Elongation factor Tu GTP binding domain containing 2-regulated alternative splicing of MutS homolog 5 drives radioresistance in recurrent isocitrate dehydrogenase-mutant glioma

BACKGROUND: Recurrent IDH-mutant gliomas frequently acquire increased radioresistance, leading to poorer outcomes. Their underlying mechanisms, however, remain largely unknown. We hypothesize that dysregulated RNA alternative splicing (AS) during IDH-mutant glioma recurrence contributes to the enhanced radioresistance by influencing critical cellular pathways. METHODS: RNA sequencing of paired primary and recurrent IDH-mutant gliomas were analyzed to identify recurrence-associated AS events. Functional effects were assessed in patient-derived glioma stem cells using RNA interference and CRISPR-dCas13-mediated isoform switching. Candidate upstream RNA-binding proteins and antisense oligonucleotide (ASO)-based therapeutic strategies were evaluated in vitro and in vivo. RESULTS: We identified differentially spliced MutS homolog 5 (MSH5) isoforms between primary and recurrent IDH-mutant gliomas. Primary gliomas predominantly expressed an exon 11/12-skipped MSH5 transcript, whereas recurrent tumors largely retained the full-length isoform. Exon 11/12 skipping introduced a premature termination codon, leading to nonsense-mediated decay and diminished MSH5 expression in primary tumors. Further analyses identified elongation factor Tu GTP binding domain containing 2 (EFTUD2) as an upstream splicing regulator that was upregulated in recurrent tumors and promoted exon 11/12 inclusion, thereby maintaining MSH5 expression and enhancing the repair of radiation-induced DNA double-strand breaks. Inducing MSH5 exon 11/12 skipping with CRISPR-dCas13 or inhibiting EFTUD2 with ASOs reduced MSH5 expression, impaired DNA repair, and sensitized recurrent IDH-mutant glioma to radiotherapy in vitro and in vivo. CONCLUSIONS: These findings identify an EFTUD2-MSH5 splicing axis that contributes to radioresistance in recurrent IDH-mutant glioma. Therapeutic disruption of this splicing program may represent a strategy to enhance the radiation response in recurrent IDH-mutant glioma.