A Selective ORAL GLYT1 Inhibitor, Improves Anemia and RED CELL Survival in a MOUSE MODEL of Beta-Thalassemia

The unbalanced hemoglobin chain synthesis in beta-thalassemias leads to hemichrome-induced damage that contributes to ineffective erythropoiesis, hemolysis and reduced red cell survival. Iron overload related to ineffective erythropoiesis and abnormally low Hepcidin (Hamp), combined with the cytotoxic effects of free heme with free-alpha-chains play a key role in the increased generation of reactive oxygen species (ROS) in beta thalassemias.[2][HA{3] Here we used a specific and selective inhibitor of the plasma membrane expressed glycine transporter GlyT1 (Ro4917838). Use of Ro4917838 has been associated with a dose-dependent decrease in MCH, Hb, soluble transferrin receptor, and increase in absolute reticulocytes and RBC counts in several animal species, attributed to reduce glycine bioavailability in erythroblasts and decreased heme synthesis. In rats, Ro4917838 reduces heme synthesis, and down-regulates erythroid transferrin receptor, but does not interfere with hepcidin regulation and systemic iron homeostasis (Winter et al. Exp Hematol, DOI: 10.1016/j.exphem.2016.07.003). We aimed to determine if reduced cellular availability of glycine in erythroblasts may reduce heme synthesis, and impact pathologic erythropoiesis in a mouse model for b-thalassemia. Wild-type control (WT) C57B6/2J, and beta-thalassemia Hbbth3/+ mice were treated with either vehicle or Ro4917838 at dosages of 3, 10, 30 mg/kg/d administered over 4 weeks once daily by gavage. [6]RO4917838 administration was associated with significant improvements of central hallmarks of the b-thalassemia pathology. Reduced erythrocyte destruction was seen bydemonstrated by [8][WM{9]as significant improvements in erythrocyte morphology and amelioration of hemoglobin reduction in reticulocytes. We also observed an impressively quick reduction of the circulating erythroblast count within 1 week of initiating treatment. This was also associated with decreased hemolysis biomarkers. Ro4917838 induced a significant reduction in extramedullary erythropoiesis and reduction in orthochromatic erythroblasts as well as insoluble alpha chain aggregates in circulating red cells. Red cell survival of b-thal mice treated with 30 mg/kg/day Ro4917838 significantly increased by more than 50%. CD71+ erythroid precursors significantly decreased in WT mice treated with Ro4917838 at 30 mg/kg and in b-thal mice at the dosage of 30 mg/kg/ d. [10]These data suggest that Ro4917838 ameliorates anemia in a b-thalassemia mouse model and positively affects ineffective erythropoiesis and red cell survival in peripheral circulation. Ro4917838 may represent a novel therapeutic approach for the treatment of anemia in b-thalassemia patients. [1] I do see the point for beta-thalassemia, but this does not apply for all thalassemias, I mean the free alpha chains. The ROS yes, they have in common. In this sentence it seems to be that the alpha chains are culprit for ROS in all thalassemias. [HA{3]I do see the point for beta-thalassemia, but this does not apply for all thalassemias, I mean the free alpha chains. The ROS yes, they have in common. In this sentence it seems to be that the alpha chains are culprit for ROS in all thalassemias. ROS was not measured, therefore I would refrain mentioning it. No data is shown. [HA{5]ROS was not measured, therefore I would refrain mentioning it. No data is shown. I propose deletion of the PK info. Although the information is correct I don't think this is the level of detail needed for the abstract. It may even be confusing as it is not the focus of the abstract to do a cross species comparison. [AK7]I propose deletion of the PK info. Although the information is correct I don't think this is the level of detail needed for the abstract. It may even be confusing as it is not the focus of the abstract to do a cross species comparison. Dosing and dosing frequency is already described above, we do not need to repeat this here. [WM{9]Dosing and dosing frequency is already described above, we do not need to repeat this here. Was the liver expression determined or the serum Hepcidin levels measured? I would rephrase it if latter is the case. [HA{11]Was the liver expression determined or the serum Hepcidin levels measured? I would rephrase it if latter is the case. Disclosures Brugnara: F. Hoffmann-La Roche Ltd, Basel, Switzerland: Consultancy. Harmeier:F. Hoffmann-La Roche Ltd, Basel, Switzerland: Employment. De Franceschi:F. Hoffmann-La Roche Ltd, Basel, Switzerland: Research Funding. Matte:F. Hoffmann-La Roche Ltd, Basel, Switzerland: Research Funding. Winter:F. Hoffmann-La Roche Ltd, Basel, Switzerland: Employment. Koerner:F. Hoffmann-La Roche Ltd, Basel, Switzerland: Employment. Khwaja:F. Hoffmann-La Roche Ltd, Basel, Switzerland: Employment. Singer:F. Hoffmann-La Roche Ltd, Basel, Switzerland: Employment. Beneduce:Roche Innovation Center Basel, F. Hoffmann-La Roche Ltd, Basel, Switzerland: Research Funding. Federti:Roche Innovation Center Basel, F. Hoffmann-La Roche Ltd, Basel, Switzerland: Research Funding. Siciliano:Roche Innovation Center Basel, F. Hoffmann-La Roche Ltd, Basel, Switzerland: Research Funding.

Ineffective Erythropoiesis Is the Major Cause of Microcytic Anemia in the TSAP6/Steap3 Null Mouse Model

STEAP3 (Six-Transmembrane Epithelial Antigen of Prostate 3) is the major ferrireductase in the erythroblast. Also named TSAP6 (Tumor Supressor Activated Pathway 6) after it had been found to play a role in cancer, its total ablation in the mouse leads to severe microcytic and hypochromic red cells with moderate anemia. The protein function appears conserved among mammals, as patients carrying a nonsense mutation in the TSAP6/STEAP3 gene have been reported with hypochromic anemia. Here, we investigated the mechanism leading to the anemia. In the present study, using the TSAP6/Steap3 knockout mice, we undertook a comprehensive hematologic characterization of the red cell compartment. Red cell indices derived using ADVIA 120 blood counter confirmed the hypochromic microcytic anemia phenotype with a marked reduction in the mean corpuscular volume (MCV; 21.5fL ± 1.3fL in knockout vs 45.2fL ± 1.5fL in wild-type X ± SD, p<0.001) and mean cell hemoglobin content (MCH; 5.8pg ± 0.1pg vs 14.5pg ± 0.1pg, p<0.001). The reticulocyte count was marginally elevated (5.7% ± 1.2% vs 2.6% ± 0.4%, p<0.05) indicating that the anemia is proliferative. By phase contrast microscopy and transmission electron microscopy, we observed marked anisocytosis as well as the presence of fragmenting erythrocytes. Consistent with this observation, we found by ektacytometry decreased membrane mechanical stability of knockout red cells. Interestingly, we were unable to document significant changes in the expression levels of the major skeletal and transmembrane proteins to account for this decrease in the membrane stability. As defects in either the production or destruction of red cells can lead to anemia, we measured red cell survival and erythropoiesis in these mice. No differences in red cell survival could be documented using biotin labeled red cells implying that decreased survival cannot account for the anemia. However, when we monitored erythropoiesis using Ter119, CD44 and Forward Scatter (FSC) as markers of terminal differentiation, we found a decreased number of proerythroblasts in the bone marrow of TSAP6/Steap3-/- animals (2.20% ± 0.49% vs 4.07% ± 0.77%, p<0.01). In addition, progression from the proerythroblastic to the orthochromatic stage was also affected, with accumulation of cells at the polychromatic stage. These findings imply that ineffective erythropoiesis is the dominant cause of anemia in these mice and that TSAP6/Steap3 may play a role during erythropoiesis beyond its role as ferrireductase. It is interesting to note that while the phenotype of microcytic red cells with decreased hemoglobin content in the TSAP6/Steap3 null mouse is similar to red cells in thalassemic and porphyria mice, there are distinct differences in the etiology of the hematologic phenotype. Disclosures No relevant conflicts of interest to declare.

Hydroxyurea up-Regulates Voltage-Dependent Anion Channels in Human Sickle Cell Erythrocytes.

Abstract 2119 Background: Hydroxyurea (HU) is clinically effective in reducing the frequency of pain crises in adults and children with sickle cell disease (SCD). In addition to increased fetal hemoglobin production, HU is known to induce macrocytosis in sickle cell erythrocytes and prolong red cell survival. However, the precise mechanism by which HU produces its varied effects is unknown. Isoelectric focusing has been applied to characterize changes in the red cell membrane following exposure to HU, but this technique has limitations in its ability to identify differences in specific membrane proteins. Mass spectrometry (MS) provides another proteomic approach in analyzing red cell proteins in SCD. Methods: Red blood cells were obtained from patients with SCD on HU therapy. After multiple wash and lysis steps, ghost membranes were obtained following ultracentrifugation. The membrane samples were then analyzed via tandem mass spectrometry and western immunoblot assay. Results: In comparison to a normal control, patients with SCD on HU therapy were found to have significantly elevated levels of voltage-dependent anion channels (VDAC) by MS, using a false discovery rate of less than five percent. The application of a stringent threshold initially identified 346 protein candidates. Out of these 346 proteins, 125 contained multiple peptides which all passed quantitation data filters according to quality parameters. Within the set of 125 proteins, 4 proteins were significantly up-regulated (up to 50-fold) in patients with SCD on HU therapy. VDAC2 and VDAC3 are members of the eukaryotic mitochondrial porin family which form channels through the mitochondrial outer membrane allowing diffusion of small hydrophilic molecules. VDAC1 can be found in both the mitochondrial outer membrane and the plasma membrane, where it is involved in cell volume regulation and apoptosis. Conclusions: Up-regulation of VDAC proteins may play an important role in altering the intracellular osmotic composition of the sickle cell erythrocyte resulting in decreased sickling and improved red cell survival in patients with SCD on HU therapy. Disclosures: No relevant conflicts of interest to declare.

Metabolomic Evaluation of Normal, In Vivo, Red Cell Aging

Abstract 2035 Red cell lifespan follows a well-defined, species-specific trajectory approaching 120 days in humans and 50 days in the mouse. At the end of this period, ‘senescent’ red cells are removed from the circulation by phagocytes, upon recognition of a surface change, or changes in the physical properties of the aged RBC. Because mature red cells have no capacity for synthesis of new proteins, and have little ability to repair damage—limitations on red cell survival are likely to relate to functional decline secondary to accumulation of damage to cell constituents. Among key targets are glycolytic enzymes, proteins involved in redox metabolism, and structural proteins, as inherited defects in these proteins can shorten red cell survival and cause hemolytic anemia. In order to further evaluate metabolic changes in normal RBC over the course of RBC lifespan, we used an in vivo biotinylation method followed by magnetic bead separation to isolate murine red cells of defined age at intervals. Purified RBC were assessed for protein oxidation, reactive oxygen species production, surface antigen profile, and the abundance of metabolic intermediates using a multiplatform mass spectrometry approach. 167 metabolites were detected in RBC extracts collected at 8, 15 and 35 days after labeling, using 6 biologic replicates for each time point. Multiple metabolites showed statistically significant changes in abundance as a function of red cell age. The figure below presents representative examples of patterns of change observed—where each line corresponds to either the biotin – (younger) or the biotin + (older) fraction. Panel A was the most common pattern observed—where a steep initial decline indicates a metabolite that may be specific for reticulocytes, found at high concentration only in the youngest cell fraction. Panel B indicates a metabolite that didn't change, panel C is an example of a metabolite with a more linear pattern of decay, and panel D is an example of a metabolite that increased with increasing red cell age. Older cells had higher levels of glucose, and lower levels of downstream metabolites from both the glycolytic and pentose phosphate pathways—consistent with a decline in hexokinase activity when comparing young and old red cells. Older cells had evidence of cumulative oxidative damage as demonstrated by increased protein carbonyl content, and had higher levels of reactive oxygen species. Surprisingly, despite this evidence of oxidative stress, reduced glutathione levels increased with cell age, and content of oxidized glutathione was unchanged. However, accumulation of alternate products of red cell gamma glutamylcysteine synthetase suggests substrate deprivation. The pattern of glutathione-related metabolites we observed in aged red cells has been described in tissues subjected to acute oxidant stress. The observed changes in metabolite concentrations support the hypothesis that metabolic decline during red cell aging impairs energy production, generation of reducing equivalents, and capacity to reverse protein oxidation. How (or whether) these changes in metabolic equilibrium are converted into a signal that results in removal of senescent red cells from the circulation remains speculative. However, we observe red cell age-dependent changes in additional metabolites, including specific lipids, that may help to bridge this gap in understanding. Disclosures: No relevant conflicts of interest to declare.