Transcriptional and splicing dysregulation by U2AF1 mutations contribute to inflammatory and migratory alterations in MDS Free

Recent genomic analyses have revealed that mutations in RNA splicing factors—including SF3B1, SRSF2, U2AF1, and ZRSR2—are among the most frequent mutations in myelodysplastic syndromes (MDS). Among these, U2AF1 mutations are enriched in MDS without ring sideroblasts and in AML with myelodysplasia-related changes, and are associated with poor clinical outcomes. U2AF1 mutations primarily affect two conserved residues, S34 and Q157, located within the N- and C-terminal zinc finger motifs. Mechanistically, U2AF1 mutant proteins preferentially recognize the ‘UAG’ motif upstream of the 3‘ splice site, causing exon skipping and aberrant 3‘ splice site selection. Among the reported targets of U2AF1 mutations are BCOR and GNAS, both of which are known drivers of MDS, as well as IRAK4, a key mediator of innate immune signaling. However, how U2AF1 mutations affect hematopoietic stem cell (HSC) function and lineage differentiation, and how they shape inflammatory responses or cell-cell interactions, remains incompletely understood.

To address these issues, we utilized a conditional knock-in mouse model independently generated to express the U2af1 S34F allele under the control of Vav1-Cre promoters. U2af1 mutant mice exhibited macrocytic anemia, leukopenia, morphological abnormalities, and myeloid-skewed hematopoiesis, resulting in shortened survival. Transplantation experiments revealed defective HSC reconstitution under competitive conditions.

Bulk RNA-seq of KSL (Kit⁺Sca-1⁺Lin⁻) cells revealed splicing abnormalities in myeloid malignancy-related genes, including Hnrnpa2b1, Csf3r and Gnas, many of which overlapped with findings from the Srsf2 P95H mutant mouse model (Kon et al., Blood 2018). In contrast, U2af1 mutant KSL cells showed specific exon skipping in genes related to inflammatory signaling and RNA metabolism. Gene set enrichment analysis revealed that both U2af1 and Srsf2 mutations upregulated pathways related to DNA repair, cell cycle regulation, and RNA processing. However, genes related to cell migration and inflammation were uniquely downregulated in U2af1 mutant KSL cells, highlighting distinct pathogenic mechanisms associated with splicing factor mutations in MDS.

To investigate the cell type-specific impact of the U2af1 S34F mutation, we performed single-cell RNA sequencing (scRNA-seq) on hematopoietic stem/progenitor cells and analyzed transcriptional changes across lineages. In monocyte-dendritic progenitors (MDPs), genes involved in migration and motility, as well as inflammatory response pathways, were significantly downregulated. Additionally, adhesion-related genes, including Cd34 and Tgfb1, were markedly reduced, suggesting diminished mobilization capacity and altered interactions with the bone marrow microenvironment. Similarly, in megakaryocyte-erythroid progenitors (MEPs), adhesion and migration-related genes, including Spn and Itga4, were downregulated, while tumor-related pathways were activated. In contrast, multipotent lymphoid progenitors (MLPs) exhibited upregulation of inflammatory genes. These findings suggest that lymphoid lineage cells, such as MLPs, contribute to the activation of immune responses and inflammatory pathways, while myeloid lineage progenitors, including MDPs and MEPs, showed impaired migration and adhesion, alongside reduced immune response capabilities, contributing to MDS pathogenesis.

Based on scRNA-seq data, we analyzed known ligand-receptor interactions and predicted that interactions related to cell migration pathways, such as Cd34–Selp, Sell–Cd34, and Icam1–Spn, were significantly altered. Notably, while a subset of inflammation-related genes exhibited splicing alterations, the majority of differentially expressed genes associated with motility and inflammation did not, suggesting that these changes are largely driven by transcriptional dysregulation rather than splicing defects. Importantly, these transcriptional changes were consistently observed in bulk RNA-seq data from U2AF1-mutant MDS patients, as well as in CRISPR-engineered MOLM13 human leukemia cells harboring the U2AF1 S34F mutation.

In conclusion, our findings demonstrate that U2AF1 mutations perturb hematopoiesis through lineage-specific transcriptional reprogramming, particularly impairing inflammatory and migratory pathways. These results highlight the mechanistic divergence among splicing factor mutations and underscore the therapeutic potential of targeting dysregulated inflammation and cell migration in MDS.