Levetiracetam reverses temozolomide resistance in glioblastoma by blocking drug efflux through RAB5A/CD63-RAB35 axis.

Glioblastoma (GBM) cells eliminate temozolomide (TMZ) through extracellular vesicles (EVs), a resistance mechanism independent of DNA damage repair that markedly reduces intracellular drug concentration. However, strategies to block this efflux pathway remain poorly explored. Levetiracetam (LEV), a first-line antiepileptic for GBM patients, suppresses seizures by inhibiting synaptic vesicle release, but its potential role in chemotherapeutic efflux has not been investigated. Chemical proteomics identified RAB5A and CD63 as novel LEV-binding targets, and analyses of TCGA datasets further supported their clinical relevance. Mechanistic studies combining co-immunoprecipitation, immunofluorescence, and in silico modeling revealed LEV-mediated disruption of endosomal trafficking and membrane fusion, thereby inhibiting TMZ efflux. Nanoparticle tracking analysis and electron microscopy were used to evaluate EV release, while LC-MS/MS was employed to quantify TMZ at the subcellular and intratumoral levels. Orthotopic GBM models were used to evaluate therapeutic efficacy. RAB5A and CD63 were identified as dual mediators of LEV activity. LEV competitively bound RAB5A, impairing endosomal maturation and TMZ trafficking. LEV also disrupted CD63-RAB35 interaction, promoting RAB35 proteasomal degradation and suppressing plasma membrane fusion. Collectively, LEV reduced TMZ efflux and synergistically enhanced TMZ cytotoxicity in vitro, while in orthotopic models the combination therapy inhibited tumor growth, was accompanied by immune microenvironment remodeling, and prolonged survival.