N6-Methyladenosine (m6A) is the most prevalent internal modification among mammalian mRNAs. Recent studies show that m6A methyltransferases, METTL3 and METTL14, play important roles in bladder carcinoma (BLCA). However, the impact of YTHDF2, a crucial m6A reader, has yet to be investigated. Here, we found that YTHDF2 is frequently up-regulated at both the RNA and protein level in bladder cancers. Functionally, YTHDF2 promotes the proliferation and tumor growth of BLCA cells in vitro and in vivo, respectively. Integrative RNA-sequencing and m6A-sequencing analyses show that RIG-I is a downstream target of YTHDF2. Mechanistically, YTHDF2 binds to the coding sequence of DDX58 mRNA and mediates its degradation in an m6A-dependent manner. Knock-down of RIG-I inhibits apoptosis and promotes the proliferation of BLCA cells. Depleting DDX58 also restores the phenotype abrogated by YTHDF2 deficiency. Moreover, bladder cancer Ythdf2-deficient cells implanted orthotopically activate an innate immune response and promote the recruitment of CD8+ T lymphocytes into the tumor bed and the urothelium. Consequently, targeting YTHDF2 may be beneficial in Bacillus Calmette-Guérin (BCG) immunotherapy. Our study reveals that YTHDF2 acts as an oncogene and RIG-I as a tumor suppressor in BLCA. These findings highlight the functional importance of the m6A modification in BLCA and implicate YTHDF2 as a potential therapeutic target of BLCA treatment.

<p>Supplementary Data clean version</p>

<div>Abstract<p>N⁶-Methyladenosine (m⁶A) is the most prevalent internal modification of mammalian mRNAs. Recent studies have shown that m⁶A methyltransferases METTL3 and METTL14 play important roles in urothelial bladder carcinoma (BLCA). To provide a more comprehensive understanding of the m⁶A regulatory landscape in bladder cancer, we investigated the role of YTHDF2, a crucial m⁶A reader, in BLCA. YTHDF2 was frequently upregulated at both the RNA and protein level in BLCA. Functionally, YTHDF2 promoted the proliferation and tumor growth of BLCA cells <i>in vitro</i> and <i>in vivo</i>, respectively. Integrative RNA sequencing and m⁶A sequencing analyses identified RIG-I as a downstream target of YTHDF2. Mechanistically, YTHDF2 bound to the coding sequence of DDX58 mRNA, which encodes RIG-I, and mediated its degradation in an m⁶A-dependent manner. Knockdown of RIG-I inhibited apoptosis and promoted the proliferation of BLCA cells. Depleting RIG-I was also able to reverse the effects of YTHDF2 deficiency. YTHDF2-deficient BLCA cells implanted orthotopically in recipient mice activated an innate immune response and promoted recruitment of CD8⁺ T lymphocytes into the tumor bed and the urothelium. Moreover, YTHDF2 deficiency enhanced the efficacy of Bacillus Calmette-Guérin immunotherapy treatment. This study reveals that YTHDF2 acts as an oncogene in BLCA. YTHDF2 inhibits RIG-I to facilitate immune evasion, supporting testing YTHDF2 inhibition in combination with immunotherapy.</p>Significance:<p>YTHDF2 regulates RIG-I–mediated innate immune signaling to support bladder cancer progression, highlighting the functional importance of m⁶A modifications in bladder cancer and uncovering therapeutic opportunities to improve patient outcomes.</p></div>

<p>Supplementary table ST3</p>