Mouse Model Of Β-thalassemia Research Articles

Preliminary screening of biomarkers and drug candidates in a mouse model of β-thalassemia based on quasi-targeted metabolomics.

β-thalassemia (β-TH) is a hereditary hemolytic anemia that results in deficient hemoglobin (Hb) synthesis. It is characterized by ineffective erythropoiesis, anemia, splenomegaly, and systemic iron overload. Exploration new potential biomarkers and drug candidates is important to facilitate the prevention and treatment of β-TH. We applied quasi-targeted metabolomics between wild type (Wt) and heterozygous β-TH mice (Th3/+), a model of non-transfusion-dependent β-TH intermedia, in plasma and peripheral blood (PB) cells. Futher data was deeply mined by Kyoto Encyclopedia of Genomes (KEGG) and machine algorithms methods. Using KEGG enrichment analysis, we found that taurine and hypotaurine metabolism disorders in plasma and alanine, aspartate and glutamate metabolism disorders in PB cells. After systematically anatomize the metabolites by machine algorithms, we confirmed that alpha-muricholic acidUP and N-acetyl-DL-phenylalanineUP in plasma and Dl-3-hydroxynorvalineUP, O-acetyl-L-serineUP, H-abu-OHUP, S-(Methyl) glutathioneUP, sepiapterinDOWN, and imidazoleacetic acidDOWN in PB cells play key roles in predicting the occurrence of β-TH. Furthermore, Sepiapterin, Imidazoleacetic acid, Methyl alpha-D-glucopyranoside and alpha-ketoglutaric acid have a good binding capacity to hemoglobin E through molecular docking and are considered to be potential drug candidates for β-TH. Those results may help in identify useful molecular targets in the diagnosis and treatment of β-TH and lays a strong foundation for further research.

Novel Anti-TMPRSS6 Monoclonal Antibody Provides a Potential Therapeutic Approach for the Treatment of Polycythemia Vera

Polycythemia vera (PV) is a myeloproliferative neoplasm (MPN) triggered by activating mutations in JAK2, an important kinase in the principal signaling pathway of the erythropoietin receptor. Most of the clinical manifestations of PV are caused by increased erythrocyte count, leading to increased risk of pulmonary hypertension and thrombosis. The mainstay of therapy for PV is therapeutic phlebotomy to reduce the hematocrit (HCT) level and minimize the risk of thrombosis, however, the effect of phlebotomy is transient until patients become iron deficient. Therefore, systemic iron restriction could be effective in treating PV patients. Transmembrane serine protease 6 (TMPRSS6) is a negative regulator hepcidin, the master regulator of iron homeostasis. Increasing genetic, preclinical and clinical evidence has demonstrated that inhibition of TMPRSS6 can potently elevated hepcidin levels, thereby achieving systemic iron restriction. Previously, we reported the generation of anti-TMPRSS6 antibody MWTx-003 and its efficacy in reducing iron overload and improving ineffective erythropoiesis in a mouse model of β-thalassemia. In this study, we explored therapeutic application of MWTx-003 in PV using a Jak2 V617F bone marrow transplantation mouse model. The mouse model exhibits typical PV phenotypes of erythrocytosis and elevated HCT levels. In the PV mice, treatment with MWTx-003 (2-10 mg/kg, i.p., every 4 days for 3 weeks) led to significantly reduced HCT levels, red blood cell (RBC) counts and hemoglobin (HGB) concentration that were comparable to that of wildtype control mice. These effects were evident in a dose-dependent manner and observed as early as two weeks into treatment. It is worth noting that in the 10 mg/kg dose group, a worsening splenomegaly and expansion of early erythroid progenitors were observed after 3 weeks of treatment, and these findings are typical of iron-restricted erythropoiesis in mice. Taken together, the anti-TMPRSS6 antibody offers a promising therapeutic approach in the management of PV, where erythrocytosis and high HCT levels are associated with poor outcomes. A Phase I clinical trial with MWTx-003 in healthy volunteers has been initiated in China.

Elevated CDKN1A (P21) mediates β-thalassemia erythroid apoptosis, but its loss does not improve β-thalassemic erythropoiesis

Abstractβ-thalassemias are common hemoglobinopathies due to mutations in the β-globin gene that lead to hemolytic anemias. Premature death of β-thalassemic erythroid precursors results in ineffective erythroid maturation, increased production of erythropoietin (EPO), expansion of erythroid progenitor compartment, extramedullary erythropoiesis, and splenomegaly. However, the molecular mechanism of erythroid apoptosis in β-thalassemia is not well understood. Using a mouse model of β-thalassemia (Hbbth3/+), we show that dysregulated expression of the FOXO3 transcription factor is implicated in β-thalassemia erythroid apoptosis. In Foxo3−/−/Hbbth3/+ mice, erythroid apoptosis is significantly reduced, whereas erythroid cell maturation, and red blood cell and hemoglobin production are substantially improved even with elevated reactive oxygen species in double-mutant erythroblasts. However, persistence of elevated reticulocytes and splenomegaly suggests that ineffective erythropoiesis is not resolved in Foxo3−/−/Hbbth3/+. We found the cell cycle inhibitor Cdkn1a (cyclin-dependent kinase inhibitor p21), a FOXO3 target gene, is markedly upregulated in both mouse and patient–derived β-thalassemic erythroid precursors. Double-mutant p21/Hbbth3/+ mice exhibited embryonic lethality with only a fraction of mice surviving to weaning. Notably, studies in adult mice displayed greatly reduced apoptosis and circulating Epo in erythroid compartments of surviving p21−/−/Hbbth3/+ mice relative to Hbbth3/+ mice, whereas ineffective erythroid cell maturation, extramedullary erythropoiesis, and splenomegaly were not modified. These combined results suggest that mechanisms that control β-thalassemic erythroid cell survival and differentiation are uncoupled from ineffective erythropoiesis and involve a molecular network including FOXO3 and P21. Overall, these studies provide a new framework for investigating ineffective erythropoiesis in β-thalassemia.

Reducing erythroblast apoptosis in β-thalassemia via unfolded protein response (UPR) signaling

Apoptosis plays an important role in ineffective erythropoiesis in β-thalassemia. This early destruction causes severe anemia and contributes significantly to patient mortality. There are several treatment options for this disorder based on the use of drugs that inhibit the destruction of immature erythrocytes in the bone marrow. However, the clinical effectiveness of these drugs has yet to be proven. Therefore, new targets for more effective treatments are needed. The unfolded protein response (UPR) is a biological process that occurs in response to endoplasmic reticulum (ER) stress. To cope with ER stress, unfolded or misfolded proteins activate the UPR pathway, thereby activating a series of cell death pathways. Here, we propose that prolonged ER stress causes apoptosis via UPR signaling during erythroid development in β-thalassemia. Our proposed hypothesis is supported by 1) the involvement of UPR signaling in apoptotic cell death, 2) changes in UPR-related proteins in β-thalassemia, and 3) the availability of UPR signaling inhibitors. In vitro and in vivo studies should be conducted to identify the proteins that participate in the UPR pathway and identify molecular targets for therapy. The β-thalassemia mouse model could be used to evaluate the efficacy of UPR mimic drugs prior to clinical trials. If verified, gaining control over cell fate determination during UPR signaling in β-thalassemia may minimize erythroblast destruction.

Ferroportin inhibitor vamifeport ameliorates ineffective erythropoiesis in a mouse model of β-thalassemia with blood transfusions.

β-thalassemia is an inherited anemia characterized by ineffective erythropoiesis. Blood transfusions are required for survival in transfusion-dependent β-thalassemia and are also occasionally needed in patients with non-transfusion-dependent β-thalassemia. Patients with transfusion-dependent b-thalassemia often have elevated transferrin saturation (TSAT) and non-transferrin-bound iron (NTBI) levels, which can lead to organ iron overload, oxidative stress, and vascular damage. Vamifeport is an oral ferroportin inhibitor that was previously shown to ameliorate anemia, ineffective erythropoiesis, and dysregulated iron homeostasis in the Hbbth3/+ mouse model of β-thalassemia, under non-transfused conditions. Our study aimed to assess the effects of oral vamifeport on iron-related parameters (including plasma NTBI levels) and ineffective erythropoiesis following blood transfusions in Hbbth3/+ mice. A single dose of vamifeport prevented the transient transfusion-mediated NTBI increase in Hbbth3/+ mice. Compared with vehicle treatment, vamifeport significantly increased hemoglobin levels and red blood cell counts in transfused mice. Vamifeport treatment also significantly improved ineffective erythropoiesis in the spleens of Hbbth3/+ mice, with additive effects observed when treatment was combined with repeated transfusions. Vamifeport corrected leukocyte counts and significantly improved iron-related parameters (serum transferrin, TSAT and erythropoietin levels) versus vehicle treatment in Hbbth3/+ mice, irrespective of transfusion status. In summary, vamifeport prevented transfusion-mediated NTBI formation in Hbbth3/+ mice. When given alone or combined with blood transfusions, vamifeport also ameliorated anemia, ineffective erythropoiesis, and dysregulated iron homeostasis. Administering vamifeport together with repeated blood transfusions additively ameliorated anemia and ineffective erythropoiesis in this mouse model, providing preclinical proof-of-concept for the efficacy of combining vamifeport with blood transfusions in β-thalassemia.

Discovery-Based Proteomics Identify Skeletal Muscle Mitochondrial Alterations as an Early Metabolic Defect in a Mouse Model of β-Thalassemia

Although metabolic complications are common in thalassemia patients, there is still an unmet need to better understand underlying mechanisms. We used unbiased global proteomics to reveal molecular differences between the th3/+ mouse model of thalassemia and wild-type control animals focusing on skeletal muscles at 8 weeks of age. Our data point toward a significantly impaired mitochondrial oxidative phosphorylation. Furthermore, we observed a shift from oxidative fibre types toward more glycolytic fibre types in these animals, which was further supported by larger fibre-type cross-sectional areas in the more oxidative type fibres (type I/type IIa/type IIax hybrid). We also observed an increase in capillary density in th3/+ mice, indicative of a compensatory response. Western blotting for mitochondrial oxidative phosphorylation complex proteins and PCR analysis of mitochondrial genes indicated reduced mitochondrial content in the skeletal muscle but not the hearts of th3/+ mice. The phenotypic manifestation of these alterations was a small but significant reduction in glucose handling capacity. Overall, this study identified many important alterations in the proteome of th3/+ mice, amongst which mitochondrial defects leading to skeletal muscle remodelling and metabolic dysfunction were paramount.

Erythroferrone Overexpression Impairs Survival and Greatly Increases Tissue Iron Loading in a Mouse Model of β-Thalassemia

Beta-thalassemia, caused by mutations in the β-globin gene, is characterized by ineffective erythropoiesis, anemia and associated multi-organ dysfunction. Like in other anemias with ineffective erythropoiesis, the erythroid hormone erythroferrone (ERFE) is pathologically overproduced. ERFE functions by suppressing the master iron regulatory hormone hepcidin, and the chronic suppression of hepcidin in β-thalassemia results in excessive iron absorption and iron overload, even in patients who do not receive erythrocyte transfusions. A major obstacle in studying the role of ERFE in β-thalassemia has been the lack of a mouse model that represents the wide range of ERFE concentrations observed in human patients (Ganz et al, Blood 2017). Our lab generated ERFE-transgenic mice with a range of ERFE overexpression (Coffey et al, Blood 2022) and showed that they developed dose-dependent iron overload and relative hepcidin deficiency. In the current study, the mid-range ERFE-overexpressing E(M) mice were crossed with HbbTh3/+ (Th3) mice, a widely used model of β-thalassemia, to generate mice with a single copy of both transgenes (T-E(M)), as well as littermate controls (Th3, WT, E(M)), to analyze the effect of ERFE on the pathophysiology of ineffective erythropoiesis. The offspring had a noticeably skewed genotypic ratio, with T-E(M) pups making up only <9% of surviving pups compared to 25% expected for each genotype. At embryonic day 18.5, however, there was an equal distribution of all expected genotypes, suggesting that the mortality in T-E(M) pups is postnatal. Surviving T-E(M) mice were analyzed at 16 weeks of age. They had similar erythrocyte parameters to the Th3 littermates , with the expected decrease in red blood cell count, hemoglobin and mean corpuscular volume. Despite the similarities in blood counts, we discovered a synergistic effect of the ERFE and Th3 transgenes on ERFE production in the T-E(M) mice as indicated by increased serum ERFE levels (Figure 1a) and increased Erfe mRNA levels in the bone marrow and spleen compared with both E(M) and Th3 littermates. The dramatic increase in serum ERFE in T-E(M) mice may result from multiplicative effects of erythroblast expansion in Th3 mice and transgenic ERFE overexpression in individual erythroblasts of T-E(M) mice. The large range of ERFE concentrations in human patients may also reflect the combined effects of erythroblast number and ERFE overproduction by erythropoietin-stimulated thalassemic erythroblasts, both of which vary depending on patient genetics. T-E(M) mice also have significantly larger spleens compared to Th3 littermates, suggesting that ERFE overexpression contributes to increased extramedullary hematopoiesis in T-E(M) spleens. In addition, T-E(M) mice had significantly higher iron levels in the liver (Figure 1b) as well as serum and kidney compared to Th3 littermates. Coupled with the inappropriately low serum hepcidin levels (compared to liver iron levels) in T-E(M) mice, even when compared to E(M) and Th3 littermates, these data indicate that excessive ERFE further increases iron loading in a mouse model of β-thalassemia. Our data demonstrate that elevated ERFE levels contribute to the pathogenesis of β-thalassemia by decreasing postnatal survival, increasing iron overload, and increasing extramedullary hematopoiesis. These results suggest that neutralizing excessive ERFE levels may have therapeutic benefits for postnatal development and prevention of iron overload in β-thalassemia. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Differential Effects of Monoferric Forms of Transferrin on Mouse Models of Anemia of Inflammation and Congenital Anemia

Transferrin (TF) is a bilobed 80kD glycoprotein with N- and C-lobe iron binding sites. TF circulates as four forms: unbound to iron (apo-TF), iron bound to the N-lobe, the C-lobe or to both lobes (diferric-TF). Most circulating TF under physiological conditions is monoferric (Dirusso et al., 1985). The TF forms interact with TF receptor-1 (TFR1), which is ubiquitously expressed and serves as the main mechanism for cellular iron delivery. TF also interacts with TF receptor-2 (TFR2) which is expressed on hepatocytes, erythroblasts, and other cells of the bone marrow and is thought to primarily influence cellular signaling events regulating hepcidin expression and erythropoiesis. In erythrocytes, loss of TFR2 results in abnormally high red blood cell production under conditions of iron-deficiency (Artuso et al., 2018), suggesting it functions as an iron sensor. Additional evidence suggests TFR2 modulates erythropoiesis via the erythropoietin (EPO) receptor (EPOR) (Forejtnikovà et al., 2010). To understand the role of monoferric TF forms in vivo, we previously generated N-lobe blocked (TfN-bl) or C-lobe blocked (TfC-bl) TF mutant mice. Characterization of these mice demonstrated dramatic differences in RBC levels, hepcidin expression relative to iron status, and EPO sensitivity (Parrow et al., 2019). More recently, we have become interested in characterizing the immune compartment of these mice. Recent studies point to iron being an important regulator of immune responses, which has important implications worldwide in understanding how to treat iron-deficient patients without exacerbating latent infections. Cells belonging to the immune branch have also been shown to express TFR2 and EPOR (Cantarelli et al., 2019; Roetto et al., 2011; Kawabata et al., 2001), however their function is largely unexplored. Therefore, in this work, we investigate the role of the two monoferric forms of TF on ineffective erythropoiesis (IE) using a mouse model of β-thalassemia (BT) intermedia (Hbbth3/+) and chronic anemia of inflammation using heat-killed Brucella Abortus (HKBA). Based on observations in BT mice treated with exogenous TF (Li et al., 2010), which led to reduced erythroid iron intake, reduced EPO requirement (as in TfC-bl mutant mice) and improved anemia, we hypothesized that Hbbth3/+ mice crossed with TfC-bl would demonstrate improved erythropoietic and iron parameters compared with Hbbth3/+TfN-bl. We measured effects on hematologic parameters, IE, and expression of relevant iron regulatory serum proteins. Hbbth3/+TfC-bl mice demonstrated significantly increased RBC counts, elevated hemoglobin, improved erythrocyte morphology, decreased splenomegaly, fewer bone marrow erythroblasts, and improvement of IE. Additionally, serum erythroferrone (ERFE) was significantly reduced and hepcidin levels were increased in Hbbth3/+TfC-bl relative to Hbbth3/+Tf+/+ controls. However, hematological parameters from Hbbth3/+TfN-bl mice did not show these improvements. In our studies examining the immune compartments of TfC-bl and TfN-bl mice we found striking differences in their expression of TFR1 and response to immunological challenge by HKBA. Our preliminary data shows that expression levels of TFR1 were elevated in both TF mutant mice relative to control at baseline on lymphocyte, monocyte and granulocyte populations of the bone marrow. When challenged with a single intraperitoneal injection of HKBA, TfC-bl-HKBA mice suffered a more severe anemic phenotype with a 30% probability of survival following HKBA treatment. On the contrary, TfN-bl-HKBA mice displayed a blunted response to the immunological challenge, with faster recovery and no death. Taken together, our work provides additional evidence that TF is not only delivering iron cargo to cells, but also functioning as a signaling molecule via the lobe that is bound to iron. Despite each mouse only possessing a TF capable of binding one atom of iron, the two mouse models show profound differences in the pathophysiology of BT and AI. We hypothesize that the monoferric TF forms exert their influence via TFR2. We are currently characterizing a new loxp flanked TFR2-FLAG mouse model generated by our lab, which has been crossed to the TF monoferric mutant mice to test this hypothesis. Additionally, we are also exploring the translation potential of the two TF forms to treat anemia in BT and AI.

Treating Iron Overload Disorders with a Novel Therapeutic Antibody Targeting TMPRSS6

Iron is an essential element for almost all living organisms as it participates in a wide variety of metabolic processes. Disorders of iron metabolism are among the most prevalent human diseases, ranging from anemia to hemochromatosis. Excessive iron accumulations in major organs of iron overload patients can lead to high mortality. Hepcidin, a HAMP-encoded liver hormone, is the master regulator of iron homeostasis. By binding to the sole iron exporter ferroportin and causing internalization and degradation of the complex, hepcidin inhibits cellular iron efflux, thereby lowers plasma iron levels. Inappropriately suppressed/low hepcidin production is central to iron overload. Transmembrane protease serine-6 (TMPRSS6), a type II transmembrane serine protease primarily expressed in liver, downregulates hepcidin expression through BMP-SMAD pathway. TMPRSS6 deficiencies have been shown to cause hepcidin overexpression in both TMPRSS6-mutant mice and in patients with iron-refractory iron deficiency anemia (IRIDA). Therefore, TMPRSS6 is a viable therapeutic target for iron overload disorders.Here we report the generation of an anti-TMPRSS6 antibody through a hybridoma campaign using a DNA-based immunization approach, followed by humanization and sequence optimization. Lead antibody, hzMWTx-003 selectively binds human TMPRSS6 with low nanomolar affinity (KD: 7.6nM), and is cross-reactive to rodent (mouse and rat) and monkey (cynomolgus and rhesus) TMPRSS6. Single-dose injection of hzMWTx-003 was able to significantly elevate serum hepcidin and liver HAMP RNA levels in wildtype mice, resulting in significantly reduced serum iron level. The Hbb th3/+ mouse model of β-thalassemia, like its human counterpart, is characterized by iron overload, ineffective erythropoiesis and splenomegaly. Treatment of Hbb th3/+mice with MWTx-003 effectively increased hepcidin expression at both protein and RNA levels, leading to significantly reduced serum iron and liver non-heme iron content. MWTx-003 also dramatically improved anemia and ineffective erythropoiesis, and alleviated splenomegaly in these mice.CMC development of hzMWTx-003 confirms outstanding biophysical properties. Preliminary studies in cynomolgus monkey using GLP-grade material demonstrated good pharmacokinetics of hzMWTx-003 and expected pharmacodynamic response where reduction of serum iron could be sustained for 21 days after single dose administration. A single dose toxicology study in cynomolgus monkey revealed no safety concerns, and no production of anti-idiotype antibodies was detected. In summary, anti-TMPRSS6 antibody MWTx-003 represents a promising therapy for iron overload disorders such as β-thalassemia, and potentially other diseases where iron restriction is beneficial. DisclosuresChen: Mabwell Therapeutics Inc: Current Employment. Wang: Mabwell Therapeutics Inc: Current Employment. Zheng: Mabwell (Shanghai) Bioscience Co. Ltd: Current Employment. Huang: Mabwell Therapeutics Inc: Current Employment. Mao: Mabwell (Shanghai) Bioscience Co. Ltd.: Current Employment. Ouyang: Mabwell (Shanghai) Bioscience Co. Ltd.: Current Employment. Du: Mabwell Therapeutics Inc: Current Employment.

Reprogramming Cell-Signaling By Delivering the Catalytic Domain of Ptpgr Ameliorates Anemia of β-Thalassemia

β-thalassemia (β-thal) is one of the most common monogenic disorders worldwide, characterized by ineffective erythropoiesis leading to a chronic, debilitating anemia associated with high morbidity and mortality. Erythroid maturation is a dynamic process tightly regulated by complex signaling mechanisms, only partially described either in normal and diseased erythropoiesis. To investigate this issue, we carried out a high throughput kinome analysis by taking advantage of Kinexus array technology (http://www.kinexus.ca), in sorted erythroid precursors from a mouse model of β-thalassemia (Hbb3th/+) compared to wildtype animals. In β-thal mice, we found differential modulation of many protein kinases. Network computational analysis unveiled common as well as erythroid precursor-specific signaling mechanisms of altered erythrocyte differentiation in beta thalassemia, suggesting a selective perturbation in protein kinase/phosphatase balance in β-thal erythropoiesis.We reasoned that balancing kinome anomalies, by increasing phosphatome activity, could normalize kinome signaling pathways, thus ameliorating erythropoiesis. We explored the expression and function of different protein phosphatase and we found reduced expression and function of protein Tyr-phosphatase receptor type gamma (PTPRG). To investigate PTPRG role in erythropoiesis, we exploited a novel Trojan-fusion protein (TAT-ICD), we recently patented, that delivers intracellularly the catalytic domain of PTPRG and up-modulates its signaling cascade, as both a research tool to map dysfunctional pathways and as a potential therapeutic agent. In β-thal mice, TAT-ICD acted on multiple abnormally activated targets identified by computational analysis. TAT-ICD significantly reduced the activation of (i) Jak2- STAT5 pathway; (ii) Bruton tyrosine kinase (BTK), that has been reported to be part of the erythropoietin cascade; (iii) Akt, that is involved in TGF-β -SMAD signaling pathway. This was associated with down-regulation of Erfe and Gdf11 gene expression in sorted erythroblasts from TAT-ICD treated β-thal mice. Collectively, TAT-ICD treatment resulted in amelioration of β-thal ineffective erythropoiesis, evaluated by multiple approaches, including the profile of erythroid maturation and the amount of Annexin-V+ erythroid cells, reticulocyte count, circulating erythroblasts and hemolytic indices (U.S. Patent #62/109,555). The improvement of anemia was also associated with reduction in alpha aggregates and membrane bound hemichromes in circulating erythrocytes. We also found a reduction of liver and spleen iron accumulation in agreement with the beneficial effects on the hematologic phenotype. It is of note that TAT-ICD treatment did not affect either peripheral leukocyte counts or spleen lymphocyte pattern.Our data unveil abnormalities in signal transduction pathways as new mechanism involved in β-thal erythropoiesis, and validate a novel, breakthrough, therapeutic approach to reset back to homeostatic equilibrium altered kinome in diseased erythropoiesis. DisclosuresNo relevant conflicts of interest to declare.

The pyruvate kinase activator mitapivat reduces hemolysis and improves anemia in a β-thalassemia mouse model

Anemia in β-thalassemia is related to ineffective erythropoiesis and reduced red cell survival. Excess free heme and accumulation of unpaired α-globin chains impose substantial oxidative stress on β-thalassemic erythroblasts and erythrocytes, impacting cell metabolism. We hypothesized that increased pyruvate kinase activity induced by mitapivat (AG-348) in the Hbbth3/+ mouse model for β-thalassemia would reduce chronic hemolysis and ineffective erythropoiesis through stimulation of red cell glycolytic metabolism. Oral mitapivat administration ameliorated ineffective erythropoiesis and anemia in Hbbth3/+ mice. Increased ATP, reduced reactive oxygen species production, and reduced markers of mitochondrial dysfunction associated with improved mitochondrial clearance suggested enhanced metabolism following mitapivat administration in β-thalassemia. The amelioration of responsiveness to erythropoietin resulted in reduced soluble erythroferrone, increased liver Hamp expression, and diminished liver iron overload. Mitapivat reduced duodenal Dmt1 expression potentially by activating the pyruvate kinase M2-HIF2α axis, representing a mechanism additional to Hamp in controlling iron absorption and preventing β-thalassemia-related liver iron overload. In ex vivo studies on erythroid precursors from patients with β-thalassemia, mitapivat enhanced erythropoiesis, promoted erythroid maturation, and decreased apoptosis. Overall, pyruvate kinase activation as a treatment modality for β-thalassemia in preclinical model systems had multiple beneficial effects in the erythropoietic compartment and beyond, providing a strong scientific basis for further clinical trials.

CYP450 Mediates Reactive Oxygen Species Production in a Mouse Model of β-Thalassemia through an Increase in 20-HETE Activity.

Oxidative damage by reactive oxygen species (ROS) is one of the main contributors to cell injury and tissue damage in thalassemia patients. Recent studies suggest that ROS generation in non-transfusion-dependent (NTDT) patients occurs as a result of iron overload. Among the different sources of ROS, the nicotinamide adenine dinucleotide phosphate (NADPH) oxidase family of enzymes and cytochrome P450 (CYP450) have been proposed to be major contributors for oxidative stress in several diseases. However, the sources of ROS in patients with NTDT remain poorly understood. In this study, Hbbth3/+ mice, a mouse model for β-thalassemia, were used. These mice exhibit an unchanged or decreased expression of the major NOX isoforms, NOX1, NOX2 and NOX4, when compared to their C57BL/6 control littermates. However, a significant increase in the protein synthesis of CYP4A and CYP4F was observed in the Hbbth3/+ mice when compared to the C57BL/6 control mice. These changes were paralleled by an increased production of 20-hydroxyeicosatetraenoic acid (20-HETE), a CYP4A and CYP4F metabolite. Furthermore, these changes corroborate with onset of ROS production concomitant with liver injury. To our knowledge, this is the first report indicating that CYP450 4A and 4F-induced 20-HETE production mediates reactive oxygen species overgeneration in Hbbth3/+ mice through an NADPH-dependent pathway.

The Oral Ferroportin Inhibitor VIT-2763 Improves Erythropoiesis without Interfering with Iron Chelation Therapy in a Mouse Model of β-Thalassemia.

In β-thalassemia, ineffective erythropoiesis leads to anemia and systemic iron overload. The management of iron overload by chelation therapy is a standard of care. However, iron chelation does not improve the ineffective erythropoiesis. We recently showed that the oral ferroportin inhibitor VIT-2763 ameliorates anemia and erythropoiesis in the Hbbth3/+ mouse model of β-thalassemia. In this study, we investigated whether concurrent use of the iron chelator deferasirox (DFX) and the ferroportin inhibitor VIT-2763 causes any pharmacodynamic interactions in the Hbbth3/+ mouse model of β-thalassemia. Mice were treated with VIT-2763 or DFX alone or with the combination of both drugs once daily for three weeks. VIT-2763 alone or in combination with DFX improved anemia and erythropoiesis. VIT-2763 alone decreased serum iron and transferrin saturation (TSAT) but was not able to reduce the liver iron concentration. While DFX alone had no effect on TSAT and erythropoiesis, it significantly reduced the liver iron concentration alone and in the presence of VIT-2763. Our results clearly show that VIT-2763 does not interfere with the iron chelation efficacy of DFX. Furthermore, VIT-2763 retains its beneficial effects on improving ineffective erythropoiesis when combined with DFX in the Hbbth3/+ mouse model. In conclusion, co-administration of the oral ferroportin inhibitor VIT-2763 and the iron chelator DFX is feasible and might offer an opportunity to improve both ineffective erythropoiesis and iron overload in β-thalassemia.

Transgenic Mice Overexpressing Erythroferrone, a Novel Erythrokine, Develop Iron Overload and Multi-Organ Iron-Independent Abnormalities

Erythroferrone (ERFE) is a hormone secreted by erythroblasts in response to endogenous or exogenous erythropoietic stimuli. ERFE suppresses the production of the iron-regulatory hormone hepcidin by hepatocytes, thereby allowing for increased iron absorption and iron mobilization to support intensified erythropoiesis. In disorders of ineffective erythropoiesis, such as β-thalassemia or congenital dyserythropoietic anemias, chronic suppression of hepcidin results in excessive iron absorption, toxic iron accumulation and a range of systemic manifestations, some of which appear unrelated to iron overload. Recent evidence suggests that ERFE is a ligand trap for select bone morphogenetic proteins (BMPs). BMPs regulate iron homeostasis, but also modulate diverse physiologic processes including embryonic development, kidney growth, and bone homeostasis. Existing mouse models of β-thalassemia used to study the effects of elevated ERFE in vivo are confounded by chronic anemia and hemolysis, complicating attribution of observed phenotypes directly to the action of ERFE. Additionally, serum ERFE levels in these mouse models are much lower than those measured in human patients with β-thalassemia, resulting in a potential underestimation of the pathophysiologic effects of elevated ERFE. To determine the specific effect of elevated ERFE levels in vivo, we generated multiple lines of novel transgenic mice that selectively overexpress graded levels of Erfe in erythroid cells. As expected, Erfe transgenic mice, relative to wild-type littermates, displayed a dose-dependent phenotype of liver iron loading with inappropriately low hepcidin expression. At 6 weeks of age liver nonheme iron levels in transgenic mice ranged from ~2 times that of wild-type littermates, in the lowest expressing line, to ~4 times that of wild-type littermates in the highest-expressing line (line H mice). However, elevated Erfe expression did not consistently alter expression of the BMP target genes Id1 or Smad7 in either the liver or bone marrow. Line-H transgenic mice had increased hemoglobin, hematocrit, mean corpuscular volume, and serum iron suggesting that elevated ERFE levels promoted erythropoiesis, at least in part, by suppressing hepcidin and mobilizing more iron, generating a phenotype similar to mouse models of hereditary hemochromatosis. We also observed phenotypic features that appeared unrelated to iron excess. Line-H breeding diverged from the expected mendelian inheritance ratio of 50% transgenic pups for male mice at weaning age, yielding 37% transgenic pups (N = 108, p =0.0035). Transgenic mice from line-H also had lower body weights and reduced kidney size, accompanied by higher serum urea levels. Additionally, approximately a third of line-H transgenic mice displayed an unusual behavior characterized by repetitive circling and an impaired righting reflex, suggesting vestibular dysfunction. These findings of stunted growth, renal impairment and behavioral abnormalities raise the possibility that elevated ERFE levels may exert iron-independent adverse effects in congenital anemias with ineffective erythropoiesis and in β-thalassemia, perhaps by inhibiting BMP-dependent developmental signals. Future studies using the graded expression of the Erfe-transgene combined with mouse models of dyserythropoiesis will facilitate analysis of the contribution of varying levels of ERFE to the pathophysiology of ineffective erythropoiesis and lay the foundation for pharmacologically targeting ERFE for the amelioration of the hematologic and nonhematologic manifestations of anemias with ineffective erythropoiesis. Disclosures Nemeth: Vifor: Consultancy; Protagonist: Consultancy; Ionis Pharmaceuticals: Consultancy; Silarus Therapeutics: Current equity holder in private company; Intrinsic LifeSciences: Current equity holder in private company. Ganz:Disc Medicine: Consultancy; Silarus Therapeutics: Current equity holder in private company; Intrinsic LifeSciences: Current equity holder in private company; Ambys: Consultancy; Sierra Oncology: Consultancy; Rockwell: Consultancy; Gossamer Bio: Consultancy; American Regent: Consultancy; Global Blood Therapeutics: Consultancy; Astellas: Consultancy; Akebia: Consultancy; Vifor: Consultancy; Ionis Pharmaceuticals: Consultancy.

The opposing roles of the mTOR signaling pathway in different phases of human umbilical cord blood-derived CD34+ cell erythropoiesis

As an indispensable, even lifesaving practice, red blood cell (RBC) transfusion is challenging due to several issues, including supply shortage, immune incompatibility, and blood‐borne infections since donated blood is the only source of RBCs. Although large‐scale in vitro production of functional RBCs from human stem cells is a promising alternative, so far, no such system has been reported to produce clinically transfusable RBCs due to the poor understanding of mechanisms of human erythropoiesis, which is essential for the optimization of in vitro erythrocyte generation system. We previously reported that inhibition of mammalian target of rapamycin (mTOR) signaling significantly decreased the percentage of erythroid progenitor cells in the bone marrow of wild‐type mice. In contrast, rapamycin treatment remarkably improved terminal maturation of erythroblasts and anemia in a mouse model of β‐thalassemia. In the present study, we investigated the effect of mTOR inhibition with rapamycin from different time points on human umbilical cord blood‐derived CD34+ cell erythropoiesis in vitro and the underlying mechanisms. Our data showed that rapamycin treatment significantly suppressed erythroid colony formation in the commitment/proliferation phase of erythropoiesis through inhibition of cell‐cycle progression and proliferation. In contrast, during the maturation phase of erythropoiesis, mTOR inhibition dramatically promoted enucleation and mitochondrial clearance by enhancing autophagy. Collectively, our results suggest contrasting roles for mTOR in regulating different phases of human erythropoiesis.

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.

Genetic disruption of KCC cotransporters in a mouse model of thalassemia intermedia

β-thalassemia (β-Thal) is caused by defective β-globin production leading to globin chain imbalance, aggregation of free alpha chain in developing erythroblasts, reticulocytes, and mature circulating red blood cells. The hypochromic thalassemic red cells exhibit increased cell dehydration in association with elevated K+ leak and increased K-Cl cotransport activity, each of which has been linked to globin chain imbalance and related oxidative stress.We therefore tested the effect of genetic inactivation of K-Cl cotransporters KCC1 and KCC3 in a mouse model of β-thalassemia intermedia. In the absence of these transporters, the anemia of β-Thal mice was ameliorated, in association with increased MCV and reductions in CHCM and hyperdense cells, as well as in spleen size. The resting K+ content of β-Thal red cells was greatly increased, and Thal-associated splenomegaly slightly decreased. Lack of KCC1 and KCC3 activity in Thal red cells reduced red cell density and improved β-Thal-associated osmotic fragility. We conclude that genetic inactivation of K-Cl cotransport can reverse red cell dehydration and partially attenuate the hematologic phenotype in a mouse model of β-thalassemia.

Bitopertin, a selective oral GLYT1 inhibitor, improves anemia in a mouse model of β-thalassemia.

Anemia of β-thalassemia is caused by ineffective erythropoiesis and reduced red cell survival. Several lines of evidence indicate that iron/heme restriction is a potential therapeutic strategy for the disease. Glycine is a key initial substrate for heme and globin synthesis. We provide evidence that bitopertin, a glycine transport inhibitor administered orally, improves anemia, reduces hemolysis, diminishes ineffective erythropoiesis, and increases red cell survival in a mouse model of β-thalassemia (Hbbth3/+ mice). Bitopertin ameliorates erythroid oxidant damage, as indicated by a reduction in membrane-associated free α-globin chain aggregates, in reactive oxygen species cellular content, in membrane-bound hemichromes, and in heme-regulated inhibitor activation and eIF2α phosphorylation. The improvement of β-thalassemic ineffective erythropoiesis is associated with diminished mTOR activation and Rab5, Lamp1, and p62 accumulation, indicating an improved autophagy. Bitopertin also upregulates liver hepcidin and diminishes liver iron overload. The hematologic improvements achieved by bitopertin are blunted by the concomitant administration of the iron chelator deferiprone, suggesting that an excessive restriction of iron availability might negate the beneficial effects of bitopertin. These data provide important and clinically relevant insights into glycine restriction and reduced heme synthesis strategies for the treatment of β-thalassemia.

Mitapivat (AG-348), an Oral PK-R Activator, in Adults with Non-Transfusion Dependent Thalassemia: A Phase 2, Open-Label, Multicenter Study in Progress

Background: Thalassemia is a group of inherited blood disorders in which genetic mutation(s) in the α- and/or β-globin locus lead to excess precipitation of β- or α-globin, respectively, and compromised red blood cell (RBC) survival. The condition is characterized by ineffective erythropoiesis and peripheral hemolysis, with resultant anemia. Adenosine triphosphate (ATP) supply appears to be insufficient in thalassemic RBCs to maintain RBC membrane fitness and clearance of globin precipitates. Mitapivat (AG-348) is an oral, small-molecule, allosteric activator of the RBC-specific form of pyruvate kinase (PK-R). PK-R is a key enzyme for maintaining energy homeostasis in RBCs, as they rely almost exclusively on the process of glycolysis to generate ATP. In healthy adults, mitapivat activates wild-type PK-R and increases ATP levels in RBCs. In adults with PK deficiency who were not regularly transfused, oral mitapivat was well tolerated and induced rapid, durable hemoglobin (Hb) increases (NCT02476916). In the Hbbth/3+ mouse model of β-thalassemia, mitapivat increased ATP levels; reduced markers of ineffective erythropoiesis; and improved anemia, RBC survival, and indices of iron overload. These data support the hypothesis that increased ATP synthesis mediated via PK-R activation by mitapivat may improve the survival of thalassemic RBCs in the bone marrow and/or peripheral circulation, and thus represents a novel mechanism to treat patients with thalassemia. Here we present the design of a phase 2, open-label study designed to test this hypothesis and assess the proof of concept of mitapivat in patients with thalassemia. Methods: This phase 2, multicenter, open-label study (NCT03692052) is evaluating the efficacy, safety, pharmacokinetics, and pharmacodynamics of mitapivat in adults with non-transfusion-dependent thalassemia (NTDT). Four sites in North America and the United Kingdom are enrolling patients. The study consists of a 24-week core period followed by a 2-year extension period (Figure). Approximately 17 subjects with NTDT, i.e., β-thalassemia with or without α-globin gene mutations, HbE β-thalassemia, or α-thalassemia (HbH disease), will be enrolled. Non-transfusion dependence is defined as ≤5 units of RBCs transfused in the preceding 24 weeks and no transfusions in the 8 weeks prior to the first day of study drug. The hemoglobin inclusion criterion is ≤10.0 g/dL, which was increased from ≤9.0 g/dL in a recent protocol amendment to better reflect the clinical representation in the NTDT patient population. All eligible patients will receive an initial mitapivat dose of 50 mg twice daily (BID). At the week 6 visit the dose may be increased to 100 mg BID, depending on safety and Hb response. The primary endpoint is the proportion of subjects who achieve an Hb response, defined as an increase in Hb of ≥1.0 g/dL from baseline at any time between week 4 and week 12 (inclusive). Key secondary and exploratory endpoints include changes in Hb and markers of hemolysis; hematopoietic activity; iron metabolism and iron overload; and assessments of safety and pharmacokinetics. The study is currently enrolling. Figure Disclosures Kuo: Agios: Consultancy; Alexion: Consultancy, Honoraria; Apellis: Consultancy; Bioverativ: Other: Data Safety Monitoring Board; Bluebird Bio: Consultancy; Celgene: Consultancy; Novartis: Consultancy, Honoraria; Pfizer: Consultancy. Layton:Cerus Corporation: Membership on an entity's Board of Directors or advisory committees; Agios: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees. Uhlig:Agios: Employment, Equity Ownership. Lynch:Agios: Employment, Equity Ownership. Liu:Agios: Employment, Equity Ownership. Vichinsky:GBT: Consultancy, Research Funding; Pfizer: Consultancy, Research Funding; Agios: Consultancy, Research Funding; bluebird bio: Consultancy, Research Funding; Novartis: Consultancy, Research Funding.

A Novel Sandwich ELISA to Quantify Erythroferrone in Mouse Serum

Erythroferrone (ERFE) is a hormone produced by erythroblasts in response to erythropoietin. ERFE acts as a regulator of hepcidin expression during stress erythropoiesis. By suppressing hepcidin expression, ERFE contributes to the mobilization of dietary and stored iron necessary for recovery from blood loss after hemorrhage. Furthermore, overproduction of ERFE plays a pathogenic role in β-thalassemia and other anemias with ineffective erythropoiesis, where it contributes to hepcidin suppression and consequent iron overload. Development of a method to quantify serum ERFE in mice would improve our ability to study the pathobiology of this erythroid regulator. A dual polyclonal sandwich ELISA was developed to quantify ERFE in mouse serum. Purified recombinant mouse ERFE was used to immunize rabbits and goats, and high titer antibodies were purified from serum via protein A. Western blotting of reduced ERFE protein demonstrated that both the capture and detection antibody specifically recognized mouse ERFE, weakly recognized recombinant human ERFE, but neither antibody recognized mouse, rabbit or human TNF-alpha. Antibody was biotinylated and screened to determine the optimal antibody pairs. ELISA optimization established the standard curve range from 0 to 4 ng/ml. With a 10% sample dilution the lower limit of quantitation (LLOQ) was 0.1 ng/ml. Average spike recovery of ERFE in 3 different mouse sera (0.75 - 24 ng/ml) ranged from 93-105% (mean 99%). Dilutional linearity of the same spiked samples ranged from 93-104% (mean 99%). Intra- and inter-assay precision was 4.9% and 3.9%, respectively, over a concentration range of 0.57 - 16.3ng/ml. The effect of phlebotomy on serum ERFE in 6- and 8-week-old male C57BL/6 mice (n=3 each) was examined 24 h after removal of 0.5 ml of blood (Figure 1). At time 0, all the mice had serum ERFE levels below the limit of detection, but serum ERFE had increased to a mean of 1.9 ng/ml at 24 hours (P=0.03). This increase in ERFE is associated with a decrease in hepcidin from 209 ng/ml to 104 ng/ml (p&lt;0.0001). In a mouse model of β-Thalassemia (th3/+) there was significantly greater ERFE (1.2 ng/ml, n=5) compared to wild type (wt, &lt;0.1 ng/ml, n=3, p &lt; 0.04). Crossing the th3/+ mice with transferrin receptor 1 (TfR1) heterozygous mice (th3/+ TfR1+/-) produced a partial rescue of ERFE (0.6 ng/ml, n=5). Erfe knockout mice (th3/+ erfe-/- and th3/+ TfR1+/- erfe-/-) produced significantly less ERFE (p&lt;0.02) than their respective single and double mutants (&lt;0.1 ng/ml [n=7] and &lt;0.1 ng/ml [n=4], respectively) (Figure 2). Collectively, this validated ELISA can quantify mouse serum ERFE in both healthy and diseased mouse models and can be used to study the pathobiology of this erythroid regulator in diseases associated with ineffective erythropoiesis. Disclosures Gutschow: Intrinsic LifeSciences: Employment, Equity Ownership. Han:Intrinsic LifeSciences: Employment, Equity Ownership. Olbina:Intrinsic LifeSciences: Employment, Equity Ownership. Westerman:Intrinsic LifeSciences: Employment, Equity Ownership. Westerman:Intrinsic LifeSciences: Employment, Equity Ownership. Ginzburg:La Jolla Pharma: Membership on an entity's Board of Directors or advisory committees. Nemeth:Silarus Therapeutics: Consultancy, Equity Ownership; Ionis Pharmaceuticals: Consultancy; Protagonist: Consultancy; La Jolla Pharma: Consultancy; Intrinsic LifeSciences: Consultancy, Equity Ownership; Keryx: Consultancy. Ganz:Intrinsic LifeSciences: Consultancy, Equity Ownership. Ostland:Intrinsic LifeSciences: Employment, Equity Ownership.