Smad2/3‐pathway ligand trap luspatercept enhances erythroid differentiation in murine β‐thalassaemia by increasing GATA‐1 availability

In β‐thalassaemia, anaemia results from ineffective erythropoiesis characterized by inhibition of late‐stage erythroid differentiation. We earlier used luspatercept and RAP‐536 protein traps for certain Smad2/3‐pathway ligands to implicate Smad2/3‐pathway overactivation in dysregulated erythroid differentiation associated with murine β‐thalassaemia and myelodysplasia. Importantly, luspatercept alleviates anaemia and has been shown to reduce transfusion burden in patients with β‐thalassaemia or myelodysplasia. Here, we investigated the molecular mechanisms underlying luspatercept action and pSmad2/3‐mediated inhibition of erythroid differentiation. In murine erythroleukemic (MEL) cells in vitro, ligand‐mediated overactivation of the Smad2/3 pathway reduced nuclear levels of GATA‐1 (GATA‐binding factor‐1) and its transcriptional activator TIF1γ (transcription intermediary factor 1γ), increased levels of reactive oxygen species, reduced cell viability and haemoglobin levels, and inhibited erythroid differentiation. Co‐treatment with luspatercept in MEL cells partially or completely restored each of these. In β‐thalassaemic mice, RAP‐536 up‐regulated Gata1 and its target gene signature in erythroid precursors determined by transcriptional profiling and gene set enrichment analysis, restored nuclear levels of GATA‐1 in erythroid precursors, and nuclear distribution of TIF1γ in erythroblasts. Bone marrow cells from β‐thalassaemic mice treated with luspatercept also exhibited restored nuclear availability of GATA‐1 ex vivo. Our results implicate GATA‐1, and likely TIF1γ, as key mediators of luspatercept/RAP‐536 action in alleviating ineffective erythropoiesis.