S1628 TARGETING THE IMMUNOPHILIN FKBP12 BY DRUG REPURPOSING OR RNASE‐BASED APPROACHES FOR BMP‐SMAD PATHWAY AND HEPCIDIN UPREGULATION IN HEREDITARY HEMOCHROMATOSIS
Authors: M. Pettinato, M. Aghajan, A. Dulja, A. Nai, V. Olivari, J. Bordini, A. Campanella, A. Pagani, S. Guo, C. Camaschella, L. Silvestri
Journal: HemaSphere (2019)

Background: The main regulator of iron homeostasis, the liver hormone hepcidin, is inappropriately low in Hereditary Hemochromatosis (HH). HH is caused by mutations in HJV , TFR2 and HFE that negatively affect the liver BMP‐SMAD pathway, the main signaling pathway regulating hepcidin, thus causing iron overload. Current phlebotomy‐based therapy is symptomatic, does not correct hepcidin deficiency and is not well tolerated by all patients. We recently identified a new function for a cytosolic protein, the immunophilin FKBP12, and demonstrated that it negatively controls the BMP‐SMAD pathway by binding the type I receptor ALK2 (Colucci et al., 2017). Therefore, FKBP12 may be a novel therapeutic target for increasing hepcidin expression. Aims: Since sequestration of FKBP12 by Tacrolimus (TAC) upregulates hepcidin in wild type (WT) mice, the aim of this work is to investigate whether reducing FKBP12 may restore hepcidin expression in HH mouse models. Methods: Hepatocytes were isolated from WT, Tfr2 and Hjv KO mice and treated with TAC. Hjv KO mice were treated for 28 days with TAC delivered by subcutaneous miniosmotic pumps. In vivo RNAse‐based Fkbp12 downregulation was achieved using antisense oligonucleotides (ASOs). Hepcidin , Id1 , Smad7 , Erfe expression were evaluated by qRT‐PCR. Iron parameters, CBC and terminal erythroid differentiation were analyzed by standard methods. Results: FKBP12 sequestration by TAC upregulates hepcidin through BMP‐SMAD pathway activation in Hjv and Tfr2 KO primary hepatocytes, suggesting that FKBP12 targeting may be effective in HH. Due to the transient effect of TAC‐mediated hepcidin upregulation, we treated Hjv KO mice with chronic TAC administration by miniosmotic pumps. TAC upregulated hepcidin through BMP‐SMAD pathway activation and caused spleen iron retention, demonstrating that the FKBP12 inhibitory function on ALK2 is also preserved in an in vivo HH model. Since TAC is an immunosuppressive drug, we are exploring Fkbp12 downregulation by ASOs. ASO‐ Fkbp12 treatment of WT mice decreased Fkbp12 expression in liver (78.4%), spleen (66.3%) and kidney (56.4%) but not in the BM. Fkbp12 inactivation mildly upregulated Hamp , Id1 and Smad7 , consistent with BMP‐SMAD pathway activation. Although hepcidin expression was not significantly increased, we observed a reduction in transferrin saturation and serum iron and increased spleen iron retention. Hemoglobin and MCV were reduced in ASO‐Fkbp12 ‐treated mice, characterized also by a trend towards a reduction in RBC number and HCT. Spleen size and EPO levels remained unchanged. Analysis of terminal erythroid differentiation demonstrated an increased percentage of immature erythroid precursors with comparable Ter119+ cells in the spleen of ASO‐Fkbp12 ‐treated mice but not in the BM, and accordingly Erfe , the erythroid hepcidin inhibitor, is upregulated. Summary/Conclusion: Fkbp12 downregulation in the liver upregulates the BMP‐SMAD pathway, although hepcidin expression is minimally affected. Fkbp12 downregulation in the spleen impairs erythropoiesis increasing Erfe expression and decreasing serum iron concentration. Considering the presence of two inhibitory signals (high Erfe, low iron), hepcidin is inappropriately high in ASO‐Fkbp12 treated mice. We conclude that to increase hepcidin expression in HH, FKBP12 must be specifically downregulated in the liver. In addition, our results reveal a potential effect of FKBP12 on erythropoiesis, uncovering a novel role for the immunophilin in the regulation of erythropoiesis.