Heme-regulated eIF2α (HRI) is required during iron restriction to alleviate ß-thalassemia via mTORC1 inhibition, autophagy activation, and suppression of protein synthesis, contributing to clearance of toxic free α-globin

Abstract In β-thalassemia, hepcidin deficiency and chronic red blood cell (RBC) transfusions cause iron overload leading to multi-organ damage. Iron overload also aggravates β-thalassemia directly via poorly understood mechanisms. In β-thalassemic mice, iron restriction with hepcidin mimetics reduces toxic free α-globin aggregates, ineffective erythropoiesis and hemolysis (PMID: 27154187). β-Thalassemic erythroblasts exhibit elevated mTORC1 activity, which inhibits ULK1-mediated autophagy of free α-globin (PMID: 37339583). Other studies have shown that iron activates mTORC1 via multiple mechanisms and that dietary iron deficiency inhibits erythroid mTORC1 via activation of the heme-regulated inhibitor of eIF2α kinase (HRI, encoded by Eif2ak1) (PMID: 29101239). Thus, we investigated whether the benefits of iron restriction in β-thalassemia are mediated by HRI and ULK1. Lethally irradiated wild-type C57BL/6 mice were transplanted with syngeneic fetal liver hematopoietic stem cells (HSCs) with genotypes Hbb+/+(WT), HbbTh3/+, HbbTh3/+ Eif2ak1-/-, HbbTh3/+ Ulk1-/-. After 60 days, reconstituted mice were treated with the hepcidin mimetic PR73 (35 nmoles SC 3x/week for 37 days). Iron restriction by PR73 alleviated β-thalassemia pathophysiology in mice reconstituted with HbbTh3/+ HSCs, with reductions in RBC α-globin precipitates (-87%, p≤0.001), reticulocyte count (-32%, p<0.05), RBC distribution width (RDW) (-26%, p≤0.001), reticulocyte reactive oxygen species (ROS) (-35%, p≤0.01), and spleen mass (-38%, p<0.05). The proportions of mature Ter119+ CD71high erythroblasts (FSChigh and FSClow) in bone marrow and spleen were increased by 62% (p<0.05) and 52% (p≤0.01), indicating reduced ineffective erythropoiesis. Across multiple stages of erythroblast maturation, iron restriction led to reductions in mTORC1 activity measured by phosphorylation of ribosomal protein S6 (-39 to -53%, p<0.05) and total protein synthesis (-27 to -49%, p=0.001-0.01) measured in vivo by puromycin incorporation. At baseline, Eif2ak1 loss worsened β-thalassemia, causing increased RBC α-globin precipitates (+127%, p<0.0001), reticulocytes (+141%, p≤0.0001), RDW (+36%, p≤0.001), reticulocyte ROS levels (+306%, p≤0.0001), spleen mass (+164%, p≤0.0001), and reduced mature erythroblasts in bone marrow and spleen (-50 to 69%, p≤0.01). Additionally, loss of Eif2ak1 inHbbTh3/+erythroblasts increased mTORC1 activity(+52 to +126%, p=0.01-0.05) and protein synthesis (+32 to +97%, p=0.0001-0.01), possibly due to impaired HRI-integrated response via pEIF2α–ATF4 signaling. In contrast to mice transplanted with HbbTh3/+ HSCs, β-thalassemic mice with disrupted Eif2ak1 genes exhibited worse erythroid pathologies after PR73 iron restriction, including increased RBC α-globin precipitates (+18%, p≤0.01), spleen mass (+24%, p=0.051) and a reduced proportion of mature splenic erythroblasts (-38%, p<0.05). Notably, Eif2ak1 loss attenuated PR73 suppression of mTORC1 activity and protein synthesis instead became more active (+34 to +54%, p=0.0001-0.01). We also compared PR73 effects in mice reconstituted with HbbTh3/+ or HbbTh3/+ Ulk1-/- HSCs. Multivariate analysis showed that loss of Ulk1 reduced the beneficial effects of PR73 on reticulocytes levels (p≤0.0001), RDW (p≤0.01), splenomegaly (p<0.05), RBC a-globin precipitates (p≤0.0001), and mature splenic erythroblasts (p≤0.0001). Notably, suppression of mTORC1 activity and erythroid protein synthesis by PR73 were not affected by loss of ULK1. Our findings delineate the regulatory hierarchy through which iron restriction can reduce the pathophysiology of β-thalassemia. At baseline, HRI protects against the toxic excess of free α-globin (PMID: 15931390). Upon iron restriction, HRI is further activated, inhibiting globin protein translation directly via eIF2α phosphorylation and suppressing mTORC1, thereby promoting ULK1-mediated autophagy and further inhibiting protein synthesis, both of which lead to reductions in free α-globin, hemolysis and ineffective erythropoiesis. Although treatment of β-thalassemia with hepcidin mimetics have not reached efficacy endpoints in clinical trials, our findings underscore the importance of minimizing iron overload in β-thalassemia. Moreover, further insights into regulation of the HRI–mTORC1 pathway in β-thalassemia may support the development of novel therapies.