Iron-loading Anemias Research Articles

Differentiating iron-loading anemias using a newly developed and analytically validated ELISA for human serum erythroferrone.

Erythroferrone (ERFE), the erythroid regulator of iron metabolism, inhibits hepcidin to increase iron availability for erythropoiesis. ERFE plays a pathological role during ineffective erythropoiesis as occurs in X-linked sideroblastic anemia (XLSA) and β-thalassemia. Its measurement might serve as an indicator of severity for these diseases. However, for reliable quantification of ERFE analytical characterization is indispensable to determine the assay's limitations and define proper methodology. We developed a sandwich ELISA for human serum ERFE using polyclonal antibodies and report its extensive analytical validation. This new assay showed, for the first time, the differentiation of XLSA and β-thalassemia major patients from healthy controls (p = 0.03) and from each other (p<0.01), showing the assay provides biological plausible results. Despite poor dilution linearity, parallelism and recovery in patient serum matrix, which indicated presence of a matrix effect and/or different immunoreactivity of the antibodies to the recombinant standard and the endogenous analyte, our assay correlated well with two other existing ERFE ELISAs (both R2 = 0.83). Nevertheless, employment of one optimal dilution of all serum samples is warranted to obtain reliable results. When adequately performed, the assay can be used to further unravel the human erythropoiesis-hepcidin-iron axis in various disorders and assess the added diagnostic value of ERFE.

Ineffective Erythropoiesis in β-Thalassaemia: Key Steps and Therapeutic Options by Drugs.

β-thalassaemia is a rare genetic condition caused by mutations in the β-globin gene that result in severe iron-loading anaemia, maintained by a detrimental state of ineffective erythropoiesis (IE). The role of multiple mechanisms involved in the pathophysiology of the disease has been recently unravelled. The unbalanced production of α-globin is a major source of oxidative stress and membrane damage in red blood cells (RBC). In addition, IE is tightly linked to iron metabolism dysregulation, and the relevance of new players of this pathway, i.e., hepcidin, erythroferrone, matriptase-2, among others, has emerged. Advances have been made in understanding the balance between proliferation and maturation of erythroid precursors and the role of specific factors in this process, such as members of the TGF-β superfamily, and their downstream effectors, or the transcription factor GATA1. The increasing understanding of IE allowed for the development of a broad set of potential therapeutic options beyond the current standard of care. Many candidates of disease-modifying drugs are currently under clinical investigation, targeting the regulation of iron metabolism, the production of foetal haemoglobin, the maturation process, or the energetic balance and membrane stability of RBC. Overall, they provide tools and evidence for multiple and synergistic approaches that are effectively moving clinical research in β-thalassaemia from bench to bedside.

Pharmacological and clinical evaluation of deferasirox formulations for treatment tailoring

Deferasirox (DFX) is the newest among three different chelators available to treat iron overload in iron-loading anaemias, firstly released as Dispersible Tablets (DT) and more recently replaced by Film-Coated Tablets (FCT). In this retrospective observational study, pharmacokinetics, pharmacodynamics, and safety features of DFX treatment were analyzed in 74 patients that took both formulations subsequently under clinical practice conditions. Bioavailability of DFX FCT compared to DT resulted higher than expected [Cmax: 99.5 (FCT) and 69.7 (DT) μMol/L; AUC: 1278 (FCT) and 846 (DT), P < 0.0001]. DFX FCT was also superior in scalability among doses. After one year of treatment for each formulation, no differences were observed between the treatments in the overall iron overload levels; however, DFX FCT but not DT showed a significant dose–response correlation [Spearman r (dose-serum ferritin variation): − 0.54, P < 0.0001]. Despite being administered at different dosages, the long-term safety profile was not different between formulations: a significant increase in renal impairment risk was observed for both treatments and it was reversible under strict monitoring (P < 0.002). Altogether, these data constitute a comprehensive comparison of DFX formulations in thalassaemia and other iron-loading anaemias, confirming the effectiveness and safety characteristics of DFX and its applicability for treatment tailoring.

Serum Hepcidin and Growth Differentiation Factor 15 In Patients with β-thalassemia and Its Relation to Blood Transfusion

Background: Serum Hepcidin level drop is a main feature of chronic hemolytic anemia like β-thalassemia as an example. It is assumed that Hepcidin is influenced by anemia, iron overload due to repeated blood infusions and inflammation due to iron disposition in tissues. Main purpose of our research: To measure plasma levels of the iron regulatory hormones; hepcidin and GDF-15 in β-thalassemic patients during intravenous blood infusion in order to assist clinical monitoring. Methodology: 30 cases previously diagnosed as a β-thalassemia major (group I) and 20 normal individuals of the same age and gender group (group II). The samples were collected immediately before and 8 days after transfusion. Hepcidin and GDF-15 levels were estimated using ELISA kit that is commercially available. Results: As regard to serum Hepcidin level in both control and patient group was similar before the transfusion. The hepcidin level increased significantly after transfusion in both groups but in was significantly higher in patient group. Hb and hepcidin increased significantly post-transfusion. The pre- and post-transfusion hepcidin showed significant correlation with Hb, Iron, and Ferritin in thalassemia patients. The pre-transfusion Growth Differentiation Factor-15 was shooting in cases with β-thalassemia than presumably healthy control. The GDF-15 level decreased significantly after transfusion and it was significantly correlated with Hb and hepcidin levels. In conclusion: Hepcidin serum level assessment can be used to monitor patients with iron-loading anemia and identify the patients prone to iron overload complications and iron toxicity.

Increased zinc and albumin but lowered copper in children with transfusion-dependent thalassemia

BackgroundMeasurements of copper and zinc in transfusion-dependent thalassemia (TDT) show contradictory results. Aim of the studyTo examine serum levels of these minerals in TDT in relation to iron overload indices and erythron variables. MethodsThis study recruited 60 children with TDT and 30 healthy controls aged 3–12 years old. ResultsZinc was significantly higher in TDT children than in controls, while copper and the copper to zinc ratio were significantly lowered in TDT. Serum zinc was significantly associated with the number of blood transfusions and iron overload variables (including serum iron and TS%) and negatively with erythron variables (including hemoglobin, mean corpuscular volume, mean corpuscular hemoglobin). Serum copper was significantly and negatively associated with the same iron overload and erythron variables. The copper to zinc ratio was significantly correlated with iron, TS%, ferritin, hemoglobin, mean corpuscular volume, and mean corpuscular hemoglobin. Albumin levels were significantly higher in TDT children than in control children. ConclusionOur results suggest that the increase in zinc in children with TDT may be explained by iron loading anemia and hemolysis and the consequent shedding of high amounts of intracellular zinc into the plasma. Increased albumin levels and treatment with Desferral may further contribute towards higher zinc levels in TDT. We suggest that the elevations in zinc in TDT are a compensatory mechanism protecting against infection, inflammation, and oxidative stress. Previous proposals for prophylactic use of zinc supplements in TDT may not be warranted.

Physiological and pathophysiological mechanisms of hepcidin regulation: clinical implications for iron disorders.

The discovery of hepcidin has provided a solid foundation for understanding the mechanisms of systemic iron homeostasis and the aetiologies of iron disorders. Hepcidin assures the balance of circulating and stored iron levels for multiple physiological processes including oxygen transport and erythropoiesis, while limiting the toxicity of excess iron. The liver is the major site where regulatory signals from iron, erythropoietic drive and inflammation are integrated to control hepcidin production. Pathologically, hepcidin dysregulation by genetic inactivation, ineffective erythropoiesis, or inflammation leads to diseases of iron deficiency or overload such as iron-refractory iron-deficiency anaemia, anaemia of inflammation, iron-loading anaemias and hereditary haemochromatosis. In the present review, we discuss recent insights into the molecular mechanisms governing hepcidin regulation, how these pathways are disrupted in iron disorders, and how this knowledge is being used to develop novel diagnostic and therapeutic strategies.

A Novel ALAS2 Missense Mutation in Two Brothers With Iron Overload and Associated Alterations in Serum Hepcidin/Erythroferrone Levels.

Iron loading anemias are characterized by ineffective erythropoiesis and iron overload. The prototype is non-transfusion dependent ß-thalassemia (NTDT), with other entities including congenital sideroblastic anemias, congenital dyserythropoietic anemias, some hemolytic anemias, and myelodysplastic syndromes. Differential diagnosis of iron loading anemias may be challenging due to heterogeneous genotype and phenotype. Notwithstanding the recent advances in linking ineffective erythropoiesis to iron overload, many pathophysiologic aspects are still unclear. Moreover, measurement of hepcidin and erythroferrone (ERFE), two key molecules in iron homeostasis and erythropoiesis, is scarcely used in clinical practice and of uncertain utility. Here, we describe a comprehensive diagnostic approach, including next-generation sequencing (NGS), in silico modeling, and measurement of hepcidin and erythroferrone (ERFE), in two brothers eventually diagnosed as X-linked sideroblastic anemia (XLSA). A novel pathogenic ALAS2 missense mutation (c.1382T>A, p.Leu461His) is described. Hyperferritinemia with high hepcidin-25 levels (but decreased hepcidin:ferritin ratio) and mild-to-moderate iron overload were detected in both patients. ERFE levels were markedly elevated in both patients, especially in the proband, who had a more expressed phenotype. Our study illustrates how new technologies, such as NGS, in silico modeling, and measurement of serum hepcidin-25 and ERFE, may help in diagnosing and studying iron loading anemias. Further studies on the hepcidin-25/ERFE axis in additional patients with XLSA and other iron loading anemias may help in establishing its usefulness in differential diagnosis, and it may also aid our understanding of the pathophysiology of these genetically and phenotypically heterogeneous entities.

Comparison of Compliance of Different Iron Chelators Including Original and Bioequivalents of Deferasirox

Objective: The current iron chelation therapy regimens include deferoxamine (DFO), deferiprone (DFP), and deferasirox (DFX) in transfusion-dependent patients. Compliance with iron-chelating therapy is one of the significant determinants of mortality and morbidities related to iron loading in chronically transfused patients. This survey aims to compare the compliance and the satisfaction of DFO, DFP, and DFX formulations in patients who have iron-overloading in three hematology centers from Turkey. Moreover, bioequivalent (generic) formulations of dispersible DFX tablet compared with the original formulation in terms of taste and treatment compliance.&#x0D; Patients and Methods: A written questionnaire with a list of pre-set questions was applied to measure patient-reported outcomes to a total of 85 patients, where 77 had beta-thalassemia major, 7 had beta-thalassemia intermedia, and 1 had sickle cell anemia diagnoses.&#x0D; Results: The patients’ median age at enrollment was 15 years (range 7 – 42). The compliance was below 50% in 8 (18.6%), 4 (16%), and 5 (6.7%) in patients receiving DFO, DFP, and DFX, respectively. Additionally the compliance was below 80% in 16 (37.2%), 9 (36%), and 17 (22.6%) in patients receiving DFO, DFP, and DFX, respectively. It was found that 39 (47%) patients had compliance problems due to the dispersible DFX tablet formulations’ taste, except combination therapies. There was no difference between the currently used oral chelators in terms of taste and treatment compliance.&#x0D; Conclusion: This study draws attention to compliance problems in patients with iron-loading anemias, partly due to the unpleasant taste of DFX. Improving patient satisfaction and compliance with iron-chelator therapy may reduce complications of iron overload.

Auranofin mitigates systemic iron overload and induces ferroptosis via distinct mechanisms

Iron homeostasis is essential for health; moreover, hepcidin-deficiency results in iron overload in both hereditary hemochromatosis and iron-loading anemia. Here, we identified iron modulators by functionally screening hepcidin agonists using a library of 640 FDA-approved drugs in human hepatic Huh7 cells. We validated the results in C57BL/6J mice and a mouse model of hemochromatosis (Hfe−/− mice). Our screen revealed that the anti-rheumatoid arthritis drug auranofin (AUR) potently upregulates hepcidin expression. Interestingly, we found that canonical signaling pathways that regulate iron, including the Bmp/Smad and IL-6/Jak2/Stat3 pathways, play indispensable roles in mediating AUR’s effects. In addition, AUR induces IL-6 via the NF-κB pathway. In C57BL/6J mice, acute treatment with 5 mg/kg AUR activated hepatic IL-6/hepcidin signaling and decreased serum iron and transferrin saturation. Whereas chronically treating male Hfe−/− mice with 5 mg/kg AUR activated hepatic IL-6/hepcidin signaling, decreasing systemic iron overload, but less effective in females. Further analyses revealed that estrogen reduced the ability of AUR to induce IL-6/hepcidin signaling in Huh7 cells, providing a mechanistic explanation for ineffectiveness of AUR in female Hfe−/− mice. Notably, high-dose AUR (25 mg/kg) induces ferroptosis and causes lipid peroxidation through inhibition of thioredoxin reductase (TXNRD) activity. We demonstrate the ferroptosis inhibitor ferrostatin significantly protects liver toxicity induced by high-dose AUR without comprising its beneficial effect on iron metabolism. In conclusion, our findings provide compelling evidence that TXNRD is a key regulator of ferroptosis, and AUR is a novel activator of hepcidin and ferroptosis via distinct mechanisms, suggesting a promising approach for treating hemochromatosis and hepcidin-deficiency related disorders.

Antibodies against the erythroferrone N-terminal domain prevent hepcidin suppression and ameliorate murine thalassemia

AbstractErythroferrone (ERFE) is produced by erythroblasts in response to erythropoietin (EPO) and acts in the liver to prevent hepcidin stimulation by BMP6. Hepcidin suppression allows for the mobilization of iron to the bone marrow for the production of red blood cells. Aberrantly high circulating ERFE in conditions of stress erythropoiesis, such as in patients with β-thalassemia, promotes the tissue iron accumulation that substantially contributes to morbidity in these patients. Here we developed antibodies against ERFE to prevent hepcidin suppression and to correct the iron loading phenotype in a mouse model of β-thalassemia [Hbb(th3/+) mice] and used these antibodies as tools to further characterize ERFE's mechanism of action. We show that ERFE binds to BMP6 with nanomolar affinity and binds BMP2 and BMP4 with somewhat weaker affinities. We found that BMP6 binds the N-terminal domain of ERFE, and a polypeptide derived from the N terminus of ERFE was sufficient to cause hepcidin suppression in Huh7 hepatoma cells and in wild-type mice. Anti-ERFE antibodies targeting the N-terminal domain prevented hepcidin suppression in ERFE-treated Huh7 cells and in EPO-treated mice. Finally, we observed a decrease in splenomegaly and serum and liver iron in anti–ERFE-treated Hbb(th3/+) mice, accompanied by an increase in red blood cells and hemoglobin and a decrease in reticulocyte counts. In summary, we show that ERFE binds BMP6 directly and with high affinity, and that antibodies targeting the N-terminal domain of ERFE that prevent ERFE–BMP6 interactions constitute a potential therapeutic tool for iron loading anemias.

Iron metabolism and iron disorders revisited in the hepcidin era.

Iron is biologically essential, but also potentially toxic; as such it is tightly controlled at cell and systemic levels to prevent both deficiency and overload. Iron regulatory proteins post-transcriptionally control genes encoding proteins that modulate iron uptake, recycling and storage and are themselves regulated by iron. The master regulator of systemic iron homeostasis is the liver peptide hepcidin, which controls serum iron through degradation of ferroportin in iron-absorptive enterocytes and iron-recycling macrophages. This review emphasizes the most recent findings in iron biology, deregulation of the hepcidin-ferroportin axis in iron disorders and how research results have an impact on clinical disorders. Insufficient hepcidin production is central to iron overload while hepcidin excess leads to iron restriction. Mutations of hemochro-matosis genes result in iron excess by downregulating the liver BMP-SMAD signaling pathway or by causing hepcidin-resistance. In iron-loading anemias, such as β-thalassemia, enhanced albeit ineffective ery-thropoiesis releases erythroferrone, which sequesters BMP receptor ligands, thereby inhibiting hepcidin. In iron-refractory, iron-deficiency ane-mia mutations of the hepcidin inhibitor TMPRSS6 upregulate the BMP-SMAD pathway. Interleukin-6 in acute and chronic inflammation increases hepcidin levels, causing iron-restricted erythropoiesis and ane-mia of inflammation in the presence of iron-replete macrophages. Our improved understanding of iron homeostasis and its regulation is having an impact on the established schedules of oral iron treatment and the choice of oral versus intravenous iron in the management of iron deficiency. Moreover it is leading to the development of targeted therapies for iron overload and inflammation, mainly centered on the manipulation of the hepcidin-ferroportin axis.

Gain-of-function mutations in PIEZO1 directly impair hepatic iron metabolism via the inhibition of the BMP/SMADs pathway.

Dehydrated hereditary stomatocytosis (DHS), or xerocytosis, is an autosomal dominant hemolytic anemia. Most patients with DHS carry mutations in the PIEZO1 gene encoding a mechanosensitive cation channel. We here demonstrate that patients with DHS have low levels of hepcidin and only a slight increase of ERFE, the erythroid negative regulator of hepcidin. We demonstrated that at the physiological level, PIEZO1 activation induced Ca2+ influx and suppression of HAMP expression in primary hepatocytes. In two hepatic cellular models expressing PIEZO1 WT and two PIEZO1 gain-of-function mutants (R2456H and R2488Q), we highlight altered expression of a few genes/proteins involved in iron metabolism. Mutant cells showed increased intracellular Ca2+ compared to WT, which was correlated to increased phosphorylation of ERK1/2, inhibition of the BMP-SMADs pathway, and suppression of HAMP transcription. Moreover, the HuH7 cells, treated with PD0325901, a potent inhibitor of ERK1/2 phosphorylation, reduced the phosphorylation of ERK1/2 with the consequent increased phosphorylation of SMAD1/5/8, confirming the link between the two pathways. Another "proof of concept" for the mechanism that links PIEZO1 to HAMP regulation was obtained by mimicking PIEZO1 activation by cell Ca2+ overload, by the Ca2+ ionophore A23187. There was strong down-regulation of HAMP gene expression after this Ca2+ overload. Finally, the inhibition of PIEZO1 by GsMTx4 leads to phenotype rescue. This is the first demonstration of a direct link between PIEZO1 and iron metabolism, which defines the channel as a new hepatic iron metabolism regulator and as a possible therapeutic target of iron overload in DHS and other iron-loading anemias.

Antibodies Against the Erythroferrone N-Terminal Domain Prevent Hepcidin Suppression and Ameliorate Murine Thalassemia

Erythroferrone (Erfe) is produced by erythroblasts in response to erythropoietin (EPO) and acts in the liver to prevent hepcidin stimulation by BMP6. Hepcidin suppression allows for the mobilization of iron to the bone marrow for the production of red blood cells. Aberrantly high circulating Erfe levels in conditions of stress erythropoiesis, such as in patients with β-thalassemia, promote the tissue iron accumulation that decisively contributes to morbidity in these patients. Here we developed neutralizing antibodies against Erfe to prevent hepcidin suppression and correct the iron loading phenotype in a mouse model of β-thalassemia (Hbb Th3/+ mice) and used these antibodies as tools to further characterize Erfe's mechanism of action. We demonstrate that Erfe binds to BMP6 with low nanomolar affinity, but also binds BMP2 and BMP4 with lower affinities. We further show that BMP6 binds the N-terminal domain of ERFE. This domain in itself was sufficient to cause hepcidin suppression in Huh7 hepatoma cells and in vivo in wildtype mice. Concurrently, anti-Erfe antibodies targeting the N-terminal domain prevented hepcidin suppression in Erfe-treated Huh7 cells and in EPO-treated mice. Crystal structure of the antibodies in contact with an N-terminal peptide of Erfe demonstrated critical contacts in the Erfe N-terminal domain imparting antibody selectivity to human and murine protein. Finally, we tested these antibodies in vivo in a mouse model of thalassemia. We observed a decrease in serum and liver iron in antibody-treated Hbb Th3/+ mice. In addition, treatment with anti-Erfe antibodies increased the number of red blood cells, hemoglobin concentration and hematocrit, while decreasing the number of reticulocytes and the red cell distribution width. These changes were more pronounced when mice are treated for eight weeks. Anti-Erfe treatment caused an increase in hepatic hepcidin mRNA expression, red blood cells, hemoglobin and hematocrit, while reticulocytes levels were lower and peripheral red cell lifespan was increased. In summary, we demonstrate that antibodies targeting the N-terminal domain of Erfe constitute a potential therapeutic tool for iron-loading anemias. Disclosures Arezes: UCB: Employment. Foy:Pfizer Inc.: Employment. Benard:pfizer: Employment. Sawant:Pfizer Inc.: Employment. Tam:Pfizer Inc.: Employment. Maben:Pfizer Inc.: Employment. LaVallie:Pfizer Inc.: Employment. Cunningham:Pfizer Inc.: Employment. Lambert:Pfizer Inc.: Employment. Pittman:Pfizer Inc.: Employment. Murphy:Pfizer Inc.: Employment. Draper:Pfizer: Research Funding. Jasuja:Pfizer Inc.: Employment. Drakesmith:Pfizer: Consultancy, Research Funding; Kymab: Other: Scientific Advistory; La Jolla Pharmaceutical: Research Funding.

Hepcidin and Anemia: A Tight Relationship

Hepcidin, the master regulator of systemic iron homeostasis, tightly influences erythrocyte production. High hepcidin levels block intestinal iron absorption and macrophage iron recycling, causing iron restricted erythropoiesis and anemia. Low hepcidin levels favor bone marrow iron supply for hemoglobin synthesis and red blood cells production. Expanded erythropoiesis, as after hemorrhage or erythropoietin treatment, blocks hepcidin through an acute reduction of transferrin saturation and the release of the erythroblast hormone and hepcidin inhibitor erythroferrone. Quantitatively reduced erythropoiesis, limiting iron consumption, increases transferrin saturation and stimulates hepcidin transcription. Deregulation of hepcidin synthesis is associated with anemia in three conditions: iron refractory iron deficiency anemia (IRIDA), the common anemia of acute and chronic inflammatory disorders, and the extremely rare hepcidin-producing adenomas that may develop in the liver of children with an inborn error of glucose metabolism. Inappropriately high levels of hepcidin cause iron-restricted or even iron-deficient erythropoiesis in all these conditions. Patients with IRIDA or anemia of inflammation do not respond to oral iron supplementation and show a delayed or partial response to intravenous iron. In hepcidin-producing adenomas, anemia is reverted by surgery. Other hepcidin-related anemias are the “iron loading anemias” characterized by ineffective erythropoiesis and hepcidin suppression. This group of anemias includes thalassemia syndromes, congenital dyserythropoietic anemias, congenital sideroblastic anemias, and some forms of hemolytic anemias as pyruvate kinase deficiency. The paradigm is non-transfusion-dependent thalassemia where the release of erythroferrone from the expanded pool of immature erythroid cells results in hepcidin suppression and secondary iron overload that in turn worsens ineffective erythropoiesis and anemia. In thalassemia murine models, approaches that induce iron restriction ameliorate both anemia and the iron phenotype. Manipulations of hepcidin might benefit all the above-described anemias. Compounds that antagonize hepcidin or its effect may be useful in inflammation and IRIDA, while hepcidin agonists may improve ineffective erythropoiesis. Correcting ineffective erythropoiesis in animal models ameliorates not only anemia but also iron homeostasis by reducing hepcidin inhibition. Some targeted approaches are now in clinical trials: hopefully they will result in novel treatments for a variety of anemias.

S1629 ERFE‐A260S ACCOUNTS FOR HEPATIC IRON METABOLISM IMPAIRMENT BY ACTING ON THE BMP‐SMAD PATHWAY

Background:Iron overload is a major clinical challenge in the management of hereditary ineffective erythropoiesis ‐related anemia (iron‐loading anemia). To coordinate iron metabolism with hemoglobin (Hb) synthesis, erythropoietin (EPO)‐stimulated erythroid precursors release erythroferrone (ERFE), a soluble protein that suppresses the expression of hepcidin. Hepcidin is a hormone that controls iron entry into the circulation by blocking the iron exporter ferroportin.Congenital dyserythropoietic anemia type II (CDAII) is a bone marrow failure anemia characterized by mono‐lineage cytopenia and morphological abnormalities of erythroid precursor cells. The marked clinical variability observed in CDAII patients is related to its major complication, the iron overload associated with reduced expression of hepatic hormone hepcidin. We recently analyzed ERFE expression in both CDAII patients and in vitro model, demonstrating that in CDAII patients ERFE is overexpressed and related to abnormal erythropoiesis.Aims:To study possible modifier genes affecting the iron overload phenotype in CDAII.Methods:t‐NGS custom panel; genotyping; cells culture and transfection assays; qRT‐PCR; immunoblotting.Results:We herein identified a recurrent low‐frequency variant, A260S, in ERFE gene in 12.5% of CDAII patients showing severe phenotype by our 81‐genes target custom panel for modifier genes. We demonstrated that ERFE‐A260S variant leads to an increased expression of ERFE. We further characterized the effect of ERFE‐A260S in an in vitro human hepatic model, HuH7 cells, showing a marked impairment of iron regulation pathways by inhibition of phosphorylation of SMAD1/5/8 and consequent downregulation of the expression of BMP/SMADs pathway target genes: HAMP, ID1, ATHO8 and SMAD6.Summary/Conclusion:We herein described for the first time an ERFE polymorphism as modifier variant with a mild effect on disease expression under a multifactorial‐like model in a condition of iron‐loading anemia due to ineffective erythropoiesis. This is a novel paradigm that paves the ways to new investigations for evaluating the role of this ERFE variant as a modifier in many other related pathological conditions, as b‐thalassemia intermedia. Moreover, the discovery of the genetic basis of CDAII and its major complication, iron overload, will lead us to expand our understanding of the biology of ineffective erythropoiesis and to provide initiative for further investigation of new therapeutics. Indeed, ERFE is currently a candidate target for therapeutic inhibition, in iron loading anemia.

Changes in Cardiac Isomyosin Composition in an Iron Loading Anemia Rat Model

Changes in Cardiac Isomyosin Composition in an Iron Loading Anemia Rat Model

Hepcidin and the BMP-SMAD pathway: An unexpected liaison.

Hepcidin, the main regulator of iron metabolism, is synthesized and released by hepatocytes in response to increased body iron concentration and inflammation. Deregulation of hepcidin expression is a common feature of genetic and acquired iron disorders: in Hereditary Hemochromatosis (HH) and iron-loading anemias low hepcidin causes iron overload, while in Iron Refractory Iron Deficiency Anemia (IRIDA) and anemia of inflammation (AI), high hepcidin levels induce iron-restricted erythropoiesis. Hepcidin expression in the liver is mainly controlled by the BMP-SMAD pathway, activated in a paracrine manner by BMP2 and BMP6 produced by liver sinusoidal endothelial cells. The BMP type I receptors ALK2 and ALK3 are responsible for iron-dependent hepcidin upregulation and basal hepcidin expression, respectively. Characterization of animal models with genetic inactivation of the key components of the pathway has suggested the existence of two BMP/SMAD pathway branches: the first ALK3 and HH proteins dependent, responsive to BMP2 for basal hepcidin activation, and the second ALK2 dependent, activated by BMP6 in response to increased tissue iron. The erythroid inhibitor of hepcidin Erythroferrone also impacts on the liver BMP-SMAD pathway although its effect is blunted by pathway hyper-activation. The liver BMP-SMAD pathway is required also in inflammation to cooperate with JAK2/STAT3 signaling for full hepcidin activation. Pharmacologic targeting of BMP-SMAD pathway components or regulators may improve the outcome of both genetic and acquired disorders of iron overload and deficiency by increasing or inhibiting hepcidin expression.

The Role of Erythropoiesis on Hepcidin Level in Polytransfused b-Thalassemia Major Patients

AbstractBackground: Hepcidin peptide hormone is a main con-troller of iron homeostasis, itbecomes elevated in case of iron overload, however despite iron overload in b-Thalassemia major (b-TM) patients a contradictory decrease in Hepcidin occurs. The incompatible Hepcidin level is the main responsible factor causing the iron overload status in iron-loading anemias such as (b-TM) which contributes to organ dysfunctionand to iron toxicity.Aim of Study: To evaluate the conflicting effect of increased erythropoiesis in contrast to the effect of iron overloading on Hepcidin serum level and its correlation with iron status in (b-TM) patients.Subjects and Methods: For all patients and controls Complete Blood Count (CBC), serum assaysof Hepcidin, iron, ferritin, transferrin, HCVAbs, HBs Ag, CRP and serum level were performed.Results: Hepcidin level was significantly lowered in (b-TM) patients compared to controls with high significant difference (p=0.00). There was no correlation between serum Hepcidin and ferritin level, neither was between Hepcidin and serum iron, transferrin, Hb level and reticulocyte count in the study group. Hepcidin/Ferritin ratio showed high significance difference (p<0.000) with mean value in the study group 0.056 vs. 4.75 in controls pointing to an incom-patible levels of Hepcidin to iron overload status. Hepcidin level wasnegative correlatedwith age (p<0.05).Conclusion: Hepcidin was markedly decreased in the study group with no significant correlation between serum Hepcidin and ferritin level as a marker of iron overload in thalassemia major. Hepcidin deficiency is the main contributing factor of iron overload in b thalassemia which results from a strong suppressive effect of the high erythropoietic activity on Hepcidin expression. Hepcidin-ferritin ratio was markedly depressed <1 in all b thalassemia major patients which indicating suppression of Hepcidin out of proportion with the degree of iron loading.

SLN124, a Galnac-siRNA Conjugate Targeting TMPRSS6, for the Treatment of Iron Overload and Ineffective Erythropoiesis Such As in Beta-Thalassemia

Accumulation of excess iron in tissues causes organ damage and dysfunction and may lead to serious clinical consequences including liver cirrhosis, diabetes, growth retardation and heart failure. Iron overload is a major health threat in iron loading anemias, like beta-thalassemia, myelodysplastic syndrome and in hereditary hemochromatosis. In patients with beta-thalassemia major, iron overload develops due to frequent blood transfusions to control the severe anemia. In addition, iron overload also occurs in patients with beta thalassemia intermedia (non-transfusion dependent beta-thalassemia). In the later cases, iron overload develops through gastrointestinal iron hyperabsorption due to stressed and ineffective erythropoiesis. Importantly, expression of the peptide hormone hepcidin, which is the key modulator in iron homeostasis, is abnormally low and unable to block ferroportin-mediated intestinal iron absorption. In hereditary hemochromatosis, gene defects in the hepcidin-ferroportin axis controlling iron homeostasis, lead to hepatic iron overload. Therefore, in these indications, iron overload is caused by dysregulation or dysfunction of the hepcidin-ferroportin axis. Hepcidin is predominantly produced by the liver and is induced by activation of the BMP/SMAD signaling pathway. Furthermore, hepcidin is under the negative control of the transmembrane protease matriptase-2, encoded by the TMPRSS6 gene.RNA interference is a natural mechanism and a powerful approach for inhibiting the expression of disease-associated genes. Silence Therapeutics has developed short interfering RNA (siRNA) conjugate technology for the selective inhibition of target gene expression in the liver. GalNAc-conjugated siRNAs bind efficiently to the asialoglycoprotein (ASGP) receptor expressed predominantly by hepatocytes thereby providing a highly specific, safe and efficient delivery technology to enable a new class of therapeutic use.Here we present the pharmacological characterization of SLN124, our GalNAc-siRNA conjugate targeting TMPRSS6 expression, in preclinical models. A single subcutaneous administration is sufficient to achieve significant modulation of target gene expression in mice and in non-human primates over several weeks. SLN124 treatment reduces systemic iron levels, transferrin saturation and tissue iron levels in a rodent model for hereditary hemochromatosis type 1. In addition, we report for the first time the therapeutic efficacy of iron restriction by SLN124 in mice with established iron overload both as monotherapy and in combination with an oral iron chelator - current standard of care- over an extended treatment period. The effects of these treatments on red blood cell parameters and tissue iron levels will be presented. In addition, we assessed the therapeutic effects of SLN124 in an animal model for beta-thalassemia intermedia, showing dose-dependent and long-lasting effects on target gene expression as well as on modulation of iron stores and normalization of erythropoiesis and anemia. Safety and tolerability studies in relevant preclinical models confirmed that SLN124 is well tolerated and shows promise as an effective and safe treatment for unmet medical need in iron loading anemias, such as beta-thalassemia. SLN124 is currently in preclinical development. The first in human study is planned to commence in 2019 in patients with beta-thalassemia and in patients with myelodysplastic syndrome. DisclosuresMuckenthaler:Novartis: Research Funding. Dames:Silence Therapeutics GmbH: Employment. Frauendorf:Silence Therapeutics GmbH: Employment. Schubert:Silence Therapeutics GmbH: Employment. Aleku:Silence Therapeutics GmbH: Employment. Zügel:Silence Therapeutics GmbH: Employment.

Erythroferrone Inhibits the Induction of Hepcidin By BMP6

Decreased hepcidin mobilizes iron, which facilitates erythropoiesis, but excess iron is pathogenic in beta-thalassemia and other iron-loading anaemias. Erythropoietin (EPO) enhances erythroferrone (ERFE) synthesis by erythroblasts, and ERFE suppresses hepatic hepcidin production, through an unknown mechanism. The BMP/SMAD pathway in the liver is critical for control of hepcidin, and we show that EPO suppressed hepcidin and other BMP target genes in vivo in a partially ERFE-dependent manner. Furthermore, recombinant ERFE suppressed the hepatic BMP/SMAD pathway independently of changes in serum and liver iron, and in vitro, ERFE decreased SMAD 1/5/8 phosphorylation and inhibited expression of BMP target genes in hepatoma cells. ERFE specifically abrogated the induction of hepcidin by BMP5, BMP6 and BMP7, but had no or very little effect on hepcidin induction by BMP2, 4, 9 or Activin B. A neutralising anti-ERFE antibody prevented the ability of ERFE to inhibit hepcidin induction by BMP5, 6 and 7. Cell-free Homogeneous Time Resolved Fluorescence assays showed that BMP5, BMP6 and BMP7 competed with anti-ERFE for binding to ERFE. Biacore analysis showed that ERFE binds to BMP6 with a higher affinity compared to its binding to BMP2, BMP4 or Activin B, and does not bind to GDF15. We propose that ERFE suppresses hepcidin by inhibiting hepatic BMP/SMAD signaling via preferentially binding and impairing the function of an evolutionarily closely related BMP sub-group consisting of BMP5, BMP6 and BMP7. These findings indicate that ERFE can act as a natural ligand trap generated by stimulated erythropoiesis in order to regulate availability of iron. DisclosuresArezes:Pfizer: Research Funding. Foy:Pfizer: Employment. McHugh:Pfizer: Research Funding. Sawant:Pfizer: Employment. Benard:Pfizer: Employment. Quinkert:Pfizer: Research Funding. Terraube:Pfizer: Employment. Brinth:Pfizer: Employment. Tam:Pfizer: Employment. LaVallie:Pfizer: Employment. Cunningham:Pfizer: Employment. Lambert:Pfizer: Employment. Draper:Pfizer: Research Funding. Jasuja:Pfizer: Employment. Drakesmith:La Jolla Pharmaceutical Company: Research Funding; Pfizer: Research Funding; Alnylam: Consultancy; Kymab: Membership on an entity's Board of Directors or advisory committees.