Rap-536, a Murine Luspatercept Analog Ameliorates Anemia and Vaso-Occlusion in Experimental Model of Sickle Cell Disease

Introduction

Sickle cell disease (SCD) is a hereditary hemoglobinopathy characterized by chronic hemolytic anemia and vaso-occlusive crises (VOC), leading to significant morbidity and mortality. Luspatercept is a ligand trap receptor indicated in thalassemia and myelodysplastic disease that restores effective erythropoiesis by reducing the inhibitory effects of transforming growth factor-beta (TGF-β) superfamily members, including GDF-11. Here we investigate the expression of GDF-11 in SCD patients and the role of RAP-536, a murine analog of luspatercept, in modulating anemia and VOC in SCD models, alone and/or in combination with hydroxyurea (HU) and epigenetic modulators Tazemetostat (TZM) and FTX6058, inducers of fetal hemoglobin (HbF).

Methods

We assessed GDF11 levels in plasma samples from SCD patients, healthy donors, and patients with other hemolytic anemias. Expression of GDF11 and pSMAD2 were evaluated in the spleen of Townes HbSS mice by immunohistochemistry. HbSS mice were treated with RAP-536 (1 or 10 mg/kg i.p. twice a week for 8 weeks), alone or in combination with hydroxyurea (100 mg/kg oral QD), TZM (250 mg/kg oral QD) and FTX6058 (20 mg/kg oral QD). Hematological, biochemical, and histological parameters were measured to assess treatment effects.

Results

Plasma GDF11 levels were significantly increased in SCD patients (27.7 pg/ml) when compared to healthy donors (5.0 pg/ml, p<0.01) and other hemolytic anemias (8.0 pg/ml). HbSS mice showed higher GDF11 expression in the spleen compared to HbAA mice, which correlated with SMAD2 phosphorylation. In HbSS mice, RAP-536 increased red blood cell (RBC) count and hemoglobin levels, decreased reticulocytes and ameliorated hemolytic markers, reducing lactate dehydrogenase (LDH), bilirubin as well as plasma free hemoglobin and plasma free heme. Likewise, tissue vascular congestion in hypoxia-induced VOC were reduced in the liver, lungs, and kidneys of HbSS mice upon RAP-536 treatment. Using ektacytometry, in vivo treatment of HSS mice by RAP-536 improved RBC deformability, as indicated by increased elongation index at 3Pa and 20Pa. This improvement correlated with enhanced oxygen carrying capacity in HbSS mice treated with RAP-536, with significant increases in total hemoglobin (+18.4%, p<0.05), oxygenated hemoglobin (+41.3%), oxygen saturation (+40.9%), oxygen content (+65.2%), and oxygen carrying capacity (+14.0%, p<0.05). All these effects were independent of HbF modulation. In contrast, RAP-536 significantly reduced blood reactive oxygen species (ROS) levels which may contribute to the reduction of VOC and improvement of erythropoiesis. RAP-536 reduced splenomegaly and improved erythropoietic maturation in spleen and bone marrow, indicated by a higher proportion of orthochromatic erythroblasts and reticulocytes (Ter-119⁺CD71⁻ Ery.C) compared to late basophilic and polychromatic erythroblasts (Ter-119⁺CD71⁺ Ery.B). ROS level reduction was associated to increase in Nrf2 levels (which regulates anti-oxidant stress) and GATA-1 expression in erythroid precursor populations.

In order to further improve the effects of RAP-536 (since it did not increase HbF), we evaluated the effects of RAP-536 combination with HU, TZM or FTX6058. First, we show that HU, TZM and FTX6058 induced HbF, with a synergistic effect observed only in RAP-536 and FTX6058 combination. Likewise, combination therapy with TZM or FTX6058 further enhanced RBC count, Ery.C cells and reduced reticulocyte counts and bilirubin levels. No additive or synergistic effect was observed to reduce ROS levels. In a hypoxia/re-oxygenation VOC model, the reduction of sickling observed with RAP-536 was additive with that observed with TZM or FTX6058.

Conclusion

These pre-clinical data highlight the potential of luspatercept (RAP-536) as a therapeutic agent in SCD, demonstrating significant improvements in ineffective erythropoiesis, reduction of hemolysis and ROS levels, and amelioration of VOC events independently of HbF induction. Effects on chronic complications are currently being investigated. The observed synergy with HbF inducers like HU, TZM and FTX6058 suggests potential combination strategies to enhance therapeutic outcomes. These findings warrant clinical evaluation of luspatercept in SCD patients, aiming to address both anemia and vaso-occlusive complications in this patient population with high unmet medical need.