Activin Receptor Type IIB Research Articles

Myostatin inhibition induces muscle fibre hypertrophy prior to satellite cell activation

Muscle fibres are multinucleated post-mitotic cells that can change dramatically in size during adulthood. It has been debated whether muscle fibre hypertrophy requires activation and fusion of muscle stem cells, the satellite cells. Myostatin (MSTN) is a negative regulator of skeletal muscle growth during development and in the adult, and MSTN inhibition is therefore a potential therapy for muscle wasting diseases, some of which are associated with a depletion of satellite cells. Conflicting results have been obtained in previous analyses of the role of MSTN on satellite cell quiescence. Here, we inhibited MSTN in adult mice with a soluble activin receptor type IIB and analysed the incorporation of new nuclei using 5-bromo-2-deoxyuridine (BrdU) labelling by isolating individual myofibres. We found that satellite cells are activated by MSTN inhibition. By varying the dose and time course for MSTN inhibition, however, we found that myofibre hypertrophy precedes the incorporation of new nuclei, and that the overall number of new nuclei is relatively low compared to the number of total myonuclei. These results reconcile some of the previous work obtained by other methods. In contrast with previous reports, we also found that Mstn null mice do not have increased satellite cell numbers during adulthood and are not resistant to sarcopaenia. Our results support a previously proposed model of hypertrophy in which hypertrophy can precede satellite cell activation. Studies of the metabolic and functional effects of postnatal MSTN inhibition are needed to determine the consequences of increasing the cytoplasm/myonuclear ratio after MSTN inhibition.

Functional redundancy of type II BMP receptor and type IIB activin receptor in BMP2‐induced osteoblast differentiation

Signaling pathways for bone morphogenetic proteins (BMPs) are important in osteoblast differentiation. Although the precise function of type I BMP receptors in mediating BMP signaling for osteoblast differentiation and bone formation has been characterized previously, the role of type II BMP receptors in osteoblasts is to be well clarified. In this study, we investigated the role of type II BMP receptor (BMPR-II) and type IIB activin receptor (ActR-IIB) in BMP2-induced osteoblast differentiation. While osteoblastic 2T3 cells expressed BMPR-II and ActR-IIB, loss-of-function studies, using dominant negative receptors and siRNAs, showed that BMPR-II and ActR-IIB compensated each other functionally in mediating BMP2 signaling and BMP2-induced osteoblast differentiation. This was evidenced by two findings. First, unless there was loss of function of both type II receptors, isolated disruption of either BMPR-II or ActR-IIB did not remove BMP2 activity. Second, in cells with loss of function of both receptors, restoration of function of either BMPR-II or ActR-IIB by transfection of the wild-type forms, restored BMP2 activity. These findings suggest a functional redundancy between BMPR-II and ActR-IIB in osteoblast differentiation. Results from experiments to test the effects of transforming growth factor β (TGF-β), activin, and fibroblast growth factor (FGF) on osteoblast proliferation and differentiation suggest that inhibition of receptor signaling by double-blockage of BMPR-II and ActR-IIB is BMP-signaling specific. The observed functional redundancy of type II BMP receptors in osteoblasts is novel information about the BMP signaling pathway essential for initiating osteoblast differentiation.

Soluble activin receptor type IIB treatment does not cause fat loss in mice with diet‐induced obesity

The growth factor myostatin (MSTN) negatively regulates skeletal muscle mass. Mstn gene deletion in mice causes increased muscle mass, reduced adiposity and resistance to genetic or diet-induced obesity (DIO). Pharmacologic MSTN inhibition in mice also causes increased muscle mass and resistance to DIO. To test whether MSTN inhibition causes weight loss in mice that are already obese, we inhibited MSTN in mice fed a high-fat diet (HFD). Mice were fed a diet containing 60% kcal from fat for 12 weeks followed by treatment with a soluble MSTN receptor derived from the activin receptor type IIB extracellular domain. During the next 12 weeks of soluble receptor treatment and HFD feeding, lean mass increased without a loss of adipose mass. Glucose metabolism was also similar between groups. Our results suggest that MSTN inhibition may be ineffective at inducing weight loss in obese patients.

RAP-536 Promotes Terminal Erythroid Differentiation and Reduces Anemia in Myelodysplastic Syndromes

Abstract 610Myelodysplatic syndromes (MDS) are a heterogeneous group of hematopoietic stem cell disorders characterized by ineffective hematopoiesis. Patients develop peripheral blood cytopenias; however, the bone marrow shows increased proliferation and apoptosis. In addition to bone marrow apoptosis, a failure of differentiation contributes to reduced terminally differentiated blood cells. A significant proportion of patients with MDS will develop anemia that are refractory to treatment with recombinant human erythropoietin (EPO) and must rely on transfusions as supportive care. The use of blood transfusions as supportive care is associated with iron overload and significant morbidity. Therefore, alternative therapies to treat anemia in MDS patients are needed.Members of the TGFβ super family of signaling molecules have been implicated in erythropoiesis and represent alternative, EPO-independent targets for the treatment of anemia. ACE-536 is a soluble receptor fusion protein consisting of a modified Activin Receptor Type IIB extracellular domain linked to a human Fc domain. ACE-536 acts as a ligand trap to modulate the activity of TGFβ ligands and promote erythroid differentiation in an EPO independent manner. Subcutaneous administration of ACE-536 to C57BL/6 mice resulted in significant increases in hematocrit, hemoglobin and red blood cells compared to vehicle treated controls within four days. These effects were observed with concurrent treatment of an EPO neutralizing antibody, indicating that EPO is not directly responsible for the initial RBC response of ACE-536. BFU-E or CFU-E colony formation assays from bone marrow or spleen of mice 48 hours after ACE-536 were normal, indicating no effect on the erythroid progenitor population. Differentiation profiling of bone marrow and splenic erythroblasts by FACS analysis following 72 hours after RAP-536 (murine version of ACE-536) treatment revealed a decrease in basophilic erythroblasts and an increase in late stage poly-, ortho-chromatophilic and reticulocytes in bone marrow and spleen compared to vehicle treated mice. The data demonstrate that while EPO treatment increases proliferation of erythroid progenitors, ACE-536 promotes maturation of terminally differentiating erythroblasts.The efficacy of ACE-536 has been demonstrated in various animal models of acute and chronic anemia. In this study we investigated the effect of ACE-536 on anemia in mouse model of MDS. The NUP98-HOXD13 (NHD13) transgenic mouse carries a common translocation found in MDS patients. NHD13 mice develop anemia, neutropenia and lymphopenia at 4–7 months of age, with normal or hypercellular bone marrow. Starting at 4 months of age, mice were treated with RAP-536 (murine homolog of ACE-536) at 10 mg/kg or vehicle control twice per week for 8 months. Wild-type littermate controls were also dosed on the same schedule. As expected, at study baseline (mice 4 months of age), NHD13 mice had reduced RBC, Hb and HCT compared to wild-type littermates. The progression of anemia over the study period was reduced by treatment with RAP-536 compared to vehicle (HCT: −8.3% v. −22%, RBC: −13% v. −30%). Based on blood smear analyses, there was no indication of increased leukemic cells with ACE-536 treatment.Our data demonstrate that RAP-536 can increase hematology parameters through enhancing maturation of terminally differentiated red blood cells and can serve as a therapeutic molecule for the treatment of anemia. As anemia contributes significantly to the morbidity of patients with MDS, a mouse model was used to test the therapeutic efficacy of ACE-536 in this disease. We have shown that systemic administration of RAP-536 to MDS mice promotes increases in red blood cell mass without enhanced progression to AML. Therefore ACE-536 may represent a novel treatment for anemia associated with MDS, particularly in patients that are refractory to EPO therapy. Disclosures:Suragani:Acceleron Pharma Inc: Employment. Mulivor:Acceleron Pharma Inc: Employment. Pearsall:Acceleron Pharma Inc: Employment. Kumar:Acceleron Pharma Inc: Employment.

Production of bioactive extracellular domain of pig and chicken activin type IIB receptors in Pichia pastoris

Myostatin (MSTN) is a potent negative regulator for skeletal muscle growth, and binds to activin type IIB receptor (ActRIIB) for its cellular signal transduction. Administration of the extracellular domain of ActRIIB (ActRIIB-ECD) improved skeletal muscle growth in laboratory animals, suggesting that ActRIIB-ECD can be a useful pharmacological agent to improve skeletal muscle growth of meat-producing animals. In the current study, pig and chicken ActRIIB-ECDs were produced in the Pichia pastoris GS115, and the recombinant proteins were purified from induced culture media by Ni-NTA affinity chromatography. The digestion of pig and chicken ActRIIB-ECDs with PNGase F and glycoprotein staining demonstrated an N-linked glycosylation of these recombinant proteins. Glycoprotein staining also indicated an additional presence of glycosylation in chicken ActRIIB-ECD. Both the pig and chicken ActRIIB-ECDs were shown to inhibit MSTN activity in a reporter gene assay system in vitro. When MSTN-inhibitory potencies were compared by analyzing EC 50 values, no difference in MSTN-inhibitory potency was observed between the glycosylated and N-deglycosylated forms of pig or chicken ActRIIB-ECD, suggesting that glycosylation does not affect the bioactivity of ActRIIB-ECD. MSTN-inhibitory potency of chicken ActRIIB-ECD was greater ( P < 0.01) than that of pig ActRIIB-ECD. Results of this study demonstrate that bioactive pig and chicken ActRIIB-ECDs can be produced from P. pastoris. In addition, the study indicates that the N-glycosylation status of ActRIIB-ECD does not affect its bioactivity in vitro.

The expression of activins, their type II receptors and follistatin in human Fallopian tube during the menstrual cycle and in pseudo-pregnancy

The Fallopian tube (FT) is the site of fertilization and early embryonic development. We have previously reported the expression of activins, their receptors and follistatin by the FT. Here, our aim was to study the expression of tubal activins, their type II receptors and follistatin during the menstrual cycle and following exposure to hCG in vivo. A set of 30 FTs were collected from cycling women (n = 12) at different stages of the cycle (n = 4 in each stage) and pseudo-pregnant women (n = 3) at the time of hysterectomy for benign disease. The pseudo-pregnant women were injected with hCG in the days leading up to hysterectomy, and pseudo-pregnancy was confirmed by the persistence of amenorrhea, the presence of corpus luteum and decidualization of the endometrium. FT specimens were examined using immunohistochemistry and quantitative RT-PCR. The expression of activin βA- and βB-subunits, activin type IIA and IIB receptors, and follistatin varied throughout the menstrual cycle, being lowest in the follicular phase and highest in the luteal phase. These results were demonstrated at the mRNA and protein level by quantitative RT-PCR and immunohistochemistry (P< 0.05). HCG injection rescued the expression of the candidate molecules from falling to the follicular stage levels but the expression remained lower than in the luteal phase. We suggest that activins play a role in tubal physiology and early embryonic development. Additionally, exposure of the tubal epithelium to hCG modulates the expression of tubal activins.

Inhibition of myostatin protects against diet-induced obesity by enhancing fatty acid oxidation and promoting a brown adipose phenotype in mice

Although myostatin-null (Mstn (-/-)) mice fail to accumulate fat in adipose tissue when fed a high-fat diet (HFD), little is known about the molecular mechanism(s) behind this phenomenon. We therefore sought to identify the signalling pathways through which myostatin regulates accumulation and/or utilisation of fat. Wild-type, Mstn (-/-) and wild-type mice treated with soluble activin type IIB receptor (sActRIIB) were fed a control chow diet or an HFD for 12 weeks. Changes in gene expression were measured by microarray and quantitative PCR. Histological changes in white adipose tissue were assessed together with peripheral tissue fatty acid oxidation and changes in circulating hormones following HFD feeding. Our results demonstrate that inactivation of myostatin results in reduced fat accumulation in mice on an HFD. Molecular analysis revealed that metabolic benefits, due to lack of myostatin, are mediated through at least two independent mechanisms. First, lack of myostatin increased fatty acid oxidation in peripheral tissues through induction of enzymes involved in lipolysis and in fatty acid oxidation in mitochondria. Second, inactivation of myostatin also enhanced brown adipose formation in white adipose tissue of Mstn (-/-) mice. Consistent with the above, treatment of HFD-fed wild-type mice with the myostatin antagonist, sActRIIB, reduced the obesity phenotype. We conclude that absence of myostatin results in enhanced peripheral tissue fatty acid oxidation and increased thermogenesis, culminating in increased fat utilisation and reduced adipose tissue mass. Taken together, our data suggest that anti-myostatin therapeutics could be beneficial in alleviating obesity.

The role of myostatin in muscle wasting: an overview

Myostatin is an extracellular cytokine mostly expressed in skeletal muscles and known to play a crucial role in the negative regulation of muscle mass. Upon the binding to activin type IIB receptor, myostatin can initiate several different signalling cascades resulting in the upregulation of the atrogenes and downregulation of the important for myogenesis genes. Muscle size is regulated via a complex interplay of myostatin signalling with the insulin-like growth factor 1/phosphatidylinositol 3-kinase/Akt pathway responsible for increase in protein synthesis in muscle. Therefore, the regulation of muscle weight is a process in which myostatin plays a central role but the mechanism of its action and signalling cascades are not fully understood. Myostatin upregulation was observed in the pathogenesis of muscle wasting during cachexia associated with different diseases (i.e. cancer, heart failure, HIV). Characterisation of myostatin signalling is therefore a perspective direction in the treatment development for cachexia. The current review covers the present knowledge about myostatin signalling pathways leading to muscle wasting and the state of therapy approaches via the regulation of myostatin and/or its downstream targets in cachexia.

Hepcidin expression in the liver of rats fed a magnesium-deficient diet

Mg deficiency accelerates Fe accumulation in the liver, which may induce various metabolic disturbances. In the present study, we examined the gene expression of Hepcidin, a peptide hormone produced in the liver to regulate intestinal Fe absorption negatively, in Mg-deficient rats. Although liver Fe concentration was significantly higher in rats fed an Mg-deficient diet for 4 weeks than in rats fed a control diet, Hepcidin expression in the liver was comparable between the dietary groups. Previous studies revealed that Fe overload up-regulated Hepcidin expression through transcriptional activation by Fe-induced bone morphogenetic protein (Bmp) 6, a growth/differentiation factor belonging to the transforming growth factor-β family, in the liver. Mg deficiency up-regulated the expression of Bmp6 but did not affect the expression of inhibition of DNA binding 1, a sensitive Bmp-responsive gene. In addition, the expression of Bmp receptors such as activin receptor-like kinase 2 (Alk2), activin receptor type IIA (Actr2a), activin receptor type IIB (Actr2b) and Bmp type II receptor (Bmpr2) was lower in the liver of Mg-deficient rats than in that of control rats. The present study indicates that accumulation of hepatic Fe by Mg deficiency is a stimulant inducing Bmp6 expression but not Hepcidin expression by blunting Bmp signalling possibly resulting from down-regulation of the receptor expression. Unresponsive Hepcidin expression may have a role in Mg deficiency-induced changes related to increased liver Fe.

Soluble activin receptor type IIB increases forward pulling tension in the mdx mouse

In this study we investigated the action of RAP-031, a soluble activin receptor type IIB (ActRIIB) comprised of a form of the ActRIIB extracellular domain linked to a murine Fc, and the NF-κB inhibitor, ursodeoxycholic acid (UDCA), on the whole body strength of mdx mice. The whole body tension (WBT) method of assessing the forward pulling tension (FPT) exerted by dystrophic (mdx) mice was used. RAP-031 produced a 41% increase in body mass and a 42.5% increase in FPT without altering the FPT normalized for body mass (WBT). Coadministration of RAP-031 with UDCA produced increases in FPT that were associated with an increase in WBT. Myostatin inhibition increases muscle mass without altering the fundamental weakness characteristic of dystrophic muscle. Cotreatment with an NF-κB inhibitor potentiates the effects of myostatin inhibition in improving FPT in mdx mice.

Targeting the Activin Type IIB Receptor to Improve Muscle Mass and Function in the mdx Mouse Model of Duchenne Muscular Dystrophy

The activin receptor type IIB (ActRIIB) is a transmembrane receptor for transforming growth factor-β superfamily members, including myostatin, that are involved in the negative regulation of skeletal muscle mass. We tested the translational hypothesis that blocking ligand binding to ActRIIB for 12 weeks would stimulate skeletal muscle growth and improve muscle function in the mdx mouse. ActRIIB was targeted using a novel inhibitor comprised of the extracellular portion of the ActRIIB fused to the Fc portion of murine IgG (sActRIIB), at concentrations of 1.0 and 10.0 mg/kg(-1) body weight. After 12 weeks of treatment, the 10.0 mg/kg(-1) dose caused a 27% increase in body weight with a concomitant 33% increase in lean muscle mass. Absolute force production of the extensor digitorum longus muscle ex vivo was higher in mice after treatment with either dose of sActRIIB, and the specific force was significantly higher after the lower dose (1.0 mg/kg(-1)), indicating functional improvement in the muscle. Circulating creatine kinase levels were significantly lower in mice treated with sActRIIB, compared with control mice. These data show that targeting the ActRIIB improves skeletal muscle mass and functional strength in the mdx mouse model of DMD, providing a therapeutic rationale for use of this molecule in treating skeletal myopathies.

Inhibition of Activin Receptor Type IIB Increases Strength and Lifespan in Myotubularin-Deficient Mice

X-linked myotubular myopathy (XLMTM) is a congenital disorder caused by deficiency of the lipid phosphatase, myotubularin. Patients with XLMTM often have severe perinatal weakness that requires mechanical ventilation to prevent death from respiratory failure. Muscle biopsy specimens from patients with XLMTM exhibit small myofibers with central nuclei and central aggregations of organelles in many cells. It was postulated that therapeutically increasing muscle fiber size would cause symptomatic improvement in myotubularin deficiency. Recent studies have elucidated an important role for the activin-receptor type IIB (ActRIIB) in regulation of muscle growth and have demonstrated that ActRIIB inhibition results in significant muscle hypertrophy. To evaluate whether promoting muscle hypertrophy can attenuate symptoms resulting from myotubularin deficiency, the effect of ActRIIB-mFC treatment was determined in myotubularin-deficient (Mtm1δ4) mice. Compared with wild-type mice, untreated Mtm1δ4 mice have decreased body weight, skeletal muscle hypotrophy, and reduced survival. Treatment of Mtm1δ4 mice with ActRIIB-mFC produced a 17% extension of lifespan, with transient increases in weight, forelimb grip strength, and myofiber size. Pathologic analysis of Mtm1δ4 mice during treatment revealed that ActRIIB-mFC produced marked hypertrophy restricted to type 2b myofibers, which suggests that oxidative fibers in Mtm1δ4 animals are incapable of a hypertrophic response in this setting. These results support ActRIIB-mFC as an effective treatment for the weakness observed in myotubularin deficiency.

Gene expression profiling of skeletal muscles treated with a soluble activin type IIB receptor

Inhibition of the myostatin signaling pathway is emerging as a promising therapeutic means to treat muscle wasting and degenerative disorders. Activin type IIB receptor (ActRIIB) is the putative myostatin receptor, and a soluble activin receptor (ActRIIB-Fc) has been demonstrated to potently inhibit a subset of transforming growth factor (TGF)-β family members including myostatin. To determine reliable and valid biomarkers for ActRIIB-Fc treatment, we assessed gene expression profiles for quadriceps muscles from mice treated with ActRIIB-Fc compared with mice genetically lacking myostatin and control mice. Expression of 134 genes was significantly altered in mice treated with ActRIIB-Fc over a 2-wk period relative to control mice (fold change > 1.5, P < 0.001), whereas the number of significantly altered genes in mice treated for 2 days was 38, demonstrating a time-dependent response to ActRIIB-Fc in overall muscle gene expression. The number of significantly altered genes in Mstn(-/-) mice relative to control mice was substantially higher (360), but for most of these genes the expression levels in the 2-wk treated mice were closer to the levels in the Mstn(-/-) mice than in control mice (P < 10⁻³⁰). Expression levels of 30 selected genes were further validated with quantitative real-time polymerase chain reaction (qPCR), and a correlation of ≥ 0.89 was observed between the fold changes from the microarray analysis and the qPCR analysis. These data suggest that treatment with ActRIIB-Fc results in overlapping but distinct gene expression signatures compared with myostatin genetic mutation. Differentially expressed genes identified in this study can be used as potential biomarkers for ActRIIB-Fc treatment, which is currently in clinical trials as a therapeutic agent for muscle wasting and degenerative disorders.

Differentiation of human rhabdomyosarcoma RD cells is regulated by reciprocal, functional interactions between myostatin, p38 and extracellular regulated kinase signalling pathways

Rhabdomyosarcoma (RMS) includes heterogeneous tumours of mesenchymal derivation which are genetically committed to the myogenic lineage, but fail to complete terminal differentiation. Previous works have reported on deregulated myostatin, p38 and extracellular regulated kinase (ERK) signalling in RMS cell lines; however, the functional link between these pathways and their relative contribution to RMS pathogenesis and/or maintenance of the transformed phenotype in vitro are unclear. Herein we show that the constitutive expression of a dominant-negative form of activin receptor type IIb (dnACTRIIb), which inhibits myostatin signalling, decreased proliferation and promoted differentiation of the human RMS RD cell line. DnACTRIIb-dependent differentiation of RD cells correlated with a reduced SMAD2/3 (small mother against decapentaplegic) and ERK signalling and the activation of p38 pathway. Conversely, the expression of a constitutively activated ALK5 (activin receptor-like kinase) (caALK5) form, activating SMAD3 and ERK pathways, led to further impairment of RD differentiation. Pharmacological blockade of ERK pathway in RD cells was sufficient to replicate the biological phenotype observed in dnACTRIIb-expressing RD cells, and also recovered the differentiation of caALK5-expressing RD cells. Conversely, deliberate activation of p38 signalling mimics the effect of dnActRIIb and overcame the differentiation block in RD cells. These data indicate the existence of a network formed by myostatin/SMAD2/3, ERK and p38 pathways that, when deregulated, might contribute to the pathogenesis of RMS. The components of this network might, therefore, be a valuable target for interventions towards correcting the malignant phenotype of RMS.

Blocking the Myostatin Signal With a Dominant Negative Receptor Improves the Success of Human Myoblast Transplantation in Dystrophic Mice

Duchenne muscular dystrophy (DMD) is a recessive disease caused by a dystrophin gene mutation. Myoblast transplantation permits to introduce the dystrophin gene in dystrophic muscle fibers. However, the success of this approach is reduced by the short duration of the regeneration following the transplantation, which reduces the number of hybrid fibers. Myostatin (MSTN) is a negative regulator of skeletal muscle development and responsible for limiting regeneration. It binds with high affinity to the activin type IIB receptor (ActRIIB). Our aim was to verify whether the success of the myoblast transplantation is enhanced by blocking the MSTN signal with expression of a dominant negative mutant of ActRIIB (dnActRIIB). In vitro, blocking MSTN activity with a lentivirus carrying dnActRIIB increased proliferation and fusion of human myoblasts because MSTN regulates the expression of several myogenic regulatory factors. In vivo, myoblasts infected with the dnActRIIB lentivirus were transplanted in immunodeficient dystrophic mice. Dystrophin immunostaining of tibialis anterior (TA) cross-sections of these mice 1 month post-transplantation revealed more human dystrophin-positive myofibers following the transplantation of dnActRIIB myoblasts than of control myoblasts. Thus, blocking the MSTN signal with dnActRIIB improved the success of myoblast transplantation by increasing the myoblast proliferation and fusion and changed the expression of myogenic regulatory factors.

RAP-536, A Soluble Activin Receptor Type IIb Murine IgG-Fc Fusion Protein, Prevents Myelodysplastic Syndrome Related Anemia

AbstractAbstract 978Myelodysplastic syndrome (MDS) is a premalignant disease characterized by ineffective hematopoiesis. MDS patients can live for extended periods of time but can develop severe anemia or may progress to acute leukemia. Current therapies require patients to receive chemotherapy and/or blood transfusions to either prevent progression to leukemia or improve anemia. The majority of therapies approved for anemia target the erythropoietin (EPO) pathway. However, recent studies suggest an increased risk of mortality associated with recombinant erythropoietin (EPO) and its derivatives, which may stimulate tumor progression and increase the occurrence of thromboembolic events. While Lenalidomide has shown efficacy in reducing red cell transfusion rates, particularly in the del(5q) subset of MDS patients, additional therapies that increase red blood cell mass would be beneficial. Proteins in the TGF-β superfamily have been reported to play a role in red blood cell (RBC) development, but they act via a different pathway from EPO. RAP-536 is a murine soluble receptor fusion protein based on the activin receptor type IIB (ActRIIB) that binds to certain TGF-β ligands and prevents signaling through the ActRIIB receptor. The purpose of the current study is to evaluate the hematopoietic effect of RAP-536 in a mouse model of MDS.To investigate the effects of RAP-536 in a model of MDS, three month old NUP98-HOX13 transgenic mice (10/dose group) were treated with Vehicle (VEH), or RAP-536 (10 mg/kg) twice per week for the duration of the study. Wild-type littermates (10/dose group) were dosed with VEH or RAP-536 (10 mg/kg) and used as controls. Blood samples and blood smears were collected monthly to conduct CBC measurements and assess changes in blood cells.On study day 0, immediately prior to the first dosing, the NUP98-HOX13 transgenic male mice had significantly decreased levels of RBC (-8.8%, P<0.05), hematocrit (-8.4%, P<0.05) and leukocytes (-67.6%, P<0.001) compared to their wild-type control littermates. Similar trends were seen in female NUP98-HOX13 transgenic mice with RBC (-1.1%) and hematocrit (-2.2%) decreased but not significantly and leukocytes (-68%, P<0.01) decreased significantly compared to the wild type controls.After 1 month of dosing, male NUP98-HOX13 transgenic mice treated with RAP-536 had significantly increased RBC counts (+9.2%, P<0.01), hemoglobin (+13.1%, P<0.01) and hematocrit (+10.8%, P<0.01) compared to their VEH controls. Female NUP98-HOX13 transgenic mice treated with RAP-536 had significant increases in RBC (+3.0%, P<0.05) and hematocrit (+4.9%, P<0.05) when compared to VEH controls. There were no significant changes in leukocyte counts between RAP-536 and VEH treated NUP98-HOX13 transgenic mice in either the male or female cohorts. RAP-536 treatment also had no effect on leukocyte number in wild-type mice. It should be noted that all of the transgenic mice had lower leukocyte counts compared to their wild-type littermates.After 4 months of dosing, NUP98-HOX13 transgenic mice treated with RAP-536 had increased RBC counts (+25.4%, P<0.01), hemoglobin (+22.8%, P<0.01) and hematocrit (+21.9%, P<0.01) in males as well as females (+16.2%, P<0.05; +16.9%, P<0.01; +18.4%, P<0.01) compared to VEH treated controls. All transgenic mice continued to have lower leukocyte counts compared to wild type controls, but there were no significant changes in leukocyte counts between RAP-536 and VEH treated NUP98-HOX13 transgenic mice in either the male or female cohorts.These data suggest that the use of a modified soluble ActRIIB receptor acts to increase red blood cell mass and represents a novel therapy for severe anemia in patients with Myelodysplastic syndrome and other hematopoietic diseases. Disclosures:Mulivor:Acceleron Pharma: Employment. Cadena:Acceleron Pharma: Employment. Suragani:Acceleron Pharma: Employment. Kumar: Acceleron Pharma: Employment. Underwood:Acceleron Pharma: Employment. Seehra: Acceleron Pharma: Employment. Pearsall:Acceleron Pharma: Employment.

RAP-536 Accelerates Recovery From Chemotherapy-Induced Anemia (CIA) without Promoting Tumor Growth In Murine Models of Cancer and CIA

AbstractAbstract 4230We have previously shown that administration of ACE-536, a soluble form of a modified activin type IIB receptor, is an effective therapy in both acute and chronic animal models of anemia. We have further demonstrated that ACE-536 induces red cell development by promoting late stage erythroblast maturation.Anemia is a debilitating complication associated with several chronic diseases, including cancer, and is exacerbated by the use of cancer chemotherapy. Recombinant human erythropoietin (EPO) is the most commonly prescribed treatment for chemotherapy induced anemia. However, there is increasing evidence to suggest that the administration of EPO may accelerate tumor growth by promoting angiogenesis. Therefore, non-EPO based strategies to correct chemotherapy induced anemia without effects on underlying tumor are needed. Several members of the TGF-β superfamily have been shown to be involved in red blood cell production. We have developed ACE-536 as a modified form of the extracellular region of the activin type IIB receptor fused to the Fc region of human IgG1, which binds to and inhibits several ligands in the TGF-β superfamily. Here we report on the effects of RAP-536, a murine analog of ACE-536 containing the modified extracellular region of the activin type IIB receptor fused to the Fc region of a murine IgG, in rodent models of chemotherapy induced anemia and tumor growth.To evaluate the efficacy of RAP-536 in a rodent model of chemotherapy induced anemia 300 g Sprague-Dawley rats were administered a single intravenous dose of carboplatin (75 mg/kg) or phosphate-buffered saline (PBS). Blood samples were taken twice weekly to assess the induction of carboplatin induced anemia. By Day 11 rats treated with carboplatin showed an average drop of 34%, 32% and 36% in red blood cells, hemoglobin and hematocrit, respectively, and were randomized to receive RAP-536 twice weekly at a dose of 10 mg/kg or an equal volume of tris-buffered (TBS). Blood samples were taken twice weekly to evaluate recovery from anemia. Rats treated with RAP-536 exhibited a more rapid rate of recovery (ANOVA; P < 0.001) than those treated with TBS. Moreover, at study termination (Day 35), red blood cells, hemoglobin and hematocrit levels of RAP-536 treated rats were 28%, 23% and 23% greater, respectively, than TBS-treated controls (P < 0.01).EPO administration has previously been shown to accelerate the rate of tumor growth in a mouse tumor bearing model; therefore, we sought to investigate the effect of RAP-536 in this context. Eight-week old mice were injected subcutaneously in the right hind flank with 3×105 Lewis lung carcinoma cells expressing a luciferase reporter. Mice were randomized to receive weekly subcutaneous injections of TBS, RAP-536 (10 mg/kg) or EPO (200 U/kg). Tumor growth was assessed twice weekly by manual external caliper measurements and once weekly by luciferase activity using an IVIS in vivo imaging system. Tumors in EPO treated mice grew at a moderately accelerated rate compared to TBS and RAP-536 treated mice; however, this was not statistically significant. At study termination tumors were excised and weighed. Mice treated with EPO showed statistically significant larger tumors compared to TBS treated animals (P = 0.0126). On the other hand, mice treated with RAP-536 did not show statistically significant increases in tumor size (P = 0.1689).In conclusion, targeting the TGF-β signaling pathway with RAP-536 is a viable therapeutic approach for the treatment of anemia induced by chemotherapy treatment and may represent a safer alternative to EPO-based therapies. Disclosures:Cadena:Acceleron Pharma: Employment. Suragani:Acceleron Pharma: Employment. Mitchell:Acceleron Pharma: Employment. Ucran:Acceleron Pharma: Employment. Liharska:Acceleron Pharma: Employment. Sako:Acceleron Pharma: Employment. Monnell:Acceleron Pharma: Employment. Underwood:Acceleron Pharma: Employment. Kumar:Acceleron Pharma: Employment. Pearsall:Acceleron Pharma: Employment.

ACE-536, a Modified Type II Activin Receptor Increases Red Blood Cells In Vivo by Promoting Maturation of Late Stage Erythroblasts

AbstractAbstract 4236Anemia is one of the most common blood disorders in several diseases including cancer, heart failure, chronic kidney disease (CKD) and Myeloid Dysplastic Syndromes (MDS) associated with a negative outcome. Administration of recombinant Erythropoietin (EPO) represents the most common treatment for anemia. However, a significant number of people remain hypo or non-responsive to EPO treatment, and in some cases its use has been linked to tumor growth, cardiovascular disease and poorer survival. The members of TGFβ super family of ligands (Activins, GDFs and BMPs) and receptors (Type I and II) regulate more than 500 target genes transcriptionally by Smad phosphorylation and are involved in many cellular functions including cell growth, adhesion, migration, differentiation and apoptosis in a concentration and context dependent manner. Members of the TGFβ family have also demonstrated a role in erythropoiesis. ACE-536, a non-ESA agent is a soluble human Fc fusion chimera of a modified Activin Type IIb receptor with a mutation in its extracellular domain. Surface Plasmon Analysis (Biacore) analysis and cell based reporter assays revealed that this mutation disrupted its binding to Activin A but not to GDF11 or GDF8. ACE-536 acts as a decoy receptor for TGFβ signaling and demonstrated potent increase in red blood cells in all the tested animals (mice, rats and monkeys). Subcutaneous administration of ACE-536 (10mg/kg) to C57BL/6 mice resulted in a significant increase in hematocrit, hemoglobin and red blood cells (RBC) over the TBS treated vehicle group after 4 days. These observations were seen even in the presence of an EPO neutralizing antibody; suggesting that EPO is not directing the initial RBC response to ACE-536 treatment. There were no increase in BFU-E or CFU-E colony formation from bone marrow and spleen after 48hrs treatment with ACE-536 over TBS treated group demonstrating that it does not have effect on erythroid progenitor population. Differentiation profiling of bone marrow and splenic erythroblasts by flow cytometric analysis revealed that ACE-536 promotes maturation of developing erythroblasts. ACE-536 treatment for 72 hours resulted in a decrease in basophilic erythroblasts and an increase in late stage poly, ortho chromatophilic erythroblasts in bone marrow and spleen compared to the TBS treated mice. Treatment of Sprague-Dawley rats with a murine analogue of ACE-536 (RAP-536; 10mg/kg) increased the reticulocyte formation in peripheral blood over vehicle treated group. ACE-536 (10mg/kg) treatment combined with recombinant human EPO (1800 units/kg) for 72 hours increased RBC, hematocrit and hemoglobin by approximately 23% over TBS treated vehicle group and 12% over EPO treatment alone. Consistent with its role in proliferation, EPO increased splenic basophilic erythroblast formation. However, ACE-536 treatment combined with EPO significantly promoted maturation of late stage erythroblasts; demonstrating a novel mechanism during erythroid differentiation. To gain further insights into its mechanism of action, C57BL/6 mice were administered with or without RAP-536 (10mg/ml twice a week) pre treated for a week with neutralizing anti-Activin A (10mg/kg) or ActRIIa (10mg/ml) or ActRIIb (10mg/ml) (does not bind ACE-536) antibodies. Anti-ActRIIa but not anti-Activin A or anti- ActRIIb antibody pre-treatment inhibited the RBC increase by RAP-536 suggesting that ActRIIa or its ligands are necessary for transducing the signal. To summarize, ACE-536 treatment results in a rapid increase in red blood cells by a novel mechanism promoting maturation of late stage erythroblasts. The efficacy of ACE-536 molecule was tested in several acute and chronic anemia animal models including blood loss anemia, chemotherapy induced anemia, chronic kidney disease (5/6 Nephrectomy) and Myeloid Dysplastic Syndrome (MDS) and found that ACE-536 treatment prevents or decreases anemia in all these models. Furthermore, unlike EPO, ACE-536 did not promote tumor progression (in Lewis Lung Carcinoma model) thus offering strong promise as alternate treatments for anemia. Disclosures:Suragani:Acceleron Pharma: Employment. Cadena:Acceleron Pharma: Employment. Mitchell:Acceleron Pharma: Employment. Sako:Acceleron Pharma: Employment. Davies:Acceleron Pharma: Employment. Tomkinson:Acceleron Pharma: Employment. Devine:Acceleron Pharma: Employment. Ucran:Acceleron Pharma: Employment. Grinberg:Acceleron Pharma: Employment. Underwood:Acceleron Pharma: Employment. Pearsall:Acceleron Pharma: Employment. Seehra:Acceleron Pharma: Employment. Kumar:Acceleron Pharma: Employment.

P3.09 The effect of combining activin receptor type IIB inhibition and prednisolone treatment in mdx mice

P3.09 The effect of combining activin receptor type IIB inhibition and prednisolone treatment in mdx mice

Reversing Cachexia

Muscle atrophy (cachexia) in cancer patients is a life-threatening condition for which therapeutic options are limited. Zhou et al. (2010) now identify a new target for treating cachexia, the activin type-2 receptor (ActRIIB). In several mouse models of cachexia, the authors reversed wasting of skeletal and cardiac muscle and increased life span by blocking ActRIIB with a decoy receptor.