Elucidating the Interplay Between DDX41 and CUX1 Mutations in Hematopoiesis in Danio Rerio and iPSCs

PROJECT SUMMARY Myelodysplastic syndromes (MDS) are a family of bone marrow failure disorders involving defective hematopoietic stem and progenitor cells (HSPCs). In MDS, there is clonal expansion of defective HSPCs over healthy cells, ineffective maturation and uni- or multilineage cytopenias of myeloid lineage cells, and increased risk of transformation to acute myeloid leukemia (AML). Although the field recognizes the effects of MDS, the origins and evolution of its pathogenesis are incompletely understood. Somatic and germline mutations contribute to MDS/AML formation by altering HSPC proliferation, differentiation, and survival. Heterozygous germline mutations of DEAD-box helicase 41 (DDX41) predispose individuals to hematologic malignancies, especially MDS/AML. Despite carrying the mutation throughout life, patients typically do not develop MDS until later in adulthood, which suggests secondary factors play key roles in driving disease. Researchers identified a strong enrichment for co-occurrence mutations in DDX41 and CUT-like homeobox 1 (CUX1), which encodes for a nonclustered homeodomain transcription factor and tumor suppressor gene and may act as a secondary somatic driver mutation in DDX41 patients. My project aims to delineate how co- occurring ddx41 and cux1 mutations cooperate to drive hematopoietic dysfunction using zebrafish and human induced pluripotent stem cells (iPSCs). In vivo, in zebrafish, I will characterize the blood system during embryogenesis by assessing changes in HSPCs, and differentiated progeny, including erythroid cells. In adult zebrafish, I will evaluate if mutations in cux1 paralogs accelerate or exacerbate hematopoietic decline and assess changes in age-associated clonal HSPC expansion, using Zebrabow, a novel method that utilizes a Cre- lox system to induce a heritable color barcoding unique to each HSPC and its progeny. To determine if our findings are conserved in humans, I will also assess HSPC levels and hematopoietic differentiation into erythrocytes in human iPSCs with a common germline DDX41 mutation in combination with CUX1 mutagenesis. These experiments will provide mechanistic insight into how these common co-occurring mutations might drive hematopoietic dysfunction and ultimately contribute to the development of MDS/AML.