Targeted m⁶A demethylation of ITGA6 mRNA by a multisite dCasRx-m⁶A editor inhibits bladder cancer development

Introduction: N6-methyladenosine (m⁶A) modification contributes to the pathogenesis and development of various cancers, including bladder cancer (BCa). In particular, integrin α6 (ITGA6) promotes BCa progression by cooperatively regulating multisite m⁶A modification. However, the therapeutic effect of targeting ITGA6 multisite m⁶A modifications in BCa remains unknown.

Objectives: We aim to develop a multisite dCasRx- m⁶A editor for assessing the effects of the multisite dCasRx-m⁶A editor targeted m⁶A demethylation of ITGA6 mRNA in BC growth and progression.

Methods: The multisite dCasRx- m⁶A editor was generated by cloning. m⁶A-methylated RNA immunoprecipitation (meRIP), luciferase reporter, a single-base T3 ligase-based qPCR-amplification, Polysome profiling and meRIP-seq experiments were performed to determine the targeting specificity of the multisite dCasRx-m⁶A editor. We performed cell phenotype analysis and used in vivo mouse xenograft models to assess the effects of the multisite dCasRx-m⁶A editor in BC growth and progression.

Results: We designed a targeted ITGA6 multi-locus guide (g)RNA and established a bidirectional deactivated RfxCas13d (dCasRx)-based m⁶A-editing platform, comprising a nucleus-localized dCasRx fused with the catalytic domains of methyltransferase-like 3 (METTL3-CD) or α-ketoglutarate-dependent dioxygenase alkB homolog 5 (ALKBH5-CD), to simultaneously manipulate the methylation of ITGA6 mRNA at four m⁶A sites. The results confirmed the dCasRx-m⁶A editor modified m⁶A at multiple sites in ITGA6 mRNA, with low off-target effects. Moreover, targeted m⁶A demethylation of ITGA6 mRNA by the multisite dCasRx-m⁶A editor significantly reduced BCa cell proliferation and migration in vitro and in vivo. Furthermore, the dCasRx-ALKBH5-CD and ITGA6 multi-site gRNA delivered to 5-week-old BALB/cJNju-Foxn1nu/Nju nude mice via adeno-associated viral vectors significantly inhibited BCa cell growth.

Conclusion: Our study proposes a novel therapeutic tool for the treatment of BC by applying the multisite dCasRx-m⁶A editor while highlighting its potential efficacy for treating other diseases associated with abnormal m⁶A modifications.