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 treated with nocodazole, a microtubule disruptor.

Microtubule stability is determined by the balance of stabilizing vs destabilizing microtubule-associated proteins (MAPs). Interrogation of human erythroid proteomic datasets highlighted selective deficiency in annotated stabilizing factors. Studies >25 yrs ago described isolation from chicken RBC of a microtubule-bundling factor syncolin, subsequently identified as ferritin heavy chain (FTH1). We thus assessed FTH1 as a potential iron-regulated stabilizing MAP. In our culture system, iron restriction rapidly suppressed, and isocitrate treatment fully restored, FTH1 protein levels. IF revealed co-localization of FTH1 with microtubules (Pearson R > 0.9). Lentiviral shRNA knockdowns of FTH1 in iron-replete progenitors recapitulated the phenotype of the erythroid iron restriction response eliciting impairments in growth, differentiation and microtubule integrity. FTH1 has numerous regulatory inputs, including multiple induction pathways for the metabolite fumarate. We examined the potential for synergy between isocitrate and fumarate in blocking the erythroid iron restriction response. In iron restricted progenitor cultures, weakly effective isocitrate doses clearly cooperated with ineffective fumarate doses to rescue differentiation (P < 0.05), FTH1 expression (P < 0.01), and microtubule integrity (P < 0.005). A prior roadblock with isocitrate treatment of anemia was poor oral efficacy. We thus tested oral efficacy of combined isocitrate and fumarate using a clinically feasible dosing regimen in murine ACDI associated with chronic injection of killed Brucella abortus. The metabolites in combination, but not separately, induced a complete and sustained correction in anemia (P < 0.005 in 2/2 trials). Thus, combined metabolite targeting of a novel FTH1-microtubule axis offers a new and effective therapy for iron restricted anemia.