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

The true abundance and distribution of mRNA ac⁴C remain among the most fundamental controversies in the field of RNA acetylation. Early antibody-enrichment studies detected ac⁴C signals across a large number of mammalian mRNAs and proposed that this modification is preferentially enriched within coding regions, where it broadly enhances RNA stability and translational efficiency. However, subsequent studies using highly purified poly(A)⁺ RNA, LC–MS/MS, and antibody-independent chemical sequencing suggested that the abundance of ac⁴C in mammalian mRNAs may be substantially lower than initially estimated. Because rRNAs and tRNAs are intrinsically enriched in ac⁴C, even minimal contamination, antibody cross-reactivity, or RNA structure-dependent enrichment can generate false-positive signals or overestimate modification abundance. Nevertheless, inconsistent detection does not necessarily indicate that mRNA ac⁴C is absent. An alternative possibility is that mRNA ac⁴C is not a constitutive and widely distributed modification but instead represents a low-abundance, condition-dependent mark that emerges in specific tissues, developmental stages, stress states, or disease contexts. Differences in NAT10 expression, adaptor-protein composition, RNA structure, and metabolic state may all alter the accessible substrate repertoire across cell types. The critical question is therefore no longer whether mRNA ac⁴C simply “exists” or “does not exist,” but rather in which biological contexts, on which RNAs, and at which sites it can be reproducibly and orthogonally validated. The concept that ac⁴C enhances mRNA stability has become the dominant model used to explain the oncogenic functions of NAT10. However, current evidence indicates that this principle is not universal. Across different studies, ac⁴C has been associated with increased RNA half-life, enhanced translational efficiency, altered translation initiation, and even reduced stability of selected transcripts. Its functional outcome is likely determined by the modification position, local sequence and RNA structure, ribosome occupancy, codon composition, and associated RBPs. ac⁴C within coding regions may directly influence ribosomal decoding and translation elongation, whereas modifications in the 5′UTR or near the start codon can alter translation-initiation-site selection. ac⁴C in the 3′UTR may instead affect RNA decay by remodeling local structure or RNA–protein interactions. ac⁴C should therefore not be defined as a single-purpose “stabilizing mark.” It is more appropriately viewed as a position- and context-dependent signal that modulates RNA fate. Future studies should move beyond transcript-wide correlative analyses toward site-level causal validation to establish the functional rules governing ac⁴C across distinct sequence and structural environments. Numerous studies have reported reduced proliferation, metabolic disruption, DNA damage accumulation, and increased therapeutic sensitivity following NAT10 knockdown, knockout, or pharmacological inhibition and have directly attributed these phenotypes to loss of RNA ac⁴C. However, NAT10 is not a single-function RNA acetyltransferase. It also participates in RNA binding, protein acetylation, protein–protein interactions, R-loop regulation, ribosome biogenesis, and phase separation. Consequently, the global phenotype resulting from NAT10 loss cannot automatically be equated with an ac⁴C catalytic effect. Future studies should distinguish among the consequences of complete NAT10 protein loss, selective loss of catalytic activity, removal of ac⁴C at a specific RNA site, and impaired acetylation of protein substrates. Parallel rescue with wild-type and catalytically inactive NAT10, site-directed RNA mutation or editing, and domain-specific manipulation of NAT10 should become standard approaches for establishing causality. A phenotype can be confidently classified as a direct RNA-acetylation effect only when restoration of the relevant ac⁴C site is sufficient to rescue it. Unlike m⁶A, ac⁴C has not yet been incorporated into a widely accepted writer–reader–eraser framework. NAT10 is currently the most clearly established mammalian ac⁴C writer, but candidate recognition proteins such as eEF2 and MYBBP1A have been identified primarily in specific cancer models and on selected RNA substrates. Their ability to directly, specifically, and broadly recognize ac⁴C has not been demonstrated. At present, they are therefore better classified as candidate or context-dependent readers rather than canonical ac⁴C readers. Likewise, no physiological RNA ac⁴C eraser has been consistently validated. Dynamic changes in ac⁴C abundance may depend mainly on regulation of NAT10 activity, RNA turnover, and synthesis of new transcripts rather than on a dedicated deacetylation reaction. ac⁴C may also function without specialized reader proteins by directly altering base pairing, RNA folding, codon decoding, or the affinity of RBPs. Applying the m⁶A regulatory model too rigidly may therefore constrain our understanding of the distinct chemical and structural functions of ac⁴C. Although most cancer studies describe NAT10 as an oncogenic factor, its function is highly dependent on substrate composition, tissue context, and cellular state. NAT10 can stabilize MYC, RAD51, SLC7A11, and multiple transcripts involved in metabolism or immune evasion, but it may also regulate DDB2, p21, or other inhibitory pathways. In selected settings, NAT10 can promote cuproptosis or maintain beneficial immune and inflammatory responses. Elevated NAT10 expression therefore does not necessarily indicate functional dependency, nor does it prove that all downstream effects are tumor promoting. Future studies should move beyond asking whether NAT10 is highly expressed and instead determine whether a tumor is addicted to a specific NAT10 functional module. Such dependency may arise from a particular RNA substrate, repair pathway, metabolic state, immune environment, or co-mutational background. Classifying tumors according to functional dependency rather than expression correlation will be essential for reconciling divergent findings and improving patient stratification. NAT10 contributes to HSC maintenance, megakaryocyte–erythroid differentiation, oocyte maturation, embryonic development, T-cell expansion, and ribosome homeostasis. Sustained systemic blockade may therefore damage normal stem cells, rapidly proliferating tissues, and immune function. This concern reflects potential on-target toxicity arising from the physiological functions of NAT10 rather than conventional off-target drug effects. The feasibility of NAT10-targeted therapy will ultimately depend on whether exploitable differences exist between tumors and normal tissues in dose dependence, timing, spatial distribution, or substrate reliance. Partial catalytic inhibition, intermittent dosing, tumor-targeted delivery, low-dose synthetic-lethal combinations, and selective disruption of specific NAT10–RNA axes may offer wider therapeutic windows than continuous, systemic, and complete inhibition. Future studies should evaluate antitumor activity in parallel with effects on normal hematopoiesis, immunity, reproduction, and tissue regeneration. Short-term tumor shrinkage in xenograft models alone is insufficient to establish clinical safety. A major priority is to establish an orthogonally validated human ac⁴C atlas. Such a resource should separately characterize mRNAs, tRNAs, rRNAs, and different classes of ncRNAs and compare their dynamic modification patterns across normal tissues, tumors, developmental stages, and treatment conditions. Antibody-enrichment datasets should be integrated with chemical sequencing, LC–MS/MS, and site-specific validation rather than treating enrichment peaks as definitive modification sites. As low-input and spatial technologies continue to advance, single-cell and spatial ac⁴C maps should help resolve modification heterogeneity among tumor cells, immune cells, and stromal populations. Manipulating NAT10 expression alone cannot establish the function of an individual ac⁴C site. Future studies should develop site-specific ac⁴C installation tools based on Cas13, programmable RNA-binding proteins, or other modular systems and should explore inducible demodification, RNA replacement, or site-blocking strategies. Such platforms must demonstrate modification specificity, low off-target activity, and reversibility. Site-level causality should then be evaluated through measurements of RNA half-life, RNA structure, ribosome occupancy, and protein output. The functional principles of ac⁴C must expand beyond a simple RNA-stability model toward an integrated framework incorporating RNA sequence, structure, and translational kinetics. Future studies should combine RNA structuromics such as SHAPE-MaP with ribosome profiling, tRNA sequencing, codon-resolution pausing analysis, and quantitative proteomics to determine how ac⁴C affects translation initiation, elongation, ribosome stalling, and RBP recruitment. It will be particularly important to distinguish site-specific effects on mRNAs from system-level translational changes caused by tRNA and rRNA acetylation. Different tumors may depend on NAT10-mediated HR repair, metabolic reprogramming, R-loop homeostasis, stemness maintenance, or immunosuppression. Future studies should integrate CRISPR screening, DepMap analyses, multi-omics, and patient-derived organoids to establish functional NAT10 dependency maps and validate them in patient-derived xenografts, genetically engineered mouse models, and immunocompetent systems. Patient selection should be based on functional dependency, specific ac⁴C substrates, and pathway state rather than solely on NAT10 expression or total ac⁴C abundance. Future therapeutic strategies should shift from broad NAT10 inhibition toward precise disruption of tumor-specific NAT10 functions. Structure-guided catalytic or allosteric inhibitors, RNA-binding-interface inhibitors, and targeted protein degraders may selectively interfere with distinct functional modules. Antisense oligonucleotides, RNA degraders, and programmable RNA-editing tools could instead directly target oncogenic ac⁴C substrates. Tumor-targeted nanodelivery, antibody- or ligand-conjugated systems, and local administration may reduce exposure of normal tissues. The central objective should not be maximal NAT10 suppression but selective interruption of tumor-specific regulatory circuits while preserving physiological RNA homeostasis. NAT10 expression and total ac⁴C abundance are unlikely to independently predict treatment response with sufficient accuracy. Future companion diagnostics should integrate specific ac⁴C substrates, NAT10 dependency scores, DDR or R-loop features, metabolic states, and immune-infiltration patterns. Dynamic changes during treatment may provide a more accurate measure of target engagement and pharmacodynamic activity than baseline levels alone. The feasibility of detecting ac⁴C in circulating RNAs, exosomal RNAs, and other liquid-biopsy samples for treatment monitoring also warrants systematic evaluation. Overall, ac⁴C research is progressing from modification discovery toward causal mechanism and precision intervention. The central questions are no longer merely whether RNA contains ac⁴C, but which RNAs are reproducibly modified in specific cellular and disease contexts, how these sites alter RNA fate, and whether they can be targeted safely and selectively.

ac⁴C is one of the earliest RNA chemical modifications identified in both prokaryotic and eukaryotic organisms and remains the only known acetylation occurring at the N4 amino group of cytidine [ 63 ]. Although ac⁴C has recently regained considerable attention owing to the rapid expansion of epitranscriptomics and the recognition of its potential regulatory functions in mRNAs, its discovery dates back to the 1960s and 1970s [ 63 ]. Initial studies, primarily based on chromatographic separation and mass spectrometric analyses, identified ac⁴C in bacterial and yeast tRNAs and rRNAs, establishing this modification as a highly conserved feature throughout evolution [ 64 ]. For several decades, ac⁴C was therefore regarded principally as a structural RNA modification involved in tRNA and rRNA maturation and in maintaining the fidelity of protein translation [ 64 ]. Only with the advent of high-throughput epitranscriptomic profiling technologies, including ac⁴C RNA immunoprecipitation sequencing (acRIP-seq) and ac⁴C-seq, has its broader regulatory landscape within mRNAs and other RNA species begun to emerge [ 65 ]. To understand the diverse biological functions of NAT10, it is first necessary to consider its biochemical properties and RNA substrate specificity. Figure 1 summarizes the structural organization of NAT10, its catalytic mechanism for ac⁴C installation, and the spectrum of currently established and proposed RNA substrates. Fig. 1 Structural organization, catalytic mechanism, and RNA substrate landscape of NAT10. NAT10 contains an RNA-binding domain, a RecD-like ATPase/helicase domain, and a GNAT acetyltransferase domain that cooperate to recognize RNA substrates and catalyze ac⁴C deposition using acetyl-CoA. ATP-dependent RNA remodeling facilitates exposure of target cytidines for acetylation. Biochemically validated substrates include rRNA and tRNA, whereas accumulating sequencing evidence supports ac⁴C modification of mRNA, lncRNA, circRNA, pri-miRNA, and viral RNA Structural organization, catalytic mechanism, and RNA substrate landscape of NAT10. NAT10 contains an RNA-binding domain, a RecD-like ATPase/helicase domain, and a GNAT acetyltransferase domain that cooperate to recognize RNA substrates and catalyze ac⁴C deposition using acetyl-CoA. ATP-dependent RNA remodeling facilitates exposure of target cytidines for acetylation. Biochemically validated substrates include rRNA and tRNA, whereas accumulating sequencing evidence supports ac⁴C modification of mRNA, lncRNA, circRNA, pri-miRNA, and viral RNA From an evolutionary perspective, the ac⁴C modification system is remarkably conserved across virtually all domains of life, including bacteria, archaea, fungi, plants, and mammals, underscoring its fundamental biological importance [ 66 , 67 ]. In prokaryotes, ac⁴C is predominantly detected in 16S rRNA and a subset of tRNAs, particularly tRNA Met , tRNA Ser , and tRNA Leu [ 68 , 69 ]. These modifications stabilize the three-dimensional architecture of the anticodon stem–loop, enhance codon–anticodon recognition, and minimize near-cognate codon mispairing, thereby ensuring translational fidelity. In budding yeast, ac⁴C is catalyzed by the RNA acetyltransferase Kre33, which plays indispensable roles in 18S rRNA processing, small ribosomal subunit assembly, and ribosome biogenesis [ 70 , 71 ]. Notably, the mammalian ortholog of Kre33, NAT10, is highly conserved in both structure and catalytic function. In addition to maintaining ac⁴C deposition in rRNAs and tRNAs, NAT10 has acquired the capacity to acetylate mRNAs, suggesting that the biological repertoire of the ac⁴C regulatory system has undergone substantial functional expansion during the evolution of higher eukaryotes. The distribution of ac⁴C differs markedly among distinct RNA classes, reflecting their specialized biological functions [ 72 , 73 ]. Within rRNAs, ac⁴C is predominantly localized to highly conserved regions of 18S rRNA, particularly in close proximity to the ribosomal decoding center. Because this region directly coordinates codon–anticodon recognition during translation, ac⁴C contributes to translational accuracy by optimizing local RNA architecture and stabilizing ribosomal conformation [ 74 ]. Collectively, these findings establish ac⁴C as far more than a structural component required for RNA maturation; instead, it represents an integral determinant of translational fidelity and ribosome function. The emergence of high-throughput epitranscriptomic technologies has fundamentally reshaped our understanding of ac⁴C biology. A landmark study by Arango and colleagues first demonstrated that ac⁴C is present in mammalian mRNAs and identified NAT10 as the only acetyltransferase currently known to catalyze mRNA ac⁴C deposition [ 34 ]. Unlike the predominantly structural functions of ac⁴C in rRNAs and tRNAs, mRNA-associated ac⁴C primarily participates in post-transcriptional gene regulation by enhancing transcript stability, promoting translational efficiency, facilitating ribosome engagement, and modulating mRNA half-life [ 20 ]. Genome-wide mapping analyses further revealed that mRNA ac⁴C is preferentially enriched within CDSs and, to a lesser extent, the 3′UTR, where its distribution closely correlates with translational output [ 20 ]. Mechanistically, ac⁴C enhances local RNA structural stability and facilitates ribosome elongation, thereby coupling RNA structural remodeling with protein synthesis. Consequently, ac⁴C should be viewed not merely as a structural RNA modification but as a multifunctional epitranscriptomic mark that integrates RNA architecture with post-transcriptional gene regulation [ 75 ]. Taken together, the ac⁴C modification system has remained evolutionarily conserved from bacteria to mammals while progressively expanding its functional repertoire. Its biological role has evolved from safeguarding RNA structural integrity and translational fidelity to orchestrating mRNA fate and dynamic gene expression programs. This evolutionary transition, from a “guardian of the translational machinery” to a versatile regulator of gene expression, highlights the remarkable adaptability of the RNA acetylation system and provides an important conceptual framework for understanding how ac⁴C contributes to tumorigenesis, metabolic reprogramming, stem cell fate determination, and therapeutic resistance. As the molecular mechanisms governing NAT10 and its downstream regulatory networks continue to be elucidated, the NAT10–ac⁴C axis has rapidly emerged as one of the most active areas of epitranscriptomics research and is increasingly recognized as a pivotal molecular bridge linking RNA structural regulation, translational control, and human disease. The expanding repertoire of NAT10 substrates has considerably broadened our understanding of ac⁴C biology. Beyond its canonical modification of rRNA and tRNA, NAT10 catalyzes ac⁴C deposition on diverse coding and non-coding RNAs, thereby regulating RNA stability, translation, RNA processing, and virus–host interactions. The currently validated RNA substrates and their biological functions are summarized in Table  1 . Table 1 NAT10-mediated ac⁴C RNA substrates and their biological functions RNA class Substrate Major function / mechanism Biological context Ref mRNA mRNAs Regulates transcript stability and protein output in a position-dependent manner. CDS ac⁴C generally supports mRNA stability, codon decoding, translational elongation, and protein synthesis, whereas 5′UTR/Kozak-site ac⁴C can attenuate canonical AUG initiation and favor uORF usage. General mammalian mRNA regulation [ 34 ] tRNA tRNAs Stabilizes the anticodon stem–loop, preserves tRNA integrity, improves codon–anticodon pairing and decoding accuracy, and supports codon-biased translation rather than uniformly increasing global protein synthesis. Translational fidelity and codon-biased proteome remodeling [ 23 ] tRNA ac⁴C-modified tRNAs linked to EGFR translation NAT10-dependent tRNA ac⁴C selectively supports translation of cognate-codon-enriched mRNAs, including EGFR; NAT10 loss reduces EGFR protein with limited effect on transcript abundance. Esophageal squamous cell carcinoma [ 76 ] tRNA Specific ac⁴C-modified tRNAs regulated by RNPS1–NAT10 Enhanced tRNA ac⁴C promotes translation of proteins associated with IL-6, IL-8, and PTEN-related signaling, thereby supporting malignant progression. Head and neck squamous cell carcinoma [ 77 ] tRNA tRNA^Ser-CGA-1-1 NAT10-mediated ac⁴C enhances decoding capacity and promotes efficient translation of KSHV lytic transcripts, increasing viral protein synthesis and virion production. KSHV reactivation [ 78 ] tRNA Chromatin-associated tRNAs ac⁴C on chromatin-associated tRNAs supports p300/CBP activity, enhancer activation, expression of genes involved in prometastatic myeloid-cell recruitment, and pulmonary metastasis. Breast cancer metastasis [ 79 ] rRNA rRNAs (including 18S rRNA-associated functions) Maintains ribosome architecture, assembly, 18S rRNA processing, small-subunit maturation, translational fidelity, nucleolar homeostasis, and stress-adaptive ribosome stability. Ribosome biogenesis and translational homeostasis [ 23 ] lncRNA CD2BP2-DT ac⁴C increases lncRNA stability; accumulated CD2BP2-DT promotes YBX1 liquid–liquid phase separation, stabilizes CDK1 mRNA, and accelerates cell-cycle progression. Breast cancer [ 30 ] lncRNA SIMALR ac⁴C stabilizes SIMALR, which binds eEF1A2, enhances its GTPase activity/phosphorylation, and promotes ITGB4/ITGA6 translation, facilitating proliferation and metastasis. Nasopharyngeal carcinoma [ 80 ] lncRNA CTC-490G23.2 ac⁴C stabilizes the lncRNA, enabling PTBP1–CD44 pre-mRNA scaffolding and a CD44s-to-CD44v8–10 isoform switch; CD44v8–10 stabilizes vimentin and promotes invasion/metastasis. Esophageal squamous cell carcinoma [ 81 ] lncRNA XIST ac⁴C stabilizes XIST; XIST recruits hnRNPK and promotes YAP1 nuclear translocation, activating TEAD4-dependent VEGFA transcription, aberrant angiogenesis, and immunosuppression. Gastric cancer [ 82 ] circRNA circANKRD12 ac⁴C promotes non-canonical translation of circANKRD12 into circANKRD12_354aa, which engages the HDAC2–c-Myc axis to enhance myeloma proliferation and suppress NK-cell cytotoxicity. Multiple myeloma / tumor–immune communication [ 32 ] pri-miRNA pri-miRNAs NAT10–THUMPD1-mediated ac⁴C strengthens pri-miRNA interaction with DGCR8, facilitating Microprocessor recognition and pri-miRNA-to-pre-miRNA processing, thereby increasing mature miRNA production. miRNA biogenesis; lung cancer models [ 31 ] Viral RNA Minute virus of canines (MVC) RNA, nt 3311 ac⁴C promotes CPSF5 binding and regulates viral RNA alternative splicing and polyadenylation, ensuring accurate RNA processing and efficient viral replication. Minute virus of canines [ 83 ] Viral lncRNA KSHV polyadenylated nuclear RNA (PAN) ac⁴C stabilizes PAN and promotes the transition from latency to lytic replication, increasing lytic gene expression and virion production; the pathway also interfaces with host inflammatory signaling. Kaposi’s sarcoma-associated herpesvirus [ 84 ] NAT10-mediated ac⁴C RNA substrates and their biological functions The discovery of ac⁴C in mRNAs has substantially broadened the functional landscape of RNA acetylation beyond its canonical roles in tRNAs and rRNAs. Current evidence suggests that NAT10-mediated ac⁴C deposition primarily regulates gene expression by modulating transcript stability and translational output [ 34 ]. However, unlike classical structural RNA modifications, the biological consequences of mRNA ac⁴C are highly context-dependent rather than universally conserved. Increasing evidence indicates that its regulatory effects are jointly determined by the modification site, local sequence and structural context, translational status, and cellular environment [ 34 ]. Early transcriptome-wide studies consistently demonstrated that mRNA ac⁴C is preferentially enriched within CDSs. Loss of NAT10 reduces the half-life and steady-state abundance of numerous ac⁴C-containing transcripts and is accompanied by diminished protein synthesis, supporting a general role for ac⁴C in maintaining transcript stability and translational efficiency [ 34 ]. Mechanistically, ac⁴C within CDSs is proposed to improve ribosomal decoding of cytidine-containing codons, particularly those harboring cytidine at the wobble position, thereby coupling codon usage with translation elongation. Through this mechanism, ac⁴C establishes a functional link between RNA stability, codon optimality, and protein production, suggesting that RNA acetylation can directly influence the efficiency of genetic decoding. Nevertheless, the function of mRNA ac⁴C cannot be simply defined as a universal translation-promoting mark. Rather, accumulating evidence indicates that its biological effects are strongly position dependent [ 85 ]. Whereas ac⁴C within CDSs generally facilitates translational elongation and protein synthesis, ac⁴C located in the 5′UTR can reshape translation initiation. Notably, when ac⁴C is deposited within the Kozak sequence surrounding the canonical AUG start codon, it disrupts interactions between mRNA cytidine residues and the modified nucleotide at position 37 of initiator tRNA, thereby attenuating canonical AUG-dependent initiation while favoring upstream open reading frame (uORF) utilization [ 85 ]. These findings demonstrate that mRNA ac⁴C functions as a position-sensitive regulatory signal capable of either enhancing or suppressing protein synthesis depending on its spatial context within individual transcripts. The physiological significance of this regulatory mechanism has been validated in multiple pathological settings. Following vascular injury, NAT10-mediated ac⁴C stabilizes integrin subunit beta 1 (ITGB1) and collagen type I alpha 2 chain (COL1A2) mRNAs, thereby potentiating integrin β1–focal adhesion kinase (FAK) signaling and promoting vascular smooth muscle cell proliferation, phenotypic switching, and neointimal hyperplasia [ 18 ]. This study illustrates how selective ac⁴C deposition can preferentially preserve transcripts involved in cell adhesion and extracellular matrix remodeling, ultimately translating post-transcriptional regulation into tissue remodeling phenotypes [ 85 ]. Similar transcript-selective mechanisms have subsequently been reported in multiple malignancies, where NAT10 preferentially stabilizes oncogenic, metabolic, stemness-associated, and immune-regulatory transcripts, highlighting the remarkable substrate specificity of the mammalian ac⁴C machinery. Importantly, regulation of mRNA fate by NAT10 is not invariably dependent on its acetyltransferase activity [ 23 ]. In HSCs, NAT10 directly recognizes AREs within extended 3′UTRs of stemness-associated transcripts and facilitates ribosome recruitment, thereby protecting these mRNAs from degradation independently of ac⁴C deposition. This non-catalytic RNA-binding function is indispensable for HSC self-renewal and long-term hematopoietic reconstitution [ 86 ]. Consequently, transcriptomic alterations observed following NAT10 depletion should not be automatically interpreted as evidence of ac⁴C-dependent regulation. Distinguishing catalytic ac⁴C-dependent mechanisms from acetylation-independent RNA-binding activities has therefore become a critical prerequisite for accurately defining the biological functions of the NAT10–ac⁴C axis. Despite growing evidence supporting regulatory roles for mRNA ac⁴C, its endogenous abundance and transcriptome-wide distribution remain among the most actively debated issues in the field. Antibody-based enrichment approaches have identified thousands of putative ac⁴C-containing mRNAs in mammalian cells, supporting a widespread distribution of this modification. In contrast, antibody-independent chemical sequencing performed at single-nucleotide resolution has failed to reproducibly detect abundant endogenous mRNA ac⁴C in either mammalian cells or yeast under physiological conditions, instead identifying only a limited number of modified sites following exogenous overexpression of the NAT10 acetyltransferase complex [ 23 ]. These discrepancies likely arise from multiple technical factors, including antibody cross-reactivity, contamination by highly abundant rRNAs and tRNAs, RNA structure-dependent detection biases, sample preparation procedures, and computational analysis pipelines. Therefore, current estimates of the mammalian mRNA ac⁴C landscape should be interpreted with appropriate caution. Collectively, available evidence supports a model in which mRNA ac⁴C modulates protein output through coordinated regulation of transcript stability, codon decoding, translational elongation, and initiation-site selection. However, these effects are highly dependent on modification position, transcript context, and cellular state rather than representing a universal regulatory principle. Future studies integrating antibody-independent single-nucleotide mapping, LC–MS/MS, site-directed mutagenesis, and catalytic-dead rescue strategies will be essential for distinguishing authentic functional ac⁴C sites from technical artifacts and for defining the genuine contribution of mRNA ac⁴C to post-transcriptional gene regulation. Unlike mRNA ac⁴C, which primarily regulates transcript fate, ac⁴C deposited on tRNAs directly influences the translational machinery by modulating tRNA stability, decoding capacity, and codon recognition. As one of the earliest identified and most evolutionarily conserved substrates of RNA acetylation, tRNA-associated ac⁴C has long been recognized as an essential determinant of translational fidelity. By stabilizing the anticodon stem–loop, preserving tRNA structural integrity, and optimizing codon–anticodon pairing, ac⁴C enhances decoding accuracy and translational efficiency. Importantly, however, its biological function extends well beyond globally promoting protein synthesis. Emerging evidence suggests that tRNA ac⁴C selectively favors the translation of transcripts enriched for specific codons, thereby reshaping the proteome in a codon-dependent manner rather than uniformly increasing translational output. This concept has been substantiated by recent studies in cancer. In esophageal squamous cell carcinoma (ESCC), NAT10 deficiency markedly reduces the abundance of multiple ac⁴C-modified tRNAs and selectively impairs the translation of mRNAs enriched for their corresponding cognate codons. Among the affected downstream targets, EGFR represents a critical effector of this translational program. Inhibition of NAT10 decreases EGFR protein expression without proportionally affecting its transcript abundance and exhibits synergistic antitumor activity when combined with the EGFR inhibitor gefitinib [ 76 ]. These findings establish a mechanistic framework in which the NAT10–tRNA ac⁴C axis couples codon usage to oncogenic protein synthesis, allowing tumor cells to preferentially produce growth-promoting proteins without requiring widespread transcriptional reprogramming. In addition to catalytic activity, the abundance of NAT10 protein itself represents an important upstream determinant of tRNA ac⁴C homeostasis. In head and neck squamous cell carcinoma (HNSCC), RNA-binding protein with serine-rich domain 1 (RNPS1) directly associates with NAT10 and protects it from ZSWIM6-mediated ubiquitin-dependent degradation. Stabilization of NAT10 consequently enhances ac⁴C deposition on specific tRNAs, thereby promoting the translation of proteins involved in interleukin-6 (IL-6), interleukin-8 (IL-8), and phosphatase and tensin homolog (PTEN)-associated signaling pathways, ultimately driving malignant progression [ 77 ]. These observations indicate that post-translational regulation of NAT10 can indirectly amplify multiple oncogenic signaling networks by remodeling the tRNA acetylation landscape. Recent studies further suggest that NAT10 activity toward tRNAs is itself dynamically regulated by post-translational modifications. During Kaposi’s sarcoma-associated herpesvirus (KSHV) reactivation, α-tubulin N-acetyltransferase 1 (ATAT1)-mediated lactylation of NAT10 enhances its catalytic activity toward tRNA Ser -CGA-1-1, resulting in increased ac⁴C deposition and more efficient translation of viral lytic transcripts. Disruption of the corresponding ac⁴C site markedly reduces viral protein synthesis and virion production [ 78 ]. This mechanism illustrates how metabolic signals can be transmitted to the translational apparatus through modification of NAT10 itself, thereby enabling selective translational reprogramming under conditions of cellular stress or viral infection. Unexpectedly, the biological consequences of tRNA ac⁴C may extend beyond conventional protein synthesis. A recent study in breast cancer demonstrated that loss of NAT10 decreases ac⁴C modification on chromatin-associated tRNAs, leading to impaired p300/CREB-binding protein (CBP) activity, reduced genome-wide enhancer activation, diminished expression of genes involved in prometastatic myeloid cell recruitment, and suppression of pulmonary metastasis [ 79 ]. These findings raise the intriguing possibility that tRNA ac⁴C may influence chromatin organization and enhancer activity through previously unrecognized RNA-dependent mechanisms, thereby functionally connecting the translational machinery with epigenetic regulation and the TIME. Although the precise molecular basis of this phenomenon remains to be elucidated, it considerably broadens the conceptual framework through which tRNA modifications are understood. Taken together, the principal function of tRNA ac⁴C is to convert enhanced tRNA stability and decoding capacity into codon-biased translational outputs, thereby selectively remodeling the cellular proteome. Rather than indiscriminately increasing global protein synthesis, tumor cells, and, in certain contexts, viruses, appear to exploit this mechanism to preferentially translate proteins required for proliferation, inflammation, metastasis, and adaptive stress responses. Moreover, the magnitude of this translational program is further shaped by NAT10 protein abundance and its post-translational modifications, highlighting multiple regulatory layers through which the tRNA ac⁴C machinery adapts protein synthesis to changing physiological and pathological demands. Unlike tRNA ac⁴C, which primarily modulates codon decoding, ac⁴C deposition in rRNAs predominantly functions to preserve ribosome architecture, assembly, and global translational homeostasis [ 23 ]. Among all known RNA substrates, rRNA represents the most evolutionarily ancient, structurally conserved, and biochemically validated target of ac⁴C modification. These acetylation sites are typically located within functionally critical regions of the ribosome, where they reinforce local RNA structure, facilitate ribosome assembly, and maintain translational fidelity. Consequently, compared with the dynamic regulatory functions of mRNA ac⁴C, rRNA ac⁴C is more appropriately regarded as a structural modification that safeguards the integrity and functionality of the translational machinery [ 23 ]. Single-nucleotide resolution analyses across multiple species have revealed abundant ac⁴C sites in archaeal rRNAs, particularly in thermophilic archaea, where ac⁴C levels increase markedly in response to elevated environmental temperatures [ 23 ]. Deletion of the corresponding RNA acetyltransferase results in pronounced growth defects under heat stress, whereas cryo-electron microscopy analyses demonstrate that ac⁴C stabilizes thermally sensitive regions within the ribosome [ 23 ]. These findings indicate that rRNA ac⁴C is not a static structural mark but rather an adaptive modification that enhances ribosome stability and preserves translational capacity under environmental stress. In eukaryotic cells, NAT10 is required for 18S rRNA processing, maturation of the small ribosomal subunit, and maintenance of nucleolar homeostasis. Accordingly, impaired rRNA ac⁴C deposition is predicted to disrupt ribosome assembly, reduce translational output, and trigger nucleolar stress. Because malignant cells generally rely on elevated ribosome biogenesis to sustain rapid proliferation and increased protein synthesis, this dependency may render tumors particularly vulnerable to NAT10 inhibition. Consequently, suppression of rRNA ac⁴C-mediated ribosome biogenesis may represent an important mechanism underlying the antitumor activity of NAT10-targeted therapies. However, rRNA ac⁴C also represents a major source of potential on-target toxicity associated with NAT10 inhibition. Normal highly proliferative tissues, including hematopoietic stem cells, intestinal epithelial cells, and germ cells, likewise require continuous ribosome biogenesis to maintain tissue renewal and physiological homeostasis. Therefore, systemic inhibition of NAT10 may compromise ribosome production in both malignant and normal cells, narrowing the therapeutic window. In this context, inhibition of ribosome biogenesis constitutes both a therapeutic opportunity and a potential limitation for NAT10-directed interventions. Another emerging concept is that distinct combinations of rRNA ac⁴C modifications may generate ribosome populations with differential translational preferences, thereby providing a theoretical basis for the concept of specialized ribosomes. Such ribosomes could preferentially translate specific subsets of mRNAs and contribute to selective gene expression programs under physiological or pathological conditions. Nevertheless, direct experimental evidence demonstrating that tumor cells establish stable and functionally distinct specialized ribosomes through NAT10-dependent rRNA ac⁴C remodeling remains lacking. Consequently, the existence and biological significance of ac⁴C-dependent specialized ribosomes in cancer require further investigation. Overall, the primary function of rRNA ac⁴C is to preserve ribosome integrity, ensure translational fidelity, and facilitate adaptive responses to cellular stress. The heightened dependence of cancer cells on robust ribosome biogenesis may constitute a therapeutically exploitable vulnerability. However, distinguishing tumor-specific ribosome dependency from the physiological requirements of normal proliferative tissues remains a central challenge for the future development of NAT10-targeted therapies. Beyond canonical coding and translation-associated RNAs, accumulating evidence indicates that ac⁴C also modifies multiple classes of non-coding RNAs and expands the functional repertoire of NAT10 by altering RNA stability, processing, translational potential, and molecular interaction networks. Studies of lncRNA ac⁴C demonstrate that NAT10 not only regulates coding transcripts but also amplifies multilayered oncogenic signaling by stabilizing selected lncRNAs [ 30 ]. Unlike mRNA ac⁴C, which directly influences protein synthesis, lncRNA ac⁴C generally prolongs RNA half-life before exerting downstream effects through molecular scaffolding, recruitment of RNA-binding proteins, or translational control. In breast cancer, NAT10-mediated ac⁴C modification increases the stability and abundance of CD2BP2-DT. Accumulated CD2BP2-DT promotes liquid–liquid phase separation of Y-box-binding protein 1 (YBX1), which subsequently stabilizes cyclin-dependent kinase 1 (CDK1) mRNA and accelerates cell-cycle progression, thereby establishing an oncogenic NAT10–ac⁴C–lncRNA–phase separation–CDK1 axis [ 30 ]. This mechanism illustrates that ac⁴C-stabilized lncRNAs can indirectly determine the fate of key mRNAs by regulating biomolecular condensates. Similarly, in nasopharyngeal carcinoma, NAT10 stabilizes SIMALR through ac⁴C deposition. SIMALR subsequently binds eEF1A2, enhances its GTPase activity and phosphorylation, and promotes the translation of integrin subunit beta 4 (ITGB4) and ITGA6, ultimately facilitating tumor-cell proliferation and metastasis [ 80 ]. Thus, lncRNA ac⁴C not only preserves RNA abundance but can also selectively enhance the synthesis of prometastatic proteins by activating the translation elongation machinery. In addition to translational regulation, lncRNA ac⁴C can reprogram alternative splicing. In esophageal squamous cell carcinoma, NAT10 stabilizes CTC-490G23.2, enabling it to function as a molecular scaffold that promotes the interaction between polypyrimidine tract-binding protein 1 (PTBP1) and CD44 pre-mRNA. This interaction drives an isoform switch from CD44 standard (CD44s) to CD44 variant 8–10 (CD44v8–10). CD44v8–10 subsequently stabilizes vimentin, enhances cytoskeletal remodeling, and promotes invasion and metastasis, whereas antisense oligonucleotide nanocomplexes targeting this lncRNA markedly suppress metastatic dissemination [ 81 ]. lncRNA ac⁴C can also connect angiogenesis with tumor immunity. In gastric cancer, NAT10 stabilizes XIST through ac⁴C modification. XIST then recruits hnRNPK and promotes nuclear translocation of YAP1, thereby activating TEA domain transcription factor 4 (TEAD4)-dependent vascular endothelial growth factor A (VEGFA) transcription. This pathway induces aberrant angiogenesis and immunosuppression. By contrast, NAT10 inhibition promotes vascular normalization, upregulates CXCL9, CXCL10, and CXCL11, enhances cytotoxic lymphocyte infiltration, and improves the response to programmed cell death protein 1 blockade [ 82 ]. Overall, a recurring feature of lncRNA ac⁴C is the stabilization of selected oncogenic lncRNAs, allowing them to persistently organize RNA-binding proteins, translation factors, splicing regulators, or transcriptional complexes. The identity of each lncRNA substrate subsequently determines whether the downstream output is directed toward cell-cycle progression, translation, alternative splicing, angiogenesis, or immune evasion. CircRNAs possess covalently closed structures and are generally more stable than linear RNAs. Accordingly, the principal consequence of ac⁴C modification in circRNAs may not be further extension of RNA half-life but rather enhanced translation initiation or more efficient utilization of embedded open reading frames [ 32 ]. In contrast to lncRNAs, which predominantly depend on RNA stabilization and molecular scaffolding, circRNA ac⁴C more prominently regulates non-canonical translational potential. In multiple myeloma, NAT10-mediated ac⁴C modification of circANKRD12 promotes its translation into a previously uncharacterized 354-amino-acid protein, circANKRD12_354aa. This protein interacts with histone deacetylase 2 (HDAC2) and stabilizes c-Myc, thereby enhancing myeloma-cell proliferation [ 32 ]. Notably, circANKRD12 can also be transferred from myeloma cells to natural killer (NK) cells, where the same HDAC2–c-Myc axis suppresses NK-cell cytotoxicity. Thus, a single ac⁴C-modified circRNA can simultaneously drive tumor-cell-intrinsic proliferation and attenuate antitumor immunity, thereby coordinating tumor expansion with immune evasion [ 32 ]. Desloratadine directly binds circANKRD12_354aa, inhibits myeloma growth, and restores NK-cell activity, suggesting that selective targeting of ac⁴C-dependent circRNA translation products may offer greater therapeutic specificity than global inhibition of NAT10 [ 32 ]. Taken together, circRNA ac⁴C expands the functional output of circRNAs by promoting non-canonical protein translation. The resulting proteins can act not only within tumor cells but also across cellular compartments to influence immune effector cells, thereby providing a new molecular basis for tumor–immune communication. Whereas ac⁴C modification of lncRNAs and circRNAs primarily affects RNA stability or translational potential, ac⁴C within pri-miRNAs directly participates in small-RNA maturation. NAT10 and its cofactor THUMP domain-containing 1 (THUMPD1) cooperatively catalyze ac⁴C deposition on pri-miRNAs. This modification strengthens the interaction between pri-miRNAs and DiGeorge syndrome critical region 8 (DGCR8), thereby facilitating recognition by the Microprocessor complex and promoting processing from pri-miRNAs to pre-miRNAs [ 31 ]. Following NAT10 depletion, pri-miRNAs accumulate whereas mature miRNA levels decline, indicating that ac⁴C primarily facilitates RNA processing in this context rather than merely maintaining pri-miRNA stability. Because a single miRNA can regulate numerous downstream transcripts, NAT10-mediated control of pri-miRNA processing can be substantially amplified through miRNA networks and thereby reprogram broad post-transcriptional regulatory circuits in tumor cells [ 31 ]. In lung cancer models, NAT10 deficiency attenuates malignant phenotypes by altering miRNA biogenesis, identifying pri-miRNA ac⁴C as an additional mechanism through which NAT10 promotes tumor progression. However, it remains unclear whether individual pri-miRNAs display selective dependence on ac⁴C and how this modification structurally enhances DGCR8 recognition. In summary, pri-miRNA ac⁴C promotes mature miRNA production by enhancing DGCR8-mediated recognition and processing. This mechanism differs fundamentally from the stabilizing effects observed in mRNAs and lncRNAs and highlights a direct role for ac⁴C in RNA processing and non-coding RNA biogenesis. Beyond host RNAs, multiple viruses also exploit the NAT10–ac⁴C system to optimize viral RNA stability, processing, and translation while simultaneously interfering with host innate immunity. Viral RNA ac⁴C is not restricted to a single molecular function. Depending on the viral species and stage of infection, it can regulate RNA stability, alternative splicing, polyadenylation, and protein translation. NAT10 therefore represents an important regulatory node linking viral RNA metabolism with host antiviral immunity. In minute virus of canines (MVC), NAT10-mediated ac⁴C does not substantially alter viral RNA stability but instead regulates alternative splicing and polyadenylation. ac⁴C at nucleotide 3311 of the viral RNA promotes the binding of cleavage and polyadenylation specificity factor 5 (CPSF5) to the target region, whereas the viral protein NP1 facilitates CPSF5 recruitment, thereby ensuring accurate viral RNA processing and efficient replication [ 83 ]. This mechanism indicates that ac⁴C can function as a structural determinant of RNA–protein interactions rather than solely as a stabilizing modification. In KSHV, NAT10 modifies the viral lncRNA polyadenylated nuclear RNA (PAN), thereby increasing its stability and promoting the transition from latency to lytic replication. Disruption of ac⁴C sites within PAN reduces lytic gene expression and virion production, identifying PAN ac⁴C as an important molecular switch controlling KSHV reactivation [ 84 ]. At the same time, ac⁴C-modified PAN promotes NAT10-dependent regulation of host interferon gamma-inducible protein 16 (IFI16) mRNA, enhancing its stability and translation and activating inflammasome signaling. Viral RNA ac⁴C can therefore simultaneously support viral replication and reshape host inflammatory responses, illustrating bidirectional coupling between viral and host RNA modification networks [ 84 ]. Beyond directly modifying viral RNAs, NAT10 can influence antiviral immunity through host transcriptional regulation and non-coding RNAs. NAT10 dynamically associates with the interferon beta (IFN-β) promoter and negatively regulates type I interferon signaling through a lncRNA that suppresses interferon regulatory factor activity. NAT10 depletion or Remodelin treatment enhances IFN-β and interferon-stimulated gene expression while restricting RNA virus replication [ 87 ]. However, whether this effect is fully dependent on RNA ac⁴C remains to be determined, given that NAT10 also possesses chromatin-binding and catalysis-independent regulatory functions. Overall, viruses exploit the NAT10–ac⁴C axis to stabilize viral RNAs, optimize RNA processing, and enhance protein synthesis, whereas host NAT10 also modulates interferon and inflammasome responses. ac⁴C therefore constitutes an important epitranscriptomic layer in the molecular contest between viral replication and host immunity. ac⁴C substrates should be classified according to the strength of supporting evidence into four categories: direct biochemical validation, support from single-nucleotide or multiplatform sequencing, predominant dependence on antibody-based enrichment, and indirect inference from functional phenotypes. Such stratification is essential to avoid equating all NAT10-associated RNA changes with direct ac⁴C modification. On this basis, the NAT10–ac⁴C axis can be organized into three interconnected regulatory layers. The first is transcript-selective regulation, in which ac⁴C modification of specific mRNAs or non-coding RNAs alters their stability, processing, or translation. The second is translational-system regulation, in which ac⁴C deposition on tRNAs and rRNAs remodels ribosome function and codon-biased protein synthesis. The third comprises NAT10-dependent but ac⁴C-independent activities mediated through protein acetylation, RNA binding, ribosome recruitment, or protein–protein interactions. Accordingly, NAT10-associated phenotypes should be interpreted along two dimensions, the strength of substrate evidence and the degree of catalytic dependence, rather than being attributed to a single ac⁴C event. NAT10 is currently considered the only mammalian acetyltransferase known to catalyze the formation of RNA ac⁴C and therefore functions as the central writer of the ac⁴C modification system. As a prominent member of the GCN5-related N-acetyltransferase (GNAT) family, NAT10 modifies tRNAs, rRNAs, and mRNAs and also regulates ribosome biogenesis, RNA stability, protein translation, DNA damage repair, cell-cycle progression, and tumor development. Recent advances in cryo-electron microscopy (cryo-EM), AlphaFold-based structural prediction, and biochemical analysis have progressively clarified the three-dimensional architecture and catalytic mechanism of NAT10, providing an essential framework for understanding substrate recognition and developing highly selective NAT10 inhibitors. Human NAT10 is a large multidomain protein of approximately 120 kDa with a modular architecture composed primarily of an N-terminal RNA-binding region, a central ATPase/RecD-like helicase domain, and a C-terminal GNAT acetyltransferase domain [ 88 ]. These domains are connected by flexible linkers and function cooperatively to mediate RNA recognition, substrate positioning, and acetyl-group transfer [ 82 ]. The N-terminal region is enriched in basic amino acids and RNA-interacting elements and can establish extensive contacts with rRNAs, tRNAs, and mRNAs. Although a canonical RNA-binding motif has not yet been defined, substantial evidence indicates that this region is essential for substrate recruitment and catalytic efficiency [ 89 ]. Deletion or mutation of the N-terminal region markedly weakens RNA binding and reduces global ac⁴C levels, demonstrating that productive RNA engagement is a prerequisite for catalysis. The central RecD-like helicase domain belongs to the superfamily 2 class of ATP-dependent RNA helicase-related domains. It binds and hydrolyzes ATP to provide the energy required for RNA conformational remodeling [ 90 ]. Although NAT10 has not been conclusively shown to possess conventional RNA helicase activity, growing evidence suggests that this domain primarily functions as an RNA chaperone [ 90 ]. By locally opening RNA secondary structures, it may expose cytidines buried within duplexes or complex folds to the catalytic center, thereby increasing acetylation efficiency. This domain therefore appears to regulate RNA accessibility rather than mediate classical processive unwinding. The C-terminal region contains the highly conserved GNAT catalytic domain, which forms the core catalytic center responsible for ac⁴C formation [ 91 ]. The GNAT domain binds acetyl-coenzyme A and transfers its acetyl group to the N4 amino group of cytidine [ 24 ]. Structural analyses indicate that this region forms a characteristic acetyl-coenzyme A-binding pocket in which multiple conserved residues stabilize cofactor binding and participate in catalysis [ 24 ]. Point mutations within the catalytic site can almost completely abolish NAT10 acetyltransferase activity without substantially affecting protein abundance, confirming that the GNAT domain is indispensable for its catalytic function. Unlike most RNA methyltransferases, NAT10-mediated RNA acetylation requires both ATP and acetyl-coenzyme A and is therefore classified as an ATP-dependent RNA acetyltransferase [ 89 ]. During catalysis, acetyl-coenzyme A enters the GNAT catalytic pocket and serves as the acetyl donor. Conserved catalytic residues facilitate precise transfer of the acetyl group to the N4 amino group of cytidine, generating ac⁴C and releasing coenzyme A [ 89 ]. In parallel, the RecD-like ATPase domain binds and hydrolyzes ATP to drive local RNA relaxation or rearrangement, thereby increasing accessibility of the target cytidine to the catalytic center. ATP does not directly participate in acetyl-group transfer but instead enhances catalytic efficiency by remodeling the RNA substrate [ 89 ]. Biochemical studies further demonstrate that inhibition of ATP binding or ATPase activity markedly reduces ac⁴C deposition even when NAT10 retains the ability to bind RNA [ 89 ]. Conversely, inactivation of the GNAT domain prevents acetyl-group transfer despite preserved RNA engagement. These findings indicate that RNA remodeling and acetyl transfer are mechanistically distinct yet interdependent steps that together determine the overall catalytic activity of NAT10. The RNA recognition–catalysis coupling model provides a useful framework for explaining how NAT10 achieves substrate specificity [ 92 , 93 ]. First, the RNA-binding region engages the target RNA through electrostatic interactions and recognition of local three-dimensional features [ 93 ]. The RecD-like ATPase domain then uses ATP hydrolysis to remodel the local RNA structure and expose the target cytidine to the catalytic center [ 94 ]. Finally, the GNAT domain binds acetyl-coenzyme A and transfers the acetyl group to the N4 position of cytidine, completing ac⁴C formation. Notably, no highly conserved ac⁴C consensus sequence analogous to the RRACH motif of m⁶A has been identified. This suggests that NAT10 substrate recognition depends more heavily on RNA secondary structure, local spatial conformation, and recruitment by RNA-binding proteins than on primary sequence alone [ 89 ]. Such a recognition strategy may explain how NAT10 modifies structurally distinct RNA classes, including rRNAs, tRNAs, mRNAs, and non-coding RNAs, while still generating tissue- and cell-type-specific ac⁴C patterns [ 95 , 96 ]. Increasing evidence further suggests that NAT10 forms dynamic complexes with THUMPD1, small nucleolar ribonucleoprotein particles, and multiple RBPs that collectively determine substrate selectivity [ 96 ]. THUMPD1 predominantly facilitates tRNA ac⁴C modification, whereas transcript-specific recruitment of NAT10 to mRNAs may depend on distinct RBPs. The complete molecular architecture and regulatory logic of these substrate-specific writer complexes remain to be fully defined. Although NAT10 is established as the only known mammalian ac⁴C writer, how it accurately selects specific substrates from thousands of cellular RNA transcripts remains one of the central unresolved questions in the field. Unlike m⁶A, which is preferentially deposited within the relatively conserved RRACH sequence context, no universal sequence feature has been identified that can account for ac⁴C deposition across different RNA classes [ 97 ]. This suggests that NAT10 substrate selection is not determined by primary sequence alone but instead emerges from an integrated recognition network involving RBPs, RNA secondary structure, and intermolecular RNA interactions [ 42 ]. Accumulating evidence identifies RBPs as key adaptors that confer substrate specificity on NAT10. A recent study characterized a mammalian mRNA ac⁴C writer complex containing NAT10, PCBP1/2, and TDP-43. PCBP1/2 preferentially recognize cytidine-rich RNA sequences, whereas TDP-43 primarily binds uridine–guanosine-rich regions [ 42 ]. Together, these RBPs recruit NAT10 to distinct RNA substrates and thereby shape ac⁴C site selection. Depletion of PCBP1/2 or TDP-43 markedly reduces global mRNA ac⁴C levels and eliminates numerous cytidine-rich ac⁴C motifs, indicating that these proteins not only facilitate RNA binding but directly determine substrate specificity [ 42 ]. These observations suggest that NAT10 itself may possess limited sequence-discrimination capacity and instead rely on distinct RBPs to assemble tissue- and cell-type-specific writer complexes. Whether different tissues harbor unique NAT10–RBP combinations and how such complexes generate disease-specific ac⁴C landscapes remain important questions for future research. In addition to RBPs, RNA primary sequence and higher-order structure influence NAT10 substrate recognition [ 42 ]. Although mRNA ac⁴C sites are often enriched in cytidine-rich regions, no consensus motif comparable to the m⁶A RRACH sequence has been established. Local RNA secondary structure may therefore be more important than sequence alone. The RecD-like ATPase/helicase domain of NAT10 can promote local RNA conformational remodeling and expose cytidines buried within duplexes or complex folds. RNA structural accessibility is therefore increasingly viewed as a critical determinant of ac⁴C deposition, whereas RBPs may further refine the specificity of this structural recognition process [ 98 ]. The markedly different spatial architectures of mRNAs, tRNAs, rRNAs, and non-coding RNAs may also explain how NAT10 modifies diverse RNA classes while generating distinct modification patterns and biological outcomes in each substrate category [ 98 ]. Beyond promoting NAT10 recruitment, certain RNAs can themselves function as NAT10 decoys and redirect substrate allocation. In cervical cancer, the tumor-suppressive circRNA circMAST1 directly binds NAT10 and competitively prevents its interaction with YAP mRNA. This reduces YAP mRNA ac⁴C levels, accelerates transcript degradation, and suppresses tumor growth and lymph-node metastasis [ 99 ]. This finding introduced the concept that circRNAs can function as competitive NAT10 adaptors by altering substrate distribution within the ac⁴C network. It also broadens the understanding of RNA–RNA-modification-enzyme interactions: RNAs are not merely passive substrates but can actively recruit, sequester, or redistribute modifying enzymes. Whether additional lncRNAs or circRNAs regulate NAT10 recruitment and substrate partitioning may prove central to explaining cell-type-specific ac⁴C heterogeneity. Altogether, NAT10 substrate selection appears to be governed by a dynamic, multilayered recognition system composed of RBPs, RNA higher-order structure, and competitive RNA adaptors rather than by a single sequence motif. This framework explains the pronounced heterogeneity of ac⁴C distribution across RNA classes and provides a conceptual basis for selectively targeting tumor-specific RNA acetylation programs. In contrast to m⁶A, for which a relatively complete writer–reader–eraser framework has been established, the ac⁴C regulatory system remains incompletely defined. Current evidence consistently supports NAT10 as the only fully validated mammalian ac⁴C writer, whereas canonical, broadly acting ac⁴C readers and physiological ac⁴C erasers have not yet been conclusively identified. The biological effects of ac⁴C therefore cannot currently be interpreted using the m⁶A paradigm without qualification. Reports of potential ac⁴C readers remain limited. One study proposed that eukaryotic translation elongation factor 2 (eEF2) recognizes an ac⁴C site within the HMGB2 coding region, strengthens its association with the mRNA, and promotes translation elongation, thereby driving hepatocellular carcinoma proliferation and metastasis [ 100 ]. Another study showed that MYBBP1A binds ac⁴C-modified PPAN and MYC mRNAs, enhances their translational efficiency, and mediates NAT10-driven colorectal cancer progression and DNA damage repair [ 58 ]. Although these findings suggest that eEF2 and MYBBP1A may possess ac⁴C-recognition capacity, the available evidence is insufficient to establish them as universal ac⁴C readers. Both studies were conducted in specific cancer models, and it remains unclear whether recognition depends on particular RNA substrates, tissue contexts, or oncogenic signaling states. Moreover, neither protein has been shown to broadly recognize ac⁴C across diverse RNA classes, and systematic structural evidence defining a dedicated ac⁴C-binding interface is lacking. They should therefore be regarded as candidate ac⁴C readers rather than established canonical readers. Similarly, there is currently no consensus evidence for a physiological ac⁴C eraser. No enzyme analogous to fat mass and obesity-associated protein or ALKB homolog 5 has been identified that directly catalyzes RNA deacetylation at ac⁴C sites. ac⁴C is therefore generally considered a relatively stable RNA modification whose dynamic regulation may depend primarily on RNA turnover, transcript renewal, and modulation of NAT10 activity rather than on classical enzymatic removal. Importantly, ac⁴C signaling may not require dedicated reader proteins in all contexts. ac⁴C can directly alter RNA physicochemical properties, stabilize local secondary structures, modulate ribosomal decoding, and change the affinity of RBPs for modified transcripts. Some ac⁴C-dependent phenotypes may therefore arise from direct changes in RNA architecture and ribosome behavior rather than from recognition by specialized binding proteins. In summary, the ac⁴C regulatory network remains centered on NAT10, whereas a definitive reader and eraser system has yet to be established. Future studies integrating structural biology, chemical biology, and high-throughput screening will be required to identify ac⁴C-binding proteins, define their recognition mechanisms, and determine whether ac⁴C participates in a genuinely reversible regulatory cycle.

NAT10 expression and activity are dynamically controlled at multiple regulatory layers. Figure  3 summarizes the major transcriptional, epigenetic, post-transcriptional, and post-translational mechanisms that modulate NAT10 abundance and function in cancer. Aberrant activation of NAT10 arises through coordinated regulation at multiple molecular levels rather than through a single genetic event. Transcriptional, epigenetic, post-transcriptional, and post-translational mechanisms collectively determine NAT10 abundance, stability, and enzymatic activity, thereby sustaining pathological ac⁴C accumulation during tumor progression. The major regulatory mechanisms identified to date are summarized in Table  3 . Fig. 3 Multilevel regulation of NAT10 expression and activity. NAT10 is regulated through transcriptional activation by oncogenic transcription factors, epigenetic control by histone acetylation, post-transcriptional regulation mediated by noncoding RNAs, and post-translational modifications including ubiquitination, deubiquitination, and lysine lactylation. These regulatory networks coordinately determine NAT10 expression, stability, and catalytic activity Multilevel regulation of NAT10 expression and activity. NAT10 is regulated through transcriptional activation by oncogenic transcription factors, epigenetic control by histone acetylation, post-transcriptional regulation mediated by noncoding RNAs, and post-translational modifications including ubiquitination, deubiquitination, and lysine lactylation. These regulatory networks coordinately determine NAT10 expression, stability, and catalytic activity Table 3 Multilevel regulatory mechanisms governing NAT10 expression, stability, and catalytic activity in cancer Regulatory layer Regulator Mechanism Biological consequence Cancer type Ref Transcriptional regulation c-Myc Directly binds the NAT10 promoter and activates transcription; NAT10 reciprocally enhances c-Myc translation through tRNA ac⁴C modification and stabilizes c-Myc protein via OTUB1, forming a positive-feedback loop Sustained NAT10 overexpression, increased global translation, tumor invasion Anaplastic thyroid carcinoma [ 57 ] Transcriptional regulation HIF-1α Directly activates NAT10 transcription under hypoxia; NAT10 stabilizes SEPT9 mRNA through ac⁴C modification, further enhancing HIF-1α signaling Hypoxia adaptation, glycolytic reprogramming, anti-angiogenic therapy resistance Gastric cancer [ 140 ] Epigenetic regulation p300-mediated H3K27ac H3K27ac enrichment at the NAT10 promoter activates NAT10 transcription Stabilization of FATP4 and CPT1A mRNAs, enhanced fatty acid uptake and β-oxidation, EGFR-TKI resistance NSCLC [ 141 ] Post-transcriptional regulation miR-330-3p Directly binds the NAT10 3′UTR and suppresses NAT10 expression Reduced ac⁴C on GPX4 mRNA, enhanced T-cell ferroptosis Tumor immunity [ 142 ] Post-transcriptional regulation miR-6716-5p Targets NAT10 mRNA Decreased NAT10 expression, enhanced colorectal cancer invasion and liver metastasis Colorectal cancer [ 143 ] Post-transcriptional regulation circTHBS1 Directly binds NAT10 and prevents ubiquitin-dependent degradation Increased NAT10 protein stability, elevated global ac⁴C, enhanced Rab8a translation and autophagy Esophageal squamous cell carcinoma [ 144 ] Post-translational regulation USP39 Interacts with NAT10 and suppresses ubiquitination Stabilizes NAT10 protein and promotes ATP6V0E1 translation, lysosomal acidification, E-cadherin degradation, and metastasis Esophageal cancer [ 145 ] Post-translational regulation K823 lysine 2-hydroxyisobutyrylation (Khib) K823 Khib enhances NAT10–USP39 interaction and suppresses ubiquitin-dependent degradation Increased NAT10 stability and stabilization of NOTCH3 mRNA Esophageal cancer [ 62 ] Post-translational regulation UBR5 E3 ubiquitin ligase mediating NAT10 ubiquitination following 5-FU treatment NAT10 degradation, decreased ERRFI1, EGFR reactivation, 5-FU resistance Colorectal cancer [ 146 ] Post-translational regulation Lactylation (K426) Lactylation at K426 enhances NAT10 catalytic activity Increased ac⁴C modification of DLAT mRNA, promotion of cuproptosis Colorectal cancer [ 147 ] Post-translational regulation SIRT1 Removes K426 lactylation (delactylation) Suppresses NAT10 catalytic activity and ac⁴C deposition Colorectal cancer [ 147 ] Multilevel regulatory mechanisms governing NAT10 expression, stability, and catalytic activity in cancer Aberrant activation of NAT10 represents an important initiating event in ac⁴C reprogramming across multiple cancer types [ 59 , 148 ]. Increasing evidence indicates that oncogenic transcription factors and hypoxia-responsive signaling can directly drive NAT10 transcription, thereby amplifying ac⁴C deposition and establishing self-sustaining positive-feedback circuits. In anaplastic thyroid carcinoma (ATC), c-Myc directly binds the NAT10 promoter and activates its transcription. In turn, NAT10 enhances c-Myc translation by increasing tRNA ac⁴C modification and further stabilizes c-Myc protein through interaction with OTUB1. This creates a c-Myc–NAT10 positive-feedback loop that continuously reinforces global protein synthesis and tumor invasiveness [ 57 ]. In addition to oncogenic transcription factors, the tumor microenvironment can also induce aberrant NAT10 expression. In gastric cancer, hypoxia-inducible factor 1 alpha (HIF-1α) directly activates NAT10 transcription. NAT10 subsequently stabilizes SEPT9 mRNA through ac⁴C modification, which further enhances HIF-1α signaling and glycolytic reprogramming. This HIF-1α–NAT10–SEPT9 positive-feedback circuit promotes hypoxia tolerance and resistance to anti-angiogenic therapy [ 140 ]. Thus, sustained transcriptional activation constitutes a major driver of NAT10 overexpression and positions ac⁴C as an important molecular node integrating oncogenic signaling with microenvironmental stress. Beyond classical transcription-factor-mediated control, epigenetic modifications also regulate NAT10 expression [ 141 ]. In NSCLC, p300-mediated enrichment of H3K27ac at the NAT10 promoter promotes its transcriptional activation. Elevated NAT10 subsequently stabilizes FATP4 and CPT1A mRNAs, thereby enhancing fatty acid uptake and fatty acid oxidation and driving resistance to EGFR-TKIs. Combined treatment with Remodelin and gefitinib markedly reverses this resistance, indicating that chromatin acetylation and RNA acetylation cooperate to establish a metabolically reprogrammed drug-resistant state [ 141 ]. NAT10 is also subject to multilayered RNA-mediated control, with miRNAs and circRNAs representing the best-supported regulatory classes. Multiple miRNAs directly bind the NAT10 3′UTR and modulate its expression [ 143 ]. For example, miR-330-3p suppresses NAT10 expression, reduces ac⁴C modification of GPX4 mRNA, and promotes T-cell ferroptosis [ 142 ]. miR-6716-5p directly targets NAT10, decreases E-cadherin expression, and promotes colorectal cancer invasion and liver metastasis [ 143 ]. These findings suggest that miRNAs constitute an important negative-feedback layer that constrains NAT10 abundance. More recent studies have expanded this regulatory network by identifying circRNA-mediated control of NAT10. In contrast to miRNAs, which reduce NAT10 expression, circTHBS1 directly binds NAT10 and prevents its ubiquitin-dependent degradation, thereby increasing NAT10 protein stability and global ac⁴C levels. Stabilized NAT10 subsequently promotes Rab8a translation and activates autophagy, driving esophageal squamous cell carcinoma progression and establishing a circTHBS1–NAT10–Rab8a positive regulatory axis [ 144 ]. Overall, post-transcriptional regulation not only determines NAT10 expression levels but also enables dynamic adaptation of NAT10 activity across distinct tissues and disease states. Accumulating evidence identifies post-translational modification as a central mechanism controlling NAT10 activity and stability and as one of the most important recent advances in the ac⁴C field [ 62 ]. First, lysine 2-hydroxyisobutyrylation (Khib) at NAT10 residue K823 enhances its interaction with USP39 and suppresses ubiquitin-dependent degradation, thereby increasing NAT10 stability. Stabilized NAT10 promotes esophageal cancer metastasis by maintaining NOTCH3 mRNA stability [ 62 ]. USP39 not only stabilizes NAT10 but also regulates its downstream functional output. NAT10 enhances lysosomal acidification and E-cadherin degradation by promoting ATP6V0E1 translation, whereas disruption of the NAT10–USP39 interaction suppresses tumor metastasis [ 145 ]. NAT10 is also dynamically regulated by ubiquitination. In colorectal cancer, 5-fluorouracil (5-FU) promotes the interaction between NAT10 and the E3 ubiquitin ligase UBR5, accelerating NAT10 ubiquitination and degradation. The resulting decrease in ERRFI1 permits reactivation of EGFR signaling and contributes to 5-FU-induced therapeutic resistance [ 146 ]. More recently, metabolism-associated post-translational modifications have revealed additional mechanisms governing NAT10 activity [ 147 ]. In colorectal cancer, lactylation of NAT10 at K426 markedly enhances its catalytic activity, increases ac⁴C modification of dihydrolipoamide S-acetyltransferase (DLAT) mRNA, and promotes cuproptosis. By contrast, sirtuin 1 (SIRT1)-mediated delactylation produces the opposite effect, establishing a direct mechanistic connection between metabolic state and RNA modification [ 147 ]. Post-translational modifications therefore regulate not only NAT10 protein stability but also its RNA acetyltransferase activity and context-specific functional output across different malignancies. Collectively, current evidence indicates that aberrant NAT10 activation is not driven by a single mechanism but instead arises through coordinated regulation at the transcriptional, epigenetic, post-transcriptional, and post-translational levels. These interconnected networks sustain elevated NAT10 expression and pathological ac⁴C accumulation through multiple positive-feedback circuits, thereby providing tumor cells with persistent advantages in translational reprogramming, metabolic adaptation, and therapeutic resistance. Several limitations remain. Most studies have focused on individual cancer types, and systematic comparisons of NAT10 regulatory networks across malignancies are lacking. Many mechanisms may also be tissue- or disease-specific, and their broader applicability remains uncertain. Furthermore, how distinct regulatory layers are integrated and how microenvironmental cues, metabolic states, and RBPs collectively determine NAT10 activity have yet to be incorporated into a unified mechanistic model. Future studies combining single-cell multi-omics, spatial transcriptomics, chromatin-interaction profiling, and dynamic proteomics will be required to construct spatiotemporal maps of the NAT10–ac⁴C regulatory network and define the hierarchical relationships among these regulatory layers, thereby providing a mechanistic basis for more precise NAT10-targeted strategies.

ac⁴C is an important epitranscriptomic layer connecting RNA structure, translational control, cell fate, and stress adaptation. By acting on mRNAs, tRNAs, rRNAs, and multiple ncRNA classes, NAT10 contributes to fundamental physiological processes including ribosome function, proteostasis, development, hematopoiesis, reproduction, and immunity. Cancer cells hijack this system to sustain proliferation and stemness, reprogram metabolism, enhance DNA damage repair, promote metastasis, and establish an immunosuppressive microenvironment. However, NAT10-associated phenotypes cannot all be equated with RNA ac⁴C effects. NAT10 also mediates RNA binding, protein acetylation, R-loop regulation, ribosome biogenesis, and protein interactions, whereas the true abundance, site distribution, and functional rules of mRNA ac⁴C remain controversial. Future studies must therefore rigorously distinguish ac⁴C-dependent effects, catalysis-independent NAT10 functions, and non-RNA-related activities through orthogonal detection, catalytic-dead rescue, and site-level RNA editing. NAT10 has clear therapeutic potential, but its essential physiological functions constrain the safety of prolonged systemic inhibition. A more feasible strategy is not to maximally suppress this fundamental RNA enzyme but to identify NAT10-dependent tumors and selectively disrupt the regulatory circuits on which malignant cells rely through tumor-targeted delivery, mechanism-guided combination therapy, and intervention against specific NAT10–RNA axes. With the continued development of high-confidence ac⁴C atlases, site-specific editing technologies, structural biology, and precision drug-delivery systems, the NAT10–ac⁴C axis may evolve from an emerging epitranscriptomic mechanism into a stratifiable, experimentally verifiable, and precisely targetable platform for cancer therapy.

Aberrant activation of NAT10 promotes multiple malignant phenotypes through coordinated regulation of RNA stability, translation, chromatin accessibility, and cellular plasticity. Figure  4 illustrates the major oncogenic programs driven by NAT10 in cancer. Accumulating studies have established NAT10 as a central regulator of multiple malignant phenotypes across diverse cancer types. By modifying distinct RNA substrates in a context-dependent manner, NAT10 regulates tumor proliferation, stemness, metabolic reprogramming, genome stability, metastasis, and therapeutic resistance. The representative molecular mechanisms identified in different malignancies are summarized in Table  4 . Fig. 4 NAT10-driven malignant phenotypes in cancer. NAT10 promotes tumor proliferation, cancer stemness, genome maintenance, and metastatic dissemination by enhancing ac⁴C-dependent RNA stability, translation efficiency, R-loop regulation, DNA repair, metabolic remodeling, and epithelial–mesenchymal transition. Representative downstream targets and signaling pathways are shown NAT10-driven malignant phenotypes in cancer. NAT10 promotes tumor proliferation, cancer stemness, genome maintenance, and metastatic dissemination by enhancing ac⁴C-dependent RNA stability, translation efficiency, R-loop regulation, DNA repair, metabolic remodeling, and epithelial–mesenchymal transition. Representative downstream targets and signaling pathways are shown Table 4 Functional mechanisms of the NAT10–ac⁴C axis across different human cancers Cancer type Direct target(s) Biological function Mechanism Ref. Prostate cancer HMGA1, KRT8 Proliferation, cell-cycle progression, EMT and migration ac⁴C stabilizes HMGA1 and KRT8 mRNAs, promoting cell-cycle progression and cytoskeletal remodeling. [ 149 ] Colorectal cancer KIF23 Proliferation Stabilizes KIF23 mRNA, activates Wnt/β-catenin signaling and promotes G2/M transition. [ 39 ] Bladder cancer BCL9L, SOX4, AKT1 Proliferation and survival Enhances mRNA stability and/or translation of oncogenic transcripts. [ 150 ] Glioblastoma PIK3R2 Cell proliferation and survival Stabilizes PIK3R2 mRNA, sustaining PI3K signaling. [ 151 , 152 ] Head and neck squamous cell carcinoma DDIT4 Cell survival and proliferation Stabilizes DDIT4 mRNA to maintain PI3K-associated signaling. [ 151 , 152 ] Hepatocellular carcinoma HMGB2 Proliferation ac⁴C within the CDS enhances eEF2 recruitment and translation rather than RNA stability. [ 100 ] NSCLC FOXA1 Proliferation, migration and stemness FOXA1 activates NAT10 transcription, while NAT10 stabilizes FOXA1 mRNA, forming a positive-feedback loop. [ 153 ] Ovarian cancer PGAM1 Cancer stemness Stabilizes PGAM1 mRNA, enhancing glycolysis and tumorsphere formation. [ 154 ] AML SLC1A4, HOXA9, MENIN Leukemia stem-cell maintenance Enhances translation of SLC1A4, HOXA9 and MENIN, promoting serine metabolism and LSC self-renewal. [ 155 ] Cervical cancer NANOGP8 Stemness and chemoresistance Stabilizes NANOGP8 mRNA, maintaining stem-like phenotype and therapy resistance. [ 156 ] Triple-negative breast cancer RAD51 DNA repair and PARP inhibitor resistance Stabilizes RAD51 mRNA and enhances homologous recombination repair. [ 157 ] Bladder cancer AHNAK DNA repair and cisplatin resistance Stabilizes AHNAK mRNA, promoting DNA damage repair. [ 158 ] Colorectal cancer PPAN, MYC Genome stability Enhances translation of PPAN and MYC, reinforcing DNA repair capacity. [ 58 ] NSCLC EMT signaling EMT and invasion Remodelin-mediated NAT10 inhibition reverses EMT and suppresses invasion. [ 159 ] Gastric cancer AKT pathway EMT and metastasis NAT10 activates PI3K/AKT signaling to induce EMT. [ 160 ] Endometriosis TGFB1 EMT and tissue invasion Stabilizes TGFB1 mRNA through ac⁴C modification. [ 161 ] Ovarian cancer CAPRIN1 Migration, invasion and stemness Stabilizes CAPRIN1 mRNA, promoting malignant progression. [ 162 ] Vascular remodeling / multiple tumors ITGB1, COL1A2 Cell adhesion and migration Stabilizes ITGB1/COL1A2 mRNAs and activates integrin–FAK signaling. [ 18 ] Breast cancer brain metastasis PHGDH, PSAT1 Brain metastasis Promotes serine biosynthesis to facilitate adaptation to the brain microenvironment. [ 163 ] Gastric cancer HK2 Glycolysis Stabilizes HK2 mRNA, enhancing glucose phosphorylation and glycolytic flux. [ 164 ] Ovarian cancer PGAM1 Glycolysis Stabilizes PGAM1 mRNA and increases glycolytic activity. [ 154 ] NSCLC ENO1 Glycolysis Stabilizes ENO1 mRNA and promotes glycolysis. [ 165 ] Retinoblastoma PFKFB3 Glycolysis Stabilizes PFKFB3 mRNA to increase glycolytic flux. [ 154 , 166 , 167 ] Triple-negative breast cancer JunB → LDHA Glycolysis and immune evasion Stabilizes JunB mRNA, increasing LDHA expression and lactate production. [ 168 ] Cervical cancer FOXP1 Glycolysis and immunosuppression Enhances FOXP1 translation, inducing GLUT4 and KHK expression. [ 169 ] Gastric cancer 9-Sep Hypoxic glycolysis Stabilizes SEPT9 mRNA and establishes the HIF-1α–NAT10–SEPT9 positive-feedback loop. [ 140 ] AML SLC1A4, HOXA9, MENIN Serine metabolism Enhances serine uptake and biosynthesis through translational regulation. [ 155 ] Lung cancer NIT2 Glutamine metabolism Stabilizes NIT2 mRNA, promoting glutamine utilization and ATP production. [ 170 ] Oral squamous cell carcinoma SLC1A5 Glutamine metabolism Stabilizes SLC1A5 mRNA and enhances glutamine uptake. [ 171 ] Osteosarcoma ATF4 Asparagine metabolism Stabilizes ATF4 mRNA, inducing ASNS transcription. [ 98 ] Multiple tumors ELOVL6, ACSL1/3/4, ACAT1, ACADSB Lipid metabolism Coordinates fatty acid synthesis, elongation and lipid homeostasis. [ 172 ] Esophageal squamous cell carcinoma FASN (in TAMs) Lipid metabolism and immune suppression Exosomal NAT10 stabilizes FASN mRNA and promotes M2 macrophage polarization. [ 173 ] Hepatocellular carcinoma Nrf2 Redox homeostasis Stabilizes Nrf2 mRNA and activates antioxidant signaling. [ 174 ] Hepatocellular carcinoma SLC7A11 Ferroptosis resistance Stabilizes SLC7A11 mRNA, suppressing ferroptosis and promoting TACE resistance. [ 175 ] Nasopharyngeal carcinoma SLC7A11 Ferroptosis resistance Stabilizes SLC7A11 and induces sorafenib resistance. [ 176 ] Colon cancer FSP1 (AIFM2) Ferroptosis resistance Stabilizes FSP1 mRNA to establish a GPX4-independent defense pathway. [ 177 ] Clear-cell renal cell carcinoma NFE2L1–GPX4 Ferroptosis resistance Maintains GPX4-dependent antioxidant defense. [ 178 ] Laryngeal squamous cell carcinoma ELANE Pyroptosis suppression Reduces ELANE mRNA stability and suppresses pyroptosis. [ 179 ] Colorectal cancer DLAT Cuproptosis Stabilizes DLAT mRNA; NAT10 lactylation enhances cuproptosis. [ 147 ] NSCLC SGK2 → EZH2 → GABARAP Autophagy inhibition Suppresses autophagic flux and promotes tumor progression. [ 180 ] Esophageal squamous cell carcinoma Rab8a Autophagy activation circTHBS1 stabilizes NAT10, promoting Rab8a translation and autophagy. [ 144 ] Functional mechanisms of the NAT10–ac⁴C axis across different human cancers Sustained proliferation is a defining feature of cancer, but it does not arise from activation of a single cell-cycle regulator [ 181 , 182 ]. Rather, it reflects coordinated reprogramming of growth-factor signaling, transcriptional networks, translational capacity, and mitotic execution [ 182 ]. Current evidence indicates that the NAT10–ac⁴C axis supports this process at multiple levels. On the one hand, it stabilizes or enhances the translation of selected oncogenic mRNAs, thereby maintaining growth signaling and cell-cycle progression. On the other hand, it remodels tRNA modification and global protein-synthesis capacity to provide the translational resources required for rapid proliferation. Importantly, the dominant mechanisms of NAT10 dependency vary across tumor types, and not all proliferative phenotypes should be attributed solely to increased mRNA stability. In many solid tumors, NAT10 promotes proliferation through selective stabilization of specific mRNAs [ 183 ]. In prostate cancer, NAT10-mediated ac⁴C modification prolongs the half-life of HMGA1 mRNA and accelerates cell-cycle progression. Simultaneous stabilization of KRT8 mRNA further promotes epithelial–mesenchymal transition (EMT), indicating that a single NAT10-dependent program can coordinate proliferative and invasive phenotypes [ 149 ]. Similarly, in colorectal cancer, NAT10 stabilizes KIF23 mRNA, activates Wnt/β-catenin signaling, and promotes nuclear translocation of β-catenin, thereby linking mitotic regulation to oncogenic transcriptional programs [ 39 ]. This substrate-selective stabilization mechanism is not restricted to canonical cell-cycle factors. In bladder cancer, BCL9L, SOX4, and AKT1 have all been identified as downstream ac⁴C targets of NAT10, although their modes of regulation differ. BCL9L and SOX4 are regulated at both the RNA-stability and translational levels, whereas AKT1 is affected primarily through enhanced translation. These findings reveal substantial heterogeneity in the functional consequences of ac⁴C among different substrates [ 150 ]. In glioblastoma and head and neck squamous cell carcinoma, NAT10 sustains PI3K-associated growth signaling and cell-survival programs by stabilizing PIK3R2 and DDIT4 mRNAs, respectively [ 151 , 152 ]. NAT10 therefore does not act through a universal “core proliferation gene.” Instead, it selectively preserves distinct growth-factor, cell-cycle, and survival transcripts according to tumor context. The shared consequence is reduced degradation of essential oncogenic mRNAs and sustained protein output. In some tumors, proliferation depends more strongly on direct enhancement of translational efficiency than on prolongation of mRNA half-life [ 100 ]. In hepatocellular carcinoma, NAT10 deposits ac⁴C within the coding region of HMGB2 mRNA and promotes binding of eEF2 to this region, thereby enhancing HMGB2 protein synthesis without merely stabilizing the transcript [ 100 ]. This mechanism suggests that coding-region ac⁴C can directly increase protein output by altering translation-factor recruitment or ribosome elongation. BCL9L, SOX4, and AKT1 in bladder cancer also exemplify this translational mode of regulation, as several of these transcripts display reduced translational efficiency in addition to altered stability following NAT10 depletion [ 150 ]. Thus, the proliferative effects of mRNA ac⁴C operate through at least two parallel mechanisms: extension of RNA lifespan and enhancement of protein output per transcript. Whether these mechanisms occur independently or simultaneously is likely determined by the modification site, local RNA structure, and associated binding proteins. This distinction has important experimental and therapeutic implications. In tumors primarily dependent on RNA stabilization, NAT10 inhibition may produce a gradual but sustained decline in protein abundance. By contrast, in tumors relying predominantly on translational enhancement, protein output may be rapidly suppressed even in the absence of major changes in mRNA levels. RNA sequencing alone may therefore underestimate the true impact of NAT10 on proliferative programs, necessitating integration with ribosome profiling and quantitative proteomics. The NAT10–ac⁴C axis can also become embedded within positive-feedback circuits involving growth factors and transcription factors, converting transient signaling into sustained oncogenic amplification [ 153 ]. In NSCLC, NAT10 stabilizes FOXA1 mRNA through ac⁴C modification, whereas FOXA1 directly activates NAT10 transcription. This FOXA1–NAT10 positive-feedback loop continuously reinforces cell proliferation, migration, and stemness [ 153 ]. This circuitry demonstrates that RNA modification is not merely a downstream effector mechanism but can also reinforce upstream transcriptional drivers. Similar feedback logic can emerge through growth-signaling pathways. Stabilization of KIF23 activates Wnt/β-catenin signaling, whereas PIK3R2 and AKT1 sustain PI3K pathway activity. HMGA1 and HMGB2 can further amplify MYC-associated or chromatin-related transcriptional programs [ 39 , 150 , 151 ]. The proliferative output of NAT10 therefore frequently reflects a cascade of RNA modification, signal activation, and transcriptional amplification rather than an isolated single-gene effect. Such feedback architecture may also explain adaptive rebound following NAT10 inhibition. In colorectal cancer, NAT10 stabilizes ERRFI1 mRNA, which would be expected to restrain EGFR signaling. NAT10 inhibition reduces ERRFI1 and consequently permits EGFR reactivation, partially offsetting growth suppression [ 146 ]. NAT10 therefore does not uniformly promote proliferation in every molecular context, because its substrate network may also include negative-feedback regulators. The final phenotype reflects the net balance between growth-promoting and growth-restraining substrates. NAT10 regulates the cell cycle not only through upstream growth signaling but also by directly controlling the mitotic machinery [ 39 ]. KIF23 is an essential regulator of cytokinesis and midbody formation, and its ac⁴C-dependent stabilization facilitates G2/M transition and mitotic completion in colorectal cancer cells [ 39 ]. HMGA1 in prostate cancer and SOX4 and AKT1 in bladder cancer similarly promote cell-cycle progression and survival through distinct mechanisms [ 149 , 150 ]. However, NAT10 does not invariably accelerate cell-cycle progression by suppressing inhibitory pathways. In bladder cancer, chloride intracellular channel 3 (CLIC3) binds NAT10 and inhibits its activity, resulting in decreased p21 mRNA ac⁴C and stability. The subsequent reduction in p21 protein promotes cell proliferation [ 184 ]. Although this result appears to contradict the prevailing model in which elevated NAT10 promotes growth, it instead reveals the bidirectional nature of the NAT10 substrate network. When the dominant substrate is a tumor suppressor, inhibition of NAT10 may paradoxically facilitate cell-cycle progression. NAT10 dependency should therefore be understood as substrate-network dependency rather than simple dependence on enzyme abundance. Beyond regulating individual mRNAs, NAT10 can remodel global translational capacity through tRNA ac⁴C, thereby supplying the protein-synthesis machinery required for rapid tumor growth. In several tumor contexts, NAT10 loss is accompanied by reduced tRNA ac⁴C, impaired global translation, and suppression of MYC-associated programs [ 54 ]. These findings indicate that the proliferative effects of NAT10 do not depend exclusively on a single mRNA substrate but may also arise from system-level changes in translation [ 55 ]. This mechanism differs fundamentally from selective mRNA stabilization. tRNA-dependent regulation affects ribosomal decoding and the preferential translation of transcripts enriched for specific codons, whereas mRNA stabilization primarily alters the half-life of individual transcripts. Both mechanisms may coexist within the same tumor, but most studies have measured only total ac⁴C or a small number of candidate targets and therefore cannot reliably distinguish transcript-specific effects from global translational reprogramming [ 56 ]. Overall, the NAT10–ac⁴C axis sustains tumor proliferation through three principal mechanisms: selective stabilization of growth- and cell-cycle-related mRNAs, direct enhancement of translation of key transcripts, and remodeling of global or codon-biased protein synthesis through tRNA and rRNA modification. The dominant mechanism varies among cancer types, and individual NAT10 substrate networks may contain both proliferative and inhibitory transcripts, thereby explaining apparently contradictory findings across studies. Current evidence nevertheless has important limitations. Many studies rely on acRIP-qPCR, total ac⁴C measurements, and rescue with a single candidate target, which are insufficient to distinguish direct modification from indirect transcriptional effects or non-catalytic NAT10 functions. Systematic comparisons of the contributions of mRNA, tRNA, and rRNA ac⁴C are also lacking. Future studies should integrate single-nucleotide ac⁴C mapping, ribosome profiling, quantitative proteomics, catalytic-dead rescue experiments, and site-specific RNA editing to establish causal substrate–mechanism–phenotype relationships across tumor types and to identify the NAT10–RNA circuits that truly determine tumor-selective dependency. Cancer stemness is not maintained by a single stemness factor but instead depends on coordinated regulation of chromatin accessibility, metabolic adaptation, transcriptional stability, and plastic reprogramming under therapeutic pressure [ 185 , 186 ]. Current evidence indicates that the NAT10–ac⁴C axis supports this state through at least three distinct mechanisms: stabilization of core stemness-associated transcripts, enhancement of translation of metabolic proteins, and direct remodeling of R-loops and chromatin architecture. ac⁴C may therefore not only preserve pre-existing cancer stem cell states but also facilitate reacquisition of stemness by non-stem tumor cells exposed to therapeutic stress. In GSCs, NAT10 functions extend beyond conventional regulation of linear RNA stability [ 61 ]. GSCs exhibit abundant and highly active promoter-proximal R-loops that are closely associated with open chromatin and active transcription [ 61 ]. NAT10 acts as a high-affinity R-loop-binding protein and deposits ac⁴C on the RNA strand within these structures, thereby stabilizing promoter-associated R-loops and maintaining local chromatin accessibility. This mechanism sustains expression of core stemness regulators such as EGR1 [ 61 ]. These findings establish a direct mechanistic link among RNA modification, R-loop stabilization, chromatin opening, and stemness-associated transcription. They further suggest that ac⁴C can maintain cancer stem cell plasticity at the chromatin level rather than merely regulating the fate of individual transcripts. Compared with classical mRNA stabilization, this mechanism may exert broader effects by simultaneously influencing multiple stemness-associated genes. Nevertheless, excessive R-loop accumulation is generally associated with replication stress and genome instability. How NAT10-mediated R-loop stabilization balances maintenance of stemness against induction of genomic damage therefore remains to be clarified. Cancer stem cells require specialized metabolic programs to sustain self-renewal, redox balance, and therapeutic resistance [ 187 , 188 ]. The NAT10–ac⁴C axis directly connects RNA modification with these metabolic demands by stabilizing or enhancing translation of metabolism-related transcripts. In ovarian cancer, NAT10 stabilizes PGAM1 mRNA through ac⁴C modification, thereby increasing glycolytic activity while promoting tumorsphere formation and expression of stemness markers. Restoration of PGAM1 reverses the reductions in glycolysis and stemness caused by NAT10 depletion, indicating that PGAM1-dependent glycolytic reprogramming is a central functional component of NAT10-mediated stemness maintenance in ovarian cancer [ 154 ]. By contrast, NAT10 in AML does not merely reinforce a single glycolytic enzyme but instead systematically remodels serine metabolism. NAT10 enhances translation of the serine transporter solute carrier family 1 member 4 (SLC1A4), as well as HOXA9 and MENIN, through ac⁴C-dependent mechanisms. HOXA9 and MENIN subsequently activate transcription of genes involved in serine biosynthesis, thereby simultaneously increasing exogenous serine uptake and endogenous serine production. This program supports leukemia stem cell self-renewal and leukemia-initiating capacity [ 155 ]. This mechanism establishes a cascade of RNA modification, translational enhancement, transcriptional amplification, and metabolic reprogramming and is therefore more system-wide than stabilization of a single metabolic enzyme. Together, these studies demonstrate that NAT10 frequently supports cancer stemness through metabolism, but the relevant metabolic dependency is tumor-type specific. Solid tumors may preferentially rely on glycolysis, whereas leukemia stem cells may depend more strongly on amino acid metabolism and one-carbon-related pathways. There is therefore no universal “stemness metabolic target” downstream of NAT10, and the relevant vulnerability must be defined according to the metabolic context of each malignancy. In addition to providing metabolic support, NAT10 can directly stabilize stemness-associated transcripts and thereby enable tumor cells to maintain or reacquire stem-like properties under chemotherapeutic pressure. In chemoresistant cervical cancer, NAT10 expression and global ac⁴C levels are increased [ 156 ]. NAT10 enhances ac⁴C modification and stability of NANOGP8 mRNA, thereby maintaining stemness and reducing chemotherapy sensitivity. Inhibition of NAT10 attenuates stem-like features and restores drug responsiveness [ 156 ]. This mechanism differs from metabolic reprogramming because its central function is direct maintenance of a stemness transcription factor. NANOGP8, a transcript derived from a NANOG-related pseudogene, reinforces pluripotency-associated and therapy-resistant programs, suggesting that NAT10 can selectively protect tumor-specific stemness RNAs. ac⁴C may therefore contribute not only to maintenance of established cancer stem cells but also to treatment-induced cell-state transitions, enabling residual tumor cells to adopt a more plastic and drug-resistant phenotype. It remains unclear, however, whether NANOGP8 stabilization initiates chemoresistance or instead functions as a secondary maintenance mechanism after resistant populations have emerged. Resolving this issue will require longitudinal single-cell profiling of ac⁴C dynamics before, during, and after treatment rather than endpoint comparisons between sensitive and resistant cells. These mechanisms are unlikely to operate independently. Open chromatin sustains transcription of stemness genes, metabolic reprogramming supplies the substrates and energy required for transcription and translation, and stable expression of core stemness factors can in turn reinforce metabolic and chromatin-regulatory programs. The NAT10–ac⁴C axis is therefore more appropriately viewed as an integrated network coordinating epigenetic plasticity, post-transcriptional regulation, and metabolic adaptation rather than as a pathway acting through a single stemness gene. Overall, the NAT10–ac⁴C axis maintains cancer stem cell self-renewal, therapeutic resistance, and state plasticity through R-loop and chromatin remodeling, stabilization of stemness-associated transcripts, and reinforcement of metabolic programs. However, the available evidence remains limited. Most studies rely on tumorsphere formation, stemness-marker expression, and xenograft growth as indirect indicators and lack rigorous serial transplantation, lineage tracing, or limiting-dilution analyses required to establish bona fide tumor-initiating capacity. In addition, several studies depend primarily on acRIP-qPCR and NAT10 knockdown, making it difficult to exclude non-catalytic functions and indirect transcriptional effects. Future work should integrate single-cell ac⁴C profiling, longitudinal treatment-response analysis, site-specific RNA editing, in situ lineage tracing, and patient-derived models to distinguish the roles of NAT10 in stemness maintenance, stemness acquisition, and therapy-induced plasticity and to identify tumor-specific NAT10–RNA dependencies that are not shared by normal stem cells. Genome stability is a fundamental determinant of tumor initiation and progression and a major factor governing the efficacy of radiotherapy, chemotherapy, and poly(ADP-ribose) polymerase (PARP) inhibitors [ 189 – 191 ]. In recent years, the NAT10–ac⁴C axis has emerged as a critical link between RNA modification and the DNA damage response (DDR) [ 158 ]. Compared with other RNA modifications, however, the role of ac⁴C in DDR is particularly complex. On the one hand, NAT10 protects the genome by increasing the expression of DNA repair factors, promoting homologous recombination (HR), and preserving chromosomal integrity. On the other hand, tumor cells exploit these same mechanisms to enhance DNA repair capacity and acquire resistance to chemotherapy and PARP inhibition. NAT10 therefore functions both as a guardian of normal genome homeostasis and as a molecular driver of therapeutic resistance in cancer. The most consistently supported mechanism is that NAT10 enhances HR by stabilizing transcripts encoding DNA repair factors. In triple-negative breast cancer, NAT10 catalyzes ac⁴C modification of RAD51 mRNA, increases its stability, and elevates RAD51 protein expression, thereby promoting HR repair. NAT10 depletion leads to persistent accumulation of DNA double-strand breaks (DSBs) and markedly enhances the antitumor activity of the PARP inhibitor olaparib, whereas combined treatment with Remodelin and a PARP inhibitor produces a pronounced synergistic effect [ 157 ]. Similarly, in bladder cancer, NAT10 stabilizes AHNAK mRNA and thereby enhances DNA damage repair, promoting cisplatin resistance. NAT10 inhibition significantly sensitizes both organoid and animal models to cisplatin treatment [ 158 ]. In colorectal cancer, NAT10 further promotes translation of PPAN and MYC, establishing a MYC–PPAN positive-feedback loop that reinforces DNA repair and cell survival. This finding suggests that NAT10 not only regulates individual DDR factors but can also sustain repair capacity by remodeling broader transcriptional networks [ 58 ]. The role of NAT10 in regulating DNA: RNA hybrids and R-loops has further expanded the mechanistic framework through which RNA modification participates in DDR. Recent work demonstrated that, following the formation of DSBs, NAT10 is rapidly recruited to damaged chromatin and, in a PARP1-dependent manner, deposits ac⁴C on the RNA strand of local DNA: RNA hybrids. This modification stabilizes the hybrid structure, facilitates RAD51 recruitment, and promotes HR repair [ 192 ]. This study provided the first direct evidence that ac⁴C is not restricted to mature RNA species but can also modify RNA molecules generated at DNA damage sites and contribute to assembly of a repair platform. In this context, controlled stabilization of DNA: RNA hybrids and R-loops supports HR, whereas NAT10 inhibition weakens HR and can restore the sensitivity of PARP inhibitor-resistant tumors. R-loops, however, are not universally protective, and their biological consequences are strongly context-dependent. Whereas transient R-loops at DNA damage sites may need to be stabilized, persistent R-loop accumulation in the nucleolus becomes a major source of replication stress and genome instability. Recent evidence indicates that NAT10 promotes nucleolar R-loop clearance through two cooperative mechanisms. First, its RNA helicase-like domain directly contributes to R-loop unwinding. Second, NAT10 acetylates the RNA helicase DDX21 and enhances its helicase activity, thereby facilitating R-loop resolution, preserving ribosomal DNA stability, and preventing DNA damage accumulation [ 88 ]. NAT10 therefore does not simply promote or suppress R-loop formation. Instead, it dynamically regulates R-loop homeostasis according to chromatin context: it stabilizes transient DNA: RNA hybrids at DNA break sites to facilitate HR while actively eliminating persistent nucleolar R-loops to relieve replication stress and nucleolar dysfunction. This dual mode of “stabilizing beneficial R-loops while clearing harmful R-loops” may represent a central mechanism through which NAT10 maintains genome integrity. Notably, findings regarding NAT10 in DDR are not entirely consistent. Most studies indicate that NAT10 promotes DNA repair by stabilizing factors such as RAD51 and AHNAK. In contrast, one study showed that, in ultraviolet B-induced DNA damage, NAT10 decreases DDB2 mRNA stability and thereby suppresses global genome nucleotide excision repair (GG-NER), whereas NAT10 depletion increases DDB2 expression and enhances repair of ultraviolet-induced lesions [ 165 ]. This result contrasts with the prevailing view that NAT10 generally stabilizes mRNAs and further supports the conclusion that ac⁴C does not invariably increase RNA stability. Its effects may instead depend on the relevant RBP, modification site, local RNA structure, and DNA repair pathway. HR primarily repairs DSBs, whereas nucleotide excision repair removes ultraviolet-induced DNA adducts, and these pathways differ substantially in their reliance on RNA metabolism. NAT10 may therefore exert distinct or even opposing functions across different DDR contexts. A more unified model is that NAT10 maintains genome homeostasis through several interconnected mechanisms: enhancing the stability and translation of HR-associated transcripts, dynamically regulating DNA: RNA hybrids and R-loops, relieving nucleolar stress and replication pressure, and preserving chromosomal integrity. In cancer cells, however, these physiologically protective mechanisms are repurposed to sustain elevated DNA repair capacity and increase resistance to chemotherapy, radiotherapy, and PARP inhibitors. NAT10 therefore exhibits a pronounced double-edged role, preserving genome integrity in normal tissues while driving treatment resistance in tumors. Overall, the NAT10–ac⁴C axis has become an emerging focus at the intersection of RNA modification, R-loop homeostasis, and DNA damage repair. Several key questions remain unresolved. The number of directly validated DDR substrates remains limited, and whether RAD51, AHNAK, PPAN, and DDB2 represent general regulatory principles across distinct repair pathways requires further investigation. The mechanisms that confer spatiotemporal specificity on R-loop stabilization at DNA damage sites versus R-loop clearance in the nucleolus also remain unclear. In addition, the determinants governing whether NAT10 promotes or suppresses RNA stability and the potential cooperation among ac⁴C, PARP1, RBPs, and other RNA modifications have yet to be defined. Future studies integrating single-nucleotide ac⁴C sequencing, R-loop profiling, DNA: RNA immunoprecipitation sequencing (DRIP-seq), spatial multi-omics, live-cell imaging, and site-specific RNA editing will be required to map the dynamic ac⁴C regulatory networks operating across different DNA repair pathways. Such approaches should also distinguish the roles of NAT10 in physiological genome protection from those in tumor treatment resistance and provide a more precise rationale for combining NAT10 inhibition with DDR-targeted therapies. Tumor metastasis is the principal cause of cancer-related mortality and does not depend solely on EMT. Rather, it encompasses a continuous sequence of events involving cytoskeletal remodeling, cell–matrix adhesion, ECM remodeling, local invasion, angiogenesis, and establishment of the pre-metastatic niche [ 193 – 195 ]. Recent studies indicate that the NAT10–ac⁴C axis operates across multiple stages of the metastatic cascade by stabilizing distinct RNA substrates and coordinating EMT, cell adhesion, metabolic adaptation, and organ-specific colonization [ 149 , 196 ]. However, the direct substrates identified across cancer types vary considerably, suggesting that NAT10 functions more as an upstream integrator of metastatic networks than as a regulator of a single EMT factor [ 149 ]. The most widely reported function of NAT10 in metastasis is promotion of EMT. Multiple studies have linked elevated NAT10 expression to reduced E-cadherin and increased N-cadherin and vimentin. In NSCLC, NAT10 knockdown markedly reverses EMT and suppresses cell migration and invasion, whereas Remodelin simultaneously inhibits EMT and malignant progression [ 159 ]. Likewise, in gastric cancer, NAT10 downregulation is accompanied by reduced global ac⁴C, decreased AKT phosphorylation, and reversal of EMT, indicating that NAT10 promotes EMT through PI3K/AKT-related signaling rather than through a single canonical EMT transcription factor [ 160 ]. In addition, in endometriosis, a benign but invasive disorder, NAT10 stabilizes TGFB1 mRNA, enhances TGF-β signaling, and induces EMT, demonstrating that ac⁴C-mediated EMT regulation is not restricted to malignant tumors but also contributes to pathological tissue invasion [ 161 ]. The pro-EMT activity of NAT10 does not appear to depend exclusively on classical EMT transcription factors. Instead, NAT10 frequently stabilizes downstream effectors with direct functions in cytoskeletal organization and cell motility. In prostate cancer, NAT10 stabilizes HMGA1 and KRT8 mRNAs. HMGA1 primarily promotes cell-cycle progression, whereas KRT8 directly contributes to cytoskeletal remodeling and EMT, thereby enhancing migratory capacity [ 149 ]. In ovarian cancer, NAT10 stabilizes the RBP CAPRIN1 and thereby promotes migration, invasion, and tumor stemness, indicating that RBPs can also serve as critical downstream effectors of NAT10-driven cellular plasticity [ 162 ]. Together, these studies suggest that NAT10 remodels the broader EMT execution network rather than merely activating canonical regulators such as SNAIL, ZEB, or TWIST. Cell migration requires not only EMT but also dynamic control of cell–matrix adhesion and ECM interactions [ 159 ]. Recent work showed that NAT10 stabilizes ITGB1 and COL1A2 mRNAs through ac⁴C modification, increases integrin β1 expression, and sustains activation of focal adhesion kinase (FAK) signaling. This enhances cell–matrix adhesion, cytoskeletal remodeling, and migratory capacity [ 18 ]. These findings directly connect ac⁴C with the integrin–FAK–focal adhesion axis and suggest that NAT10 not only promotes acquisition of a mesenchymal phenotype but also provides the mechanical support required for persistent migration by reinforcing focal adhesion formation and ECM remodeling. The role of NAT10 in metastasis therefore extends beyond conventional EMT to encompass broader remodeling of the cellular motility apparatus. Successful distant metastasis also requires adaptation to a new metabolic environment. A recent study of breast cancer brain metastasis identified NAT10 as an important driver of cerebral colonization. Unlike most studies focused on EMT, this work showed that NAT10 increases phosphoglycerate dehydrogenase and PSAT1 expression and thereby enhances serine biosynthesis. This metabolic program allows tumor cells to maintain a proliferative advantage within the serine- and glycine-poor environment of the brain, facilitating brain colonization and distant metastasis [ 163 ]. These findings indicate that metabolic plasticity is itself an integral component of metastatic competence. NAT10 can enhance the ability of tumor cells to adapt to distinct organ microenvironments through metabolic reprogramming rather than by promoting migration alone. An increasing number of studies further suggest that the prometastatic functions of NAT10 extend beyond stabilization of individual mRNAs. NAT10 has been reported to regulate stress tolerance, inflammatory signaling, cell adhesion, and the metastatic microenvironment through diverse downstream axes involving GLMP, HSP90AA1, CXCL5, NFE2L3, and HNRNPUL1. However, the dominant substrates differ among cancer types. This pronounced heterogeneity supports the view that NAT10 functions as a post-transcriptional regulatory platform that selectively stabilizes distinct RNA networks according to tissue context, thereby coordinating EMT, ECM remodeling, angiogenesis, and pre-metastatic niche formation rather than relying on a universal “metastasis gene.” Overall, current evidence supports a model in which the NAT10–ac⁴C axis promotes local invasion and distant metastasis by coordinating EMT, cytoskeletal remodeling, cell–matrix adhesion, ECM reorganization, and metabolic adaptation. Several important limitations remain. Most studies have focused primarily on tumor-cell-intrinsic motility, whereas direct evidence that NAT10 regulates angiogenesis, vascular permeability, or pre-metastatic niche formation remains limited. Direct substrates also differ substantially across cancer types, and no unified mechanism currently explains organ-specific metastatic tropism. In addition, most available evidence derives from experimental metastasis models and lacks systematic analysis of the full natural metastatic process and its spatial dynamics. Future studies should integrate single-cell spatial transcriptomics, spatial ac⁴C profiling, intravital tracing, and organ-specific metastasis models to determine how NAT10 coordinates EMT, cell adhesion, ECM remodeling, metabolic adaptation, and remodeling of the tumor microenvironment throughout the metastatic cascade. Such studies will also be essential for evaluating the clinical potential of NAT10 as a target for antimetastatic therapy. One of the emerging functions of NAT10 is the coordination of metabolic reprogramming, allowing tumor cells to satisfy biosynthetic and energetic demands. Figure  5 summarizes the major metabolic pathways regulated by NAT10-mediated ac⁴C modification. Fig. 5 NAT10-mediated metabolic reprogramming in cancer. NAT10 remodels amino acid, glucose, lipid, and redox metabolism through stabilization and enhanced translation of ac⁴C-modified transcripts. These metabolic adaptations promote anabolic metabolism, antioxidant defense, ferroptosis resistance, and establishment of an immunosuppressive tumor microenvironment NAT10-mediated metabolic reprogramming in cancer. NAT10 remodels amino acid, glucose, lipid, and redox metabolism through stabilization and enhanced translation of ac⁴C-modified transcripts. These metabolic adaptations promote anabolic metabolism, antioxidant defense, ferroptosis resistance, and establishment of an immunosuppressive tumor microenvironment Glycolytic reprogramming is a central feature of tumor metabolism [ 197 , 198 ]. It not only supplies ATP and biosynthetic precursors for rapid proliferation but also remodels the immune microenvironment through lactate accumulation and contributes to therapeutic resistance [ 199 – 201 ]. A growing body of evidence indicates that the NAT10–ac⁴C axis participates in multiple critical steps of the glycolytic program, including glucose uptake, expression of rate-limiting enzymes, adaptation to hypoxia, and lactate-driven immunosuppression [ 164 ]. However, the metabolic dependence on NAT10 varies across tumor types. Its downstream targets include glycolytic enzymes, transcription factors, and upstream signaling molecules, which collectively converge on reinforcement of the Warburg effect. The most consistently supported mechanism is that NAT10 enhances glycolytic flux by increasing the stability of mRNAs encoding key glycolytic enzymes. In gastric cancer, NAT10 directly modifies the junction between the coding region and 3′UTR of HK2 mRNA, thereby increasing transcript stability, promoting glucose phosphorylation, and driving glycolysis and tumorigenesis [ 164 ]. In ovarian cancer, NSCLC, and retinoblastoma, NAT10 stabilizes PGAM1, ENO1, and PFKFB3 mRNAs, respectively, leading to increased glucose uptake, lactate production, and extracellular acidification rate and ultimately promoting tumor growth [ 154 , 166 , 167 ]. These studies suggest that, although different tumors depend on distinct glycolytic nodes, NAT10 generally reinforces glycolytic throughput by maintaining the expression of rate-limiting enzymes rather than by activating a single universal metabolic target. Beyond directly regulating metabolic enzymes, NAT10 can amplify glycolytic programs through upstream transcriptional networks. In triple-negative breast cancer, NAT10 stabilizes JunB mRNA, increases downstream lactate dehydrogenase A (LDHA) expression, enhances lactate production, and suppresses antitumor T-cell activity [ 168 ]. In cervical cancer, the transcription factor HOXC8 activates NAT10 expression, after which NAT10 enhances FOXP1 translation and induces GLUT4 and KHK expression, thereby sustaining glucose uptake and lactate secretion. The lactate-rich tumor microenvironment further promotes immunosuppressive regulatory T-cell activity and reduces the efficacy of PD-L1 blockade [ 169 ]. Compared with direct stabilization of glycolytic enzymes, this transcription factor–metabolic program has a broader amplifying effect and more directly links tumor metabolism with immune evasion. NAT10 is also an important mediator of hypoxic adaptation. In gastric cancer, HIF-1α directly activates NAT10 transcription, whereas NAT10 stabilizes SEPT9 mRNA through ac⁴C modification and further reinforces HIF-1α signaling. This HIF-1α–NAT10–SEPT9 positive-feedback loop persistently activates glycolysis and promotes resistance to anti-angiogenic therapy [ 140 ]. Thus, NAT10 not only responds to hypoxia but also amplifies hypoxic signaling, converting a transient adaptive response into a stable metabolic dependency. Notably, NAT10 itself is dynamically regulated by nutrient availability. Under glucose deprivation, the autophagy–lysosome pathway promotes SQSTM1/LC3-mediated degradation of NAT10, resulting in reduced global ac⁴C levels and suppression of HK2 expression, thereby limiting glycolytic activity [ 164 ]. This finding indicates that NAT10 is not a constitutively active metabolic driver but rather a nutrient-responsive regulator that participates in bidirectional feedback between metabolic stress and RNA modification. The glycolytic dependence on NAT10 also provides opportunities for therapeutic intervention. In addition to Remodelin, GNE-493 directly binds NAT10, reduces ac⁴C modification of HK2 mRNA, and suppresses glycolysis in cisplatin-resistant gastric cancer cells [ 202 ]. In triple-negative breast cancer and cervical cancer models, NAT10 inhibition also enhances the efficacy of CTLA-4- or PD-L1-directed immunotherapy, respectively, suggesting that blocking NAT10 can simultaneously restrain tumor metabolism and reverse lactate-driven immunosuppression [ 168 , 169 ]. Overall, the NAT10–ac⁴C axis drives glycolytic reprogramming by stabilizing key glycolytic enzymes, enhancing metabolic transcriptional networks, and amplifying hypoxic signaling. Several limitations nevertheless remain. First, direct substrates identified across studies differ substantially, and it is unclear whether HK2, PFKFB3, ENO1, PGAM1, and LDHA represent tissue-specific dependencies or components of a shared core metabolic network. Second, most studies focus on individual enzymes and provide limited information on global metabolic flux or carbon-source utilization. Third, it remains uncertain whether NAT10-dependent glycolytic regulation is entirely mediated by mRNA ac⁴C or also involves tRNA-dependent translational reprogramming and non-catalytic functions. Future studies integrating stable-isotope tracing, single-cell metabolomics, ribosome profiling, and site-specific RNA editing will be required to define both the common mechanisms and tissue-specific metabolic vulnerabilities of NAT10-driven glycolysis and to provide a more precise foundation for combining NAT10-targeted therapy with metabolic or immune-based interventions. Beyond glycolysis, amino acid metabolic reprogramming provides an essential foundation for rapid tumor growth, redox homeostasis, and therapeutic resistance [ 203 – 205 ]. Recent studies indicate that the NAT10–ac⁴C axis broadly regulates serine, glutamine, and asparagine metabolism. However, it does not act uniformly on all metabolic enzymes. Instead, depending on tumor context, NAT10 selectively stabilizes amino acid transporters, metabolic enzymes, or upstream transcriptional regulators, thereby establishing tissue-specific metabolic dependencies. The strongest evidence currently concerns serine metabolism. In AML, NAT10 enhances the translational efficiency of the serine transporter SLC1A4 and the transcriptional regulators HOXA9 and MENIN through ac⁴C-dependent mechanisms. HOXA9 and MENIN subsequently activate transcription of the serine biosynthetic genes PHGDH, PSAT1, and PSPH, thereby promoting both exogenous serine uptake and endogenous de novo synthesis. This coordinated program sustains leukemia stem cell self-renewal and leukemogenesis [ 155 ]. Unlike conventional mechanisms that directly stabilize a metabolic enzyme, this pathway establishes a cascade of RNA modification, translational enhancement, transcriptional amplification, and metabolic reprogramming, rendering serine metabolism a major metabolic vulnerability underlying the high dependence of AML on NAT10. By contrast, solid tumors appear more likely to rely on glutamine metabolism. In lung cancer, NAT10 stabilizes NIT2 mRNA through ac⁴C modification, thereby promoting glutamine consumption, α-ketoglutarate (α-KG) production, and ATP generation and consequently enhancing cell proliferation and migration [ 170 ]. In oral squamous cell carcinoma, NAT10 directly modifies and stabilizes the glutamine transporter SLC1A5 mRNA, increasing glutamine uptake and driving tumor progression. Upstream, SP1 directly activates NAT10 expression, establishing an SP1–NAT10–SLC1A5 regulatory axis [ 171 ]. Together, these studies indicate that NAT10 can regulate both glutamine transport and downstream glutamine utilization, although the critical dependency differs among tumor types. Asparagine metabolism illustrates yet another regulatory mode. In osteosarcoma, NAT10 does not directly stabilize the mRNA of a metabolic enzyme [ 206 – 208 ]. Instead, it stabilizes ATF4 mRNA through ac⁴C modification, increases ATF4 protein expression, and thereby promotes transcriptional activation of ASNS, enhancing asparagine biosynthesis and tumor growth [ 98 ]. Thus, unlike the translationally driven mechanism observed in AML, NAT10 in osteosarcoma remodels amino acid metabolism indirectly through a transcription factor–metabolic enzyme axis, further underscoring the heterogeneity of NAT10-dependent metabolic networks. Current evidence therefore suggests that NAT10 regulates amino acid metabolism at multiple levels. In some pathways, it directly stabilizes mRNAs encoding transporters or metabolic enzymes. In others, it preferentially enhances the expression of upstream transcriptional regulators such as ATF4, HOXA9, and MENIN, which then reprogram broader metabolic networks. This multilayered architecture enables NAT10 to establish tumor-specific amino acid metabolic programs adapted to distinct tissue environments rather than imposing a uniform metabolic state. Overall, the NAT10–ac⁴C axis coordinates amino acid uptake, de novo synthesis, and transcriptional control to generate context-dependent metabolic programs across diverse cancers. Important limitations remain. Existing studies have focused primarily on serine, glutamine, and asparagine metabolism, whereas branched-chain amino acids, arginine, methionine, and one-carbon metabolism remain comparatively understudied. Most investigations validate only one principal substrate and lack comprehensive flux analyses capable of revealing coordinated rewiring across amino acid pathways. It is also unclear whether NAT10 can further influence the global synthesis of amino acid metabolic proteins through tRNA ac⁴C-dependent codon-biased translation. Future studies combining stable-isotope tracing, single-cell metabolomics, multi-omics integration, and site-specific RNA editing will be needed to define both the shared mechanisms and tissue-specific metabolic dependencies of NAT10-driven amino acid reprogramming and to support the development of precision metabolic therapies. Lipid metabolic reprogramming provides tumor cells with membrane components, energy substrates, signaling molecules, and adaptive capacity within the tumor microenvironment [ 209 – 211 ]. Recent studies indicate that the NAT10–ac⁴C axis regulates not only fatty acid synthesis but also fatty acid elongation, β-oxidation, cholesterol metabolism, and lipid storage and can further modulate metabolic interactions between tumor and immune cells [ 172 ]. As in glycolysis and amino acid metabolism, NAT10 controls lipid metabolism through a network of targets rather than a single metabolic enzyme. The most direct evidence derives from global metabolomic and lipidomic analyses [ 172 ]. Following NAT10 depletion, fatty acid metabolism emerges as one of the most significantly altered pathways. Multiple fatty acid metabolism-related genes, including elongation of very ELOVL6, acyl-CoA synthetase long-chain family members 1, 3, and 4 (ACSL1, ACSL3, and ACSL4), ACADSB, and ACAT1, require NAT10-mediated ac⁴C to maintain their expression. Correspondingly, intracellular total lipid, triglyceride, and cholesterol levels are markedly reduced, while lipidomic profiling reveals widespread remodeling of hundreds of lipid species. These findings indicate that NAT10 preserves tumor-cell lipid homeostasis by coordinating multiple nodes of fatty acid metabolism rather than by regulating a single substrate [ 172 ]. Beyond fatty acid synthesis, NAT10 also regulates mitochondrial fatty acid metabolism. Untargeted metabolomics following Remodelin treatment revealed substantial changes in metabolites associated with mitochondrial fatty acid elongation and β-oxidation, including lauroyl-CoA, (S)-3-hydroxyhexadecanoyl-CoA, and nicotinamide adenine dinucleotide (NAD⁺), together with reductions in cholesterol and triglyceride levels. Expression of key mitochondrial fatty acid metabolic enzymes, including enoyl-CoA hydratase short-chain 1 (ECHS1) and mitochondrial trans-2-enoyl-CoA reductase (MECR), is also decreased. These observations suggest that NAT10 supports not only lipid synthesis but also mitochondrial fatty acid utilization and metabolic flexibility [ 212 ]. More recently, the role of NAT10 in lipid metabolism has been extended from tumor cells to the tumor microenvironment. In esophageal squamous cell carcinoma, METTL3-mediated m⁶A modification increases NAT10 expression, whereas tumor-derived exosomes transport NAT10 into TAMs. Within macrophages, NAT10 stabilizes FASN mRNA through ac⁴C modification, enhances fatty acid synthesis, and promotes M2 polarization, thereby establishing an immunosuppressive microenvironment favorable to tumor growth. NAT10 inhibition not only suppresses M2 polarization but also markedly enhances the efficacy of PD-1 blockade, identifying NAT10-mediated lipid metabolism as an important link between metabolic reprogramming and immune evasion [ 173 ]. Current findings therefore place NAT10-mediated lipid regulation at three interconnected levels. First, NAT10 stabilizes mRNAs encoding fatty acid metabolic proteins and thereby sustains lipid synthesis and fatty acid activation. Second, it coordinates mitochondrial fatty acid elongation and β-oxidation to support energy production and metabolic plasticity. Third, it remodels macrophage lipid metabolism through exosome-mediated intercellular communication, promoting formation of an immunosuppressive tumor microenvironment. Together, these mechanisms indicate that NAT10 is not only an intracellular metabolic regulator but also a coordinator of tumor–immune metabolic crosstalk. Overall, current evidence supports a role for NAT10 in fatty acid synthesis, mitochondrial fatty acid metabolism, and lipid-mediated immune regulation, although this field remains at an early stage. The number of directly validated ac⁴C substrates is still limited, and much of the available evidence derives from RNA sequencing, lipidomics, or metabolomics without systematic single-nucleotide ac⁴C validation. How fatty acid synthesis, β-oxidation, and cholesterol metabolism are coordinately regulated by NAT10 also remains unclear. In addition, whether NAT10 remodels the global translation of lipid metabolic proteins through tRNA or rRNA ac⁴C and how it regulates metabolism in distinct immune-cell populations require further investigation. Future work integrating stable-isotope lipid tracing, spatial lipidomics, single-cell metabolomics, and site-specific RNA editing will be essential for defining the molecular network of NAT10-driven lipid reprogramming and for evaluating its potential as a target for combined metabolic and immunotherapeutic strategies. Rapidly proliferating tumor cells are continuously exposed to oxidative stress resulting from the accumulation of ROS [ 213 , 214 ]. Establishing a stable redox state is therefore essential for tumor survival and therapeutic resistance [ 213 ]. Recent evidence indicates that the NAT10–ac⁴C axis not only regulates glucose, lipid, and amino acid metabolism but also coordinates antioxidant defense and ferroptosis sensitivity, thereby enabling tumor cells to adapt to metabolic stress [ 215 ]. Compared with glycolysis and other metabolic pathways, however, the mechanisms linking NAT10 to redox metabolism remain less extensively characterized and are currently centered mainly on nuclear factor erythroid 2-related factor 2 (Nrf2) signaling and the glutathione (GSH) metabolic network. The most direct evidence indicates that NAT10 stabilizes Nrf2 mRNA through ac⁴C modification, enhances its translational efficiency, and reinforces downstream antioxidant programs [ 174 ]. In hepatocellular carcinoma, elevated NAT10 expression is accompanied by sustained activation of Nrf2 signaling and increased expression of canonical antioxidant genes, including SLC7A11, GPX4, NAD(P)H quinone dehydrogenase 1 (NQO1), and heme oxygenase 1 (HO-1). This reduces intracellular ROS accumulation and increases resistance to oxidative stress [ 174 ]. NAT10 depletion decreases Nrf2 expression, markedly elevates ROS levels, and increases lipid peroxidation, indicating that NAT10 functions as an upstream regulator of tumor redox homeostasis. Because Nrf2 is also a major transcriptional determinant of ferroptosis resistance, the NAT10-dependent antioxidant program directly influences ferroptotic sensitivity. Activation of Nrf2 by NAT10 enhances cystine uptake, GSH synthesis, and GPX4-dependent detoxification of lipid peroxides, thereby suppressing ferroptosis and reducing the efficacy of ferroptosis-inducing agents such as sorafenib. Conversely, NAT10 inhibition weakens Nrf2-dependent antioxidant defenses, increases ROS and lipid peroxidation, and restores tumor-cell sensitivity to sorafenib [ 174 ]. The regulation of redox homeostasis by NAT10 is unlikely to be restricted to Nrf2 itself. As discussed above, NAT10 promotes glycolysis, serine metabolism, and lipid metabolism, all of which provide substrates required for GSH synthesis, NADPH production, and membrane-lipid renewal. NAT10 also regulates amino acid transporters such as SLC1A4 and SLC1A5 and multiple fatty acid metabolic enzymes, thereby indirectly influencing cellular antioxidant capacity. The impact of NAT10 on redox homeostasis is therefore more likely to reflect the integrated effects of glucose, amino acid, and lipid metabolism together with Nrf2 signaling than the activity of a single antioxidant pathway. Redox regulation is also closely connected to tumor immune evasion. Altered ROS levels can influence not only tumor-cell survival but also immune-cell activation and the response to immune checkpoint therapy. NAT10-mediated metabolic reprogramming and antioxidant defense may therefore simultaneously regulate tumor-intrinsic oxidative stress and immunometabolism within the tumor microenvironment, providing a rationale for combining NAT10 inhibition with ferroptosis inducers or immunotherapy. Overall, available evidence supports a model in which the NAT10–ac⁴C axis maintains tumor redox homeostasis and suppresses ferroptosis through Nrf2-dependent antioxidant programs together with broader metabolic reprogramming. Several important gaps remain. Current evidence is derived predominantly from Nrf2-centered studies, and whether GSH cycling, NADPH metabolism, mitochondrial ROS clearance, and lipid-peroxidation networks are directly regulated by ac⁴C has not been systematically examined. It also remains unclear whether NAT10 remodels the global translation of antioxidant proteins through tRNA or rRNA ac⁴C, and comparative analyses of ferroptosis dependency across tumor types are lacking. Future studies integrating metabolic flux analysis, lipid-peroxidation profiling, single-cell metabolomics, and site-specific RNA editing will be required to define the molecular network linking NAT10 to redox homeostasis and ferroptosis resistance and to assess the translational potential of combining NAT10 inhibitors with ferroptosis-inducing agents, radiotherapy, or immunotherapy. Programmed cell death constitutes a critical barrier against tumorigenesis and is essential for maintaining tissue homeostasis [ 216 , 217 ]. With the identification of emerging forms of regulated cell death, including ferroptosis, pyroptosis, and cuproptosis, it has become increasingly clear that tumor cells evade not only classical apoptosis but also multiple death programs by remodeling metabolism, redox homeostasis, and inflammatory signaling. Recent studies have identified the NAT10–ac⁴C axis as an important link between RNA modification and cell-death regulation. However, unlike its generally protumorigenic effects on proliferation and metastasis, the role of NAT10 varies across different cell-death modalities and depends strongly on the specific death pathway and tissue context. NAT10 should therefore not be simplistically defined as a universal inhibitor of cell death; rather, its biological functions must be considered separately within each mode of regulated cell death. Besides regulating tumor growth, NAT10 also modulates multiple stress-response pathways that determine cancer cell survival and therapeutic sensitivity. Figure 6 summarizes the diverse roles of NAT10 in regulated cell death and autophagy. Fig. 6 Roles of NAT10 in regulated cell death pathways and autophagy. NAT10 regulates ferroptosis, pyroptosis, cuproptosis, and autophagy through ac⁴C-dependent stabilization of key regulators involved in oxidative stress, inflammasome activation, mitochondrial metabolism, and autophagic flux. These context-dependent effects influence tumor progression and treatment response Roles of NAT10 in regulated cell death pathways and autophagy. NAT10 regulates ferroptosis, pyroptosis, cuproptosis, and autophagy through ac⁴C-dependent stabilization of key regulators involved in oxidative stress, inflammasome activation, mitochondrial metabolism, and autophagic flux. These context-dependent effects influence tumor progression and treatment response Current research on NAT10-mediated cell-death regulation has focused predominantly on ferroptosis, for which the supporting evidence is most extensive. Across multiple tumor models, NAT10 suppresses ferroptosis by maintaining the system Xc⁻–GSH–GPX4 antioxidant axis, thereby promoting tumor-cell survival and therapeutic resistance [ 175 , 217 ]. Among the reported downstream targets, SLC7A11 is the most consistently validated direct substrate. In hepatocellular carcinoma, NAT10 stabilizes SLC7A11 mRNA through ac⁴C modification, increases cystine uptake and GSH synthesis, and reduces lipid peroxidation and Fe²⁺ accumulation. These effects suppress ferroptosis and promote tumor progression, whereas NAT10 inhibition markedly enhances ferroptosis and sensitizes tumors to transarterial chemoembolization (TACE) [ 175 ]. Similarly, in nasopharyngeal carcinoma, NAT10 stabilizes SLC7A11 and confers resistance to sorafenib-induced ferroptosis, whereas combined treatment with Remodelin and sorafenib substantially enhances antitumor activity [ 176 ]. Beyond SLC7A11, NAT10 can coordinately regulate multiple components of the GSH metabolic network. Systematic analyses have shown that NAT10 deficiency simultaneously reduces the expression of several ferroptosis-related genes, including glutamate-cysteine ligase catalytic subunit (GCLC), solute carrier family 39 member 8 (SLC39A8), and microtubule-associated protein 1 light chain 3 alpha (MAP1LC3A). These changes are accompanied by decreased cysteine uptake, GSH depletion, and sustained accumulation of ROS and oxidized phospholipids, ultimately inducing a canonical ferroptotic phenotype [ 215 ]. NAT10 therefore does not act exclusively through SLC7A11 but instead remodels a broader antioxidant metabolic network. Distinct tumors may additionally rely on tissue-specific downstream protective axes. In colon cancer, NAT10 suppresses ferroptosis by stabilizing ferroptosis suppressor protein 1 (FSP1/AIFM2) mRNA, thereby establishing a GPX4-independent ferroptosis-defense pathway [ 177 ]. In clear-cell renal cell carcinoma, NAT10 primarily sustains ferroptosis resistance through the NFE2L1–GPX4 axis [ 178 ]. Although the downstream targets differ, these pathways converge functionally on preservation of lipid peroxide detoxification and redox homeostasis. Overall, current evidence supports a model in which the NAT10–ac⁴C axis enables tumor cells to evade ferroptosis by stabilizing ferroptosis suppressors and reinforcing antioxidant metabolic networks, thereby contributing to resistance to therapies such as sorafenib and TACE. Several important questions remain unresolved. The hierarchical relationships among the SLC7A11, FSP1, and GPX4-centered protective pathways remain unclear. It is also unknown whether NAT10 can reshape the global translation of ferroptosis-related proteins through tRNA or rRNA ac⁴C and why different tumor types depend on distinct ferroptosis-defense mechanisms. Compared with ferroptosis, evidence regarding NAT10-mediated regulation of pyroptosis remains limited, but available studies indicate pronounced cell-type specificity [ 218 ]. In sepsis models, reduced NAT10 expression decreases ULK1 mRNA stability and subsequently activates the STING–IRF3–NLR family pyrin domain-containing 3 (NLRP3) inflammasome pathway, thereby promoting neutrophil pyroptosis. Restoration of NAT10 expression markedly alleviates sepsis-associated lung injury and improves animal survival [ 218 ]. These findings indicate that NAT10 can restrain excessive pyroptosis and preserve immune homeostasis in inflammatory settings. By contrast, in laryngeal squamous cell carcinoma, NAT10 suppresses pyroptosis by reducing the stability of ELANE mRNA, thereby promoting malignant progression [ 179 ]. NAT10-mediated pyroptotic regulation is therefore not unidirectional but instead depends on the direct substrate and the tissue or disease context in which it operates. Cuproptosis is a recently identified form of mitochondria-dependent regulated cell death. Current studies linking NAT10 to cuproptosis have focused mainly on colorectal cancer [ 219 – 221 ]. In contrast to its predominantly anti-ferroptotic activity, NAT10 promotes cuproptosis. NAT10 stabilizes DLAT mRNA through ac⁴C modification and increases cellular sensitivity to cuproptosis inducers. Lactylation of NAT10 at K426 further enhances its acetyltransferase activity, whereas SIRT1-mediated delactylation suppresses this process. DLAT and NAT10 also form a positive-feedback loop that amplifies the cuproptotic response [ 147 ]. This study provides the first mechanistic link among RNA modification, metabolic post-translational modification, and cuproptosis and demonstrates that NAT10 does not invariably support tumor-cell survival but can exert bidirectional effects depending on the regulated death program. Autophagy is both a major adaptive response to stress and an important regulator of tumor-cell survival and metabolic homeostasis [ 222 – 224 ]. Current evidence indicates that NAT10 frequently supports tumor growth by suppressing autophagy. In NSCLC, NAT10 stabilizes serum- and SGK2 mRNA and subsequently stabilizes EZH2 protein. This suppresses GABARAP expression and autophagosome–lysosome fusion, thereby blocking autophagic flux and promoting tumor progression [ 180 ]. In contrast, in esophageal squamous cell carcinoma, circTHBS1 stabilizes NAT10, enhances Rab8a translation, promotes autophagy, and increases tumor aggressiveness [ 144 ]. These findings demonstrate that the effect of NAT10 on autophagy is strongly context-dependent and that distinct tumors may rely on different autophagic states or programs. Cellular energy status can also regulate NAT10 in the opposite direction. Glucose deprivation activates SQSTM1/LC3-mediated autophagy–lysosome degradation, promotes NAT10 turnover, and reduces global ac⁴C levels, thereby establishing a negative-feedback circuit between nutrient stress and RNA modification [ 164 ]. Earlier work further showed that SIRT1-mediated deacetylation of NAT10 suppresses rRNA synthesis while relieving Che-1-mediated autophagy inhibition, thereby facilitating a cellular transition from anabolism to catabolism [ 225 ]. Collectively, the NAT10–ac⁴C axis has emerged as an upstream regulator of multiple forms of programmed cell death, but its functions are highly dependent on the death modality and tissue context. In ferroptosis, NAT10 primarily suppresses lipid peroxidation through stabilization of SLC7A11, FSP1, and GPX4-associated networks. Its effects on pyroptosis vary across inflammatory and malignant settings, whereas in cuproptosis NAT10 uniquely promotes cell death. Autophagic regulation is likewise bidirectional. These findings indicate that NAT10 is not simply a “cell-death suppressor” but rather a central coordinator of metabolic stress, RNA homeostasis, and cell-fate determination. Several important questions remain. It is unclear whether different forms of regulated cell death share a common ac⁴C-dependent regulatory network, whether RNA substrate selection is tissue-specific, and how catalytic and non-catalytic NAT10 functions contribute to each death program. Future studies combining single-cell multi-omics, site-specific RNA editing, metabolic flux analysis, and spatiotemporal live-cell imaging will be required to define the shared and distinct functions of NAT10 across regulated cell-death pathways and to evaluate its therapeutic potential in combination with radiotherapy, chemotherapy, immunotherapy, and emerging cell-death inducers. RNA modifications do not function independently but instead form a highly dynamic and coordinated epitranscriptomic regulatory network [ 226 , 227 ]. RNA modifications rarely function in isolation but instead form integrated regulatory networks with other RNA processing pathways. Figure 7 summarizes the current evidence supporting crosstalk between ac⁴C and other epitranscriptomic regulators. Unlike the conventional writer–reader–eraser framework, ac⁴C may act as an upstream organizer within RNA regulatory networks by modulating the expression, splicing, and function of m⁶A regulators and thereby amplifying their effects on post-transcriptional regulation in cancer [ 48 ]. The most direct evidence comes from NAT10-mediated regulation of m⁶A readers. In osteosarcoma, NAT10 stabilizes YTHDC1 mRNA through ac⁴C modification and enhances its translation, thereby maintaining YTHDC1 protein expression. YTHDC1 subsequently recognizes m⁶A modifications on phosphofructokinase, muscle (PFKM), and LDHA mRNAs and increases their stability, promoting glycolysis and tumor progression [ 48 ]. This study established an ac⁴C→m⁶A reader→m⁶A target RNA cascade, demonstrating that ac⁴C can act upstream of m⁶A signaling and indirectly amplify m⁶A-dependent biological effects rather than being restricted to regulation of its own modified substrates. In addition to controlling m⁶A-reader abundance, NAT10 can influence alternative splicing of m⁶A-related transcripts. In gastric cancer, NAT10 forms LLPS condensates through its C-terminal intrinsically disordered region, promotes acetylation of the splicing factor SRSF2, and increases its stability. Acetylated SRSF2 subsequently promotes exon 4 skipping in the m⁶A reader YTHDF1, generating a shorter isoform with stronger oncogenic activity and thereby enhancing gastric cancer proliferation and metastasis [ 228 ]. This finding indicates that NAT10 can reshape the epitranscriptome through a three-tiered network involving protein acetylation, RNA splicing, and RNA modification, further expanding the complexity of ac⁴C–m⁶A crosstalk. Importantly, the relationship between ac⁴C and m⁶A is not unidirectional. m⁶A can also regulate NAT10. In esophageal squamous cell carcinoma, METTL3-mediated m⁶A modification increases NAT10 mRNA expression. NAT10 is subsequently transferred through exosomes to TAMs, where it stabilizes FASN mRNA through ac⁴C modification, promotes lipid metabolic reprogramming and M2 macrophage polarization, and ultimately enhances resistance to PD-1-directed immunotherapy [ 173 ]. This finding establishes an m⁶A→ac⁴C sequential regulatory mode in which m⁶A acts upstream of NAT10 expression and ac⁴C amplifies downstream metabolic and immunological effects. More recently, ac⁴C and m⁶A were shown to form a mutually reinforcing positive-feedback loop. In adenoid cystic carcinoma (ACC), the transcription factor MYB first activates NAT10 expression. NAT10 then stabilizes IGF2BP3 mRNA through ac⁴C modification, whereas IGF2BP3, a canonical m⁶A reader, stabilizes NAT10 mRNA through m⁶A-dependent recognition. This establishes a MYB–NAT10–IGF2BP3 self-reinforcing circuit that persistently activates MAPK signaling and promotes tumor proliferation, invasion, and metastasis [ 229 ]. This study demonstrates that an ac⁴C writer and an m⁶A reader can form an interdependent bidirectional regulatory network rather than a conventional linear hierarchy. Current evidence supports at least three levels of ac⁴C–m⁶A crosstalk. The first is hierarchical regulation, in which NAT10 stabilizes m⁶A regulators such as YTHDC1 and thereby influences downstream m⁶A signaling. The second is reciprocal regulation, in which m⁶A factors such as METTL3 or IGF2BP3 regulate NAT10 expression or stability and establish positive-feedback circuits. The third is network integration, in which NAT10 coordinates RNA modification and RNA processing through LLPS, protein acetylation, and alternative splicing. Together, these findings demonstrate that RNA modifications form a highly coupled epitranscriptomic network rather than acting as isolated regulatory marks. Fig. 7 Crosstalk between ac⁴C and other RNA regulatory pathways. NAT10-mediated ac⁴C modification cooperates with m⁶A readers, liquid–liquid phase separation (LLPS), alternative splicing, and protein acetylation to establish multilayered RNA regulatory networks. Reciprocal regulation and positive feedback loops further amplify oncogenic signaling Crosstalk between ac⁴C and other RNA regulatory pathways. NAT10-mediated ac⁴C modification cooperates with m⁶A readers, liquid–liquid phase separation (LLPS), alternative splicing, and protein acetylation to establish multilayered RNA regulatory networks. Reciprocal regulation and positive feedback loops further amplify oncogenic signaling Nevertheless, current studies of ac⁴C crosstalk with other RNA modifications remain focused predominantly on m⁶A and on a limited group of regulators, including YTHDF1, YTHDC1, IGF2BP3, and METTL3. Whether ac⁴C engages in comparable interactions with m⁵C, m⁷G, pseudouridine (Ψ), adenosine-to-inosine editing, or RNA terminal modifications remains largely unexplored. It is also unclear whether different RNA modifications act cooperatively on the same RNA molecule or regulate separate substrate pools that converge on common cell-fate programs. Future studies integrating single-molecule multimodification sequencing, multi-omics analysis, spatial epitranscriptomics, and site-specific RNA editing will be required to define the spatiotemporal coordination among RNA modifications and to establish a unified cross-epitranscriptomic network model that can inform combination strategies targeting multiple RNA modification pathways. Non-coding RNAs are integral components of epitranscriptomic regulatory networks, and their dysregulation contributes broadly to tumorigenesis, metastasis, and therapeutic resistance [ 230 , 231 ]. Recent studies have revealed a bidirectional regulatory relationship between ac⁴C and ncRNAs [ 81 ]. On the one hand, NAT10 directly modifies lncRNAs and circRNAs, altering their stability, translational potential, and molecular scaffold functions. On the other hand, multiple ncRNAs regulate NAT10 expression, protein stability, or substrate selection, thereby forming complex feedback networks [ 81 ]. Increasing evidence indicates that noncoding RNAs function both as direct ac⁴C substrates and as important regulators of NAT10 activity. Figure 8 summarizes the bidirectional interactions between NAT10 and noncoding RNAs in cancer. ncRNAs therefore function not only as important ac⁴C substrates but also as key determinants of NAT10 specificity. The best-supported mechanism is direct NAT10-mediated regulation of lncRNA stability. Early work first demonstrated that lncRNAs can undergo ac⁴C modification. In esophageal squamous cell carcinoma, NAT10 catalyzes ac⁴C deposition on CTC-490G23.2 and increases its stability. CTC-490G23.2 subsequently acts as a molecular scaffold that facilitates binding of PTBP1 to CD44 pre-mRNA and promotes an isoform switch from the standard CD44s isoform to the prometastatic CD44v8–10 isoform. CD44v8–10 further stabilizes vimentin and promotes EMT and distant metastasis [ 81 ]. Notably, this study not only achieved single-nucleotide-resolution identification of an lncRNA ac⁴C site but also demonstrated that ac⁴C can promote tumor progression by regulating alternative splicing rather than through conventional RNA stabilization alone. Subsequent studies extended this regulatory paradigm. In breast cancer, NAT10-mediated ac⁴C modification increases the stability and abundance of CD2BP2-DT. Elevated CD2BP2-DT promotes YBX1 LLPS, enhances CDK1 mRNA stability, and stimulates cell proliferation [ 30 ]. In nasopharyngeal carcinoma, NAT10 stabilizes the lncRNA SIMALR, which binds eEF1A2, enhances its GTPase activity, accelerates ITGB4 and ITGA6 translation, and promotes invasion and metastasis [ 80 ]. These studies demonstrate that NAT10 does not simply increase lncRNA abundance but selectively stabilizes lncRNAs with scaffold, translational-regulatory, or phase-separation functions, thereby amplifying their downstream networks. In addition to serving as direct substrates, ncRNAs can regulate NAT10 expression. The most common mechanism is ceRNA regulation. In gastric cancer, the lncRNA DARS-AS1 competitively binds miR-330-3p and relieves its repression of NAT10, thereby establishing a DARS-AS1/miR-330-3p/NAT10 axis that promotes proliferation, migration, and invasion [ 232 ]. Unlike direct modification of an RNA substrate, this ceRNA mechanism positions the lncRNA upstream of NAT10 and enables it to influence the broader ac⁴C landscape through regulation of NAT10 abundance. Fig. 8 Bidirectional crosstalk between NAT10 and noncoding RNAs. NAT10 acetylates multiple lncRNAs to regulate RNA stability and function, whereas lncRNAs and circRNAs modulate NAT10 expression, stability, ubiquitination, or substrate accessibility. Competitive binding and positive feedback loops further diversify NAT10-dependent regulatory mechanisms Bidirectional crosstalk between NAT10 and noncoding RNAs. NAT10 acetylates multiple lncRNAs to regulate RNA stability and function, whereas lncRNAs and circRNAs modulate NAT10 expression, stability, ubiquitination, or substrate accessibility. Competitive binding and positive feedback loops further diversify NAT10-dependent regulatory mechanisms Recent studies further show that lncRNAs can directly regulate NAT10 protein stability rather than merely altering its transcript levels. In thyroid cancer, DLEU2 simultaneously binds NAT10 and USP39 and maintains the stability of both proteins while exerting minimal effects on their mRNA abundance. DLEU2 depletion markedly reduces NAT10 protein expression and suppresses tumor growth, suggesting that certain lncRNAs function as protein scaffolds that coordinate deubiquitination and proteostasis to sustain NAT10 activity [ 233 ]. This mechanism further broadens the range of ncRNA–ac⁴C interactions. Compared with lncRNAs, circRNAs more frequently regulate NAT10 through protein interaction and substrate competition rather than acting as direct modification substrates. In cervical cancer, the tumor-suppressive circRNA circMAST1 directly binds NAT10 and competitively blocks its interaction with YAP mRNA. This decreases YAP mRNA ac⁴C, accelerates transcript degradation, and suppresses tumor growth and lymph-node metastasis [ 99 ]. A similar mechanism operates in HER2-positive breast cancer. Nuclear circ-PPID binds NAT10 and prevents it from modifying HER2 mRNA, thereby reducing HER2 mRNA stability and restoring trastuzumab sensitivity. In resistant cells, however, XPO4-mediated export of circ-PPID to the cytoplasm eliminates its ability to compete with NAT10, leading to sustained HER2 expression and drug resistance [ 234 ]. These studies establish a regulatory mode in which circRNAs function as NAT10 molecular decoys and determine the targeting specificity of ac⁴C deposition through competitive substrate binding. Not all circRNAs inhibit NAT10. In esophageal squamous cell carcinoma, circTHBS1 directly binds NAT10 and prevents its ubiquitin-mediated degradation, thereby increasing NAT10 protein stability and global ac⁴C levels. NAT10 subsequently enhances Rab8a translation, promotes autophagy, and drives tumor progression [ 144 ]. circRNAs can therefore function either as competitive inhibitors or as stabilizing factors for NAT10, depending on the nature of their molecular interaction. Current evidence supports at least four major modes of crosstalk between ac⁴C and ncRNAs. First, lncRNAs can serve as direct ac⁴C substrates whose stability and function are controlled by NAT10. Second, lncRNAs can act upstream of NAT10 through ceRNA mechanisms or protein-stability regulation. Third, circRNAs can function as competitive NAT10 decoys that alter substrate recognition and ac⁴C deposition. Fourth, circRNAs can stabilize NAT10 and enhance global ac⁴C activity. Together, these interactions establish a NAT10-centered post-transcriptional regulatory network in which ncRNAs participate broadly as substrates, adaptors, competitors, and stabilizers rather than conforming to a unidirectional writer–substrate relationship.Despite rapid progress, several limitations remain. Existing studies focus on a small number of lncRNAs and circRNAs, whereas the potential regulation of miRNAs, small nucleolar RNAs, PIWI-interacting RNAs, and tRNA-derived small RNAs by ac⁴C remains largely unexplored. Most studies also emphasize changes in RNA stability, and direct evidence that ncRNAs determine NAT10 substrate specificity or organelle localization remains limited. It is additionally unclear whether multiple ncRNAs cooperate to regulate NAT10 at the network level. Future studies integrating single-nucleotide ac⁴C sequencing, crosslinking immunoprecipitation sequencing, spatial transcriptomics, RNA interactomics, and live-cell imaging will be required to define the integrated epitranscriptomic network jointly constructed by ac⁴C and ncRNAs and to evaluate the therapeutic potential of targeting lncRNA/circRNA–NAT10 interactions.

Given its broad oncogenic functions, NAT10 has emerged as an attractive therapeutic target. Figure  10 outlines current and future strategies for targeting NAT10 and highlights major challenges toward clinical translation. Fig. 10 Therapeutic strategies targeting NAT10 in cancer. Current approaches include catalytic inhibition, targeted protein degradation, rational combination therapies, biomarker-guided precision medicine, and optimization of therapeutic selectivity. Integrating molecular stratification with improved drug delivery may maximize antitumor efficacy while minimizing toxicity Therapeutic strategies targeting NAT10 in cancer. Current approaches include catalytic inhibition, targeted protein degradation, rational combination therapies, biomarker-guided precision medicine, and optimization of therapeutic selectivity. Integrating molecular stratification with improved drug delivery may maximize antitumor efficacy while minimizing toxicity As the only well-established mammalian RNA acetyltransferase responsible for ac⁴C formation, NAT10 represents the most direct pharmacological target for developing ac⁴C-directed anticancer strategies [ 155 ]. In recent years, several compounds capable of suppressing NAT10 activity have been reported and have demonstrated encouraging antitumor effects across diverse cancer models [ 155 ]. However, compared with clinically mature targets such as mTOR, PARP, and CDKs, the development of NAT10 inhibitors remains at an early stage. Their precise mechanisms of catalytic inhibition, target selectivity, pharmacokinetic properties, and translational potential require further validation. Existing compounds should therefore be evaluated according to an evidence hierarchy rather than being indiscriminately classified as specific NAT10 inhibitors [ 155 ]. Remodelin is currently the most widely used pharmacological inhibitor of NAT10 [ 169 ]. It was initially identified in studies of Hutchinson–Gilford progeria syndrome as a small molecule capable of improving nuclear-envelope abnormalities and was subsequently shown to inhibit NAT10 activity. Since then, it has been extensively used to investigate the biological functions of NAT10. Numerous studies have demonstrated that Remodelin reduces intracellular ac⁴C levels, inhibits tumor-cell proliferation, invasion, EMT, metabolic reprogramming, DNA damage repair, and cancer stemness, and enhances the therapeutic efficacy of chemotherapy, PARP inhibitors, and ICIs. It has consequently become the most widely used pharmacological tool compound for studying NAT10 [ 155 , 212 ]. Nevertheless, whether Remodelin can be regarded as a genuinely highly selective NAT10 inhibitor remains uncertain. Many studies infer its mechanism from phenotypic similarities between NAT10 depletion and Remodelin treatment, whereas direct evidence of target engagement, binding kinetics, and in vivo specificity remains relatively limited. A recently resolved 2.9-Å cryo-EM structure of the NAT10–Remodelin complex revealed, for the first time, the binding mode of Remodelin within the NAT10 catalytic domain. This structural evidence supports direct binding and provides a foundation for subsequent structure-guided optimization [ 192 ]. Even so, systematic studies are still lacking regarding potential in vivo off-target effects, whether its antitumor activity is entirely dependent on NAT10 inhibition, and the safety of long-term administration. At present, Remodelin is therefore more appropriately regarded as a chemical probe for validating NAT10 biology than as a clinically ready drug candidate. High-throughput virtual screening and drug repurposing have further expanded the repertoire of candidate NAT10 inhibitors. In AML, molecular docking identified fludarabine, fosaprepitant, folinic acid, and dantrolene as compounds with potential NAT10-binding activity. Among them, fludarabine displayed the strongest antileukemic effect. Its half-maximal inhibitory concentration was substantially lower in AML cells than in normal peripheral blood mononuclear cells, and it effectively suppressed colony formation in patient-derived leukemia cells and MLL–AF9 leukemia models [ 155 ]. Similarly, in ovarian cancer, fludarabine reduces ac⁴C modification and expression of acyl-CoA thioesterase 7 (ACOT7) mRNA, suppresses fatty acid metabolism, and restores ferroptosis sensitivity. It also demonstrates marked antitumor activity in patient-derived organoids and xenograft models [ 252 ]. In addition to repurposed drugs, new small-molecule candidates have been identified. In osteosarcoma, virtual screening revealed that paliperidone and AG-401 directly bind NAT10 and inhibit ATF4–ASNS-mediated asparagine metabolism, thereby suppressing tumor growth in patient-derived xenograft models. Notably, combined administration of the two compounds produced stronger synergistic antitumor activity than either agent alone, suggesting considerable scope for further structural optimization of NAT10-directed inhibitors [ 98 ]. Taken together, currently available small-molecule NAT10 inhibitors appear to exert antitumor activity primarily by suppressing ac⁴C catalytic activity. Their common effect is a reduction in RNA ac⁴C levels, followed by alterations in RNA stability, translational efficiency, and downstream oncogenic signaling. However, substantial differences remain among these compounds in binding mode, catalytic inhibitory potency, and pharmacological properties. More importantly, most studies assess drug activity mainly through phenotypic improvement, whereas systematic biochemical enzyme assays, intracellular target-engagement measurements, proteome-wide off-target profiling, and pharmacokinetic evaluation remain insufficient. The field is therefore still transitioning from the use of chemical probes toward the development of bona fide clinical candidates. Overall, catalytic inhibition remains the most mature and best-supported strategy for targeting NAT10, but several limitations must be addressed. First, most existing inhibitors primarily block catalytic activity and do not suppress non-catalytic functions such as RNA binding, protein acetylation, phase separation, or protein–protein interactions. Second, next-generation inhibitors with high selectivity, favorable pharmacokinetics, and clearly demonstrated in vivo target engagement are still lacking. Third, because NAT10 contributes broadly to normal hematopoiesis, embryonic development, and tissue homeostasis, prolonged systemic inhibition may produce substantial toxicity. Future efforts should integrate cryo-EM-guided structural analysis, artificial intelligence-assisted drug design, covalent inhibitor development, and medicinal chemistry optimization to generate next-generation NAT10 catalytic inhibitors with improved selectivity, in vivo activity, and clinical translatability. Although small-molecule catalytic inhibitors have established the druggability of NAT10, increasing evidence demonstrates that its biological functions extend beyond ac⁴C catalysis. In addition to RNA acetyltransferase activity, NAT10 possesses RNA-binding capacity, protein acetyltransferase activity, RNA helicase-like activity, LLPS properties, and extensive protein–protein interaction functions [ 59 , 253 ]. These non-catalytic activities also contribute to RNA processing, R-loop homeostasis, ribosome biogenesis, DNA damage repair, and tumor progression. Consequently, blockade of the catalytic center alone may not completely suppress the oncogenic functions of NAT10, prompting a conceptual shift from enzyme inhibition toward targeted protein degradation (TPD). TPD has become an important direction in small-molecule drug development [ 254 , 255 ]. Proteolysis-targeting chimeras (PROTACs) simultaneously engage a target protein and an E3 ubiquitin ligase, inducing ubiquitination and proteasomal degradation of the target [ 256 ]. Unlike conventional inhibitors, PROTACs do not require continuous occupancy of a catalytic site. Instead, a transient binding event can trigger degradation, producing event-driven pharmacology and potentially enabling sustained activity at lower drug concentrations. For NAT10, targeted degradation offers several theoretical advantages. First, degradation would eliminate not only ac⁴C catalytic activity but also non-catalytic functions involving RNA binding, protein acetylation, R-loop regulation, and LLPS [ 228 ]. For example, NAT10-mediated gastric cancer progression depends partly on its ability to form LLPS condensates, a function that is not determined solely by the catalytic domain and may therefore persist despite catalytic inhibition [ 228 ]. Likewise, in the nucleolus, NAT10 promotes R-loop clearance by acetylating DDX21, a function that extends beyond conventional RNA acetylation [ 88 ]. Mechanistically, complete degradation of NAT10 could therefore produce broader biological effects than inhibition of its catalytic activity alone. NAT10 also functions as a structural component of multiple regulatory complexes. It recruits PCBP1/2 and TDP-43 to form an mRNA ac⁴C writer complex and establishes dynamic regulatory networks with RNA-processing factors such as SRSF2 and YBX1. Many of these interactions do not depend on the catalytic center [ 30 , 228 ]. In principle, targeted degradation may therefore be required to dismantle entire NAT10-containing regulatory complexes, whereas conventional catalytic inhibitors may leave these scaffolding functions intact. In addition to PROTACs, molecular-glue degraders represent another promising strategy. Unlike bifunctional PROTACs, molecular glues stabilize otherwise weak interactions between a target protein and an E3 ligase, enabling degradation through a structurally simpler small molecule [ 257 ]. NAT10 contains several conformationally flexible regions, including its RNA helicase-like domain and extended C-terminal intrinsically disordered region, which may provide suitable surfaces for molecular-glue development. However, no NAT10-directed molecular glue has yet been reported. Emerging degradation platforms, including lysosome-targeting chimeras, autophagy-targeting chimeras, autophagosome-tethering compounds, and ribonuclease-targeting chimeras, have further broadened the conceptual landscape of targeted degradation. Because NAT10 is predominantly localized to the nucleus and nucleolus, several of these platforms may not yet be directly applicable. Nevertheless, advances in RNA degraders and RNA-binding small molecules provide alternative strategies for disrupting NAT10–RNA interactions [ 42 , 258 ]. Rather than eliminating the entire NAT10 protein, it may be possible to selectively degrade oncogenic RNAs that depend on NAT10. Such an approach could preserve essential physiological functions of NAT10 in normal tissues while selectively disrupting tumor-specific regulatory circuits and may therefore offer a superior safety profile.Importantly, all proposed NAT10 degradation strategies remain theoretical. The major challenges are therefore not merely whether NAT10 degraders should be developed, but how to define the structural degradability of NAT10, identify suitable E3 ligases, and optimize pharmacokinetic properties. Because NAT10 contributes broadly to normal tissue homeostasis, whether complete protein elimination offers a superior therapeutic index to partial catalytic inhibition also requires careful evaluation. Overall, targeted protein degradation represents an important future direction for NAT10-directed therapy. Its principal theoretical advantage is the simultaneous elimination of catalytic and non-catalytic functions and the comprehensive disruption of NAT10-mediated RNA regulatory networks. However, this field remains at an early stage. Future studies should combine cryo-EM-guided structural analysis, artificial intelligence-assisted PROTAC design, high-throughput E3-ligase screening, and structural biology to develop clinically translatable NAT10 degraders. Direct comparisons between catalytic inhibition and protein degradation across distinct tumor contexts will be essential for determining which strategy provides the optimal balance of antitumor activity, selectivity, and safety. Although NAT10 inhibitors have demonstrated substantial antitumor activity across multiple cancer models, their efficacy as monotherapies may be limited by tumor heterogeneity, adaptive compensation, and toxicity in normal tissues [ 18 , 98 ]. Similar to other precision therapies, NAT10 is therefore more likely to serve as a central node in combination treatment rather than as an isolated therapeutic target. Notably, the rationale underlying current combination strategies is not defined primarily by drug class but by the specific tumor vulnerabilities created by NAT10-dependent DNA repair, metabolic adaptation, immune evasion, and adaptive resistance [ 192 ]. Mechanism-based combination strategies may therefore better capture the therapeutic value of NAT10 as a network regulator than combinations organized solely by pharmacological category. DDR represents the best-supported setting for NAT10-based combination therapy. Numerous studies have shown that NAT10 enhances HR repair by stabilizing transcripts such as RAD51, AHNAK, and PPAN, thereby enabling tumor cells to resist chemotherapy and PARP inhibition [ 157 ]. NAT10 inhibition can therefore induce an HR-deficient state, increase DNA damage accumulation, and create a synthetic-lethal interaction with PARP inhibitors. In hepatocellular carcinoma, Remodelin destabilizes DNA: RNA hybrids, impairs HR repair, and markedly enhances the efficacy of PARP inhibition [ 192 ]. Similarly, in triple-negative breast cancer, NAT10 stabilizes RAD51 mRNA and sustains HR, whereas combined Remodelin and olaparib treatment causes persistent accumulation of DSBs and produces substantially stronger antitumor effects than either agent alone [ 157 ]. In bladder cancer, colorectal cancer, and other models, NAT10 inhibition likewise enhances the activity of cisplatin and additional DNA-damaging agents. These observations support a mechanistic framework in which NAT10 blockade converts repair-proficient tumors into repair-vulnerable states and thereby broadens the therapeutic scope of DDR-targeted agents. Nevertheless, because NAT10 also contributes to genome maintenance in normal tissues, the dose, timing, and duration of such combinations will require careful optimization to avoid excessive genomic toxicity. Metabolic reprogramming has also emerged as an important basis for NAT10-directed combination therapy. NAT10 broadly regulates serine metabolism, glycolysis, lipid metabolism, GSH metabolism, and ferroptosis sensitivity, suggesting that tumors with distinct metabolic dependencies may represent particularly suitable populations for NAT10-based combinations. AML provides a representative example. NAT10 enhances translation of HOXA9, MENIN, and SLC1A4, thereby increasing serine synthesis and uptake and establishing a metabolic dependency required for leukemia stem cell maintenance. Fludarabine-mediated NAT10 inhibition disrupts this program and induces leukemia-cell death [ 155 ]. In multiple solid tumors, NAT10 also regulates SLC7A11-, FSP1-, and GPX4-associated networks and thereby determines ferroptosis sensitivity. NAT10 inhibitors may therefore be combined with ferroptosis inducers, system Xc⁻ inhibitors, or agents that promote lipid peroxidation. Because NAT10 also supports glycolysis and lactate production, combinations with LDHA inhibitors, PHGDH inhibitors, fatty acid metabolic inhibitors, or mitochondrial metabolic inhibitors have a strong mechanistic rationale. Rather than targeting a single metabolic pathway, NAT10 inhibition may function upstream of several metabolic networks and thereby improve the overall efficacy of metabolism-directed therapies. However, such combinations should be matched to the dominant metabolic dependency of each tumor rather than applied uniformly across cancer types. A large body of recent evidence further identifies NAT10 as a promising target for combination immunotherapy. NAT10 not only increases PD-L1 expression but also establishes immunosuppression through enhanced glycolysis, lactate accumulation, TGF-β signaling, M2 macrophage polarization, and CD8⁺ T-cell exhaustion. NAT10 inhibition can therefore relieve several layers of immune suppression simultaneously rather than merely reducing PD-L1 abundance. Multiple tumor models have shown that Remodelin combined with anti-PD-1, anti-PD-L1, or anti-CTLA-4 therapy produces stronger antitumor activity than either treatment alone. Such effects have been reported in esophageal squamous cell carcinoma, cervical cancer, pancreatic cancer, and colorectal cancer [ 169 , 259 ]. Because NAT10 also contributes to TGF-β-driven immunosuppression, combinations with TGF-β inhibitors, anti-angiogenic therapies, or macrophage-reprogramming strategies may offer additional therapeutic opportunities [ 41 , 169 ]. Importantly, normal T-cell activation also depends on NAT10. Combination immunotherapy must therefore account for drug dose, treatment sequence, tissue selectivity, and exposure duration. Intermittent or tumor-targeted NAT10 inhibition may prove preferable to sustained systemic blockade. Beyond DDR, metabolism, and immunity, increasing evidence identifies NAT10 as a shared regulator of adaptive resistance. In different tumor types, NAT10 stabilizes MYC, ETS2, AHNAK, DUSP1, HER2, and other resistance-associated transcripts and thereby facilitates compensatory signaling after treatment. NAT10 inhibition may therefore delay or prevent the emergence of acquired resistance to targeted therapies [ 54 ]. Future combination strategies may involve EGFR-, HER2-, KRAS, BRAF, or MYC-directed agents. Rather than intervening only after resistance has developed, a more rational strategy may be to inhibit NAT10 at treatment initiation, thereby restricting adaptive post-transcriptional remodeling and improving the durability of therapeutic responses. Overall, the central concept of NAT10 combination therapy is shifting from combining different drugs to combining interventions against distinct tumor vulnerabilities. DDR dependence, metabolic adaptation, immune evasion, and adaptive resistance represent different manifestations of tumor reliance on NAT10. Future treatment design should therefore be mechanism-driven and tailored to the dominant biological dependency of each tumor rather than based on a fixed universal regimen. Functional genomic screening and patient-derived models will be essential for identifying optimal combinations and advancing NAT10-directed therapy toward precision oncology. With the development of precision oncology, biomarker-guided patient stratification has become essential for the successful clinical translation of targeted therapies. Although extensive evidence demonstrates that the NAT10–ac⁴C axis contributes broadly to cancer progression, the degree of NAT10 dependency varies substantially among cancer types and even among patients with the same malignancy [ 254 ]. NAT10-targeted therapy is therefore unlikely to benefit all patients equally. Future clinical application of NAT10 inhibitors should be based on precision targeting rather than broad inhibition [ 254 ]. The central objective should be to identify patients with genuine NAT10 dependency, thereby maximizing therapeutic efficacy while minimizing toxicity to normal tissues. The most direct predictive marker is NAT10 expression. Multiple studies have associated elevated NAT10 with poor prognosis, tumor invasion, therapeutic resistance, and immune evasion in hepatocellular carcinoma, colorectal cancer, pancreatic cancer, gastric cancer, breast cancer, AML, and other malignancies [ 260 , 261 ]. Nevertheless, patient selection based solely on NAT10 protein or mRNA abundance has important limitations. First, NAT10 expression does not necessarily reflect catalytic activity. Second, NAT10 performs different physiological functions across tissues, meaning that comparable expression levels may not indicate equivalent therapeutic dependency. Third, NAT10 possesses extensive non-catalytic functions, and expression measurements cannot fully capture its biological state. Direct assessment of ac⁴C may provide greater predictive value than NAT10 expression alone. Advances in acRIP-seq [ 50 , 262 ], and single-nucleotide ac⁴C detection increasingly enable mapping of ac⁴C landscapes across normal tissues and tumors [ 18 ]. Global ac⁴C burden and substrate-specific ac⁴C patterns may therefore serve as direct indicators of NAT10 activity and treatment sensitivity. The relative distribution of ac⁴C across different RNA classes, including mRNAs, lncRNAs, circRNAs, rRNAs, and tRNAs, may also reveal distinct biological dependencies. For example, tumors dominated by mRNA ac⁴C may rely primarily on transcript stabilization, whereas tumors with prominent tRNA or rRNA ac⁴C alterations may depend more strongly on translational reprogramming or ribosome biogenesis. Such RNA-class-specific profiles could enable more refined patient stratification. Functional pathway signatures are also likely to be informative. Because NAT10 regulates DDR, R-loop homeostasis, metabolism, and immunity, functional scores reflecting these processes could be incorporated into predictive models. Potential examples include HR repair scores, R-loop burden, replication-stress scores, serine-metabolism scores, ferroptosis scores, and immune-suppression scores. Compared with single-gene expression, process-based functional signatures more accurately reflect the role of NAT10 as a network regulator and may be more transferable across cancer types. Integrating such signatures with direct ac⁴C profiling could help distinguish tumors dependent on the catalytic activity of NAT10 from those relying primarily on non-catalytic functions. Immune subtyping provides another strategy for precision NAT10 targeting. Elevated NAT10 is frequently associated with increased PD-L1, enrichment of Tregs and M2 macrophages, and CD8⁺ T-cell exhaustion [ 241 , 243 , 244 ]. Immune-cold tumors may therefore depend strongly on NAT10-mediated immunosuppression, whereas some immune-hot tumors may rely predominantly on its DDR or metabolic functions. NAT10-associated immune subtypes generated through spatial transcriptomics, single-cell RNA sequencing, and spatial proteomics may help identify patients most likely to benefit from NAT10–ICI combinations and distinguish them from those better suited to combinations involving DDR- or metabolism-targeted therapies [ 237 ]. Metabolic subtypes may likewise determine sensitivity to NAT10 inhibition. AML is highly dependent on serine metabolism, hepatocellular carcinoma may rely on the SLC7A11-mediated antioxidant system, and breast cancer brain metastases depend on PHGDH-driven serine biosynthesis [ 163 ]. Patient selection based on metabolic dependency may therefore be more informative than classification by tissue of origin alone [ 163 ]. A clinically meaningful precision strategy should ultimately distinguish NAT10-dependent tumors from merely NAT10-high tumors. CRISPR screens, the Cancer Dependency Map, and patient-derived organoids can be used to construct a NAT10 dependency atlas across malignancies and determine which tumors require NAT10 for survival and which display only correlative overexpression. Functional dependency-based patient selection is more consistent with modern precision oncology than expression-based classification alone. It may also reveal tumors with moderate NAT10 expression but profound reliance on a specific NAT10-regulated pathway. Overall, the key question for future NAT10-directed therapy is shifting from whether NAT10 should be inhibited to which patients are most likely to benefit from its inhibition. Integration of NAT10 expression, ac⁴C profiles, DDR status, R-loop burden, metabolic features, immune subtypes, and functional dependency may enable development of a comprehensive NAT10 precision-oncology framework. Artificial intelligence, multi-omics integration, and prospective clinical cohorts will be required to establish clinically actionable biomarker systems that guide patient selection and optimize combination therapy. Although extensive evidence supports the NAT10–ac⁴C axis as a promising anticancer target, its broad involvement in RNA metabolism, translational control, and tissue homeostasis creates substantial challenges related to safety, tissue selectivity, and therapeutic window. Unlike oncogenic drivers such as EGFR or BRAF, which are frequently aberrantly activated in tumors, NAT10 regulates fundamental cellular processes. A central translational question is therefore how an enzyme involved in essential RNA biology can be safely targeted in cancer while preserving normal tissue function [ 228 ]. The most immediate safety concern arises from the physiological requirement for NAT10 in normal stem cells. Recent genetic studies have shown that Nat10 deletion impairs HSC self-renewal, blocks MEP differentiation, and ultimately causes fetal and postnatal hematopoietic failure. Mechanistically, NAT10 promotes translation of key hematopoietic transcription factors such as Nfix through ac⁴C modification and thereby sustains normal hematopoiesis [ 50 ]. NAT10 also maintains HSC function through ac⁴C-independent mechanisms. It binds AREs within the 3′UTRs of essential HSC transcripts, recruits ribosomes, and stabilizes these RNAs [ 86 ]. These findings imply that complete catalytic inhibition may preserve some non-catalytic functions, whereas targeted degradation of NAT10 could eliminate both. Catalytic inhibition and protein degradation may therefore differ fundamentally in toxicity profile. The reproductive system is likewise highly dependent on NAT10. Multiple genetic studies consistently show that Nat10 loss causes defective oocyte maturation, meiotic abnormalities, arrested follicular development, and complete female infertility. NAT10 participates in maternal mRNA degradation, ZGA, and blastocyst formation and regulates oocyte maturation by maintaining the stability or processing of key transcripts associated with OGA and the CCR4–NOT complex [ 130 – 132 ]. Prolonged systemic inhibition may therefore carry substantial reproductive toxicity, particularly in patients of reproductive age. Because NAT10 broadly supports ribosome biogenesis and protein translation, rapidly proliferating normal tissues may also be vulnerable. The intestinal epithelium, bone marrow, hair follicles, and skin all have high requirements for protein synthesis and tissue renewal. Sustained NAT10 inhibition could therefore cause mucosal injury, myelosuppression, impaired wound healing, or regenerative failure. These toxicities may be amplified when NAT10 inhibitors are combined with DNA-damaging agents or PARP inhibitors. Dose intensity, treatment sequence, and recovery intervals will therefore be particularly important in combination regimens. Growing evidence also suggests that NAT10 contributes to nervous-system and cardiovascular homeostasis. In the nervous system, NAT10 regulates neurogenesis, axonal development, synaptic maturation, R-loop homeostasis, and RNA translation. Long-term inhibition could therefore affect neural plasticity or cognitive function. In vascular smooth muscle cells, NAT10 stabilizes ITGB1 and COL1A2 mRNAs and regulates vascular remodeling and FAK signaling, suggesting that prolonged inhibition may impair vascular repair and tissue regeneration [ 18 ]. Although systematic toxicological data remain limited, these potential risks should be carefully investigated during preclinical and clinical development. The immune system further illustrates the duality of NAT10 function. Tumor cells exploit NAT10 to establish an immunosuppressive microenvironment, increase PD-L1 expression, and induce T-cell exhaustion. In contrast, activated normal T cells require NAT10-mediated Myc translation for rapid clonal expansion. T-cell-specific Nat10 deletion impairs adaptive immune responses, and age-associated NAT10 decline is linked to immune dysfunction [ 56 ]. Prolonged systemic NAT10 inhibition may therefore enhance antitumor immunity in some contexts while simultaneously impairing normal immune surveillance and increasing susceptibility to infection. This concern is particularly relevant when NAT10 inhibitors are combined with immunotherapies. Given these challenges, future NAT10-directed strategies should prioritize selectivity rather than complete inhibition. Intermittent dosing may allow sufficient suppression of tumor dependency while permitting recovery of normal tissues and thereby widening the therapeutic window. Tumor-targeted delivery systems may further reduce systemic exposure. Lipid nanoparticles, polymeric nanocarriers, tumor-microenvironment-responsive materials, and exosome-based delivery platforms could increase local drug concentrations within tumors. Antibody–drug conjugates, ligand-directed delivery, and tumor-selective prodrugs may provide additional tissue specificity. A more advanced therapeutic concept is to shift from inhibiting NAT10 itself toward disrupting tumor-specific NAT10 dependency networks. Rather than suppressing the entire protein, interventions could selectively block oncogenic regulatory axes such as NAT10–RAD51. Targeting individual tumor-specific RNA substrates or NAT10–RNA interfaces may preserve essential physiological functions in normal tissues. CRISPR functional screens, dependency-map resources, and patient-derived organoids can further identify tumor subtypes that are genuinely dependent on NAT10 and help restrict treatment to patients with the most favorable therapeutic index. Overall, NAT10 performs both fundamental physiological functions and tumor-promoting activities, creating substantial therapeutic potential but also considerable safety challenges. Expanding the therapeutic window will depend less on developing increasingly potent inhibitors than on achieving greater precision and selectivity. Optimized dosing schedules, tumor-targeted delivery, functional selection of NAT10-dependent patients, and selective disruption of tumor-specific NAT10–RNA circuits may maximize antitumor efficacy while minimizing irreversible injury to normal tissue homeostasis.

RNA is no longer regarded as a passive intermediate that merely conveys genetic information from DNA to proteins. Instead, it is subject to multilayered regulation through alternative splicing, RNA editing, chemical modifications, structural remodeling, and RNA–protein interactions [ 1 – 4 ]. To date, more than 170 distinct RNA chemical modifications have been identified, collectively constituting the epitranscriptome, which dynamically converts intracellular and extracellular cues into context-dependent gene expression programs by regulating RNA folding, processing, localization, stability, translation, and decay [ 5 – 10 ]. Compared with DNA and histone modifications, RNA modifications provide a substantially more rapid and reversible layer of gene regulation, enabling cells to respond promptly to nutrient availability, metabolic stress, hypoxia, inflammation, and DNA damage [ 11 – 14 ]. Consequently, epitranscriptomic regulation has emerged as a critical interface linking cellular states to proteome remodeling. Dysregulation of RNA modifications not only perturbs normal development and tissue homeostasis but is also frequently exploited by cancer cells to sustain uncontrolled proliferation, cellular plasticity, metabolic adaptation, and therapeutic resistance [ 15 – 17 ]. Among the growing repertoire of RNA modifications, N⁴-acetylcytidine (ac⁴C) is unique because it represents the only known acetylated form of cytidine identified in RNA [ 18 , 19 ]. Formed through acetylation of the N4 amino group of cytidine, ac⁴C was initially discovered in transfer RNA (tRNA) and ribosomal RNA (rRNA), and its remarkable evolutionary conservation across bacteria, archaea, and eukaryotes strongly suggests indispensable physiological functions [ 1 , 20 – 22 ]. In tRNA, ac⁴C stabilizes the anticodon stem–loop, restricts near-cognate codon misreading, and enhances decoding fidelity [ 23 , 24 ]. In rRNA, ac⁴C contributes to local structural stabilization, 18S rRNA processing, maturation of the small ribosomal subunit, and maintenance of translational accuracy [ 25 , 26 ]. Accordingly, ac⁴C was originally regarded as a structural RNA modification that safeguards the integrity and performance of the translational machinery [ 27 – 29 ]. Advances in high-throughput sequencing and epitranscriptomic profiling have substantially expanded the putative substrate repertoire of ac⁴C beyond tRNAs and rRNAs to include messenger RNAs (mRNAs), long non-coding RNAs (lncRNAs), circular RNAs (circRNAs), primary microRNAs (pri-miRNAs), and viral RNAs [ 30 – 33 ]. Correspondingly, its biological functions have evolved from maintaining RNA structural integrity to regulating RNA stability, translation initiation and elongation, alternative splicing, non-canonical translation, and the assembly of ribonucleoprotein complexes [ 20 , 21 , 34 , 35 ]. These discoveries have fundamentally reshaped our understanding of ac⁴C, suggesting that it is not merely a constitutive “housekeeping” RNA modification but rather a dynamic regulatory node that integrates RNA fate with diverse cellular phenotypes. N-acetyltransferase 10 (NAT10) is currently the best-characterized ac⁴C writer in mammals. NAT10 contains an RNA-binding region, an ATPase/RecD-like helicase domain, and a GCN5-related N-acetyltransferase (GNAT) catalytic domain [ 36 – 38 ]. Its catalytic activity requires ATP-dependent RNA conformational remodeling coupled with acetyl-group transfer from acetyl-coenzyme A (acetyl-CoA) [ 39 , 40 ]. Notably, substrate recognition by NAT10 is not dictated solely by linear sequence motifs. Instead, accumulating evidence indicates that substrate selectivity is determined by multiple layers of regulation, including local RNA secondary structure, cytidine-enriched sequence contexts, accessory cofactors such as THUMPD1 [ 41 ], RNA-binding proteins including poly(rC)-binding proteins 1 and 2 (PCBP1/2) and TAR DNA-binding protein 43 (TDP-43), as well as the recruitment or competitive sequestration of NAT10 by distinct non-coding RNAs [ 42 ]. This multilayered substrate recognition strategy enables NAT10 to establish highly context-dependent ac⁴C modification programs across different tissues and physiological states, providing a mechanistic explanation for the remarkable heterogeneity of the mammalian ac⁴C epitranscriptome. Importantly, the biological significance of ac⁴C extends far beyond the frequent dysregulation of NAT10 observed in human diseases [ 43 – 45 ]. NAT10 and ac⁴C represent two overlapping yet biologically distinct regulatory layers. On the one hand, ac⁴C directly influences the physicochemical properties of RNA molecules, local RNA architecture, codon decoding, and the binding affinity of RNA-binding proteins (RBPs) [ 46 , 47 ]. On the other hand, NAT10 performs numerous functions independent of RNA acetylation, including protein acetylation, RNA binding, ribosome recruitment, ribosome biogenesis, R-loop regulation, liquid–liquid phase separation (LLPS), and assembly of macromolecular protein complexes [ 48 , 49 ]. Therefore, phenotypes resulting from NAT10 knockdown, knockout, or pharmacological inhibition should not be automatically interpreted as ac⁴C-dependent unless supported by catalytic-dead rescue experiments together with site-specific validation of RNA acetylation. From a physiological perspective, the NAT10–ac⁴C axis serves as a fundamental regulatory system that coordinates translational fidelity, proteostasis, cell fate determination, reproductive biology, embryogenesis, and hematopoietic homeostasis [ 50 – 52 ]. In parallel, ac⁴C modification of selected mRNAs and ncRNAs fine-tunes the expression of pluripotency-associated factors, lineage-specifying regulators, and transcripts required for tissue homeostasis [ 53 ]. Genetic studies further demonstrate that Nat10 deficiency compromises hematopoietic stem cell (HSC) self-renewal, impairs megakaryocyte–erythroid lineage commitment, disrupts oocyte maturation and folliculogenesis, abolishes zygotic genome activation (ZGA), and ultimately causes early embryonic developmental arrest [ 50 ]. Collectively, these observations establish the NAT10–ac⁴C axis as an essential physiological regulatory network rather than a cancer-specific signaling pathway. Malignant cells, however, co-opt this evolutionarily conserved RNA regulatory system to satisfy the diverse biological demands associated with tumor progression. At the transcript level, NAT10 selectively stabilizes or enhances the translation of numerous oncogenic transcripts, including MYC, and multiple regulators of stemness, metabolism, and immune signaling [ 54 – 58 ]. At the level of the translational machinery, ac⁴C modification of tRNAs and rRNAs promotes codon-biased translation, ribosomal adaptation, and global protein synthesis, thereby supporting the elevated biosynthetic capacity required for rapid tumor growth [ 59 ]. Beyond RNA metabolism, NAT10 also governs chromatin and genome dynamics through regulation of R-loop formation and resolution, homologous recombination (HR)-mediated DNA repair, and nucleolar homeostasis [ 60 , 61 ]. Importantly, these biological processes are not independent. Instead, they are integrated through positive-feedback circuits, metabolic–immune crosstalk, and extensive interactions with other epitranscriptomic pathways, thereby reinforcing tumor plasticity and conferring persistent adaptive advantages throughout cancer evolution [ 62 ]. Despite the rapid expansion of this field, several fundamental questions remain unresolved. First, although the presence of ac⁴C in rRNAs and specific tRNAs is supported by compelling biochemical and structural evidence, the genuine abundance, transcriptome-wide distribution, and site authenticity of mRNA ac⁴C in mammals remain controversial. Early antibody-based enrichment studies proposed that ac⁴C is broadly distributed across mammalian mRNAs; however, subsequent chemical sequencing approaches and analyses of highly purified RNA fractions suggested that a substantial proportion of these signals may reflect rRNA or tRNA contamination, antibody cross-reactivity, or technical background noise. Second, ac⁴C should not be regarded as a universal RNA-stabilizing modification. Instead, its functional consequences appear to depend on multiple contextual determinants, including the modified nucleotide position, local RNA secondary structure, codon usage, translational status, and interactions with RBPs. Third, neither canonical ac⁴C readers nor physiologically validated ac⁴C erasers have yet been conclusively identified. Emerging evidence instead suggests that ac⁴C may exert many of its biological effects by altering the physicochemical properties of RNA molecules, modulating ribosome dynamics, and reshaping RNA structural conformations, rather than operating through the classical writer–reader–eraser paradigm established for N⁶-methyladenosine (m⁶A). These unresolved issues also have direct implications for therapeutic development. Nevertheless, because NAT10 is indispensable for normal hematopoiesis, reproduction, immune function, and ribosome homeostasis, systemic, continuous, or complete inhibition is likely to produce substantial on-target toxicities. Therefore, the clinical potential of NAT10-directed therapies should not be evaluated solely by tumor regression but also by their pharmacological selectivity, substrate specificity, tissue dependency, and long-term therapeutic window. These considerations suggest that future therapeutic strategies should shift from indiscriminate NAT10 inhibition toward identifying NAT10-dependent tumor subtypes and selectively disrupting tumor-specific NAT10–RNA regulatory circuits while preserving its essential physiological functions in normal tissues. In this review, we comprehensively summarize the discovery and evolutionary conservation of ac⁴C, its chemical characteristics, RNA substrate landscape, the structural and catalytic mechanisms of NAT10, and cofactor-mediated substrate recognition. We subsequently discuss the physiological functions of the NAT10–ac⁴C axis in translational fidelity, cell fate determination, reproductive development, embryogenesis, and hematopoietic homeostasis before focusing on how malignant cells hijack this regulatory network to promote proliferation, cancer stemness, metabolic reprogramming, DNA damage repair, resistance to regulated cell death, invasion and metastasis, and immune evasion. Furthermore, we establish a hierarchical framework for evaluating ac⁴C substrates based on biochemical validation, sequencing evidence, antibody-dependent detection, and functional inference, thereby clearly distinguishing ac⁴C-dependent biological effects from NAT10 functions that are independent of its catalytic activity. Finally, we critically assess current NAT10 inhibitors, targeted protein degradation strategies, rational combination therapies, biomarker-guided patient stratification, and therapeutic windows. By integrating physiological functions, tumor-specific hijacking, mechanistic controversies, and translational opportunities into a unified conceptual framework, this Review aims to define the authentic biological boundaries of the NAT10–ac⁴C axis and provide a theoretical foundation for translating epitranscriptomic discoveries into precision cancer therapeutics.

Proteostasis depends on the precise coordination of RNA processing, accurate codon decoding, ribosome maturation, and cellular stress responses [ 101 , 102 ]. In this context, ac⁴C is not merely a static structural mark that preserves RNA integrity. Instead, by acting on tRNAs, rRNAs, and potentially selected mRNAs, ac⁴C connects translational fidelity, ribosome function, and cellular adaptation to stress [ 103 ]. In particular, tRNA ac⁴C primarily ensures accurate codon–anticodon pairing and prevents ribosome stalling, whereas rRNA ac⁴C contributes to small ribosomal subunit maturation and structural stability. Together, these functions sustain normal protein synthesis and tissue homeostasis. The tRNA anticodon loop is a central structural determinant of codon-recognition accuracy, and its chemical modification therefore directly influences translational fidelity [ 104 – 106 ]. In bacterial elongator tRNA Met , ac⁴C is positioned at the wobble nucleotide C34, where it stabilizes recognition of the AUG codon and restricts erroneous decoding of the near-cognate AUA codon. tRNA Me t cytidine acetyltransferase A (TmcA) uses ATP and acetyl-coenzyme A to catalyze ac⁴C formation at this site and distinguishes structurally similar tRNA Met and tRNA Ile molecules by recognizing specific base pairs within the anticodon stem, demonstrating that ac⁴C deposition is governed by stringent structural substrate selectivity [ 107 ]. Similarly, in Bacillus subtilis , TmcA-like protein-mediated ac⁴C34 modification of tRNA Met cooperates with a lysine-derived modification on tRNA Ile to maintain the decoding boundary between AUG and AUA codons. When ac⁴C34 is lost and the tRNA Ile modification is simultaneously impaired, AUA codons can be mistranslated as methionine. This finding demonstrates that ac⁴C directly preserves protein-sequence accuracy by suppressing near-cognate codon misreading [ 108 ]. Thus, the principal physiological function of tRNA ac⁴C is not to broadly accelerate translation but to establish a stringent codon-recognition threshold that minimizes amino acid misincorporation and the generation of aberrant proteins. In mammals, the function of tRNA ac⁴C further extends to tRNA stability and the control of ribosomal stress. THUMPD1 is an essential adaptor for NAT10-mediated acetylation of selected cytoplasmic tRNAs. THUMPD1 deficiency reduces tRNA Leu abundance, increases ribosome stalling, and activates eIF2α phosphorylation. THUMPD1-deficient mice exhibit growth retardation and infertility, whereas combined deletion of the stress-sensing kinase GCN2 causes pronounced postnatal lethality. These observations indicate that translational stress arising from defective tRNA ac⁴C must be buffered by the GCN2–eIF2α integrated stress-response pathway [ 69 ]. This work links a single-site tRNA modification to organismal development, fertility, and stress survival, establishing tRNA ac⁴C as a functional determinant of mammalian proteostasis rather than a dispensable structural mark. Beyond preventing decoding errors, tRNA ac⁴C can shape codon-biased translational outputs by altering the abundance and decoding capacity of specific tRNA species [ 109 – 111 ]. When a particular ac⁴C-modified tRNA becomes limiting, mRNAs enriched for its cognate codons are more likely to undergo ribosome pausing or reduced translational efficiency, thereby selectively remodeling the proteome without substantially changing transcript abundance [ 76 ]. This principle has been clearly demonstrated in esophageal cancer [ 76 ]. NAT10 deficiency reduces the abundance of ac⁴C-modified tRNAs and selectively suppresses the translation of mRNAs enriched for the corresponding codons, with EGFR emerging as a major downstream target. Although this study focused on a malignant setting, the underlying mechanism is also physiologically relevant: tRNA ac⁴C coordinates cellular protein-synthesis demands by matching tRNA modification states with codon-usage frequencies [ 76 ]. Accordingly, tRNA ac⁴C may constitute an important regulatory layer linking cellular state, codon preference, and proteomic output. Whereas tRNA ac⁴C primarily controls codon decoding, rRNA ac⁴C is more closely associated with ribosome biogenesis and structural maturation. In yeast, Kre33/Rra1 catalyzes ac⁴C formation at specific sites within 18S rRNA [ 112 ]. Depletion of Rra1 causes pronounced accumulation of 23S precursor rRNA together with loss of mature 18S rRNA and 40S small ribosomal subunits, indicating that the NAT10 homolog participates in early pre-rRNA processing and small-subunit production [ 113 ]. This reaction requires both ATP and acetyl-coenzyme A and can be influenced by nuclear acetyl-coenzyme A availability, suggesting that rRNA ac⁴C may couple cellular metabolic state to ribosome biogenesis [ 113 ]. In human cells, NAT10 catalyzes ac⁴C modification at C1842 within the terminal helix region of mammalian 18S rRNA. NAT10 depletion leads to accumulation of 30S precursor rRNA, reduced cell growth, and impaired ribosome biogenesis, further supporting its role in 18S rRNA processing and small-subunit maturation [ 114 ]. Mechanistically, rRNA ac⁴C may stabilize local RNA folding, facilitate the binding of processing factors, or coordinate ribosome-assembly steps to ensure formation of mature 40S subunits. However, the essential role of NAT10 in ribosome biogenesis does not imply that every individual rRNA ac⁴C site is indispensable. SNORD13 guides ac⁴C deposition at a single 18S rRNA site in humans and zebrafish, yet SNORD13 deletion does not substantially affect human-cell growth, translation, ribosome biogenesis, or embryonic development. Moreover, Caenorhabditis elegans lacks this 18S rRNA modification despite retaining an essential NAT10 homolog [ 115 ]. These findings suggest that some NAT10-associated ribosomal phenotypes may arise from other rRNA sites, tRNA acetylation, or non-catalytic NAT10 functions rather than from a single conserved rRNA ac⁴C residue. Although rRNA modifications have traditionally been regarded as stable marks installed once during ribosome maturation, recent evidence indicates that rRNA ac⁴C may undergo site-specific dynamic regulation [ 114 ]. In yeast, two conserved rRNA ac⁴C sites accumulate with distinct kinetics during 18S rRNA maturation and display differential changes across growth phases and during heat stress. Stress-associated alterations in ac⁴C coincide with changes in translational efficiency and fidelity and are influenced by neighboring 2′-O-methylation, suggesting that ribosomal ac⁴C is embedded within an interdependent rRNA-modification network rather than functioning in isolation [ 116 ]. In thermophilic archaea, ac⁴C levels increase broadly as environmental temperature rises, whereas deletion of the relevant acetyltransferase produces temperature-dependent growth defects. Cryo-EM analyses indicate that ac⁴C sites are distributed within heat-sensitive ribosomal regions and may preserve ribosome integrity and translational capacity at elevated temperatures by strengthening local RNA structure [ 23 ]. Thus, rRNA ac⁴C may serve both structural maintenance and environmental adaptation, allowing ribosomes to adjust their modification states according to growth conditions. Defects in tRNA and rRNA ac⁴C ultimately converge on shared cellular consequences, including ribosome stalling, reduced translational fidelity, and accumulation of aberrant proteins [ 116 , 117 ]. Loss of tRNA ac⁴C can limit the availability of specific tRNAs, prolong codon-decoding times, and activate the GCN2–eIF2α pathway. Abnormal rRNA ac⁴C, by contrast, may impair small-subunit maturation or weaken ribosome structural stability. Both mechanisms reduce productive protein synthesis and increase the burden of misfolded proteins on cellular quality-control systems. Some studies further suggest that mRNA ac⁴C can enhance protein output by increasing transcript stability and improving decoding within coding regions, particularly for codons containing cytidine at the wobble position [ 34 ]. However, because the widespread occurrence of mammalian mRNA ac⁴C remains technically controversial, its contribution to physiological proteostasis should be distinguished from the more firmly established roles of tRNA and rRNA ac⁴C. A more conservative unified model is therefore that tRNA ac⁴C controls decoding accuracy and ribosome transit, rRNA ac⁴C supports ribosome maturation and structural adaptation, and potential mRNA ac⁴C may further fine-tune transcript stability and translational output [ 116 ]. Current evidence supports a continuous regulatory framework in which ac⁴C maintains translational fidelity and proteostasis through coordinated control of tRNA decoding, rRNA maturation, and ribosomal stress responses. Several limitations nevertheless remain. NAT10 loss simultaneously affects multiple RNA classes and non-catalytic functions, making it difficult to assign individual phenotypes to a single substrate. The requirement for specific rRNA ac⁴C sites is not uniform, direct causal evidence linking stress-induced modification changes to translational phenotypes remains limited, and the authenticity and abundance of mammalian mRNA ac⁴C continue to depend heavily on detection methodology. Future studies should combine RNA-class-specific genetic models, site-specific RNA editing, ribosome profiling, quantitative mistranslation assays, protein-aggregation measurements, and multiplatform ac⁴C validation to distinguish the individual contributions of tRNA, rRNA, and mRNA ac⁴C to translational homeostasis and to determine which modification changes represent genuine adaptive mechanisms rather than secondary consequences of ribosome dysfunction. Cell-fate determination depends on the precise coordination of transcriptional networks, epigenetic regulation, and translational programs [ 118 – 120 ]. Increasing evidence indicates that ac⁴C not only regulates RNA stability and translational efficiency but also selectively enhances the expression of lineage-determining factors, thereby participating in stem-cell maintenance, lineage specification, tissue remodeling, and developmental plasticity [ 50 , 52 , 121 ]. ac⁴C has therefore emerged as an important regulatory layer linking epitranscriptomic regulation to cell-fate transitions. Beyond its molecular function as an RNA acetyltransferase, NAT10 participates in numerous physiological processes across development and tissue homeostasis. Figure 2 summarizes the current understanding of NAT10-mediated ac⁴C regulation under physiological conditions. Emerging evidence indicates that NAT10-mediated ac⁴C modification is essential for normal development and tissue homeostasis. Through selective regulation of key developmental transcripts, NAT10 controls stem-cell maintenance, lineage specification, embryogenesis, hematopoiesis, reproduction, and skeletal development. Representative physiological functions and their underlying molecular mechanisms are summarized in Table 2 . Fig. 2 Physiological functions of NAT10-mediated RNA ac⁴C modification. NAT10 regulates RNA stability and translation during embryonic development, reproduction, hematopoiesis, neural development, metabolism, immune regulation, and tissue homeostasis. These activities collectively support cell fate determination, lineage commitment, metabolic adaptation, and maintenance of normal tissue function Physiological functions of NAT10-mediated RNA ac⁴C modification. NAT10 regulates RNA stability and translation during embryonic development, reproduction, hematopoiesis, neural development, metabolism, immune regulation, and tissue homeostasis. These activities collectively support cell fate determination, lineage commitment, metabolic adaptation, and maintenance of normal tissue function Table 2 Physiological functions and molecular mechanisms of NAT10-mediated ac⁴C modification in normal biological processes Physiological process Target(s) Biological function Molecular mechanism Ref Stem cell identity and pluripotency OCT4, ESRRB, ZFP42 mRNAs Maintains embryonic stem cell (ESC) self-renewal and pluripotency NAT10-mediated ac⁴C increases the stability and translational efficiency of core pluripotency factor mRNAs, thereby sustaining the pluripotency network and preventing premature differentiation. [ 122 , 123 ] Pluripotent–totipotent state equilibrium KAP1 mRNA Maintains ESC identity and suppresses 2-cell-like (2C) transition ac⁴C stabilizes KAP1 expression, preserving the H3K9me3-repressive chromatin landscape and preventing aberrant activation of the totipotency program. [ 122 ] Neuroectoderm lineage specification NR2F1 mRNA Promotes neuroectoderm differentiation NAT10 deposits ac⁴C on NR2F1 mRNA, enhancing its translation; increased NR2F1 drives YAP1 cytoplasmic translocation and neuroectoderm commitment. [ 52 ] Preimplantation embryonic lineage specification Nop2 mRNA Promotes morula-to-blastocyst transition and trophectoderm specification ac⁴C stabilizes Nop2 mRNA, maintains NOP2 expression, enhances TEAD4 translation, and induces Cdx2 expression to establish the first embryonic lineage decision. [ 124 ] Developmental plasticity and somatic-cell reprogramming ANP32B mRNA Regulates lineage differentiation and cellular plasticity NAT10-mediated ac⁴C enhances ANP32B translation, leading to chromatin remodeling of developmental pathways including Wnt and TGF-β signaling. [ 125 ] Osteogenic differentiation (BMSCs) Runx2 mRNA Promotes osteoblast differentiation and bone formation ac⁴C stabilizes Runx2 mRNA, increasing RUNX2 expression and osteogenic differentiation. [ 126 ] BMP-dependent osteogenesis Gremlin1 mRNA Enhances osteogenesis NAT10 promotes Gremlin1 mRNA degradation, relieving BMP/Smad inhibition and facilitating osteoblast differentiation. [ 127 ] Periodontal ligament stem-cell osteogenesis VEGFA mRNA Promotes osteogenic differentiation ac⁴C stabilizes VEGFA mRNA and activates AKT signaling, enhancing osteogenic differentiation. [ 128 ] Osteoclast differentiation Fos mRNA Promotes osteoclastogenesis and bone resorption ac⁴C stabilizes Fos mRNA, activating the MAPK–AP-1 signaling pathway during osteoclast differentiation. [ 129 ] Oocyte maturation and meiotic progression CCR4–NOT complex component mRNAs Coordinates maternal mRNA clearance and oocyte maturation NAT10-mediated ac⁴C promotes CCR4–NOT-dependent poly(A)-tail-mediated degradation of maternal transcripts, enabling transcriptome remodeling during oocyte maturation. [ 130 ] Oocyte maturation OGA mRNA Supports meiotic maturation ac⁴C stabilizes OGA mRNA, preventing excessive O-GlcNAc accumulation and maintaining GPCR signaling, chromatin regulation, and maturation-associated gene expression. [ 131 ] Folliculogenesis and female fertility Multiple oocyte-derived factor mRNAs Supports follicular development and female fertility NAT10-mediated ac⁴C maintains the stability and translation of oocyte-derived transcripts, promoting both intrinsic oocyte growth and granulosa-cell development. [ 132 ] Maternal-to-zygotic transition (MZT) and zygotic genome activation (ZGA) Nanog mRNA; maternal transcripts; Nop2 mRNA Drives early embryogenesis and blastocyst development NAT10-mediated ac⁴C promotes maternal RNA clearance, stabilizes Nanog/Nop2 mRNAs, supports TEAD4 translation and Cdx2 expression, thereby ensuring successful ZGA and blastocyst formation. [ 133 ] Oocyte quality under pathological conditions Zar1 (translation) Maintains oocyte developmental competence Reduced ac⁴C selectively impairs translation of developmental regulators such as Zar1; restoration of NAT10-mediated ac⁴C improves oocyte quality and fertility in SLE models. [ 134 ] HSC self-renewal and lineage commitment Nfix mRNA Maintains hematopoietic stem-cell homeostasis and megakaryocyte–erythroid differentiation NAT10 deposits ac⁴C on Nfix mRNA, enhancing its translation and activating downstream hematopoietic regulators (e.g., MPL), thereby promoting HSC fate determination and lineage commitment. [ 50 ] Physiological functions and molecular mechanisms of NAT10-mediated ac⁴C modification in normal biological processes Maintenance of pluripotency is fundamental to the stable identity of embryonic stem cells (ESCs). NAT10-mediated ac⁴C preferentially modifies mRNAs encoding core pluripotency transcription factors, including OCT4, ESRRB, and zinc finger protein 42 (ZFP42), thereby increasing their stability and translational efficiency to sustain the self-renewal capacity of both human and mouse ESCs. Loss of NAT10 results in reduced expression of these master regulators, collapse of the pluripotency network, and premature differentiation of stem cells [ 122 , 123 ]. Beyond directly maintaining pluripotency factors, NAT10 also stabilizes transcripts encoding chromatin regulators such as KAP1, thereby preserving the repressive H3K9me3 chromatin landscape. This suppresses aberrant activation of the 2-cell-like (2C) totipotency program and maintains the dynamic equilibrium between pluripotent and totipotent states [ 122 ]. These findings indicate that ac⁴C safeguards stem-cell identity not only by sustaining transcription-factor expression but also by preserving the epigenetic landscape that defines cellular identity. As embryogenesis proceeds, cells must rapidly commit to distinct developmental lineages. ac⁴C provides post-transcriptional support for these lineage decisions by selectively enhancing the translation of lineage-determining factors. During differentiation of human ESCs toward the ectodermal lineage, NAT10 catalyzes ac⁴C deposition on NR2F1 mRNA, thereby increasing its translational efficiency. Elevated NR2F1 subsequently promotes the translocation of YAP1 from the nucleus to the cytoplasm, driving commitment to the neuroectodermal lineage and demonstrating that ac⁴C directly regulates early lineage-specification programs [ 52 ]. Similarly, during mouse preimplantation development, NAT10 stabilizes nucleolar protein 2 (Nop2) mRNA through ac⁴C modification, thereby maintaining Nop2 expression, enhancing translation of TEAD4, and inducing expression of the trophoblast determinant Cdx2. Consequently, NAT10 ensures the transition from the morula to the blastocyst and trophectoderm lineages. Nat10 deficiency disrupts the first cell-fate decision, preventing most embryos from forming normal blastocysts, whereas ectopic Nop2 expression partially rescues these developmental defects [ 124 ]. Collectively, these studies demonstrate that ac⁴C contributes to lineage establishment throughout embryogenesis by selectively promoting the expression of key developmental determinants. Cell-fate transitions involve not only transcription-factor networks but also extensive chromatin remodeling. Recent studies have revealed that NAT10 preferentially enhances the translation of a subset of chromatin regulators involved in developmental decisions, among which ANP32B represents a key example. The NAT10–ac⁴C–ANP32B axis subsequently modulates chromatin accessibility within developmental signaling pathways, including Wnt and TGF-β, thereby influencing lineage differentiation of human ESCs and the efficiency of somatic-cell reprogramming [ 125 ]. This work establishes a direct mechanistic link between epitranscriptomic regulation and chromatin remodeling, suggesting that ac⁴C extends beyond the regulation of individual RNA molecules to reshape entire gene-regulatory networks through translational control of chromatin regulators, thereby conferring greater developmental plasticity upon cells. Beyond embryonic development, ac⁴C also governs cell-fate decisions in adult stem cells and contributes to the maintenance of tissue homeostasis [ 128 ]. In the skeletal system, NAT10 regulates both bone formation and bone resorption through distinct downstream substrates. In bone marrow-derived mesenchymal stem cells, NAT10 stabilizes Runx2 mRNA to promote osteogenic differentiation [ 126 ]. It also facilitates degradation of Gremlin1 mRNA, thereby relieving inhibition of bone morphogenetic protein (BMP)/Smad signaling and further enhancing osteogenesis [ 127 ]. Likewise, in human periodontal ligament stem cells, NAT10 stabilizes VEGFA mRNA and activates AKT signaling to promote osteogenic differentiation [ 128 ]. Conversely, during osteoclast differentiation, NAT10 stabilizes Fos proto-oncogene (Fos) mRNA and activates the MAPK–AP-1 pathway, thereby promoting osteoclastogenesis. Pharmacological inhibition of NAT10 with Remodelin markedly attenuates inflammation-associated bone resorption [ 129 ]These findings indicate that ac⁴C does not uniformly promote or suppress differentiation. Instead, it selectively stabilizes lineage-determining transcripts in a cell-type-specific manner, thereby directing the overall outcome of tissue remodeling. Overall, current evidence supports a model in which ac⁴C coordinates stem-cell maintenance, lineage commitment, and tissue remodeling through the combined regulation of RNA stability and translation, preferentially enhancing the expression of lineage-determining transcription factors, chromatin regulators, and tissue-specific effector molecules. Nevertheless, several important challenges remain. Most available studies rely on whole-gene NAT10 knockout models, making it difficult to distinguish ac⁴C-dependent catalytic functions from the non-catalytic activities of NAT10. In addition, current knowledge is largely derived from ESCs and a limited number of tissues, whereas the dynamic ac⁴C landscape, substrate-selection mechanisms, and cell-type-specific regulatory programs across diverse organs remain poorly understood. Future investigations integrating single-cell and spatial epitranscriptomics, site-specific RNA editing, developmental lineage tracing, and genetic rescue models will be essential for systematically defining the spatiotemporal regulatory network of ac⁴C during development and tissue homeostasis and for elucidating how ac⁴C cooperates with chromatin modifications, RNA-binding proteins, and translational programs to govern cell fate, thereby providing a conceptual foundation for targeting developmental disorders and tumor cell plasticity. Germ-cell development and early embryogenesis rely on exquisitely coordinated post-transcriptional regulation [ 135 , 136 ]. Because oocyte maturation, meiosis, and preimplantation embryonic development depend predominantly on the stability, translation, and timely degradation of maternal mRNAs, RNA modifications play indispensable roles throughout these processes. Recent studies have demonstrated that NAT10-mediated ac⁴C not only maintains maternal transcriptome homeostasis but also coordinates meiotic progression, zygotic genome activation (ZGA), lineage specification, and folliculogenesis, thereby emerging as an important regulatory layer connecting RNA metabolism with reproductive development. Oocyte maturation is a prerequisite for developmental competence and is characterized by dynamic maternal mRNA degradation coupled with selective translation [ 130 ]. Genetic studies have shown that Nat10 deficiency markedly impairs meiotic prophase progression, oocyte growth, and maturation. Mechanistically, NAT10 promotes ac⁴C modification of mRNAs encoding key components of the CCR4–NOT complex, facilitating poly(A)-tail-dependent degradation of maternal transcripts and thereby driving the transcriptomic remodeling required for oocyte maturation [ 130 ]. Further studies revealed that NAT10 also regulates in vitro oocyte maturation by stabilizing O-GlcNAcase (OGA) mRNA. Loss of NAT10 decreases OGA transcript stability, resulting in excessive accumulation of O-linked N-acetylglucosamine (O-GlcNAc) modification and disruption of G protein-coupled receptor signaling, chromatin regulation, and multiple maturation-associated genes. These findings reveal a previously unrecognized layer of crosstalk between RNA ac⁴C modification and protein O-GlcNAcylation during oocyte development [ 131 ]. In addition to oocyte maturation, ac⁴C is indispensable for normal follicular development. Oocyte-specific Nat10 deletion arrests folliculogenesis at the secondary follicle stage, ultimately causing premature ovarian insufficiency (POI) and complete female infertility [ 132 ]. Mechanistically, NAT10-mediated ac⁴C maintains the stability and translational efficiency of multiple oocyte-derived factor mRNAs, thereby promoting not only intrinsic oocyte growth but also the development of surrounding granulosa cells through cell non-autonomous mechanisms, ultimately sustaining normal folliculogenesis [ 132 ]. These observations indicate that ac⁴C determines not only the fate of individual germ cells but also bidirectional communication between oocytes and somatic cells, thereby contributing to maintenance of the ovarian microenvironment. Preimplantation embryonic development critically depends on successful transition from maternal RNA-driven regulation to activation of the embryonic genome. Recent studies demonstrate that NAT10 is indispensable for both ZGA and the morula-to-blastocyst transition in mice. Nat10 knockdown arrests most embryos at the morula stage, accompanied by reduced expression of Nanog and Cdx2, with Nanog mRNA displaying markedly decreased stability [ 133 ]. More strikingly, embryos derived from oocyte-specific Nat10 knockout females almost uniformly arrest at the two-cell stage. These embryos exhibit defective maternal mRNA clearance and failed initiation of ZGA, ultimately resulting in complete developmental arrest [ 133 ]. These findings establish NAT10-mediated ac⁴C as a critical molecular event driving the maternal-to-zygotic transition (MZT), extending beyond maintenance of maternal RNA homeostasis. In addition to global transcriptome remodeling, ac⁴C also controls early lineage specification through stabilization of specific developmental regulators. NAT10 catalyzes ac⁴C deposition on Nop2 mRNA, thereby increasing transcript stability and promoting translation of TEAD4 together with expression of the trophoblast lineage determinant Cdx2. Consequently, ac⁴C drives the transition from the morula to the blastocyst and trophectoderm lineages. Restoration of Nop2 expression partially rescues the developmental defects caused by Nat10 deficiency, identifying Nop2 as a direct and functionally important ac⁴C substrate during early embryogenesis [ 124 ]. An increasing body of evidence links dysregulated ac⁴C to disease-associated reproductive dysfunction [ 134 ]. In systemic lupus erythematosus (SLE) models, oocyte ac⁴C levels are markedly reduced and accompanied by globally impaired translational activity. Single-cell transcriptomic and translatomic analyses demonstrate that loss of ac⁴C selectively impairs translation of key developmental regulators such as Zar1, thereby compromising oocyte quality.Notably, nicotinamide treatment restores NAT10-mediated ac⁴C levels, increases both the number and quality of oocytes, and improves fertility in SLE model animals, suggesting that modulation of ac⁴C may represent a promising therapeutic strategy for immune-associated infertility [ 134 ]. Although the role of ac⁴C in the male reproductive system remains comparatively understudied, recent work has identified an mRNA ac⁴C writer complex composed of NAT10, PCBP1/2, and TDP-43 [ 42 ]. Importantly, this complex is stably present in the mouse testis, suggesting that RBP-mediated substrate recruitment may contribute to spermatogenesis and male reproductive function. However, its direct targets and physiological significance remain to be elucidated [ 42 ]. Taken together, current evidence demonstrates that ac⁴C coordinates maternal mRNA stability, selective translation, and programmed degradation to integrate oocyte maturation, meiotic progression, folliculogenesis, ZGA, and early lineage specification into a unified post-transcriptional regulatory program during reproduction. Nevertheless, several important knowledge gaps remain. Most studies rely on global NAT10-deficient models, making it difficult to distinguish catalytic ac⁴C-dependent effects from non-catalytic functions of NAT10. Furthermore, only a limited number of direct substrates have been identified, dynamic ac⁴C maps across developmental stages and reproductive cell types are still unavailable, and the roles of ac⁴C in male reproduction, meiotic recombination, and spermatogenesis remain largely unexplored. Future studies integrating single-cell multi-omics, spatial epitranscriptomics, site-specific RNA editing, and conditional genetic models will be required to define the spatiotemporal dynamics and substrate-selection mechanisms of ac⁴C during gametogenesis and early embryogenesis and to evaluate its translational potential as a therapeutic target for infertility and assisted reproductive technologies. Hematopoietic homeostasis depends on the ability of HSCs to maintain a dynamic balance among self-renewal, lineage differentiation, and stress responses [ 137 – 139 ]. Recent studies have shown that NAT10-mediated ac⁴C is not only involved in malignant hematopoiesis but also serves as an essential regulator of normal blood-cell development [ 50 ]. Notably, NAT10 operates through two mechanistically distinct pathways in the hematopoietic system: an ac⁴C-dependent translational mechanism that directs lineage commitment and an ac⁴C-independent RNA-stabilizing mechanism that preserves HSC homeostasis. Together, these activities establish a multilayered post-transcriptional network required for normal hematopoietic function. HSCs must maintain a precise equilibrium between long-term self-renewal and continuous differentiation, a process that requires sustained expression of lineage-determining transcription factors [ 50 ]. Using ultra-low-input ac⁴C sequencing (ULAC-seq), investigators generated the first ac⁴C landscape of normal HSPCs. ac⁴C displayed pronounced cell-type-specific distribution and reached its highest abundance in megakaryocyte–erythroid progenitors (MEPs), closely paralleling dynamic Nat10 expression [ 50 ]. Genetic studies further demonstrated that Nat10 deficiency markedly impairs HSC self-renewal and blocks MEP differentiation, ultimately resulting in both fetal and postnatal hematopoietic failure [ 50 ]. Mechanistically, NAT10 deposits ac⁴C on mRNAs encoding key hematopoietic transcription factors, including nuclear factor I X (Nfix), thereby increasing their translational efficiency. Elevated Nfix subsequently activates downstream hematopoietic regulators such as myeloproliferative leukemia protein (Mpl), promoting HSC fate determination and megakaryocyte–erythroid differentiation. Restoration of Nfix expression partially rescues the colony-forming capacity of Nat10-deficient HSPCs, demonstrating that ac⁴C-dependent translational control provides a direct mechanistic bridge between RNA modification and the hematopoietic transcriptional network [ 50 ]. Unlike many RNA modifications that predominantly influence stem-cell maintenance, NAT10 exerts particularly prominent effects on megakaryocytic and erythroid development. ac⁴C is strongly enriched in MEPs, whereas Nat10 deficiency markedly impairs their differentiation into mature megakaryocytes and erythroid cells, indicating that this lineage exhibits heightened dependence on NAT10 activity [ 50 ]. These findings suggest that ac⁴C does not uniformly support all hematopoietic lineages. Instead, it preferentially reinforces megakaryocyte–erythroid developmental programs by enhancing translation of selected lineage-specifying factors [ 50 ]. Because erythrocyte and platelet production requires rapid and sustained protein synthesis, the elevated ac⁴C dependency of MEPs may reflect functional coupling between high translational demand and RNA modification. Importantly, the hematopoietic functions of NAT10 are not entirely dependent on its ac⁴C catalytic activity [ 86 ]. Recent work demonstrated that key HSC-maintenance genes in humans and mice frequently contain extended 3′UTRs enriched in AREs. NAT10 directly binds these ARE-containing regions and promotes ribosome recruitment to the 3′UTRs, thereby protecting the corresponding mRNAs from degradation and maintaining their stable expression [ 86 ]. This function is independent of NAT10-mediated ac⁴C deposition. Nat10 deficiency substantially compromises long-term HSC reconstitution, whereas loss of catalytic activity alone does not fully account for this phenotype, indicating that NAT10 also functions as an RNA-binding regulator of transcript turnover and stem-cell maintenance [ 86 ]. These findings identify NAT10 not only as an RNA acetyltransferase but also as a catalysis-independent determinant of RNA fate. Current evidence supports at least two complementary mechanisms through which NAT10 maintains normal hematopoiesis. First, NAT10-mediated ac⁴C enhances translation of key hematopoietic transcription factors, thereby directing HSC fate decisions and megakaryocyte–erythroid differentiation. Second, NAT10 directly binds AREs within the extended 3′UTRs of essential HSC transcripts, recruits ribosomes, and stabilizes these mRNAs independently of ac⁴C catalysis, thereby sustaining long-term self-renewal capacity [ 50 , 86 ]. Accordingly, NAT10 integrates the epitranscriptomic landscape with the hematopoietic transcriptional network while simultaneously coordinating RNA stability and translational control. This dual mechanism also indicates that hematopoietic phenotypes caused by NAT10 loss cannot be attributed solely to reduced ac⁴C levels and must be interpreted in the context of its non-catalytic RNA-regulatory activities [ 50 , 86 ]. Overall, available evidence indicates that NAT10 maintains HSC self-renewal, lineage differentiation, and megakaryocyte–erythroid development through a dual-layered regulatory system comprising ac⁴C-dependent translational control and ac⁴C-independent RNA stabilization. Several key questions, however, remain unresolved. The relative contributions of these catalytic and non-catalytic mechanisms across distinct hematopoietic cell types are unclear, and whether they act cooperatively or compensate for one another has not been systematically examined. In addition, only a limited number of direct ac⁴C substrates have been identified, and it remains unknown whether other RNAs controlling lineage choice are similarly regulated by NAT10. The dynamic functions of NAT10 during stress hematopoiesis induced by infection, blood loss, inflammation, or aging also remain largely unexplored. Future studies integrating single-cell ac⁴C profiling, site-specific RNA editing, conditional genetic models, and stress-hematopoiesis systems will be required to define the spatiotemporal actions of NAT10 across hematopoietic stages and microenvironments and to distinguish the relative contributions of RNA acetylation and catalysis-independent RNA binding to normal hematopoiesis and hematologic disease.

Recent studies have uncovered a critical role of NAT10 in shaping the tumor immune microenvironment and determining responses to cancer immunotherapy. Figure  9 summarizes the mechanisms linking NAT10 to immune regulation. Recent studies have expanded the role of NAT10 beyond tumor-cell intrinsic regulation to include extensive modulation of the tumor immune microenvironment and responses to cancer immunotherapy. Through regulation of immunometabolism, cytokine and chemokine networks, immune-cell communication, and immune-checkpoint signaling, the NAT10–ac⁴C axis profoundly influences antitumor immunity and therapeutic efficacy. Representative immune-regulatory mechanisms are summarized in Table  5 . Fig. 9 NAT10-mediated regulation of tumor immunity and immunotherapy response. NAT10 remodels the immune microenvironment by regulating cytokine production, immune cell recruitment, immune checkpoint expression, metabolic competition, and interferon signaling. These mechanisms collectively influence immune evasion and the efficacy of immune checkpoint blockade NAT10-mediated regulation of tumor immunity and immunotherapy response. NAT10 remodels the immune microenvironment by regulating cytokine production, immune cell recruitment, immune checkpoint expression, metabolic competition, and interferon signaling. These mechanisms collectively influence immune evasion and the efficacy of immune checkpoint blockade Table 5 Functional mechanisms of the NAT10–ac⁴C axis in tumor immune regulation and immunotherapy Cancer type / Model Direct target(s) Immune function Molecular mechanism Ref Cervical cancer FOXP1 Establishes an immunosuppressive tumor microenvironment and reduces PD-L1 blockade efficacy HOXC8 activates NAT10; NAT10 enhances FOXP1 translation through ac⁴C modification, inducing GLUT4 and KHK, increasing glycolysis and lactate production, which promotes Treg-mediated immunosuppression. [ 169 ] Cisplatin-resistant tumors GDF6, INHBA Remodels the immunosuppressive TIME NAT10-mediated ac⁴C enhances translation of GDF6 and INHBA, activating TGF-β signaling and promoting infiltration of MDSCs, M2 macrophages, Tregs, and exhausted CD8⁺ T cells. [ 235 ] Multiple myeloma circANKRD12 → circANKRD12₃₅₄aa Suppresses NK-cell cytotoxicity NAT10 acetylates circANKRD12 and promotes its translation into circANKRD12₃₅₄aa, which is transferred into NK cells via extracellular vesicles and suppresses NK-cell function through the HDAC2–c-Myc axis. [ 32 ] Prostate cancer CCL25 Reduces CD8⁺ T-cell recruitment NAT10 suppresses the CCL25–CCR9 chemokine axis, generating an immune-desert phenotype and reducing antitumor T-cell infiltration. [ 236 ] Colorectal cancer DKK2 Induces CD8⁺ T-cell exhaustion NAT10 stabilizes DKK2 mRNA; secreted DKK2 activates LRP6–AKT–mTOR signaling in CD8⁺ T cells, causing cholesterol accumulation and impaired cytotoxicity. [ 236 ] Pancreatic cancer ETS2, KRT8 Promotes immune escape NAT10 stabilizes ETS2 mRNA, establishing an ETS2–NAT10 positive-feedback loop that upregulates PD-L1 and decreases CD8⁺ T-cell infiltration while promoting invasion through KRT8. [ 237 ] Triple-negative breast cancer JunB → LDHA Suppresses antitumor T-cell activity NAT10 stabilizes JunB mRNA, increases LDHA expression and lactate accumulation, thereby impairing T-cell activation; NAT10 inhibition synergizes with CTLA-4 blockade. [ 168 ] Normal T cells Myc Promotes physiological T-cell activation NAT10 enhances ac⁴C modification and translation of Myc mRNA, supporting T-cell proliferation and clonal expansion. This represents the physiological function of NAT10 in immune cells. [ 56 ] Lung adenocarcinoma CXCL5, DEK Promotes immunosuppressive inflammation NAT10 stabilizes CXCL5 and DEK mRNAs, enhancing recruitment of neutrophils and MDSCs while promoting metastasis. [ 238 ] Macrophages (inflammatory model) ETS2 Activates inflammatory macrophages USP39 stabilizes NAT10; NAT10 enhances ETS2 mRNA stability and translation, promoting inflammatory cytokine production. [ 239 ] NSCLC GAS5 Enhances antitumor immunity NAT10 stabilizes lncRNA GAS5, activating the MYBBP1A–p53–IRF1 pathway, increasing CXCL10 and CCL5, and promoting T-cell/macrophage infiltration. [ 240 ] Multiple tumors MYC-CDK2-DNMT1 pathway Activates innate immunity NAT10 suppresses dsRNA accumulation through the MYC–CDK2–DNMT1 pathway; NAT10 inhibition restores type I interferon signaling, enhances CD8⁺ T-cell immunity, and improves PD-1 therapy. [ 54 ] Cervical cancer FOXP1 Improves response to PD-L1 blockade NAT10 inhibition reduces glycolysis and lactate production, reversing Treg-mediated immune suppression and enhancing anti-PD-L1 efficacy. [ 169 ] Pancreatic cancer ETS2 Enhances anti-PD-L1 therapy NAT10 inhibition reduces PD-L1 expression and restores CD8⁺ T-cell infiltration, synergizing with PD-L1 blockade. [ 237 ] Breast cancer HDAC4 Promotes immune-checkpoint signaling NAT10 stabilizes HDAC4 mRNA, activating NF-κB signaling and increasing PD-L1 expression. [ 241 ] Multiple cancers NPM1 acetylation Enhances PD-L1 expression NAT10-mediated NPM1 acetylation increases PD-L1 expression; Remodelin synergizes with anti-CTLA-4 therapy. [ 242 ] Gastric cancer DUSP1 -FOSB Promotes immune escape and PD-1 resistance NAT10 stabilizes DUSP1 mRNA, activates JNK/ERK–FOSB signaling, increases PD-L1 expression, and promotes cisplatin resistance; NAT10 inhibition synergizes with anti-PD-1 therapy. [ 243 ] Colorectal cancer DKK2 Enhances response to PD-1 blockade NAT10 inhibition or DKK2-neutralizing antibody restores CD8⁺ T-cell cytotoxicity and synergizes with anti-PD-1 therapy. [ 236 ] Multiple cancers MYC-CDK2-DNMT1 pathway Restores tumor immunogenicity NAT10 inhibition restores dsRNA sensing and type I IFN signaling, markedly enhancing PD-1 immunotherapy. [ 54 ] Multiple cancers Kynurenine-NAT10 Immunometabolic immune escape Kynurenine enhances NAT10 activity and promotes PD-L1-associated immune suppression; NAT10 inhibition reverses this effect. [ 244 ] Functional mechanisms of the NAT10–ac⁴C axis in tumor immune regulation and immunotherapy The immunosuppressive TIME is a major barrier to effective antitumor immunity and immune-checkpoint therapy [ 245 – 247 ]. Increasing evidence indicates that the NAT10–ac⁴C axis not only promotes tumor-cell-intrinsic malignant phenotypes but also drives the formation of an immunosuppressive microenvironment by remodeling tumor metabolism, cytokine networks, and intercellular communication. Notably, NAT10 does not appear to regulate a single immune-cell population directly [ 248 ]. Instead, it first alters post-transcriptional programs within tumor cells and subsequently reshapes the composition and function of immune cells through metabolites, cytokines, and paracrine signals [ 169 ]. NAT10 should therefore be regarded as an important molecular hub linking tumor-intrinsic programs with remodeling of the immune microenvironment. One of the best-defined mechanisms is NAT10-mediated induction of immunosuppression through metabolic reprogramming. In cervical cancer, HOXC8 directly activates NAT10 expression. NAT10 subsequently enhances FOXP1 translation through ac⁴C modification and induces GLUT4 and KHK expression, thereby sustaining glycolysis and lactate secretion. The resulting lactate-rich microenvironment further reinforces the immunosuppressive activity of Tregs and ultimately reduces the efficacy of PD-L1 blockade. NAT10 inhibition not only decreases lactate accumulation but also markedly enhances the antitumor activity of PD-L1-targeting antibodies [ 169 ]. These findings establish a glycolysis–lactate–Treg axis through which NAT10 creates a metabolically driven immunosuppressive niche rather than merely promoting tumor-cell proliferation. Beyond metabolic reprogramming, NAT10 can shape the TIME by regulating immunosuppressive cytokines. In the context of cisplatin resistance, aberrant NAT10-mediated ac⁴C remodeling enhances the translation of growth differentiation factor 6 (GDF6) and INHBA, leading to sustained activation of TGF-β signaling. This promotes extensive infiltration of MDSCs, M2 macrophages, exhausted CD8⁺ T cells, and Tregs, thereby establishing a profoundly immunosuppressive microenvironment [ 235 ]. Notably, this study further developed a luteinizing hormone-releasing hormone receptor-targeted TGF-β nanodelivery system, NanoTi, which in combination with cisplatin markedly reversed the immunosuppressive microenvironment. These findings indicate that NAT10-mediated remodeling of the TIME is therapeutically actionable. More recent studies have extended the immunological functions of NAT10 from T cells to the innate immune system. In multiple myeloma, NAT10 catalyzes ac⁴C modification of circANKRD12 and promotes its translation into the novel protein circANKRD12₃₅₄aa. This protein not only enhances myeloma-cell proliferation but can also be transferred together with circANKRD12 into NK cells, where it suppresses NK-cell cytotoxicity through the HDAC2–c-Myc axis and thereby promotes immune evasion [ 32 ]. This study provided the first mechanistic link among ac⁴C modification, circRNA translation, and tumor–immune-cell communication and further demonstrated that RNA modification can participate in TIME remodeling through extracellular vesicle-mediated intercellular signaling. Current evidence therefore reveals a multilayered architecture of NAT10-mediated immunosuppression. NAT10 increases glycolysis and lactate production, activates immunosuppressive pathways such as TGF-β, promotes enrichment of MDSCs, M2 macrophages, and Tregs, and weakens NK-cell cytotoxicity. These mechanisms cooperate to shift the tumor microenvironment from an inflammatory, immune-active state toward immune tolerance, thereby laying the foundation for CD8⁺ T-cell dysfunction and resistance to immune-checkpoint therapy. NAT10 is therefore not merely an RNA-modifying enzyme but also an important coordinator of metabolic, inflammatory, and immune-cell remodeling. Overall, current evidence supports a model in which the NAT10–ac⁴C axis shapes an immunosuppressive TIME through metabolic reprogramming, activation of TGF-β signaling, and intercellular communication. Several important questions remain unresolved. Most studies have focused on individual immune-cell populations, and how different immune subsets coordinately respond to NAT10-driven signals remains poorly understood. It is also unclear whether lactate, TGF-β, and exosomal circRNAs operate within a hierarchical network or represent distinct tumor-type-specific strategies. In addition, the direct ac⁴C substrates responsible for TIME remodeling must be distinguished from secondary downstream effects. Future studies integrating spatial transcriptomics, spatial proteomics, single-cell ac⁴C profiling, and multicellular interaction analyses will be required to define the spatiotemporal dynamics of NAT10-driven immunosuppression and provide a more precise rationale for combining NAT10-targeted therapy with immunotherapy. Beyond remodeling the immunosuppressive microenvironment, increasing evidence indicates that the NAT10–ac⁴C axis can directly impair antitumor T-cell immunity through tumor-intrinsic programs [ 169 ]. These effects extend beyond simple induction of PD-L1 expression and encompass chemokine remodeling, metabolic competition, dysregulated cholesterol metabolism, and immune-checkpoint activation [ 169 ]. Together, these mechanisms reduce CD8⁺ T-cell recruitment, promote functional exhaustion, and impair cytotoxicity. NAT10 has therefore emerged as an important molecular bridge linking tumor-cell RNA modification with T-cell dysfunction. The most direct evidence concerns regulation of CD8⁺ T-cell recruitment by tumor cells. In prostate cancer, NAT10 markedly suppresses the CCL25–CCR9 chemokine axis, reduces recruitment of CD8⁺ T cells into tumor tissue, and further impairs their cytotoxic activity, thereby generating a typical immune-desert phenotype. An ac⁴C score derived from NAT10 downstream targets can also predict immune-cell infiltration and response to immunotherapy, suggesting that NAT10 may have biomarker value in addition to its mechanistic role in immune evasion. NAT10 can also directly induce CD8⁺ T-cell exhaustion. In colorectal cancer, NAT10 stabilizes DKK2 mRNA through ac⁴C modification and increases DKK2 secretion. DKK2 then binds low-density lipoprotein receptor-related protein 6 (LRP6) on CD8⁺ T cells, activates AKT–mTOR signaling, and induces pathological cholesterol accumulation, thereby markedly suppressing CD8⁺ T-cell cytotoxicity. Genetic Nat10 deletion or pharmacological NAT10 inhibition restores CD8⁺ T-cell function and synergizes with anti-PD-1 therapy [ 236 ]. This study established a previously unrecognized NAT10–DKK2–cholesterol metabolism–CD8⁺ T-cell exhaustion axis and demonstrated that tumor cells can reprogram immune-cell metabolism through RNA modification. A related mechanism operates in pancreatic cancer. NAT10 stabilizes ETS2 mRNA through ac⁴C modification and establishes an ETS2–NAT10 positive-feedback circuit that further enhances PD-L1 expression while simultaneously stabilizing KRT8 to promote tumor invasion. Single-cell RNA sequencing revealed stronger abnormal interactions between NAT10-high tumor cells and T cells. NAT10 depletion not only reduced pulmonary metastasis but also increased CD8⁺ T-cell infiltration and enhanced the efficacy of anti-PD-L1 therapy [ 237 ]. These findings further indicate that NAT10 regulates not only immune-cell abundance but also tumor–T-cell communication networks. Tumor-cell NAT10 can additionally suppress T-cell function through metabolic competition rather than through classical immune checkpoints alone. In triple-negative breast cancer, NAT10 stabilizes JunB mRNA, upregulates LDHA, and enhances glycolysis, leading to sustained lactate accumulation. Remodelin-mediated NAT10 inhibition reduces tumor glycolysis and enhances CTLA-4 antibody-induced T-cell activation, underscoring the close relationship between tumor metabolism and T-cell dysfunction [ 168 ]. A particularly important issue is that NAT10 performs different functions in tumor cells and normal T cells. During physiological immune activation, NAT10 is rapidly induced in T cells. It increases ac⁴C modification and translational efficiency of Myc mRNA, promotes rapid MYC accumulation, and thereby drives T-cell proliferation and clonal expansion. T-cell-specific Nat10 deletion causes severe cell-cycle arrest and defective expansion and impairs antiviral immune responses. NAT10 downregulation in T cells from older individuals is also closely associated with immune dysfunction [ 56 ]. These findings reveal the dual identity of NAT10 in tumor immunity: tumor cells exploit NAT10 to promote immune evasion, whereas normal effector T cells require NAT10 to sustain proliferation and function. Systemic NAT10 inhibition may therefore relieve tumor-mediated immunosuppression while simultaneously compromising normal T-cell responses. This tension represents a major challenge for NAT10-targeted therapy and emphasizes the need for tumor-specific delivery, conditional inhibition, or therapeutic strategies that exploit tumor-selective NAT10 dependencies rather than indiscriminate systemic blockade. Overall, current studies support a model in which the NAT10–ac⁴C axis establishes tumor-intrinsic immune-evasion programs by suppressing CD8⁺ T-cell recruitment, inducing functional exhaustion, remodeling immunometabolism, and enhancing abnormal tumor–immune-cell interactions. Several key questions remain. The direct substrates identified across studies, including CCL25, DKK2, ETS2, and JunB, differ substantially and may represent tumor-type-specific dependencies. How NAT10-mediated immunometabolic reprogramming cooperates with canonical immunosuppressive signals such as PD-L1 and TGF-β also remains unclear. Most importantly, selectively inhibiting tumor-cell NAT10 while preserving normal T-cell immunity is a central translational challenge. Future studies integrating spatial single-cell multi-omics, conditional genetic models, and tumor-selective delivery systems will be required to define the distinct functions of NAT10 in tumor and immune cells and support the development of more precise combination immunotherapies. Beyond directly regulating immune-cell function, the NAT10–ac⁴C axis coordinates dynamic interactions between tumor and immune cells by remodeling cytokine and chemokine networks [ 238 ]. Although this study primarily focused on invasion and dissemination, CXCL5 is also a major chemokine that recruits neutrophils and MDSCs, suggesting that NAT10 may simultaneously promote inflammatory remodeling and immunosuppression. Similarly, in acute kidney injury, NAT10 increases the stability of CCL2 and CXCL1 mRNAs through ac⁴C modification, promotes macrophage and neutrophil recruitment, and aggravates local inflammation. Genetic deletion or pharmacological inhibition of NAT10 markedly reduces inflammatory-cell infiltration [ 249 ]. Although this study was conducted in a non-malignant disease model, it reveals a general NAT10–chemokine–immune-cell recruitment mechanism that may also operate within tumors and underscores the broader capacity of NAT10 to reshape immune-cell composition. NAT10 can also regulate inflammatory transcriptional networks. In macrophages, inflammatory stimulation induces USP39-mediated stabilization of NAT10. NAT10 subsequently enhances ETS2 mRNA stability and translational efficiency through ac⁴C modification, thereby promoting proinflammatory cytokine expression and macrophage activation [ 239 ]. This study demonstrates that NAT10 can act directly within innate immune cells. However, whereas acute inflammation is protective during infection and tissue injury, chronic inflammation in tumors can promote malignant progression and immunosuppression. NAT10-driven inflammatory signaling may therefore have opposing consequences depending on disease context. Importantly, NAT10 does not invariably promote immunosuppression. In NSCLC, the lncRNA growth arrest-specific 5 (GAS5) is an important ac⁴C substrate of NAT10. Stabilized GAS5 activates type I interferon signaling through the MYBBP1A–p53–IRF1 axis, induces CXCL10 and CCL5 expression, enhances T-cell and macrophage infiltration, and is associated with improved immunotherapy response [ 240 ] This finding demonstrates that ac⁴C modification itself does not inevitably drive immune escape; the biological outcome depends strongly on the function of the modified RNA substrate. Increasing evidence further indicates that NAT10 regulates broader inflammatory networks rather than isolated cytokines. In tumor cells, NAT10 suppresses double-stranded RNA (dsRNA) accumulation through the MYC–CDK2–DNMT1 pathway and thereby attenuates type I interferon responses. NAT10 inhibition restores dsRNA-induced antiviral-like immunity, enhances tumor-specific CD8⁺ T-cell responses, and markedly improves the efficacy of PD-1 blockade [ 54 ]. NAT10 therefore regulates not only chemokine expression but also innate immune sensing and interferon signaling, positioning it as an important link between RNA modification and innate immunity. Current evidence thus reveals bidirectional regulation of cytokine and chemokine networks by NAT10. On the one hand, NAT10 enhances CXCL5, CCL2, and CXCL1 expression and promotes immunosuppressive inflammation and aberrant immune-cell recruitment. On the other hand, in selected contexts, it stabilizes GAS5, activates type I interferon signaling, increases CXCL10 and CCL5 expression, and strengthens antitumor immunity. These apparently contradictory findings indicate that NAT10 does not predetermine the direction of inflammation. Rather, it functions as an RNA-modification platform that selectively reshapes the immune ecosystem according to cell type and substrate identity. Overall, the NAT10–ac⁴C axis broadly participates in remodeling the tumor immune microenvironment by coordinating chemokine expression, inflammatory transcriptional networks, and type I interferon signaling. Important limitations remain. Most studies focus on individual cytokines or chemokines and lack systems-level analysis of the broader secretory network and intercellular communication. The determinants that cause NAT10 to promote immunosuppression in some tissues while enhancing antitumor inflammation in others remain unclear. It is also unknown how these inflammatory programs cooperate with metabolic reprogramming, DDR, and EMT. Future studies integrating spatial transcriptomics, single-cell secretome profiling, spatial proteomics, and cell–cell interaction analysis will be required to determine how NAT10-dependent cytokine networks reshape tumor immune ecology and to evaluate NAT10 as a target for immune modulation. Immune-checkpoint inhibitors (ICIs) have substantially improved clinical outcomes in multiple malignancies, yet most patients exhibit primary or acquired resistance [ 250 , 251 ]. Recent studies indicate that the NAT10–ac⁴C axis not only contributes to the formation of an immunosuppressive microenvironment but also directly regulates PD-L1 expression, immune-checkpoint signaling, and antitumor immune activation, thereby influencing the response to ICIs. Importantly, NAT10-mediated immunotherapy resistance does not arise from a single pathway. Instead, it reflects coordinated regulation of PD-L1 expression, immunometabolic reprogramming, immune-cell exhaustion, and innate immune activation. NAT10 should therefore be viewed as an upstream determinant of immunotherapy sensitivity rather than merely as a regulator of PD-L1. The strongest evidence concerns NAT10-dependent regulation of PD-L1 expression. In different tumor types, NAT10 sustains PD-L1 through distinct molecular mechanisms. In cervical cancer, the HOXC8–NAT10–FOXP1 axis enhances glycolysis and lactate accumulation and thereby indirectly reinforces Treg-mediated immunosuppression. NAT10 depletion markedly improves the efficacy of PD-L1 blockade [ 169 ]. In pancreatic cancer, NAT10 stabilizes ETS2 mRNA, establishes an ETS2–NAT10 positive-feedback loop, and promotes PD-L1 expression, resulting in reduced CD8⁺ T-cell infiltration. Combined treatment with a NAT10 inhibitor and an anti-PD-L1 antibody produces stronger antitumor activity than either agent alone [ 237 ]. In breast cancer, NAT10 stabilizes HDAC4 mRNA and establishes a NAT10–HDAC4 positive-feedback loop that activates NF-κB signaling and promotes PD-L1 transcription [ 241 ]. Across multiple tumor types, NAT10 can also increase PD-L1 expression by promoting NPM1 acetylation, and combined Remodelin and anti-CTLA-4 treatment displays pronounced therapeutic synergy [ 242 ]. Beyond direct regulation of PD-L1, NAT10 can promote immunotherapy resistance through additional signaling networks. In gastric cancer, NAT10 stabilizes DUSP1 mRNA through ac⁴C modification, enhances JNK/ERK signaling, and promotes cisplatin resistance. NAT10 further increases PD-L1 expression through FOSB, thereby creating a shared regulatory axis connecting chemotherapy resistance with immune escape. In animal models, combined NAT10 inhibition and anti-PD-1 treatment markedly improves therapeutic responses in cisplatin-resistant gastric cancer [ 243 ]. Similar dual roles in tumor progression, immunosuppression, and ICI resistance have been observed in pancreatic and cervical cancer, indicating that RNA modification is an important link between resistance to conventional therapy and resistance to immunotherapy. Recent studies have expanded this framework beyond the conventional view that NAT10 acts mainly through PD-L1. In colorectal cancer, NAT10 stabilizes DKK2 mRNA and induces pathological cholesterol accumulation in CD8⁺ T cells, leading to cytotoxic exhaustion. Remodelin or a DKK2-neutralizing antibody restores CD8⁺ T-cell function and synergizes with PD-1 blockade [ 236 ] NAT10 inhibition can also relieve MYC–CDK2–DNMT1-mediated suppression of dsRNA, restore type I interferon signaling and tumor-specific CD8⁺ T-cell immunity, and further enhance the efficacy of PD-1 therapy [ 54 ]. NAT10 therefore affects ICI responsiveness through multiple processes, including innate immune activation, tumor immunogenicity, and restoration of effector T-cell function, rather than through PD-L1 expression alone. Metabolic factors also contribute to NAT10-mediated immunotherapy resistance. Kynurenine, a tryptophan metabolite, enhances NAT10 activity and promotes PD-L1 mRNA-associated regulation, thereby further suppressing T-cell activation. NAT10 inhibition reverses kynurenine-induced immune escape [ 244 ]. This finding further highlights the close interplay among metabolic reprogramming, RNA modification, and immune checkpoints and identifies NAT10 as a potential molecular link between immunometabolism and treatment response. Current evidence supports at least four levels of NAT10-mediated resistance to ICIs: enhancement of PD-L1 expression and immune-checkpoint signaling; remodeling of glycolysis and lactate metabolism to establish an immunosuppressive microenvironment; induction of CD8⁺ T-cell exhaustion and loss of cytotoxicity; and suppression of innate immune programs such as dsRNA–type I interferon signaling. Together, these mechanisms determine tumor sensitivity to PD-1/PD-L1 or CTLA-4 blockade and explain why NAT10 inhibition enhances ICI efficacy across multiple preclinical models. Overall, the NAT10–ac⁴C axis is an important regulator of immune-checkpoint therapy response, but several critical questions remain. Most studies focus on PD-L1 and PD-1 signaling, whereas direct evidence that NAT10 regulates antigen processing and presentation, MHC class I expression, dendritic-cell cross-presentation, or neoantigen immunogenicity remains limited. The downstream substrates driving immune escape also differ across tumor types, and a unified ac⁴C signature capable of predicting ICI response has not yet been established. Moreover, systemic NAT10 inhibition may impair normal T-cell expansion and antiviral immunity, and this potential safety concern requires careful evaluation. Future studies integrating spatial single-cell multi-omics, immune lineage tracing, integrated multi-omics, and clinical cohorts should establish NAT10-associated immune subtypes and predictive models and develop precise combination strategies involving NAT10 inhibitors together with PD-1/PD-L1, CTLA-4, TGF-β, or metabolic therapies.