The Inherited MDS Gene DDX41 Is Required for Ribosome Biogenesis and Cell Viability
Authors: Chlon Timothy M , Stepanchick Emily , Choi Kwangmin , Zheng Yi , Hueneman Kathleen , Davis Ashley , Starczynowski Daniel T. 
Published: February 2020

Myelodysplastic syndromes (MDS) are hematopoietic stem cell (HSC) disorders in which myeloid cell differentiation is impaired, causing blood lineage cytopenias and potentially leading to acute myeloid leukemia (AML) through malignant transformation. MDS occurs in adults with a median age of 71 years, and is associated with multiple cytogenetic and genetic abnormalities in the diseased HSC. Younger patients can also have MDS as a result of underlying congenital diseases or secondary effects from cancer therapy. It has recently been discovered that some families with high rates of MDS incidence bear heterozygous inherited mutations in DDX41, a member of the DEAD box RNA helicase family of genes. These patients typically have normal hematopoietic indices into adulthood and present with MDS at a median age of 61 years, slightly younger than the general population. Inherited DDX41 mutations are always heterozygous and are typically frame-shift mutations, indicating they are likely loss of function. Approximately half of MDS patients with inherited DDX41 mutations acquire a second-hit, often R525H, in the healthy DDX41 allele in their disease clones. This mutation is also observed in 1-2% of de novo AML patients, suggesting it causes gain of function or dominant negative activity. Multiple functions have been ascribed to DDX41, such as functioning as an innate immune sensor and as an RNA splicing regulator, but its role in the pathogenesis of MDS remains unknown. We set out to model DDX41 mutations by generating conditional DDX41 knockout and R525H-knock-in mice. Combining these alleles and crossing to Rosa-Cre-ERT expressing mice allowed for tamoxifen-inducible acquisition of knockout (KO), heterozygous (HET), heterozygous knock-in (KI/+) and knock-in alone (KI/-) HSPC. The KO and KI/- HSPC were incapable of engrafting into recipient mice and underwent rapid cell cycle arrest and apoptosis, indicating that Ddx41 is required for HSPC cell viability and that the R525H mutation causes loss of the required function. In contrast, the HET and KI/+ HSPC survived and proliferated normally in culture and successfully engrafted irradiated recipient mouse bone marrow. HET and KI/+ transplanted mice had increased numbers of LSK cells, and subset of mice developed a myeloid malignancy, resembling the human disease. To determine the function of DDX41 that is critical for hematopoiesis, we performed a tandem-pulldown followed by mass spectrometry analysis to identify relevant DDX41 interacting proteins in human AML cells. We found that DDX41 interacts with multiple proteins in the small ribosomal subunit, including RPS3 and RPS14. Consistent with disruption of the assembly and function of the small ribosomal subunit, KO and KI/-HSPC exhibited rapid and robust impairment of global protein translation. Polysome profiling indicated an increase in monosomes and a decrease in polysomes in KO cells, consistent with an inability of ribosomes to initiate translation and move along the mRNA. To determine the role of the translation defect in the cell growth deficiency of DDX41-deficient cells, we treated WT, HET, and KO HSPC with the translation inhibitor puromycin and determined that KO cells were relatively more sensitive to translation inhibition, indicating that DDX41-deficient cells are specifically sensitive to further reduction in protein translation. This data supports the conclusion that the cell lethality caused by DDX41 loss is related to ribosome dysfunction. Mechanistically, we find that DDX41-deficient cells have stalled ribosomal RNA (rRNA) processing, characterized by increased unprocessed rRNA and decreased processed rRNA intermediates. In conclusion, we identify a novel function of DDX41 in regulating rRNA processing and ribosome formation that is essential for the survival and proliferation of HSPC. The loss of DDX41 may contribute to MDS as a result of impaired ribosome function, as has been previously reported in patients bearing mutations in other ribosome regulators.

Disclosures

Starczynowski: Kurome Therapeutics: Consultancy.