DDX41 Is Required to Activate and Modulate cGAS-STING Activation

Upon binding double-stranded (ds)DNA, cGAS is activated and initiates the cGAS-STING-type I interferon pathway; thus, the availability of dsDNA ligand is critical for cGAS activation. DDX41 is a DEAD-box helicase and mutations in DDX41 cause myelodysplastic syndromes (MDS) and acute myeloid leukemia (AML). Here, we found that DDX41-knockout (KO) human cells and mice primary cells had reduced type I interferon production after viral infection, which is consistent with previous reports. Unexpectedly, cGAS and STING activation were affected in DDX41 KO cells, suggesting that DDX41 functions upstream of cGAS. The recombinant DDX41 protein exhibited ATP-dependent DNA unwinding activity and ATP-independent strand annealing activity. The MDS/AML-derived mutant R525H had reduced unwinding activities but retained normal strand annealing activity and stimulated greater cGAS dinucleotide synthesis activity than WT-DDX41. Overexpression of R525H in either DDX41-deficient or -proficient cells resulted in higher type I interferon production, indicating that DDX41 inactivates cGAS-STING-type I interferon pathway by unwinding dsDNA to single-stranded (ss)DNA. Our results demonstrate that DDX41 utilizes its unwinding and annealing activities to regulate the homeostasis of dsDNA and ssDNA, which in turn modulates the cGAS-STING pathway; dysregulation of this pathway leads to MDS/AML. Funding Information: This work was supported by grants from the Natural Sciences and Engineering Research Council of Canada (NSERC, RGPIN 2019-04578) to YZ, the Canada Foundation for Innovation (CFI33364) and Cancer Research Society (CRS-22493) to FJV, the National Institutes of Health (R01-AI-085015) to SRR, and NSERC (RGPIN-2019-05487), the Canadian Breast Cancer Foundation (CBCF-R1636), and the Cancer Research Society (CRS-24139) to YW. Declaration of Interests: The authors declare no conflicts of interest. Ethics Approval Statement: All studies were performed following the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. The experiments performed with mice in this study were approved by the UIC ACC (protocol no. 15-222). Bone marrow aspirates were collected from 23 patients with AML or MDS who participated in the study according to the protocol approved by the ethics committee on human genome research at Hiroshima University.