Targeted RNA m5C demethylation of NFE2L2 mRNA by dCas13b-TET2 restores ferroptosis and overcomes cisplatin resistance in bladder cancer

Abstract

Cisplatin-based chemotherapy remains a cornerstone for the treatment of advanced bladder cancer; however, the emergence of resistance severely limits its clinical efficacy. Ferroptosis suppression has been implicated in chemotherapy resistance, yet the upstream epitranscriptomic mechanisms remain unclear. Here, we investigated the role of RNA m5C modification in cisplatin resistance and evaluated the therapeutic potential of targeted RNA demethylation. NFE2L2 mRNA and NRF2 protein expression were analyzed using public datasets, clinical specimens, and cisplatin-resistant bladder cancer cell models. Mechanistic studies included m5C-RIP, RNA immunoprecipitation (RIP), RNA pull-down, NRF2 chromatin immunoprecipitation followed by qPCR (ChIP-qPCR), promoter and RNA reporter assays, and Actinomycin D chase assays. A programmable dCas13b–TET2 RNA demethylation system was used to target NFE2L2 transcripts. Cisplatin sensitivity and ferroptosis-associated endpoints were assessed in vitro and in xenograft models. NFE2L2 mRNA and NRF2 protein were upregulated in bladder cancer tissues and cisplatin-resistant cells and were associated with poor prognosis. Reduced TET2 expression was associated with increased m5C enrichment on NFE2L2 mRNA, YBX1-dependent transcript stabilization, and higher NRF2 expression. TET2-RIP recovery controls showed comparable amounts of immunoprecipitated TET2 protein in parental and resistant cells. NRF2 ChIP-qPCR and wild-type versus mutant SLC7A11 promoter reporters supported NRF2 occupancy at the tested promoter region and sequence-dependent promoter activation. Targeted NFE2L2 mRNA demethylation reduced m5C enrichment, accelerated transcript decay, lowered NRF2 expression, enhanced lipid peroxidation, depleted glutathione, and increased cisplatin sensitivity. SLC7A11 restoration partially reversed these effects. In vivo, targeted NFE2L2 mRNA demethylation enhanced cisplatin efficacy without detectable changes in body weight or overt organ histopathology under the tested conditions. This study identifies a TET2–m5C–YBX1–NRF2–SLC7A11 regulatory axis that drives ferroptosis resistance and cisplatin resistance in bladder cancer. Targeted RNA demethylation of NFE2L2 mRNA using a dCas13b–TET2 system restores ferroptosis sensitivity and enhances cisplatin response, providing a proof-of-concept strategy for RNA epigenetic therapy in bladder cancer.