METTL3 Increases Temozolomide Resistance in MGMT ‐Negative Glioblastoma by Enhancing CBX5 mRNA Stability in an m 6 A ‐ IGF2BP2 ‐Dependent Manner

Aims We examined whether chromobox protein 5 (CBX5) contributes to temozolomide (TMZ) resistance in O 6 ‐methylguanine‐DNA methyltransferase (MGMT)‐negative glioblastoma (GBM) and whether it is regulated by methyltransferase‐like 3 (METTL3)‐dependent N 6 ‐methyladenosine (m 6 A) 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, m 6 A enrichment, and wild‐type reporter activity but did not significantly affect the m 6 A 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 m 6 A modification and IGF2BP2‐associated stabilization help maintain CBX5 expression, contributing to TMZ resistance and ATM–RAD51‐associated signaling in MGMT‐negative GBM models.