GDF11/SMAD Regulated Splicing of GATA1 Is Associated with Response to Luspatercept in Lower-Risk Myelodysplastic Syndromes (LR-MDS)

Introduction: Luspatercept is an erythroid maturation agent that promotes late-stage erythropoiesis in patients with LR-MDS. While it is known that luspatercept, a modified activin receptor ligand trap, binds to GDF11 and Activin B, the mechanism by which these ligands trigger a decrease in red blood cell (RBC) formation is unknown. Luspatercept was approved by the FDA following the phase 3 MEDALIST study, but biomarkers of response are currently unknown. Results:In a large cohort of primary MDS CD34+ samples, GDF11 expression was significantly higher in MDS samples than in age-matched healthy controls (183 MDS samples vs 17 controls, P < 0.05). GDF11 was detected in primary MDS sera and was substantially elevated in the refractory anemia (RA) and RA with ring sideroblasts (RARS) MDS subgroups. Activin receptor 2B (ACVR2B), the receptor of GDF11, was also overexpressed in MDS samples, and its expression was confirmed by immunohistochemistry (IHC) in the bone marrows of primary MDS patients. GDF11/ACVR2B activates SMAD2, and we found that the overexpression of SMAD2 in CD34+ cells was associated with more severe anemia in MDS patients. RNA-seq and SMAD2 ChIP-seq analyses were then performed to determine the downstream effects of GDF11 in erythroid progenitors. Human CD34+ primary cells were utilized to generate BFU-E stage erythroid progenitors. We discovered that GDF11 promotes an abnormal erythroid gene expression program characterized by increased expression of pro-apoptotic and early erythroid differentiation-related genes. SMAD2 binding was detected in regulatory regions of early erythroid (including GATA1), proinflammatory, and apoptotic transcripts using ChIP-seq analysis with p-SMAD2. In a zebrafish model, administration of GDF11 caused a decrease in erythroid cells, which was rescued by treatment with luspatercept (Wobus et al. Leukemia 2021 35(10):2936-47). Luspatercept inhibited the phosphorylation and activation of SMAD2 in response to GDF11 stimulation in primary hematopoietic cells and leukemia cell lines. Importantly, luspatercept could reverse the growth-inhibiting effects of MDS-derived sera on primary hematopoietic progenitors. Finally, to identify biomarkers associated with clinical responses, we analyzed phase 3 MEDALIST study samples collected before and after treatment with lupatercept. RNA-seq analysis was performed on bone marrow samples from responders (R; n = 14) and non-responders (NR; n = 9) (Figure). Analysis of pre-treatment/sc