Background: N6-methyladenosine (m6A), a type of RNA modification, influences the stability and translation of oncogene transcripts and plays a crucial role in glioblastoma (GBM) stemness and drug resistance. However, its role in epidermal growth factor receptor type III mutation (EGFRvIII) and temozolomide (TMZ)-resistant GBM remains unclear. This study explores the regulatory impact of m6A modification in TMZ-resistant GBM with EGFRvIII, with a focus on resistance genes and tumor stemness.

Methods: Stable EGFRvIII-expressing glioma cell lines were established and treated with TMZ for varying durations. Transcriptome sequencing was then performed to identify resistance-associated genes. The findings were further validated using publicly available transcriptomic datasets from TMZ-resistant GBM. Key genes related to prognosis were identified using Cox regression. The m6A modification patterns and mRNA expression-based stemness index (mRNAsi) were evaluated based on 27 m6A regulators and a one-class logistic regression model. Spearman correlation analysis assessed relationships between candidate genes, m6A scores, and mRNAsi. Cox regression analyzed prognostic differences in GBM patients with varying m6A and mRNAsi levels. U251 and A172 cells stably expressing EGFRvIII (U251OE, A172OE) were cultured, and the expression levels of GARNL3 and RBM15 were examined by fluorescent polymerase chain reaction and Western blotting. Furthermore, the cell proliferative capacity, viability, and apoptosis in A172OE and U251OE cells were assessed following modulation of RBM15 expression in conjunction with TMZ treatment.

Results: GARNL3 was identified as a drug-resistant gene in EGFRvIII-positive GBM, with high expression correlating with better overall survival (p < 0.05). GARNL3 correlated with m6A scores (p < 0.01, r = 0.38) and RBM15 (p < 0.001, r = 0.992). RBM15 promoted GARNL3 protein expression in A172OE and U251OE cells. Concurrently, knockdown of RBM15 not only reduced GARNL3 expression but also enhanced the proliferation and viability of A172OE and U251OE cells under TMZ treatment, while suppressing apoptosis. Conversely, overexpression of RBM15 promoted apoptosis in these cells and enhanced the tumor-inhibitory effects of TMZ. Additionally, in TMZ-resistant EGFRvIII GBM, m6A scores were significantly decreased (p < 0.01), while mRNAsi scores were increased (p < 0.001). Low m6A and high mRNAsi levels correlated with poorer prognosis (p < 0.05).

Conclusion: GARNL3 may contribute to TMZ resistance through RBM15-mediated m6A modification, thereby enhancing tumor cell stemness. However, this mechanism requires further validation.