Dysregulation of the RNA modification m6A promotes cancer progression and provides therapeutic opportunities

The objective of this study is to determine how sensitive detection and quantitation of the RNA modification N 6-methyladenosine (m6A) can enable cancer detection, mechanistic insights into tumor biology, and improved monitoring of treatment responses. RNA modifications, which are co- and post-transcriptional alterations to nucleobases or ribose, are key regulators of nearly all aspects of RNA metabolism, including transcription, splicing, localization, translation, stability, and interactions with RNA-binding proteins. Among these, m6A is the most abundant modification in human mRNA and long noncoding RNA and is frequently dysregulated in cancer. To enable comprehensive analysis of RNA modifications, we applied a multiplexed proximity-barcoding assay that enriches for modified RNA fragments, encodes modification sites during cDNA synthesis, and quantifies their abundance at each locus using synthetic spike-in controls for calibration. This approach allows concurrent detection of m6A, inosine, and pseudouridine, providing a multidimensional, high-sensitivity view of the cancer epitranscriptome. Application of this assay to neuroblastoma samples and cell line-derived xenograft tumor models revealed consistent increases in m6A abundance across all tumor types examined. Notably, m6A peak patterns also distinguished MYCN-amplified (MNA⁺) from ALT-activated (ALT⁺) and low-risk neuroblastomas, demonstrating that the method can resolve biologically and clinically distinct subgroups. Treatment of neuroblastoma cell lines and xenograft-derived samples with STC-15, a small-molecule inhibitor targeting the primary m6A writer enzyme METTL3, led to a dose-dependent reduction in m6A levels measurable by this assay. METTL3 inhibition was associated with decreased tumor cell viability and increased sensitivity to chemotherapeutics such as doxorubicin. Sensitive, quantitative measurement of m6A and related RNA modifications provides a powerful strategy to detect cancer-associated epitranscriptomic dysregulation, to dissect molecular heterogeneity in neuroblastoma, and to monitor therapeutic responses to METTL3-targeted interventions.