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Investigating the regulation of iron homeostasis by erythroferrone and therapeutic applications

Periodic Reporting for period 4 - INVESTIGERFE (Investigating the regulation of iron homeostasis by erythroferrone and therapeutic applications)

Reporting period: 2021-11-01 to 2023-02-28

Iron is essential for most living organisms. As a functional component of red blood cells, iron binds oxygen and ensures the proper transport and storage of oxygen throughout the body. An estimated 1/3 of the worldwide population suffers from anemia, a condition defined as an insufficient amount of circulating red blood cells. Iron deficiency or restricted iron availability for red cells synthesis is a major cause of anemia. On the contrary, excess iron is highly toxic and leads to severe clinical complications. Hepcidin is the central regulator of iron homeostasis. During the recovery from hemorrhage-induced anemia or the anemia that accompanies chronic inflammatory diseases, hepatic synthesis of hepcidin is repressed by the erythroid hormone erythroferrone (ERFE). ERFE regulates the adequate supply of iron supply for new red blood cells synthesis to promote the recovery. At the other end of the spectrum, increased production of ERFE causes iron overload in pathologies associated with ineffective red cell production such as β-thalassemia. Therefore, delineating the mechanism of hepcidin suppression by ERFE is of high biomedical importance as the pathway could be targeted to develop novel treatments for various forms of anemia. The design of new therapeutic approaches for the treatment of anemia directly answers an unmet need in public health. Indeed, Iron-restrictive anemias affect patients with chronic infections, inflammatory diseases, chronic kidney disease and hematologic and other malignancies and current treatments are largely inefficient. At the other end of the spectrum, increased ERFE concentrations cause iron overload in β-thalassemia intermedia, which is the major cause of morbidity and mortality and is controlled by parenteral or oral chelators but their limitations have highlighted the need for new strategies. Prevention of iron overload by the use of ERFE antagonists would improve the patients’ quality of life and survival. Identification of the receptor and signaling pathway are a prerequisite to envision this possibility. The objectives of this project was therefore to identify the receptor(s) for ERFE and the signal transduction pathways for hepcidin regulation, to develop a diagnostic tool to monitor the contrition of ERFE in human pathologies, to search for potential other regulators and to examine the potential of ERFE agonists or antagonists to treat iron-restrictive anemias and β-thalassemia in mice.
Most of the work performed during the project has not yet published but significant results have been obtained.

1) Develop an assay to measure ERFE levels in human pathologies and in mice. The development of an assay for human ERFE has been undertaken in collaboration with an academic laboratory in the US and published in Blood (Ganz, 2017). A second collaborative assay was developed in Europe (Diepveen, 2021). The production of antibodies to mouse ERFE has been subcontracted and we are using this assay in every aspect of the project. We identified a new variant that is specific for the clonal hematopoiesis observed in myelodysplastic syndromes (Bondu, 2019). This variant is a promising tool to diagnose and monitor the treatment efficacy of various forms of myelodysplastic syndromes. We therefore developed a 3-antibodies based assay that is still under validation.

2) Identify the receptor for ERFE, the signaling pathways triggered by ERFE, and molecules with agonist/antagonist effects. At the beginning of the project, a study suggested that ERFE acted as a ligand trap for BMP molecules. In contrast with this hypothesis, our work indicated that ERFE interacted with a receptor complex. We first tested the candidate receptors and demonstrated that ERFE does not bind any receptor of the TNFR or PAQR superfamilies. In parallel, we initiated an unbiased approach to isolate the ERFE interactors and we identified a candidate receptor complex and confirmed its interaction with ERFE. However, we generated murine models and cell lines invalidated for these candidate receptors to show that this receptor complex was not involved in hepcidin regulation. We performed a transcriptomic and a proteomic analysis to determine the signaling pathway triggered by ERFE in mouse liver and hepatoma cell line respectively. We tested a large number of signaling pathway induced by ERFE but none of those contributed to hepcidin regulation. Finally, we showed that, upon binding to its receptor, ERFE is internalized and translocated into the nucleus. We used proteomic approaches to identify the intracellular binding partners of ERFE in the cells. We are currently deciphering this mechanism, its contribution in liver metabolism and the function of the receptor complex. The manuscript describing this work is in preparation.

3) Search for potential other erythroid regulators. We first demonstrated that ERFE is not the sole regulator of hepcidin and iron metabolism during anemia. We searched for potential regulators of hepcidin during anemia by microarray. We identified the hepatokine FGL1 as a new regulator of iron homeostasis and characterized its mechanism of action. The manuscript has been deposited in BioRxiv and is now in revision in Blood journal (Sardo, 2023). Two patent applications have been deposited in February 2023. We developed a specific immunoassay to examine FGL1 levels in human diseases.

4) Study the potential of ERFE manipulation in therapy in the mouse. We first investigated the contribution of ERFE during inflammatory bowel disease. While ERFE is necessary for the resolution of anemia, we found that the absence of ERFE was accompanied by a much more severe inflammation. We further demonstrated that ERFE is a critical regulator of intestinal homeostasis. The manuscript is in preparation. We further extended this task to the potential of FGL1 for the treatment of anemia. We developed potent recombinant proteins (ERFE and FGL1) that are capable of repressing hepcidin expression in vitro and in vivo in mice. We validated antisense oligo-nucleotides (ASO) to target FGL1 in the liver. Targeting ERFE by this approach proved more difficult as it is expression preferentially in the bone marrow but alternative modifications of these ASOs to target the marrow are under consideration. A combinatory approach targeting ERFE and FGL1 might prove more effective than targeting a single molecule.
When we started this project, ERFE had emerged as the erythroid regulator of iron homeostasis and targeting its pathway was presumably the solution to treat various forms of anemia. However, we provided evidence that another mechanism was taking place in this regulation and we identified a new iron regulatory hepatokine: FGL1. Its identification was not unplanned as its potential existence was supported by previous observations. The identification of FGL1 as a critical regulator of iron homeostasis is certainly a major breakthrough for the iron and hematology community. A completely unexpected breakthrough is the identification of a function for ERFE in intestinal homeostasis. While investigating the mechanism involved in anemia associated with inflammatory bowel disease in mice, we observed that ERFE was not only important to resolve anemia but also to ensure a proper healing from colitis. We therefore investigated this axis further and ERFE might prove a new critical player in gut homeostasis and microbiota maintenance.
The erythroid regulator erythroferrone