vascular occlusion. Gene expression changes caused by hypoxia and other factors during reticulocytosis may impact SCD outcomes. We detected 1226 differentially expressed genes in SCD reticulocyte transcriptome compared to normal Black controls. To assess the role of hypoxia-mediating HIFs from other regulation of changes of the SCD reticulocyte transcriptome, we compared differential expression in SCD to that in Chuvash erythrocytosis (CE), a disorder characterized by constitutive upregulation of HIFs in normoxia. Of the SCD differentially expressed genes, 28% were shared between CE and SCD and thus classified as HIF-mediated. The HIF-mediated changes were generally in genes promoting erythroid maturation. We found that genes encoding the response to endoplasmic reticulum stress generally lacked HIF mediation.We then investigated the clinical correlation of erythroid gene expression for the 1226 differentially expressed genes detected in SCD reticulocytes, using clinical measures and gene expression data previously profiled in peripheral blood mononuclear cells (PBMCs) of 157 SCD patients at the University of Illinois at Chicago (UIC). Normal PBMCs contain only a small number of erythroid progenitors, but in SCD or CE PBMCs the erythroid transcriptome is enriched due to elevated circulating erythroid progenitors from heightened erythropoiesis (PMID: 32399971). We applied deconvolution analysis to assess the clinical correlation of erythroid gene expression, using a 16-gene expression signature of erythroid progenitors previously identified in SCD PBMCs. Deconvolution analysis uses the proportion of cell/tissue or specific marker genes (here the erythroid specific 16-gene signature) to dissect gene expression variation in biological samples with cell/tissue type heterogeneity. We correlated, in the 157 UIC patients, erythroid gene expression with i) degree of anemia as indicated by hemoglobin concentration, ii) vaso-occlusive severe pain episodes per year, and iii) degree of hemolysis measured by a hemolysis index. The analysis identified 231 genes associated with at least one of the complications. Increased expression of 40 erythroid specific genes, including 15 HIF-mediated genes, was associated with all three complications. These 40 genes are all upregulated in SCD reticulocytes and correlated with low hemoglobin concentration, frequent severe pain episodes, and high hemolysis index, suggesting that these manifestations may share a relationship to stress erythropoiesis-driven transcriptional activity.Expression quantitative trait loci (eQTL) contain genetic polymorphisms that associate with gene expression level, which can be viewed as a natural experiment to investigate the causal relations between gene expression change and phenotypic outcomes. To assess the causal effect of erythroid gene expression, we tested association between erythroid eQTL and the clinical manifestations in 906 SCD patients from the Walk-PHaSST and PUSH cohorts. We first mapped erythroid eQTL in the 157 UIC patients, who were previously genotyped by array, applying deconvolution algorithm on the same PBMC data for the 1226 differential genes in SCD reticulocytes, and detected 54 distinct eQTL for 30 genes at 5% false discovery rate. After adjusting for multiple comparisons, we found that the C allele of rs16911905, located in the β-globin cluster and associated with increased erythroid expression of HBD (encodes δ-globin of hemoglobin A 2), significantly correlated with lower hemoglobin concentration (β=-0.064, 95% CI -0.092 - -0.036, P=6.7×10 -6). The C allele was also associated with higher hemolytic rate (P=0.031), less frequent pain episodes (P=0.045), and increased erythroid expression of HBB here encoding sickle β-globin (P=5.1x10 -5). The association of the C allele with lower hemoglobin concentration was then validated in 242 patients from the UIC cohort (β=-0.071, 95% CI -0.13 - -0.011, P=0.023), as was the trend of association with higher hemolytic rate (P=0.0031) and less pain episodes (P=0.034).Our findings reveal HIF- and non-HIF-mediated genes in SCD stress erythropoiesis, and identify novel clinical associations for a HBD eQTL. Our study highlights the correlation of altered erythroid gene expression with SCD hemolytic and vaso-occlusive manifestations. DisclosuresSaraf: Global Blood Therapeutics: Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Membership on an entity's Board of Directors or advisory committees, Research Funding; Pfizer: Research Funding. Gordeuk: Modus Therapeutics: Consultancy; Novartis: Research Funding; Incyte: Research Funding; Emmaus: Consultancy, Research Funding; Global Blood Therapeutics: Consultancy, Research Funding; CSL Behring: Consultancy.

Lack of IL6 Improves Recovery from Anemia of Inflammation Which Gets Hampered in Presence of Excess Iron

Abstract Anemia of inflammation (AI) is the second most common anemia after iron deficiency anemia. The predominant regulators of AI are the cytokine interleukin 6 (IL6) and the hormone hepcidin (HAMP). IL6 is an inflammatory cytokine which also limits iron absorption by inducing HAMP, which promotes the degradation of the iron exporter ferroportin. We hypothesized that knocking down both HAMP and IL6 simultaneously will help us to understand if their mode of action in AI is uniquely limited to iron absorption and erythroid iron intake or if they also have independent roles. Henceforth, we generated IL6/HampKO (DKO) mice and, unexpectedly, observed that IL6KO mice showed the best recovery in bone marrow (BM) erythropoiesis (using flow cytometry analysis and looking at the absolute number of erythroid progenitors) after BA administration when compared to wild type (WT), HampKO and DKO mice. The best differences were observed at 14 days post BA administration. In contrast, the extramedullary erythropoiesis in the spleen was more pronounced in HampKO and DKO mice compared to WT and IL6KO animals, indicating that the mechanism impairing erythropoiesis in the BM did not affect erythroid progenitors in the spleen. These observations suggest that HAMP and IL6 proteins contribute independently to AI, with IL6 having some effect on the erythropoiesis in the BM independent from the IL6-HAMP axis leading to iron restriction. Furthermore, these observations raised the question why both HampKO and DKO mice showed reduced BM erythropoiesis compared to IL6KO animals. We investigated inflammatory cytokines and altered iron parameters as potential mediators of impaired erythropoiesis. We compared several inflammatory cytokines, including IL6, TNFa and INFg following BA administration: cytokine levels were elevated 6 hrs, reduced 48hrs after BA administration and moderately increased again two weeks later. Interestingly, among all the cytokines the levels of IL1b were significantly attenuated in IL6KO mice at day 14 compared to WT and HampKO animals. Moreover, transferrin saturation and NTBI levels were higher in HampKO and DKO animals compared to IL6KO mice. These observations strongly suggested that BM erythropoiesis is more sensitive to inflammatory insult in presence of an excess of iron, while extramedullary erythropoiesis is mildly affected and can eventually thrive under supra-physiological transferrin saturation levels. To test if increased iron affects BM erythropoiesis in presence of inflammation, we treated both WT and IL6KO mice with combination of iron dextran and BA. Both WT and IL6KO mice were treated with a combination of BA and iron at day 0 followed by alternate day of iron injections showed the poorest erythropoiesis in the BM and became rapidly sick, although the effect was significantly more pronounced in WT animals, as suggested by their survival curve. Since mycobacterium infections lead to NLPR3 inflammasome activation and Caspase1 upregulation (Marim et al. Semin Immunopathology 2017), we investigated how erythroid progenitors were affected. By flow cytometry analyses, we observed a significantly higher upregulation of the Caspase1 protein in WT and DKO mice compared to IL6KO animals. This was also reproduced by culturing WT or IL6KO BM progenitor erythroid cells in presence of mouse serum derived from WT or IL6KO mice treated with BA. Most importantly, IL6KO mice treated with BA and iron showed the highest levels of Caspase1 compared to only BA treated IL6KO mice, indicating that excess of iron abrogates the beneficial effect of IL6 deficiency on erythropoiesis under conditions of AI. Furthermore, using flow cytometry, we observed in WT mice treated with BA or BA and iron a significant increase in mitochondrial mass, which is an indicator of mitochondrial stress. The mitochondrial mass was reduced in IL6KO mice treated with BA, but again increased in IL6KO mice treated with BA and iron. We have also observed an increase of mitochondrial superoxide by confocal microscopy in WT mice compared to IL6KO mice treated with BA. Altogether, these data support a model where inflammation in presence of an excess of iron impairs BM erythropoiesis through mechanisms at least in part mediated by Caspase1 and mitochondrial dysfunction, while iron excess itself is sufficient to boost extramedullary erythropoiesis to compensate and sustain RBC production. Disclosures Vinchi: PharmaNutra: Research Funding; Vifor Pharma: Research Funding; Silence Therapeutics: Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Research Funding. Rivella: Ionis Pharmaceuticals: Consultancy; Meira GTx: Consultancy.

A Pharmacologic Approach Using Pomalidomide and Dexamethasone to Study Stage-Specific Regulation of Erythroid Progenitors

The erythroid progenitors, burst forming unit-erythroid (BFU-E) and colony forming unit-erythroid (CFU-E), control red cell production through the integration of a variety of self-renewal and differentiation signals. The identification of these signals and their effects on erythroid progenitor populations are of therapeutic interest for human disorders characterized by decreased red cell production such as Diamond Blackfan Anemia (DBA). We have previously documented that pomalidomide acts at the BFU-E stage and dexamethasone at the CFU-E stage in in vitro models of human erythropoiesis. Therefore, these two agents represent potential pharmacologic interventions for the treatment of human diseases with disordered erythropoiesis. Here, we studied the mechanisms by which pomalidomide and dexamethasone enhance erythroid progenitor numbers in cord blood and adult bone marrow-derived CD34+ hematopoietic stem and progenitor cells (HSPC).Pomalidomide increased adult and fetal BFU-E numbers in both serum-free and serum-containing in vitro erythroid culture systems. CFU-E numbers and terminal erythroblasts were also transiently attenuated suggesting that pomalidomide mediates increased BFU-E in part through delayed progression to erythropoietin-sensitive progenitors and precursor stages. Transforming growth factor-β (TGF-β) has previously been shown to regulate the transition from BFU-E to CFU-E, and therefore, we investigated whether pomalidomide modulates TGF-β signaling. We noted accelerated differentiation as assessed by glycophorin A (GPA) expression of CD34+ HSPCs treated with TGF-β1, and the addition of pomalidomide completely abrogated TGF-β1 mediated differentiation. This effect was not due to inhibition of TGF-β1 receptors or kinases as treatment of NIH 3T3 cells with TGF-β1 ± pomalidomide failed to prevent SMAD2 phosphorylation. Only CD34+ HSPCs differentiated in the presence of pomalidomide exhibited reduced sensitivity to TGF-β1. These results suggest that pomalidomide alters erythroid and/or earlier hematopoietic progenitor differentiation to enrich a population of TGF-β1 insensitive cells and/or affects the activity of downstream TGF-β1 effectors.Dexamethasone, in contrast to pomalidomide, was found to specifically increase the number of CFU-E progenitors. Elevated CFU-E self-renewal was associated with an accumulation of cells in G0/G1 of the cell cycle, which suggests that alterations to cell cycle progression may contribute to the regulation of self-renewal genes in CFU-E. In contrast to adult-derived CFU-E, dexamethasone failed to affect CFU-E numbers or cell cycle status in cord blood derived CFU-Es. We investigated the expression of glucocorticoid receptor isoforms (GRα and GRβ) in erythroid progenitors, which revealed that BFU-E and CFU-E differentially express GR isoforms. BFU-E expressed comparable levels of GRα and the hypothesized dominant-negative GRβ, where as only GRα transcript was measured in CFU-E. These expression patterns were similar between adult and cord blood-derived progenitors. Despite similar GR levels, adult-derived erythroid progenitors, but not cord blood derived progenitors, were shown to translocate GRα to the nucleus after a 24hr pulse of dexamethasone suggesting that post-translational mechanisms may govern dexamethasone response in erythroid progenitors.These findings strongly indicate that pharmacologic modulation of the TGF-β and glucocorticoid-signaling pathways can effectively stimulate erythroid progenitor self-renewal. We are currently elucidating specific targets for pomalidomide and dexamethasone in BFU-E and CFU-E, respectively, through proteomics and transcriptome approaches, and investigating the efficacy of mono- and combination therapies using peripheral blood-derived CD34+ HSPCs from DBA patients to study their therapeutic efficacy. DisclosuresNo relevant conflicts of interest to declare.

Splenic erythroid progenitors decrease TNF-α production by macrophages and reduce systemic inflammation in a mouse model of T cell-induced colitis.

In inflammatory bowel disease (IBD), inflammation can occur beyond the intestine and spread systemically causing complications such as arthritis, cachexia, and anemia. Here, we determine the impact of CD45, a pan-leukocyte marker and tyrosine phosphatase, on IBD. Using a mouse model of T cell transfer colitis, CD25- CD45RBhigh CD4+ T cells were transferred into Rag1-deficient mice (RAGKO) and CD45-deficient RAGKO mice (CD45RAGKO). Weight loss and systemic wasting syndrome were delayed in CD45RAGKO mice compared to RAGKO mice, despite equivalent inflammation in the colon. CD45RAGKO mice had reduced serum levels of TNF-α, and reduced TNF-α production by splenic myeloid cells. CD45RAGKO mice also had increased numbers of erythroid progenitors in the spleen, which had previously been shown to be immunosuppressive. Adoptive transfer of these erythroid progenitors into RAGKO mice reduced their weight loss and TNF-α expression by splenic red pulp macrophages. In vitro, erythroid cells suppressed TNF-α expression in red pulp macrophages in a phagocytosis-dependent manner. These findings show a novel role for erythroid progenitors in suppressing the pro-inflammatory function of splenic macrophages and cachexia associated with IBD.

Ibrutinib for Pure Red Cell Aplasia after Allogeneic Hematopoietic Stem Cell Transplant with Major ABO Incompatibility

<h3>Introduction</h3> Since Human leukocyte antigen (HLA) and ABO blood group system are inherited independently and with up to 50% of allogeneic hematopoietic cell transplantations (HCT) are performed with donor-recipient ABO incompatibility, recipients are at increased risk for acute and delayed hemolytic reaction and delayed RBC precursors engraftment; i.e. pure RBC aplasia (PRCA). PRCA is a sever consequence of major and bi-directional ABO mismatch after alloHCT, leading to frequent transfusions, iron overload and secondary complications. Risk factors for PRCA after Major ABO incompatibility include; stem cell source, conditioning intensity, and titer of iso-agglutinins. Treatment is mostly supportive with transfusions and growth factors as well as withdrawing immunosuppressive therapy (IS). Targeting recipient immune system with high-dose steroids, DLI, and rituximab have been reported with variable results. Here we report a case series of a novel method of targeting recipient iso-agglutinins-producing B cells by Ibrutinib. <h3>Methods</h3> We report 3 cases of PRCA refractory to conventional therapy who responded to ibrutinib, a BTK inhibitor targeting recipient B cells to allow engraftment of donor RBCs. Patient, transplant and disease characteristic including prior therapies, time to transfusion independence, and switch of blood group (BG) are summarized in Table 1. First patient's day 30 post-transplant bone marrow biopsy (BMBx) showed complete remission (CR) with full donor chimera, but BG remained O. He developed severe anemia requiring transfusions and Day 100 BMBx showed CR with markedly decreased number of erythroid progenitors. He did not respond to IS withdrawal, prednisone, rituximab or bortezomib. But BMBx 4 weeks after starting ibrutinib showed trilineage hematopoiesis. Second patient remained RBC transfusion dependent post-HCT with Day 100 biopsy showing PRCA and full donor chimera. BG remained O. He developed severe GVHD of skin and eyes after DLI and His PRCA was refractory to multiple lines of therapies but responded to ibrutinib. Third patient underwent HCT for refractory AML with MDS; Anti-A isoagglutinin level pre-HCT was high and patient underwent plasma exchange. Day 30 BMBx showed CR and full donor chimera. Day +73 BMBx showed CR with near absent erythropoiesis. PRCA was refractory to rituximab and responded to ibrutinib. <h3>Conclusion</h3> All three patients with PRCA refractory to multiple lines of therapy responded to ibrutinib with median time to transfusion independence of 4 weeks and median time to BG switch of 6 weeks. All patients tolerated therapy well. Our experience at City of Hope shows that ibrutinib could offer a safe and efficacious treatment option for cases of refractory PRCA. Prospective studies are needed to assess if early therapy with ibrutinib could reduce the morbidity resulting from frequent transfusions, and GVHD complications.

Non Invasive Evaluation of Bone Marrow Activity in Patients with Sickle Cell Disease: Correlation with Disease Features, Genotype, Markers of Erythropoiesis, Iron Metabolism and Hydroxyurea Treatment

Background: Sickle cell disease (SCD) is an inherited hemoglobinopathy characterized by pathological polymerization of hemoglobin, increased red cell rigidity and poor microvascular blood flow with consequent tissue ischemia and infarction. Thus, hemolytic anemia, vaso-occlusion and vasculopathy are the hallmarks of its clinical presentation. The transferrin receptor (TfR) mediates the transport of iron into cells and the circulating TfR can be measured as soluble transferrin receptor (sTfR). sTfR levels are frequently used to establish the diagnosis of iron deficiency anemia, especially in the context of inflammation, but they also reflect bone marrow erythropoietic activity (BMA) and mass. Erythropoietic activity has been found to be the most important determinant of sTfR levels. In this context, we aimed to study and evaluate bone marrow activity in patients with compound heterozygous HbS and beta-thalassemia (HbS/βthal) based in sTfR measurements and explore possible correlations with of key features of the disease such as: the hemolytic component, vaso-occlusive crises (VOC), acute chest syndrome, venous thrombosis, arterial thrombosis including stroke, avascular necrosis, pulmonary hypertension, hydroxyurea therapy, inflammation and renal injury. along with other biomarkers of erythropoiesis and iron metabolism such as Placental Growth Factor (PlGF), Growth Differentiation Factor-15 (GDF-15), Ferritin and Hepcidin-25. Patients and Methods: Ninety adult Caucasian patients with HbS/βthal [49 patients under hydroxyurea (HU+) treatment and 41 patients without hydroxyurea (HU-) treatment], were included in this study, while 22 apparently healthy individuals of similar age and gender served as controls. None of the patients has received any transfusions at least 6-monthes before enrollment in the study. Along with hematologic and blood chemistry parameters determination, levels of circulating sTfR, PlGF, GDF-15 and Hepcidin-25 were measured in patients with HbS/βthal and controls using RUO and IVD immunoenzymatic techniques. BMA activity was calculated from the established formula: patient-sTFR/meanControl-sTFR. Results: We found that: sTfR levels were markedly elevated in all patients with HbS/βthal compared to controls (4.8±2.2 vs. 1.0±0.2 mg/L, p&lt;0.001), resulting in a 1.6-11.9 fold increase of BMA. No correlation was found between BMA and disease features as well as regarding hydroxyurea treatment BMA (p&gt;0.434). BMA correlated significantly with the markers of the erythropoietic and hemolytic component such as: Hemoglobin (r=-0.434, p&lt;0.001); Reticulocyte Production Index (r=0.645, p&lt;0.001); LDH (r=0.570, p&lt;0.001); Billirubin (r=0.540, p&lt;0.001), PlGF (r=0.597, p&lt;0.001) and Hb A levels (r=-0.493, p&lt;0.001), while no correlation was found between BMA and Hb F levels. Furthermore, BMA values correlated significantly only with GDF-15 (r=0.466, p&lt;0.001), while interestingly no correlation was found between BMA and Ferritin and Hepcidin-25 levels alone (r=0.101, p&gt;0.351 and r=-0.043, p&gt;0.710, respectively), but a negative correlation was found between BMA and Hepcidin-25/Ferritin ratio, (r=-0.330, p=0.005). Conclusions: Our findings demonstrate that all patients with HbS/βthal studied have a significantly increased degree of erythroid BMA as assessed by measurements of sTfR levels. Erythroid BMA correlated significantly with Hepcidin/Ferritin ratio, which is an index of the degree of Hepcidin expression relative to iron overload. The correlation of erythroid BMA with Hb A levels, indicate the important role of βthal genotype in HbS/βthal disease. Furthermore, BMA is not related to hydroxyurea therapy and/or iron metabolism parameters in these patients. This implicates a likely complex action of hydroxyurea, which causes intermittent cytotoxic suppression of erythroid progenitors and cell stress signaling. The latter affects erythropoiesis, leading to recruitment of erythroid progenitors with increased HbF levels, although the number of erythroid progenitors -the main source of sTfR- remains stable. Disclosures Voskaridou: Genesis: Consultancy, Research Funding; Protagonist: Research Funding; Celgene Corporation: Consultancy, Research Funding; Acceleron: Consultancy, Research Funding; Addmedica: Membership on an entity's Board of Directors or advisory committees.

Chromatin occupancy and epigenetic analysis reveal new insights into the function of the GATA1 N terminus in erythropoiesis

AbstractMutations in GATA1, which lead to expression of the GATA1s isoform that lacks the GATA1 N terminus, are seen in patients with Diamond-Blackfan anemia (DBA). In our efforts to better understand the connection between GATA1s and DBA, we comprehensively studied erythropoiesis in Gata1s mice. Defects in yolks sac and fetal liver hematopoiesis included impaired terminal maturation and reduced numbers of erythroid progenitors. RNA-sequencing revealed that both erythroid and megakaryocytic gene expression patterns were altered by the loss of the N terminus, including aberrant upregulation of Gata2 and Runx1. Dysregulation of global H3K27 methylation was found in the erythroid progenitors upon loss of N terminus of GATA1. Chromatin-binding assays revealed that, despite similar occupancy of GATA1 and GATA1s, there was a striking reduction of H3K27me3 at regulatory elements of the Gata2 and Runx1 genes. Consistent with the observation that overexpression of GATA2 has been reported to impair erythropoiesis, we found that haploinsufficiency of Gata2 rescued the erythroid defects of Gata1s fetuses. Together, our integrated genomic analysis of transcriptomic and epigenetic signatures reveals that, Gata1 mice provide novel insights into the role of the N terminus of GATA1 in transcriptional regulation and red blood cell maturation which may potentially be useful for DBA patients.

Generation of an Immortalized Erythroid Progenitor Cell Line Directly from Peripheral Blood Mononuclear Cells without Using Mobilized CD34+ Cells

A reliable stable human erythroid progenitor cell line that can differentiate to the later stages of erythropoiesis is an important cellular model for studying molecular mechanisms of human erythropoiesis in physiological and pathological situations. An erythroid progenitor cell line (HUDEP) was derived from cord blood haematopoietic stem cells (HSCs) by doxycycline inducible expression of HPV E6/E7 gene (Kurita et al., 2013). This cell line could be differentiated further to terminally differentiated red cells, and it has been extensively used for studying transcriptional regulation of human erythropoiesis. Using the same strategy, immortalized erythroid progenitors could also be generated from adult HSCs (Trakarnsanga et al, 2017). However, generation of immortalized erythroid cells from patients using this protocol is challenging as obtaining sufficient number of adult HSCs requires mobilization of HSCs using GCSF. Peripheral blood mononuclear cells (PBMNCs) contain a small number of erythroid progenitors, which can be expanded and differentiated in culture. Till date, there are no reports on the generation of immortalized erythroid progenitors directly from PBMNCs. In this study, we established a protocol for the generation of immortalized erythroid progenitors from PBMNCs of a normal donor. The PBMNCs isolated from 10ml of blood from a normal donor were cultured for 24 hours in the primary erythroid expansion medium as described earlier (Trakarnsanga et al, 2017). These cells were then transduced with lentiviruses to express HPV E6/E7 gene and a fluorescent protein hKO1. After 3 days, the cells were cultured in a serum free medium containing the cytokines (stem cell factor and erythropoietin) and dexamethasone in the presence of doxycycline for 15 days. The cells that expressed hKO1 were sorted by FACS, and they were cultured in the same medium till the immortalization was complete. We continuously monitored the cells for the kinetics in the expression of erythroid cell surface markers, CD36, CD71 and CD235a, till >95% of the cells expressed all these markers. On day 50, all the cells expressed high levels of the erythroid markers and the cell morphology analysis using Giemsa staining showed that 65% of the cells were pronormoblasts, 22% were basophilic normoblasts and the rest of the cells were at the later stages of differentiation. To evaluate the differentiation potential of these cells, the cells were cultured using the media and conditions described by Hawksworth et al, 2018. After the removal of doxycycline from the culture medium, the cells showed haemoglobinization and the morphology analysis showed that 10% of the cells were in the polychromatic stage and 88% of the cells were in the orthochromatic stage, suggesting robust erythroid differentiation of the immortalized erythroid progenitors with the suitable cell culture conditions. These data showed that immortalized erythroid progenitors with differentiation potential could be generated directly from peripheral blood without using mobilized haematopoietic stem cells. This protocol is suitable for the generation of immortalized erythroid cells from the patients with rare red cell genetic disorders for studying disease mechanisms. DisclosuresNo relevant conflicts of interest to declare.

A High-Throughput Screening Approach to Identify Therapeutics for the Treatment of Diamond-Blackfan Anaemia

Diamond-Blackfan Anaemia (DBA) is a rare blood cell aplasia that presents clinically at approximately 2-3 months of age and its main characteristic is reduced erythroid precursors in the bone marrow, i.e. anemia. Mutations in different ribosomal protein (RP) genes have been associated with DBA, with mutations in RPS19 accounting for 20-25% of all cases. It has been proposed that RPS19 deficiency causes perturbations in ribosome biogenesis, thus activation of the p53-dependent Nucleolar Surveillance Pathway (NSP). In this context free RPs (predominantly L5 and L11) in a complex with 5S rRNA sequester the E3 ubiquitin ligase murine double minute 2 (MDM2), leading to the accumulation of p53 and subsequent activation of its transcriptional targets mediating cell cycle arrest or apoptosis. In DBA, one of the molecular mechanisms impairing the proliferation and thus reducing the number of erythroid progenitors that can progress to mature red blood cells is an elevation of p53 protein mediating activation of the NSP.In order to identify potential therapeutics that could be repurposed to prevent the activation of NSP in DBA patients, we have screened compound libraries of clinically approved therapeutics to identify pathways implicated in the p53-dependent NSP due to RPS19 deficiency. We quantitated both cell number and the level of p53 expression, identifying compounds that can result in low and high expression of p53, the latter for potential use in cancer therapy. Using an RPS19 depleted A549 cell line as a model system, the screen successfully identified different therapeutic groups. In the DBA context, we were most interested in the compounds that reduced p53 and had no negative effect on cell number. A selection of 22 molecules were re-evaluated in vitro, again using RPS19 depleted A549 cells, through the quantification of p53 protein expression and densitometry analysis. From this, 10candidates were evaluated ex vivofor their effects on proliferation using bone marrow obtained from an inducible Rps19 knockdown (DBA) mouse model.While we are currently testing a number of compounds in vivo using the Rps19 DBA mouse model (as described), one of the compounds tested thus far has demonstrated a partial rescue of the cKit+ population, no changes in erythroid precursors but interestingly a reversal of the defect in the Granulocyte-Monocyte Progenitor (GMP) population. Impairment in lineage progression in the GMP compartment has also been reported to present in bone marrow failure Shwachman-Diamond Syndrome. We are currently evaluating the mechanism by which this drug is rescuing the c-Kit+ and GMP populations in these mice.In summary, our high-throughput screening approach and follow up studies have identified a suite of novel therapies that may be beneficial for repurposing for the treatment of bone marrow failure by increasing hematopoietic progenitor cells. We plan to evaluate this, and potentially other therapies, in a clinical trial with DBA patients. DisclosuresFlygare:LU Holding: Patents & Royalties: Patent.

Global Chromatin Occupancy and Epigenetic Signature Analysis Reveal New Insights into the Function of GATA1 N-Terminus in Erythropoiesis

Mutations in GATA1 are seen in rare cases of dyserythropoietic anemia and in a subset of patients with Diamond Blackfan Anemia (DBA). Of note the truncation mutations in DBA, known as GATA1s, closely resemble those that are more commonly associated with acute megakaryoblastic leukemia in children with Down syndrome (DS). Studies with a mouse model of the Gata1s mutation revealed that replacement of the full-length protein by the shortened isoform led to a marked yet transient enhancement in megakaryopoiesis, similar in some respects to transient myeloproliferative disorder in DS. Furthermore, these mutant mice displayed impaired embryonic erythropoiesis but ostensibly no defects in adult hematopoiesis. In our efforts to better understand the connection between GATA1s and DBA, we comprehensively studied erythropoiesis in the Gata1s mouse strain. We observed a striking impairment in erythropoiesis in fetuses at E10.5 though E12.5, but saw improvement as the animals progressed through E14.5 and beyond. Defects included impaired terminal maturation and reduced numbers of erythroid progenitors, likely at the expense of expanded megakaryopoiesis. RNA-sequencing revealed that both erythroid genes and megakaryocytic genes were altered by the Gata1s mutation. Epiproteomic histone modification analysis further revealed there was an accumulation of H3K27 methylation in the R3 (CD71hiTer119hi) erythroid progenitor population, which suggests that GATA1 has a link to the epigenetic machinery that is altered in Gata1s mutant cells. Despite a global increase in H3K27me3, critical Gata2 regulatory elements in Gata1s mutant erythroid progenitors were marked by substantially less H3K27me3 than in wild-type littermates. Given that overexpression of GATA2 has been reported to impair erythropoiesis, we investigated whether reducing the GATA2 levels would ameliorate the phenotype. Indeed, we observed that haploinsufficiency for Gata2 rescued the erythroid defects of Gata1s fetuses. Next, to comprehensively study the effect of absence of the GATA1 N-terminus genome-wide, we performed Cleavage Under Targets and Release Using Nuclease (CUT&RUN) with H3K27me3, GATA1 or GATA1s antibodies on wild-type versus Gata1s expressing fetal erythroid cells. Our data indicated that there is a substantial reduction in H3K27me3 along regulatory elements of the Runx1 gene at the late stage (R3) of fetal erythropoiesis in Gata1s mice. Along with an increase in Runx1 expression we observed strong downregulation of Klf1, a repressive target of RUNX1. Thus, failure of GATA1s to facilitate trimethylation of Runx1 and Gata2 regulatory elements appears to cause the defects in erythroid cell and megakaryocyte development. In parallel, we performed an in-depth analysis of the phenotype of adult Gata1s mice and discovered that they have reduced red cell counts, lower hemoglobin and hematocrit, increased extramedullary hematopoiesis and impaired stress erythropoiesis compared to control littermates. Although there were significantly more megakaryocyte erythrocyte progenitors (MEPs, Lin-c-Kit+Sca-1-CD34-FcgR-) in Gata1s mouse bone marrow, there were fewer pre-colony-forming unit erythroid cells (preCFU-E, Lin-c-Kit+Sca-1-CD41-FcgR-CD150hiCD105hi), likely at the expense of expanded megakaryocyte progenitors (MkP, Lin-c-Kit+Sca-1-CD41+CD150hi).Gata1s mice also developed an MDS-like disease with age. Together, our integrated genomic analysis of transcriptome, GATA1/GATA1s chromatin binding profile and chromatin signature reveal that, although Gata1s mice do not precisely model DBA, they provide novel insights into the role of the N-terminus of GATA1 in gene transcriptional regulation, lineage determination and red blood cell maturation. DisclosuresCrispino:Scholar Rock: Research Funding; Forma Therapeutics: Research Funding.

Identification of a Multipotent Progenitor Population in the Spleen That Is Regulated by NR4A1.

The developmental fate of hematopoietic stem and progenitor cells is influenced by their physiological context. Although most hematopoietic stem and progenitor cells are found in the bone marrow of the adult, some are found in other tissues, including the spleen. The extent to which the fate of stem cells is determined by the tissue in which they reside is not clear. In this study, we identify a new progenitor population, which is enriched in the mouse spleen, defined by cKit+CD71lowCD24high expression. This previously uncharacterized population generates exclusively myeloid lineage cells, including erythrocytes, platelets, monocytes, and neutrophils. These multipotent progenitors of the spleen (MPPS) develop from MPP2, a myeloid-biased subset of hematopoietic progenitors. We find that NR4A1, a transcription factor expressed by myeloid-biased long term-hematopoietic stem cells, guides the lineage specification of MPPS. In vitro, NR4A1 expression regulates the potential of MPPS to differentiate into erythroid cells. MPPS that express NR4A1 differentiate into a variety of myeloid lineages, whereas those that do not express NR4A1 primarily develop into erythroid cells. Similarly, in vivo, after adoptive transfer, Nr4a1-deficient MPPS contribute more to erythrocyte and platelet populations than do wild-type MPPS. Finally, unmanipulated Nr4a1-/- mice harbor significantly higher numbers of erythroid progenitors in the spleen compared with wild-type mice. Together, our data show that NR4A1 expression by MPPS limits erythropoiesis and megakaryopoeisis, permitting development to other myeloid lineages. This effect is specific to the spleen, revealing a unique molecular pathway that regulates myeloid bias in an extramedullary niche.

Myeloid Precursors in the Bone Marrow of Mice after a 30-Day Space Mission on a Bion-M1 Biosatellite.

The content of myeloid stem CFU in bone marrow karyocytes from the tibial bone of C57Bl/6 mice was evaluated after a 30-day Bion-M1 pace flight/ground control experiment and subsequent 7-day recovery period. After the space flight, we observed a significant decrease in the number of erythroid progenitors in the bone marrow, including common myeloid precursor - granulocyte, erythrocyte, monocyte/macrophage, megakaryocyte CFU. After 7-day readaptation, CFU level in flight animals did not recover completely. In the ground control, the count of erythroid burst-forming units was higher than in vivarium animals. Comparison of the changes observed in fight and ground experiments demonstrated effects associated space flight factors and manifesting in suppression of the bone marrow erythropoiesis.

Potential Therapeutic Applications of Jak2 Inhibitors and Hif2a-ASO for the Treatment of β-Thalassemia Intermedia and Major

β-Thalassemia is one of the most common genetic red blood cell (RBC) disorders characterized by reduced (such as in non-transfusion dependent thalassemia or NTDT) or absent (such as in transfusion dependent thalassemia or TDT) production of β-globin chains. Ineffective erythropoiesis (IE) with consequent anemia leads to extra-medullary hematopoiesis (EMH), splenomegaly and iron overload mediated by low levels of hepcidin. We previously demonstrated that IE in β-thalassemia is associated with increased proliferation and reduced differentiation of erythroid progenitors (Libani et al, 2008, Blood). This is mediated by increased production of erythropoietin (EPO), which activates the downstream JAK2 kinase in erythroid progenitors. As a consequence, hepatosplenomegaly may result, often requiring splenectomy to prevent serious morbidities and mortality. The increased synthesis of EPO in thalassemia is the result of hypoxia. Hypoxia Inducible Factor-2a (HIF2a) is a central mediator of cellular adaptation to hypoxia and stimulates renal and hepatic EPO synthesis. Furthermore, splenomegaly in thalassemia also exacerbates the anemia, as a large proportion of the circulating RBC are engulfed and eliminated by an enlarged spleen.Therefore, we hypothesized that targeting the EPO/JAK2 pathway limits the number of erythroid progenitor cells and reduces the splenomegaly, and it could be utilized as an alternative to splenectomy. We utilized JAK2 inhibitors or HIF2a antisense oligonucleotides (HIF2a-ASO) to target the EPO/JAK2 pathway. Moreover, we hypothesized that combination of these drugs with blood transfusion therapy will further reduce the splenomegaly and, eventually, improve the blood transfusion regimen.We tested two commercially available JAK2 inhibitors in mice affected by NTDT (Hbbth3/+): INCB018424 (Ruxolitinib) and TG101348 (Fedratinib, SAR302503). Both drugs were administered for 10 days, twice daily by oral gavage at a dose of 180 and 120 mg/Kg respectively. A mild reduction in hemoglobin (Hb) levels, (in the range of 9%), was observed in animals treated with both inhibitors when compared to vehicle- treated mice. Splenomegaly was significantly reduced with both inhibitors (up to 56% in reduction). Flow cytometry studies on spleen cells revealed that animals receiving the inhibitors exhibited a reduction in the number of erythroid progenitors compared to the placebo-treated animals . In parallel, we performed pharmacokinetic[BM1] studies using Hif2a-ASO. Animals received Hif2a-ASO at a dose of 25 mg/kg twice weekly for 10 days or 3 weeks by IP injections. After 10 days of treatment the spleen weight was reduced 58%, while the Hb level was reduced in the range of 27%%. After 3 weeks the effect observed on the anemia was more pronounced, with a reduction of more that 40% in Hb levels, while the spleen reduction was 36%. We then compared and combined these drugs with blood transfusion using Hbbth3/+ mice. Blood transfusion reduced splenomegaly 49% and 53% when compared, respectively, to non-transfused controls and animals treated with JAK2 inhibitors alone. When transfusion was combined with the administration of JAK2 inhibitors for 10 days, the spleen size was further reduced (72% when compared with non-transfused controls). Combination of Hif2a-ASO and blood transfusion is in progress. We then tested the JAK2 inhibitors in mice affected by TDT. TDT animals are generated by engrafting WT mice with Hbbth3/th3 fetal liver cells. Following engraftment, these mice show a large splenomegaly and rapidly become dependent on chronic blood transfusion for survival (Gardenghi et al, 2007, Blood). Preliminary studies suggest that administration of JAK2 inhibitors for 10 days, together with blood transfusion, further reduces spleen weight by 71% compared to transfusion alone. Combination of Hif2a-ASO and blood transfusion in these animals is also in progress. In summary, JAK2 inhibitors and Hif2a-ASO reduce splenomegaly by targeting the EPO/JAK2 pathway and limiting the excessive proliferation of erythroid cells. Therefore, these drugs could be effective in reversing the splenomegaly and may offer an important approach to splenectomy. Additional studies are in progress to evaluate the outcome of combination therapy on the efficacy of transfusions.[BM1]These aren't pharmacokinetic studies. Suggest just striking the word DisclosuresCasu:Medgenics LLC: Research Funding; Ionis Pharmaceuticals: Research Funding; Merganser Biotech: Research Funding. Aghajan:Ionis Pharmaceuticals: Employment, Equity Ownership. Guo:Ionis Pharmaceuticals: Employment, Equity Ownership. Melchiori:Adaptimmune Ltd: Employment. Ramos:Novartis: Employment.

Expansion and Biased Commitment of Hematopoietic Stem Cells Towards Erythroid Cells upon Acute Anemia Induction

The majority of adult hematopoietic stem cells (HSCs) are maintained in a dormant state under homeostatic conditions. In contrast, under stressed conditions such as myeloablation and infection, HSCs are known to proliferate and rapidly give rise to downstream progeny. However, it is unclear whether and how HSCs respond to severe anemic conditions. Here we report that HSCs rapidly expand with a biased differentiation towards erythroid cells upon the induction of acute anemia.Injection of 60 mg/kg of phenylhydrazine (PHZ) was used to induce hemolytic anemia, after which the peripheral blood (PB), bone marrow (BM) and spleen of the mice were analyzed for the blood profiles and stem/progenitor cell content. The red blood cell (RBC) count of the PHZ treated mice was at its lowest at day 6 post injection. BM analysis showed that the number of HSCs (CD150+CD34-c-kit+Sca-I+Lineage-) immediately started increasing, as well as megakaryocyte-erythroid progenitors (MEP, CD34-FcγIII/IIR-c-kit+Sca-I-Lineage-) with a peak at day 3-4 (3.0 and 3.4 fold increase, respectively). Interestingly, the number of common myeloid progenitors (CMP, CD34+FcγIII/IIR-c-kit+Sca-I-Lineage-) did not show a clear increase over time and the number of erythroid progenitors (Ter119+) started increasing at a later time point than the HSC/MEP expansion, suggesting that the expansion of primitive cells is a primary response to the anemic condition that possibly skips some of the regular stages that are observed in the normal differentiation towards erythrocytes.In contrast to the BM, in the spleen HSC expansion was modest while MEP and CMP were robustly expanded (5.7 and 6.6 fold increase, respectively). These findings indicate that the BM and spleen have distinct roles in the response to the anemic conditions.In order to accurately evaluate the lineage potential of HSCs in vitro, we developed a combined assay utilizing colony formation and flow cytometry analysis (CFU-FACS), with which all generated colonies were analyzed for the morphology and the frequency of each lineage. The result showed that HSCs isolated from control mice had a balanced differentiation towards megakaryocyte and erythroid cells with 20-25% of the colonies containing only granulocytes/macrophages and megakaryocytes, but not erythroid cells (GMMk colonies). In contrast, HSCs isolated from PHZ treated mice showed significantly increased the number of colonies containing a higher content of erythroid cells, whereas the ratio of GMMk colonies was decreased. Furthermore, 3-dimensional analysis of the three lineage potentials (myeloid, megakaryocyte and erythroid) in the colonies revealed an imbalanced lineage potential of HSCs from anemic mice, showing higher erythroid potential instead of the megakaryocyte potential.As an alternative method, phlebotomy was performed to induce acute anemia. Although phlebotomized mice did not display a clear expansion of the HSC population, CFU-FACS analysis showed an erythroid-biased lineage potential of the HSCs, indicating that the HSC expansion and the lineage bias may be caused by independent mechanisms.To demonstrate if the alterations in the HSCs affect the in vivo function of these cells, 50 HSCs isolated from control or PHZ injected Kusabira Orange (KuO) mice were transplanted into lethally irradiated mice. Two weeks after the transplantation, the ratio of KuO+ RBCs against KuO+ platelets was higher in the PHZ-HSC transplanted mice than control-HSC transplanted mice. This difference was not seen four weeks after transplantation and the long-term reconstitution (>12 weeks) levels did not differ between both groups, suggesting that the enhanced erythropoiesis is a transient event that does not reduce the stem cell capacity.In summary, we demonstrated that not only progenitor cells but also HSCs respond to severe anemic conditions and contribute to erythropoiesis through rapid expansion and a transient fate change, depicting a novel model of stress response. DisclosuresNo relevant conflicts of interest to declare.

The Thrombopoietin Receptor (MPL) Is a Selective Marker for the Identification of Hematopoietic Stem Cells from Myelofibrosis Patients

▪Recently, it has been shown that both JAK2V617F and mutated calreticulin require MPL for oncogenic transformation (Sangkhae et al., Blood 2014; Elf et al., Cancer Discov. 2016). We have reported that treatment of myelofibrosis (MF) CD34+ cells with a thrombopoietin (TPO) receptor antagonist, LCP4, selectively depletes MF hematopoietic stem cell (HSC) and hematopoietic progenitor cells (HPC) (Wang et al., Blood 2016). Since MF is thought to originate at the level of the HSC, we hypothesize that MF HSCs expressing MPL might differ in function from those lacking MPL and might be associated with disease initiation and progression. To test this hypothesis, we have studied the nature of these two types of MF HSCs and HPCs.Initially, using flow cytometry we showed that MPL+ cells accounted for 45.0% and 17.7% of CD34+ cells isolated from the spleens of 2 JAK2V617F+MF patients (anti-MPL mAb, Clone REA250, Miltenyi Biotec). CD34+MPL+ or CD34+MPL- cells were then isolated and assayed for HPCs. As expected, in the presence of TPO, megakaryocyte (MK) progenitors were generated by MF CD34+MPL+ cells (24-96/1250 cells), but rarely from CD34+MPL- cells (1-4.5/1250 cells). Greater numbers of common myeloid (4.3-fold) and myeloid progenitors (2.2-fold), but smaller numbers of erythroid progenitors (4.5-fold less) were generated from CD34+MPL+ cells as compared with CD34+MPL- cells. These data indicate that MF CD34+MPL+ cells primarily give rise to common myeloid, MK and myeloid progenitors whereas MF CD34+MPL- cells contain erythroid progenitors.The marrow repopulating potential of MF CD34+MPL+ or CD34+MPL- cells was then assessed by transplanting these cell populations into NSG mice. Four months after transplantation, as few as 1×103 CD34+MPL+ cells were able to engraft recipient mice to a similar degree as 1×105 CD34+MPL- cells. Moreover, there was a clear relationship between the numbers of CD34+MPL+ cells but not CD34+MPL- cells infused and the degree of human cell chimerism achieved in the bone marrow and spleen of these mice. When 1 × 104 and 1× 105 CD34+MPL+ cells were transplanted, significant numbers of human cells belonging to various hematopoietic lineages were detected in recipient mice. Furthermore, only CD34+MPL+ cells were able to engraft secondary recipients. The frequency of SCID repopulating cells within CD34+MPL+ cells was 1,400-fold greater than that calculated for CD34+MPL- cells. These findings indicate that long-term MF HSCs are primarily present in CD34+ cells expressing MPL.The influence of TPO/MPL signaling on the proliferation of MF HSCs and HPCs was then examined by culturing single MF CD34+MPL+ or CD34+MPL- cells. Single CD34+MPL+ cells generated greater numbers of clones of various sizes (<50: 1.7-fold; 50-200:0.8-fold; >200 cells: 2.1-fold) than an equal number of single CD34+MPL- cells. Next, the number of progenitors generated after replating of the primary clones was assessed. Clones derived from single CD34+MPL+ cells were able to generate 55±20 secondary CFU-GM, while clones from CD34+MPL- cells were incapable of generating secondary HPC. These data show that the proliferation of MF HSCs expressing and lacking MPL differs markedly in their contribution to the clonal expansion of MF HSCs/HPCs.Finally, the inhibitory effect of the TPO receptor antagonist, LCP4, on the proliferation of MF MPL+ or MPL- CD34+ cells was evaluated. The production of CFU-GEMM was eliminated and a 43.8-50% reduction in the number of CFU-MK was achieved following the treatment of CD34+MPL+ cells by LCP4. Surprisingly, the treatment of CD34+MPL- cells from one patient with LCP4 also resulted in a 100% and 48.7% reduction in the number of CFU-GEMM and CFU-MK, respectively. Moreover, LCP4 treatment reduced the percentage of total JAK2V617F+ and homozygous JAK2V617F+ CFU-GM generated from CD34+MPL+ cells. The number of clones derived from single MF CD34+MPL+ cells was reduced by 61.5-66.7% in the presence of LCP4, but LCP4 did not alter the proliferation of MF CD34+ cells lacking MPL. These data indicate that the depletion of malignant HSCs/HPCs by LCP4 is achieved by interrupting TPO stimulation of MPL expressing HSC/HPC.We conclude that CD34+MPL+ cells represent a subset of malignant HSCs that are responsible for the origin of MF, and that MPL antagonist therapy represents a promising therapeutic strategy which substantially alters the natural history of MF. DisclosuresNo relevant conflicts of interest to declare.

Inhibition of Human Erythropoiesis during Inflammation Is Mediated By High Mobility Group Box Protein 1 (HMGB1) through Decreased Commitment of Hematopoietic Stem Cells to the Erythroid Lineage and By Increased Apoptosis of Terminally Differentiating Erythroblasts

Anemia plays a significant role in the morbidity and mortality of chronic inflammatory diseases and post-sepsis recovery. Inflammation-associated anemia is further complicated as these conditions are typically refractory to erythropoietin (EPO) administration, which suggests that circulating inflammatory molecules prevent effective erythropoiesis. High mobility group box protein 1 (HMGB1), a pro-inflammatory molecule involved in sepsis has been shown to mediate anemia in a murine model of sepsis survivors. However, the effects of soluble HMGB1 on human erythropoiesis remain poorly understood. To address this issue, we studied the effect of increasing concentrations of HMGB1 on human erythropoiesis in vitro using CD34+ hematopoietic stem and progenitor cells (CB HSPCs) and a previously described 3-phase erythroid differentiation culture system to asses the effects on erythroid cell proliferation as well as the semisolid methylcellulose assay to study effects on erythroid progenitors. HMGB1 markedly reduced erythroid cell proliferation in the 3-phase culture system in a dose-dependent manner. We evaluated the contributions of early- and late-stage erythropoiesis to decreased erythroid proliferation using using flow cytometric analyses of cell surface markers interleukin-3 receptor (IL-3R), glycophorin A (GPA), CD36, CD34 (BFU-E and CFU-E) and GPA, α4-integrin and band 3 (ProEB-orthoEB). HMGB1 increased the percentage of IL-3Rpos cells (vehicle v HMGB1: 8.6% vs 15.5%), and reduced the percentage of GPApos cells (vehicle v HMGB1: 32.2% v 12.8%) implying decreased commitment of CB HSPCs to erythroid lineage in the presence of HMGB1. Flow cytometric analysis confirmed an increased commitment to myeloid lineage and Giemsa-stained cytospins revealed increased numbers of myelo-monocytic cells in HMGB1 cultures. Furthermore, the decreased number of erythroid progenitors generated in culture was blocked in terminal differentiation leading to increased apoptosis of erythroblasts.Since we found increased numbers of myelo-monocytic cells and increased erythroblast apoptosis in HMGB1 treated cultures, we performed additional studies to investigate the role of soluble HMGB1 on HSPC lineage commitment and erythroblast survival signaling (i.e. EPO signaling). CB CD34+ HSPCs were seeded in fully enriched methylcellulose ± HMGB1, and hematopoietic colony numbers were quantified based on morphologic characteristics. HMGB1 increased the number of CFU-GM colonies and decreased the number of erythroid colonies (CFU-E, BFU-E). Further, erythroblasts treated with 3IU/mL EPO ± HMGB1 revealed that HMGB1 significantly reduced EPO-dependent STAT5 phosphorylation in a dose-dependent manner.In order to begin to identify the HMGB1 receptors responsible for HSPC lineage skewing and erythroblast apoptosis we used transcriptome-wide RNA-sequencing data to examine the expression patterns of various HMGB1 receptors during differentiation of HSPCs in our culture system. Toll-like receptor 2 (TLR2), TLR4 and CXCR4 are expressed during the early-stages of erythroid differentiation while the receptor for advanced glycation end products (RAGE) is expressed only during late stages of erythroid differentiation. These findings suggest that two discrete HMGB1-receptor signaling axes may underlie the noted changes in early- and late-stage cellular differentiation in our culture system.Taken together, our results identify HMGB1 as a potent inhibitor of human erythropoiesis. HMGB1 exerts this effect at two levels of erythroid differentiation, the first during the commitment of HSPC to erythroid differentiation and subsequently during terminal erythroid differentiation. The erythroid differentiation defect appears to be partially dependent on EPO signaling while it has no effect on the HSPC defect. We proposed that the elevated HMGB1 during inflammation could explain the persistent erythroid defect observed in patients with chronic inflammatory diseases and sepsis survivors. DisclosuresNo relevant conflicts of interest to declare.

Pomalidomide and Dexamethasone Regulate Human Erythroid Progenitor Signaling through Two Distinct Pathways

With the exception of cytokines, corticosteroids such as dexamethasone (Dex) and the immunomodulatory drugs (IMiDs) such as pomalidomide (Pom) are the primary pharmacologic agents that directly improve red cell production in patients with disordered erythropoiesis. Corticosteroids are the standard of care for patients with Diamond Blackfan anemia (DBA) while the IMiDs are used for patients with myelodysplastic syndrome (MDS) to improve the profound anemia seen in these patients. While it is generally accepted that loss of erythroid progenitors such as burst forming unit-erythroid (BFU-E) and colony forming unit-erythroid (CFU-E) underlie congenital and acquired bone marrow failure syndromes such as DBA and MDS, our limited understanding of the regulatory programs governing erythroid progenitor self-renewal and differentiation hinder development of more specific therapies aimed at increasing erythroid progenitor numbers in these diseases. To investigate the dynamics of erythroid progenitors in normal and diseased states, we used an in vitro culture system consisting of 3 phases to study human erythropoiesis in the presence of Pom or Dex.Our earlier work provided evidence that Pom transcriptionally reprograms adult erythroid progenitors, and delays the BFU-E to CFU-E transition; this delay is through a yet to be characterized mechanism. To elucidate the effects of Pom on erythroid progenitors we differentiated adult CD34+ hematopoietic stem and progenitor cells (HSPCs) and performed gene expression microarray analyses on erythroid cells at day 4. We found that Pom induced significant expression changes in 20 genes including TEA domain transcription factor 2 (TEAD2), a transcription factor involved in stem cell self-renewal and hippo signaling. To date, a function for TEAD2 in human erythropoiesis has not been identified. qRT-PCR and western blot studies validated that Pom induces a greater than 100-fold increase in TEAD2 expression compared to vehicle control. Previous studies have shown that members of the TEAD family cooperate with cytokines such as transforming growth factor-β (TGF-β) and Bone Morphogenic Protein 4 (BMP4), two known modulators of erythropoiesis. We observed that Pom did not affect the levels of phospho-SMAD2 by western blot analysis, a downstream effector of TGF-β. In marked contrast, Pom-treated cells exhibited higher levels of p-SMAD1/5/9 suggesting that Pom may enhance or activate the BMP4 pathway. These data suggest that Pom leads to the self-renewal of BFU-E through the activation of TEAD2. We are currently investigating the downstream signaling pathways involved.The role of Dex in human erythropoiesis is more controversial. Previous studies, particularly in murine models have suggested that the effects of Dex occur at the BFU-E stage. However, in our studies, we found that Dex acts at the level of CFU-E in models of human erythropoiesis. We found that Dex promotes both the self-renewal as well as the differentiation of CFU-E. We found that treatment with dexamethasone resulted in a 4-fold increase in the percentage of CFU-E in culture when compared to vehicle control. Interestingly, these effects vary with the type of HSPC used (peripheral blood vs. cord blood). Indeed, while Dex induced extensive proliferation of adult CFU-Es, it had no effects on CFU-E from cord blood. These results suggest a specificity for corticosteroids during development and suggest that these drugs may prove more useful in certain types of anemia. Finally, clinical data from three different DBA patients demonstrated a response to steroids within a week of treatment, as measured by an increase in reticulocytes suggesting that the population responding to treatment is indeed the CFU-E, reinforcing our in vitro findings. We are now investigating the molecular mechanisms involving the effects of dexamethasone on erythroid progenitors.Together, these results suggest that Pom and Dex may influence erythroid progenitor self-renewal and differentiation through independent mechanisms and that combination therapy may be beneficial for a variety of diseases characterized by disordered erythropoiesis. DisclosuresNo relevant conflicts of interest to declare.