Receptor Type IIA Research Articles

Activins and their related proteins in colon carcinogenesis: insights from early and advanced azoxymethane rat models of colon cancer.

BackgroundActivin-A may exert pro- or anti-tumorigenic activities depending on cellular context. However, little is known about its role, or the other mature activin proteins, in colorectal carcinoma (CRC). This study measured the expression of activin βA- & βB-subunits, activin type IIA & IIB receptors, smads 2/3/4/6/7 and follistatin in CRC induced by azoxymethane (AOM) in rats. The results were compared with controls and disseminated according to the characteristics of histopathological lesions.MethodsEighty male Wistar rats were allocated into 20 controls and the remaining were equally divided between short ‘S-AOM’ (15 weeks) and long ‘L-AOM’ (35 weeks) groups following injecting AOM for 2 weeks. Subsequent to gross and histopathological examinations and digital image analysis, the expression of all molecules was measured by immunohistochemistry and quantitative RT-PCR. Activin-A, activin-B, activin-AB and follistatin were measured by ELISA in serum and colon tissue homogenates.ResultsColonic pre-neoplastic and cancerous lesions were identified in both AOM groups and their numbers and sizes were significantly (P < 0.05) greater in the L-AOM group. All the molecules were expressed in normal colonic epithelial cells. There was a significantly (P < 0.05) greater expression of βA-subunit, IIB receptor and follistatin in both pre-neoplastic and cancerous tissues. Oppositely, a significant (P < 0.05) decrease in the remaining molecules was detected in both AOM groups. Metastatic lesions were only observed within the L-AOM group and were associated with the most significant alterations of all molecules. Significantly higher concentrations of activin-A and follistatin and lower activin-AB were also detected in both groups of AOM. Tissue and serum concentrations of activin-A and follistatin correlated positively, while tissue activin-AB inversely, and significantly with the numbers and sizes of colonic lesions.ConclusionsNormal rat colon epithelial cells are capable of synthesising, controlling as well as responding to activins in a paracrine/autocrine manner. Colonic activin systems are pathologically altered during tumorigenesis and appear to be time and lesion-dependent. Activins could also be potential sensitive markers and/or molecular targets for the diagnosis and/or treatment of CRC. Further studies are required to illustrate the clinical value of activins and their related proteins in colon cancer.Electronic supplementary materialThe online version of this article (doi:10.1186/s12885-016-2914-9) contains supplementary material, which is available to authorized users.

Testing for the erythropoiesis-stimulating agent Sotatercept/ACE-011 (ActRIIA-Fc) in serum by means of Western blotting and LC-HRMS.

Sotatercept (formerly ACE-011) is a glycosylated, dimeric fusion protein composed of the extracellular domain of the human activin receptor type IIA (ActRIIA) and the Fc region of human IgG1. The protein-based drug candidate acts as a ligand trap which competitively binds to activin A and other members of the transforming growth factor beta superfamily, thus blocking signalling through ActRIIA. Since the inhibition of activin A was found to significantly increase bone formation and quality, Sotatercept was originally developed for the treatment of diseases involving bone loss. But as the protein therapeutic also stimulates erythropoiesis by a mechanism independent of the EPO receptor, it has been evaluated for the treatment of anaemia in rare blood diseases such as beta thalassemia. Due to its positive effects on erythropoiesis and bone formation, Sotatercept may also be misused as performance-enhancing agent in sports. Within this study, two complementary detection assays for Sotatercept and related ActRIIA-Fc fusion proteins in serum samples were developed. While the first assay combines affinity purification and Western blotting to generically detect ActRIIA-Fc fusion proteins irrespective of their amino acid sequence, the liquid chromatography-high resolution mass spectrometry (LC-HRMS) method is highly specific for proteolytic peptides originating from the receptor and Fc domain of Sotatercept. Both approaches can readily be modified to include other pharmaceutical proteins such as therapeutic antibodies, and serve as proof-of-concept for the capability of the approach to detect TGF-β inhibitors and Fc fusion proteins in doping control serum samples. Copyright © 2016 John Wiley & Sons, Ltd.

Ligand trap of the activin receptor type IIA inhibits osteoclast stimulation of bone remodeling in diabetic mice with chronic kidney disease

Dysregulation of skeletal remodeling is a component ofrenal osteodystrophy. Previously, we showed that activinreceptor signaling is differentially affected in various tissues in chronic kidney disease (CKD). We tested whether a ligand trap for the activin receptor type 2A (RAP-011) is an effective treatment of the osteodystrophy of the CKD-mineral bone disorder. With a 70% reduction inthe glomerular filtration rate, CKD was induced at 14weeks of age in the ldlr-/- high fat-fed mouse modelofatherosclerotic vascular calcification and diabetes. Twenty mice with CKD, hyperphosphatemia, hyperparathyroidism, and elevated activin A were treated with RAP-011, wherease 19 mice were given vehicle twice weekly from week 22 until the mice were killed at 28 weeks of age. The animals were then evaluated by skeletal histomorphometry, micro-computed tomography, mechanical strength testing, and exvivo bone cell culture. Results in the CKD groups were compared with those of the 16 sham-operated ldlr-/- high fat-fed mice. Sham-operated mice had low-turnover osteodystrophy and skeletal frailty. CKD stimulated bone remodeling with significant increases in osteoclast and osteoblast numbers and bone resorption. Compared with mice with CKD andsham-operated mice, RAP-011 treatment eliminated the CKD-induced increase in these histomorphometric parameters and increased trabecular bone fraction. RAP-011 significantly increased cortical bone area and thickness. Activin A-enhanced osteoclastogenesis was mediated through p-Smad2 association with c-fos and activation of nuclear factor of activated T cells c1 (NFATc1). Thus, an ActRIIA ligand trap reversed CKD-stimulated bone remodeling, likely through inhibition ofactivin-A induced osteoclastogenesis.

Molecular characterization of Activin Receptor Type IIA and its expression during gonadal maturation and growth stages in rohu carp.

Activin receptor type IIA (ActRIIA), a transmembrane serine/threonine kinase receptor is an important regulator of physiological traits, viz., reproduction and body growth in vertebrates including teleosts. However, existing knowledge of its role in regulating fish physiology is limited. To address this, we have cloned and characterized the ActRIIA cDNA of Labeo rohita (rohu), an economically important fish species of the Indian subcontinent. Comparative expression profiling of the receptor gene at various reproductive and growth stages supports to its role in promoting oocyte maturation, spermatogenesis and skeletal muscle development via interaction with multiple ligands of transforming growth factor-β (TGF-β) family. The full-length cDNA of rohu ActRIIA was found to be of 1587bp length encoding 528 amino acids. The three-dimensional structure of the intracellular kinase domain of rohu ActRIIA has also been predicted. Phylogenetic relationship studies showed that the gene is evolutionarily conserved across the vertebrate lineage implicating that the functioning of the receptor is more or less similar in vertebrates. Taken together, these findings could be an initial step towards the use of ActRIIA as a potential candidate gene marker for understanding the complex regulatory mechanism of fish reproduction and growth.

Activin A increases arterial pressure in the hypothalamic paraventricular nucleus in rats by angiotension II.

Activin A, a member of the transforming growth factor β superfamily, plays an important role in the central nervous system as a neurotrophic and neuroprotective factor. The hypothalamic paraventricular nucleus (PVN) in the central nervous system is characterized as an important integrative site to regulate arterial pressure (AP). However, whether activin A in the PVN is involved in the regulation of AP is not well characterized. This study aimed to determine the effect of activin A on AP in the PVN in rats. The results showed that activin βA, activin type IIA and IIB receptors (ActRIIA and ActRIIB), and Smad2 and Smad3 mRNA expressions were detectable in the PVN of WKY rats by reverse-transcription PCR, and the expression of ActRIIA protein in the PVN was further confirmed by immunohistochemical staining. A microinjection of angiotensin II (AngII) (0.1 nmol/100 nl) or activin A (2 ng/100 nl) into the PVN increased AP significantly in WKY rats (P<0.05). Moreover, activin A (5 ng/ml) promoted AngII release from the primary cultured PVN neurons that can increase AP and upregulated the expressions of ActRIIA and Smad3 mRNA in the primary cultured PVN neurons (P<0.05). These data suggest that activin A can regulate AP in the PVN in an autocrine or a paracrine manner, which is related to AngII release and the ActRIIA-Smad3 signal pathway.

NB‐3 signaling mediates the cross‐talk between post‐traumatic spinal axons and scar‐forming cells

Little is known about the molecules mediating the cross-talk between post-traumatic axons and scar-forming cells after spinal cord injury. We found that a sustained NB-3 induction was simultaneously present in the terminations of post-traumatic corticospinal axons and scar-forming cells at the spinal lesion site, where they were in direct contact when axons tried to penetrate the glial scar. The regrowth of corticospinal axons was enhanced invivo with NB-3 deficiency or interruption of NB-3 trans-homophilic interactions. Biochemical, invitro and invivo evidence demonstrated that NB-3 homophilically interacted in trans to initiate a growth inhibitory signal transduction from scar-forming cells to neurons by modulating mTOR activity via CHL1 and PTPσ. NB-3 deficiency promoted BMS scores, electrophysiological transmission, and synapse reformation between regenerative axons and neurons. Our findings demonstrate that NB-3 trans-homophilic interactions mediate the cross-talk between post-traumatic axons and scar-forming cells and impair the intrinsic growth ability of injured axons.

Expression of receptor protein tyrosine phosphatase δ, PTPδ, in mouse central nervous system

Protein tyrosine phosphate δ (PTPδ), one of the receptor type IIa protein tyrosine phosphates, is known for its roles in axon guidance, synapse formation, cell adhesion, and tumor suppression. Alternative splicing of this gene generates at least four (A–D) isoforms; however, the major isoform in vivo is yet to be determined. The protein localization has neither been revealed. We have generated anti-mouse PTPδ-specific monoclonal antibody and analyzed the protein expression in wild-type and Ptpδ knockout mice. Immunoblot analysis of various organs revealed that neuronal tissues express both C-and D-isoforms of PTPδ, whereas non-neuronal tissues express only C-isoform. Immunohistochemistry of wild-type or Ptpδ heterozygous sections showed that olfactory bulb, cerebral cortex, hippocampus, cerebellum, and several nuclei in brain stem exhibit moderate to strong positive signals. These signals were absent in Ptpδ knockout specimens. Higher magnification revealed differences between expression patterns of PTPδ mRNA and its protein product. In hippocampus, weak mRNA expression in CA1 stratum pyramidale but strong immunostaining in the stratum lacunosum moleculare was observed, suggesting the axonal expression of PTPδ in the entorhinal cortical afferents. Olfactory mitral cells exhibited mRNA expression in cell bodies and protein localization in their dendritic fields, glomerular and external plexiform layers. Nissl staining showed that the external plexiform layer was reduced in Ptpδ knockout mice. Golgi-impregnation confirmed the poor dendritic growth of homozygous mitral cells. These results suggest that PTPδ may localize in axons as well as in dendrites to regulate their elaboration in the central nervous system.

Ligand trap for the activin type IIA receptor protects against vascular disease and renal fibrosis in mice with chronic kidney disease

The causes of cardiovascular mortality associated with chronic kidney disease (CKD) are partly attributed to the CKD-mineral bone disorder (CKD-MBD). The causes of the early CKD-MBD are not well known. Our discovery of Wnt (portmanteau of wingless and int) inhibitors, especially Dickkopf 1, produced during renal repair as participating in the pathogenesis of the vascular and skeletal components of the CKD-MBD implied that additional pathogenic factors are critical. In the search for such factors, we studied the effects of activin receptor type IIA (ActRIIA) signaling by using a ligand trap for the receptor, RAP-011 (a soluble extracellular domain of ActRIIA fused to a murine IgG-Fc fragment). In a mouse model of CKD that stimulated atherosclerotic calcification, RAP-011 significantly increased aortic ActRIIA signaling assessed by the levels of phosphorylated Smad2/3. Furthermore, RAP-011 treatment significantly reversed CKD-induced vascular smooth muscle dedifferentiation as assessed by smooth muscle 22α levels, osteoblastic transition, and neointimal plaque calcification. In the diseased kidneys, RAP-011 significantly stimulated αklotho levels and it inhibited ActRIIA signaling and decreased renal fibrosis and proteinuria. RAP-011 treatment significantly decreased both renal and circulating Dickkopf 1 levels, showing that Wnt activation was downstream of ActRIIA. Thus, ActRIIA signaling in CKD contributes to the CKD-MBD and renal fibrosis. ActRIIA signaling may be a potential therapeutic target in CKD.

Phase 1 Dose-Escalation Study of Sotatercept (ACE-011) in Combination with Lenalidomide and Dexamethasone in Patients with Relapsed and/or Refractory Multiple Myeloma

IntroductionAnemia and bone disease are hallmarks of multiple myeloma (MM). Sotatercept (ACE-011) is a novel, first-in-class activin type IIA receptor fusion protein that binds with high affinity to activin A and GDF11, and it acts during late-stage erythropoiesis to increase the production of mature erythrocytes through a mechanism independent of erythropoietin. Sotatercept has shown promising activity in clinical trials for anemia in myelodysplastic syndromes (Komrokji et al., ASH 2014) and in thalassemia (Cappellini et al., EHA 2015). Additionally, we have shown that targeting activin A through an analog of sotatercept reverses osteoblast inhibition and improves MM bone disease in a mouse model (Vallet et al., PNAS 2010). Lenalidomide increases activin A secretion with consequent inhibition of osteoblastogeneis, and this can be abrogated by treatment with an activin A neutralizing antibody (Scullen et al., Leukemia 2013). Sotatercept has been previously studied with melphalan, prednisolone, and thalidomide in MM (Abdulkadyrov et al., Br J Haematol 2014). Based on these findings, we evaluated sotatercept in combination with lenalidomide and dexamethasone in MM (NCT01562405).MethodsPatient with relapsed and/or refractory MM with at least one prior line of therapy, anemia with hemoglobin <13 g/dL, lytic bone disease, and otherwise adequate organ function were eligible to participate. Sotatercept 10, 15, 30, or 45 mg was given s.c. q28 days along with lenalidomide and weekly dexamethasone on a standard 28 day schedule, with dose escalation following a 3 + 3 design. Sotatercept was held for hemoglobin ≥13 g/dL or for ≥ grade 3 hypertension. Bone mineral density by DEXA was assessed after four cycles. Bisphosphonates were not permitted during the study; prior bisphosphonate therapy was allowed.ResultsThirteen patients with a median age of 62 years (range 49-77) and a median of 2 prior lines (range 1-5) of therapy have been enrolled to date (July 31, 2015). Median duration of treatment is 8.1 months (range 0.5, 27 months); five patients continue on study. The MTD has not been reached, and the current dosing level is sotatercept 45 mg with lenalidomide 25 mg. Grade 3-4 adverse events included anemia (38%), diarrhea (15%), fatigue (15%), hypophosphatemia (15%), and thrombocytopenia (38%). Grade 3 hypertension occurred in one patient receiving sotatercept 15 mg (hemoglobin at the time, 11.5 g/dL). There was one death on study that was unrelated to treatment. In patients who completed at least two cycles of treatment, there was a significant mean increase in hemoglobin on study of 0.94 g/dL (N = 10, p = 0.0048) from a mean starting hemoglobin 10.27 g/dL (range 8.6, 12.2). Mean maximal increase in hemoglobin was 2.11 g/dL (range 1.1, 4). Bone density by DEXA was assessed after four cycles. In patients who received a cumulative dose of sotatercept over 45 mg (N = 6), total lumbar spine BMD increased by a mean of 2.0% after four cycles; 83% had increase in BMD. ORR for this combination was 60% (CR = 1, VGPR = 1, PR = 4, SD = 4) in patients evaluable for response.ConclusionSotatercept in combination with lenalidomide and dexamethasone is well tolerated with expected toxicities related to lenalidomide in MM. Preliminary data from this ongoing study suggest that sotatercept leads to early increases in both hemoglobin and bone mineral density, and it is the first agent that may address both of these significant causes of morbidity in MM. DisclosuresLaubach:Novartis: Research Funding; Onyx: Research Funding; Celgene: Research Funding; Millennium: Research Funding. Nooka:Onyx Pharmaceuticals: Consultancy; Spectrum Pharmaceuticals: Consultancy. O'Donnell:Millennium: Consultancy. Puccio-Pick:Celgene Corp.: Employment. Laadem:Celgene Corporation: Employment. Sherman:Acceleron Pharma: Employment. Raje:Acetylon: Research Funding; AstraZeneca: Research Funding; Onyx: Consultancy; Takeda: Consultancy; Eli Lilly: Research Funding; Millenium: Consultancy; Novartis: Consultancy; Amgen: Consultancy; BMS: Consultancy; Celgene Corporation: Consultancy.

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.

Ability of Interleukin-33- and Immune Complex-Triggered Activation of Human Mast Cells to Down-Regulate Monocyte-Mediated Immune Responses.

Mast cells have been implicated in the pathogenesis of rheumatoid arthritis (RA). In particular, their activation by interleukin-33 (IL-33) has been linked to the development of arthritis in animal models. The aim of this study was to evaluate the functional responses of human mast cells to IL-33 in the context of RA. Human mast cells were stimulated with IL-33 combined with plate-bound IgG or IgG anti-citrullinated protein antibodies (ACPAs), and their effects on monocyte activation were evaluated. Cellular interactions of mast cells in RA synovium were assessed by immunofluorescence analysis, and the expression of messenger RNA (mRNA) for mast cell-specific genes was evaluated in synovial biopsy tissue from patients with early RA who were naive to treatment with disease-modifying antirheumatic drugs. IL-33 induced the up-regulation of Fcγ receptor type IIa and enhanced the activation of mast cells by IgG, including IgG ACPAs, as indicated by the production of CXCL8/IL-8. Intriguingly, mast cell activation triggered with IL-33 and IgG led to the release of mediators such as histamine and IL-10, which inhibited monocyte activation. Synovial mast cells were found in contact with CD14+ monocyte/macrophages. Finally, mRNA levels of mast cell-specific genes were inversely associated with disease severity, and IL-33 mRNA levels showed an inverse correlation with the levels of proinflammatory markers. When human mast cells are activated by IL-33, an immunomodulatory phenotype develops, with human mast cells gaining the ability to suppress monocyte activation via the release of IL-10 and histamine. These findings, together with the presence of synovial mast cell-monocyte interactions and the inverse association between the expression of mast cell genes at the synovial level and disease activity, suggest that these newly described mast cell-mediated inhibitory pathways might have a functional relevance in the pathogenesis of RA.

RAP-011 improves erythropoiesis in zebrafish model of Diamond-Blackfan anemia through antagonizing lefty1

AbstractDiamond-Blackfan Anemia (DBA) is a bone marrow failure disorder characterized by low red blood cell count. Mutations in ribosomal protein genes have been identified in approximately half of all DBA cases. Corticosteriod therapy and bone marrow transplantation are common treatment options for patients; however, significant risks and complications are associated with these treatment options. Therefore, novel therapeutic approaches are needed for treating DBA. Sotatercept (ACE-011, and its murine ortholog RAP-011) acts as an activin receptor type IIA ligand trap, increasing hemoglobin and hematocrit in pharmacologic models, in healthy volunteers, and in patients with β-thalassemia, by expanding late-stage erythroblasts through a mechanism distinct from erythropoietin. Here, we evaluated the effects of RAP-011 in zebrafish models of RPL11 ribosome deficiency. Treatment with RAP-011 dramatically restored hemoglobin levels caused by ribosome stress. In zebrafish embryos, RAP-011 likely stimulates erythropoietic activity by sequestering lefty1 from erythroid cells. These findings identify lefty1 as a signaling component in the development of erythroid cells and rationalize the use of sotatercept in DBA patients.

15 HLA-MATCHED ALLOGENEIC STEM CELL TRANSPLANTATION IMPROVES OVERALL SURVIVAL OF HIGHER RISK MYELODYSPLASTIC SYNDROME

sotatercept (ACE-011), a novel first-in-class activin type IIA receptor fusion protein, in anemic patients with IPSS Low/Intermediate-1risk MDS or non-proliferative CMML. ClinicalTrials.gov identifier: NCT01736683. Methods: The primary endpoint of this study is erythroid hematological improvement (HI-E; defined by modified IWG 2006 criteria). Secondary endpoints include rate of RBC-transfusion independence (RBC-TI) ≥8 weeks and safety. Eligible patients have lower-risk MDS or non-proliferative CMML and anemia, with no response, loss of response, or low chance of response to ESAs. Patients receive subcutaneous sotatercept at 0.1, 0.3, 0.5, or 1.0 mg/kg every 3 weeks. Results: As of May 22, 2014, 54 MDS patients were enrolled: 7, 6, 21, and 20 in the sotatercept 0.1, 0.3, 0.5, and 1.0 mg/kg groups, respectively. Median age 71 years, median time from diagnosis 4 years, and 70% of patients were male. Patients received a median 6 RBC units (range 0–16) in the 8 weeks before starting treatment; 46 received ≥4 RBC units (high transfusion burden; HTB) and 8 received. Of 53 patients evaluable for efficacy, 24 achieved HI-E: 0, 4, 8, and 12 patients in the sotatercept 0.1, 0.3, 0.5, and 1.0 mg/kg groups, respectively. Five of 46 (11%) HTB patients achieved RBCTI ≥8 weeks; duration of response ranged from 59–345+ days. Five of 8 (63%) LTB patients achieved RBC-TI ≥8 weeks with a mean hemoglobin increase of ≥1.5 g/dL sustained for ≥8 weeks; duration ranged from 76–233+ days. Sotatercept was generally well tolerated. Twenty (37%) patients reported ≥1 suspected treatment-related adverse event. The most common were: fatigue/asthenia (13%), headache (9%), decreased appetite (7%), and nausea (7%). Three patients discontinued due to suspected treatment-related adverse events: 1 each with grade 2 hemolytic anemia, grade 3 hypertension, and grade 2 muscular weakness in the sotatercept 0.3, 0.5, and 1.0 mg/kg groups, respectively. Conclusions: Sotatercept was well tolerated, with promising evidence of clinical activity in this cohort of anemic, lower-risk MDS patients. Further exploration of sotatercept treatment is ongoing. PF and AFL contributed equally to this abstract as senior coauthors.

An Open-Label, Phase 2, Dose-Finding Study of Sotatercept (ACE-011) in Patients with Low or Intermediate-1 (Int-1)-Risk Myelodysplastic Syndromes (MDS) or Non-Proliferative Chronic Myelomonocytic Leukemia (CMML) and Anemia Requiring Transfusion

Introduction: Anemia, a hallmark of MDS, is challenging to treat, particularly after failure of erythropoiesis-stimulating agents (ESAs). Sotatercept (ACE-011) is a novel and first-in-class activin type IIA receptor fusion protein that acts on late-stage erythropoiesis to increase mature erythrocyte release into the circulation (Carrancio et al. Br J Haematol 2014;165:870-82). Treatment with sotatercept stimulated erythropoiesis and significantly increased hemoglobin (Hb) levels in healthy volunteers (Sherman et al. J Clin Pharmacol 2013;53:1121-30), supporting its clinical development for the treatment of anemia in patients (pts) with lower-risk MDS. Methods: The primary objective of this phase 2, open-label, dose-finding study is to determine a safe, tolerable, and effective dose of sotatercept resulting in erythroid hematological improvement (HI-E; modified IWG 2006 criteria) in pts with anemia and IPSS-defined Low or Int-1-risk MDS or non-proliferative CMML (white blood cells 500 mIU/mL). Pts received subcutaneous sotatercept at dose levels of 0.1, 0.3, 0.5, or 1.0 mg/kg once every 3 weeks. ClinicalTrials.gov identifier: NCT01736683. Results: As of May 22, 2014, a total of 54 MDS pts were enrolled: 7, 6, 21, and 20 in the sotatercept 0.1, 0.3, 0.5, and 1.0 mg/kg dose groups, respectively. Median age was 71 years (range 56–86) and median time from diagnosis was 4 years (range 0–31); most pts were male (70%). Pts received a median of 6 RBC units (range 0–18) in the 8 weeks prior to treatment start. Forty-five pts (83%) received ≥ 4 RBC units in the 8 weeks prior to treatment start (high transfusion burden; HTB), and 9 pts (17%) received 500 mIU/mL. Of the 53 pts evaluable for efficacy, HI-E was observed in 21 pts (40%) overall: 0, 4 (67%), 8 (40%), and 9 pts (45%) in the sotatercept 0.1, 0.3, 0.5, and 1.0 mg/kg dose groups, respectively. Nineteen of 44 HTB pts responded with a ≥ 4 RBC units/8 weeks transfusion burden reduction; duration of transfusion response appeared to be dose-dependent. Five HTB pts achieved RBC-TI ≥ 8 weeks, with RBC-TI duration ranging from 59–345+ days. Eight of 9 LTB pts showed Hb increases, not influenced by transfusion, ranging from 1.3–3.8 g/dL. Of these, 2 pts had a Hb increase ≥ 1.5 g/dL sustained for ≥ 8 weeks. Pts with Hb > 11.0 g/dL were subject to dose delay per protocol, which may have impacted Hb increase sustainability. RBC-TI ≥ 8 weeks was achieved in 6 LTB pts. Increases in platelet and neutrophil levels were seen in pts with baseline thrombocytopenia and pts with baseline neutropenia, respectively. Sotatercept was generally well tolerated. Twenty pts (37%) reported ≥ 1 suspected treatment-related adverse event (AE); fatigue (11%), headache (9.3%), decreased appetite (7.4%), and nausea (7.4%) were the most common. Of 35 pts (65%) who discontinued treatment, 28 discontinued due to lack of therapeutic effect and 4 due to AEs. Of those AEs leading to discontinuation, 3 were suspected to be treatment-related: 1 pt with grade 2 hemolytic anemia, 1 pt with grade 3 hypertension, and 1 pt with grade 2 muscular weakness in the sotatercept 0.3, 0.5, and 1.0 mg/kg dose groups, respectively. Other reasons for discontinuation were withdrawal of consent (n = 2; 4%) and pt decision (n = 1; 2%). Conclusions: Sotatercept was well tolerated in lower-risk MDS pts at the dose levels tested, with promising evidence of clinical activity in this largely HTB cohort of ESA-refractory, anemic, lower-risk MDS pts. Further exploration of higher sotatercept dose levels and longer-term treatment is planned. PF and AFL contributed equally to this abstract as senior co-authors. Disclosures Komrokji:Celgene Corporation: Consultancy, Research Funding. Off Label Use: Sotatercept (ACE-011) is an investigational agent that is being assessed for efficacy and safety in myelodysplastic syndromes.. Garcia-Manero:Celgene Corporation: Research Funding. Ades:Novartis: Research Funding; Celgene Corporation: Research Funding. Laadem:Celgene Corporation: Employment, Equity Ownership. Vo:Celgene Corporation: Employment. Prebet:Celgene Corporation: Honoraria. Boyd:US Oncology: Research Funding. Sekeres:Celgene: Membership on an entity9s Board of Directors or advisory committees; Amgen Corporation: Membership on an entity9s Board of Directors or advisory committees; Boehringer-Ingelheim Corporation: Membership on an entity9s Board of Directors or advisory committees. Beyne-Rauzy:Novartis: Research Funding; Celgene: Research Funding. Zou:Celgene: Employment. Attie:Acceleron Pharma: Employment. Sherman:Acceleron Pharma: Employment, Equity Ownership. Fenaux:Novartis: Research Funding; Janssen: Research Funding; Celgene: Research Funding. List:Celgene: Consultancy.

RAP-011, an activin receptor ligand trap, increases hemoglobin concentration in hepcidin transgenic mice.

Over expression of hepcidin antimicrobial peptide is a common feature of iron-restricted anemia in humans. We investigated the erythroid response to either erythropoietin or RAP-011, a "murinized" ortholog of sotatercept, in C57BL/6 mice and in hepcidin antimicrobial peptide 1 over expressing mice. Sotatercept, a soluble, activin receptor type IIA ligand trap, is currently being evaluated for the treatment of anemias associated with chronic renal disease, myelodysplastic syndrome, β-thalassemia, and Diamond Blackfan anemia and acts by inhibiting signaling downstream of activin and other Transforming Growth Factor-β superfamily members. We found that erythropoietin and RAP-011 increased hemoglobin concentration in C57BL/6 mice and in hepcidin antimicrobial peptide 1 over expressing mice. While erythropoietin treatment depleted splenic iron stores in C57BL/6 mice, RAP-011 treatment did not deplete splenic iron stores in mice of either genotype. Bone marrow erythroid progenitors from erythropoietin-treated mice exhibited iron-restricted erythropoiesis, as indicated by increased median fluorescence intensity of transferrin receptor immunostaining by flow cytometry. In contrast, RAP-011-treated mice did not exhibit the same degree of iron-restricted erythropoiesis. In conclusion, we have demonstrated that RAP-011 can improve hemoglobin concentration in hepcidin antimicrobial peptide 1 transgenic mice. Our data support the hypothesis that RAP-011 has unique biologic effects which prevent or circumvent depletion of mouse splenic iron stores. RAP-011 may, therefore, be an appropriate therapeutic for trials in human anemias characterized by increased expression of hepcidin antimicrobial peptide and iron-restricted erythropoiesis.

Inhibition of Activin A Ameliorates Skeletal Muscle Injury and Rescues Contractile Properties by Inducing Efficient Remodeling in Female Mice

Activin A, a member of the transforming growth factor-β superfamily, provides pleiotropic regulation of fibrosis and inflammation. We aimed at determining whether selective inhibition of activin A would provide a regenerative benefit. The introduction of activin A into normal muscle increased the expression of inflammatory and muscle atrophy genes Tnf, Tnfrsf12a, Trim63, and Fbxo32 by 3.5-, 10-, 2-, and 4-fold, respectively. The data indicate a sensitive response of muscle to activin A. Two hours after cardiotoxin-induced muscle damage, local activin A protein expression increased by threefold to ninefold. Neutralization of activin A with a specific monoclonal antibody in this muscle injury model decreased the muscle protein levels of lymphotoxin α and Il17a by 32% and 42%, respectively. Muscle histopathological features showed that activin A antibody-treated mice displayed an increase in muscle degradation, with the concomitant 9.2-fold elevation in F4/80-positive cells 3 days after injury. At the same time, the number of Pax7/Myod1-positive cells also increased, indicative of potentiated muscle precursor activation. Ultimately, activin A inhibition resulted in rapid recovery of muscle contractile properties indicated by a restoration of maximum and specific force. In summary, selective inhibition of activin A with a monoclonal antibody in muscle injury leads to the early onset of tissue degradation and subsequent enhanced myogenesis, thereby accelerating muscle repair and functional recovery.

Receptor protein tyrosine phosphatase σ binds to neurons in the adult mouse brain

The role of type IIA receptor protein tyrosine phosphatases (RPTPs), which includes LAR, RPTPσ and RPTPδ, in the nervous system is becoming increasingly recognized. Evidence supports a significant role for these RPTPs during the development of the nervous system as well as after injury, and mutations in RPTPs are associated with human disease. However, a major open question is the nature of the ligands that interact with type IIA RPTPs in the adult brain. Candidates include several different proteins as well as the glycosaminoglycan chains of proteoglycans. In order to investigate this problem, we used a receptor affinity probe assay with RPTPσ-AP fusion proteins on sections of adult mouse brain and to cultured neurons. Our results demonstrate that the major binding sites for RPTPσ in adult mouse brain are on neurons and are not proteoglycan GAG chains, as RPTPσ binding overlaps with the neuronal marker NeuN and was not significantly altered by treatments which eliminate chondroitin sulfate, heparan sulfate, or both. We also demonstrate no overlap of binding of RPTPσ with perineuronal nets, and a unique modulation of RPTPσ binding to brain by divalent cations. Our data therefore point to neuronal proteins, rather than CSPGs, as being the ligands for RPTPσ in the adult, uninjured brain.

A Phase 2a, Open-Label, Dose-Finding Study To Determine The Safety and Tolerability Of Sotatercept (ACE-011) In Adults With Beta (β)-Thalassemia: Interim Results

BackgroundBeta (β)-thalassemia is characterized by ineffective erythropoiesis leading to anemia, bone marrow erythroid hyperplasia, iron overload, and organ failure. Sotatercept (ACE-011) is a novel and first-in-class activin type IIA receptor (ActRIIA) fusion protein that increases the release of mature erythrocytes into circulation by acting mainly on late-stage erythropoiesis (Carrancio S, et al. Blood. 2012;120:abstract 372). Clinical data in healthy volunteers have shown that treatment with sotatercept results in increased red blood cell (RBC) parameters, including hemoglobin level (Ruckle J, et al. J Bone Miner Res. 2009;24:744-52). RAP-011, a murine ortholog of sotatercept, was efficacious in a mouse model of β-thalassemia intermedia, reducing ineffective erythropoiesis as well as significantly improving anemia and decreasing bilirubin levels, supporting the clinical development of sotatercept (Dussiot M et al. Blood 2012;120:abstract 247). MethodsThis is an ongoing phase 2a, multicenter, open-label, dose-finding study to determine a safe and active dose level of sotatercept in adult patients with RBC-transfusion dependent (TD) β-thalassemia major or patients with β-thalassemia intermedia who are either TD or non-transfusion dependent (NTD). The dose levels of sotatercept studied to date are 0.1, 0.3, and 0.5 mg/kg, given subcutaneously once every 3 weeks. Safety is assessed according to NCI-CTC grading. Efficacy is assessed by hemoglobin increase from baseline and/or reduction in transfusion burden. Secondary endpoints include assessment of biomarkers for erythropoiesis, hemolysis, iron metabolism, and bone metabolism, as well as in vitro dyserythropoiesis. Dose escalation to higher dose levels is planned contingent on data review and favorable safety profile as determined by the Steering Committee. ResultsPatient demographics. A total of 25 patients have been enrolled as of July 26, 2013; 8 in the 0.1 mg/kg cohort, 9 in the 0.3 mg/kg cohort, and 8 in the 0.5 mg/kg cohort. Treatment and analysis for the 0.5 mg/kg cohort is underway and will be updated and presented. In the 0.1 and 0.3 mg/kg cohorts, 3 (18%) patients had β-thalassemia major and 14 (82%) had β-thalassemia intermedia (12 of whom were NTD and 2 of whom were TD). Of the 12 NTD β-thalassemia intermedia patients, 6 were treated at the 0.1 mg/kg dose level and 6 at the 0.3 mg/kg dose level. Median baseline hemoglobin for these NTD patients was 8.6 g/dL (range 5.8 to 10.7 g/dL). Median number of sotatercept doses administered was 4 (range 2 to 7) in the 0.1 mg/kg cohort and 8 (range 3 to 9) in the 0.3 mg/kg cohort; 13/17 (76%) patients remained on treatment.Safety.Sotatercept was generally well tolerated. There were no dose-limiting toxicities reported. Two serious adverse events were reported in the 0.1 mg/kg cohort: a grade 2 phlebitis in an NTD patient with a history of high D-dimer at baseline, and a worsening grade 3 bone pain in a TD β-thalassemia major patient with a history of osteoporosis; both were considered possibly study drug-related.Hemoglobin levels/transfusion requirements. Among NTD patients, preliminary data showed that 1 (17%) patient in the 0.1 mg/kg cohort and all 6 (100%) patients in the 0.3 mg/kg cohort had at least a 1 g/dL increase in hemoglobin level from baseline; among these, 1 patient treated with sotatercept 0.3 mg/kg showed a 2 g/dL hemoglobin level increase from baseline as well as a decrease in total bilirubin level from 2.7 × upper limit of normal (ULN) at baseline to 1.8 × ULN. No other relevant decrease in total bilirubin level was reported at the lower dose levels (0.1 mg/kg or 0.3 mg/kg).Three TD patients were still receiving treatment (2 β-thalassemia intermedia and 1 β-thalassemia major). There had been no appreciable reduction in transfusion burden in the 0.1 and 0.3 mg/kg cohorts to date; however further follow up is warranted and an update will be presented. ConclusionBased on these preliminary data, sotatercept administered subcutaneously every 3 weeks may improve anemia via a novel mechanism of action with a favorable safety profile, thereby addressing a significant unmet medical need for patients with NTD β-thalassemia intermedia. The current data suggest a dose-dependent response that supports further evaluation of the exposure–effect relationship of sotatercept in patients with NTD β-thalassemia intermedia.The first, second, and last authors contributed equally to this abstract. Disclosures:Cappellini:Genzyme: Consultancy, Speakers Bureau; Novartis: Consultancy, Speakers Bureau. Off Label Use: Sotatercept is an investigational agent that is being assessed for efficacy and safety in beta-thalassemia. Porter:Novartis: Consultancy, Research Funding; Shire: Consultancy; Celgene: Honoraria. Forni:Celgene: Research Funding; Shire: Research Funding; Novartis Pharma: Research Funding. Laadem:Celgene Corp.: Employment, Equity Ownership. Galacteros:Celgene: Consultancy. Miteva:Celgene Corp.: Employment. Sung:Celgene Corp.: Employment, Equity Ownership. Chopra:Celgene Corp.: Employment, Equity Ownership. Klesczewski:Celgene Corp.: Employment. Attie:Acceleron Pharma: Employment. Hermine:Celgene Corporation: Consultancy, Research Funding.

Endogenous Protection Derived from Activin A/Smads Transduction Loop Stimulated via Ischemic Injury in PC12 Cells

Activin A (ActA), a member of transforming growth factor-beta (TGF-b) super- family, affects many cellular processes, including ischemic stroke. Though the neuroprotective effects of exogenous ActA on oxygen-glucose deprivation (OGD) injury have already been reported by us, the endogenous role of ActA remains poorly understood. To further define the role and mechanism of endogenous ActA and its signaling in response to acute ischemic damage, we used an OGD model in PC12 cells to simulate ischemic injury on neurons in vitro. Cells were pre-treated by monoclonal antibody against activin receptor type IIA (ActRII-Ab). We found that ActRII-Ab augments ischemic injury in PC12 cells. Further, the extracellular secretion of ActA as well as phosphorylation of smad3 in PC12 cells was also up-regulated by OGD, but suppressed by ActRII-Ab. Taken together, our results show that ActRII-Ab may augment ischemic injury via blocking of transmembrane signal transduction of ActA, which confirmed the existence of endogenous neuroprotective effects derived from the ActA/Smads pathway. ActRIIA plays an important role in transferring neuronal protective signals inside. It is highly possible that ActA transmembrance signaling is a part of the positive feed-back loop for extracellular ActA secretion.