Abstract 3908: M6A RNA modifications regulate expression of transcripts that promote transition to cisplatin resistance in bladder cancer

Abstract BACKGROUND: Chemotherapy resistance is recognized to occur not only through selection of pre-existing genetically resistant clones, but also through rapid phenotypic plasticity mechanisms. We previously reported that bladder cancer cells can rapidly transition to and from a chemo-resistant phenotype. Recently, N6-methyladenosine (m6A) RNA modifications were shown to dynamically and reversibly regulate mRNA processing, differentiation, and cell fate. Here, we tested the hypothesis that m6A modifications regulate phenotypic plasticity and transition to cisplatin resistance in bladder cancer (BC). METHODS: We utilized methyl-RNA-immunoprecipitation followed by sequencing (MeRIP-seq) and by RNA-seq to identify transcripts that were both differentially methylated and differentially expressed between cisplatin-sensitive and cisplatin-resistant BC cell lines. Candidate transcripts were annotated using Gene Ontology (GO) enrichment analysis and Gene Set Enrichment Analysis (GSEA). Clinical relevance was evaluated using databases such as The Cancer Genome Atlas (TCGA) and the Oncology Research Information Network (ORIEN) avatar. Cancer-relevant genes were then validated in vitro by targeted immunoprecipitation and PCR (MeRIP-PCR) and qPCR. Candidate genes were then functionally validated in cell lines and in patient derived organoids (PDOs) by siRNA-mediated knockdown and cisplatin treatment, as well as by m6A measurement after short-term cisplatin treatment. RESULTS: MeRIP-seq and RNA-seq revealed that cisplatin-sensitive and cisplatin-resistant BC cells have distinct m6A profiles, with 130 transcripts that were both differentially methylated and differentially expressed. Using clinical and functional database tools, we filtered this list to 37 transcripts and ranked them based on clinical relevance in BC. Of the top 15 candidates, eight were successfully validated in vitro via repeat qPCR and MeRIP-PCR. In cisplatin-resistant cells, one candidate, SLC7A11, was found to have decreased m6A level, which was associated with increased RNA stability and elevated transcript and protein levels. Consistent with this, 48 hr cisplatin treatment of cisplatin-sensitive cells led to SLC7A11 hypomethylation, increased RNA stability, and elevated transcript and protein levels. Furthermore, depletion of SLC7A11 by siRNA-knockdown re-sensitized BC cell lines and PDOs to cisplatin. CONCLUSIONS: Using unbiased transcriptome-wide m6A profiling followed by targeted validation, we demonstrated that m6A modifications regulate expression of clinically relevant gene transcripts in BC, and that a subset of these modifications may promote resistance to chemotherapy Collectively, these results implicate epitranscriptomic plasticity as a driver in potentiating cisplatin resistance in bladder cancer. Citation Format: Emmanuelle Hodara, Aubree Mades, Tong Xu, Amir Goldkorn, Suhn Rhie. M6A RNA modifications regulate expression of transcripts that promote transition to cisplatin resistance in bladder cancer. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3908.