THE PYRUVATE KINASE ACTIVATOR AG-348 IMPROVES MURINE ?-THALASSEMIC ANEMIA AND CORRECTS INEFFECTIVE ERYTHROPOIESIS
(Abstract release date: 05/19/16)
EHA Library. De Franceschi L. 06/10/16; 135168; S135
Prof. Lucia De Franceschi
Contributions
Contributions
Abstract
Abstract: S135
Type: Oral Presentation
Presentation during EHA21: On Friday, June 10, 2016 from 12:15 - 12:30
Location: Hall C15
Background
β-thalassemias (β-thal) are worldwide distributed red cell disorders, characterized by ineffective erythropoiesis and reduced red blood cell (RBC) lifespan. Increased levels of reactive oxygen species (ROS) have been reported to play a key role in anemia of β thal, targeting both erythropoiesis and circulating RBCs. Pyruvate kinase (PK) is an important enzyme in the glycolytic pathway, responsible for conversion of phosphoenolpyruvate to pyruvate, with concomitant formation of the energy carrier adenosine triphosphate (ATP). As mature erythrocytes lack mitochondria, they rely almost exclusively on glycolysis to generate ATP, as well as the interlinked pentose phosphate pathway shunt to generate the reducing agent NADPH. The possible impact of PK activity on erythropoiesis is supported by evidence of ineffective erythropoiesis in human subjects with PK deficiency as well as mouse models of PK deficiency. The PK activator AG-348 has been evaluated in Phase I trials in healthy human subjects (NCT02149966) and is currently in Phase II studies in PK deficiency patients (NCT02476916). In the Phase I studies, AG-348 was shown to decrease levels of an upstream metabolite 2,3-diphosphoglycerate (2,3-DPG) and increased levels of ATP in whole blood, consistent with in vivo activation of PK.
Aims
To evaluate the impact of AG-348 on anemia and ineffective erythropoiesis in a mouse model of β thal intermedia.
Methods
Mouse strains C57B6/2J, as wildtype (WT) controls, and Hbbth3/+, as a mouse model of β-thal intermedia, were used. Female mice aged between 2-3 months were treated with either vehicle or AG-348 at 50 mg/kg bid by oral gavage. Hematologic parameters, RBC indices, morphology, and reticulocyte count were evaluated at baseline, 7, 14, 21 days of treatment. Mouse erythropoiesis was studied using the CD44/TER119 gating strategy by FACS. ROS levels and the amount of Annexin-V+ cells were evaluated in erythroblast populations. Liver iron accumulation was evaluated by Pearl’s staining and expression of liver hepcidin was measured by RT-PCR.
Results
In Hbb3th/+ mice, 21 days of AG-348 treatment was associated with (i) a marked amelioration of anisopoykylocytosis; (ii) significantly increased Hb levels, MCV and MCH; (iii) a significant reduction in circulating erythroblasts (Es) and reticulocyte count; and (iv) reduction of ROS levels in circulating RBCs. In addition, AG-348 significantly decreased the amount of membrane precipitated α-globin chains and increased the amount of soluble Hb compared to the vehicle treated Hbb3th/+ group. Consistent with these findings, we observed (i) a reduction of extramedullar erythropoiesis as indicated by both a decrease in spleen weight/mouse weight ratio and total Es (CD44TER119 Fsc high cells); (ii) a significant increase in pro-Es and basophilic Es, associated with reduction in orthochromatic Es; (iii) a reduction in ROS levels of Hbb3th/+ Es and the amount of apoptotic orthochromatic Es compared to vehicle treated Hbb3th/+ mice, suggesting an amelioration of β thal ineffective erythropoiesis.. The amelioration of ineffective erythropoiesis was paralleled by a reduction in liver iron overload and up-regulation of hepcidin mRNA in liver from AG-348 treated Hbb3th/+ mice.
Conclusion
Our data show that the PK activator AG-348 beneficially affects ineffective erythropoiesis in a mouse model of β thal and might represent a novel therapeutic tool in clinical management of anemia in β thalassemic syndromes.
Session topic: Red blood cells and iron
Keyword(s): Red blood cell, Thalassemia
Type: Oral Presentation
Presentation during EHA21: On Friday, June 10, 2016 from 12:15 - 12:30
Location: Hall C15
Background
β-thalassemias (β-thal) are worldwide distributed red cell disorders, characterized by ineffective erythropoiesis and reduced red blood cell (RBC) lifespan. Increased levels of reactive oxygen species (ROS) have been reported to play a key role in anemia of β thal, targeting both erythropoiesis and circulating RBCs. Pyruvate kinase (PK) is an important enzyme in the glycolytic pathway, responsible for conversion of phosphoenolpyruvate to pyruvate, with concomitant formation of the energy carrier adenosine triphosphate (ATP). As mature erythrocytes lack mitochondria, they rely almost exclusively on glycolysis to generate ATP, as well as the interlinked pentose phosphate pathway shunt to generate the reducing agent NADPH. The possible impact of PK activity on erythropoiesis is supported by evidence of ineffective erythropoiesis in human subjects with PK deficiency as well as mouse models of PK deficiency. The PK activator AG-348 has been evaluated in Phase I trials in healthy human subjects (NCT02149966) and is currently in Phase II studies in PK deficiency patients (NCT02476916). In the Phase I studies, AG-348 was shown to decrease levels of an upstream metabolite 2,3-diphosphoglycerate (2,3-DPG) and increased levels of ATP in whole blood, consistent with in vivo activation of PK.
Aims
To evaluate the impact of AG-348 on anemia and ineffective erythropoiesis in a mouse model of β thal intermedia.
Methods
Mouse strains C57B6/2J, as wildtype (WT) controls, and Hbbth3/+, as a mouse model of β-thal intermedia, were used. Female mice aged between 2-3 months were treated with either vehicle or AG-348 at 50 mg/kg bid by oral gavage. Hematologic parameters, RBC indices, morphology, and reticulocyte count were evaluated at baseline, 7, 14, 21 days of treatment. Mouse erythropoiesis was studied using the CD44/TER119 gating strategy by FACS. ROS levels and the amount of Annexin-V+ cells were evaluated in erythroblast populations. Liver iron accumulation was evaluated by Pearl’s staining and expression of liver hepcidin was measured by RT-PCR.
Results
In Hbb3th/+ mice, 21 days of AG-348 treatment was associated with (i) a marked amelioration of anisopoykylocytosis; (ii) significantly increased Hb levels, MCV and MCH; (iii) a significant reduction in circulating erythroblasts (Es) and reticulocyte count; and (iv) reduction of ROS levels in circulating RBCs. In addition, AG-348 significantly decreased the amount of membrane precipitated α-globin chains and increased the amount of soluble Hb compared to the vehicle treated Hbb3th/+ group. Consistent with these findings, we observed (i) a reduction of extramedullar erythropoiesis as indicated by both a decrease in spleen weight/mouse weight ratio and total Es (CD44TER119 Fsc high cells); (ii) a significant increase in pro-Es and basophilic Es, associated with reduction in orthochromatic Es; (iii) a reduction in ROS levels of Hbb3th/+ Es and the amount of apoptotic orthochromatic Es compared to vehicle treated Hbb3th/+ mice, suggesting an amelioration of β thal ineffective erythropoiesis.. The amelioration of ineffective erythropoiesis was paralleled by a reduction in liver iron overload and up-regulation of hepcidin mRNA in liver from AG-348 treated Hbb3th/+ mice.
Conclusion
Our data show that the PK activator AG-348 beneficially affects ineffective erythropoiesis in a mouse model of β thal and might represent a novel therapeutic tool in clinical management of anemia in β thalassemic syndromes.
Session topic: Red blood cells and iron
Keyword(s): Red blood cell, Thalassemia
Abstract: S135
Type: Oral Presentation
Presentation during EHA21: On Friday, June 10, 2016 from 12:15 - 12:30
Location: Hall C15
Background
β-thalassemias (β-thal) are worldwide distributed red cell disorders, characterized by ineffective erythropoiesis and reduced red blood cell (RBC) lifespan. Increased levels of reactive oxygen species (ROS) have been reported to play a key role in anemia of β thal, targeting both erythropoiesis and circulating RBCs. Pyruvate kinase (PK) is an important enzyme in the glycolytic pathway, responsible for conversion of phosphoenolpyruvate to pyruvate, with concomitant formation of the energy carrier adenosine triphosphate (ATP). As mature erythrocytes lack mitochondria, they rely almost exclusively on glycolysis to generate ATP, as well as the interlinked pentose phosphate pathway shunt to generate the reducing agent NADPH. The possible impact of PK activity on erythropoiesis is supported by evidence of ineffective erythropoiesis in human subjects with PK deficiency as well as mouse models of PK deficiency. The PK activator AG-348 has been evaluated in Phase I trials in healthy human subjects (NCT02149966) and is currently in Phase II studies in PK deficiency patients (NCT02476916). In the Phase I studies, AG-348 was shown to decrease levels of an upstream metabolite 2,3-diphosphoglycerate (2,3-DPG) and increased levels of ATP in whole blood, consistent with in vivo activation of PK.
Aims
To evaluate the impact of AG-348 on anemia and ineffective erythropoiesis in a mouse model of β thal intermedia.
Methods
Mouse strains C57B6/2J, as wildtype (WT) controls, and Hbbth3/+, as a mouse model of β-thal intermedia, were used. Female mice aged between 2-3 months were treated with either vehicle or AG-348 at 50 mg/kg bid by oral gavage. Hematologic parameters, RBC indices, morphology, and reticulocyte count were evaluated at baseline, 7, 14, 21 days of treatment. Mouse erythropoiesis was studied using the CD44/TER119 gating strategy by FACS. ROS levels and the amount of Annexin-V+ cells were evaluated in erythroblast populations. Liver iron accumulation was evaluated by Pearl’s staining and expression of liver hepcidin was measured by RT-PCR.
Results
In Hbb3th/+ mice, 21 days of AG-348 treatment was associated with (i) a marked amelioration of anisopoykylocytosis; (ii) significantly increased Hb levels, MCV and MCH; (iii) a significant reduction in circulating erythroblasts (Es) and reticulocyte count; and (iv) reduction of ROS levels in circulating RBCs. In addition, AG-348 significantly decreased the amount of membrane precipitated α-globin chains and increased the amount of soluble Hb compared to the vehicle treated Hbb3th/+ group. Consistent with these findings, we observed (i) a reduction of extramedullar erythropoiesis as indicated by both a decrease in spleen weight/mouse weight ratio and total Es (CD44TER119 Fsc high cells); (ii) a significant increase in pro-Es and basophilic Es, associated with reduction in orthochromatic Es; (iii) a reduction in ROS levels of Hbb3th/+ Es and the amount of apoptotic orthochromatic Es compared to vehicle treated Hbb3th/+ mice, suggesting an amelioration of β thal ineffective erythropoiesis.. The amelioration of ineffective erythropoiesis was paralleled by a reduction in liver iron overload and up-regulation of hepcidin mRNA in liver from AG-348 treated Hbb3th/+ mice.
Conclusion
Our data show that the PK activator AG-348 beneficially affects ineffective erythropoiesis in a mouse model of β thal and might represent a novel therapeutic tool in clinical management of anemia in β thalassemic syndromes.
Session topic: Red blood cells and iron
Keyword(s): Red blood cell, Thalassemia
Type: Oral Presentation
Presentation during EHA21: On Friday, June 10, 2016 from 12:15 - 12:30
Location: Hall C15
Background
β-thalassemias (β-thal) are worldwide distributed red cell disorders, characterized by ineffective erythropoiesis and reduced red blood cell (RBC) lifespan. Increased levels of reactive oxygen species (ROS) have been reported to play a key role in anemia of β thal, targeting both erythropoiesis and circulating RBCs. Pyruvate kinase (PK) is an important enzyme in the glycolytic pathway, responsible for conversion of phosphoenolpyruvate to pyruvate, with concomitant formation of the energy carrier adenosine triphosphate (ATP). As mature erythrocytes lack mitochondria, they rely almost exclusively on glycolysis to generate ATP, as well as the interlinked pentose phosphate pathway shunt to generate the reducing agent NADPH. The possible impact of PK activity on erythropoiesis is supported by evidence of ineffective erythropoiesis in human subjects with PK deficiency as well as mouse models of PK deficiency. The PK activator AG-348 has been evaluated in Phase I trials in healthy human subjects (NCT02149966) and is currently in Phase II studies in PK deficiency patients (NCT02476916). In the Phase I studies, AG-348 was shown to decrease levels of an upstream metabolite 2,3-diphosphoglycerate (2,3-DPG) and increased levels of ATP in whole blood, consistent with in vivo activation of PK.
Aims
To evaluate the impact of AG-348 on anemia and ineffective erythropoiesis in a mouse model of β thal intermedia.
Methods
Mouse strains C57B6/2J, as wildtype (WT) controls, and Hbbth3/+, as a mouse model of β-thal intermedia, were used. Female mice aged between 2-3 months were treated with either vehicle or AG-348 at 50 mg/kg bid by oral gavage. Hematologic parameters, RBC indices, morphology, and reticulocyte count were evaluated at baseline, 7, 14, 21 days of treatment. Mouse erythropoiesis was studied using the CD44/TER119 gating strategy by FACS. ROS levels and the amount of Annexin-V+ cells were evaluated in erythroblast populations. Liver iron accumulation was evaluated by Pearl’s staining and expression of liver hepcidin was measured by RT-PCR.
Results
In Hbb3th/+ mice, 21 days of AG-348 treatment was associated with (i) a marked amelioration of anisopoykylocytosis; (ii) significantly increased Hb levels, MCV and MCH; (iii) a significant reduction in circulating erythroblasts (Es) and reticulocyte count; and (iv) reduction of ROS levels in circulating RBCs. In addition, AG-348 significantly decreased the amount of membrane precipitated α-globin chains and increased the amount of soluble Hb compared to the vehicle treated Hbb3th/+ group. Consistent with these findings, we observed (i) a reduction of extramedullar erythropoiesis as indicated by both a decrease in spleen weight/mouse weight ratio and total Es (CD44TER119 Fsc high cells); (ii) a significant increase in pro-Es and basophilic Es, associated with reduction in orthochromatic Es; (iii) a reduction in ROS levels of Hbb3th/+ Es and the amount of apoptotic orthochromatic Es compared to vehicle treated Hbb3th/+ mice, suggesting an amelioration of β thal ineffective erythropoiesis.. The amelioration of ineffective erythropoiesis was paralleled by a reduction in liver iron overload and up-regulation of hepcidin mRNA in liver from AG-348 treated Hbb3th/+ mice.
Conclusion
Our data show that the PK activator AG-348 beneficially affects ineffective erythropoiesis in a mouse model of β thal and might represent a novel therapeutic tool in clinical management of anemia in β thalassemic syndromes.
Session topic: Red blood cells and iron
Keyword(s): Red blood cell, Thalassemia
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