Transcriptome-wide mapping reveals an RNA-dependent mechanism of platinum cancer drugs

Small molecules developed to target proteins or DNA may also bind RNA, but the extent and biological significance of such interactions among oncology drugs remain poorly defined. Here, we systematically profiled RNA interactions of a cohort of clinically approved anticancer agents and uncovered widespread RNA off-targeting. Cisplatin, a frontline chemotherapeutic agent for solid tumors, has emerged as a prominent RNA-binding drug. While the primary mechanism of action of cisplatin has been attributed to DNA damage-induced apoptosis, it has also been shown to bind RNA molecules. However, the extent of RNA binding in cancer cells and its functional relevance in platinum-based chemotherapy remained unknown. To map specific RNA targets of cisplatin <i>in vivo</i>, we developed PlatRNA-seq, a click-chemistry-enabled transcriptome-wide assay. Using this approach and integrated genomic, biophysical, and computational analysis, we show that cisplatin binding is enriched at guanine-rich regions of transcripts, with a pronounced affinity for RNA G-quadruplexes (rG4s) secondary structures. Cisplatin accumulates preferentially near the 5' ends of transcripts associated with R-loop formation and RNA pol II stalling. Mechanistically, cisplatin binding to rG4s modulates their formation and stability. Importantly, we provide evidence that cisplatin-induced cytotoxicity is mediated in part through its binding to RNA, revealing a noncanonical RNA-based mechanism of action. Analysis of single-cell RNA-seq data from tumor biopsies of treatment-naïve ovarian cancer patients further shows that the expression of rG4-enriched cisplatin-RNA targets predicts platinum sensitivity, underscoring the prognostic and clinical relevance of drug-RNA interactions. Together, these results demonstrate that RNA off-targeting by small molecules is not passive but can modulate therapeutic outcomes and may be leveraged to overcome current limitations of chemotherapeutic agents. Our findings highlight the importance of systematically investigating RNA interactions of clinically used small molecules to better inform therapeutic and prognostic strategies.