Probing Aberrant Splicing in a Novel Model of SF3B1-Mutant Myelodysplastic Syndromes

Splicing is a fundamental process by which introns are removed from primary RNA transcripts. Alternative splicing is widely appreciated to be a major mechanism of gene regulation by which eukaryotic cells expand their transcriptional repertoire. By contrast, aberrant splicing generates novel transcripts not found in normal cells. Heterozygous mutations in a core U2 spliceosome factor SF3B1 are strongly associated with MDS with ring sideroblasts (MDS-RS). MDS-RS is characterized by iron retention in mitochondria and ineffective erythropoiesis. How aberrant splicing induced by mutant SF3B1 causes ring sideroblasts and erythroid defects remains poorly understood.

There are no genetically accurate models of MDS-RS that recapitulate ring sideroblast formation. Our lab has established an iPSC reprogramming approach to generate an in vitro model of MDS-RS. This method captures genetically distinct MDS subclones as well as normal cells in individual patients enabling internal normalization to the patient's isogenic normal clone. By differentiating iPSCs we generated inducibly immortalized MDS-RS CD34⁺ progenitor cell lines with robust expansion and erythroid differentiation potential enabling extensive analysis of SF3B1-mutant aberrant splicing throughout erythroid differentiation. We show that SF3B1-mutant lines form abundant ring sideroblasts compared to normal isogenic controls. We next performed RNA-sequencing and splicing analysis at different stages of erythroid differentiation. We show that global splicing patterns are maintained in SF3B1-mutant cells, while revealing distinct subsets of aberrantly spliced transcripts in CD34⁺ progenitors and erythroblast populations. This suggests that different aberrant splicing events contribute to erythroid progenitor expansion and inefficient erythropoiesis and iron dysregulation in MDS-RS.

Taken together, this novel iPSC model of MDS-RS is a robust tool for studying the role of aberrant splicing in SF3B1-mutant erythroid progenitor expansion, inefficient erythropoiesis and ring sideroblast formation.