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

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.