DNA methylation driven clonal splicing neoantigens predict immunotherapy response in lung cancer

Immunotherapy has transformed the treatment landscape for previously untreatable cancers, yet its effectiveness is typically associated with high tumour mutational burden. However, additional biological processes can generate immunogenic antigens independently of DNA mutations. Alternative splicing, a major mechanism of transcriptome diversification, can produce cancer-specific isoforms that give rise to neoantigens. Emerging evidence suggests that splicing-derived neoantigens may arise from coordinated dysregulation of splicing factors, including epigenetic alterations such as DNA methylation. Here, we developed an integrated multi-omics framework that combines paired methylome and transcriptome data to identify cancer-specific, splicing-derived epitopes driven by DNA-methylation changes at gene loci. We applied this framework to the DARWIN II cohort of non-small cell lung cancer (NSCLC) patients treated with immune checkpoint inhibitors (CPI), leveraging multi-region paired RRBS and RNA-seq data from 256 tumour regions across 72 patients. Using this approach, we uncovered a compendium of recurrent clonal splicing-derived neoantigens linked to DNA-methylation alterations, several of which were predictive of clinical benefit from CPI therapy. Our findings demonstrate that DNA methylation-mediated splicing regulation represents a source of clonal neoantigens in lung cancer, providing new avenues for patient stratification and informing future vaccine and immunotherapy development.