Iron Restriction Alleviates β-Thalassemia By Stimulating ULK1-Mediated Autophagy of Free α-Globin
Authors: Fernandez Alfonso G. , Christakopoulos Georgios E. , Michael Dudley E. , Sweileh Razan B. A. , Telange Rahul , Sheppard Heather , Yao Yu , Zhang Jingjing , Manolova Vania , Nemeth Elizabeta , Ganz Tomas , Weiss Mitchell J. 
Published: December 2023

In β-thalassemia, mutations in the HBB gene cause reduced β-globin synthesis with accumulation of toxic free α-globin, leading to ineffective erythropoiesis and hemolysis. We showed previously that β-thalassemic erythroblasts can eliminate free α-globin by ULK1-mediated autophagy and that this process is stimulated by rapamycin inhibition of mTORC1, which phosphorylates ULK1 to inhibit its activity. Several studies have shown that iron restriction can reduce the accumulation of free α-globin precipitates to alleviate the pathophysiology of β-thalassemia. Considering that iron stimulates mTORC1 activity in numerous cell types including erythroblasts, we tested the hypothesis that iron restriction alleviates β-thalassemia by inhibiting mTORC1, thereby stimulating ULK1-mediated autophagy of free α-globin.

Hbb Th3/+ β-thalassemic mice or HbbTh3/+Ulk1-/- mice were treated with the clinical stage oral ferroportin inhibitor vamifeport (known also as VIT-2763) for 45 days to cause systemic iron restriction. In HbbTh3/+ mice, vamifeport administration led to 66% and 40% reductions in the levels of serum iron (p&lt;0.001) and ferritin (p=0.001), respectively, as expected. Iron restriction by vamifeport alleviated the pathophysiology of β-thalassemia compared to vehicle-treatment in HbbTh3/+, as evidenced by a 21% increase in RBC count (p&lt;0.001), 32% reduction in insoluble α-globin in red blood cells (RBC) (p=0.002), 41% reduction in reticulocyte count (p&lt;0.001), 27% reduction in spleen mass (p=0.034) and enhanced maturation of spleen and bone marrow erythroid precursors, indicating reduced ineffective erythropoiesis. Multivariate analysis showed that beneficial effects of iron restriction in β-thalassemic mice were attenuated by concomitant disruption of both Ulk1 alleles. For example, vamifeport treatment of HbbTh3/+Ulk1-/- mice caused only a 12% increase in RBC counts (p=0.015 compared to HbbTh3/+). Additionally, iron restriction improved erythroid maturation in HbbTh3/+Ulk1-/- mice, but to a significantly lesser extent than in thalassemic mice with intact Ulk1alleles. We obtained similar results by inducing iron deprivation with PR73, a peptide hepcidin mimetic.

Mechanistically, long-term administration of vamifeport was associated with inhibition of mTORC1, as evidenced by 32% reductions in the phosphorylation of its substrate, ribosomal protein S6 kinase (p=0.004), and activation of the heme-regulated eIF2α kinase (HRI), indicated by increased phosphorylation of eIF2α and/or accumulation of the HRI effector transcription factor ATF4. Similar results were obtained with PR73. Administration of a single dose of PR73 to HbbTh3/+ mice led to activation of HRI in Ter119 + erythroblasts after 8 hours, which preceded the downregulation of mTORC1 activity, detected at 24 hours. This is consistent with prior studies showing that HRI inhibits mTORC1. Loss of ULK1 had no effect on suppression of mTORC1 with either drug, consistent with ULK1 being the substrate of mTORC1; we are currently investigating whether loss of ULK1 alters the HRI pathway. Together, our data support a mechanism through which iron restriction alleviates the pathophysiology of β-thalassemia in part by stimulating ULK1-mediated autophagy of free α-globin. This process is likely mediated by iron restriction that activates HRI which in turn inhibits mTORC1 to block its negative effects on ULK1. Additional mechanisms for iron regulation of mTORC1 activity are also possible. Our findings provide new insight into the mechanisms by which iron restriction alleviates β-thalassemia and illustrate how metabolic regulators of erythropoiesis impact the severity of β-thalassemia by eliminating free α-globin through a key protein quality control pathway.