The functional relevance of adenine mRNA methylation for the progression of bladder cancer

Urothelial carcinoma of the bladder (UCB) presents a major health challenge, and increasing evidence indicates that epitranscriptomic reprogramming contributes to tumor development. N6-methyladenosine (m6A) is the most abundant internal modification in mammalian mRNA and regulates transcripts across the mRNA life cycle. The mark is installed by the m6A writer complex, in which METTL3 serves as the catalytically active component. Although METTL3 has been implicated in many cancers, its role in UCB remains controversial, and quantitative, high-resolution maps of m6A are lacking. Thus, this study aimed to reevaluate the role of METTL3 in UCB and to generate the first quantitative, base-resolution map of m6A in cancer, thereby resolving the mechanisms underlying malignant epitranscriptomic reprogramming. METTL3 knockout and inhibition impaired the oncogenic phenotype and global m6A levels in UCB cells. A complete loss of METTL3 could not be achieved due to the selection of cells expressing aberrant METTL3 isoforms. Dependency analyses and inhibitor response assays indicated that both UCB and uroepithelial cells require METTL3 for viability. Clinically, METTL3 is upregulated in UCB. However, liquid chromatography-tandem mass spectrometry (LC-MS/MS) analyses of patient tissues found a global decrease in m6A, emphasizing the need for rigorous m6A mapping to resolve this contradiction. Accordingly, GLORI-seq was implemented to compare UCB with paratumoral tissue and showed systematic changes in the m6A profile of UCB. Two central m6A signatures were identified: a global methylation dilution driven by the upregulation of unmethylated transcripts and the downregulation of highly methylated transcripts. Conversely, differential methylation analyses found local stop-codon-proximal hypermethylation associated with an upregulation of the m6A writer complex component VIRMA. VIRMA knockdown reduced stop-codon-proximal m6A levels and impaired the oncogenic phenotype of UCB cells, suggesting a functional role for VIRMA in UCB development. Taken together, this study suggests a narrow therapeutic window for systemic METTL3 inhibition in UCB due to its essential character. However, the systematic alterations of the UCB m6A profile emphasize the diagnostic potential of m6A profiling. Finally, this work also provides first insights into the mechanisms that drive m6A epitranscriptomic reprogramming in cancer.