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The hallmark of β-thalassemias is ineffective erythropoiesis leading to anemia and tissue hypoxia. Activin has been shown to affect the erythropoiesis in the late-stage of maturation. Sotatercept [ACE-011; Celgene Corporation (Summit, NJ, USA)], a recombinant activin receptor type IIA ligand trap, binds with high affinity activin A/B and other members of the transforming growth factor β-superfamily. In animal models, RAP-011, a murine ortholog of ACE-011, reverses bone loss and increases hemoglobin and hematocrit by acting indirectly on the late stages of erythropoiesis and in a mouse model of β-thalassemia, RAP-011 was shown to improve anemia and the quality of erythroid cells. 

The aim of our study is to further explore the molecular mechanisms underlying the effect of RAP-011 on erythropoiesis in  erythroid progenitor cell cultures from β-thalassemia patients at different stages of differentiation and maturation.

CD34⁺-enriched cells were isolated from peripheral blood of β-thalassemia patients and healthy donors by immunoselection. CD34⁺ cells were cultured in the presence or absence of RAP-011 (50 and 100 µg/mL) for 14 days in two conditions: liquid standard cultures and HS5 stromal cell line co-cultured with CD34⁺ cells. CD34⁺ liquid cultures in medium from HS5 cells conditioned with RAP-011 (CM) were also set up. Conditioned medium was assayed for apoptosis activity and cytokine content by  ELISA. In the co-cultures, the erythroid cells were rescued as non-adherent cells in supernatant (NAC), phase-bright cells adhering to the surface of HS5 cells (PBC) and phase-dim cells beneath the stromal cells (PDC). At day 14 erythroid cells were evaluated for cell number and viability, differentiation (Glycophorin/CD71/CD34) and gene expression profile.

At day 14, there were no significant differences in cell number, viability and immunophenotype between untreated liquid cultures and those treated with RAP-011 (either derived from β-thalassemia  and control subjects). In β-thalassemia co-cultures, no relevant differences in cell number and viability of the three cell fractions, in presence or absence of RAP-011 were observed; whereas the cell surface marker Glycophorin was more highly expressed in NAC (1.5-fold, p<0.05) and PDC (3.6-fold, p<0.001) treated with RAP-011 in comparison to untreated fractions. Similar results were observed in controls. In CM cultures, RAP-011-treated erythroid precursors from β-thalassemic patients expanded significantly compared to untreated cells (6.5-fold vs 3.1-fold). No significant differences were found in controls. High levels of anti-inflammatory, anti-apoptotic cytokines (SICAM-1, Bcl-2 and Bcl-xL) and factors that favour erythroid differentiation (MCP-1 and GRO-a) were detected in CM. At day 14 in presence of RAP-011, GATA1 expression increased (p<0.005) while GATA2 and α-globin expression decreased in erythroid thalassemic cells. In control subjects, no significant differences were observed. In β-thalassemic CM and co-cultures treated with RAP-011, GATA1 mRNA production was strongly induced (p<0.001), while the levels of GATA2 and α-globin mRNA were significantly lower (p<0.005). Similar results were observed in controls.

Our results suggest that RAP-011 didn’t directly affect erythroid maturation, but probably acted  through bone marrow-derived factors. Furthermore, RAP-011 seemed to recruit quiescent CD34⁺ cells with more primitive properties (NAC and PDC), stimulating them to differentiate with an especially marked effect on erythroid maturation.