Regulations of m⁶A and other RNA modifications and their roles in cancer

Abstract

RNA modification is an essential component of the epitranscriptome, regulating RNA metabolism and cellular functions. Several types of RNA modifications have been identified to date; they include N⁶-methyladenosine (m⁶A), N¹-methyladenosine (m¹A), 5-methylcytosine (m⁵C), N⁷-methylguanosine (m⁷G), N⁶,2′-O-dimethyladenosine (m⁶A_m), N⁴-acetylcytidine (ac⁴C), etc. RNA modifications, mediated by regulators including writers, erasers, and readers, are associated with carcinogenesis, tumor microenvironment, metabolic reprogramming, immunosuppression, immunotherapy, chemotherapy, etc. A novel perspective indicates that regulatory subunits and post-translational modifications (PTMs) are involved in the regulation of writer, eraser, and reader functions in mediating RNA modifications, tumorigenesis, and anticancer therapy. In this review, we summarize the advances made in the knowledge of different RNA modifications (especially m⁶A) and focus on RNA modification regulators with functions modulated by a series of factors in cancer, including regulatory subunits (proteins, noncoding RNA or peptides encoded by long noncoding RNA) and PTMs (acetylation, SUMOylation, lactylation, phosphorylation, etc.). We also delineate the relationship between RNA modification regulator functions and carcinogenesis or cancer progression. Additionally, inhibitors that target RNA modification regulators for anticancer therapy and their synergistic effect combined with immunotherapy or chemotherapy are discussed.

Keywords

RNA modification / writers / erasers / readers / regulatory subunits / PTMs / cancer

1 Introduction

Cancer is a disease affecting population worldwide that causes a large public health care burden [1]. Dysregulated gene expression is an intrinsic factor in carcinogenesis and cancer progression [2]. In addition to alterations in DNA sequences, epigenetic regulation of DNA and RNA contributes to dysregulated gene expression in cancer cells.

From an epigenome perspective, a diverse set of RNA modifications play a vital role in the post-transcriptional regulation of gene expression. With the development of high-throughput sequencing technology, more than 170 types of RNA modifications have been discovered [3]. According to their different chemical structures, the main RNA modifications are classified into several types, including N⁶-methyladenosine (m⁶A), N¹-methyladenosine (m¹A), 5-methylcytosine (m⁵C), N⁷-methylguanosine (m⁷G), N⁶,2'-O-dimethyladenosine (m⁶A_m), N⁴-acetylcytidine (ac⁴C), etc. RNA modifications are mediated by three groups of proteins: writers which catalyze the deposition of specific modification marks, erasers which remove specific modification marks, and readers which recognize specific modification marks and mediate downstream effects. Writers and erasers are involved in modulating RNA modification levels, while readers mostly contribute to the biological function mediated by RNA modifications. An increasing number of studies have demonstrated that RNA modifications are involved in mediating the acquisition of cancer phenotypes, such as cancer cell proliferation, survival, metastasis, metabolism reprogramming, immunosuppression, and drug resistance [4]. Great attention has been given to research on potential therapeutic targets for RNA modification with which to treat cancers because RNA modification is extensive in various cancers and is reversible [4]. With the deeper understanding of RNA modification, a novel perspective is gradually being recognized: the action of RNA modification regulators can be modulated via a series of factors, including regulatory subunits (proteins, noncoding RNAs (ncRNAs) or peptides encoded by long noncoding RNAs (lncRNAs)) and post-translational modifications (PTMs) (acetylation, SUMOylation, lactylation, phosphorylation, etc.), which is also closely associated with carcinogenesis and cancer progression [5–8].

Although many review papers have summarized the functional roles of RNA modifications (especially RNA m⁶A methylation) in cancer [4,9,10], the factors associated with regulators of RNA modification and their biological function have not been systematically summarized and categorized; these factors, which have been gradually discovered and attracted widespread attention, include regulatory subunits and PTMs.

In this review, a concise overview of six different RNA modifications including m⁶A, m¹A, m⁵C, m⁷G, m⁶A_m, and ac⁴C, is provided. The review mainly focuses on the function of regulatory subunits (proteins, ncRNAs or peptides encoded by lncRNAs) and PTMs (acetylation, SUMOylation, lactylation, phosphorylation, etc.) that modulate RNA modification regulators (writers, erasers, and readers) in cancer. We also describe the relationship between the functions of RNA modification regulators and their involvement in carcinogenesis or cancer progression. In addition, inhibitors that target RNA modification regulators for anticancer therapy and their synergistic effect combined with immunotherapy or chemotherapy are discussed. This review contributes to a more comprehensive understanding of the overall system of RNA modifications in the context of cancer.

2 m⁶A modification

2.1 Overview of m⁶A

N6-methyladenosine (m⁶A) is generated when a methyl group is added to the amino group at the N⁶ position of an adenine nucleobase [11]. It is the most prevalent and well-characterized internal modification of RNA in various species including viruses, bacteria, plants, and mammals [12]. Approximately 0.1%–0.4% of adenosine residues in total RNA are modified by m⁶A marks [13]. The m⁶A modification is evident in a variety of RNAs, including mRNA, rRNA, tRNA, long noncoding RNA (lncRNA), and circular RNA (circRNA) [14]. Increasing evidence has proven that in the context of abnormal m⁶A levels, m⁶A plays a critical role in numerous diseases, including cancer (Fig.1) [15].

2.2 Regulators of m⁶A

The m⁶A modification is a dynamic and reversible process triggered in response to various stimuli and is regulated by two distinct classes of protein factors: methyltransferases (“writers”) and demethylases (“erasers”). Writers deposit m⁶A marks on RNA, while erasers remove them. The biological functions of m⁶A mostly depend on the recognition and recruiting ability of RNA m⁶A reader proteins (“readers”).

2.2.1 Writers

Writers recognize a conserved consensus site and catalyze methylation of a substrate by transferring a methyl group from S-adenosylmethionine (SAM) to the N⁶ position of adenosine [16]. The core component of m⁶A writers comprises three proteins: methyltransferase-like 3 (METTL3), methyltransferase-like 14 (METTL14) and Wilms tumor 1-associated protein (WTAP). METTL3 was the first methyltransferase to be discovered, and it consists of a sole catalytic unit, and METTL14 exhibits a substrate recognition function [17,18]. METTL3/METTL14 complex, a stable heterodimer formed by METTL3 and METTL14 at a ratio of 1:1, is recruited by WTAP to specific RNA sequence. Other components, including RNA binding motif protein 15 (RBM15) [19], zinc finger CCCH-type containing 13 (ZC3H13) [20], virus-like m⁶A methyltransferase associated (VIRMA, also known as KIAA1429) [21] and Hakai (also known as CBLL1) [22], contribute to the full methyltransferase activity of m⁶A writers by forming an m⁶A-METTL-associated complex in conjunction with the METTL3/METTL14 heterodimer. In addition to the aforementioned methyltransferases, METTL5 [23], METTL16 [24], and ZCCHC4 [25] have been reported to show methyltransferase effects on mRNAs and ncRNAs.

2.2.2 Erasers

Demethylases, as m⁶A erasers, can directly remove an m⁶A modification mark from RNA. Fat mass and obesity-associated protein (FTO) was the first m⁶A demethylase identified in vivo [26]. AlkB homolog 5 (ALKBH5) is another m⁶A eraser in nuclear speckles that regulates RNA metabolism by reducing m⁶A levels [27].

2.2.3 Readers

The m⁶A readers specifically recognize and selectively bind to m⁶A sites, thereby modulating certain biological functions in an m⁶A-dependent manner [28]. According to the manner by which they bind to different RNAs, readers can be classified into two types: direct readers and indirect readers. Direct readers have direct RNA binding ability which is realized by their m⁶A recognition and binding domain. One of the classic m⁶A readers are proteins containing the YT521-B homology (YTH) domain, including YTHDF1-3 and YTHDC1-2 [29,30]. Insulin-like growth factor 2 mRNA binding protein 1–3 (IGF2BP1–3) are additional m⁶A readers that preferentially bind m⁶A-containing RNAs via their RNA binding domains, namely RNA recognition motifs and K homology domains [31]. Moreover, eIF3 binds to m⁶A sites within the 5′ untranslated region (5′UTR) and functions as a direct N⁶-methyladenosine binding reader protein [32]. Fragile X mental retardation protein (FMRP) can also serve as an m⁶A reader to bind and interpret m⁶A to regulate RNA function [33]. Prrc2a binds to the consensus GGACU motif in the Olig2 coding sequence [34]. In addition, m⁶A alters the local structure of RNA, facilitating the interactions of nearby RNAs with proteins; these RNA–protein interactions are considered an “m⁶A-switch” mechanism [35]. Several HNRNPs bind indirectly to RNAs through this mechanism; these HNRNPs include HNRNPC [35], HNRNPA2/B1 [36], and HNRNPG [37].

Through the m⁶A reader, different regulatory or functional machines are recruited to m⁶A-containing mRNAs to direct the fate of the target RNA. The metabolism of mRNA, including translation [29,31,32,38–40], decay [29,41–48], stabilization [31,49], splicing [35,39,50], and nuclear export [35,39,51], is regulated by this mechanism. In addition, m⁶A affects the activity [19], decay [52], processing [53], nuclear export [54], and translation [55] of noncoding RNAs. Thus, readers determine the fate and function of m⁶A-methylated RNAs.

2.3 Regulatory subunits and post-translational modification of m⁶A writers, erasers, and readers

m⁶A regulators (writers, erasers, and readers) exert their influence on tumor progression in an m⁶A-dependent manner. Recent evidence has indicated that m⁶A regulators can also be modulated via a series of regulatory subunits (proteins, ncRNAs or peptides encoded by lncRNAs) and PTMs (acetylation, SUMOylation, lactylation, phosphorylation, etc.). Thus, through the regulation mediated by these factors, m⁶A regulators exhibit different functions that are closely related to tumor progression and treatment.

2.4 Regulatory subunits that modulate the functions of m⁶A writers, erasers, and readers

As mentioned above, m⁶A regulators play an important role in both the m⁶A pathway and the development of tumors. Recent studies have revealed that regulatory subunits modulate the functions of m⁶A-modifying enzymes and m⁶A readers. Some proteins, ncRNAs, and peptides encoded by lncRNAs have been proven to interact with m⁶A writers, erasers, and readers to modulate their functions (Fig.2).

2.5 Proteins that modulate m⁶A regulator functions

WTAP, RBM15, ZC3H13, VIRMA, and Hakai, interact with METTL3/METTL14 heterodimer, contributing to the overall methyltransferase activity of m⁶A writers; the regulatory mechanisms underlying the functions of these subunits have been extensively studied and elaborated [19–22,56]. In addition, eukaryotic translation initiation factor 3 subunit h (eIF3h) directly interacts with METTL3, facilitating the enhanced translation by METTL3. METTL3-eIF3h interaction promotes the translation of a majority of oncogenic mRNAs in human primary lung cancer in an m⁶A-dependent manner [57]. It has also been reported that peptidyl-prolyl cis-trans isomerase NIMA-interacting 1 (PIN1) interacts with METTL3 and prevents its ubiquitin-dependent and lysosomal degradation, leading to the stabilization of METTL3. This, in turn, promotes the m⁶A-dependent translation of transcriptional coactivator with PDZ binding motif (TAZ) and epidermal growth factor receptor (EGFR) mRNAs, inducing the progression of breast cancer (BCa) [58]. In addition, METTL5, as the enzyme critical for 18S rRNA m⁶A modification, interacts with TRMT112 and is thus metabolically stable in colon carcinoma cells [23].

Our research team first discovered a regulatory subunit of ALKBH5, namely, Paraspeckle component 1 (PSPC1), which is an RNA binding protein [7]. As a regulatory subunit of ALKBH5, PSPC1 preferentially interacts with K235-acetylated ALKBH5 but not nonacetylated ALKBH5, subsequently recruiting and facilitating the recognition of m⁶A mRNA by ALKBH5. This process promotes the m⁶A demethylation activity of ALKBH5, inducing tumorigenesis and tumor progression. In addition, RNA binding motif protein 33 (RBM33) is an m⁶A binding protein that recruits ALKBH5 to its m⁶A-marked substrate, inhibiting its SUMOylation and activating its demethylase activity by interacting with SENP1, which is involved in the maintenance of tumorigenesis in human head and neck squamous cell carcinoma (HNSCC) [59]. SFPQ is another RNA binding protein that is closely related to FTO throughout the transcriptome. SFPQ interacts with FTO and mediates FTO substrate specificity by recruiting FTO to specific RNA sites [60].

In addition, a study has shown that FBW7 interacts with YTHDF2 and induces the proteasomal degradation of the latter to counteract the tumor-promoting effects of YTHDF2; this mechanism reduces the survival time and proliferation rate of ovarian cancer cells in an m⁶A-dependent manner [61].

2.6 ncRNAs that modulate m⁶A regulator functions

Several reports have highlighted the roles of ncRNAs in modulating m⁶A regulator functions in cancer. The latest research has demonstrated that the FTO-IT1 lncRNA directly binds and inhibits RBM15, a critical component of the m⁶A methyltransferase complex, and then suppresses m⁶A methylation and reduces the stability of p53 transcriptional target mRNAs (e.g., FAS, TP53INP1, and SESN2) [62]. Depletion of FTO-IT1 increased the mRNA m⁶A level and expression of the p53 tumor suppression signaling gene and inhibited prostate cancer (PCa) growth in mice.

In addition, lncRNA-CBSLR interacted with YTHDF2 to augment the binding of YTHDF2 with m⁶A-modified CBS mRNA, subsequently decreasing the stability of CBS mRNA and promoting the progression of gastric cancer [63]. Recent research has also shown that the lncRNA STEAP3-AS1 interacted competitively with YTHDF2, leading to the disassociation of YTHDF2 from STEAP3 mRNA, which prevented m⁶A-modified STEAP3 mRNA from being degraded by YTHDF2 in an m⁶A-dependent manner, facilitating the progression of colorectal cancer (CRC) [64]. The lncRNA DMDRMR interacts with IGF2BP3 to enhance its activity, subsequently stabilizing target genes, including CDK4, COL6A1, LAMA5, and FN1, in an m⁶A-dependent manner. This process ultimately promoted clear cell renal cell carcinoma tumor growth and metastasis [5]. ncRNAs interact with m⁶A reader proteins and regulate their stability mediated through the ubiquitination system in an m⁶A-dependent manner. For example, circEZH2 interacts with IGF2BP2 and blocks its ubiquitination-dependent degradation. This process enhances the stability of m⁶A-modified CREB1 mRNA through the action of IGF2BP2, thereby exacerbating CRC progression [65].

2.7 Peptides encoded by lncRNAs that modulate m⁶A regulator functions

Interestingly, the peptides encoded by lncRNAs have attracted considerable attention and have been found to exert potential effects by modulating m⁶A regulator functions. Our research team discovered a 71-amino acid peptide encoded by the lncRNA LINC00266-1. This peptide is named “RNA-binding regulatory peptide” (RBRP) because its main interaction is with RNA binding proteins, including the m⁶A reader IGF2BP1 [6]. The RBRP peptide, but not the lncRNA LINC00266-1 itself, functions as a regulatory subunit of the m⁶A reader IGF2BP1. Mechanistically, RBRP binds to IGF2BP1, enhancing its recognition of m⁶A marks on target RNAs such as c-Myc mRNA. This recognition, in turn, increases the stability and expression of c-Myc mRNA, thereby promoting tumorigenesis.

2.8 PTMs that modulate the functions of m⁶A writers, erasers, and readers

In addition to regulatory subunits, m⁶A regulator functions can be regulated by PTMs that affect their enzyme activity, subcellular localization, affinity for substrates, and stability. These PTMs play important roles in regulating gene expression, cellular responses, and key cellular processes, ultimately influencing critical processes such as tumorigenesis [66].

2.8.1 Acetylation

Lysine acetylation is catalyzed reversibly by protein acetyltransferases and deacetylases. Acetylation is widely linked to diverse cellular processes, altering protein activity, interactions, translation rate or stability [67]. Our research team has discovered that K235 acetylation regulates the m⁶A demethylation activity of ALKBH5 and contributes to tumorigenesis [7]. K235 acetylation of ALKBH5 promotes a significant increase in its m⁶A demethylation activity by enhancing its binding and recognition of RNA m⁶A mark, resulting in the efficient removal of the RNA m⁶A mark. Furthermore, K235 acetylation positively influences the RNA binding affinity of ALKBH5 to RNA m⁶A, contributing to its increased demethylation capacity. Additionally, K235-acetylated ALKBH5 preferentially interacts with PSPC1, which facilitates the recruitment and recognition of ALKBH5 to RNA m⁶A, thereby facilitating the removal of RNA m⁶A modification by ALKBH5 and promoting tumorigenesis (Fig.3) [7]. Moreover, studies have shown that IL-6 promotes METTL3 deacetylation, leading to its nuclear translocation. This nuclear localization of METTL3 enhances its m⁶A methyltransferase activity, contributing to increased global m⁶A abundance in BCa cells and triggering BCa metastasis (Fig.4) [68].

2.8.2 SUMOylation

SUMOylation is catalyzed by the dimer E1 SAE1/UBA2, E2 Ubc9, and E3 ligases, and a SUMO group is removed by sentrin-specific proteases [69]. SUMOylation is related to various cellular processes and is an important mechanism in the cellular stress response [70,71]. SUMOylation of METTL3 significantly represses its m⁶A methyltransferase activity, reducing the levels of m⁶A on mRNAs and promoting the colony formation and tumor growth in human non-small cell lung carcinoma cells (Fig.4) [72]. Another study revealed that SUMOylation of METTL3 was increased upon mitogen stimulation, inhibiting the METTL3 m⁶A methyltransferase activity, altering Snail mRNA homeostasis and promoting hepatocellular carcinoma (HCC) progression (Fig.4) [73]. In addition, ROS-induced stress promotes SUMOylation (lysine K86 and K321) and phosphorylation (serine S87 and S325) of ALKBH5 by activating the ERK/jnk signaling pathway, thereby inhibiting the RNA m⁶A demethylase activity of ALKBH5, increasing the global mRNA m⁶A level and suppressing tumorigenesis (Fig.3 and 3C) [59,66]. FTO K216 SUMOylation promotes FTO instability possibly via its effects on ubiquitination pathway. FTO K216 SUMOylation increased the RNA m⁶A level of the HCC tumor suppressor guanine nucleotide binding protein G (o) subunit alpha and decreased its mRNA level, thereby promoting HCC tumorigenesis [74]. YTHDF2 was modified by SUMO1 at the main SUMOylation site, K571, under hypoxic conditions. The SUMOylation of YTHDF2 greatly increased its affinity for m⁶A-modified mRNAs, leading to dysregulated gene expression and tumor progression (Fig.5) [75].

2.8.3 Lactylation

Lactylation, an emerging recently described epigenetic modification of histone lysine residues, directly stimulates the transcription of genes in chromatin. Recently, a study revealed that lactate stimulates the expression of METTL3. METTL3 is lactylated at K281 and K345, which increases its RNA binding affinity, thereby increasing RNA m⁶A levels (Fig.4). Moreover, lactylation of METTL3 in the METTL3-JAK1-STAT3 regulatory axis induced immunosuppressive effects on tumor-infiltrating myeloid cells in the CRC context [76].

2.8.4 Phosphorylation

Phosphorylation is an extensively studied PTM type, and phosphate groups added to proteins exert a significant effect on the stability of the marked protein [77]. Activated ERK phosphorylates METTL3 and WTAP, stabilizing the m⁶A methyltransferase complex (Fig.4). Mouse embryonic stem cells, with loss of METTL3/WTAP phosphorylation, retained pluripotency via the reduced degradation rate of m⁶A-labeled pluripotent factor transcripts. In ERK-activated tumor cells, METTL3 phosphorylation contributed to CRC tumorigenesis [78]. Besides, growth factor signaling induces phosphorylation of METTL3, resulting in METTL3-mediated methylation of small nuclear RNA 7SK. The methylation of 7SK enhances its binding to heterogeneous nuclear ribonucleoproteins, leading to the release of the HEXIM1 P-TEFb complex subunit 1 (HEXIM1)/P-TEFb complex and promoting transcriptional elongation (Fig.4) [79]. In addition, phosphorylation of YTHDF2 at serine 39 and threonine 381 by ERK1/2 led to increased stability of YTHDF2 and facilitated the mRNA degradation of LXRA and HIVEP2 in an m⁶A-dependent manner, resulting in tumorigenesis leading to glioblastoma (Fig.5) [80].

2.8.5 Ubiquitination

Ubiquitination is a highly conserved protein modification process that plays an important role in regulating the degradation of various substrates [81,82]. USP38 mediates METTL14 protein deubiquitination, leading to attenuating bladder cancer cell malignancy. METTL14 enhances the stability of USP38 mRNA through the m⁶A modification with the involvement of YTHDF2. Through this regulatory mechanism, METTL14 suppressed bladder cancer progression and established a feedback loop with USP38 [83]. FTO ubiquitination was promoted by glycogen synthase kinase-3 β, which induced FTO degradation and then regulated the m⁶A modification of the downstream gene MZF1 and finally inhibited CRC cell proliferation [84]. In addition, deubiquitination of FTO mediated by USP18 increased the stability of the FTO protein, resulting in reduced m⁶A levels on PYCR1, thus promoting the initiation and progression of bladder cancer [85].

2.8.6 Other PTMs

Arginine methylation is a critical PTM that regulates protein functions. Protein arginine methyltransferase 1 methylates the C terminus of METTL14, which enhances its binding affinity for RNA substrates and promotes its RNA methylation activity and interaction with RNA polymerase II, thereby increasing RNA m⁶A levels [86].

O-GlcNAcylation is a PTM that connects nutrient flux and gene transcription during the replication of viruses and tumor growth [87,88]. A study has revealed that serine 263 is a critical site for the O-GlcNAc modification of YTHDF2; this modification inhibited the ubiquitination of YTHDF2, enhanced its protein stability, and promoted liver cancer (Fig.5) [89].

As mentioned above, a new perspective on the m⁶A modification is clearly gaining recognition: in addition to their regulation of m⁶A methylation, m⁶A regulators are also influenced by various other factors. Notably, these regulatory factors (regulatory subunits and PTMs) may not act independently but rather in a multifactorial manner to modulate the functions of these regulators. Notably, the factors that influence the function of regulators also show great potential to become drug targets.

2.9 m⁶A regulators and cancers

2.9.1 m⁶A writers/erasers

The expression of known m⁶A writers and erasers jointly contributes to the m⁶A modification abundance [90]. The homeostasis of the m⁶A system is under strict control; otherwise, the chaos caused by methylation might initiate or exacerbate various diseases, especially cancer [91,92]. An increasing number of studies have investigated the m⁶A modification levels in different types of cancers. The biological functions and recent advances in studies on m⁶A writers and erasers in the contexts of cancers have been well reviewed by Deng [93], Wang [94], Flamand [95], etc. Therefore, this review does not cover these research directions.

2.9.2 m⁶A readers

Various m⁶A readers are involved in multiple processes of RNA metabolism in an m⁶A-dependent manner and function as direct executors of m⁶A-dependent bioprocesses [96], emphasizing the crucial role of readers as a bridge between the m⁶A modification and cancer. Therefore, in a variety of tumor biological processes, such as tumorigenicity, tumor immune infiltration, evasion of apoptosis, and reprogramming metabolism, in addition to considering the overall abnormal m⁶A level, changes to the m⁶A reader itself need to be taken into account. Recently, researchers have begun to focus on the relationship between dynamic protein level changes in reader proteins and tumor progression.

Readers are highly expressed in certain cancers and are involved in cancer progression in an m⁶A-dependent manner. YTHDF1, which is overexpressed in CRC [97,98], esophageal squamous cell carcinoma (ESCC) [99], bladder cancer, and HCC [100], mediates tumorigenicity, attenuates antitumor immunity, and promotes cell proliferation and metastasis by enhancing mRNA translation. YTHDF2 expression has been shown to be abnormally upregulated in lung cancer [101] and HCC [102], augmenting the protein expression of oncogenes to facilitate tumor growth and metastasis. In addition, studies in lung adenocarcinoma [103], glioma [104], and acute myeloid leukemia (AML) [105] have shown that YTHDF2 promotes the degradation of m⁶A-containing transcripts, enhancing tumorigenesis and cell proliferation and migration and protecting cancer cells from apoptosis. The overexpression of YTHDF3 in cancer stem-like cells in ocular melanoma [106] and brain metastases of BCa [107] was directly correlated with tumorigenicity and metastasis because it promoted the translation of m⁶A-marked transcripts. IGF2BP1, which is overexpressed in endometrial cancer [108] and HCC [109], enhanced cancer cell proliferation and alleviated immune cell infiltration by stabilizing downstream target genes. In clinical pancreatic cancer tissues, high expression of HNRNPC was correlated with metastasis because HNRNPC mediated the alternative splicing of TAF8 in an m⁶A-dependent manner [110]. HNRNPA2B1 was highly overexpressed in PCa, modulating PCa cell proliferation and invasion by accelerating the maturation of miR-93-5p [111].

In addition, it has been reported that low expression of readers affects the functions of cancer cell. The downregulation of YTHDF2 specifically induced by hypoxia in HCC cells contributed to the continuation of cell proliferation, tumor growth, and activation of MEK and ERK by attenuating the degradation of EGFR mRNA [112]. A portion of lung adenocarcinoma cells exhibited YTHDC2 downregulation, which decreased the degradation rate of SLC7A11 in an m⁶A-dependent manner and thus promoted the tumorigenesis [113].

The crosstalk among different types of m⁶A readers increases the diversity of m⁶A-marked mRNA fates. Interestingly, YTHDF1 and YTHDF2 have been shown to interact competitively with YTHDF3 to regulate YAP expression. YTHDF1 promotes YAP mRNA translation by interacting with eIF3a, while YTHDF2 promotes YAP mRNA decay through the Argonaute 2 (Ago2) system, influencing non-small cell lung cancer progression [114]. Different readers exert synergistic [99,115] or discordant [112,113,116,117] effects on identical downstream targets in specific cancers.

In addition to the influence of m⁶A reader expression, it is also necessary to consider that changes to the expression of different downstream targets may lead to different outcomes. The same types of reader can produce the opposite effect in identical cancers because they exert effects on different targets [97,118–120], which is determined by the heterogeneity of the tumor and the cellular environments.

2.10 Targeting m⁶A regulators for anticancer therapy

Most m⁶A regulators are overexpressed in tumor cells, making them potential targets for treatments that inhibit tumorigenesis. By inhibiting m⁶A regulators, multiple processes, such as RNA translation, decay, stability, splicing, and nuclear export, can be affected, ultimately affecting the acquisition of a malignant phenotype, such as the proliferation and invasion of cancer cells. In recent years, significant progress has been made in the development of small molecule inhibitors targeting m⁶A regulators (Tab.1) [121]. As RNA modifications have been shown to be involved in cancer development and are potential biomarkers for patient stratification, inhibitors that target specific RNA modification regulators show potential to modulate cancer treatment and drive advancements in personalized medicine and the realization of personalized and precision treatment approaches.

2.10.1 Targeting m⁶A writers

m⁶A methyltransferases play a crucial role in various cancers and are considered to be promising targets for anticancer drugs. STM2457, the first METTL3 inhibitor, directly binds to the SAM site of the METTL3/METTL14 complex. By reducing the m⁶A enrichment of METTL3-dependent substrates, such as HOXA10 and MYC, STM2457 led to the inhibition of AML growth and induced cellular differentiation and apoptosis [122]. METTL3 inhibitors have been optimized using medicinal chemistry optimization methods based on protein crystallography, and this strategy led to the discovery of the lead compound UZH2, which showed favorable pharmacokinetic properties. In AML MOLM-13 and PC-3 cell lines, UZH2 exerted targeted effects by reducing m⁶A levels in polyadenylated RNAs [123]. An effective and selective METTL3 inhibitor, UZH1a, was discovered by screening using a homogeneous time-resolved fluorescence enzyme inhibition assay. Although UZH1a only slightly inhibited METTL3 expression, it significantly reduced m⁶A levels in AML and osteosarcoma cells [124]. In addition, Eltrombopag selectively inhibited the most active catalytic form of the METTL3/METTL14 complex by directly binding it. Eltrombopag demonstrated antiproliferative effects on relevant AML cell lines, and its effect was accompanied by a decrease in m⁶A levels [125]. Other studies have reported on several METTL3 inhibitors but they showed low specificity; these inhibitors include the general nucleoside analogs sinefungin and S-adenosine homocysteine [126,127].

2.10.2 Targeting m⁶A erasers

FTO is an important target for m⁶A regulation, and rhein was identified as the first cell-based inhibitor of FTO [128]. Additionally, a selective FTO inhibitor, meclofenamic acid (MA), was discovered through high-throughput fluorescence polarization (FP) screening, and MA competes with FTO for binding m⁶A-containing nucleic acids [129]. Another potential FTO inhibitor, FB23-2, showed significantly enhanced antiproliferative activity and inhibited FTO m⁶A demethylase activity by directly binding it, subsequently upregulating the expression of RARA and ASB2, downregulating the expression of MYC and CEBPA, and ultimately attenuating AML in model animals [130]. Dac51 is a novel and effective FTO inhibitor that inhibited glycolysis in multiple tumor cell lines through its effects on the FTO-m⁶A-Jun/Cebpb signaling pathway [131]. Additionally, the tumor metabolite R-2HG has been shown to exhibit significant inhibition of FTO, thereby increasing the global m⁶A levels in R-2HG-sensitive leukemia cells, leading to the degradation of MYC/CEBPA transcripts and impeding the progression of non-IDH mutant AML [132]. Recently, two potent FTO inhibitors, CS1 and CS2, were shown to bind directly to FTO and effectively limit its m⁶A demethylase activity, suggesting their anticancer effects, especially for targeted therapy of AML stem cells [133].

In addition, inhibitors of the m⁶A demethylase ALKBH5 have been identified from a large number of compounds. Two compounds, namely, 2-[(1-hydroxy-2-oxo-2-phenylethyl)sulfanyl]acetic acid and 4-{[(furan-2-yl)methyl]amino}-1,2-diazinane-3,6-dione, inhibited cell proliferation when administered at low micromolar doses to leukemia cell lines [134,135]. Furthermore, through three-dimensional protein array screening, MV1035 was shown to inhibit the activity of the m⁶A demethylase ALKBH5, increase RNA m⁶A levels, and exert potent anticancer effects against glioblastoma cells [136].

2.10.3 Targeting m⁶A readers

Recently, a series of inhibitors targeting RNA m⁶A readers have been discovered. Researchers discovered that the organic selenium compound ebselen inhibited YTHDF protein function by disrupting the interaction between m⁶A-modified mRNA and YTH domains, which reduced the viability of PCa cells [137]. In addition, YTHDF1 is a promising immunotherapeutic target because it weakened antitumor immunity through an m⁶A-p65-CXCL1/CXCR2 axis to promote CRC. Thus, a research team developed a nanoparticle-encapsulated YTHDF1-siRNA delivery system. When administered with addition of anti-programmed death 1 (anti-PD-1) therapy, the YTHDF1-siRNA delivery system showed good efficacy in microsatellite instability-high CRC and overcame anti-PD-1 resistance in microsatellite stable CRC [98]. Additionally, it has been revealed that CpG oligodeoxynucleotides combined with the m⁶A reader YTHDF2 siRNA specifically targeted YTHDF2 in tumor-associated macrophages (TAMs) and reprogrammed TAMs to induce its acquisition of an antitumoral phenotype, which promoted CD8⁺ T cell-mediated antitumor immunity. In combined with an anti-PD-L1 antibody, this treatment significantly inhibited the proliferation and metastasis of mouse CRC cells, demonstrating powerful antitumor effects [138]. The small molecule DC-Y13 was confirmed to be a YTHDF2 inhibitor using a high-throughput screening approach. The compound DC-Y13-27, obtained via structural modification, directly bound to the YTHDF2 protein and inhibited its binding to m⁶A-RNA. In addition, DC-Y13-27 increased the antitumor effect of a combination therapy consisting of ionizing radiation and PD-L1 on mouse CRC cells [139]. IGF2BP1 also functions as a novel pharmacological allosteric target. Cucurbitacin B, a natural active antitumor small-molecule, directly targeted IGF2BP1 at a unique site (Cys253) in the K homology 1-2 domain to block IGF2BP1 recognition of the m⁶A mRNA target c-MYC, thereby inducing apoptosis, blocking PD-L1 expression, and activating antitumor immunity in HCC cells [109]. In addition, CWI 1-2 selectively bound to IGF2BP2 and inhibited its interaction with m⁶A-modified target transcripts such as MYC, GPT2, and SLC1A5, thereby inhibiting the development of AML [140].

2.11 Synergistic effect from inhibitors of m⁶A regulators and immunotherapy or chemotherapy

Targeting m⁶A regulators is an emerging and effective way to inhibit cancer cell proliferation and cancer stem cell self-renewal and improve antitumor immunity. Combined with immune therapy or chemotherapy, some inhibitors of m⁶A regulators, such as STM2457 (targeting METTL3/METTL14), MA (targeting FTO), and Dac51 (targeting FTO), etc., have shown synergistic effects in preclinical models (Tab.2).

STM2457 exhibits a satisfactory therapeutic effect in nonalcoholic fatty liver disease-related HCC by inhibiting METTL3-mediated cholesterol biosynthesis and restoring cytotoxic CD8⁺ T cell function. Combining STM2457 with anti-PD-1 therapy enhances tumor regression and overcomes resistance [141]. Dac51 effectively impaired glycolytic metabolism by inhibiting FTO activity and restored the function of CD8⁺ T cells, resulting in the inhibition of tumor growth. Furthermore, combining Dac51 with anti-PD-L1 blockade therapy achieved significant synergistic effects, leading to enhanced tumor control compared to monotherapy [131]. Targeting the FTO/m⁶A axis by CS1/CS2 suppressed the expression of intrinsic immune checkpoint genes, impaired the immune evasion phenotype, and ultimately increased the sensitivity of AML cells to T cell treatment [133]. The YTHDF2 inhibitor DC-Y13-27 overcame immunosuppressive myeloid-derived suppressor cell-induced immunosuppression and improved combined IR and/or anti-PD-L1 treatment by restraining the IR-YTHDF2-NF-κB circuit. This combined therapeutic strategy not only improved local tumor control but also suppressed distant metastasis [139]. Furthermore, Xiao et al. suggested that MA, an inhibitor of FTO, enhanced the efficiency of temozolomide in suppressing the proliferation of glioma cells by negatively regulating the MYC-miR-155/23a cluster-MXI1 loop [142].

3 m¹A modification

3.1 Overview of m¹A

The N¹-methyladenosine (m¹A) modification was discovered on tRNA and rRNA in eukaryotes. With the advancement of detection technology, the m¹A modification in mammalian mRNA was discovered, further promoting the advancement of epigenetic research. Functionally, the m¹A modification is present mainly in the mRNA 5′UTR and coding sequence (CDS), especially near a start codon, and it regulates mRNA stability and translation [143–146]. m¹A on tRNA and rRNA exerts an effect on ribosome assembly and stability [147–153].

3.2 Regulators of m¹A

3.2.1 Writers

Writers of the m¹A modification on mRNA include TRMT6, TRMT61A, TRMT61B, and TRMT10C. Writers for the m¹A modification on tRNA are TRMT6/61A/61B/10B and TRMT10C/SDR5C1. In addition, RRP8 and TRMT61B as writers have been confirmed to be involved in the rRNA m¹A modification [144,148–152].

3.2.2 Erasers

ALKBH3 is the demethylase of m¹A on mRNA [146], while FTO and ALKBH1/3 are erasers of m¹A in tRNA [144,147–149,154]. The identity of rRNA m¹A demethylase has not yet been confirmed.

3.2.3 Readers

In the past, YTHDF1, YTHDF2, and YTHDF3 were considered to be reader proteins involved in the m⁶A modifications. Researchers have gradually discovered that YTHDF1, YTHDF2, and YTHDF3 are also reader proteins of m¹A in mRNA [145,155], involving in the translation and stabilization of mRNA [156]. No reader protein of m¹A in tRNA and rRNA has been found.

3.3 Regulatory factors that modulate m¹A regulator functions

Although many m¹A regulators have been identified, no regulatory factors that modulate the functions of these regulators have been discovered to date. However, notably, m¹A and m⁶A share some identical regulators, such as FTO and YTHDF2. Whether their regulatory factors are shared is unclear, and the mechanisms of their regulation remain unknown; these aspects of m¹A regulators warrant further exploration.

3.4 m¹A regulators and cancers

An abnormal level of m¹A and the corresponding regulators may directly lead to the progression of tumors. Given that m¹A regulators govern the metabolism of various types of RNA, they play a crucial role in multiple biological processes associated with tumors. Thus, further functional research on m¹A regulators in tumors can meaningfully deepen our understanding of tumor progression.

3.4.1 m¹A writers

Recent research has shown that the level of m¹A is abnormally elevated in liver cancer cells. A study showed that TRMT6/TRMT61A, as an m¹A methyltransferase complex, increases the level of m¹A on some tRNAs to augment the peroxisome proliferator-activated receptor delta translation, inducing the activation of Hedgehog signaling through cholesterol synthesis. This process ultimately promotes self-renewal of liver cancer stem cells and tumorigenesis [149].

3.4.2 m¹A erasers

ALKBH3, an m¹A demethylase, enhances colony-stimulating factor 1 (CSF-1) mRNA stability by demethylating m¹A on CSF-1 mRNA, which increases CSF-1 expression and the invasion of breast and ovarian cancer cells [146]. In addition, ALKBH3 is viewed as a tRNA demethylase that selectively catalyzes the demethylation of m¹A on tRNA, triggering the generation of tDRs, which can enhance ribosome assembly and inhibit cancer cell apoptosis [147].

3.4.3 m¹A readers

Notably, YTHDF3 binds directly to m¹A-methylated transcripts, such as insulin-like growth factor 1 receptor (IGF1R), promoting the degradation of IGF1R mRNA. This biological process strongly affects the downstream matrix metallopeptidase 9 signaling pathway, thereby diminishing trophoblast migration and invasion [155].

In general, the m¹A modification exhibits a strong association with the cancer progression, and regulators of m¹A may be potential targets for cancer therapy.

3.5 Targeting m¹A regulators for anticancer therapy

Considering the clear association between m¹A modification regulator activity and tumor progression, the development of inhibitors targeting m¹A regulators is important. In fact, inhibitors that target m¹A regulators have been identified. For example, Thiram has been shown to play an effective therapeutic role by antagonizing the activity of the TRMT6/TRMT61A complex and reducing the level of m¹A marks in HCC cells, effectively attenuating the proliferation of liver cancer cells [149].

4 m⁵C modification

4.1 Overview of m⁵C

In recent years, the 5-methylcytosine (m⁵C) RNA modification has been shown to play a vital role in regulating the function of RNA, particularly tRNA and rRNA but also mRNA [157]. Specifically, m⁵C controls tRNA self-stability and translation efficiency [158,159]. On rRNA, the m⁵C mark modulates the maturation of rRNA and protein synthesis [153,160–162]. Moreover, the m⁵C RNA modification is present mainly on GC-rich sequences, the 3′UTR, and near start codons, regulating mRNA stability and translation [163–166].

4.2 Regulators of m⁵C

4.2.1 Writers

NSUN1–6, members of the NOL1/NOP2/SUN (NSUN) domain protein family, and the DNA methyltransferase (DNMT) homolog DNMT2 are viewed as writers of m⁵C RNA modification in RNA, coregulating various RNA stabilities, translation, and ribosome assembly [160,162,163,166–170].

4.2.2 Erasers

TET1 is the eraser of mRNA m⁵C [171]. TET2 and ALKBH1 exert their demethylation effects on tRNA m⁵C, promoting translation efficiency [172,173]. The rRNA m⁵C demethylase has not yet been found.

4.2.3 Readers

In recent years, a series of readers of the m⁵C modification have been identified; they include YBX1, ALYREF, RAD52, LIN28B, FMRP, and YTHDF2. These proteins modulate RNA stability, synthesis, maturation, and translation [161,165,167,174–176].

4.3 Regulatory factors that modulate m⁵C regulator functions

NSUN2, the writer protein of m⁵C, has been reported to be modulated by SUMOylation. Specifically, the SUMOylation of NSUN2 modified by small ubiquitin-like modifier (SUMO)-2/3 makes NSUN2 more stable and increases m⁵C mark abundance on oncogene mRNAs, subsequently promoting gastric cancer progression [177] (Fig.6).

4.4 m⁵C regulators and cancers

Increasing evidence indicates that the alterations of the regulators-mediated m⁵C level are involved in the progression of various cancers. In-depth research into the functionality of each m⁵C regulator provides novel insights into the role of RNA m⁵C in tumor progression.

4.4.1 m⁵C writers

NSUN2, a writer of m⁵C, enhances the stability of H19 lncRNA, which promotes the development of HCC [178]. Further study demonstrated that NSUN2 induced the instability of the p57^Kip2 mRNA in an m⁵C-dependent manner, promoting gastric cancer cell proliferation [179]. Recent studies have also shown that NSUN2, mediated by LIN28B, stabilizes the mRNA of growth factor receptor-bound protein 2 through m⁵C modification, inducing the activation of the PI3K/AKT and ERK/MAPK signaling pathways. This process promotes the tumorigenesis and progression of ESCC [165].

4.4.2 m⁵C erasers

FMRP binds to m⁵C-modified mRNA, which induces TET1 to remove the m⁵C mark from mRNA. This process augments mRNA-dependent repair and cancer cell survival [171].

4.4.3 m⁵C readers

ALYREF, an m⁵C reader, promotes the proliferation of bladder cancer cells induced by PKM2-mediated glycolysis [174]. Moreover, YBX1 and NSUN2 have been demonstrated to maintain the stability of their target mRNAs, facilitating the proliferation and migration of bladder urothelial carcinoma cells [167].

In general, the aberrant expression of m⁵C regulators is closely related to tumorigenesis and cancer progression in humans because it modulates the m⁵C modification level on RNA. These regulators of m⁵C are potential therapeutic targets for cancers.

4.5 Targeting m⁵C regulators for anticancer therapy

Some proof-of-concept studies have indicated that small-molecule inhibitors targeting m⁵C regulators show potential for cancer therapy. However, to date, specific m⁵C inhibitors have not yet been developed [180]. Therefore, research on specific drug targets for m⁵C regulators remains a relatively novel field. Considering the important association between m⁵C regulators and tumor progression, the development of these inhibitors is worth further exploration.

5 m⁷G modification

5.1 Overview of m⁷G

The N⁷-methylguanosine (m⁷G) modification is one of the most prevalent RNA modifications, and the marks are located mainly on the rRNA, tRNA, and 5′ cap structure of mRNA [181–184]. It plays important roles in regulating RNA processing, metabolism and function [183]. Recent reports indicate that the m⁷G modification is not limited to the 5′ cap of mRNA but also within an mRNA sequence. The proportion of m⁷G/G marks on internal mRNA sequences ranges from 0.01% to 0.03%, depending on the cell line [185].

5.2 Regulators of m⁷G

5.2.1 Writers

METTL1/WDR4 and RNMT/RAM are writers of the m⁷G modification of mRNA, affecting corresponding RNA export, stability, and translation efficiency [186–188]. In addition, METTL1/WDR4 regulates the m⁷G modification of tRNA, facilitating translation efficiency [189–192]. The WBSCR22/TRMT112 methyltransferase complex is critical for the m⁷G modification of 18S rRNA, which promotes biogenesis [153,193,194].

5.2.2 Erasers

Notably, no eraser of m⁷G marks has been identified thus far, which means there is still much work to be done.

5.2.3 Readers

Some early reports have indicated that the cap binding complex, eukaryotic translation initiation factor 4E (eIF4E), and Ago2 are readers of m⁷G in the 5′ cap structure of mRNA, modulating the translation of mRNA [195,196]. Notably, Ago2 competes with eIF4E by binding to the m⁷G cap of mRNA targets, subsequently impeding the translation of mRNA [196]. In addition, a recent report indicated that Quaking proteins (QKIs) were the first reader proteins of internal mRNA sequences and selectively recognized m⁷G marks of internal mRNA [185].

5.3 Regulatory factors that modulate m⁷G regulator functions

Early reports claimed that the promyelocytic leukemia (PML) protein was the first example of an eIF4E regulatory subunit. PML directly interacted with eIF4E to attenuate the affinity of eIF4E for the m⁷G cap, and the translation of cyclin D1 mRNA was significantly dampened, which suppressed oncogenic transformation [197] (Fig.6).

It has been reported that RNMT, a writer protein of m⁷G in mRNA, is modulated by phosphorylation. CDK1-cyclin B1 phosphorylated the regulatory domain T77 of RNMT. Interestingly, the phosphorylation of RNMT inhibited the interaction of RNMT with the RNMT inhibitor KPNA2, increasing its m⁷G-cap methyltransferase activity and thereby increasing the mRNA transcription level. In addition, inhibition of T77 phosphorylation reduced the proliferation rate of HeLa cells [198] (Fig.6).

In addition, it has been discovered that the phosphorylation of eIF4E enhances its binding ability to the m⁷G cap in many oncogene mRNAs, subsequently facilitating their translation and inducing PCa progression [199] (Fig.6).

5.4 m⁷G regulators and cancers

There is increasing evidence suggesting that certain tumors are closely associated with m⁷G marks. The aberrant expression of m⁷G regulators leads to the activation of tumor-related genes through the m⁷G modification of RNA. In particular, the recent discovery of novel m⁷G reader proteins has provided new insights into the role of the m⁷G modification in tumor progression [185].

5.4.1 m⁷G writers

METTL1/WDR4-mediated tRNA m⁷G modification enhanced the translation of certain oncogenic mRNAs in lung cancer cells, HCC, and bladder cancer. Gene Ontology and pathway analyses have indicated that these types of mRNAs are enriched in cell cycle regulation and cancer pathways [189,190,200]. Increased tRNA m⁷G level mediated by METTL1/WDR4 promoted the translation of the transcripts of genes related to the phosphatidylinositol-3-kinase/protein kinase B/mammalian target of rapamycin (PI3K/AKT/mTOR) signaling pathway, thereby facilitating the progression of HNSCC [191]. In addition, METTL1 increased m⁷G tRNA modification, which led to enhanced codon recognition during mRNA translation, therefore facilitating the translation efficiencies of mRNAs with higher m⁷G-marked codons. This process promoted WNT/β-catenin signaling pathway activation and thus facilitated the nasopharyngeal carcinoma cell epithelial-to-mesenchymal transition (EMT) [192]. In general, METTL1/WDR4, writers of the m⁷G modification, regulate tumor-associated gene expression, leading to the progression of certain cancers.

5.4.2 m⁷G erasers

The m⁷G erasers are not still reported to date. The m⁷G erasers and their roles in cancer are valuable to be explored in the future.

5.4.3 m⁷G readers

QKIs, novel readers of internal mRNA sequence m⁷G marks, have only recently been discovered. Specifically, QKI7, a QKI isoform, interacts with the stress granule (SG) core protein G3BP1 and traffics transcripts modified with m⁷G marks into SGs to modulate mRNA stability and translation under stress conditions. Further research has revealed that QKI7 can dampen the translation efficiency of mRNAs via Hippo signaling pathways, sensitizing cancer cells to chemotherapy [185].

Indeed, the functions of other regulators of m⁷G and their underlying molecular mechanisms are worthy of further study.

5.5 Targeting m⁷G regulators for anticancer therapy

Since m⁷G marks are ubiquitous on RNA and have been demonstrated to have close associations with tumorigenesis, the development of inhibitors targeting m⁷G regulators may be very beneficial in impeding tumor progression. For example, eIF4E exhibits carcinogenic properties that are directly related to its ability to bind to the m⁷G cap of mRNA. Researchers have found that ribavirin, which specifically binds to eIF4E via physical mimicry of an m⁷G-marked cap, can antagonize the transport and translation functions of eIF4E-sensitive mRNAs, reduce the expression level of oncogenes such as cyclin D1, and play an antitumor role [201].

6 m⁶A_m modification

6.1 Overview of m⁶A_m

The N⁶,2′-O-dimethyladenosine (m⁶A_m) modification is deposited on the first nucleotide downstream of an m⁷G-marked cap of mRNA. Specifically, this nucleotide is likely to be methylated at the 2′-hydroxyl group of most mRNA. When the first nucleotide is 2′-O-methyladenosine, it can be further methylated at the N⁶-position to generate the m⁶A_m mark [202]. Notably, the m⁶A_m modification is found on small nuclear RNA (snRNA) [203]. Functionally, the m⁶A_m modification has been proven to modulate snRNA biogenesis, mRNA stability, and translation [203–207]. The findings on the roles of this mark in translation promotion or repression and transcript stabilization profoundly vary, and further studies are needed to elucidate the biological function of m⁶A_m of the mRNA cap.

6.2 Regulators of m⁶A_m

6.2.1 Writer

Phosphorylated CTD interacting factor 1 (PCIF1) has been identified as the mRNA m⁶A_m methyltransferase; it functions in an m⁷G cap-dependent manner to modulate mRNA stability and translation [204–207].

6.2.2 Eraser

FTO is believed to be a demethylase involved in the m⁶A modification. However, as research on FTO has been advanced, FTO has been increasingly viewed as the demethylase of m⁶A_m of mammalian mRNA, exerting its demethylating effects in the cytoplasm [208]. In addition, FTO exerts its demethylating effect selectively during snRNA biogenesis [203].

6.2.3 Reader

Recently, it has been reported that the RNA binding protein KHSRP is a novel potential m⁶A_m reader in mRNA and is related to RNA degradation [209]. Further experiments are needed to validate KHSRP as a potential reader of m⁶A_m.

6.3 Regulatory factors that modulate m⁶A_m regulator functions

Given that research on the functions of m⁶A_m regulators is still in the early stages, no relevant regulatory factor of m⁶A_m regulators has yet been discovered.

6.4 m⁶A_m regulators and cancers

The roles of m⁶A_m in carcinogenesis and cancer progression has been recently proposed. Several studies have reported that regulators of m⁶A_m are associated with cancers in an m⁶A_m-dependent manner, but research in this area performed to date is limited in scope.

6.4.1 m⁶A_m writer

TM9SF1 functions as a tumor suppressor gene in gastric cancer, and it has been reported that PCIF1, a writer of m⁶A_m, suppresses the translation of TM9SF1 through the m⁶A_m modification, leading to the proliferation and invasion of gastric cancer cells [210]. Recent studies have also shown that overexpression of PCIF1 inhibits cell cycle progression and promotes apoptosis in glioma cells [211]. PCIF1 also stabilizes the mRNA of FOS in an m⁶A_m-dependent manner and promotes the progression of CRC through FOS-dependent TGF-β regulation [212].

6.4.2 m⁶A_m eraser

FTO, acting as the demethylase of m⁶A_m, represses cancer stem cell properties including sphere formation, in vivo tumorigenesis and chemoresistance in CRC, through its m⁶A_m demethylase activity, which might affect relative mRNA translation efficiency [208].

6.4.3 m⁶A_m reader

KHSRP, a novel potential m⁶A_m reader, recognizes both m⁶A marked on enhancer RNA (eRNA) and m⁶A_m marked on the 5′UTR of related mRNA to inhibit RNA degradation by XRN2, enhancing the proliferation and radiotherapy resistance sensitization of bone metastatic PCa cells [209].

As in the cases mentioned above, the functions and mechanisms of m⁶A_m regulators in tumors have not been fully explained, and their roles remain unclear, deserving of further research.

6.5 Targeting m⁶A_m regulators for anticancer therapy

The preceding reports have demonstrated that PCIF1, functioning as an m⁶A_m methyltransferase, is significantly associated with carcinogenesis and cancer progression. Thus, lipid nanoparticles and chemically modified small interfering RNAs were used to silence PCIF1 in vivo, and these PCIF1 inhibitors combined with anti-PD-1 treatment showed efficacy in the treatment of CRC [212].

7 ac⁴C modification

7.1 Overview of ac⁴C

N⁴-acetylcytidine (ac⁴C) is a highly conserved RNA modification found on tRNA and rRNA and has recently been studied on eukaryotic mRNA, where it is enriched mainly in CDS regions, with small amounts in 5′UTR and 3′UTR regions. Notably, a certain portion of ac⁴C marks is present on polyadenylated (poly(A)) RNAs [213]. Functionally, the mammalian mRNA ac⁴C modification, especially that of coding sequences, promotes mRNA translation and stability [213].

7.2 Regulator of ac⁴C

NAT10 is the only known regulator of the ac⁴C modification. Specifically, the ac⁴C modification is regulated by the NAT10 monoenzyme system, which has both acetylation catalytic function and RNA binding activity. Nevertheless, human NAT10 cannot function in the absence of THUMPD1. Moreover, whether the same mechanism underlies the action of NAT10 on mRNA is unknown, and the identification of potential deacetylases deserves further exploration [214].

7.3 Regulatory factors that modulate ac⁴C regulator functions

Recent studies have revealed that the 2-hydroxyisobutyrylation (Khib) modification of lysine 823 in NAT10 enhanced its interaction with the deubiquitinase USP39, thereby increasing the stability of the NAT10 protein. NAT10, in turn, increased the stability of NOTCH3 mRNA in an ac⁴C-dependent manner to promote metastasis of ESCC [8] (Fig.6).

7.4 ac⁴C regulator and cancers

The relationship between the ac⁴C modification and cancers is an area of ongoing research, and dysregulation of the ac⁴C regulator has been implicated in various types of cancer. Several studies have shown that the ac⁴C modification pattern is different between cancer cells and normal cells. For example, NAT10 promoted the mRNA stability of ferroptosis suppressor protein 1 through ac⁴C acetylation in colon cancer cells, resulting in the proliferation and metastasis of these cells [215]. In addition, NAT10 directed the ac⁴C modification of the COL5A1 mRNA 3′UTR, which maintained its stability without affecting mRNA translation efficiency. Previous studies have revealed that COL5A1 is a marker of EMTII in tumor cells, directly promoting the EMT and metastasis in gastric cancer [216]. Further research indicated that NAT10 stabilized MDM2 mRNA through ac⁴C acetylation, resulting in gastric cancer progression [217]. In addition, NAT10 mediates the ac⁴C modification on a series of mRNAs, such as BCL9L, SOX4, and AKT1, increasing their translation efficiency and stability in bladder urothelial carcinoma cells. These target mRNAs are probably involved in facilitating the bladder urothelial carcinoma progression [218]. Recent research findings have revealed that NAT10 mediated the ac⁴C modification of SEPT9 mRNA. This modification, in turn, activated the HIF-1 pathway and metabolic reprogramming of glucose, inducing glycolytic addiction in gastric cancer cells. This process enabled these cells to withstand the hypoxic microenvironment induced by antiangiogenic therapy. Therefore, combining antiangiogenic agents with ac⁴C inhibitors may be a novel therapeutic approach to gastric cancer [219]. In conclusion, NAT10-mediated ac⁴C modification is closely associated with tumor progression and metastasis and might be a potential and effective therapeutic target for certain cancers.

7.5 Targeting the ac⁴C regulator for anticancer therapy

As previous reports have confirmed the association of NAT10 with tumor progression in an ac⁴C-dependent manner, the development of inhibitors targeting NAT10 may be highly beneficial to tumor therapy. Small-molecule compounds #7586-3507 showed significant binding affinity for NAT10 and hindered the Khib modification of NAT10. The stabilization of NAT10 was reduced, thereby reducing the level of ac⁴C in NOTCH3 mRNA in ESCC cells and exerting anticancer biological effects [8].

We summarized the latest findings on other RNA modifications, including m¹A, m⁵C, m⁷G, m⁶A_m, and ac⁴C, even though some regulators associated with these modifications remain undiscovered (Tab.3). We emphasized that regulatory subunits and PTMs play roles in modulating the biological functions of regulators in these RNA modifications, which are also associated with carcinogenesis and cancer progression (Fig.6). Additionally, relevant inhibitors that target regulators of m¹A, m⁷G, m⁶A_m, and ac⁴C have been discovered (Tab.4). In general, there remains a lack of comprehensive and systematic researches on RNA modifications other than m⁶A, especially regulatory subunits and PTMs of their associated regulators; thus, further exploration is worthwhile.

8 Future prospects

Different RNA modifications and regulators responsible for their deposition, removal, and recognition play special roles in cancer in different contexts. Considering some shared regulators of different RNA modifications, it will be interesting to investigate the comprehensive interactions of RNA modifications in the context of tumorigenesis and tumor progression. Nevertheless, to date, no related publications have discussed the collaborative or competitive effects of multiple RNA modifications in cancer.

Inhibitors that target RNA modification regulators have shown potential in cancer therapy and provide a basis for personalized medicine approaches. Notably, a phase 1, multicenter, open-label, first-in-human clinical study is being performed with STC-15, an orally bioavailable small molecule inhibiting METTL3, to assess its safety and tolerability in patients with advanced malignancies (NCT05584111). Clinical evidence that supports the selection of cancer patients for RNA modification-targeting drug administration is lacking; however, clinical and genetic biomarkers, such as the regulators of RNA modifications, must be considered to select optimal patients and assess treatment response.

9 Conclusions

Regarding the study of RNA modifications, especially m⁶A, researchers initially focused on the mechanisms of downstream target genes modulated by regulators. However, the regulatory factors that modulate the functions of RNA modification regulators have received increasing attention. Nevertheless, studies are still in the early stage, and in-depth and extensive research is required.

In this review, we summarized that writers, erasers, and readers of RNA modification can be modulated by a series of factors, including regulatory subunits (proteins, ncRNAs or peptides encoded by lncRNAs) and PTMs (acetylation, SUMOylation, lactylation, phosphorylation, etc.), which is also closely related to carcinogenesis and cancer progression. In addition, inhibitors that target RNA modification regulators for anticancer therapy and their synergistic effect combined with immunotherapy or chemotherapy have been summarized.

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