RAP-536 (MURINE ANALOG OF ACE-536/LUSPATERCEPT) INHIBITS SMAD2/3 SIGNALING AND PROMOTES ERYTHROID DIFFERENTIATION BY RESTORING GATA-1 FUNCTION IN A MURINE MODEL OF ?-THALASSEMIA
(Abstract release date: 05/19/16)
EHA Library. Martinez P. 06/10/16; 135169; S136
Dr. Pedro Martinez
Contributions
Contributions
Abstract
Abstract: S136
Type: Oral Presentation
Presentation during EHA21: On Friday, June 10, 2016 from 12:30 - 12:45
Location: Hall C15
Background
Previously, we reported elevated Smad2/3 signaling in diseases characterized by ineffective erythropoiesis such as myelodysplastic syndromes (MDS) and β-thalassemia (Suragani et al. 2014). Luspatercept (modified ActRIIB receptor-Fc fusion protein), a Smad 2/3 ligand trap, has demonstrated efficacy in correcting ineffective erythropoiesis and the resulting anemia in murine models of MDS and β-thalassemia. RAP-536 (murine version of ACE-536/luspatercept) treatment also alleviated disease pathology in β-thalassemic mice.
Aims
In this study, we investigated the molecular mechanism of action of RAP-536 in a murine model of β-thalassemia.
Methods
Wildtype and β-thalassemic mice were used in this study. β-thalassemic mice (Hbbth3/+) were administered a single bolus of vehicle (VEH) or RAP-536 (30 mg/kg, i.p) (N=2/group). At 16 hours, splenic basophilic erythroblasts (CD71+Ter119+FSChigh) were sorted by flow cytometry. RNA was isolated and subjected to genome wide transcriptome profiling using RNA sequencing analysis.Mouse erythroid leukemic (MEL) cells, primary fetal liver erythroid and β-thalassemic erythroid precursors were treated with GDF11 in the presence or absence of ACE-536 as described below.
Results
Transcriptome analysis of β-thalassemic erythroblasts identified 74 genes that were differentially expressed (absolute fold change >1.5, false discovery rate adjusted P value <0.05) in RAP-536 treated samples vs. VEH treatment. To identify molecular mechanisms, we performed gene set enrichment analysis (GSEA) (Subramanian et al., 2005) on these samples. The analysis depicted significant upregulation of target genes of multiple transcriptional regulators including GATA-1. Previously, multiple studies have established GATA-1 as a master transcriptional regulator of terminal erythroid differentiation. Further GSEA of GATA-1 activator signatures against RAP-536 treatment data revealed a significant up-regulation of 158 activated genes (Normalized Enrichment Score=2.7, P=0) involved in heme biosynthesis (such as Ppox, Fech, and Abcb10) and terminal erythroid differentiation (such as Fog1, Klf1 and Bcl-xl).ACE-536 is known to bind and inhibit Smad2/3 ligands such as GDF8, GDF11 and activin B but not activin A (Suragani et al 2014). Consistent with this data, treatment of MEL and fetal liver erythroid cells with GDF11 (50ng/mL) induced Smad2/3 phosphorylation and ACE-536 co-treatment inhibited the increase in pSmad2/3. In differentiating erythroid cells, GDF11 treatment displayed reduced nuclear GATA-1 protein levels by both western blotting and immunofluorescence studies. Additionally, reactive oxygen species (ROS) levels indicative of oxidative stress were elevated in MEL and primary fetal liver cells following GDF11 treatment. Consistent with the increase in ROS, we found decreased mitochondrial transmembrane potential (Δψm) indicative of unhealthy cells and increased caspase 3/7 activity in erythroid cells treated with GDF11. Importantly, treatment of erythroid cells with ACE-536 and GDF11 decreased ROS, restored Δψm and GATA-1 levels to control levels. Immunofluorescence studies demonstrated decreased GATA-1 levels in the nucleus of erythroid precursors from b-thalassemic cells compared to wild type mice. Treatment of these b-thalassemic erythroid precursors with ACE-536 prevented the decrease in GATA-1 levels.
Conclusion
Together, these data done with RAP-536 provide a potential mechanistic role for luspatercept as a novel treatment of β-thalassemia. By inhibiting pSmad2/3 signaling, RAP-536 treatment decreases ROS, prevents caspase 3/7 activation and GATA-1 cleavage. Thus by restoring GATA-1 availability and functional activity, RAP-536 treatment causes upregulation of genes involved in promoting terminal erythroid maturation, and consequently corrects anemia in b-thalassemia. Luspatercept currently completed phase 2 clinical trials and has initiated phase 3 studies in patients with MDS and b-thalassemia.
Session topic: Red blood cells and iron
Keyword(s): Erythroid differentiation, GATA-1, Reactive oxygen species, Thalassemia
Type: Oral Presentation
Presentation during EHA21: On Friday, June 10, 2016 from 12:30 - 12:45
Location: Hall C15
Background
Previously, we reported elevated Smad2/3 signaling in diseases characterized by ineffective erythropoiesis such as myelodysplastic syndromes (MDS) and β-thalassemia (Suragani et al. 2014). Luspatercept (modified ActRIIB receptor-Fc fusion protein), a Smad 2/3 ligand trap, has demonstrated efficacy in correcting ineffective erythropoiesis and the resulting anemia in murine models of MDS and β-thalassemia. RAP-536 (murine version of ACE-536/luspatercept) treatment also alleviated disease pathology in β-thalassemic mice.
Aims
In this study, we investigated the molecular mechanism of action of RAP-536 in a murine model of β-thalassemia.
Methods
Wildtype and β-thalassemic mice were used in this study. β-thalassemic mice (Hbbth3/+) were administered a single bolus of vehicle (VEH) or RAP-536 (30 mg/kg, i.p) (N=2/group). At 16 hours, splenic basophilic erythroblasts (CD71+Ter119+FSChigh) were sorted by flow cytometry. RNA was isolated and subjected to genome wide transcriptome profiling using RNA sequencing analysis.Mouse erythroid leukemic (MEL) cells, primary fetal liver erythroid and β-thalassemic erythroid precursors were treated with GDF11 in the presence or absence of ACE-536 as described below.
Results
Transcriptome analysis of β-thalassemic erythroblasts identified 74 genes that were differentially expressed (absolute fold change >1.5, false discovery rate adjusted P value <0.05) in RAP-536 treated samples vs. VEH treatment. To identify molecular mechanisms, we performed gene set enrichment analysis (GSEA) (Subramanian et al., 2005) on these samples. The analysis depicted significant upregulation of target genes of multiple transcriptional regulators including GATA-1. Previously, multiple studies have established GATA-1 as a master transcriptional regulator of terminal erythroid differentiation. Further GSEA of GATA-1 activator signatures against RAP-536 treatment data revealed a significant up-regulation of 158 activated genes (Normalized Enrichment Score=2.7, P=0) involved in heme biosynthesis (such as Ppox, Fech, and Abcb10) and terminal erythroid differentiation (such as Fog1, Klf1 and Bcl-xl).ACE-536 is known to bind and inhibit Smad2/3 ligands such as GDF8, GDF11 and activin B but not activin A (Suragani et al 2014). Consistent with this data, treatment of MEL and fetal liver erythroid cells with GDF11 (50ng/mL) induced Smad2/3 phosphorylation and ACE-536 co-treatment inhibited the increase in pSmad2/3. In differentiating erythroid cells, GDF11 treatment displayed reduced nuclear GATA-1 protein levels by both western blotting and immunofluorescence studies. Additionally, reactive oxygen species (ROS) levels indicative of oxidative stress were elevated in MEL and primary fetal liver cells following GDF11 treatment. Consistent with the increase in ROS, we found decreased mitochondrial transmembrane potential (Δψm) indicative of unhealthy cells and increased caspase 3/7 activity in erythroid cells treated with GDF11. Importantly, treatment of erythroid cells with ACE-536 and GDF11 decreased ROS, restored Δψm and GATA-1 levels to control levels. Immunofluorescence studies demonstrated decreased GATA-1 levels in the nucleus of erythroid precursors from b-thalassemic cells compared to wild type mice. Treatment of these b-thalassemic erythroid precursors with ACE-536 prevented the decrease in GATA-1 levels.
Conclusion
Together, these data done with RAP-536 provide a potential mechanistic role for luspatercept as a novel treatment of β-thalassemia. By inhibiting pSmad2/3 signaling, RAP-536 treatment decreases ROS, prevents caspase 3/7 activation and GATA-1 cleavage. Thus by restoring GATA-1 availability and functional activity, RAP-536 treatment causes upregulation of genes involved in promoting terminal erythroid maturation, and consequently corrects anemia in b-thalassemia. Luspatercept currently completed phase 2 clinical trials and has initiated phase 3 studies in patients with MDS and b-thalassemia.
Session topic: Red blood cells and iron
Keyword(s): Erythroid differentiation, GATA-1, Reactive oxygen species, Thalassemia
Abstract: S136
Type: Oral Presentation
Presentation during EHA21: On Friday, June 10, 2016 from 12:30 - 12:45
Location: Hall C15
Background
Previously, we reported elevated Smad2/3 signaling in diseases characterized by ineffective erythropoiesis such as myelodysplastic syndromes (MDS) and β-thalassemia (Suragani et al. 2014). Luspatercept (modified ActRIIB receptor-Fc fusion protein), a Smad 2/3 ligand trap, has demonstrated efficacy in correcting ineffective erythropoiesis and the resulting anemia in murine models of MDS and β-thalassemia. RAP-536 (murine version of ACE-536/luspatercept) treatment also alleviated disease pathology in β-thalassemic mice.
Aims
In this study, we investigated the molecular mechanism of action of RAP-536 in a murine model of β-thalassemia.
Methods
Wildtype and β-thalassemic mice were used in this study. β-thalassemic mice (Hbbth3/+) were administered a single bolus of vehicle (VEH) or RAP-536 (30 mg/kg, i.p) (N=2/group). At 16 hours, splenic basophilic erythroblasts (CD71+Ter119+FSChigh) were sorted by flow cytometry. RNA was isolated and subjected to genome wide transcriptome profiling using RNA sequencing analysis.Mouse erythroid leukemic (MEL) cells, primary fetal liver erythroid and β-thalassemic erythroid precursors were treated with GDF11 in the presence or absence of ACE-536 as described below.
Results
Transcriptome analysis of β-thalassemic erythroblasts identified 74 genes that were differentially expressed (absolute fold change >1.5, false discovery rate adjusted P value <0.05) in RAP-536 treated samples vs. VEH treatment. To identify molecular mechanisms, we performed gene set enrichment analysis (GSEA) (Subramanian et al., 2005) on these samples. The analysis depicted significant upregulation of target genes of multiple transcriptional regulators including GATA-1. Previously, multiple studies have established GATA-1 as a master transcriptional regulator of terminal erythroid differentiation. Further GSEA of GATA-1 activator signatures against RAP-536 treatment data revealed a significant up-regulation of 158 activated genes (Normalized Enrichment Score=2.7, P=0) involved in heme biosynthesis (such as Ppox, Fech, and Abcb10) and terminal erythroid differentiation (such as Fog1, Klf1 and Bcl-xl).ACE-536 is known to bind and inhibit Smad2/3 ligands such as GDF8, GDF11 and activin B but not activin A (Suragani et al 2014). Consistent with this data, treatment of MEL and fetal liver erythroid cells with GDF11 (50ng/mL) induced Smad2/3 phosphorylation and ACE-536 co-treatment inhibited the increase in pSmad2/3. In differentiating erythroid cells, GDF11 treatment displayed reduced nuclear GATA-1 protein levels by both western blotting and immunofluorescence studies. Additionally, reactive oxygen species (ROS) levels indicative of oxidative stress were elevated in MEL and primary fetal liver cells following GDF11 treatment. Consistent with the increase in ROS, we found decreased mitochondrial transmembrane potential (Δψm) indicative of unhealthy cells and increased caspase 3/7 activity in erythroid cells treated with GDF11. Importantly, treatment of erythroid cells with ACE-536 and GDF11 decreased ROS, restored Δψm and GATA-1 levels to control levels. Immunofluorescence studies demonstrated decreased GATA-1 levels in the nucleus of erythroid precursors from b-thalassemic cells compared to wild type mice. Treatment of these b-thalassemic erythroid precursors with ACE-536 prevented the decrease in GATA-1 levels.
Conclusion
Together, these data done with RAP-536 provide a potential mechanistic role for luspatercept as a novel treatment of β-thalassemia. By inhibiting pSmad2/3 signaling, RAP-536 treatment decreases ROS, prevents caspase 3/7 activation and GATA-1 cleavage. Thus by restoring GATA-1 availability and functional activity, RAP-536 treatment causes upregulation of genes involved in promoting terminal erythroid maturation, and consequently corrects anemia in b-thalassemia. Luspatercept currently completed phase 2 clinical trials and has initiated phase 3 studies in patients with MDS and b-thalassemia.
Session topic: Red blood cells and iron
Keyword(s): Erythroid differentiation, GATA-1, Reactive oxygen species, Thalassemia
Type: Oral Presentation
Presentation during EHA21: On Friday, June 10, 2016 from 12:30 - 12:45
Location: Hall C15
Background
Previously, we reported elevated Smad2/3 signaling in diseases characterized by ineffective erythropoiesis such as myelodysplastic syndromes (MDS) and β-thalassemia (Suragani et al. 2014). Luspatercept (modified ActRIIB receptor-Fc fusion protein), a Smad 2/3 ligand trap, has demonstrated efficacy in correcting ineffective erythropoiesis and the resulting anemia in murine models of MDS and β-thalassemia. RAP-536 (murine version of ACE-536/luspatercept) treatment also alleviated disease pathology in β-thalassemic mice.
Aims
In this study, we investigated the molecular mechanism of action of RAP-536 in a murine model of β-thalassemia.
Methods
Wildtype and β-thalassemic mice were used in this study. β-thalassemic mice (Hbbth3/+) were administered a single bolus of vehicle (VEH) or RAP-536 (30 mg/kg, i.p) (N=2/group). At 16 hours, splenic basophilic erythroblasts (CD71+Ter119+FSChigh) were sorted by flow cytometry. RNA was isolated and subjected to genome wide transcriptome profiling using RNA sequencing analysis.Mouse erythroid leukemic (MEL) cells, primary fetal liver erythroid and β-thalassemic erythroid precursors were treated with GDF11 in the presence or absence of ACE-536 as described below.
Results
Transcriptome analysis of β-thalassemic erythroblasts identified 74 genes that were differentially expressed (absolute fold change >1.5, false discovery rate adjusted P value <0.05) in RAP-536 treated samples vs. VEH treatment. To identify molecular mechanisms, we performed gene set enrichment analysis (GSEA) (Subramanian et al., 2005) on these samples. The analysis depicted significant upregulation of target genes of multiple transcriptional regulators including GATA-1. Previously, multiple studies have established GATA-1 as a master transcriptional regulator of terminal erythroid differentiation. Further GSEA of GATA-1 activator signatures against RAP-536 treatment data revealed a significant up-regulation of 158 activated genes (Normalized Enrichment Score=2.7, P=0) involved in heme biosynthesis (such as Ppox, Fech, and Abcb10) and terminal erythroid differentiation (such as Fog1, Klf1 and Bcl-xl).ACE-536 is known to bind and inhibit Smad2/3 ligands such as GDF8, GDF11 and activin B but not activin A (Suragani et al 2014). Consistent with this data, treatment of MEL and fetal liver erythroid cells with GDF11 (50ng/mL) induced Smad2/3 phosphorylation and ACE-536 co-treatment inhibited the increase in pSmad2/3. In differentiating erythroid cells, GDF11 treatment displayed reduced nuclear GATA-1 protein levels by both western blotting and immunofluorescence studies. Additionally, reactive oxygen species (ROS) levels indicative of oxidative stress were elevated in MEL and primary fetal liver cells following GDF11 treatment. Consistent with the increase in ROS, we found decreased mitochondrial transmembrane potential (Δψm) indicative of unhealthy cells and increased caspase 3/7 activity in erythroid cells treated with GDF11. Importantly, treatment of erythroid cells with ACE-536 and GDF11 decreased ROS, restored Δψm and GATA-1 levels to control levels. Immunofluorescence studies demonstrated decreased GATA-1 levels in the nucleus of erythroid precursors from b-thalassemic cells compared to wild type mice. Treatment of these b-thalassemic erythroid precursors with ACE-536 prevented the decrease in GATA-1 levels.
Conclusion
Together, these data done with RAP-536 provide a potential mechanistic role for luspatercept as a novel treatment of β-thalassemia. By inhibiting pSmad2/3 signaling, RAP-536 treatment decreases ROS, prevents caspase 3/7 activation and GATA-1 cleavage. Thus by restoring GATA-1 availability and functional activity, RAP-536 treatment causes upregulation of genes involved in promoting terminal erythroid maturation, and consequently corrects anemia in b-thalassemia. Luspatercept currently completed phase 2 clinical trials and has initiated phase 3 studies in patients with MDS and b-thalassemia.
Session topic: Red blood cells and iron
Keyword(s): Erythroid differentiation, GATA-1, Reactive oxygen species, Thalassemia
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