3020 – SPLICING OF A MASTER ERYTHROID TRANSCRIPTION FACTOR GOVERNS THERAPEUTIC TARGETING OF ANEMIA IN MDS

While anemia is the primary clinical manifestation in myelodysplastic syndromes (MDS), the molecular pathogenesis of ineffective erythropoiesis remains poorly understood. Although, Luspatercept, an activin receptor 2B (ACVR2B) ligand trap, is approved for treating anemia, the precise mechanism of action and response biomarkers in LR-MDS are not clearly defined. We found that ACVR2B, its ligand GDF11 and downstream effector, SMAD2 are all upregulated in MDS patient samples. GDF11 inhibited human erythropoiesis in vitro and caused anemia in zebrafish, and its effects were abrogated by Luspatercept treatment. Upon GDF11 stimulation of human erythroid progenitors, SMAD2 binding was seen to occur in regulatory regions of erythroid genes, including at an intronic sequences of GATA1, the master erythroid transcription factor. Intronic SMAD2 binding led to skipping of exon 2 of GATA1, resulting in a shorter, functionally hypomorphic isoform, (GATA1s). CRISPR deletion of the SMAD2 binding intronic region abrogated the expression of GATA1s. Enforced expression of GATA1s in a mouse model led to anemia, that was rescued by a murine ACVR2B ligand trap. Finally, RNAseq analysis of bone marrow samples from the Phase 3 MEDALIST trial showed that clinical responders to Luspatercept at baseline have a greater proportion of GATA1s when compared to non-responders and that clinical increases in red cells are associated with relative decreases in the shorter GATA1 isoforms. Our findings reveal that GDF11 mediated SMAD2 activation leads to relative increase in functionally deficient GATA1 isoforms, thus driving anemia and responses to Luspatercept in MDS.