Elucidating the Interplay between ddx41 and cux1 mutations in Hematopoiesis in Danio Rerio

Myelodysplastic syndromes (MDS) are a family of myeloid cancers involving defective hematopoietic stem and progenitor cells (HSPCs). In MDS, there is clonal expansion of defective HSPCs over healthy cells, which impacts cell maturation and increases the risk of transformation to acute myeloid leukemia (AML). Somatic and germline mutations contribute to MDS/AML formation by altering HSPC proliferation, differentiation, and inflammatory signaling. Heterozygous germline mutations of DEAD-box helicase 41 (DDX41) predispose individuals to hematologic malignancies, especially MDS/AML. However, patients typically do not develop MDS until later in life. This clinical observation suggests secondary factors play key roles in driving disease. Researchers identified a strong enrichment for co-occurrence of mutations in DDX41 and CUT-like homeobox 1 (CUX1), which encodes for a non-clustered homeodomain transcription factor involved in regulating gene expression and DNA repair. To decipher if co-mutations in DDX41 and CUX1 could promote hematopoietic dysfunction, we generated a zebrafish system that mimics germline ddx41 mutations and somatic cux1 mutations. In humans, the CUX1 locus has two divergent splice isoforms that encode for two distinct proteins, CUX1 and CASP. In zebrafish, these factors are encoded by two paralogous genes, cux1a and cux1b. As zebrafish ddx41 homozygous loss-of-function mutant embryos expand their HSPCs and develop macrocytic anemia, we assessed these two cell types in ddx41 and cux1-paralog co-mutated embryos. Transient mutagenesis of cux1a or cux1b elevated cmyb+ HSPC levels in ddx41 homozygous mutants beyond non-mutagenized mutants. Similarly, mutagenesis of cux1a or cux1b worsened anemia in ddx41 homozygous mutant embryos and induced anemia in ddx41 heterozygous embryos. Of note, cux1a mutagenesis had no impact on erythrocyte levels in ddx41 wildtype embryos, suggesting genotype-selective effects of mutating this CUX1 homolog. These findings indicate that combined insufficiencies in DDX41 and CUX1 act synergistically or additively to promote hematopoietic dysfunction and provide a platform to discover the mechanism underlying how this might contribute to the development of MDS/AML.