SF3B1 mutations K700E, K666N, and R625H: Gene expression and aberrant splicing consequences

Abstract Myelodysplastic syndromes (MDS) are a group of clonal hematopoietic stem cell disorders characterized by ineffective hematopoiesis and an increased risk of transformation to acute myeloid leukemia (AML). Somatic mutations in splicing factor 3B subunit 1 (SF3B1) are among the most prevalent in MDS and are pathognomonic for subtypes with ring sideroblasts. SF3B1 encodes a core component of the U2 snRNP complex involved in branchpoint recognition during the initial stages of pre-mRNA splicing. The K700E hotspot mutation is associated with a more favorable prognosis, however recent reports suggest other common mutations such as K666N, and R625H, which also cluster within the Huntingtin, Elongation factor 3, A subunit of PP2A, TOR1 (HEAT) repeat domain of SF3B1 may be associated with shorter overall survival and earlier progression to AML. Our group and others have shown that the SF3B1 K700E mutation perturbs canonical branchpoint recognition and promotes aberrant RNA splicing. These differences in splicing are thought to lead to the expression of novel antigens and aberrant protein isoforms that expose new therapeutic vulnerabilities in SF3B1-mutant cells. To investigate whether different SF3B1 mutations have similar or distinct effects on gene expression and alternative splicing during hematopoiesis, three mouse models, each carrying a distinct SF3B1 mutation, were generated. Following Poly(I:C) injections to activate the mutations, Lin⁻Sca-1⁺c-Kit⁺(LSK), Lin⁻c-Kit⁺(LK), and terminal erythroblast populations separated by forward scatter, CD44 and Ter119 staining were isolated by cell sorting and subjected to RNA sequencing. Differential gene expression was analyzed using DESeq2, alternative splicing (AS) events were identified with the rMATSpipeline, and isoform switching was examined usingIsoformSwitchAnalyzeR. The LSK differential gene expression analysis showed that each mutation clustered separately with very few shared differentially expressed genes. Pathway analysis showed that K700E upregulated cell adhesion and migration, while K666N activated inflammatory and myeloid pathways and downregulated cellular organization. R625H upregulated immune response and complement activation, with concurrent downregulation of adaptive immune response pathways. In LK cells, K666N and R625H clustered together, whereas K700E aligned closely with wild-type. Approximately 15% of differentially expressed genes were shared among all mutants. K700E upregulated immune-related pathways and downregulated cell cycle and transcriptional programs. K666N induced autophagy and intracellular transport while suppressing innate immune and inflammatory responses. R625H upregulated interferon-mediated signaling and downregulated transcriptional regulators and NF-κB signaling, with immune effector genes represented in both directions. AS analysis in LSK cells showed the highest number of events in K666N, followed by K700E and R625H. Isoform switches in LSKs affected genes regulating transcription (Zfp688), immune signaling (Ifngr1), metabolism (Cyp27a1), and autophagy (Atg2a). In LK cells, AS events were highest in R625H, followed by K700E and K666N. Isoform switches in LK were linked to transcriptional regulation (Rara), cell cycle (Csnk1g1), and NF-κB signaling (Commd4). Tight regulation of AS is required for terminal erythroid maturation. Thus, we sorted terminal erythroblasts from anemic Sf3b1 mutant mice and found that differential gene expression was driven by the stage of maturation rather than mutant Sf3b1 expression. R625H mice developed the most severe anemia and had the highest number of AS events at each stage. K666N erythroblasts had more AS events than K700E erythroblasts, but K666N mice were the least anemic. Shared aberrant splicing events were identified in immune-related genes, including Fkbp1a and Ccl25, and genes in cytokine signaling pathways. Taken together these findings provide critical insights into how different SF3B1 mutations drive lineage- and stage-specific splicing alterations, which may inform future therapeutic strategies for hematologic malignancies.