Late-stage Erythroid Differentiation Research Articles

A comparative study of two routinely used protocols for ex vivo erythroid differentiation

BackgroundErythropoiesis is a complex developmental process in which a hematopoietic stem cell undergoes serial divisions and differentiates through well-defined stages to give rise to red blood cells. Over the last decades, several protocols have been developed to perform ex vivo erythroid differentiation, allowing investigation into erythropoiesis and red cell production in health and disease. ResultsIn the current study, we compared the two commonly used protocols by assessing the differentiation kinetics, synchronisation, and cellular yield, using molecular and cellular approaches. Peripheral blood CD34+ cells were cultured in a two-phase (2P) or a four-phase (4P) liquid culture (LC) and monitored for 20 days. Both protocols could recapitulate all stages of erythropoiesis and generate reticulocytes, although to different extents. Higher proliferation and viability rates were achieved in the 4P-LC, with a higher degree of terminal differentiation and enucleation, associated with higher levels of the erythroid-specific transcription factors GATA-1, KLF-1, and TAL-1. Although the 2P-LC protocol was less efficient regarding terminal erythroid differentiation and maturation, it showed a higher yield of erythroid progenitors in the erythropoietin (EPO)-free expansion phase. ConclusionsWe provide data supporting the use of one protocol or the other to study the biological processes occurring in the early or late stages of erythroid differentiation, depending on the physiological process or pathological defect under investigation in a given study.

Fine-Tuning of Cholesterol Homeostasis Controls Erythroid Differentiation.

Lipid metabolism is essential for stemness maintenance, self‐renewal, and differentiation of stem cells, however, the regulatory function of cholesterol metabolism in erythroid differentiation is poorly studied. In the present study, a critical role for cholesterol homeostasis in terminal erythropoiesis is uncovered. The master transcriptional factor GATA1 binds to Sterol‐regulatory element binding protein 2 (SREBP2) to downregulate cholesterol biosynthesis, leading to a gradual reduction in intracellular cholesterol levels. It is further shown that reduced cholesterol functions to block erythroid proliferation via the cholesterol/mTORC1/ribosome biogenesis axis, which coordinates cell cycle exit in the late stages of erythroid differentiation. The interaction of GATA1 and SREBP2 also provides a feedback loop for regulating globin expression through the transcriptional control of NFE2 by SREBP2. Importantly, it is shown that disrupting intracellular cholesterol hemostasis resulted in defect of terminal erythroid differentiation in vivo. These findings demonstrate that fine‐tuning of cholesterol homeostasis emerges as a key mechanism for regulating erythropoiesis.

Mutational Profile and Analysis of Lower-Risk Myelodysplastic Syndromes (MDS) Patients Treated with Luspatercept: Phase 2 PACE-MDS Study

Background: Management of anemia is a common therapeutic challenge in patients (pts) with MDS. Luspatercept (ACE-536), a fusion protein containing modified activin receptor type IIB, is being developed for treatment of anemia in lower-risk MDS. Luspatercept acts as a ligand trap for GDF11 and other TGF-β family ligands to suppress Smad2/3 signaling, promote late-stage erythroid differentiation, and increase hemoglobin (Hgb) levels (Suragani R, Nat Med, 2014; Attie K, Am J Hematol, 2014). Luspatercept has demonstrated clinical activity in ring-sideroblast positive (RS+,≥15% in bone marrow) MDS pts and comparable responses in non-RS+ pts with EPO<500 U/L. While RS+ pts have distinct clinical and molecular features including SF3B1 splicing mutations, non-RS+ pts have a more heterogeneous population of mutations.Aims: This ongoing, phase 2, multicenter, open-label study followed by a long-term extension (ext) study evaluates the effects of luspatercept in pts with lower-risk MDS. Endpoints include long-term safety and tolerability, erythroid response (IWG HI-E), RBC transfusion independence (RBC-TI,≥8 weeks), and duration of HI-E. New analyses focus on clinical, molecular, and bone marrow morphological features of disease and factors associated with luspatercept response.Methods: Inclusion criteria: MDS IPSS low or int-1, age ≥18 yr, Hgb<10 g/dL (if <4U RBC/8 wks), no prior HMA, no current lenalidomide or erythropoiesis-stimulating agent (ESA). The dose-escalation phase of the study is completed. An expansion cohort was added to this phase 2 study to evaluate response in pts not qualified for the phase 3 MEDALIST trial (for regularly transfused RS+ pts refractory to ESA or EPO>200 U/L). These include pts with low transfusion burden (<4U RBC/8 weeks) and either 1) RS+ with baseline EPO ≤200 U/L or 2) non-RS+ and any EPO level. Non-RS+ pts allowed up to 6U RBC/8 wks. Pts treated every 3 weeks subcutaneously for up to 5 doses (titration up to 1.75 mg/kg) in the base study (NCT01749514) and are then eligible for long-term treatment up to 5 additional years (NCT02268383).Results: Data (as of 13Apr2017) were available for 88 pts treated at therapeutic dose levels (≥0.75 mg/kg) in both the base and ext studies. 88 pts were evaluable for IWG HI-E and 60 pts were evaluable for RBC-TI (baseline transfusions of ≥2 units/8 wks). 75 pts were evaluable for mutational data analysis.Baseline characteristics are shown in Table 1. In pts with mutational data, the most common mutations were SF3B1 (57%), TET2 (39%), DNMT3A (21%), ASXL1 (17%), and SRSF2 (11%). Response by subgroup is shown in Table 2.Responses were observed in both RS+ and non-RS+ pts of various mutational backgrounds. Many pts possessed mutations in multiple genes; pts carrying SF3B1 mutations tended to have increased response rates compared to SF3B1 WT pts. Other commonly observed (>10%) mutations (TET2, DNMT2A, ASXL1, and SRSF2) demonstrated similar responses in pts when either mutated or not. Longer-term data on all pts plus additional exploratory analyses of molecular, cellular, and morphologic parameters and their relationship to response will be presented at the meeting.Luspatercept has been well tolerated, with seven possibly related grade 3 non-serious adverse events (AE): ascites, blast cell count increase, blood bilirubin increase, bone pain, hypertension, platelet count increase, and pleural effusion and four possibly related SAEs as of 15June2017: ataxia, general physical health deterioration, muscle weakness, and myalgia. Related AEs in >2 pts were headache, fatigue, hypertension, bone pain, diarrhea, arthralgia, injection site erythema, myalgia, and peripheral edema.Conclusions: Lower-risk MDS pts treated long-term with luspatercept demonstrated robust and sustained increases in Hgb and decreases in transfusion burden and a high rate of RBC-TI. Encouraging responses were seen across EPO levels up to 500 U/L in both RS+ and non-RS+ patients. The presence of an SF3B1 mutation was associated with a higher response in this initial cohort of pts and responses were also observed in SF3B1 WT pts with mutations typically associated with poor prognosis (e.g., ASXL1). The data support continued study of luspatercept in MDS pts, including aPhase 3 study in regularly-transfused RS+ pts with lower-risk MDS according to IPSS-R (MEDALIST study; NCT02631070; fully enrolled) and a future Phase 3 study planned in first-line lower-risk MDS pts. [Display omitted] DisclosuresPlatzbecker:Acceleron: Consultancy, Honoraria, Research Funding; Novartis: Consultancy, Research Funding; Celgene: Consultancy, Honoraria, Research Funding; Janssen: Consultancy, Honoraria, Research Funding. Kiewe:Acceleron: Consultancy, Honoraria, Research Funding. Germing:Novartis: Honoraria, Research Funding; Janssen: Honoraria; Celgene: Honoraria, Research Funding. Götze:Celgene: Honoraria; Novartis: Honoraria; BMS: Honoraria; Abbvie: Honoraria. Radsak:Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Travel support, Research Funding; Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Travel support; Gilead: Other: Travel support; Acceleron: Research Funding. Wilson:Acceleron: Employment, Equity Ownership. Zhang:Acceleron: Employment, Equity Ownership. Rovaldi:Acceleron: Employment, Equity Ownership. Laadem:Celgene: Employment, Equity Ownership. Sherman:Acceleron: Employment, Equity Ownership. Attie:Acceleron: Employment, Equity Ownership. Reynolds:Acceleron: Employment, Equity Ownership. Linde:Acceleron: Employment, Equity Ownership. Giagounidis:Celgene: Consultancy; Acceleron: Consultancy.

Identification of characteristic proteins at late-stage erythroid differentiation in vitro.

The production of red blood cells in vitro, which is useful for basic or clinical research, has been improved. Further optimization of culture protocols may facilitate erythroid differentiation from hematopoietic stem cells to red blood cells. However, the details of erythropoiesis, particularly regarding the behaviors of differentiation-related proteins, remain unclear. Here, we performed erythroid differentiation using two independent bone marrow- or cord blood-derived CD34+ cell sources and identified proteins showing reproducible differential expression in all groups. Notably, most of the proteins expressed at the early stage were downregulated during erythroid differentiation. However, seven proteins showed upregulated expression in both bone marrow cells and cord blood cells. These proteins included alpha-synuclein and selenium-binding protein 1, the roles of which have not been clarified in erythropoiesis. There is a possibility that these factors contribute to erythroid differentiation as they maintained a high expression level. These findings provide a foundation for further mechanistic studies on erythropoiesis.

Coordinated Mis-Splicing of Multiple Mitochondrial Iron Metabolism Genes Causes Ring Sideroblast Formation in SF3B1-Mutant MDS

Splicing is a fundamental process by which introns are removed from primary RNA transcripts. Alternative splicing is a major mechanism of gene regulation by which eukaryotic cells expand their transcriptional repertoire. By contrast, aberrant splicing generates novel transcripts not found in normal cells. Heterozygous gain-of-function mutations in a core U2 spliceosome factor SF3B1 are present in ~25% of myelodysplastic syndrome (MDS) patients. Strikingly, SF3B1 mutations are present in ~80% of MDS with ring sideroblasts (MDS-RS) patients suggesting a causal connection between mutant SF3B1 and ring sideroblasts (RS), erythroid precursors with iron-laden mitochondria (Yoshida et al. Nature 2011, Papaemmanuil et al.NEJM 2011). However, the mechanism by which SF3B1 mutations cause RS formation remains poorly understood since existing models of SF3B1-mutant MDS do not recapitulate RS formation in vitro. We have established an induced pluripotent stem cell (iPSC) model of MDS-RS that recapitulates mutant SF3B1-mediated mis-splicing and in vitro ring sideroblast formation. We reprogrammed SF3B1-mutant and SF3B1-wild-type iPSCs from individual MDS-RS patients enabling internal normalization to the isogenic normal clone (Hsu et al. Blood 2019). We established expandable multipotential HPC lines by conditional expression of five transcription factors (5F-HPC), followed by an 18-day erythroid differentiation. We monitored RS formation during the erythroid differentiation of 5F-HPCs using Prussian blue histological staining. RS formation increased during terminal erythropoiesis and peaked at 25-40% on day 18 in SF3B1-mutant lines, whereas SF3B1-wild-type lines showed no detectable RS formation. The frequency of RS was similar in isogenic SF3B1-only and SF3B1/EZH2 co-mutant cells suggesting that the mutant SF3B1 is sufficient to drive RS formation. To identify mis-splicing events that contribute to RS formation, we performed RNA-sequencing and splicing analysis of SF3B1-mutant and SF3B1-wild-type iPSCs at three stages of erythroid differentiation: CD34+ progenitor, CD71+ early erythroblast, and CD71+Glycophorin A+ erythroblast. Global isoform usage was dramatically altered during erythropoiesis, but was more similar in stage-matched SF3B1-mutant and SF3B1-wild-type cells suggesting that mutant SF3B1 selectively mis-splices a subset of transcripts. We identified 2300 transcripts with &amp;gt;10% mis-splicing in SF3B1-mutant lines, and only 120 transcripts with more significant &amp;gt;40% mis-splicing. Of these, TMEM14C and PPOX, inner mitochondrial membrane components of the heme synthesis pathway were strongly mis-spliced throughout erythroid differentiation, consistent with previous studies (Conte et al.BJHaem 2015, Shiozawa et al. Nat Commun 2018). The transcript levels of PPOX but not TMEM14C were reduced in SF3B1-mutant lines as a result of mis-splicing. The expression of ABCB7, a mitochondrial iron sulfur cluster biogenesis component mutated in inherited X-linked sideroblastic anemia (Allikmets et al. Hum Mol Genet 1999), was also reduced in SF3B1-mutant cells as expected (Shiozawa et al. Nat Commun 2018, Dolatshad et al. Leukemia 2016). To investigate the role of these mis-splicing events in ring sideroblast formation, we performed lentiviral overexpression of TMEM14C, PPOX, and ABCB7, in SF3B1-mutant 5F-HPCs and quantified RS formation during late stages of erythroid differentiation. Overexpression of TMEM14C and ABCB7 in SF3B1-mutant cells partially but not completely rescued RS formation compared to luciferase control. These findings confirm the long-standing hypothesis that mis-splicing of mitochondrial iron metabolism genes causes RS formation. Furthermore, these findings suggest that RS formation in MDS is a multigenic event caused by coordinated but incomplete mis-splicing of several critical iron metabolism genes. Disclosures No relevant conflicts of interest to declare.

Smad2/3-pathway ligand trap luspatercept enhances erythroid differentiation in murine β-thalassaemia by increasing GATA-1 availability.

In β‐thalassaemia, anaemia results from ineffective erythropoiesis characterized by inhibition of late‐stage erythroid differentiation. We earlier used luspatercept and RAP‐536 protein traps for certain Smad2/3‐pathway ligands to implicate Smad2/3‐pathway overactivation in dysregulated erythroid differentiation associated with murine β‐thalassaemia and myelodysplasia. Importantly, luspatercept alleviates anaemia and has been shown to reduce transfusion burden in patients with β‐thalassaemia or myelodysplasia. Here, we investigated the molecular mechanisms underlying luspatercept action and pSmad2/3‐mediated inhibition of erythroid differentiation. In murine erythroleukemic (MEL) cells in vitro, ligand‐mediated overactivation of the Smad2/3 pathway reduced nuclear levels of GATA‐1 (GATA‐binding factor‐1) and its transcriptional activator TIF1γ (transcription intermediary factor 1γ), increased levels of reactive oxygen species, reduced cell viability and haemoglobin levels, and inhibited erythroid differentiation. Co‐treatment with luspatercept in MEL cells partially or completely restored each of these. In β‐thalassaemic mice, RAP‐536 up‐regulated Gata1 and its target gene signature in erythroid precursors determined by transcriptional profiling and gene set enrichment analysis, restored nuclear levels of GATA‐1 in erythroid precursors, and nuclear distribution of TIF1γ in erythroblasts. Bone marrow cells from β‐thalassaemic mice treated with luspatercept also exhibited restored nuclear availability of GATA‐1 ex vivo. Our results implicate GATA‐1, and likely TIF1γ, as key mediators of luspatercept/RAP‐536 action in alleviating ineffective erythropoiesis.

Evaluating Metabolic Regulation of Human Erythropoiesis By Profiling Nutrient Transporter Expression

Recent studies have documented that cell metabolism regulates hematopoietic stem cell (HSC) renewal and lineage commitment. However, the detailed metabolic changes that occur during human erythropoiesis remain to be defined. As erythroid cell differentiation is likely to be associated with changes in metabolic requirements, we hypothesized that progenitors adapt to these metabolic modulations by altering their nutrient transporter expression profile. Using an in vitro erythroid-inducing cell culture system employing CD34+ cells from human bone marrow and peripheral blood as well as primary erythroid cells isolated from fresh bone marrow samples, we assessed the cell surface nutrient transporter profiles of progenitors at different stages of erythroid development. Quantification of cell surface nutrient transporter expression was performed using a novel scaffold of retroviral envelope receptor binding domains (RBDs) that function as specific ligands of solute carrier (SLC) nutrient transporters. This bank allowed an evaluation of diverse metabolite transporters including GLUT1/SLC2A1 glucose transporter, the PiT1/SLC20A1 and PiT2/SLC20A2 phosphate importers, the XPR1/SLC53A1 phosphate exporter, the FLVCR1 heme exporter, the RFVT1/2 (SLC52A1/SLC52A2) riboflavin importers, the CAT1/SLC7A1 arginine importer, the ASCT2/SLC1A5 glutamine transporter and the SMVT/SLC5A6 sodium-dependent multivitamin transporter. Notably, the cell surface expression profiles of these nutrient transporters, as evaluated by flow cytometry, revealed marked changes as a function of the stage of erythroid differentiation, as shown in the figure below. Specifically, while FLVCR1, RFVT1/2, and SMVT are highly expressed on erythroid progenitors, the levels of these transporters decrease starting at the proerythroblast stage. In addition, PiT1, PiT2, XPR1, and CAT1 are expressed highly during the erythroid colony forming unit (CFU-E) stage while GLUT1 gradually increases and reaches a peak at late stages of erythroid differentiation, remaining elevated on mature red cells. The noted distinct changes in transporter expression are likely a reflection of the changing demands of various nutrients during human erythropoiesis. In summary, we have established a comprehensive metabolite transporter profile at distinct stages of normal human erythropoiesis using a novel experimental strategy. These original findings form a strong foundation for future studies aiming at elucidating the metabolic requirements of normal erythropoiesis, evaluating diseases affecting red blood cell maturation due to aberrant metabolic regulation, and for identifying new therapeutic targets. Figure Disclosures Bitaudeau: Metafora-biosystems: Employment. Petit:Metafora-biosystems: Equity Ownership, Other: CEO and co-founder. Sitbon:Metafora-biosystems: Membership on an entity's Board of Directors or advisory committees, Other: Co-founder.

Enasidenib Drives Maturation of Human Erythroid Precursors Independently of IDH2

Acute Myeloid Leukemia (AML) remains one of the most difficult cancers to treat, with a 30% 2-year survival rate. High-throughput sequencing of AML patients has identified mutations, including FLT3, IDH1, and IDH2, for which targeted therapies have been developed. Enasidenib is an FDA-approved, first-in-class agent that preferentially inhibits IDH2-mutant activity and reduces levels of the oncometabolite 2-HG, inducing differentiation of IDH2-mutated blasts. Interestingly, greater than 50% of enasidenib-treated patients who had no objective clinical response still demonstrated improvement in their peripheral blood counts and reached RBC transfusion independence. The mechanism underlying this phenomenon is unknown but is of great clinical relevance given the high transfusion dependence and anemia-associated complications universally associated with AML. Thus, we sought to investigate how enasidenib drives normal hematopoiesis to improve quality of life and reduce morbidity in AML patients. In this study, we demonstrate that enasidenib enhances erythropoiesis from normal CD34+ hematopoietic stem and progenitor cells (HSPCs) derived from cord blood (CB) and bone marrow. Enasidenib doubled the proportion and total number of mature CD71+/GPA+ erythroblasts after 8 days of culture with EPO, SCF, and IL-3. In the presence of EPO, enasidenib induced a gene signature characteristic of maturing erythrocytes, with increased expression of GATA1 (1.3 fold), EPOR (2 fold), and KLF1 (1.4 fold), and decreased PU.1 (0.5 fold) and GATA2 (0.7 fold). Enasidenib-treated progenitor cells further demonstrated increased hemoglobin production (1.9 fold) and morphologic characteristics of increased erythroid maturation. Next, we sought to determine if enasidenib augments erythroid differentiation through IDH2 and IDH2-dependent pathways. First, we found that other IDH inhibitors (AG-120, AGI-6780, and AG-881) did not increase erythropoiesis at doses ranging from 1-10μM. As expected for normal HSPCs, 2-HG was not present at detectable levels in either the DMSO or enasidenib-treated conditions, and addition of 2-HG (50, 200μM) did not affect the ability of enasidenib to increase the proportion of CD71+GPA+ cells. Because it is possible that enasidenib acts through inhibition of wild-type IDH2, we generated CRISPR-Cas9 engineered IDH2 knockout (KO) CD34+ cells and treated them with enasidenib. Similar to wildtype cells, IDH2 KO CB CD34+ cells demonstrated a 3.4-fold increase in %CD71+GPA+ erythroid cells. Thus, enasidenib augments erythropoiesis independently of both mutant and wildtype IDH2 pathways. We then investigated the progenitor population that enasidenib acts on to drive erythroid maturation. Enasidenib did not increase the number of BFU-E or CFU-E colonies or the proportion of BFU-E (IL3R-CD34+CD36-) and CFU-E (IL3R-CD34-CD36+) progenitors in colony forming or liquid culture assays, respectively, leading us to conclude that enasidenib acts on more mature erythroid progenitors. Indeed, treating sorted mature CD71+ erythroid progenitors with enasidenib increased %CD71+GPA+ cells compared to DMSO control, whereas enasidenib treatment of CD71- early erythroid progenitors showed no effect. These observations provide evidence that enasidenib acts on CD71+ erythroid progenitors to increase late-stage erythroid differentiation. Given that CD71 allows for iron uptake into erythroid progenitors, we hypothesized that enasidenib modulates the heme biosynthesis pathway. Enasidenib inhibited the ABCG2 transporter, which effluxes protoporphyrin IX (PPIX), the direct precursor to heme, from the mitochondria and cytosol. Inhibition of ABCG2 by enasidenib could lead to PPIX accumulation within the cell, driving increased heme synthesis. To investigate this hypothesis, we treated cells with 20μM Ko143, a potent ABCG2 inhibitor, and observed a similar increase in %CD71+GPA+ cells as seen with enasidenib. Measurement of PPIX autofluorescence by flow cytometry and microscopy revealed an increase of PPIX in enasidenib-treated cells by 1.2-fold. Together, our data suggests that enasidenib drives maturation of CD71+ erythroid precursors independently of wildtype or mutant IDH2. Our results position enasidenib as a promising therapy to stimulate erythropoiesis and provide the basis for a clinical trial using enasidenib to improve anemia in a wide array of clinical contexts. Disclosures Majeti: Forty Seven Inc.: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Patents &amp; Royalties; BioMarin: Consultancy.

Investigation of FoxO3 dynamics during erythroblast development in β-thalassemia major

The FoxO3 transcription factor is a key regulator of oxidative stress and erythroid maturation during erythropoiesis. In this study, we explored the involvement of FoxO3 in severe β-thalassemia. Using primary CD34+ hematopoietic progenitor cells from patients with β-thalassemia major, we successfully developed an in vitro model of ineffective erythropoiesis. Based on this model, FoxO3 activity was quantified in single cells using high throughput imaging flow cytometry. This study revealed a significant reduction of FoxO3 activity during the late stage of erythroblast differentiation in β-thalassemia, in contrast to erythropoiesis in normal cells that maintain persistent activation of FoxO3. In agreement with the decreased FoxO3 activity in β-thalassemia, the expression of FoxO3 target genes was also found to decrease, concurrent with elevated phosphorylation of AKT, most clearly at the late stage of erythroid differentiation. Our findings provide further evidence for the involvement of FoxO3 during terminal erythropoiesis and confirm the modulation of the PI3K/AKT pathway as a potential therapeutic strategy for β-thalassemia.

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.

Pharmacokinetics and Exposure-Response of Luspatercept in Patients with Beta-Thalassemia: Preliminary Results from Phase 2 Studies

▪Background: In β-thalassemia, imbalanced production of α and β globin chains in erythroid precursors inhibits late-stage erythroid differentiation, leading to ineffective erythropoiesis and anemia. Luspatercept is a modifiedactivin receptor type IIB-IgG Fc fusion protein that promotes terminal erythroid differentiation and mitigates ineffective erythropoiesis in murine β-thalassemia. In phase 2 studies, luspatercept led to long-term increases in hemoglobin (Hb) levels and reduction in transfusion burden in patients (pts) with β-thalassemia.Aims: To characterize the pharmacokinetics (PK) of luspatercept, and to explore the exposure-response relationship for efficacy and safety in pts with β-thalassemia, thereby informing selection of the starting dose for phase 3 studies of luspatercept in β-thalassemia.Methods: PK, safety, and efficacy data were collected from two phase 2 studies (base and extension). In the base study, luspatercept was administered once every 3 weeks by subcutaneous injection to sequential cohorts for up to 5 doses. The base study included a dose-finding phase (at fixed doses ranging from 0.2 mg/kg to 1.25 mg/kg) and an expansion cohort (at a starting dose of 0.8 mg/kg followed by individual dose titration up to 1.25 mg/kg). Pts completing the base study were eligible to enroll in an extension study, where they continued to receive luspatercept every 3 weeks for up to 24 months. Pts who had treatment interruption for ≥ 3 months before enrolling in the extension study received a starting dose of 0.8 mg/kg (followed by dose titration) and were treated as "new" pts in the exposure-response analysis. The main exposure endpoint was area under the luspatercept serum concentration-time curve (AUC). Clinical endpoints included Hb increase, transfusion reduction, and drug-related adverse events (AEs) in weeks 1-15. Responders were defined as non-transfusion-dependent (NTD) pts with a mean Hb increase ≥ 1.0 g/dL/15 weeks, and transfusion-dependent (TD) pts with a transfusion reduction ≥ 33%/15 weeks.Results: As of July 20, 2016, preliminary data were available for 89 pts, including 49 NTD pts (baselineHb 6.5-9.8 g/dL) and 40 TD pts (baseline transfusion burden 4-18 units/12 weeks). Median age was 37 years (range 20-62), and 47% were female. A total of 49 pts were eligible for individual dose titration, with ~71% having at least 1 dose escalation (to 1 mg/kg) and 27% having 2 dose escalations (up to 1.25 mg/kg) within the first 5 cycles.Luspatercept PK was adequately described by a 1-compartment PK model with linear absorption and elimination. The half-life of luspatercept in serum was~10 days across doses. Body weight positively correlated with luspatercept clearance and its volume of distribution. TD pts had~23% lower volume of distribution than NTD pts.In NTD pts, higher luspatercept serum AUC correlated with a greater increase in Hblevels (P < 0.01). Most TD pts had a reduction in transfusion burden; however, an exposure-dependent trend was not apparent for relative or absolute reduction in units transfused, possibly due to the narrow exposure range tested. The median AUC was similar for NTD responders and TD responders (~100d·μg/mL vs ~120d·μg/mL). As AUC increased, the frequency of responders increased for NTD pts, TD pts, and the 2 groups combined (Figure). Population PK simulation predicted that a starting dose of 1.0 mg/kg luspatercept would result in > 90% of NTD pts and ~50% of TD pts achieving the median AUC concentration observed in responders. By contrast, < 50% pts (NTD or TD) were predicted to achieve the target AUC at the 0.8 mg/kg dose.Grade 2-3 drug-related AEs (all types) were more frequent with higher AUC (P < 0.05). In both NTD and TD pts, bone and muscle pain were the most common AEs; however, these AES did not have a significant relationship with luspatercept serum exposure.Conclusions: Higher luspatercept serum exposure correlated with greater erythroid hematopoietic response as well as more frequent grade 2-3 related AEs. Exposure-response modeling and PK simulation support a phase 3 starting dose of 1.0 mg/kg and intra-patient dose escalation up to 1.25 mg/kg according to pts' response. A phase 3 study of luspatercept in regularly transfused adults with β-thalassemia is ongoing (BELIEVE study; ClinicalTrials.gov NCT02604433). [Display omitted] DisclosuresChen:Celgene Corporation: Employment, Equity Ownership. Laadem:Celgene Corporation: Employment, Equity Ownership. Wilson:Acceleron Pharma: Employment, Equity Ownership. Zhang:Acceleron Pharma: Employment. Sherman:Acceleron Pharma: Employment, Equity Ownership, Patents & Royalties. Ritland:Celgene Corporation: Employment, Equity Ownership. Attie:Acceleron Pharma: Employment, Equity Ownership.

Luspatercept Increases Hemoglobin, Decreases Transfusion Burden and Improves Iron Overload in Adults with Beta-Thalassemia

▪Background.Luspatercept (ACE-536) is a modified activin receptor type IIB fusion protein that promotes late-stage erythroid differentiation. In beta-thalassemia, imbalanced production of alpha and beta globin chains in erythroid precursors causes ineffective erythropoiesis (IE) leading to anemia and dysregulated iron homeostasis. In a mouse model of beta-thalassemia, luspatercept corrected the effects of ineffective erythropoiesis and in a phase 1 clinical study with healthy volunteers, was well tolerated and increased hemoglobin (Suragani R, Nat Med, 2014; Suragani R, Blood, 2014; Attie K, Am J Hematol, 2014).Aims.This is an ongoing, phase 2, multicenter, open-label, dose-finding study followed by a long-term extension study to evaluate the effects of luspatercept in patients (pts) with either transfusion-dependent (TD) or non-transfusion dependent (NTD) beta-thalassemia with the following key endpoints: erythroid response (including Hgb increase) and patient-reported quality-of-life (QoL) measures in NTD patients, and reductions in RBC transfusion burden in TD patients.Methods.Inclusion criteria included age ≥ 18 yr and either TD (defined as ≥ 4 RBC units transfused in the 8 weeks prior to first dose, confirmed over 6 months) or NTD (defined as < 4 RBC units transfused in the 8 weeks prior to first dose with baseline Hgb < 10.0 g/dL). Luspatercept was administered subcutaneously every 3 weeks for up to 5 doses over 12 weeks with an 8 week follow-up (20 weeks total). Six cohorts (n=35) were treated at dose levels from 0.2-1.25 mg/kg. Pts in the expansion cohort (n=29) and all pts who rolled over to the extension study (n=51) were treated at ≥ 0.8 mg/kg with escalation up to 1.25 mg/kg.Results. A total of 30 TD pts enrolled in the base study and, of those, 24 enrolled in the extension study (data as of 11 March 2016). Data summarized are for the long-term extension study. Median age was 38 yr (range 22-55 yr), 67% had prior splenectomy. At baseline, median transfusion burden was 8 units/12 weeks (range 4-15 units) and mean (SD) liver iron concentration (LIC) was 5.1 (5.3) mg/g dw. A total of 20/24 (83%) and 16/24 (67%) TD pts achieved a ≥ 33% and ≥ 50% decrease in transfusion burden over any 12-week period compared to baseline, respectively. Duration of response ranged from 12 to 48+ weeks.A total of 34 NTD pts enrolled in the base study and, of those, 27 enrolled in the extension study (data as of 11 March 2016). Data summarized are for the long-term extension study. Median age was 37 yr (range 23-62 yr); 67% had prior splenectomy. At baseline, median Hgb was 8.7 g/dL (range 7.6-9.8 g/dL) and mean (SD) LIC was 4.9 (3.4) mg/g dw. A total of 21/27 (78%) and 15/27 (56%) NTD pts achieved ≥ 1.0 g/dL and ≥ 1.5 g/dL increases, respectively, in mean Hgb over any 12-week period compared to baseline. Duration of response ranged from 16 to 72+ weeks, with no trend for lower Hgb response over time. Increases in mean hemoglobin over a 12-week period correlated with increases in a pt-reported QoL questionnaire, FACIT-F (r=0.67, p=0.001). 3/5 (60%) pts with baseline LIC ≥ 5 mg/g dw had a decrease in LIC ≥ 2 mg/g dw after at least 6 months of treatment; 8/9 (89%) patients with baseline LIC < 5 mg/g dw maintained LIC < 5 mg/g dw.Luspatercept was generally well tolerated, with no related SAEs. AEs were mostly mild-moderate. The most frequent related AEs (≥ 10% in base + extension studies) in TD pts were bone pain, myalgia, arthralgia, headache, asthenia, and musculoskeletal pain. The most frequent related AEs (≥ 10% in base + extension studies) in NTD pts were bone pain, headache, musculoskeletal pain, and arthralgia.Conclusions: Luspatercept treatment in pts with beta-thalassemia had a favorable safety profile. Efficacy was clinically relevant in both NTD pts (increased Hgb levels, decreased LIC, and improved quality of life) and TD pts (decreased RBC transfusions). A Phase 3, double-blind, placebo-controlled study of luspatercept in regularly transfused adults with beta-thalassemia is ongoing (NCT02604433). DisclosuresZhang:Acceleron Pharma: Employment. Wilson:Acceleron Pharma: Employment, Equity Ownership. Leneus:Acceleron Pharma: Employment. Laadem:Celgene Corporation: Employment, Equity Ownership. Sherman:Acceleron Pharma: Employment, Equity Ownership, Patents & Royalties. Attie:Acceleron Pharma: Employment, Equity Ownership.

Luspatercept Response in ESA-NaïVe/RS+ Patients and RS- Patients with Low-Intermediate Risk Myelodysplastic Syndromes (MDS)

Background: Management of anemia is a common therapeutic challenge in patients with myelodysplastic syndromes (MDS). Luspatercept (ACE-536), a fusion protein containing modified activin receptor type IIB, is being developed for treatment of anemia in lower-risk MDS. Luspatercept binds GDF11 and other TGF-β superfamily ligands to promote late-stage erythroid differentiation and increase hemoglobin (Hgb) levels (Suragani R, Nat Med, 2014 and Attie K, Am J Hematol, 2014).Aims: This is an ongoing, phase 2, multicenter, open-label study to evaluate the effects of luspatercept in patient (pts) with low-intermediate risk MDS. Endpoints included erythroid response (IWG HI-E), RBC transfusion independence (RBC-TI, ≥ 8 weeks), duration of HI-E, pharmacodynamic and iron metabolism biomarkers, safety, and pt-reported QoL.Methods: Inclusion criteria included age ≥ 18 yr, Hgb < 10 g/dL (if < 4U RBC/8 weeks), no prior HMA, and no current lenalidomide or erythropoiesis-stimulating agent (ESA). An expansion cohort of up to 56 patients was added to this phase 2 study to evaluate response to luspatercept in pts who do not qualify for the phase 3 MEDALIST trial (for RS+ positive patients with baseline EPO ≥ 200 U/L and ≥ 2U RBC/8 weeks). These include pts with low transfusion burden (< 4U RBC/8 weeks) who are either 1) ring sideroblast (RS)+ (≥ 15% RS in bone marrow) with baseline EPO ≤ 200 U/L and no prior ESA use, or 2) RS- with any baseline EPO level and any prior ESA use. Patients are treated with 1.0 mg/kg of luspatercept every 3 weeks for up to 5 doses, with titration up to 1.75 mg/kg. Patients may rollover to an open-label extension study for up to an additional 2 years of treatment.Results: Results for the initial patient cohorts have demonstrated a high proportion of HI-E and RBC-TI responses in RS+ patients. Data for the additional ESA-naïve RS+ patients with low EPO levels and RS- patients with 3 months of treatment will be presented at the meeting.Conclusions: Erythroid response to luspatercept has been demonstrated in RS+ patients with lower-risk MDS and is being explored in ESA-naïve RS+ patients with low EPO levels and RS- patients. A Phase 3 study of luspatercept in regularly-transfused RS+ patients with lower-risk MDS according to IPSS-R is ongoing (MEDALIST study; NCT02631070). DisclosuresDonovan:Acceleron Pharma: Employment. Wilson:Acceleron Pharma: Employment, Equity Ownership. Zhang:Acceleron Pharma: Employment. Laadem:Celgene Corporation: Employment, Equity Ownership. Sherman:Acceleron Pharma: Employment, Equity Ownership, Patents & Royalties. Attie:Acceleron Pharma: Employment, Equity Ownership. Giagounidis:Celgene Corporation: Consultancy.

Luspatercept Increases Hemoglobin and Reduces Transfusion Burden in Patients with Low-Intermediate Risk Myelodysplastic Syndromes (MDS): Long-Term Results from Phase 2 PACE-MDS Study

▪Background: Management of anemia is a common therapeutic challenge in patients with MDS. Luspatercept (ACE-536), a fusion protein containing modified activin receptor type IIB, is being developed for treatment of anemia in lower-risk MDS. Luspatercept binds GDF11 and other TGF-β superfamily ligands to promote late-stage erythroid differentiation and increase hemoglobin (Hgb) levels (Suragani R, Nat Med, 2014 and Attie K, Am J Hematol, 2014).Aims: This is an ongoing, phase 2, multicenter, open-label, long-term extension study to evaluate the effects of luspatercept in patients (pts) with low-intermediate risk MDS. Endpoints include long-term safety and tolerability, erythroid response (IWG HI-E), RBC transfusion independence (RBC-TI, ≥ 8 weeks), duration of HI-E, pharmacodynamic and iron metabolism biomarkers, and pt-reported QoL.Methods: Inclusion criteria included age ≥ 18 yr, Hgb < 10 g/dL (if < 4U RBC/8 weeks), ESA refractory or EPO > 500 U/L, no prior HMA, and no current lenalidomide or ESA. Luspatercept was administered SC every 3 wks for up to 5 doses in the base study (NCT01749514), including 7 dose escalation cohorts (n=27 total, 0.125 to 1.75 mg/kg) and an expansion cohort (n=31, starting dose 1.0 mg/kg, max 1.75 mg/kg). A 2-year extension study (n=32) is ongoing (NCT02268383).Results: Data (as of 4 Mar 2016) were available for the 32 extension study pts. Of these, 13 pts received < 4U RBC/8 weeks pretreatment (low transfusion burden, LTB) and 19 pts received ≥ 4U RBC/8 weeks (high transfusion burden, HTB). Median age was 72 yr (range 29-90 yr), 59% had prior ESA. Median Hgb for LTB pts was 8.5 g/dL (range 6.4-10.1 g/dL) and median RBC transfusion burden for HTB pts was 6 U/8 weeks (range 4-14 units). 91% pts were RS+ (≥ 15% RS in bone marrow).IWG HI-E was achieved in 11/13 (85%) LTB pts and 15/19 (79%) HTB pts. 11/22 (50%) pts with at least 2 units transfused in 8 weeks prior to dosing with luspatercept achieved RBC transfusion independence for at least 8 weeks. The range of transfusion independence was 9 to 80+ weeks, with most responders still receiving treatment. IWG HI-E response rates were 83% for RS+ pts, 90% for EPO < 200 U/L, 86% for EPO 200-500 U/L, and 50% for EPO > 500 U/L; 85% for ESA-naïve and 79% for those who had prior ESA treatment. RBC transfusion independence was achieved in 58% for EPO < 200 U/L, 50% for EPO 200-500 U/L, and 33% for EPO > 500 U/L.Luspatercept was well tolerated, with 3 related grade 3 adverse events of myalgia, worsening of general condition, and blast cell count increase. The most common related AEs (≥ 2 pts in both base and extension studies) were fatigue, bone pain, diarrhea, myalgia, headache, hypertension, and injection site erythema.Conclusions: Long-term treatment with luspatercept was well tolerated and led to erythroid response in 81% of low-intermediate risk MDS pts who enrolled into the extension study. A Phase 3 study of luspatercept in regularly-transfused RS+ patients with lower-risk MDS according to IPSS-R is ongoing (MEDALIST study; NCT02631070). DisclosuresPlatzbecker:Onconova, Teva, Celgene, Janssen, Novartis, Amgen: Honoraria, Research Funding. Donovan:Acceleron Pharma: Employment. Wilson:Acceleron Pharma: Employment, Equity Ownership. Zhang:Acceleron Pharma: Employment. Laadem:Celgene Corporation: Employment, Equity Ownership. Sherman:Acceleron Pharma: Employment, Equity Ownership, Patents & Royalties. Attie:Acceleron Pharma: Employment, Equity Ownership. Giagounidis:Celgene Corporation: Consultancy.

Luspatercept (ACE-536) Reduces Disease Burden, Including Anemia, Iron Overload, and Leg Ulcers, in Adults with Beta-Thalassemia: Results from a Phase 2 Study

Background. Luspatercept is a fusion protein (modified activin receptor IIB-IgG Fc) being investigated for the treatment of anemias with ineffective erythropoiesis. In beta-thalassemia, imbalanced production of alpha and beta globin chains in erythroid precursors inhibits late-stage erythroid differentiation, leading to anemia, ineffective erythropoiesis (IE), and dysregulated iron homeostasis. Luspatercept promotes late-stage erythroid differentiation, corrects ineffective erythropoiesis, and reduces alpha globin aggregates, hemolysis, and disease complications including iron overload in a beta-thalassemia mouse model (Suragani R, Blood 2014). Aims. This is an ongoing, phase 2, multicenter, open-label, dose-finding study to evaluate luspatercept in adults with transfusion-dependent (TD) or non-transfusion dependent (NTD) beta-thalassemia. Efficacy outcomes include erythroid response including Hb increase in NTD patients and reduction in RBC transfusion burden in TD patients, and reduction in liver iron concentration (LIC) by MRI in both TD and NTD patients. Methods. Inclusion criteria included age ≥ 18 yr and either TD (defined as ≥4 RBC units transfused in the 8 weeks prior to first dose, confirmed over 6 months) or NTD (defined as &lt;4 RBC units transfused in the 8 weeks prior to first dose) with baseline Hb &lt;10.0 g/dL. Luspatercept was administered SC every 3 weeks for up to 5 doses (12 weeks) with an 8-week follow-up (20 weeks total). Six sequential cohorts (n=up to 6 each) were treated at escalating dose levels from 0.2 to 1.25 mg/kg and an expansion cohort (n=30) is ongoing with starting dose level 0.8 mg/kg and individual dose titration. Patients who completed the core study may have been eligible to enroll in an ongoing 24-month extension study. Results. Preliminary data (as of 10-Apr-2015) were available for 39 patients, 35 patients from dose escalation, and 4 patients from the expansion. Median age was 40 yr, ranging from 20-57 yr, and 82% had prior splenectomy. TD patients (n=14). Transfusion burden prior to treatment ranged from 4 to 12 units/12 weeks. Ten of 14 (71%) patients were treated for ≥12 weeks and were evaluable for the primary endpoint of ≥20% reduction in transfusion burden. All 10 of these patients responded with &gt;40% reduction in transfusion burden over any 12-week period during the study. Reduction in transfusion burden correlated with reduction in LIC. Two of the 3 TD patients with baseline LIC ≥7 mg/g dw (all on iron chelation therapy) had a reduction in LIC at 16 weeks (-2.0 and -4.7 mg/g dw). NTD patients (n=25). Median baseline Hb was 8.4 g/dL (range 6.5-9.6 g/dL). Four of 8 (50%) patients in the 0.8-1.25 mg/kg dose groups achieved the primary endpoint of increase in Hb ≥1.5 g/dL sustained for ≥2 weeks, compared with 0 of 17 (0%) patients in the 0.2-0.6 mg/kg dose groups. Four of eight (50%) patients in the 0.8-1.25 mg/kg dose groups had a mean increase in Hb ≥1.0 g/dL sustained for ≥12 weeks, compared with 5 of 17 (29%) patients in the 0.2-0.6 mg/kg dose groups. Eight of 12 (67%) NTD patients with baseline LIC ≥5 mg/g dw (including 6 patients on iron chelation therapy) had a reduction of at least 1 mg/g dw in LIC at 16 weeks; reduction in LIC correlated with increase in hemoglobin. All 3 patients with chronic leg ulcers at baseline (2 NTD, 1 TD) had substantial, rapid healing, evident within 6 weeks after the first dose of luspatercept. Luspatercept was generally well tolerated, with no related serious adverse events reported to date. Adverse events were mostly mild-moderate; most frequent related adverse events (&gt;10% patients) were bone pain, myalgia, headache and asthenia. Longer-term treatment data from the extension study for TD and NTD patients will be presented. Conclusions. Luspatercept treatment for up to 3 months was well-tolerated, increased Hb levels in NTD patients, and decreased transfusion requirement in TD patients with beta-thalassemia. Both TD and NTD patients also had decreases in LIC, and healing of leg ulcers occurred in 3 of 3 patients. These treatment effects represent a significant reduction in disease burden for patients with beta-thalassemia. Phase 3 studies of luspatercept in beta-thalassemia are planned. Disclosures Piga: Celgene Corporation: Honoraria; Acceleron Pharma: Research Funding. Zhang:Acceleron: Employment. Bellevue:Acceleron: Employment. Wilson:Acceleron: Employment. Laadem:Celgene Corporation: Employment. Sherman:Acceleron Pharma: Employment. Attie:Acceleron: Employment.

Biomarkers of Ineffective Erythropoiesis Predict Response to Luspatercept in Patients with Low or Intermediate-1 Risk Myelodysplastic Syndromes (MDS): Final Results from the Phase 2 PACE-MDS Study

Background: Luspatercept is a fusion protein (modified activin receptor IIB-IgG Fc) being investigated for the treatment of anemias with ineffective erythropoiesis. MDS patients have increased Smad2/3 signaling in the bone marrow, leading to ineffective erythropoiesis. Luspatercept inhibits Smad2/3 signaling and promotes late-stage erythroid differentiation, thereby correcting ineffective erythropoiesis.Aims: This completed, 3-month, phase 2, multicenter, open-label study evaluated the effects of luspatercept on anemia in patients with low/int-1 risk MDS (IPSS classification). Study outcomes include erythroid response of increased hemoglobin (Hb) in low transfusion burden (LTB) patients (<4 RBC units/8 weeks), reduced transfusions in high transfusion burden (HTB) patients (≥4 RBC units/8 weeks), transfusion independence, safety, PK, and PD biomarkers.Methods: Inclusion criteria include age ≥ 18 yr, anemia defined as being HTB or LTB with Hb <10.0 g/dL, EPO >500 U/L or nonresponsive/refractory to ESAs, no prior azacitidine or decitabine, and no current treatment with ESA, G-CSF, GM-CSF, or lenalidomide. Luspatercept was administered once every 3 weeks by SC injection in sequential cohorts (n=3-6 each) at dose levels ranging from 0.125 to 1.75 mg/kg for up to 5 doses with a 3-month follow-up. An expansion cohort with a starting dose level of 1.0 mg/kg was enrolled with individual patient dose titration allowed. Patients completing this study were eligible to enroll into the PACE-MDS extension study.Results: Enrollment is complete for 27 patients in the dose escalation cohorts and 31 patients in the expansion cohort (total n=58). Preliminary safety and efficacy data (as of 17-Apr-2015) were available for 49 patients (22 female/27 male, 17 LTB/32 HTB). Final 3-month data for all 58 patients will be presented. Median age was 71 years, 61% had prior ESA therapy, and 18% had prior lenalidomide. Forty (83%) patients had ≥15% ring sideroblasts (RS+) in the bone marrow. The median baseline Hb for LTB patients was 8.7 g/dL (range 6.8-10.1 g/dL). The median number of RBC units transfused over 8 weeks prior to treatment for HTB patients was 6 units (range 4-14 units). Luspatercept was generally well-tolerated.In patients (n=40) receiving 0.75-1.75 mg/kg, 48% of patients responded per IWG HI-E (Hb increase ≥1.5 g/dL for LTB patients or reduction of ≥ 4 RBC units/8 weeks for HTB patients). Higher response rates were observed in RS+ patients, including patients with EPO ≥ 200 U/L; patients with splicing factor (SF) mutations present (primarily SF3B1) had 60% response rate (see Table). Of the patients who received RBC transfusions prior to luspatercept treatment (range 2-14 units/8 weeks), 11 of 30 (37%) patients were transfusion-free for ≥8 weeks during the 12 week treatment period. Neutrophil responses (IWG HI-N) were seen in 4 of 8 (50%) patients with baseline neutrophil count <1.0 X 10^9/L. TablePatient SubgroupIWG HI-E Response Rate (0.75-1.75 mg/kg)RS positive119/35 (54%)EPO < 200 U/L14/23 (61%)EPO ≥ 200 U/L5/12 (42%)RS negative10/4 (0%)SF mutation2 present18/30 (60%)SF mutation2 absent1/9 (11%)1RS: Ring sideroblasts ≥15% in bone marrow; data available for n=392SF: Splicing factor mutations: SF3B1, SRSF2, U2AF1, or ZRSR2; data available for n=39The relationship of erythroid response to bone marrow erythroid cellularity, morphology and characteristics, comprehensive gene mutation profile, GDF15, and other biomarkers of ineffective erythropoiesis will be presented.Conclusions: Based on these data, treatment with luspatercept in lower risk MDS patients at therapeutic dose levels for 3 months led to a high response rate for increased Hb levels or decreased transfusion burden, including transfusion independence, with a favorable safety profile. Ring sideroblasts, splicing factor gene mutations and other biomarkers of ineffective erythropoiesis may help define a MDS population most likely to respond to luspatercept treatment. Phase 3 studies in patients with anemia due to lower risk MDS are planned. DisclosuresPlatzbecker:Celgene: Honoraria, Research Funding; Novartis: Honoraria, Research Funding; Boehringer: Research Funding. Ottmann:BMS: Honoraria, Research Funding; Novartis: Consultancy. Hankin:Acceleron: Employment. Wilson:Acceleron: Employment. Zhang:Acceleron: Employment. Laadem:Celgene Corporation: Employment. Sherman:Acceleron Pharma: Employment. Attie:Acceleron: Employment.

Luspatercept Treatment Leads to Long Term Increases in Hemoglobin and Reductions in Transfusion Burden in Patients with Low or Intermediate-1 Risk Myelodysplastic Syndromes (MDS): Preliminary Results from the Phase 2 PACE-MDS Extension Study

Background: Luspatercept is a fusion protein (modified activin receptor IIB-IgG Fc) being investigated for the treatment of anemias with ineffective erythropoiesis. MDS patients have increased Smad2/3 signaling in the bone marrow, leading to ineffective erythropoiesis. Luspatercept inhibits Smad2/3 signaling and promotes late-stage erythroid differentiation, thereby correcting the ineffective erythropoiesis. Aims: This is an ongoing, phase 2, multicenter, open-label, 24-month extension study (following a 3-month base study) to evaluate the longer-term effects of luspatercept on anemia in patients (pts) with low/int-1 risk MDS (IPSS classification). Study outcomes include erythroid response of increased Hb in low transfusion burden (LTB) pts (&lt;4 RBC units/8 weeks), reduced transfusions in high transfusion burden (HTB) patients (≥4 RBC units/8 weeks), transfusion independence, safety, PK, and PD biomarkers. Methods: In the base study, eligibility criteria included age ≥18 yr; anemia defined as being HTB or LTB with Hb &lt;10.0 g/dL; EPO &gt;500 U/L or nonresponsive/refractory to ESAs; no prior azacitidine or decitabine; and no current treatment with ESA, G-CSF, GM-CSF, or lenalidomide. Luspatercept was administered once every 3 weeks by SC injection in sequential cohorts (n=3-6 each; total n=27) at dose levels ranging from 0.125 to 1.75 mg/kg for up to 5 doses with a 3-month follow-up. An expansion cohort (n=31) with a starting dose level of 1.0 mg/kg was enrolled with individual pt dose titration allowed. Patients completing the 3-month base study were eligible to enroll into this extension study (with or without interruption ≥3 months) where they continue to receive luspatercept every 3 weeks for up to 24 months. Results: Enrollment is complete for this extension study (n=32). Preliminary safety and efficacy data (as of 17-Apr-2015) were available for 22 of 32 patients (9 LTB/13 HTB) with continuous luspatercept treatment for a median of 8 cycles (range 5-12). Median age was 70.5 yr, 64% had prior ESA therapy, and 18% had prior lenalidomide. All 22 patients had ≥15% ring sideroblasts (RS+) in bone marrow. Eight of 9 (89%) of LTB patients achieved an IWG HI-E response for hemoglobin increase (≥1.5 g/dL for ≥8 weeks). Mean change in hemoglobin in these patients exceeded 1.5 g/dL for a median duration of 17 weeks (range 5-22 weeks) as of the data cutoff date in this ongoing study. Ten of 13 (77%) of HTB patients achieved an IWG HI-E response (reduction of ≥4 RBC units transfused over 8 weeks). Of the 14 patients who received transfusion of ≥2 RBC units over 8 weeks prior to the start of treatment, 6 (43%) patients achieved transfusion independence for at least 8 weeks (median 23 weeks, range 10-30 weeks as of the data cutoff date). All 6 patients were continuing to receive treatment as of the data cutoff date; updated data for duration of response will be presented. Luspatercept has been generally well-tolerated in the extension study, with no grade 3 or higher related adverse events. Conclusions: Based on preliminary data from the 24-month extension study in lower risk MDS patients, luspatercept treatment led to sustained HI-E response for increased Hb levels, decreased transfusion requirement or transfusion independence in the majority of patients, with a favorable safety profile. Phase 3 studies in patients with anemia due to lower risk MDS are planned. Disclosures Platzbecker: Boehringer: Research Funding; Celgene: Honoraria, Research Funding; Novartis: Honoraria, Research Funding. Ottmann:BMS: Honoraria, Research Funding; Novartis: Consultancy. Hankin:Acceleron: Employment. Wilson:Acceleron: Employment. Zhang:Acceleron: Employment. Laadem:Celgene Corporation: Employment. Sherman:Acceleron Pharma: Employment. Attie:Acceleron: Employment.

53 LUSPATERCEPT INCREASES HEMOGLOBIN AND REDUCES TRANSFUSION BURDEN IN PATIENTS WITH LOW OR INTERMEDIATE-1 RISK MYELODYSPLASTIC SYNDROMES (MDS): PRELIMINARY RESULTS FROM A PHASE 2 STUDY

Introduction. ACE-536, a recombinant fusion protein containing modified activin receptor type IIB and IgG Fc, is being developed for the treatment of anemias due to ineffective erythropoiesis, such as myelodysplastic syndromes (MDS). Patients with MDS often have elevated levels of erythropoietin (EPO) and may be non-responsive or refractory to erythropoiesis-stimulating agents (ESAs). MDS patients have also been shown to have increased serum GDF11 levels (Suragani R et al., Nature Medicine 2014) and increased Smad 2/3 signaling in the bone marrow (Zhou L et al., Blood 2008). ACE-536 binds to ligands in the TGF-s superfamily, including GDF11, inhibits Smad 2/3 signaling, and promotes late-stage erythroid differentiation via a mechanism distinct from ESAs. RAP-536 (murine version of ACE-536) reduced Smad 2 signaling, increased hemoglobin (Hb) levels and decreased bone marrow erythroid hyperplasia in a mouse model of MDS (Suragani R et al., Nature Medicine 2014). In a healthy volunteer study, ACE-536 was well-tolerated and increased Hb levels (Attie K et al., Am J Hematol 2014). Aims. This is an ongoing, phase 2, multicenter, open-label, dose-finding study to evaluate the effects of ACE-536 on anemia in patients with Low or Int-1 risk MDS who have either high transfusion burden (HTB, defined as ≥4 units RBCs/8 weeks prior to baseline) or low transfusion burden (LTB, defined as Methods.Inclusion criteria included Low or Int-1 risk MDS, age ≥ 18 yr, anemia (defined as either being HTB patient or having baseline Hb 500 U/L or nonresponsive/refractory to ESAs, no prior azacitidine or decitabine, and no current treatment with ESA, G-CSF, GM-CSF, or lenalidomide. In the dose escalation phase, ACE-536 was administered by subcutaneous (SC) injection once every 3 weeks in 7 sequential cohorts (n=3-6) at dose levels of 0.125, 0.25, 0.5, 0.75, 1.0, 1.33 and 1.75 mg/kg for up to 5 doses with a 3-month follow-up. An expansion cohort (n=30) is planned, and all patients completing this study may enroll in a 12-month extension study. Results. Preliminary data were available for 26 patients (7 LTB/19 HTB) as of 18 Jul 2014. Median age was 71 yr (range: 27-88 yr), 50% were female, 54% had prior EPO therapy and 15% had prior lenalidomide. 69% were WHO subtype RCMD, and the remaining patients were del(5q), RARS, or RAEB-1. Mean (SD) baseline Hgb for the LTB patients (n=7) was 9.1 (0.4) g/dL. Mean (SD) units RBC transfused in the 8 weeks prior to treatment was 0.9 (1.1) units for the LTB patients and 6.3 (2.4) units for the HTB patients. Two of the 7 LTB patients had an increase in mean Hb ≥1.5 g/dL over 8 weeks compared to baseline. Mean maximum Hb increase in the LTB patients was 0.8, 1.0, 2.2, and 2.7 g/dL in the 0.125 (n=1), 0.25 (n=1), 0.75 (n=3), and 1.75 (n=2) mg/kg dose groups, respectively. Six of the 7 LTB patients achieved RBC transfusion independence (RBC-TI) for ≥8 weeks during the study. Six of the 19 HTB patients had a ≥4 unit or ≥50% reduction in RBC units transfused over an 8-week interval during the treatment period compared to the 8 weeks prior to treatment; five of these 6 patients achieved RBC-TI ≥ 8 weeks during the study (range 71-152 days). Increases in neutrophil count following study drug administration were observed in some patients. ACE-536 was generally well tolerated. No related serious adverse events have been reported to date. The most frequent adverse events regardless of causality were: diarrhea (n=4, grade 1/2), bone pain, fatigue, muscle spasms, myalgia, and nasopharyngitis (n=3 each, grade 1/2). Conclusions. Based on preliminary data in Low or Int-1 MDS patients, ACE-536 administered SC every 3 weeks for up to 5 doses increased Hb levels or decreased transfusion requirement, with a favorable safety profile. These data strongly support further evaluation of longer-term treatment with ACE-536 in patients with MDS. Disclosures Platzbecker:Celgene: Honoraria, Research Funding. Germing:Celgene: Honoraria, Research Funding. Goetze:Celgene Corp: Honoraria; Novartis Pharma: Honoraria. Radsak:Celgene: Research Funding. Hankin:Acceleron Pharma: Employment, Equity Ownership. Wilson:Acceleron Pharma: Employment, Equity Ownership. Sherman:Acceleron Pharma: Employment, Equity Ownership. Attie:Acceleron Pharma: Employment, Equity Ownership.

ACE-536 Increases Hemoglobin and Reduces Transfusion Burden in Patients with Low or Intermediate-1 Risk Myelodysplastic Syndromes (MDS): Preliminary Results from a Phase 2 Study

▪Introduction. ACE-536, a recombinant fusion protein containing modified activin receptor type IIB and IgG Fc, is being developed for the treatment of anemias due to ineffective erythropoiesis, such as myelodysplastic syndromes (MDS). Patients with MDS often have elevated levels of erythropoietin (EPO) and may be non-responsive or refractory to erythropoiesis-stimulating agents (ESAs). MDS patients have also been shown to have increased serum GDF11 levels (Suragani R et al., Nature Medicine 2014) and increased Smad 2/3 signaling in the bone marrow (Zhou L et al., Blood 2008). ACE-536 binds to ligands in the TGF-ß superfamily, including GDF11, inhibits Smad 2/3 signaling, and promotes late-stage erythroid differentiation via a mechanism distinct from ESAs. RAP-536 (murine version of ACE-536) reduced Smad 2 signaling, increased hemoglobin (Hb) levels and decreased bone marrow erythroid hyperplasia in a mouse model of MDS (Suragani R et al., Nature Medicine 2014). In a healthy volunteer study, ACE-536 was well-tolerated and increased Hb levels (Attie K et al., Am J Hematol 2014).Aims. This is an ongoing, phase 2, multicenter, open-label, dose-finding study to evaluate the effects of ACE-536 on anemia in patients with Low or Int-1 risk MDS who have either high transfusion burden (HTB, defined as ≥4 units RBCs/8 weeks prior to baseline) or low transfusion burden (LTB, defined as <4 units RBCs/8 weeks prior to baseline). Study outcomes include erythroid response (either Hb increase in LTB patients or reduced transfusion burden in HTB patients), safety, tolerability, PK, and PD biomarkers.Methods.Inclusion criteria included Low or Int-1 risk MDS, age ≥ 18 yr, anemia (defined as either being HTB patient or having baseline Hb < 10.0 g/dL in LTB patient), EPO >500 U/L or nonresponsive/refractory to ESAs, no prior azacitidine or decitabine, and no current treatment with ESA, G-CSF, GM-CSF, or lenalidomide. In the dose escalation phase, ACE-536 was administered by subcutaneous (SC) injection once every 3 weeks in 7 sequential cohorts (n=3-6) at dose levels of 0.125, 0.25, 0.5, 0.75, 1.0, 1.33 and 1.75 mg/kg for up to 5 doses with a 3-month follow-up. An expansion cohort (n=30) is planned, and all patients completing this study may enroll in a 12-month extension study.Results. Preliminary data were available for 26 patients (7 LTB/19 HTB) as of 18 Jul 2014. Median age was 71 yr (range: 27-88 yr), 50% were female, 54% had prior EPO therapy and 15% had prior lenalidomide. 69% were WHO subtype RCMD, and the remaining patients were del(5q), RARS, or RAEB-1. Mean (SD) baseline Hgb for the LTB patients (n=7) was 9.1 (0.4) g/dL. Mean (SD) units RBC transfused in the 8 weeks prior to treatment was 0.9 (1.1) units for the LTB patients and 6.3 (2.4) units for the HTB patients.Two of the 7 LTB patients had an increase in mean Hb ≥1.5 g/dL over 8 weeks compared to baseline. Mean maximum Hb increase in the LTB patients was 0.8, 1.0, 2.2, and 2.7 g/dL in the 0.125 (n=1), 0.25 (n=1), 0.75 (n=3), and 1.75 (n=2) mg/kg dose groups, respectively. Six of the 7 LTB patients achieved RBC transfusion independence (RBC-TI) for ≥8 weeks during the study.Six of the 19 HTB patients had a ≥4 unit or ≥50% reduction in RBC units transfused over an 8-week interval during the treatment period compared to the 8 weeks prior to treatment; five of these 6 patients achieved RBC-TI ≥ 8 weeks during the study (range 71-152 days). Increases in neutrophil count following study drug administration were observed in some patients.ACE-536 was generally well tolerated. No related serious adverse events have been reported to date. The most frequent adverse events regardless of causality were: diarrhea (n=4, grade 1/2), bone pain, fatigue, muscle spasms, myalgia, and nasopharyngitis (n=3 each, grade 1/2).Conclusions. Based on preliminary data in Low or Int-1 MDS patients, ACE-536 administered SC every 3 weeks for up to 5 doses increased Hb levels or decreased transfusion requirement, with a favorable safety profile. These data strongly support further evaluation of longer-term treatment with ACE-536 in patients with MDS. DisclosuresPlatzbecker:Celgene: Honoraria, Research Funding. Germing:Celgene: Honoraria, Research Funding. Goetze:Celgene Corp: Honoraria; Novartis Pharma: Honoraria. Radsak:Celgene: Research Funding. Hankin:Acceleron Pharma: Employment, Equity Ownership. Wilson:Acceleron Pharma: Employment, Equity Ownership. Sherman:Acceleron Pharma: Employment, Equity Ownership. Attie:Acceleron Pharma: Employment, Equity Ownership.

ACE-536 Increases Hemoglobin and Decreases Transfusion Burden and Serum Ferritin in Adults with Beta-Thalassemia: Preliminary Results from a Phase 2 Study

▪Introduction. ACE-536, a recombinant fusion protein containing modified activin receptor type IIB and IgG Fc, is being developed for the treatment of anemias due to ineffective erythropoiesis (IE), such as beta-thalassemia (β-thal). In β-thal, IE is due to intracellular α-globin aggregates causing premature death of late-stage erythroid precursors. Patients with β-thal often have elevated levels of EPO and are unresponsive to erythropoiesis-stimulating agents (ESAs). ACE-536 binds to ligands in the TGF-β superfamily, such as GDF11, and promotes late-stage erythroid differentiation via a mechanism distinct from ESAs. In a healthy volunteer study, ACE-536 was well-tolerated and increased hemoglobin (Hb) levels (Attie K et al., Am J Hematol 2014). Murine ACE-536 (RAP-536) increased Hb levels and decreased RBC inclusion bodies and hemolysis in a mouse model of β-thal (Suragani R et al., Blood 2014).Aims. This is an ongoing, phase 2, multicenter, open-label, dose-finding study to evaluate the effects of ACE-536 in adults with transfusion-dependent (TD, defined as ≥4 units RBCs/8 weeks prior to baseline) or non-transfusion dependent (NTD, defined as <4 units RBCs/8 weeks prior to baseline) β-thalassemia. Study outcomes include erythroid response (either Hb increase in NTD patients or reduced RBC transfusion burden in TD patients), safety, tolerability, PK, and pharmacodynamic biomarkers.Methods. Inclusion criteria included age ≥ 18 yr and anemia, defined as either being TD patient or having baseline Hb <10.0 g/dL in NTD patient. Transfusion burden in TD patients for any 12 weeks on treatment was compared with the 12 weeks prior to treatment. ACE-536 was administered by subcutaneous injection once every 3 weeks for up to 5 doses with a 2-month follow-up. Study design includes sequential cohorts (n=6 patients/cohort) at dose levels of 0.2, 0.4, 0.6, 0.8, 1.0, 1.25 and 1.5 mg/kg. An expansion cohort (n=30) is planned, and all patients completing this study may be eligible to enroll in a 12-month extension study.Results. Preliminary data were available for the 30 patients (23 NTD/7 TD) enrolled in the first 5 cohorts as of 18 Jul 2014. Median age was 34.5 yr (range: 20-57 yr), 16 (53%) were male and 83% had prior splenectomy. Mean (SD) baseline Hb for the NTD patients was 8.3 (0.9) g/dL.Efficacy data for the NTD patients in the 0.6 to 1.0 mg/kg cohorts demonstrated a maximum increase from baseline in Hb ≥ 1.5 g/dL in 7 of 11 (64%) patients. Efficacy data for the 7 TD patients treated in the 0.6 to 1.0 mg/kg cohorts demonstrated a >50% reduction in transfusion burden for all 7 patients. This was accompanied by a maximum decrease from baseline in serum ferritin levels ranging from 12-60%. Reductions in liver iron concentration by MRI were observed in both NTD and TD patients. Two patients with long-standing leg ulcers at baseline (one NTD, one TD) healed within approximately 3 months of ACE-536 treatment.ACE-536 was generally well tolerated. No related serious adverse events have been reported to date. The most frequent related adverse events included bone pain, headache and myalgia. No notable changes in platelets or WBC were observed.Conclusions. Based on preliminary data, ACE-536 administered SC every 3 weeks for up to 5 doses increased Hb levels in NTD patients and decreased transfusion requirement and serum ferritin levels in TD patients with β-thalassemia via a novel mechanism of action, and demonstrated a favorable safety profile. These data strongly support further evaluation of ACE-536 in patients with β-thalassemia. DisclosuresPiga:Acceleron Pharma: Research Funding; Celgene: Honoraria; Novartis: Research Funding; ApoPharma: Research Funding. Voskaridou:Celgene Coporation: Membership on an entity’s Board of Directors or advisory committees. Condon:Acceleron Pharma: Employment, Equity Ownership. Wilson:Acceleron Pharma: Employment, Equity Ownership. Sherman:Acceleron Pharma: Employment, Equity Ownership. Attie:Acceleron Pharma: Employment, Equity Ownership.