METTL3 Increases Temozolomide Resistance in MGMT-Negative Glioblastoma by Enhancing CBX5 mRNA Stability in an m6A-IGF2BP2-Dependent Manner.

AIMS: We examined whether chromobox protein 5 (CBX5) contributes to temozolomide (TMZ) resistance in O6-methylguanine-DNA methyltransferase (MGMT)-negative glioblastoma (GBM) and whether it is regulated by methyltransferase-like 3 (METTL3)-dependent N6-methyladenosine (m6A) and insulin-like growth factor 2 mRNA-binding protein 2 (IGF2BP2).

METHODS: Public datasets, clinical specimens, MGMT-negative cells, and subcutaneous xenografts were examined using functional, mechanistic, and rescue assays.

RESULTS: In the CGGA693 cohort of the Chinese Glioma Genome Atlas, higher CBX5 expression remained associated with shorter survival after multivariable adjustment (hazard ratio 1.178, 95% confidence interval 1.050-1.322; p = 0.005). CBX5 depletion increased TMZ sensitivity, apoptosis, and DNA damage, reduced ATM phosphorylation and RAD51 abundance, and restricted TMZ-treated xenograft growth; overexpression produced reciprocal effects in vitro. METTL3 depletion reduced CBX5 expression, mRNA stability, m6A enrichment, and wild-type reporter activity but did not significantly affect the m6A motif-mutant reporter. IGF2BP2 depletion reduced CBX5 protein and mRNA stability, whereas RNA immunoprecipitation supported an IGF2BP2-CBX5 mRNA association. CBX5 re-expression partially reversed METTL3 depletion-induced effects on viability and DNA damage.

CONCLUSION: METTL3-dependent m6A modification and IGF2BP2-associated stabilization help maintain CBX5 expression, contributing to TMZ resistance and ATM-RAD51-associated signaling in MGMT-negative GBM models.