2.1 m⁶A writers

m⁶A modification of mRNA is catalyzed by a multicomponent writer complex in a highly specific manner (Figure 1), including methyltransferase-like 3 (METTL3), METTL14, Willms tumor 1 associated protein (WTAP), RNA-binding motif protein 15 (RBM15) and its paralogue RBM15B, Vir like m⁶A methyltransferase associated (VIRMA), zinc finger CCCH domain-containing protein 13 (ZC3H13), METTL16, zinc finger CCHC domain-containing protein 4 (ZCCHC4) and METTL5 [33-35]. Mechanistically, METTL3 and METTL14 form a stable heterodimeric core complex. METTL3 is the catalytically active subunit, while METTL14 plays a structural role critical for binding the target RNA [8, 36, 37]. WTAP is critical for the recruitment of the m⁶A methyltransferase complex to mRNA targets and is considered as a regulatory subunit that stabilizes the core complex [9]. RBM15 and its paralogue RBM15B can bind the m⁶A methylation complex and recruit it to specific sites, resulting in the methylation of adenosine nucleotides in m⁶A consensus motifs [38]. VIRMA recruits other methyltransferases to guide regioselective methylation and mediates preferential mRNA methylation in 3' UTRs and near stop codons [39]. ZC3H13 is an essential component for the translocation of WTAP, Virilizer and Hakai to the cytoplasm, which drives ZC3H13-WTAP-Virilizer-Hakai complex formation [40]. METTL16 contains a methyltransferase domain furnished with an extra N-terminal module, which forms a deep groove that is vital for RNA binding. METTL16 independently catalyzes m⁶A modification of U6 small nuclear RNA (snRNA) and a small number of mRNAs and non-coding RNAs [41-44]. In addition, ZCCHC4 plays a vital role in modifying human 28S rRNA and also interacts with a subset of mRNAs [45]. METTL5 is a recently reported m⁶A RNA methyltransferase with a significantly different RNA-binding mode from others. It catalyzes m⁶A modification of the 18S rRNA [46]. In addition to their well-known role in m⁶A RNA methylation, recent studies have shown that m⁶A methyltransferases have methyltransferase-independent functions [47, 48]. For instance, METTL3 and METTL16 have been shown to promote translation in the cytosol, independent of their methyltransferase activity [49, 50]. Similarly, METTL16 has been found to be involved in regulating DNA damage, regardless of their methyltransferase activity [51]. These findings highlight the complexity of m⁶A regulation and suggest that there may be additional, as yet undiscovered, functions of m⁶A methyltransferases beyond their traditional role in RNA methylation.

2.2 m⁶A erasers

m⁶A demethylases can remove m⁶A methylation and affect the function of m⁶A modification in a dynamic, rapid, and signal-dependent manner (Figure 1) [7]. Unlike the m⁶A methyltransferase and m⁶A-binding protein groups, only FTO and ALKBH5 have been identified as m⁶A demethylases. FTO, the first demethylase discovered, has efficient oxidative demethylation activity. It works with methyltransferase components to determine the modification status of mRNAs [52, 53]. FTO-mediated RNA m⁶A demethylation is not specific. It also occurs with the cap 2-O-dimethyl adenosine (m⁶A_m) modification of mRNAs, internal m⁶A modification of U6 RNA, internal and cap m⁶A_m modification of snRNAs, and N¹-methyladenosine (m¹A) modification of tRNAs [54]. FTO can influence mRNA processing mediated by m⁶A through binding the intronic regions of pre-mRNAs in the proximity of alternatively spliced exons and poly(A) sites [52]. ALKBH5, the second demethylase to be discovered, has profound effects on mRNA export and the assembly of mRNA processing factors in nuclear speckles [11]. Another study revealed that ALKBH5-directed m⁶A demethylation is critical for correct splicing and the production of longer 3'-UTR mRNAs [55].

2.3 m⁶A readers

The recruitment of m⁶A-specific binding proteins is the main mechanism by which m⁶A modification affects the fate of RNAs. m⁶A “readers” can recognize and bind m⁶A sites, thereby mediating a series of biological changes, such as RNA degradation, processing, splicing and translation (Figure 1) [56]. YT521-B homology (YTH) RNA-binding domain is highly conserved and has an exquisite pocket for specific recognition of the m⁶A [57]. YTH domain-containing protein 1 (YTHDC1) as a nuclear m⁶A reader can influence mRNA splicing and nuclear export [13, 14]. YTHDC2 plays critical roles during spermatogenesis by accelerating both translation and decay of mRNA [24]. YTH domain-containing family proteins (YTHDFs) accelerate the metabolism of m⁶A-modified mRNAs in the cytoplasm. YTHDF1 increases the translation efficiency of m⁶A-modified mRNAs, and YTHDF2 selectively recognizes m⁶A-modified mRNAs that promote their degradation [15, 16]. YTHDF3 promotes protein production through its interaction with YTHDF1 and affects methylated mRNA decay mediated through YTHDF2 [58]. Studies have shown that YTHDFs contain a high degree of functional redundancy. This functional redundancy is important for allowing YTHDFs to recognize and bind to a wide variety of m⁶A-modified RNAs [59]. In addition, insulin-like growth factor 2 mRNA-binding proteins 1, 2 and 3 (IGF2BP1/2/3), as a family of m⁶A readers, can recognize thousands of mRNA transcripts by identifying consistent GG(m⁶A)C sequences [60]. IGF2BP1/2/3 can promote stability and facilitate translation efficiency in an m⁶A-dependent manner [60-63]. Heterogeneous nuclear ribonucleoproteins (HNRNPs) are abundant nuclear proteins that alter the secondary structure of m⁶A-modified RNAs. To promote primary miRNA processing, HNRNPA2B1 directly binds a set of nuclear transcripts and interacts with the microprocessor complex protein DiGeorge syndrome critical region 8 (DGCR8) [17]. Importantly, HNRNPC and HNRNPG can affect the abundance and alternative splicing of target mRNAs [18, 64]. Furthermore, eukaryotic initiation factor 3 (eIF3) binds m⁶A sites in the 5’ UTRs of mRNAs and promotes their cap-dependent translation [65].

3.1 m⁶A methylation and cancer metabolic reprogramming

Tumorigenesis and progression are closely linked to the metabolic reprogramming of cancer cells. The reprogramming of energy metabolism promotes rapid cell growth, survival, proliferation, and long-term maintenance, and is considered a hallmark of tumor cells [66, 67].

Unlike normal differentiated cells, most cancer cells undergo metabolic reprogramming to accelerate aerobic glycolysis for cellular processes, even under non-hypoxic conditions. This unique metabolic property is known as the “Warburg effect” [68, 69]. Increasing evidence suggests that m⁶A methylation can regulate a number of key metabolic enzymes, thereby influencing the Warburg effect (Figure 2 and Table 1) [70]. The initial and crucial step of glucose metabolism is catalyzed by hexokinase 2 (HK2). In colorectal cancer (CRC), METTL3 has been observed to stabilize the expression of HK2 and glucose transporter type 1 (GLUT1) through an m⁶A-IGF2BP2/3-dependent mechanism, thereby activating the subsequent glycolysis pathway and regulating tumor growth [71]. In cervical cancer, METTL3 has been found to target the 3’-UTR of HK2 mRNA and recruit the m⁶A reader YTHDF1, which enhances the stability of HK2, ultimately promoting the Warburg effect [72]. Meanwhile, WTAP facilitates the Warburg effect of gastric cancer (GC) by enhancing the stability of HK2 mRNA [73]. Furthermore, METTL3 has been found to induce m⁶A modification of hepatoma-derived growth factor (HDGF) mRNA. The m⁶A reader IGF2BP3 recognizes the m⁶A site on HDGF mRNA, thereby enhancing the stability of HDGF mRNA. This activation, in turn, promotes glycolysis and tumor angiogenesis in GC by stimulating GLUT4 and enolase 2 (ENO2) expression [74]. Furthermore, METTL3 could increase the m⁶A level of APC mRNA and the m⁶A reader YTHDF2 to promote APC mRNA degradation in esophageal squamous cell carcinoma (ESCC) cells. Reduced APC increases the expression of pyruvate kinase M2 (PKM2), thereby leading to enhanced aerobic glycolysis [75]. In addition to influencing these key metabolic enzymes in glycolysis, m⁶A modifications can also regulate several proteins and non-coding RNAs related to the glycolysis involved in tumor progression. In CRC, IGF2BP2 as an m⁶A reader stabilizes long non-coding RNA (lncRNA) zinc finger antisense 1 (ZFAS1) [76]. Then ZFAS1 directly binds to obg-like ATPase 1 (OLA1) to enhance the ATPase activity of OLA1, which enhances the ATP hydrolysis capacity and activates the Warburg effect [76]. In GC cells, it has been demonstrated that METTL3 promotes the methylation of NADH:ubiquinone oxidoreductase subunit A4 (NDUFA4) mRNA, which is stabilized by IGF2BP1 [77]. This leads to increased NDUFA4 expression, promoting GC development by enhancing glycolysis and mitochondrial fission [77]. However, it is worth noting that m⁶A methylation can also suppress aerobic glycolysis. In pancreatic ductal adenocarcinoma, the m⁶A reader YTHDC1 facilitates the maturation of miR-30d through m⁶A-mediated regulation of mRNA stability [78]. Subsequently, miR-30d induces the downregulation of SLC2A1 and HK1 by directly targeting runt-related transcription factor 1 (RUNX1), which ultimately inhibits the Warburg effect [78]. Numerous studies have demonstrated that the tumor glucose aerobic glycolytic pathway holds great potential as a target for tumor therapy [79, 80]. Additionally, it has been revealed that m⁶A methylation plays a significant role in the process of tumor metabolic reprogramming [76, 81]. Therefore, targeting m⁶A-related regulators may be a promising approach to regulate metabolic reprogramming in the future.

In addition to glucose metabolism, fatty acid oxidation is an extremely relevant energy source for tumor cells [82]. Recently, reports have indicated that m⁶A modifications regulate lipid metabolism to support tumor progression [83] (Figure 2 and Table 1). In ESCC, ATP citrate lyase (ACLY) and acetyl-CoA carboxylase 1 (ACC1), the de novo fatty acid synthetic enzymes, can be upregulated by HNRNPA2B1, thus accelerating cellular lipid accumulation, which contributes to tumor growth and metastasis [83]. In glioblastoma, YTHDF2 depletion decreases cellular cholesterol levels and inhibits cell proliferation, invasion, and tumorigenesis [84]. This is because YTHDF2 can mediate m⁶A-dependent mRNA decay to restrain liver X receptor alpha (LXRA) and human immunodeficiency virus type I enhancer binding protein 2 (HIVEP2) expression [84]. Moreover, in hepatocellular carcinoma (HCC), METTL5 has been shown to promote both de novo lipogenesis and β-oxidation, which contribute to cancer growth and progression [85]. The mechanism behind this involves the depletion of METTL5-mediated 18S rRNA m⁶A modification, which impairs 80S ribosome assembly and decreases the translation of acyl-CoA synthetase (ACSL) family mRNAs in HCC cells [85]. The aforementioned discoveries highlight the crucial connections between m⁶A modification and fatty acid metabolism, underscoring their fundamental physiological roles in tumorigenesis. Hence, disrupting fatty acid metabolism that depends on m⁶A methylation could provide novel approaches for anti-tumor therapies.

3.2 m⁶A methylation and cancer programmed cell death

Programmed cell death, which encompasses apoptosis, autophagy, pyroptosis, necroptosis, and ferroptosis, has been found to play a critical role in tumorigenesis, the tumor microenvironment, and cancer treatment [86]. In recent years, several studies have linked m⁶A modification to various programmed cell death processes (Figure 3 and Table 1) [87]. For example, in myeloid leukemia cell lines, depletion of METTL3 inhibits the translation of c-MYC, B-cell lymphoma 2 (BCL-2) and phosphatase and tensin homolog (PTEN) mRNAs, which markedly induces cell differentiation and increases levels of apoptosis [88]. However, IGF2BP3 overexpression can have the opposite effect, dramatically suppressing apoptosis and promoting the proliferation and tumorigenesis of acute myeloid leukemia (AML) by stabilizing the regulator of chromosome condensation 2 (RCC2) mRNA in an m⁶A-dependent manner [89]. Dysregulation of METTL3 can also affect the apoptosis and proliferation phenotype of lung adenocarcinoma cells. Specifically, METTL3 contributes to the m⁶A modification of the coding region of F-box and WD repeat domain-containing 7 (FBXW7) mRNA, which enhances FBXW7 translation and decreases the protein levels of Mcl-1 and c-Myc, ultimately leading to decreased proliferation and increased apoptosis [90]. Interestingly, in MYC-driven breast cancer, YTHDF2 can stabilize several mRNAs in mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK/ERK) signaling pathways and cause endoplasmic reticulum stress through unfolded protein accumulation, contributing to an apoptotic phenotype [91].

Autophagy is a highly conserved catabolic process that involves salvaging and reusing degraded proteins, lipids, and organelles through the lysosomal degradation pathway [92]. Recent studies have linked m⁶A-induced autophagy to increased tumor cell migration and invasion. Under hypoxic conditions, hypoxia-inducible factor 1 alpha (HIF-1α) can upregulate the YTHDF1 transcription by directly binding to its promoter region. YTHDF1 can promote the translation of autophagy related 2A (ATG2A) and ATG14, which facilitates autophagy and autophagy-related malignancy in HCC [93]. In CRC, during glutaminolysis inhibition, upregulation of FTO stabilizes activating transcription factor 4 (ATF4) mRNA by reducing its m⁶A modification [94]. This results in the upregulation of DNA damage-inducible transcript 4 (DDIT4) expression, which in turn leads to the inactivation of mammalian target of rapamycin (mTOR) signaling and induces a pro-survival autophagy response [94]. On the contrary, another study has reported that m⁶A-induced autophagy has a negative relationship with tumorigenesis. In oral squamous cell carcinoma cells, knockdown of FTO can enhance autophagic flux and inhibit malignant progression by promoting eukaryotic initiation factor 4 gamma 1 (eIF4G1) mRNA degradation in an m⁶A-YTHDF2-dependent manner [95].

Ferroptosis is a novel form of non-apoptotic cell death that is driven by iron-dependent lipid peroxidation [96]. Researches have shown that m⁶A modification is closely associated with ferroptosis. For example, in breast cancer, fibroblast growth factor receptor 4 (FGFR4) accelerates cystine uptake and Fe2⁺ efflux via the β-catenin/transcription factor 4 (TCF4)-SLC7A11/ferroportin 1 (FPN1) axis, which confers anti-human epidermal growth factor receptor 2 (HER2) resistance in part by suppressing ferroptosis. Knockdown of METTL14 promotes FGFR4 mRNA stability and upregulates its expression [97]. In hepatoblastoma, METTL3-mediated m⁶A modification enhances SLC7A11 stability and expression in an m⁶A-IGF2BP1-dependent manner [98]. SLC7A11 promotes tumorigenesis by enhancing ferroptosis resistance [98]. In addition, FTO inhibits the development of prevents papillary thyroid carcinoma by downregulating the expression of SLC7A11 through ferroptosis [99].

Despite several studies showing that m⁶A modification is critical to programmed cell death, its potential molecular mechanisms in cancer have not been fully established. Therefore, deciphering the m⁶A and programmed cell death signaling pathways in various cancers is imperative, as it not only provides new insights into the pathogenesis but also helps in the development of new targeted anticancer therapeutic strategies.

3.3 m⁶A methylation and cancer metastasis

Metastasis is the leading cause of poor prognosis in cancer patients [100]. Recent studies have demonstrated that m⁶A methylation is associated with tumor invasion and metastasis by regulating epithelial-mesenchymal transition (EMT) states [101] and angiogenesis (Table 1) [102].

EMT is a crucial developmental process wherein cells lose epithelial polarization and gain mesenchymal features and is closely related to tumor metastasis [103]. For example, in head and neck squamous cell carcinoma, EMT enhances cell motility and invasiveness, promoting the lymphatic metastatic process by regulating snail family zinc finger 2 (Snai2) mRNA stability, a key EMT-related transcription factor, in an m⁶A modification-dependent manner [104]. Additionally, YTHDC1 promotes the nuclear export of methylated SMAD family member 3 (SMAD3) mRNA, affecting its protein production and leading to enhanced EMT and promoting lung metastasis of triple-negative breast cancer (TNBC) cells [105]. In GC, METTL3 facilitates the EMT process and metastasis by enhancing the stability of zinc finger MYM type-containing 1 (ZMYM1) via the m⁶A reader human antigen R (HuR), which mediates the repression of E-cadherin by recruiting the C-terminal binding protein (CtBP)/ lysine-specific demethylase 1 (LSD1)/co-repressor for element-1-silencing transcription factor (CoREST) complex [106]. Conversely, in non-small cell lung cancer (NSCLC), the m⁶A demethylase ALKBH5 can reduce YTHDF-mediated YAP expression and inhibit miR-107/ large tumor suppressor kinase 2 (LATS2)-mediated YAP activity, thereby inhibiting tumor growth and metastasis [107]. Overexpression of METTL14 markedly inhibits the EMT process and CRC cell metastasis by repressing SRY-box transcription factor 4 (SOX4) expression through YTHDF2-dependent mRNA degradation. Furthermore, it can elevate the expression of N-cadherin and Vimentin, while reducing E-cadherin expression levels [108].

Angiogenesis plays a prominent role in the development and metastasis of tumors. In patients who have breast cancer with brain metastasis, YTHDF3 promotes cancer cell interactions with brain endothelial cells and astrocytes, blood-brain barrier extravasation, angiogenesis, and outgrowth [109]. Mechanistically, YTHDF3 mediates multiple steps of brain metastasis, primarily by enhancing the translation of m⁶A-enriched transcripts for ST6 N-acetylgalactosaminide alpha-2,6-sialyltransferase 5 (ST6GALNAC5), gap junction alpha-1 protein (GJA1), and epidermal growth factor receptor (EGFR), all of which are associated with brain metastasis [109]. Furthermore, it has been shown that Mettl3, an m⁶A methyltransferase, can enhance the splicing of precursor miR-143-3p, facilitating its biogenesis. This, in turn, leads to activation of the miR-143-3p/vasohibin-1 (VASH1) axis, which promotes brain metastasis of lung cancer by regulating angiogenesis and microtubules and increasing the degradation of vascular endothelial growth factor A (VEGFA) [110]. In addition, METTL14-mediated m⁶A modification has been shown to negatively regulate the mRNA expression of basic leucine zipper ATF-like transcription factor 2 (BATF2), and suppress growth, metastasis and angiogenesis of tongue squamous cell carcinoma (TSCC) by inhibiting VEGFA [111].

While a growing number of studies have evaluated the role of m⁶A methylation in tumor metastasis, it is important to recognize that this is a complex biological process. The specific role of m⁶A methylation modifications in the development of primary tumor cells and their subsequent metastasis requires further investigation to gain a deeper understanding of the underlying mechanisms.

4.1 m⁶A methylation and cancer therapeutic resistance

Therapeutic resistance is a well-known phenomenon in cancer treatment and has become a significant hurdle to overcome in the management of tumor patients. There are multiple mechanisms that contribute to therapeutic resistance in cancer, including specific genetic and epigenetic changes in the cancer cell and its microenvironment [112]. Recently, m⁶A methylation has emerged as a novel epigenetic regulatory mechanism that plays a crucial role in the progression of drug resistance. A growing body of evidence suggests that m⁶A methylation is closely associated with chemoresistance, radio-resistance, and resistance to immunotherapy in cancer [113].

m⁶A methylation mediates the development of resistance to many classical chemotherapeutic agents, such as cisplatin [114-118], 5-fluorouracil (5-FU) [119, 120] and gemcitabine [121-123]. In NSCLC, the writer METTL3 and the reader YTHDF1 play distinct roles in rendering cancer cells resistant to cisplatin treatment [114]. While METTL3 enhances sensitivity to cisplatin by increasing YAP expression and activity, it has also been found to promote YAP mRNA translation through the recruitment of YTHDF1/3 and eIF3b to the translation initiation complex. In addition, METTL3 regulates the metastasis-associated lung adenocarcinoma transcript 1 (MALAT1)-miR-1914-3p-YAP axis, which leads to increased YAP mRNA stability [114]. Depletion of YTHDF1 mediates cisplatin resistance through the kelch-like ECH-associated protein 1 (KEAP1)/nuclear factor erythroid 2-related factor 2 (NRF2)/aldo-keto reductase family 1 member C1 (AKR1C1) axis, and higher expression of YTHDF1 is correlated with better clinical outcomes in NSCLC patients [115]. High levels of ALKBH5 contribute to cisplatin resistance in oral squamous cell carcinoma by demethylating forkhead box M1 (FOXM1) and the nanog homeobox protein (NANOG) nascent transcript [117]. Similarly, the ALKBH5-homeobox A10 (HOXA10) loop promotes cancer cell cisplatin resistance by demethylating janus kinase 2 (JAK2) in epithelial ovarian cancer [118]. Upregulation of METTL3 in CRC cells increases the expression of lactate dehydrogenase A (LDHA) to promote glucose metabolism-mediated 5-FU resistance and tumor progression [119]. In pancreatic cancer, an m⁶A-dependent mechanism promotes gemcitabine resistance by regulating the lncANRIL splicing process [121]. Additionally, METTL14 promotes gemcitabine resistance by regulating the stability of cytidine deaminase transcripts [122].

Radiotherapy is a type of ionizing irradiation that works by damaging cancer cells’ DNA, which can cause them to stop dividing or die. In glioblastoma multiforme, research has shown that METTL3-dependent m⁶A modification is critical for glioblastoma stem cell (GSC) maintenance and radiation sensitivity [124]. High levels of METTL3 have been found to induce radio-resistance in GSCs through SOX2-dependent enhanced DNA repair [124]. Furthermore, studies have revealed that ALKBH5 can alter DNA damage repair and radiation sensitivity by regulating several homologous recombination genes in GSCs [125]. Additionally, YTHDC2 has been shown to promote radiotherapy resistance in nasopharyngeal carcinoma cells by activating the insulin-like growth factor 1 receptor (IGF1R)/protein kinase B (ATK)/ribosomal protein S6 (S6) signaling axis [126]. In hypopharyngeal squamous cell carcinoma, METTL3 mediates the m⁶A methylation of circCUX1, which stabilizes its expression and confers radio-resistance through the caspase-1 pathway [127].

In recent years, tumor immunotherapy has become a hot spot in the field of oncology treatment. Although immunotherapy is regarded as a promising approach to combat cancer, the occurrence of immune evasion limits its effectiveness. In CRC, when METTL3 is silenced, there is a reduction in the accumulation of myeloid-derived suppressor cells (MDSCs) [128]. This reduction promotes the activation and proliferation of CD4⁺ and CD8⁺ T cells. Mechanistically, METTL3 promotes basic helix-loop-helix family member e41(BHLHE41) expression in an m⁶A-dependent manner. This promotion of BHLHE41 expression subsequently induces C-X-C motif chemokine ligand 1 (CXCL1) transcription, which enhances MDSC migration in vitro [128]. In melanoma and CRC, ALKBH5 deficiency reduces monocarboxylate transporter 4 (Mct4) expression and lactate content of the tumor microenvironment and the composition of tumor-infiltrating Treg and myeloid-derived suppressor cells [129]. Intriguingly, FTO plays a critical role in regulating the immune surveillance that tumors use to evade detection. FTO-mediated m⁶A demethylation in tumor cells results in increased levels of transcription factors such as c-Jun, JunB, and CCAAT/enhancer-binding protein beta (C/EBPβ) [130]. This enhancement promotes glycolysis in tumors and also reduces T cell effector functions [130]. Moreover, in melanoma, the knockdown of FTO has been shown to sensitize melanoma cells to interferon-gamma (IFN-γ) and increase their sensitivity to anti-programmed cell death protein 1 (PD-1) treatment [131].

Overall, further research to understand the mechanisms of m⁶A modification in the development of therapy resistance is particularly important, which can improve the clinical outcomes of cancer patients.

4.2 m⁶A methylation and cancer targeted therapy

Current research demonstrates that RNA m⁶A modification is involved in tumorigenesis and development, and some related essential regulators have been used as new pharmacological targets for anti-tumor drug development [132]. The RNA m⁶A demethylase FTO is a member of the 2-oxoglutarate (2OG) and iron-dependent nucleic acid oxygenase (NAOX) family. Analyzing the substrate specificity and catalytic domain of FTO provides a new way to develop highly specific and efficient inhibitors [133, 134]. The first FTO inhibitor was rhein, which globally increased the cellular mRNA m⁶A levels by binding the FTO catalytic domain and preventing the recognition of m⁶A substrates [135]. However, rhein shows little selectivity for the alkB family demethylases [136]. Meclofenamic acid (MA), a non-steroidal anti-inflammatory drug, is a highly selective inhibitor of FTO that specifically inhibits FTO over ALKBH5. MA2 is an ester derivative of MA that might aid the inhibitor in penetrating cells [137]. Of note, the FTO inhibitor MA2 dramatically inhibits the growth and self-renewal of GSCs. Consistent with the effect on GSCs, in mice treated with MA2, the growth of tumors was shown to be slowed, and survival was significantly prolonged [138]. Furthermore, MO-I-500 acts as a pharmacological inhibitor of FTO and could effectively inhibit cell survival and/or colony formation in a TNBC cell line [139]. R-2-hydroxyglutarate (R-2HG), a metabolite produced by the mutant isocitrate dehydrogenase 1/2 (IDH1/2) enzyme, plays an anti-tumor role in leukemia by inhibiting cell proliferation and promoting cell cycle arrest and apoptosis [140]. Mechanistically, R-2HG directly targets the m⁶A demethylase FTO and inhibits its catalytic activity, increasing the level of m⁶A-modified RNA in R-2HG-sensitive leukemia cells. In addition, the effects of R-2HG on the treatment of leukemia were improved when it was used in combination with various first-line anticancer drugs, including all-trans retinoic acid (ATRA), azacitidine, decitabine and daunorubicin [140, 141]. FB23 and FB23-2 are two newly discovered small-molecule inhibitors of FTO based on structure-guided design that directly bind to FTO and selectively inhibit FTO's m⁶A demethylase activity [142]. Moreover, FB23-2 suppresses leukemia progression and prolongs survival. FB23-2 also displays therapeutic efficacy in targeting a patient-derived xenograft AML mouse model [142]. Additionally, CS1 and CS2 are highly efficacious FTO inhibitors screened from the 260,000 compounds [143]. They can selectively bind to and occupy the catalytic pocket of FTO, thus inhibiting FTO's demethylase activity. Surprisingly, CS1 and CS2 exhibit strong anti-tumor effects in multiple types of cancers, and they are highly feasible for clinical application [143].

In addition to FTO inhibitors, several studies have shown that other m⁶A protein inhibitors may be promising targets for treating m⁶A-related human cancers. ALK-04, a specific inhibitor of ALKBH5, enhances the efficacy of immunotherapy in combination with GVAX and PD-1 antibodies [129]. The first-in-class catalytic inhibitor of METTL3, STM2457, is a highly potent and selective inhibitor. Its in vivo activity and therapeutic efficacy represent a significant milestone, as it is the first demonstration of an RNA methyltransferase inhibitor's effectiveness against cancer [144]. BTYNB, a novel IGF2BP1 inhibitor, suppresses the cell cycle and cancer progression by impairing IGF2BP1-dependent stabilization of mRNA-encoding factors. Moreover, BTYNB acts in an additive manner or even synergistically with palbociclib, a cell cycle inhibitor targeting key E2 promoter binding factor (E2F)-activating kinases [145, 146]. The small-molecule inhibitor CWI1-2 has been shown to effectively bind to IGF2BP2 and inhibit its interaction with m⁶A-modified target transcripts. This is a promising development, as CWI1-2 has been shown to have significant anti-leukemia effects both in vitro and in vivo. Additionally, it has been found to exhibit synergistic effects when used in combination with other AML therapeutic agents such as daunorubicin and homoharringtonine [147].

Collectively, these results reveal that targeting m⁶A methylation enzymes is a promising approach for anticancer therapy. However, it is important to note that current m⁶A methylation inhibitors alter the overall level of m⁶A methylation by targeting the enzymes responsible for this process. It remains unclear whether targeting gene-specific m⁶A methylation will lead to better therapeutic outcomes. Further research is needed to explore this possibility.

5.1 m⁶A methylation and cancer immunity

m⁶A modification not only regulates the fate of tumor cells by targeting specific genes in various cancers but also affects the anti-tumor functions of immune cells. Recent studies have highlighted the critical role of m⁶A methylation in tumor immunity [128, 148]. These findings suggest that m⁶A methylation may represent a promising target for developing novel immunotherapeutic strategies to enhance anti-tumor immunity and improve the outcomes of cancer treatment.

Natural killer (NK) cells are the prototypical innate lymphoid immune cells and play a vital role in tumor surveillance [149]. Recently, researchers have found that YTHDF2 is required for NK cell survival, proliferation, and effector functions [150]. Deletion of YTHDF2 was found to significantly inhibit interleukin-15 (IL-15)/signal transducer and activator of transcription 5 (STAT5) signaling in activated NK cells, which downregulated STAT5 activation, thereby impairing NK cells anti-tumor and antiviral activity. YTHDF2 can also inhibit the mRNA stability of transactive response DNA binding protein (Tardbp), regulating NK cells proliferation and division [150]. In addition, one study showed that METTL3-mediated mRNA m⁶A methylation promotes the anti-tumor immunity of NK cells [151]. Mechanistically, METTL3 can promote src homology 2 domain-containing protein tyrosine phosphatase-2 (SHP-2) expression, AKT-mTOR and MAPK-ERK signaling pathways, leading NK cells to respond to IL-15 [151].

Tumor-associated macrophages (TAMs) can polarize into classically activated macrophages with anti-tumor responses (M1 type) or alternatively activated macrophages with pro-tumor functions (M2 type), contributing to dynamic and heterogeneous tumor immunity [152, 153]. Studies have shown that m⁶A methylation can regulate the polarization of macrophages in multiple ways. Overexpression of METTL3 greatly facilitates M1 macrophage polarization by upregulating STAT1 expression through enhancing mRNA stability [154]. FTO knockdown impedes macrophage activation by inhibiting the nuclear factor kappa B (NF-κB) signaling pathway and reducing the mRNA stability of STAT1 and peroxisome proliferator-activated receptor gamma (PPAR-γ) [155]. Deletion of METTL14 diminishes suppressor of cytokine signaling 1 (SOCS1) expression and leads to overactivation of toll-like receptor 4 (TLR4)/NF-κB signaling, which blunts the negative feedback control of macrophage activation in response to bacterial infection [156]. Moreover, the loss of METTL3 in macrophages has been found to establish an immunosuppressive microenvironment by increasing the infiltration of M1- and M2-like TAM and Treg cells in tumors [157]. This effect is mediated by the impairment of YTHDF1-mediated translation of sprouty-related EVH1 domain-containing protein 2 (SPRED2) and the subsequent downregulation of ERK, NF-κB and STAT3 phosphorylation [157]. IGF2BP2 can switch M1 macrophages to M2 activation by targeting tuberous sclerosis complex 1 (TSC1)-mechanistic target of rapamycin complex 1 (mTORC1) pathway and PPARγ-mediated fatty acid uptake [158].

Dendritic cells (DCs) are a critical component of the immune system and play a key role in stimulating T cell responses by presenting antigens [159]. Specifically, a recent study showed that Mettl3-mediated m⁶A modification enhances the translational expression of CD80 and CD40, leading to increased antigen presentation and T-cell stimulation by DCs [160]. Additionally, Mettl3 promotes the translational expression of Tirap, which strengthens TLR4/NF-κB signaling and increases the secretion of proinflammatory cytokines [160]. Another study demonstrated that mRNA m⁶A methylation and YTHDF1 in DCs control anti-tumor immunity [148]. Loss of YTHDF1 in classical DCs was found to enhance the cross-presentation of tumor antigens and the cross-priming of CD8⁺ T cells, while YTHDF1 promotes the translation of lysosomal cathepsins for excessive antigen degradation [148].

T cells, as a key component of the adaptive immune system, play a central role in cancer immunology due to their ability to directly mediate cancer cell killing. mRNA m⁶A modification has emerged as an important regulator of T cell homeostasis and differentiation. In mouse T cells, loss of Mettl3 has been shown to increase the half-lives and protein levels of Socs1, Socs3, and cytokine-inducible SH2-containing protein (Cish) mRNAs, which in turn suppresses the IL-7/STAT5 signaling pathway, ultimately disrupting T cell homeostatic proliferation and differentiation [161]. ALKBH5-mediated m⁶A demethylation in CD4⁺ T cells increases the transcript stability and protein expression of CXCL2 and IFN-γ, which controls the pathogenicity of CD4⁺ T cells during autoimmunity [162]. Interestingly, tumor-intrinsic FTO restricts the activation and effector states of CD8⁺ T cells. FTO knockdown impairs the glycolytic activity of tumor cells by elevating the transcription factors c-Jun, JunB, and C/EBPβ, which suppresses the function of CD8⁺ T cells [130].

5.2 m⁶A methylation and cancer immunotherapy

In recent years, immune checkpoint blockade (ICB) therapy has led to a significant breakthrough in the treatment of various types of cancer. However, resistance to ICB remains a major challenge for the future of cancer treatment. To address this challenge, some research groups are exploring the potential of combining checkpoint blockade with m⁶A inhibitors as a new therapeutic strategy to improve outcomes in patients with a low response to checkpoint blockade. This approach shows promise and may represent a significant step forward in the fight against cancer. The deletion of the m⁶A RNA demethylase ALKBH5 has been found to increase the sensitivity of tumors to anti-PD-1 immunotherapy while decreasing the populations of MDSC and Treg suppressive immune cells [129]. The mechanism underlying this effect involves the inhibition of ALKBH5 mRNA demethylation, which increases m⁶A in MCT4/SLC16A3, a lactate transporter. This, in turn, decreases the mRNA levels of MCT4/SLC16A3 and leads to a reduction in lactate in the tumor interstitial fluid [129]. Similarly, YTHDF1 depletion elevates the antigen-specific CD8⁺ T cell anti-tumor response and enhances the therapeutic efficacy of programmed death ligand 1 (PD-L1) checkpoint blockade [148]. In melanoma, the level of FTO is significantly increased and reduces the response to PD-1-blocking immunotherapy [131]. Knockdown of FTO in mice has been shown to the m⁶A methylation of the proto-oncogenes PD-1, C-X-C chemokine receptor type 4 (CXCR4), and SOX10, rendering melanoma cells more sensitive to interferon-gamma (IFN-γ) and improving the anti-PD-1 treatment response of melanoma in mice [131]. In CRC and melanoma, depletion of methyltransferases, Mettl3 and Mettl14, can augment the response to anti-PD-1 treatment promoted by enhancing IFN-γ-Stat1-interferon regulatory factor 1 (Irf1) signaling through stabilizing the Stat1 and Irf1 mRNA via Ythdf2 [163].

AUTHORS CONTRIBUTIONS

LXZ, XDH and JLZ designed and wrote the manuscript. DXL and JZ revised and supervised manuscript preparation. All authors read and approved the final manuscript.