Decoding the Paradox of DDX41-mutant MDS

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Project Summary Inherited mutations in the RNA helicase gene DDX41 cause predisposition to adult-onset Myelodysplastic Syndrome and related myeloid malignancies, accounting for up to 5% of MDS cases. These mutations are always heterozygous and are often frameshifts in the N-terminal region of the protein, suggesting that they cause loss of protein function. The DDX41 protein has described functions in mRNA splicing, innate immune signaling and ribosome biogenesis, but the precise mechanism by which DDX41 mutations contribute to myeloid malignancy is not understood. In approximately 70% of MDS patients with germline DDX41 mutations, a mutation in the other allele of DDX41 is acquired in the diseased bone marrow (BM) cells. This somatic mutation almost always causes the specific amino acid substitution Arginine-525 to Histidine (R525H). In a recent publication, we demonstrated that murine hematopoietic stem and progenitor cells (HSPC) bearing two loss-of-function Ddx41 mutations (Ddx41-/-) or one loss-of-function mutation and one Ddx41R525H mutation (Ddx41R525H/-) undergo cell cycle arrest and apoptosis. This indicates that one copy of wild-type DDX41 is required for hematopoiesis and that the R525H mutant is deficient for the required function. In our mouse model, we found that hematopoietic stem cells (HSC), which are mostly quiescent, can survive with biallelic Ddx41 mutations but do not expand in vivo. These findings raise an apparent paradox in that the precise combination of DDX41 mutations that is present in many patients is selected against in the proliferative HSPC pool and thus clones bearing these mutations would be unlikely to survive and expand sufficiently to contribute to disease. Our proposed studies will elucidate the mechanistic basis of selection for the acquired DDX41R525H mutation in germline DDX41-mutant patients. Our overall hypothesis is that HSC, which have obligatory low protein translation activity, are the cell type in which DDX41R525H mutations arise, persist, and expand due to decreased function of the DDX41 protein causing reduced ribosome synthesis. The selection for DDX41R525H mutant HSC, possibly with the assistance of other co-mutations, causes hematopoietic inefficiency due to the limited proliferative capacity of their differentiating progenitor cells, leading to MDS. The inability of Ddx41R525H/- mouse HSC to expand in BM necessitates examination of patient cells bearing this combination of mutations to understand how they survive and expand. Using single-cell sequencing approaches, we propose to analyze MDS patient BM cells to determine the differentiation state, transcriptomic changes, and co-mutations present in cells bearing the acquired R525H mutation. To determine the molecular basis for the selective advantage of cells bearing the R525H mutation, we will use genetically-engineered cell models to quantitatively assess the function of the R525H mutant protein compared to wild-type. With an improved understanding of the effect of acquired DDX41 mutations on MDS pathogenesis, we will then test genetic or chemical inhibitors of rationally-chosen targets for therapeutic eradication or mechanistic rescue of HSPC bearing biallelic DDX41 mutations.