AARS2‐SIRT5‐Driven DLD K445 Lactylation Promotes Cuproptosis Resistance in Glioblastoma Stem Cells

ABSTRACT Therapy‐resistant glioblastoma stem cells (GSCs) drive tumor progression and recurrence in glioblastoma (GBM). Cuproptosis is a copper‐dependent form of regulated cell death triggered by aggregation of lipoylated mitochondrial proteins, but the mechanisms underlying cuproptosis resistance in GSCs remain unclear. Lactylome profiling identifies dihydrolipoamide dehydrogenase (DLD) as a key lactylation target regulating cuproptosis. Mechanistically, lactate‐dependent alanyl‐tRNA synthetase 2 (AARS2) catalyzes DLD K445 lactylation, while SIRT5 reverses this modification via NAD + ‐dependent delactylation. DLD K445 lactylation does not disrupt pyruvate dehydrogenase (PDH) complex assembly or DLD stability, but suppresses PDH activity and DLAT lipoylation, limiting copper‐triggered DLAT aggregation and cuproptosis. Genetic disruption of DLD K445 lactylation restores DLAT lipoylation and sensitizes GSCs to elesclomol‐copper (ES‐Cu). Inhibiting lactate metabolism, activating SIRT5, or a cell‐penetrating peptide targeting DLD K445 all boost ES‐Cu efficacy in patient‐derived glioblastoma organoids and intracranial xenografts. Our work establishes DLD K445 lactylation as a metabolic driver of cuproptosis resistance and validates the AARS2–DLD–SIRT5 axis as a druggable therapeutic target for GBM.