DDX41 resolves G-quadruplexes to maintain erythroid genome integrity and prevent cGAS-mediated cell death.

Deleterious germline DDX41 variants constitute the most common inherited predisposition disorder linked to myeloid neoplasms (MNs), yet their role in MNs remains unclear. Here we show that DDX41 is essential for erythropoiesis but dispensable for other hematopoietic lineages. Ddx41 knockout in early erythropoiesis is embryonically lethal, while knockout in late-stage terminal erythropoiesis allows mice to survive with normal blood counts. DDX41 deficiency induces a significant upregulation of G-quadruplexes (G4), which co-distribute with DDX41 on the erythroid genome. DDX41 directly binds to and resolves G4, which is significantly compromised in MN-associated DDX41 mutants. G4 accumulation induces erythroid genome instability, ribosomal defects, and p53 upregulation. However, p53 deficiency does not rescue the embryonic death of Ddx41 hematopoietic-specific knockout mice. In parallel, genome instability also activates the cGas-Sting pathway, impairing survival, as cGas deficiency rescues the lethality of hematopoietic-specific Ddx41 knockout mice. This is supported by data from a DDX41-mutated MN patient and human iPSC-derived bone marrow organoids. Our study establishes DDX41 as a G4 resolvase, essential for erythroid genome stability and suppressing the cGAS-STING pathway.