The Fanconi anemia pathway restrains MLL-rearranged leukemogenesis through suppressing non-homologous end joining-mediated genomic instability.

Mixed lineage leukemia (MLL) rearrangements drive approximately 10% of acute leukemias, including acute myeloid leukemia (AML) and acute lymphoblastic leukemia (ALL). Individuals with Fanconi anemia (FA), a hereditary DNA damage repair (DDR) disorder, are at markedly increased risk of AML, yet the interplay between FA deficiency and MLL-rearranged (MLL-r) leukemia remains unclear. Using murine models and human MLL-r leukemia cells, we show that the FA pathway suppresses leukemogenesis by limiting error-prone non-homologous end joining (NHEJ)-mediated genomic instability. Loss of FA components accelerates leukemia development, promotes leukemic stem cell expansion, and increases DNA damage through hyperactive NHEJ. Pharmacological NHEJ inhibition selectively impairs the survival of FA-deficient MLL-r leukemia cells, revealing a synthetic lethal vulnerability. Consistent with these findings, reduced FA pathway gene expression in patient samples correlates with poor prognosis and increased sensitivity to NHEJ inhibition. These findings identify NHEJ blockade as a promising targeted therapeutic strategy for high-risk MLL-r leukemia.