A Chemical Modifier Screen in Zebrafish Mutants for the Spliceosomal Component Sf3b1reveals Genotype-Selective Vulnerabilities

Myelodysplastic syndrome (MDS) is a disorder arising from hematopoietic stem and progenitor cell (HSPC) dysfunction resulting in ineffective hematopoiesis. Mutations in the spliceosomal component SF3B1 (Splicing Factor 3B, subunit 1) are prevalent in MDS, but how this leads to aberrant hematopoiesis is unclear. We elucidated the in vivo consequences of sf3b1 misregulation on hematopoiesis and splicing using a zebrafish sf3b1 (sf3b1^hi3394) loss-of-function mutant. Primitive erythropoiesis initiates normally in sf3b1 mutants, as evidenced by normal expression of scl (stem cell leukemia), gata1 and beta-globin, but maturation is defective as shown by decreased hemoglobinization and an increase in immature erythroblasts at 48 hours post fertilization (hpf). Similarly, primitive myelopoiesis initiates normally as early expression of scl and pu.1 are normal, but expression of the differentiation markers l-plastin and myeloperoxidase are diminished in mutants at 24 and 28 hpf. Hemogenic endothelial and HSPC markers runx1 and gata2b are also diminished, while the pan-endothelial marker kdrl (kinase insert domain receptor like) and aorta-specific markers notch1b and notch3 are normally expressed at 24 hpf, indicating hematopoietic induction is more sensitive to sf3b1 loss than vascular formation. RNA-seq analysis of sf3b1 mutants revealed large-scale splicing defects and alterations, particularly affecting introns with suboptimal splice sites. Comparison of RNA-seq data from zebrafish sf3b1 mutants and MDS patients harboring SF3B1 mutations revealed a significant overlap in misspliced genes and in the corresponding affected pathways, including many pathways implicated in MDS pathology such as TGFβ (Transforming Growth Factor-Beta) and immune signaling. Together, these results show conservation between zebrafish mutants and human cells with SF3B1 mutations. We next took a chemical genetic approach and discovered sf3b1 mutants were more sensitive than wild type siblings to treatment with E7107, a small molecule inhibitor of the spliceosome, indicating a potential therapeutic window for spliceosome modulators in MDS. Motivated by this finding, we performed a large-scale chemical modifier screen and identified many new lead compounds (6 out of 562 screened) that also show synthetic lethality in sf3b1 mutants. These small molecules are enriched in those with anti-inflammatory properties, suggesting aberrant immune signaling could underlie some of the defects in sf3b1 mutants. Together, our data demonstrate the molecular and cellular consequences of sf3b1 loss in vivo and establish sf3b1 zebrafish mutants as a novel model for discovery of therapeutics for MDS.