Exploitation of a Newly Characterized Metabolic-Cytoskeletal Axis for Effective Combination Therapy of Iron Restricted Anemia

The erythroid iron restriction response suppresses erythropoiesis in a lineage-selective manner. This response underlies two common types of anemia: anemia of chronic disease and inflammation (ACDI) and iron deficiency anemia (IDA). These anemias confer a major global disease burden, with no optimal therapies available for ACDI. An early step in this cellular response consists of inactivation of the iron-dependent aconitase enzymes. Supplying cells with the downstream product isocitrate abrogates the erythropoietic blockade caused by iron restriction. In several rodent anemia models isocitrate treatment has shown promise, but high intraperitoneal doses were needed, and benefits were transient. A critical aspect of the erythroid iron restriction response comprises progenitor resistance to erythropoietin (Epo), clinically reflected in poor patient responses to Epo treatment. Mechanistically, we have shown that iron deprivation decreases cell surface delivery of the Epo receptor (EpoR) and associated chaperones (Scribble and TfR2), in a manner reversible by isocitrate. To assess vesicular trafficking defects in erythroid iron restriction, primary human progenitors cultured in iron replete or restricted erythroid media underwent analysis of Golgi structure. Immunofluorescence microscopy (IF) for the markers Golgin97 and Giantin revealed Golgi dispersion in iron-restricted erythroid but not granulocytic cells, a defect reversed by isocitrate treatment. Supporting clinical relevance, Giantin immunostaining of human marrow samples identified erythroid Golgi disruption (>50% of cells) in 9/10 cases of ACDI versus 1/5 of non-anemic controls (P < 0.05 by Fisher's exact two-tailed test). The microtubule cytoskeleton, a key determinant of Golgi integrity, next was characterized in our culture system. IF for beta-tubulin showed extensive microtubule disassembly occurring within the first 16 hours of iron restriction, prior to changes in proliferation, differentiation and viability. Isocitrate treatment did not prevent the initial collapse at 16 hours but induced microtubule recovery between 24 and 48 hours of iron restriction. For clinical corroboration, red cells (RBC) from patients with IDA and non-anemic controls underwent IF for remnant microtubule fragments that have been identified in human erythrocytes. IDA RBC displayed loss of microtubule remnants in most cells (81% vs 19% of controls, P < 0.005, Student t), recapitulating changes described in human RBC treate