β-thalassemia, the most common congenital anemia, is caused by mutations in β-globin gene resulting in partial or complete absence of β-globin protein chains. In the absence of properly paired α- and β-globin chains, the α-globin protein accumulates causing proteotoxicity and apoptosis of erythroid cells. Hemolysis and ineffective erythropoiesis together cause severe anemia in thalassemia syndromes. Increased proliferation with arrest of terminal erythroid differentiation and accelerated apoptosis is the hallmark of ineffective erythropoiesis in β-thalassemia. In chronic patients, blood transfusions are required for survival, but result in severe iron overloading. Non-transfusion dependent thalassemia (NTDT) patients however, are also affected by ineffective erythropoiesis, anemia and iron overload. Recombinant EPO therapy is ineffective and rarely used for β-thalassemia patients, as it does not affect the later stages of erythroid differentiation. Therefore, a pharmacological approach is necessary that can increase hemoglobin levels, prevent splenomegaly, bone abnormalities and iron overloading in β-thalassemia patients.
Several members of the TGFβ-superfamily are involved in erythropoiesis. ACE-536 is a modified activin type IIb (ActRIIb) receptor fusion protein that acts as a ligand trap. Unlike wild type ActRIIb, ACE-536 does not inhibit activin A induced signaling but inhibits signaling induced by other members of the TGF-β superfamily such as GDF11. While EPO increases proliferation of erythroid progenitors, ACE-536 promotes maturation of terminally differentiating erythroblasts. We hypothesized that ACE-536 treatment will promote terminal erythroid differentiation, as well as reduce anemia, ineffective erythropoiesis and associated co-morbidities in β-thalassemia.
We investigated the efficacy of RAP-536 (murine ortholog of ACE-536) in a mouse model of β-thalassemia intermedia (Hbbth1/th1). β-thalassemic mice were severely anemic and had significantly decreased RBC (−31.6% p<0.001), hemoglobin (−35.0% p<0.001) and hematocrit (−34.8% p<0.001) compared to wild type littermates. β-thalassemic mice were treated subcutaneously twice a week with RAP-536 (1 mg/kg) or TBS vehicle (VEH) control for two months (N=7 per treatment group). Wild-type littermates were dosed with VEH or RAP-536 (1 mg/kg) and used as controls (N=13 per treatment group).
Following two months of treatment, RAP-536 treated β-thalassemic mice had significantly increased RBC (+32.9%, p<0.01), hemoglobin (+17.4%, p<0.01) hematocrit (+11.0%, p<0.01) and displayed reduced reticulocytosis (−30.07%, p<0.05) compared to VEH treated β-thalassemic mice. Terminal erythroid differentiation analyses of bone marrow and spleen from β-thalassemic mice treated with RAP-536 revealed significant decreases in basophilic erythroblasts while increasing late stage orthochromatic erythroblasts. RAP-536 treated β-thalassemic mice had significantly decreased serum EPO levels (639.7±111 vs. 1769.7± 517 pg/mL, p<0.05), bone marrow erythroid precursors and spleen weights (418.3± 28 vs. 677.1± 65 mgs, p<0.01) compared to VEH treatment indicating decreased erythroid hyperplasia and extramedullary erythropoiesis. RAP-536 treatment also restored bone mineral density in β-thalassemic mice to levels observed in wild type mice. Furthermore, RAP-536 treatment resulted in decreased splenic, liver and kidney iron levels by Perl's Prussian blue staining indicating decreased iron overloading. Interestingly, serum bilirubin (0.41± 0.01 vs. 0.72± 0.09 mg/dL, p<0.05) and lactate dehydrogenase levels (334.6± 33 vs. 424.6± 76 IU/mL) were lower in β-thalassemic mice treated with RAP-536 compared to VEH treated mice demonstrating decreased hemolysis. Morphological assessment of blood smears also displayed decreased hemolysis, reduced α-globin inclusions and poikilocytosis compared to VEH treatment. RAP-536 treatment also extended RBC life span in β-thalassemic mice compared to VEH treated mice.
In summary, these data demonstrate that RAP-536 attenuates ineffective erythropoiesis, ameliorates anemia and improved associated co-morbidities in a murine model of β-thalassemia. ACE-536 represents a novel potential therapy for patients with β-thalassemia and these preclinical data provide a rationale for clinical studies of ACE-536 in β-thalassemia patients.
Suragani:Acceleron Pharma Inc: Employment, Equity Ownership. Li:Acceleron Pharma Inc: Employment, Equity Ownership. Cawley:Acceleron Pharma Inc: Employment. Rivella:Novartis Pharmaceuticals: Consultancy; Biomarin: Consultancy; Merganser Biotech: Consultancy, Equity Ownership, Research Funding; Isis Pharma: Consultancy, Research Funding. Pearsall:Acceleron Pharma Inc: Employment, Equity Ownership. Kumar:Acceleron Pharma Inc: Employment, Equity Ownership.
Asterisk with author names denotes non-ASH members.