Contributions
Abstract: S842
Type: Oral Presentation
Presentation during EHA23: On Saturday, June 16, 2018 from 11:30 - 11:45
Location: Room K2
Background
Previously, we reported elevated Smad2/3 signaling in diseases characterized by erythroid maturation defects (Ineffective Erythropoiesis; IE) such as MDS and β-thalassemia. Luspatercept (an erythroid-maturation agent [EMA]) binds and inhibits signaling by certain Smad2/3 ligands such as GDF11, activin B, and corrects IE/anemia in a murine model of thalassemia and MDS (Suragani et. al, 2014). However, the molecular mechanism through which Smad2/3 ligands negatively regulate erythroid maturation is not completely understood.
Aims
We investigated the molecular mechanism of action through which Smad2/3 ligands such as GDF11 inhibit erythroid maturation in vitro, in vivo, and how luspatercept prevents this inhibition.
Methods
β-thalassemic mice (Hbbth3/+) were used to obtain splenic basophilic erythroblasts to be used for RNA-seq. MEL cells were treated with GDF11 in the presence or absence of luspatercept. In vivo studies were conducted using transgenic mice with inducible GDF11 overexpression under a ROSA26Cre promoter. CBC and erythroid differentiation analysis were carried out following 3-weeks of inducing GO (N= 5).
Results
To investigate the effect of GDF11 in vivo, we used transgenic mice with inducible GDF11 overexpression (GO). RBC and hemoglobin (Hgb) levels were significantly decreased in GO mice. RBC levels were 6.83 x 106/μl in GO mice vs. 8.7 x 106/μl in WT (p<0.05). Hgb levels were 9.92 g/dl in GO mice vs. 12.96 g/dl in WT (p<0.05). Additionally, bone marrow TER119+ cells were significantly reduced in GO mice (5.294%) compared to WT (29.16%, p<0.001) suggesting IE. Furthermore, we found that GATA1+ cells in splenic TER119+ erythroid cells was significantly reduced in GO mice (2.876%) compared to WT (7.342%; p<0.05).
Transcriptome analysis of sorted β-thalassemic erythroblasts identified 74 genes that were differentially expressed in RAP-536 (murine version of luspatercept) treated samples vs. VEH. Analysis of the significantly upregulated genes by RAP-536 revealed increased activity of GATA1. We investigated whether elevated pSmad2/3 levels by GDF11 affects GATA1 expression using MEL cells and found that GATA1 was decreased in both mRNA and protein levels, and importantly luspatercept treatment prevented the decrease. We also found increased nuclear accumulation of GATA1 in these cells consistent with increased transcriptional activity in β-thalassemic mice in vivo. Furthermore, we found that the expression of Transcription Intermediary Factor (TIF) 1γ in β-thalassemic erythroid tissues was significantly lower in VEH vs. RAP-536 treated mice. TIF1γ has been reported to compete with Smad4, binding pSmad2/3 and leading to erythroid differentiation (Wei et. al, 2006), and may act as a molecular link between pSmad2/3 and GATA1.
Conclusion
In this study, we showed that pSmad2/3 negatively regulates the levels of GATA1 protein, and prevents RBC maturation. We have emerging data to show TIF1γ as a link between pSmad2/3 and GATA1. Thus by preventing elevated pSmad2/3, and restoring GATA1 availability through TIF1γ, luspatercept treatment causes upregulation of genes involved in promoting terminal erythroid maturation, and consequently corrects anemia in β-thalassemia. Luspatercept is currently in phase 3 studies in patients with MDS and β-thalassemia.
Session topic: 28. Thalassemias
Keyword(s): Anemia, Beta thalassemia, GATA-1
Abstract: S842
Type: Oral Presentation
Presentation during EHA23: On Saturday, June 16, 2018 from 11:30 - 11:45
Location: Room K2
Background
Previously, we reported elevated Smad2/3 signaling in diseases characterized by erythroid maturation defects (Ineffective Erythropoiesis; IE) such as MDS and β-thalassemia. Luspatercept (an erythroid-maturation agent [EMA]) binds and inhibits signaling by certain Smad2/3 ligands such as GDF11, activin B, and corrects IE/anemia in a murine model of thalassemia and MDS (Suragani et. al, 2014). However, the molecular mechanism through which Smad2/3 ligands negatively regulate erythroid maturation is not completely understood.
Aims
We investigated the molecular mechanism of action through which Smad2/3 ligands such as GDF11 inhibit erythroid maturation in vitro, in vivo, and how luspatercept prevents this inhibition.
Methods
β-thalassemic mice (Hbbth3/+) were used to obtain splenic basophilic erythroblasts to be used for RNA-seq. MEL cells were treated with GDF11 in the presence or absence of luspatercept. In vivo studies were conducted using transgenic mice with inducible GDF11 overexpression under a ROSA26Cre promoter. CBC and erythroid differentiation analysis were carried out following 3-weeks of inducing GO (N= 5).
Results
To investigate the effect of GDF11 in vivo, we used transgenic mice with inducible GDF11 overexpression (GO). RBC and hemoglobin (Hgb) levels were significantly decreased in GO mice. RBC levels were 6.83 x 106/μl in GO mice vs. 8.7 x 106/μl in WT (p<0.05). Hgb levels were 9.92 g/dl in GO mice vs. 12.96 g/dl in WT (p<0.05). Additionally, bone marrow TER119+ cells were significantly reduced in GO mice (5.294%) compared to WT (29.16%, p<0.001) suggesting IE. Furthermore, we found that GATA1+ cells in splenic TER119+ erythroid cells was significantly reduced in GO mice (2.876%) compared to WT (7.342%; p<0.05).
Transcriptome analysis of sorted β-thalassemic erythroblasts identified 74 genes that were differentially expressed in RAP-536 (murine version of luspatercept) treated samples vs. VEH. Analysis of the significantly upregulated genes by RAP-536 revealed increased activity of GATA1. We investigated whether elevated pSmad2/3 levels by GDF11 affects GATA1 expression using MEL cells and found that GATA1 was decreased in both mRNA and protein levels, and importantly luspatercept treatment prevented the decrease. We also found increased nuclear accumulation of GATA1 in these cells consistent with increased transcriptional activity in β-thalassemic mice in vivo. Furthermore, we found that the expression of Transcription Intermediary Factor (TIF) 1γ in β-thalassemic erythroid tissues was significantly lower in VEH vs. RAP-536 treated mice. TIF1γ has been reported to compete with Smad4, binding pSmad2/3 and leading to erythroid differentiation (Wei et. al, 2006), and may act as a molecular link between pSmad2/3 and GATA1.
Conclusion
In this study, we showed that pSmad2/3 negatively regulates the levels of GATA1 protein, and prevents RBC maturation. We have emerging data to show TIF1γ as a link between pSmad2/3 and GATA1. Thus by preventing elevated pSmad2/3, and restoring GATA1 availability through TIF1γ, luspatercept treatment causes upregulation of genes involved in promoting terminal erythroid maturation, and consequently corrects anemia in β-thalassemia. Luspatercept is currently in phase 3 studies in patients with MDS and β-thalassemia.
Session topic: 28. Thalassemias
Keyword(s): Anemia, Beta thalassemia, GATA-1