Receptor Type IIB Research Articles

Rap-536 (Murine ACE-536/Luspatercept) Inhibits Smad2/3 Signaling and Promotes Erythroid Differentiation By Restoring GATA-1 Function in Murine b-Thalassemia

We have previously reported that Smad2/3 signaling (of the TGFβ superfamily) is elevated in myelodysplastic syndromes (MDS) and β-thalassemia, diseases that are characterized by ineffective erythropoiesis (Suragani et al. 2014). Smad2/3 pathway inhibition using RAP-536 (murine version of ACE-536/luspatercept), a modified activin receptor type IIB ligand trap, decreased ineffective erythropoiesis (IE) and alleviated disease pathology in a murine model of β-thalassemia. In this study, we investigated the a) potential role of different Smad2/3 ligands that bind to luspatercept in the regulation of erythropoiesis and b) molecular mechanism of RAP-536 therapy in the murine model of β-thalassemia.Wild-type (WT) mice were treated with neutralizing antibodies against activin B, GDF8 or GDF8/11 (10mg/kg, s.c, twice weekly for 2- weeks, N=5/group) either as a single agent or in combination, and compared with RAP-536 (10 mg/kg, s.c) treatment. β-thalassemic mice (Hbbth3/+) were administered a single bolus of vehicle (VEH) or RAP-536 (30 mg/kg, i.p) (N=2/group). At 16 hours following administration the splenic basophilic erythroblasts (CD71+ Ter119+ FSChigh) were sorted by flow cytometry and RNA was isolated and subjected to genome wide transcriptome profiling using RNA sequencing analysis.a) Surface plasmon analysis revealed that ACE-536 binds Smad2/3 signaling ligands GDF11 and GDF8 with high affinity and activin B with lower affinity. There was minimal binding detected with Activin A, TGFβ1 or TGFβ3 ligands. Wt mice treated with RAP-536 increased RBC (+19%, P<0.001) and Hgb (+15.2%, P<0.001) compared to VEH treated mice. Treatment with anti-GDF8 or anti-activin B antibodies marginally affected RBC parameters (~2-4%, N.S) where as anti-GDF8/11 treatment alone increased RBC (+6.1%, P<0.05) and Hgb (6.9%, P<0.05) compared to VEH treatment. A combination treatment of anti-GDF8/11 and activin B antibodies synergistically increased RBC (10.7%, P<0.001) and Hgb (11%, P<0.001) compared to VEH treated mice. These data suggests that in addition to GDF11 and activin B, other TGFβ superfamily ligands are possibly involved in the stimulation of erythropoiesis by luspatercept.b) Transcriptome analysis of β-thalassemic erythroblasts revealed a total of 74 genes that were differentially expressed (absolute fold change >1.5, false discovery rate adjusted P value <0.05) in RAP-536 treated samples compared to VEH treatment. To identify molecular mechanisms, we performed gene set enrichment analysis (GSEA) (Subramanian et al., 2005) on data from RAP-536 and VEH treated samples. The analysis depicted significant upregulation of target genes of multiple transcriptional regulators including GATA-1 (erythroid differentiation), NFE2 and heat shock factor (involved in globin expression and protein quality-control). Previously, multiple studies established GATA-1 as a master transcriptional regulator of terminal erythroid differentiation. The individual gene symbols based comparative analysis revealed up-regulation of 53/478 GATA-1 activators and down regulation of 9/342 GATA-1 repressors. The GATA-1 target genes that were up regulated by RAP-536 treatment are involved in heme biosynthesis (such as Ppox, Fech, Alas2 and Abcb10) and erythroid differentiation (such as Klf1, Nfe2, Gypa, Bcl2l, Bnip3l, Bach1, and Ank1). Further GSEA of GATA-1 activator and repressor signatures against RAP-536 treatment data revealed a significant up-regulation of 158/328 activated genes (Normalized Enrichment Score=2.7, P=0) involved in heme biosynthesis, and cell cycle regulation whereas there was no statistically significant down regulation of GATA-1 repressed genes. Consistent with this data, our preliminary results in differentiating mouse erythroleukemic (MEL) cells showed increased Smad2/3 phosphorylation that is correlated with reduced GATA-1 protein levels suggesting that pSmad2/3 may negatively regulate terminal erythroid differentiation by decreasing GATA-1 availability.These data provide a potential mechanistic role for luspatercept treatment in β-thalassemia, by transcriptionally upregulating genes that promote erythroid differentiation and processing of unpaired α-globins. By inhibiting SMAD2/3 signaling, luspatercept relieves the block of terminal erythroid maturation and decreases ineffective erythropoiesis in diseases such as β-thalassemia and MDS. DisclosuresMartinez:Acceleron Pharma: Employment. Suragani:Acceleron Pharma Inc: Employment, Equity Ownership, Patents & Royalties: No royalties. Li:Acceleron Pharma: Employment, Equity Ownership. Pearsall:Acceleron Pharma Inc: Employment, Equity Ownership, Patents & Royalties. Kumar:Acceleron Pharma: Employment, Equity Ownership, Patents & Royalties.

Growth and differentiation factor 11 (GDF11): Functions in the regulation of erythropoiesis and cardiac regeneration.

Members of the TGF-β superfamily transduce their signals through type I and II receptor serine/threonine kinases. The regulation of members of the TGF-β family is known to be complex, because many proteins able to bind the ligands and inhibit their activities have been identified. Growth and differentiation factor 11 (Gdf11) as activins belong to the TGF-β family. GDF11, like other members of the TGF-β superfamily, is produced from precursor proteins by proteolytic processing. The binding of activins to activin type IIA (ActRIIA) or type IIB (ActRIIB) receptors induces the recruitment and phosphorylation of an activin type I receptor which then phosphorylates the Smad2 and Smad3 intracellular signaling proteins. GDF11 signal through the ActRIIB pathway. Recent studies have reported that GDF11-ActRIIB-Smad2/3-dependent signaling is a key regulatory mechanism in proliferating erythroid precursors as it controls their late-stage maturation. The administration of GDF11 is effective in experimental cardiac hypertrophy, and the identification of GDF11 as a "rejuvenating factor" opens up perspectives for the treatment of age-related cardiac dysfunction. Recent studies of the heart indicate that exposure to young blood reverses age-related impairments. GDF11 could be one of the circulating molecules that influence the aging of different tissues. Is GDF11 an "elixir of youth"?

Muscle-bone interactions: From experimental models to the clinic? A critical update

Bone is a biomechanical tissue shaped by forces from muscles and gravitation. Simultaneous bone and muscle decay and dysfunction (osteosarcopenia or sarco-osteoporosis) is seen in ageing, numerous clinical situations including after stroke or paralysis, in neuromuscular dystrophies, glucocorticoid excess, or in association with vitamin D, growth hormone/insulin like growth factor or sex steroid deficiency, as well as in spaceflight. Physical exercise may be beneficial in these situations, but further work is still needed to translate acceptable and effective biomechanical interventions like vibration therapy from animal models to humans. Novel antiresorptive and anabolic therapies are emerging for osteoporosis as well as drugs for sarcopenia, cancer cachexia or muscle wasting disorders, including antibodies against myostatin or activin receptor type IIA and IIB (e.g. bimagrumab). Ideally, increasing muscle mass would increase muscle strength and restore bone loss from disuse. However, the classical view that muscle is unidirectionally dominant over bone via mechanical loading is overly simplistic. Indeed, recent studies indicate a role for neuronal regulation of not only muscle but also bone metabolism, bone signaling pathways like receptor activator of nuclear factor kappa-B ligand (RANKL) implicated in muscle biology, myokines affecting bone and possible bone-to-muscle communication. Moreover, pharmacological strategies inducing isolated myocyte hypertrophy may not translate into increased muscle power because tendons, connective tissue, neurons and energy metabolism need to adapt as well. We aim here to critically review key musculoskeletal molecular pathways involved in mechanoregulation and their effect on the bone-muscle unit as a whole, as well as preclinical and emerging clinical evidence regarding the effects of sarcopenia therapies on osteoporosis and vice versa.

AB044. Update in the management of thalassemia.

Treatment of thalassemia depends on the clinical diagnosis. Almost all severe alpha thalassemia, Hb Bart’s hydrops fetalis, die intra utero or a few minute after birth. However, pregnant woman with Hb Bart’s hydrops fetus may develop toxemia of pregnancy and antepartum and postpartum hemorrhage. Those homozygous beta thalassemias (transfusion dependent thalassemia, TDT) are severely anemic after 6 months. At birth the baby is asymptomatic because Hb F level is high. As Hb F production waning off, replaced by inefficient beta globin chain production, at the age of 6 to 12 months, the baby begins to be anemic with hepatosplenomegaly. A group of non transfusion dependent thalassemia (NTDT) includes the majority of beta thalassemia/Hb E, Hb H disease and a few cases of homozygous beta thalassemias. The thalassemia minor including both alpha and beta thalassemia carriers and some homozygous state like homozygous alpha+ thalassemia, homozygous Hb E are asymptomatic and does not need any regular treatment. All thalassemia major, TDT, patients need regular blood transfusion, every 3-4 weeks, to maintain the hemoglobin around 10-12 g/dL. For those NTDT blood is given only when necessary. Usually patients with hemoglobin level higher than 6 g/dL require no blood transfusion. In infants and children if the hemoglobin levels can be maintained at 7 g/dL or above, defective physical development and bone changes can be prevented until they reached third or fourth decades of life that osteoporosis is almost always presented in those with low hemoglobin level. Iron overload occurs in moderate and severe case without exception. This will lead to dark skin, liver cirrhosis, cardiac arrhythmia and congestive heart failure. Diabetes mellitus secondary to iron deposition in the pancreas and other endocrine dysfunction does develop if the patients live long enough. Iron chelation with 1-2 gm/day of desferrioxamine intravenously or intramuscularly every day, at least 5 days a week, is recommended for those patients who have hemochromatosis. Oral iron chelator such as deferiprone and deferasirox is recently available. This helps patients to have better compliance with the iron chelator. Stem cell transplantation has been tried with a very good result in class I cases. Because some difficulty in finding appropriated HLA matched donor lately people try to perform haploidentical stem cell transplantation with some good results. Lupstaercept (ACE-536) and Sotatarcept (ACE-011), a recombinant fusion protein containing modified activin receptor type IIB and IgG Fc, is being developed for the treatment of anemia due to ineffective erythropoiesis. Preliminary data showed that the compound could increase hemoglobin levels 1.5 g/dL after two weeks of treatment in NTDT case and decrease blood transfusion in 60% of cases with TDT. Research is in progress to find better agents to enhance Hb F production.

Muscle hypertrophy induced by myostatin inhibition accelerates degeneration in dysferlinopathy.

Myostatin is a secreted signaling molecule that normally acts to limit muscle growth. As a result, there is extensive effort directed at developing drugs capable of targeting myostatin to treat patients with muscle loss. One potential concern with this therapeutic approach in patients with muscle degenerative diseases like muscular dystrophy is that inducing hypertrophy may increase stress on dystrophic fibers, thereby accelerating disease progression. To investigate this possibility, we examined the effect of blocking the myostatin pathway in dysferlin-deficient (Dysf−/−) mice, in which membrane repair is compromised, either by transgenic expression of follistatin in skeletal muscle or by systemic administration of the soluble form of the activin type IIB receptor (ACVR2B/Fc). Here, we show that myostatin inhibition by follistatin transgene expression in Dysf−/− mice results in early improvement in histopathology but ultimately exacerbates muscle degeneration; this effect was not observed in dystrophin-deficient (mdx) mice, suggesting that accelerated degeneration induced by follistatin transgene expression is specific to mice lacking dysferlin. Dysf−/− mice injected with ACVR2B/Fc showed significant increases in muscle mass and amelioration of fibrotic changes normally seen in 8-month-old Dysf−/− mice. Despite these potentially beneficial effects, ACVR2B/Fc treatment caused increases in serum CK levels in some Dysf−/− mice, indicating possible muscle damage induced by hypertrophy. These findings suggest that depending on the disease context, inducing muscle hypertrophy by myostatin blockade may have detrimental effects, which need to be weighed against the potential gains in muscle growth and decreased fibrosis.

Internalization of rituximab and the efficiency of B Cell depletion in rheumatoid arthritis and systemic lupus erythematosus.

ObjectiveRituximab, a type I anti‐CD20 monoclonal antibody (mAb), induces incomplete B cell depletion in some patients with rheumatoid arthritis (RA) and systemic lupus erythematosus (SLE), thus contributing to a poor clinical response. The mechanisms of this resistance remain elusive. The purpose of this study was to determine whether type II mAb are more efficient than type I mAb at depleting B cells from RA and SLE patients, whether internalization influences the efficiency of depletion, and whether Fcγ receptor type IIb (FcγRIIb) and the B cell receptor regulate this internalization process.MethodsWe used an in vitro whole blood B cell–depletion assay to assess the efficiency of depletion, flow cytometry to study cell surface protein expression, and surface fluorescence–quenching assays to assess rituximab internalization, in samples from patients with RA and patients with SLE. Paired t‐test or Mann‐Whitney U test was used to compare groups, and Spearman's rank correlation test was used to assess correlation.ResultsWe found that type II mAb internalized significantly less rituximab than type I mAb and depleted B cells from patients with RA and SLE at least 2‐fold more efficiently than type I mAb. Internalization of rituximab was highly variable between patients, was regulated by FcγRIIb, and inversely correlated with cytotoxicity in whole blood B cell–depletion assays. The lowest levels of internalization were seen in IgD– B cells, including postswitched (IgD–CD27+) memory cells. Internalization of type I anti‐CD20 mAb was also partially inhibited by anti‐IgM stimulation.ConclusionVariability in internalization of rituximab was observed and was correlated with impaired B cell depletion. Therefore, slower‐internalizing type II mAb should be considered as alternative B cell–depleting agents for the treatment of RA and SLE.

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.

Upregulation of activin-B and follistatin in pulmonary fibrosis - a translational study using human biopsies and a specific inhibitor in mouse fibrosis models.

BackgroundActivins are members of the TGF-ß superfamily of growth factors. First, we identified by expression array screening that activin-B and follistatin are upregulated in human idiopathic pulmonary fibrosis (IPF). Next, we wanted to clarify their specific role in lung fibrosis formation.MethodsWe used specific antibodies for activin-A and -B subunits and follistatin to measure and localize their levels in idiopathic pulmonary fibrosis and control lung biopsies. To inhibit activin signaling, we used soluble activin type IIB receptor fused to the Fc portion of human IgG1 (sActRIIB-Fc) in two different mouse models of pulmonary fibrosis.ResultsActivin-B and follistatin mRNA levels were elevated in the human IPF lung. Immunoreactivity to activin-A, -B and follistatin localized predominantly to the hyperplastic, activated alveolar epithelium, but was also seen in inflammatory cells. Mice treated with sActRIIB-Fc showed increased skeletal muscle mass and a clear reduction in alveolar cell counts in bronchoalveolar lavage fluid, but no significant antifibrotic effect in the lung was observed.ConclusionsThe upregulation of activin-B and follistatin in IPF is a novel finding. Our results indicate that activin inhibition is not an efficient tool for antifibrotic therapy, but could be useful in reducing alveolar cellular response to injury. Activin-B and follistatin levels may be useful as biomarkers of IPF.

MicroRNA-21 Mediates Stretch-Induced Osteogenic Differentiation in Human Periodontal Ligament Stem Cells

microRNAs (miRNAs) are short 20- to 22-nucleotide noncoding RNAs that negatively regulate the expression of target genes at the post-transcriptional level. The expression of specific miRNAs and their roles in the osteogenic differentiation of human periodontal ligament stem cells (PDLSCs) exposed to mechanical stretch remain unclear. Here, we found that stretch induced both osteogenic differentiation and the differential expression of miR-21 in PDLSCs. Furthermore, we identified activin receptor type IIB (ACVR2B) as a target gene of miR-21. Luciferase reporter assays showed that miR-21 interacts directly with the 3'-untranslated repeat sequence of ACVR2B mRNA. Mechanical stretch suppressed ACVR2B protein levels in PDLSCs, and this suppressive effect was modulated when endogenous miR-21 levels were either enhanced or inhibited. Both stretch and the expression of miR-21 altered endogenous ACVR2B protein levels and thus the osteogenic differentiation of PDLSCs. In addition, gain- and loss of function of ACVR2B mediated the osteogenic differentiation of PDLSCs. This study demonstrates that miR-21 is a mechanosensitive gene that plays an important role in the osteogenic differentiation of PDLSCs exposed to stretch.

G.O.25: Follistatin Gene Therapy for Becker Muscular Dystrophy

Becker muscular dystrophy (BMD), a dystrophinopathy with milder skeletal muscle manifestations compared to Duchenne muscular dystrophy, characteristically demonstrates weakness of knee extensors (KE), accompanied by muscle atrophy and fibrosis. The weakness predisposes to gait impairment and loss of ambulation. Myostatin, a member of the TGF-<i>β</i> superfamily of signal peptides, is expressed in skeletal muscle, acting through the activin type IIB (ACTRIIB) receptor. Follistatin, a myostatin antagonist, inhibits this pathway resulting in muscle hypertrophy, and improved muscle strength. FS315 and FS288 are two isoforms of follistatin generated by alternative splicing. FS315 has no cell-surface affinity providing a safety margin avoiding pituitary–gonadal axis complications. FS344 is the precursor cDNA to FS315. When FS344 is delivered to mdx mice in adeno-associated virus (AAV1. CMV. FS344), muscle mass and strength increase. Similar findings are seen in non-human primates without safety concerns. Six BMD patients, 18years and older with proven in-frame DMD gene mutations were enrolled in a clinical trial. The first 3 subjects received 12 intramuscular (IM) injections of AAV1. CMV. FS344 at a total dose of 6e11 vg/kg in the quadriceps muscles bilaterally. A second cohort received the same number of IM injections at higher total dose (1.2e12 vg/kg). Studies extending beyond one year in Cohort 1 and for six months in Cohort 2 showed no adverse events. The distance walked (meters) on the 6-min walk test (6MWT) was the primary functional outcome measure. At one year the distance walked by two subjects in Cohort 1 increased by 125m and 58m; subject 3 improved modestly by 9m. In Cohort 2, one patient reaching the 6-month time point increased by 108 meters. For the entire group of BMD patients only a single patient declined in distance. Preliminary analysis of muscle biopsies favor increased muscle regeneration contributing to improved function.

Goat activin receptor type IIB knockdown by muscle specific promoter driven artificial microRNAs

Activin receptor type IIB (ACVR2B) is a transmembrane receptor which mediates signaling of TGF beta superfamily ligands known to function in regulation of muscle mass, embryonic development and reproduction. ACVR2B antagonism has shown to enhance the muscle growth in several disease and transgenic models. Here, we show ACVR2B knockdown by RNA interference using muscle creatine kinase (MCK) promoter driven artificial microRNAs (amiRNAs). Among the various promoter elements tested, the ∼1.26kb MCK promoter region showed maximum transcriptional activity in goat myoblasts cells. We observed up to 20% silencing in non-myogenic 293T cells and up to 32% silencing in myogenic goat myoblasts by MCK directed amiRNAs by transient transfection. Goat myoblasts stably integrated with MCK directed amiRNAs showed merely 8% silencing in proliferating myoblasts which was increased to 34% upon induction of differentiation at transcript level whereas up to 57% silencing at protein level. Knockdown of ACVR2B by 5′-UTR derived amiRNAs resulted in decreased SMAD2/3 signaling, increased expression of myogenic regulatory factors (MRFs) and enhanced proliferation and differentiation of myoblasts. Unexpectedly, knockdown of ACVR2B by 3′-UTR derived amiRNAs resulted in increased SMAD2/3 signaling, reduced expression of MRFs and suppression of myogenesis. Our study offers muscle specific knockdown of ACVR2B as a potential strategy to enhance muscle mass in the farm animal species.

Goat activin receptor type IIB knockdown by artificial microRNAs in vitro.

Activin receptor type IIB (ACVR2B) has been known to negatively regulate the muscle growth through mediating the action of transforming growth factor beta superfamily ligands. Recently, the artificial microRNAs (amiRNAs) which are processed by endogenous miRNA processing machinery have been proposed as promising approach for efficient gene knockdown. We evaluated amiRNAs targeting goat ACVR2B in HEK293T and goat myoblasts cells. The amiRNAs were designed based on the miR-155 backbone and cloned in 5'- and 3'-UTR of GFP reporter gene under the CMV promoter. Although both 5'- and 3'-UTR-amiRNAs vectors showed efficient synthesis of GFP transcripts, amiRNAs in 5'-UTR drastically affected GFP protein synthesis in transfected goat myoblast cells. Among the four amiRNAs targeting ACVR2B derived from either 5'- or 3'-UTR, ami318 showed highest silencing efficiency against exogenously co-expressed ACVR2B in both 293T and goat myoblast cells whereas ami204 showed highest silencing efficiency against endogenous ACVR2B in goat myoblasts cells. The 3'-UTR-derived amiRNA exerted higher knockdown efficiency against endogenous ACVR2B at transcript level whereas 5'-UTR-derived amiRNAs exerted higher knockdown efficiency at protein level. The expression of ACVR2B showed positive correlation with the expression of MYOD (r = 0.744; p = 0.009) and MYOG (r = 0.959; p = 0.000) in the amiRNA-transfected myoblasts. Although both 5'- and 3'-UTR-amiRNA vectors led to substantial induction of interferon response, the magnitude of the response was found to be higher with the 3'-UTR-amiRNA vectors.

Modified activin receptor IIB ligand trap mitigates ineffective erythropoiesis and disease complications in murine β-thalassemia

AbstractIn β-thalassemia, unequal production of α- and β-globin chains in erythroid precursors causes apoptosis and inhibition of late-stage erythroid differentiation, leading to anemia, ineffective erythropoiesis (IE), and dysregulated iron homeostasis. Here we used a murine model of β-thalassemia intermedia (Hbbth1/th1 mice) to investigate effects of a modified activin receptor type IIB (ActRIIB) ligand trap (RAP-536) that inhibits Smad2/3 signaling. In Hbbth1/th1 mice, treatment with RAP-536 reduced overactivation of Smad2/3 in splenic erythroid precursors. In addition, treatment of Hbbth1/th1 mice with RAP-536 reduced α-globin aggregates in peripheral red cells, decreased the elevated reactive oxygen species present in erythroid precursors and peripheral red cells, and alleviated anemia by promoting differentiation of late-stage erythroid precursors and reducing hemolysis. Notably, RAP-536 treatment mitigated disease complications of IE, including iron overload, splenomegaly, and bone pathology, while reducing erythropoietin levels, improving erythrocyte morphology, and extending erythrocyte life span. These results implicate signaling by the transforming growth factor-β superfamily in late-stage erythropoiesis and reveal potential of a modified ActRIIB ligand trap as a novel therapeutic agent for thalassemia syndrome and other red cell disorders characterized by IE.

Activation of activin type IB receptor signals in pancreatic β cells leads to defective insulin secretion through the attenuation of ATP-sensitive K+ channel activity

In studies of gene-ablated mice, activin signaling through activin type IIB receptors (ActRIIB) and Smad2 has been shown to regulate not only pancreatic β cell mass but also insulin secretion. However, it still remains unclear whether gain of function of activin signaling is involved in the modulation of pancreatic β cell mass and insulin secretion. To identify distinct roles of activin signaling in pancreatic β cells, the Cre-loxP system was used to activate signaling through activin type IB receptor (ActRIB) in pancreatic β cells. The resultant mice (pancreatic β cell-specific ActRIB transgenic (Tg) mice; ActRIBCAβTg) exhibited a defect in glucose-stimulated insulin secretion (GSIS) and a progressive impairment of glucose tolerance. Patch-clamp techniques revealed that the activity of ATP-sensitive K+ channels (KATP channels) was decreased in mutant β cells. These results indicate that an appropriate level of activin signaling may be required for GSIS in pancreatic β cells, and that activin signaling involves modulation of KATP channel activity.

Assessment of goat activin receptor type IIB knockdown by short hairpin RNAs in vitro

Background: Targeted knockdown of ACVR2B, a receptor for TGF beta superfamily, has been seen as a potential candidate to enhance the muscle mass through RNAi approach. Methods: We have evaluated the potential short hairpin RNAs targeting goat ACVR2B in human HEK293T cells and goat myoblasts cells by transient transfection and measured their knockdown efficiency and possible undesired interferon response by quantitative real-time PCR. Results: We observed a significant silencing (64–81%) of ACVR2B in 293T cells with all seven shRNAs (sh1 to sh7) constructs and 16–46% silencing with maximum of 46% by sh6 (p = 0.0318) against endogenous ACVR2B whereas up to 66% (p = 0.0002) silencing by sh6 against exogenously expressed ACVR2B in goat myoblasts cells. Transient knockdown of ACVR2B in goat myoblasts cells by shRNAs did not show significant correlation with the expression of MyoD (r = 0.547; p = 0.102), myogenin (r = 0.517; p = 0.126) and Myf5 (r = 0.262; p = 0.465). As reported earlier, transfection of plasmid DNA induced potent interferon response in 293T and goat myoblasts cells. Conclusions: The present study demonstrates the targeted knockdown of ACVR2B by shRNAs in HEK293T and goat myoblasts cells in vitro. The transient knockdown of ACVR2B by shRNAs in goat myoblasts did not alter the myogenic gene expression program. However, shRNAs showing significant knockdown efficiency in our study may further be tested for long term and stable knockdown to assess their potential to use for enhancing muscle mass in vivo. As reported earlier, expression of shRNAs through plasmid expression vectors induces potent interferon response raising the concern of safety of its application in vivo.

A phase 1 study of ACE‐536, a regulator of erythroid differentiation, in healthy volunteers

ACE-536, a recombinant protein containing a modified activin receptor type IIB, is being developed for the treatment of anemias caused by ineffective erythropoiesis, such as thalassemias and myelodysplastic syndromes. ACE-536 acts through a mechanism distinct from erythropoiesis-stimulating agents to promote late-stage erythroid differentiation by binding to transforming growth factor-β superfamily ligands and inhibiting signaling through transcription factors Smad 2/3. The goal of this Phase 1 study was to evaluate the safety, tolerability, pharmacokinetics, and pharmacodynamic effects of ascending dose levels of ACE-536 in healthy volunteers. Thirty-two postmenopausal women were randomized in sequential cohorts of eight subjects each to receive up to two doses of either ACE-536 (0.0625–0.25 mg/kg) or placebo (3:1 randomization) given subcutaneously every 2 weeks. Mean baseline age was 59.4 years, and hemoglobin was 13.2 g/dL. ACE-536 was well tolerated at dose levels up to 0.25 mg/kg over the 1-month treatment period. There were no serious or severe adverse events, nor clinically meaningful changes in safety laboratory measures or vital signs. Mean ACE-536 AUC0–14d and Cmax increased proportionally after first dose; mean t½ was 15–16 days. Dose-dependent increases in hemoglobin concentration were observed, beginning 7 days after initiation of treatment and maintained for several weeks following treatment. The proportion of subjects with a hemoglobin increase ≥1.0 g/dL increased in a dose-dependent manner to 83.3% of subjects in the highest dose group, 0.25 mg/kg. ACE-536 was well tolerated and resulted in sustained increases in hemoglobin levels in healthy postmenopausal women. Am. J. Hematol. 89:766–770, 2014. © 2014 Wiley Periodicals, Inc.

Differential Muscle Hypertrophy Is Associated with Satellite Cell Numbers and Akt Pathway Activation Following Activin Type IIB Receptor Inhibition in Mtm1 p.R69C Mice

X-linked myotubular myopathy is a congenital myopathy caused by deficiency of myotubularin. Patients often present with severe perinatal weakness, requiring mechanical ventilation to prevent death from respiratory failure. We recently reported that an activin receptor type IIB inhibitor produced hypertrophy of type 2b myofibers and modest increases of strength and life span in the severely myopathic Mtm1δ4 mouse model of X-linked myotubular myopathy. We have now performed a similar study in the less severely symptomatic Mtm1 p.R69C mouse in hopes of finding greater treatment efficacy. Activin receptor type IIB inhibitor treatment of Mtm1 p.R69C animals produced behavioral and histological evidence of hypertrophy in gastrocnemius muscles but not in quadriceps or triceps. The ability of the muscles to respond to activin receptor type IIB inhibitor treatment correlated with treatment-induced increases in satellite cell number and several muscle-specific abnormalities of hypertrophic signaling. Treatment-responsive Mtm1 p.R69C gastrocnemius muscles displayed lower levels of phosphorylated ribosomal protein S6 and higher levels of phosphorylated eukaryotic elongation factor 2 kinase than were observed in Mtm1 p.R69C quadriceps muscle or in muscles from wild-type littermates. Hypertrophy in the Mtm1 p.R69C gastrocnemius muscle was associated with increased levels of phosphorylated ribosomal protein S6. Our findings indicate that muscle-, fiber type-, and mutation-specific factors affect the response to hypertrophic therapies that will be important to assess in future therapeutic trials.

Transforming growth factor-β superfamily ligand trap ACE-536 corrects anemia by promoting late-stage erythropoiesis.

Erythropoietin (EPO) stimulates proliferation of early-stage erythrocyte precursors and is widely used for the treatment of chronic anemia. However, several types of EPO-resistant anemia are characterized by defects in late-stage erythropoiesis, which is EPO independent. Here we investigated regulation of erythropoiesis using a ligand-trapping fusion protein (ACE-536) containing the extracellular domain of human activin receptor type IIB (ActRIIB) modified to reduce activin binding. ACE-536, or its mouse version RAP-536, produced rapid and robust increases in erythrocyte numbers in multiple species under basal conditions and reduced or prevented anemia in murine models. Unlike EPO, RAP-536 promoted maturation of late-stage erythroid precursors in vivo. Cotreatment with ACE-536 and EPO produced a synergistic erythropoietic response. ACE-536 bound growth differentiation factor-11 (GDF11) and potently inhibited GDF11-mediated Smad2/3 signaling. GDF11 inhibited erythroid maturation in mice in vivo and ex vivo. Expression of GDF11 and ActRIIB in erythroid precursors decreased progressively with maturation, suggesting an inhibitory role for GDF11 in late-stage erythroid differentiation. RAP-536 treatment also reduced Smad2/3 activation, anemia, erythroid hyperplasia and ineffective erythropoiesis in a mouse model of myelodysplastic syndromes (MDS). These findings implicate transforming growth factor-β (TGF-β) superfamily signaling in erythroid maturation and identify ACE-536 as a new potential treatment for anemia, including that caused by ineffective erythropoiesis.