Epitranscriptomic regulation of NVP-BEZ235 resistance in ccRCC via the YTHDF3-SYNM regulatory pathway

NVP-BEZ235 is a dual PI3K/mTOR inhibitor with promising antitumor activity in clear cell renal cell carcinoma (ccRCC). However, the mechanisms underlying acquired drug resistance remain incompletely defined.

To investigate whether epitranscriptomic regulation-specifically N6-methyladenosine (m6A) modification and its reader YTHDF3-drives reversible, non-genetic resistance to NVP-BEZ235 in ccRCC, and to identify key downstream effector genes.

NVP-BEZ235-resistant models were established from 786-O and PTEN-deficient Caki-1ko ccRCC cells via chronic drug exposure. Drug withdrawal assays were performed to evaluate the reversibility of resistance. Expression of m6A regulators was assessed by qRT-PCR and Western blotting. YTHDF3 was silenced using lentiviral shRNA, and changes in NVP-BEZ235 sensitivity were evaluated by CCK-8 and cell-cycle assays. Genome-wide m6A-RIP-seq and RNA-seq were integrated to define transcripts with coordinated changes in m6A methylation and expression, and core causal genes for ccRCC were prioritized by summary-based Mendelian randomization (SMR). YTHDF3-RNA interactions and dynamic m6A changes were validated by RIP-qPCR and MeRIP-qPCR, and the functional role of SYNM was tested by loss-of-function studies.

NVP-BEZ235-resistant cells displayed a reversible, nonheritable phenotype, partially regaining drug sensitivity after short-term drug withdrawal. YTHDF3 was selectively upregulated in resistant cells, and its knockdown significantly lowered the IC50 of NVP-BEZ235 and suppressed proliferation. Integrated m6A-RIP-seq/RNA-seq analyses revealed extensive reprogramming of m6A methylation, and SMR identified SYNM as a core m6A-regulated gene associated with increased ccRCC risk. SYNM was overexpressed in ccRCC tissues and positively correlated with YTHDF3 expression. YTHDF3 bound SYNM mRNA in resistant cells, while m6A levels and expression of both YTHDF3 and SYNM were dynamically reversible upon drug withdrawal. Silencing SYNM robustly resensitized resistant cells to NVP-BEZ235.

Reversible resistance to NVP-BEZ235 in ccRCC is driven, at least in part, by an m6A-dependent YTHDF3-SYNM axis. Targeting YTHDF3 or SYNM may provide a rational strategy to overcome PI3K/mTOR inhibitor resistance in ccRCC.