Elevated p21 (CDKN1a) mediates β-thalassemia erythroid apoptosis but its loss does not improve β-thalassemic erythropoiesis

β-thalassemias are common hemoglobinopathies due to mutations in the β-globin gene that lead to hemolytic anemias. Premature death of β-thalassemic erythroid precursors results in ineffective erythroid maturation, increased production of erythropoietin (Epo), expansion of erythroid progenitor compartment, extramedullary erythropoiesis and splenomegaly. However, the molecular mechanism of erythroid apoptosis in β-thalassemia is not well understood. Using a mouse model of β-thalassemia ( Hbb th3/+ ), we show that dysregulated expression of Foxo3 transcription factor and its upstream pro-apoptotic regulator TP53 is implicated in β-thalassemia erythroid apoptosis. In Foxo3 -/- /Hbb th3/+ mice, erythroid apoptosis is significantly reduced while erythroid cell maturation, red blood cell and hemoglobin production are substantially improved. However, persistence of elevated reticulocytes and splenomegaly suggests that ineffective erythropoiesis is not resolved in Foxo3 -/- /Hbb th3/+ . We next focused on cell cycle inhibitor Cdkn1a (p21) and show that p21 that is a target of both Foxo3 and TP53 is markedly upregulated in both mouse and patients-derived β-thalassemic erythroid precursors. To address the contribution of p21 to β-thalassemia pathophysiology, we generated p21 -/- /Hbb th3/+ mice. Double mutant p21 /Hbb th3/+ mice exhibited embryonic lethality with only a fraction of mice surviving to weaning. Notably, studies in adult mice showed apoptosis and circulating Epo were greatly reduced in erythroid compartments of surviving p21 -/- /Hbb th3/+ relative to Hbb th3/+ mice, while ineffective erythroid cell maturation, extramedullary erythropoiesis and splenomegaly were not modified. These combined results indicate that while lack of Foxo3 reduces apoptosis and improves anemia, diminished p21-mediated apoptosis is insufficient to improve red blood cell production in Hbb th3/+ mice. They also suggest that a molecular network constituted by p21, FOXO3 and TP53, control erythroid cell survival and differentiation in β-thalassemia. Overall, these studies provide a new framework for investigating ineffective erythropoiesis in β-thalassemia. Key Points ### Competing Interest Statement The authors have declared no competing interest.