RNA N4-acetylcytidine modification in health and cancer: molecular mechanisms, cellular plasticity, and therapeutic opportunities.

N⁴-acetylcytidine (ac⁴C) is an evolutionarily conserved RNA modification that was initially identified in transfer RNA (tRNA) and ribosomal RNA (rRNA). Increasing evidence has subsequently expanded its functional landscape to messenger RNA (mRNA) and multiple classes of non-coding RNAs (ncRNAs). As the best-characterized ac⁴C writer in mammals, N-acetyltransferase 10 (NAT10) orchestrates RNA structure, stability, processing, and translation, thereby governing fundamental physiological processes, including ribosome biogenesis, translational fidelity, proteostasis, cell fate determination, hematopoiesis, reproduction, and embryonic development. Cancer cells exploit the NAT10-ac⁴C axis to selectively stabilize or enhance the translation of oncogenic transcripts, reprogram tRNA and rRNA-dependent translational programs, and cooperate with non-canonical NAT10 functions, including R-loop regulation, protein acetylation, and RNA binding, to sustain uncontrolled proliferation, cancer stemness, metabolic reprogramming, DNA damage repair, invasion and metastasis, resistance to regulated cell death, and immune evasion. Consequently, the NAT10-ac⁴C axis has emerged as a central molecular hub linking RNA fate, the translational machinery, and adaptive tumor reprogramming. Therefore, phenotypes resulting from NAT10 depletion or pharmacological inhibition should not be indiscriminately attributed to reduced ac⁴C deposition in the absence of catalytic rescue experiments and site-specific validation. In this Review, we comprehensively summarize the molecular basis of ac⁴C, its substrate landscape, catalytic mechanisms, and determinants of substrate selectivity, as well as its physiological functions in normal tissues and pathological roles in cancer. We further compare the shared and context-dependent mechanisms by which the NAT10-ac⁴C axis drives malignant progression and therapeutic resistance across diverse cancer types. In addition, we critically evaluate current ac⁴C detection technologies, the hierarchy of substrate evidence, NAT10 inhibitors, targeted protein degradation strategies, rational combination therapies, biomarker-guided patient stratification, and therapeutic windows. Finally, we propose that future research should move beyond broad NAT10 inhibition toward identifying tumor-specific NAT10 dependencies and selectively disrupting oncogenic NAT10-RNA regulatory circuits through high-confidence ac⁴C atlases, site-specific RNA editing technologies, and precision delivery strategies.