Novel insight into RNA modifications in tumor immunity: Promising targets to prevent tumor immune escape

Abstract

An immunosuppressive state is a typical feature of the tumor microenvironment. Despite the dramatic success of immune checkpoint inhibitor (ICI) therapy in preventing tumor cell escape from immune surveillance, primary and acquired resistance have limited its clinical use. Notably, recent clinical trials have shown that epigenetic drugs can significantly improve the outcome of ICI therapy in various cancers, indicating the importance of epigenetic modifications in immune regulation of tumors. Recently, RNA modifications (N⁶-methyladenosine [m⁶A], N¹-methyladenosine [m¹A], 5-methylcytosine [m⁵C], etc.), novel hotspot areas of epigenetic research, have been shown to play crucial roles in protumor and antitumor immunity. In this review, we provide a comprehensive understanding of how m⁶A, m¹A, and m⁵C function in tumor immunity by directly regulating different immune cells as well as indirectly regulating tumor cells through different mechanisms, including modulating the expression of immune checkpoints, inducing metabolic reprogramming, and affecting the secretion of immune-related factors. Finally, we discuss the current status of strategies targeting RNA modifications to prevent tumor immune escape, highlighting their potential.

Graphical abstract

Public summary

• •RNA modification is a novel hotspot of epigenetic research, affecting a wide range of physiological and pathological processes.
• •RNA modification plays an important role in tumor immunity.
• •RNA modification may be a potential clinical therapeutic target to prevent tumor immune escape.

Introduction

The immune system plays a crucial role in host defense against tumors. Antitumor responses mainly rely on activated CD8⁺ T cells recognizing tumor antigens. In addition, innate immune cells are pivotal in eliminating tumors. Despite the immune system aiming to eliminate tumor cells, tumor cells with reduced immunogenicity in an immunosuppressive tumor microenvironment (that is, one that includes immunosuppressive cells and immunosuppression-related molecules) can escape T cell attack. Immune checkpoint inhibitor (ICI) therapy can overcome immune escape and provide significant benefits to patients, and its application has revolutionized the field of tumor therapy. However, despite ICI effectiveness, most patients have a poor response to ICIs (primary resistance), and some patients who initially respond develop resistance after a period of treatment (secondary resistance), highlighting the necessity to explore novel treatment strategies to enhance the anticancer potency of immunotherapies.

RNA modification, a crucial epigenetic change, has become a research hotspot since its discovery in the 1950s.¹ To date, more than 100 chemical modifications of RNA have been identified;² these modifications include methylation to generate N1-methyladenosine (m¹A),³ N6-methyladenosine (m⁶A),⁴ 5-methylcytosine (m⁵C),⁵ and N7-methylguanosine (m⁷G);⁶ RNA cap methylation;⁷ pseudouridine formation;⁸ and uridylation.⁹ The enzymes that catalyze the addition and removal of these modifications from RNA are called “writers” and “erasers,” respectively, and the RNA-binding proteins that identify modified RNA modifications are called “readers.” All types of RNA modifications are regulated by “readers,” “writers,” and “erasers.” These modifications play multifunctional roles in the RNA life cycle, affecting factors such as translation efficiency, transcript stabilization, pre-mRNA splicing, nuclear export, and mRNA storage under stress.^{4,10,11,12,13,14,15} Hence, RNA modifications affect a wide range of physiological and pathological processes.^13,16,17

Notably, the combination of epigenetic drugs with immunotherapy may be a useful strategy to circumvent ICI resistance. For example, the combination of pembrolizumab (a PD-L1 inhibitor) plus entinostat (a histone deacetylase inhibitor) provides a clinically meaningful benefit to non-small cell lung cancer (NSCLC) patients who have shown resistance to PD-L1 inhibitors.¹⁸ In addition, administration of guadecitabine (a DNA methylation inhibitor) combined with pembrolizumab to patients with an advanced solid tumor can reverse previous resistance to ICIs.¹⁹ The positive results of these clinical trials indicate the importance of epigenetics in tumor immune regulation. Notably, RNA modifications have also been found to play a critical role in tumor immunity. For example, methyltransferase-like protein 3 (METTL3), a typical m⁶A reader, inhibits tumor growth and metastasis by enhancing the YTHDF1-mediated translation of SPRED2.²⁰ Moreover, YTHDF1, a typical m⁶A reader, impairs tumor antigen cross-presentation of dendritic cells (DCs) by enhancing the translation of lysosomal cathepsin transcripts.²¹ Although numerous studies have been performed, details regarding the roles of RNA modifications in regulating tumor immunity remain unclear. Some reviews have summarized the critical roles of m⁶A in tumorigenesis, progression,²² and cancer metabolism.²³ However, the roles of m⁶A and other RNA modifications (including m¹A and m⁵C) in tumor immunity and the underlying mechanisms have not been systematically described, and we attempt to provide an up-to-date and comprehensive overview of these topics in this review.

In this review, we outline the evidence showing that m⁶A, m¹A, and m⁵C modifications participate in tumor immunity by directly regulating different immune cells as well as by indirectly regulating tumor cells. Our review emphasizes the essential roles played by RNA modifications in shaping tumor immunity and highlights potential therapeutic strategies to prevent immune escape of tumor cells by targeting RNA modifications.

The m⁶A modification in tumor immunity

Overview of the m⁶A RNA modification

The m⁶A modification is the most prevalent RNA modification on mRNA and regulates the fate of mRNA by affecting its stability, splicing, translation, and export. It plays an important role in tumour immunity (Table 1). M⁶A was first discovered in 1974²⁴ and is now considered the most representative type of RNA modification, accounting for 0.1%–0.4% of all adenosine molecules in total cellular RNA, with an average of 3–5 m⁶A modifications per mRNA.²⁵ RRACH (R = G or A; H = A, C, or U) is the consensus sequence in mRNA on which the m⁶A modification is found, and a combination of immunoprecipitation and high-throughput sequencing results has revealed that the m⁶A modification is enriched at stop codons and 3′ UTRs.²⁶ However, the consensus sequence for the m⁶A modification is different on noncoding RNAs (ncRNAs), including long ncRNAs (lncRNAs), microRNAs (miRNAs), circular RNAs (circRNAs), small nuclear RNAs (snRNAs), and ribosomal RNAs (rRNAs).^27,28 Based on the known m⁶A modification sites, several RNA modification databases have been established (Table 2), and various m⁶A modification site prediction algorithms have been proposed (Table 3), such as WHISTLE,²⁹ SRAMP,³⁰ BERMP,³¹ iRNA-PseColl,³² and Gene2vec.³³ Despite these advances, the specificities of m⁶A modification sites are still poorly understood.

Table 1.

Functions of m⁶A regulators in tumor immunity

	Regulators	Cancer type	Function	References
Writers	METTL3	breast cancer	enhance PD-L1 expression	Wan et al.104
		bladder cancer	enhance PD-L1 expression	Ni et al.105
		NSCLC	reduce degradation of PD-L1	Liu et al.106
		colorectal carcinoma	reduce recruitment of CD8+ T cells and the levels of IFN-γ, CXCL9, and CXCL10	Wang et al.121
		colorectal cancer	facilitate MDSC accumulation by promoting CXCL1 expression	Chen et al.122
	RBM-15	ccRCC	increase macrophage infiltration and M2 polarization by promoting CXCL11 expression	Zeng et al.123
	METTL14	CCA	reduce degradation of PD-L1	Zheng et al.107
		HCC	enhance PD-L1 expression	Peng et al.108
		colorectal carcinoma	reduce recruitment of CD8+ T cells and the levels of IFN-γ, CXCL9, and CXCL10	Wang et al.121
Readers	IGF2BP1	HCC	enhance PD-L1 expression	Liu et al.109
	YTHDF1	colon cancer	enhance PD-L1 expression	Li et al.110
		GC	reduce DC recruitment and T cell infiltration by reducing expression of IFNGR1	Bai et al.115
	YTHDF3		downregulate PD-L1 expression	Zhao et al.113
Erasers	ALKBH5	ICC	enhance PD-L1 expression	Qiu et al.111
		melanoma/colon cancer	reduce sensitivity to immunotherapy	Li et al.118
		GBM	promote secretion of CXCL8/IL8 and recruitment of TAMs	Dong et al.124
		HCC	increase PD-L1+ macrophage recruitment	You et al.125
			Promote tumor progression by reducing RIG-I expression	Jin et al.126
	FTO	melanoma	enhance PD-1 (PDCD1), CXCR4, and SOX10 expression	Yang et al.112
		leukemia	maintain immune evasion by enhancing LILRB4 expression	Su et al.114
		melanoma	suppress infiltration and function of CD8+ T cells	Liu et al.119

Table 2.

RNA modification databases

Database	Resources	Website	References
m6A2Target	m6A modifies the associated writers/erasers/readers	http://m6a2target.canceromics.org/#/	Bao et al.220
m6A-Atlas	m6A sites and quantitative epitranscriptome profiles	http://180.208.58.66/m6A-Atlas/index.html	Tang et al.221
REPIC	m6A-seq and MeRIP-seq data	https://repicmod.uchicago.edu/repic	Liu et al.222
DirectRMDB	Oxford Nanopore Technologies (ONT)-based database of quantitative RNA modification profiles	http://www.rnamd.org/directRMDB/	Zhang et al.223
MODOMICS	Modified residues, enzymes, guide RNAs, RNA modification pathways, sequences of modified RNAs, diseases	https://iimcb.genesilico.pl/modomics/	Boccaletto et al.202
RMVar	Multiple modification types, lncRNA/circRNA/miRNA methylation-associated SNPs/SNVs, RNA bing proteins (RBPs), diseases	http://rmvar.renlab.org/	Luo et al.224
RMDisease V2.0	1,366,252 RM-associated variants that may affect 16 different types of RNA modifications, diseases	www.rnamd.org/rmdisease2	Song et al.225
RMBase	Multiple methylation types, SNP/SNVs, RBPs, miRNA modifications	http://rna.sysu.edu.cn/rmbase/	Xuan et al.226
m5C-Atlas	m5C sites, associated SNPs, relevance to RNA secondary structure, RBPs	https://www.xjtlu.edu.cn/biologicalsciences/m5c-atlas	Ma et al.227

Table 3.

RNA modification site prediction methods

Tool	Modification	Algorithm	Website	References
WHISTLE	m6A	SVM	www.xjtlu.edu.cn/biologicalsciences/whistle; http://whistle-epitranscriptome.com	Chen et al.29
SRAMP	m6A	RF	http://www.cuilab.cn/sramp/	Zhou et al.30
BERMP	m6A	BGRU	http://www.bioinfogo.org/bermp	Huang et al.31
iRNA-PseColl	m6A	SVM	http://lin-group.cn/server/iRNA-PseColl/	Feng et al.32
Gene2vec	m6A	CNN	http://server.malab.cn/Gene2vec/	Zou et al.33
iRNA-3typeA	m1A m6A	SVM	http://lin-group.cn/server/iRNA-3typeA/	Chen et al.228
RAMPred	m1A	SVM	http://lin-group.cn/server/RAMPred	Chen et al.134
M1ARegpred	m1A	SVM	https://github.com/SXWuFJMU/m1ARegpred.	Yao et al.133
iRNA-m5C	m5C	RF	http://lin-group.cn/server/iRNA-m5C/service.html	Lv et al.229
RNAm5Cfinder	m5C	RF	http://www.rnanut.net/rnam5cfinder	Li et al.230
M5C-HPCR	m5C	HPCR	http://cslab.just.edu.cn:8080/M5C-HPCR/	Zhang et al.231
m6AmPred	m6Am	XgbDart	https://www.xjtlu.edu.cn/biologicalsciences/m6am	Jiang et al.232
MultiRM	Multiple types	OHEM	www.xjtlu.edu.cn/biologicalsciences/multirm	Song et al.233

To understand the roles of RNA modifications, appropriate analytical methods or techniques are essential. The lack of specific antibodies limits functional exploration to some extent. However, considerable improvements in methods for deciphering RNA modifications have been achieved in the last decade.^34,35,36 The methods for detection of RNA modifications have evolved from liquid chromatography,³⁷ reverse-transcriptase polymerase chain reaction (RT-PCR),³⁸ and thin-layer chromatography (TLC)³⁹ to mass spectrometry (MS)⁴⁰ and sequencing analysis.⁴¹ Liquid chromatography (LC) coupled to MS (LC-MS) or tandem MS (LC-MS/MS) are the most commonly used MS methods to quantify RNA modifications on single nucleosides.³⁶ Recently, an LC-electrospray ionization (ESI)-MS/MS method was developed. It was used to profile and characterize the RNA modifications in plant 18S rRNA and 25S rRNA.⁴² In addition, this method was also used to detect modifications of RNA in rat peripheral blood during alcohol exposure⁴³ and in thyroid carcinoma tissue.⁴⁴

m⁶A modifications are reversible and regulated by three types of enzymes: m⁶A methyltransferases (“writers”), m⁶A demethylases (“erasers”), and m⁶A-binding proteins (“readers”). The deposition of a methyl group to form m⁶A relies on the m⁶A methyltransferase complex (MTC), comprising the METTL3-methyltransferase-like protein 14 (METTL14) heterodimer and other adaptor proteins, including WT1-associated protein (WTAP), RNA-binding motif protein 15 (RBM15), vir-like m⁶A methyltransferase associated (VIRMA) subunits, and zinc-finger CCCH domain-containing protein 13 (ZC3H13).^45,46 METTL3, discovered in 1999, was the first identified m⁶A methyltransferase.⁴⁷ An S-adenosyl methionine (SAM)-dependent methyltransferase catalyzes RNA methylation using SAM, a prevalent methyl donor in vivo, and is critical for almost all m⁶A modifications on mRNA. In 2014, METTLE14 and WTAP were identified as other important MTC subunits.^48,49 In the complex, METTL3 is the catalytic core, METTL14 is the RNA-binding platform, and WTAP is crucial for recruitment of the MTC.^45,49 In addition to the aforementioned complexes and factors, the functions of methyltransferase-like protein 16 (METTL16) and the METTL5-TRMT112 complex have also been validated.⁵⁰ m⁶A demethylases can remove a methyl group from m⁶A modifications on mRNA and cooperate with m⁶A methyltransferases to maintain the dynamic balance of m⁶A modifications. Fat mass and obesity-associated protein (FTO) and alkB homolog 5 (ALKBH5) are two well-known m⁶A demethylases.^51,52 In general, m⁶A modifications are involved in functions via their recognition by m⁶A-binding proteins, including five members of the YTH domain-containing family (YTHDF1, YTHDF2, YTHDF3, YTHDC1, and YTHDC2),⁵³ insulin-like growth factor 2 mRNA-binding proteins (IGF2BP1, IGF2BP2, and IGF2BP3),¹⁵ heterogeneous nuclear ribonucleoprotein proteins (HNRNPA2B1 and HNRNPC),^54,55 leucine-rich pentatricopeptide repeat-containing (LRPPRC),⁵⁶ fragile X mental retardation 1 (FMR1),⁵⁷ and proline-rich coiled-coil 2 C (Prrc2c).⁵⁸ Their roles are described in detail below.

m⁶A modifications affect the fate of mRNAs by regulating and controlling gene expression in various ways in different subcellular locations, as facilitated by reader proteins. (Figure 1) YTH domain-containing family members are the principal readers. The YTH structural domain recognizes and binds m⁶A modification sites. YTHDF1 interacts with translation initiation factor complex 3 (eIF3) and enhances mRNA translation efficiency,⁴ whereas YTHDF2 accelerates target transcript degradation in two ways: deadenylation mediated by carbon catabolite repression 4 (CCR4)-negative on TATA-less (NOT)¹⁰ complex action and endoribonucleolytic cleavage mediated by heat-responsive protein 12 (HRSP12).¹¹ YTHDF3 cooperates with YTHDF1 to enhance protein synthesis by interacting with ribosomal proteins and increases YTHDF2-mediated mRNA decay,⁵⁹ suggesting that YTHDF proteins interact with each other to synthetically influence fundamental biological processes associated with m⁶A RNA modification.⁶⁰ Moreover, multiple m⁶A-modified mRNAs form phase-separated YTHDF-m⁶A-mRNA complexes by binding to YTHDF1–YTHDF3, which, in turn, form phase-separated structures in cells and may function during cellular stress to induce storage of certain mRNAs encoding cellular repair proteins.⁶¹ YTHDC1 facilitates exon inclusion in targeted mRNAs by recruiting arginine/serine-splicing factor 3 (SRFS3) and blocking serine/arginine-splicing factor 10 (SRSF10) binding, thus promoting mRNA translation.⁶² In addition, YTHDC1 affects mRNA density by splicing pre-mRNA transcripts¹³ and mediating the export of mRNA.¹⁴ YTHDC2 also enhances the translation efficiency of target mRNAs, shows 3'→5′ RNA helicase activity, and recruits XRN1, which is a 5'→3′ exoribonuclease, by inserting ankyrin repeats.⁶³ In contrast to the decay-promoting function of YTHDF2, IGF2BPs (IGF2BP1–IGF2BP3) have been shown to stabilize target mRNAs in a manner facilitated by their cofactors (HuR and MATR3) and to promote the storage of target mRNAs under stress.¹⁵ In a previous study, FMR1 was found to bind to m⁶A to enhance mRNA stability.¹² However, a recent study revealed that Drosophila FMR1 bound preferentially to mRNAs containing the m⁶A-tagged “AGACU” motif, forming FMR1-ribonucleoprotein (RNP) granules that then underwent a dynamic phase switch to promote mRNA decay.⁶⁴ FMR1 has also been proven to promote nuclear export of m⁶A-modified mRNAs.⁶⁵ As mentioned above, m⁶A-binding proteins play different or even contradictory roles in mRNA modification. YTHDF2 promotes mRNA degradation, but IGF2BPs have the opposite effect. In addition, in older and more recent studies, FMR1 has been reported to exert the opposite effect on mRNA stability. In addition, interactions between different RNA-binding proteins can be either cooperative or competitive.^57,59 These complex regulatory roles may explain the diversity of m⁶A mechanisms of action and biological effects.

Figure 1.

Regulators of RNA modifications and their molecular functions

All RNA modifications are catalyzed by writers and removed by erasers. They can be recognized by their respective reader proteins and affect the fate of target RNAs by regulating their nuclear export, splicing, translation, and degradation.

In addition to influencing the fate of mature mRNAs, m⁶A modifications regulate the generation and function of ncRNAs, which are also of physiological importance. Among miRNAs, HNRNPA2B1 is involved in regulating variable shearing of exons and subsequent processing of certain primary miRNAs (pri-miRNAs) by binding to the microprocessor complex DGCR8.⁵⁴ Some lncRNAs are closely related to cancers and are regulated by RNA-modification-related enzymes, such as DMDRMR and MALAT1.⁶⁶ Moreover, recent studies on m⁶A have focused on its interaction with the nucleus and chromatin as well as its role in nascent RNA processes, by which it can regulate gene transcription. m⁶A modifications on the lncRNA X-inactive-specific transcript (XIST) placed by RBM15 participate in transcriptional repression of X chromosome gene expression.⁶⁷ m⁶A modifications on chromosome-associated regulatory RNAs (carRNAs) deposited by METTL3 and YTHDC1 accelerate the decay of these RNAs.⁶⁸ This process is involved in regulating the state of nearby chromatin and downstream transcription.⁶⁸ Regarding enhancer RNAs (eRNAs) and nascent transcripts, m⁶A-modified eRNAs can recruit the nuclear m⁶A reader YTHDC1 and promote formation of BRD4, which is related to enhancer and gene activation,⁶⁹ and the m⁶A modification of nascent transcripts protects them from termination mediated by the integrator complex, thus enhancing gene expression.⁷⁰ Moreover, m⁶A can bind to YTHDC1, and YTHDC1 can recruit KDM3B via physical interaction, which promotes histone dimethylated histone H3 Lys9 (H3K9me2) demethylation and gene expression.⁷¹

Overall, m⁶A modification of mRNAs and ncRNAs is involved in regulation of almost all important life processes. Recently, m⁶A modifications have been shown to regulate tumor immunity, and details regarding the regulatory network housing tumor immune factors and m⁶A modifications are described below. Collectively, m⁶A modifications regulate the immune response through two main processes: by directly affecting the function of immune cells and by indirectly regulating tumor cells. In the following sections, both of these mechanisms are outlined.

m⁶A regulates tumor immunity by directly affecting immune cells

Macrophages

Macrophages, vital components of innate immunity, can be polarized into different phenotypes depending on their genetic background and stimuli in the environment. A general classification of macrophages places these cells into the classically activated macrophage (M1) and alternatively activated macrophage (M2) categories.⁷² M1 macrophages show high antigen-presenting and T cell activation abilities, and M2 macrophages are related to immunosuppression.⁷³ Recent studies have revealed that m⁶A is involved in regulating polarization of macrophages under physiological and inflammatory conditions. METTL3 drives M1 polarization by enhancing the stability of signal transducer and activator of transcription 1 (STAT1) mRNA.⁷⁴ However, IGF2BP2 facilitates the switch of M1 macrophages into M2 macrophages by stabilizing tuberous sclerosis complex 1 (TSC1) and peroxisome proliferator-activated receptor γ (PPARγ).⁷⁵ In addition to affecting polarization, m⁶A regulates the activation of macrophages. METTL3 has been identified as a positive regulator. For example, deletion of METTL3 in macrophages impairs their ability to eliminate pathogens because of reduced degradation of Irakm transcripts and subsequent inhibition of the Toll-like receptor 4 (TLR4) signaling pathway.⁷⁶ However, FTO, a demethylase, activates macrophages by enhancing the stability of STAT1 and PPARγ in a YHTDF2-dependent manner.⁷⁷ Moreover, m⁶A inhibits activation of certain cellular programs, with METTL14 and FTO preventing their hyperactivation. METTL14 mediates m⁶A methylation of suppressor of cytokine signaling 1 (SOCS1) mRNA and promotes translation of SOCS1 by binding to YTHDF1, an interaction that is essential for sustaining the negative feedback loop established via macrophage activation.⁷⁸ In addition, knocking out FTO in macrophages mediates similar inflammation inhibition and leads to increased m⁶A abundance on SOCS1 mRNA, thereby facilitating transcript stability and subsequent translation in a YHTDF1-dependent manner⁷⁹ (Figure 2).

Figure 2.

The mechanisms by which m⁶A modification regulates macrophages

METTL3 (a writer) promotes production of TNF-α by enhancing degradation of Irakm (an inhibitor of TRL4) mRNA, and it also inhibits tumor growth by targeting SPRED2 (an inhibitor of the ERK pathway). However, METTL3 enhances immunosuppression through the Jak1/STAT3 pathway. METTL14 (a writer) increases degradation of Ebi3 mRNA, promoting the antitumor function of CD8⁺ T cells. FTO (an eraser) reduces degradation of STAT1/PPAR-γ mRNA, promoting macrophage activation.

Tumor-associated macrophages (TAMs) acquire an M2 phenotype and either infiltrate tumors or reside near tumor tissues. TAMs promote tumor progression and subvert antitumor immunity.⁸⁰ Recently, the mechanisms by which m⁶A regulates the function of TAMs have been further elucidated. METTL3 and METTL14 are crucial for maintaining the antitumor function of macrophages. Deletion of METTL3 in myeloid cells creates an environment conducive to tumor growth and metastasis by increasing infiltration of immunosuppressive cells.²⁰ Further research revealed that METTL3 deficiency in macrophages leads to a reduction in YTHDF1-mediated translation of SPRED2 and inhibition of the nuclear factor κB (NF-κB) pathway.²⁰ Another study highlighted that C1q⁺ TAMs with high expression of METTL14 and YTHDF2 express diverse immunomodulatory ligands, which enhances the recruitment and function of CD8⁺ effector T cells (TEFFs).⁸¹ Moreover, METTL14-deficient C1q⁺ TAMs show elevated Ebi3, a cytokine subunit that negatively regulates T cell function, which results in CD8⁺ T cell dysfunction.⁸¹ On the other hand, m⁶A readers, including IGF2BP2 and YTHDF1, have been shown to play a significant inhibitory role in macrophage antitumor ability. By binding to IGF2BP2, the lncRNA PACERR induces pro-oncogenic effects and promotes M2 polarization in pancreatic cancer by stabilizing KLF12 and c-myc in the cytoplasm.⁸² In addition, METTL3 enhances the immunosuppressive function of tumor-infiltrating myeloid cells (TIMs) through the Jak1/STAT3 pathway by promoting Jak1 translation through the m⁶A-YTHDF1 axis.⁸³ This process may be driven by lactate accumulation in the form of H3K18 lactylation marks⁸³ (Figure 2).

DCs

DCs are antigen-presenting cells (APCs) that process antigenic information, thereby connecting innate and adaptive immune responses.⁸⁴ Recent studies have provided some clues about how m⁶A regulates stimulation, migration, and presentation of DCs under physiological conditions. METTL3-mediated mRNA m⁶A methylation enhances DC-mediated stimulation of T cell activation and cytokine production by enhancing the translation efficiency of CD40, CD80, and TLR4 signaling adaptor (Tirap) mRNA.⁸⁵ In addition to affecting stimulation, m⁶A regulates DC migration. CC-chemokine receptor 7 (CCR7) is critical for DC migration to draining lymph nodes,⁸⁶ and a feedback mechanism that inhibits CCR7-mediated DC migration in a manner mediated via m⁶A modification has been identified.⁸⁷ Demethylation of the lncRNA Dpf3 induced by CCR reduces Dpf3 decay. Dpf3 binds to hypoxia-inducible factor 1 alpha (HIF-1α), impairing the glycolytic metabolism and migratory capacity of DCs by attenuating expression of the glycolytic gene Ldha⁸⁷ (Figure 3).

Figure 3.

The mechanisms by which m⁶A modification regulates DCs

METTL3 positively regulates the function of dendritic cells (DCs) by increasing CD40, CD80, and Tirap (TLR4 signaling adaptor) translation. YTHDF1 inhibits the cross-priming ability of DCs targeting lysosomal proteases, suppressing the CD8⁺ T cell antitumor response.

Other studies have focused on the tumor background. For example, on the basis of positive regulation of DC function by m⁶A, researchers have designed nanomedicines containing an FTO inhibitor, and such agents promote DC maturation and antigen presentation after thermal ablation of hepatocellular carcinoma (HCC) cells.⁸⁸ However, contrary to previous findings, YTHDF1 has been found to be a negative regulator of DC antitumor effects. YTHDF1-silenced mice show improved tumor antigen cross-presentation by DCs and antitumor responses by CD8⁺ T cells.²¹ Mechanistically, YTHDF1 enhances translation of lysosomal cathepsin transcripts, which contributes to antigen degradation, thereby reducing DC cross-priming capacity²¹ (Figure 3).

Natural killer cells

Natural killer (NK) cells constitute an important part of the innate immune system and are essential effector cells in antitumor immunity because they exert cytotoxic effects on tumor cells through surface receptors.^89,90 They also regulate the antitumor immune response by expressing cytokines and chemokines that influence the recruitment and action of other immune cells, such as T cells and DCs.^91,92 Recent studies have shown that regulators of m⁶A consistently activate NK cells. YTHDF2 deficiency profoundly impairs the antitumor and antiviral capacities of NK cells.⁹³ YTHDF2 plays multifaceted roles in NK cells, including maintaining homeostasis, regulating terminal maturation, and favoring response to interleukin-15 (IL-15) by enhancing the stability of Tardbp mRNA.⁹³ In addition, METTL3 is an important positive regulator of NK cell antitumor effects because it maintains homeostasis and infiltration of NK cells.⁹⁴ Conditional ablation of METTL3 in NK cells weakened the immune response to IL-15 exposure, and this mitigated response was related to reduced expression of Src-homology phosphotyrosine phosphatase-2 (SHP-2) mediated by a reduction in m⁶A abundance on Ptpn11 mRNA⁹⁴ (Figure 4).

Figure 4.

The mechanisms by which m⁶A modification regulates NK cells

METTL3 enhances the antitumor function of NK cells by promoting SHP-2 (a critical mediator of IL-15-induced activation) translation. YTHDF2 (a writer) positively regulates NK cell division/proliferation by increasing Tardbp mRNA degradation. The STAT5–YTHDF2 positive feedback loop, which is downstream of IL-15, controls NK cell effector functions.

T cells

T cells, including CD4⁺ T cells and CD8⁺ T cells, play essential roles in adaptive immunity. Regulatory T cells (Tregs) constitute a special T cell subpopulation that controls self-tolerance and is associated with immunosuppression.⁹⁵ Recent studies have provided some clues to explain the association between m⁶A regulators and the differentiation, homeostasis, survival, and pathogenicity of T cells. First, deleting METTL3 in T cells disrupts CD4⁺ T cell homeostasis and differentiation by upregulating the expression of SOCS family members (Socs1, Socs3, and Cish), which encode proteins that inhibit STAT signaling and thus downregulate IL-7-mediated STAT5 activation⁹⁶ (Figure 5). Similarly, in Tregs, METTL3 has been shown to contribute to immune suppression through the IL-2/STAT5 signaling pathway by regulating the expression of SOCS family members (Cish, Socs1, Socs2, Socs3, and Asb2)⁹⁷ (Figure 6). Second, after targeted knockdown of WTAP, cell death is induced by activation of the T cell receptor (TCR) signaling pathway.⁹⁸ WTAP knockdown stabilizes Orai1 and Ripk1 mRNAs, and subsequent cell death is mediated by more intense and sustained Ca²⁺ signaling.⁹⁸ Third, ALKBH5 controls the pathogenicity of CD4⁺ T cells.⁹⁹ Ablation of ALKBH5 in naive CD4⁺ T cells suppresses development of colitis and autoimmune diseases by reducing interferon-γ (IFN-γ) and CXCL2 mRNA stability⁹⁹ (Figure 5).

Figure 5.

The mechanisms by which m⁶A modification regulates T cells

METTL3 promotes proliferation and differentiation of T cells via the JAK/STAT5 pathway by decreasing the expression of SOCS family proteins (SOCS1, SOCS3, and CISH). ALKBH5 (an eraser) enhances production of CXCL-2 and IFN-γ by targeting CXCL-2 and IFN-γ mRNA degradation. METTL3 also promotes T follicular helper (Tfh) development by stabilizing Tcf-7 mRNA to increase TCF-1 (a regulator of Tfh differentiation) expression. The VHL-HIF-1α axis regulates Tfh cell development in an m⁶A-dependent manner.

Figure 6.

The mechanisms by which m⁶A modification regulates T cells

METTL3 mediates immune suppression via the JAK/STAT5 pathway by decreasing the expression of SOCS family proteins (SOCS1, SOCS2, SOCS3, CISH, and Asb2).

T follicular helper (Tfh) cells are another specific subpopulation of CD4⁺ T cells; they initiate formation of germinal centers and promote secretion of high-affinity antibodies by B cells.¹⁰⁰ Knocking out METTLE3 in CD4⁺ T cells leads to poor differentiation of Tfh cells and, thus, impaired germinal center responses.¹⁰¹ Further experiments elucidated that m⁶A modification of the 3′ UTR of Tcf7 mRNA is mediated by METTL3 and that this modification stabilizes Tcf7 transcripts and subsequently ensures activation of Tfh cells.¹⁰¹ However, in another study, m⁶A modifications have been shown to exert the opposite effects on Tfh cell development. Specifically, m⁶A modification induced by glyceraldehyde-3-phosphate dehydrogenase (GAPDH) accelerates degeneration of inducible costimulator (ICOS) mRNA and, thus, impairs Tfh cell initiation when von Hippel-Lindau (VHL) activity is impaired¹⁰² (Figure 5).

In addition to its effect on CD4αβ T cells, m⁶A regulates the development of γδ T cells. γδ T cells are involved in intrinsic immunity and are abundant in the mucosae, where they function in immune surveillance. m⁶A has been identified as a developmental checkpoint for immature γδ T cell development, and the number of γδ T cells has been shown to increase significantly when ALKBH5 is absent.¹⁰³ Moreover, ALKBH5-mediated m⁶A demethylation decreases the stability and expression of Jagged1 and Notch2 mRNAs, inactivating the Jagged1/Notch2 signaling pathway.¹⁰³

Above all, different m⁶A modification regulators play a variety of roles in different cells. For example, METTL3, the most classic m⁶A writer, has the most extensively studied function in immune cells, and its knockdown in macrophages, DCs, NK cells, and T cells impairs their functions. However, other m⁶A regulators, such as YTHDF2 in DCs, negatively regulate antitumor functions; therefore, inhibitors of these m⁶A regulators may be effective antitumor agents. Despite the diversity of roles of m⁶A modifications, there are common mechanisms regulating functions across different immune cells. First, m⁶A, as the most abundant modification on mRNA, regulates gene expression by regulating the stability, splicing, translation, and export of mRNAs. In immune cells, m⁶A mainly influences the translation efficiency and stability of mRNAs of related genes. Second, although the regulatory pathways are different, m⁶A modifications generally influence similar cellular processes in different immune cells, such as cell proliferation, differentiation, and secretion of cytokines, which ultimately affects their antitumor capacity.

In addition, interactions and synergies between different immune cells are worthy of attention. For example, CD8⁺ T cells, as the main antitumor effector cells, are downstream of many immune cell functions. Therefore, regulation of macrophage and DC function by m⁶A ultimately affects their antitumor function by influencing the recruitment, function, and activation of CD8⁺ T cells.^21,81,85

m⁶A regulates the immune response by affecting tumor cells

In addition to its direct regulatory role in a variety of immune cells, m⁶A regulates antitumor immunity by indirectly regulating tumor cell function. As recent studies have shown, three main mechanisms, surface molecule expression, cytokine secretion, and metabolism reprogramming, are involved in m⁶A regulation of tumor cells, and these mechanisms are described below.

Expression of surface molecules

Tumor cells express surface molecules associated with tumor immunity, especially immune checkpoints such as PD-L1, CTLA-4, and CD276, which play important roles in regulating antitumor immunity by costimulating or coinhibiting the killing effect of T cells and are thus key targets for immunotherapy.

Several studies have demonstrated that PD-L1 expression in tumor cells is closely correlated with m⁶A modification of RNA. METTL3, a typical m⁶A methyltransferase, suppresses tumor immune surveillance in breast cancer.¹⁰⁴ Knocking out METTL3 results in decreased m⁶A modification abundance on PD-L1 mRNA and, thus, reduced PD-L1 mRNA stability, leading to less IGF2BP3 binding to PD-L1 mRNA and downregulating PD-L1 expression.¹⁰⁴ Similarly, in bladder cancer, METTL3 induced c-Jun N-terminal kinase (JNK) signaling activation to stabilize PD-L1 mRNA by promoting the interaction between the m⁶A site and IGF2BP1.¹⁰⁵ In addition, METTL3 also mediates immune escape by affecting ncRNA modifications. METTL3 mediates the m⁶A modification of circIGF2BP3 in NSCLC and promotes cyclization of circIGF2BP3 by binding to YTHDC1, thereby increasing the content of circIGF2BP3.¹⁰⁶ This increase in the circIGF2BP3 level in NSCLC contributes to tumor cell immune evasion by increasing PD-L1 levels through reduced PD-L1 ubiquitination and subsequent proteasomal degradation.¹⁰⁶ METTL14 is another important m⁶A writer. METTL14 reduces the degradation of PD-L1 via the ubiquitination-dependent pathway by destabilizing Siah2 mRNA in a YTHDF2-dependent manner in cholangiocarcinoma (CCA).¹⁰⁷ In addition, in HCC, LPS-induced METLL14 expression increased m⁶A modification abundance on the lncRNA MIR155HG, thereby stabilizing MIR155HG by binding to ELAVL1 (HuR) and subsequently increasing PD-L1 expression via the miR-223/STAT1 axis.¹⁰⁸ Regarding reader proteins, knockdown of IGF2BP1 reduced PD-L1 expression and promoted immune infiltration in HCC.¹⁰⁹ In addition to the abovementioned regulators, methionine and YTHDF1 have been shown to promote PD-L1 expression.¹¹⁰ FTO and ALKBH5 are the two main m⁶A demethylases. Conditional deletion of the ALKBH5 gene in intrahepatic CCA (ICC) cells increases m⁶A abundance on PD-L1 mRNA and increases PD-L1 mRNA decay by binding to YTHDF2.¹¹¹ Knocking down FTO in melanoma enhances sensitivity to anti-PD-1 and IFN therapies by suppressing PD-1 (PDCD1), CXCR4, and SOX10 expression through YTHDF2-mediated mRNA decay, but it does not affect PD-L1 (CD274).¹¹² In general, METTL3, METTL14, IGF2BP1, FTO, and ALKBH increase the level of PD-L1 on the membrane and thereby promote tumor cell immune escape. In summary, these mechanisms of PD-L1 regulation can either directly upregulate PD-L1 expression or reduce PD-L1 protein degradation. Therefore, speculating that inhibitors of METTL3, IGF2BP, FTO, and ALKBH may be promising targets for cancer therapy is reasonable, and moreover, combination of these inhibitors with PD-1 inhibitors may enhance the efficacy of anti-PD-1 therapy to overcome drug resistance. This possibility has been confirmed by numerous studies.¹⁰⁹¹¹⁰ However, m⁶A on CBX1 mRNA decreases its stability via YTHDF3 recognition, and CBX1 upregulates PD-L1 expression via the IFN-γ-STAT1 pathway,¹¹³ suggesting diverse functions of m6A in tumor immunity (Figure 7A; Table 1).

Figure 7.

The mechanisms by which m⁶A modification induces immune evasion in tumor cells

(A) FTO promotes ILIRB4 (an immune checkpoint) expression by targeting its mRNA. METTL3 and ALKBH5 upregulate the expression of PD-L1 by targeting PD-L1 mRNA, thus enhancing immunosuppression. METTL3 also regulates ncRNAs (circIGF2BP3 and lncMIR155HG). METTL14 negatively regulates Siah2 (a RING E3 ubiquitin ligase that enhances PD-L1 degradation) to increase PD-L1.

(B) FTO increases the metabolic barrier for T cell activation and inhibits the function of CD8⁺ T cells targeting JunB and C/EBPb (glycolysis regulators). ALKBH5 enhances lactate content in tumor-infiltrating Tregs and MDSCs by targeting Mct4/Slc16a3.

(C) ALKBH5 inhibits production of IFN-γ and IL-8 via the ALKBH5/RIG-I/IFNα axis, the ALKBH5/paraspeckle/CXCL8 axis, and the ALKBH5/MAP3K8 axis. METTL3 promotes CXCL1 production by targeting BHLHE4 (which binds to the promoter region of CXCL1) and induces immunosuppression. METTL3/14 also inhibits IFN-γ, CXCL9, and CXCL10 by inhibiting IFN-γ-STAT1-IRF1 signaling. RBM-15 promotes CXCL11 production, thus inducing M2 polarization.

In addition to that of PD-1/PD-L1, the expression of other immune checkpoint ligands is regulated by m⁶A modifications. FTO has been shown to play a role in maintaining immune evasion in leukemia.¹¹⁴ LILRB4 is an important immune checkpoint, and reduced LILRB4 expression enhances T cell toxicity and antitumor immune responses. Inhibition or depletion of FTO increases degradation of m⁶A-modified LILRB4 mRNA, primarily by promoting m⁶A-modified LILRB4 mRNA binding to YTHDF2¹¹⁴ (Table 1).

In addition to the checkpoint ligands discussed above, expression of many other surface molecules regulates the immune response. YTHDF1 has been found to be overexpressed in gastric cancer (GC) and has been identified as an oncogene because of its induction of cell proliferation, and it has also been found to be associated with the antitumor immune response.¹¹⁵ Deletion of YTHDF1 in GC cells led to increased DC recruitment and T cell infiltration because of increased levels of IFNγ receptor 1 (IFNGR1) on the surface of tumor cells.¹¹⁵ However, it is not clear whether this effect is mediated by m⁶A.

Metabolic reprogramming

Metabolic reprogramming of tumor cells can reshape the tumor microenvironment and thus affect the immune response; moreover, the vital role played by m⁶A in cancer metabolism has been fully proven.^116,117 Notably, m⁶A controls tumor immunity via metabolic reprogramming, which is discussed below. Deletion of the m⁶A demethylase ALKBH5 has been found to increase the sensitivity of melanoma and colon cancer to immunotherapy.¹¹⁸ ALKBH5 deletion alters the metabolic profile of tumor cells and reduces lactate levels in the tumor immune microenvironment by altering the splicing and expression of target genes (Mct4/Slc16a3) via an m⁶A-dependent mechanism.¹¹⁸ This altered metabolism, in turn, leads to attenuation of recruitment of immunosuppressive cells such as Tregs and myeloid-derived suppressor cells (MDSCs).¹¹⁸ Another m⁶A demethylase, FTO, has also been found to be associated with tumor cell escape from immune surveillance through its effect on glycolytic metabolism.¹¹⁹ Notably, the FTO gene reduces the levels of basic leucine zipper (bZIP) transcription factors (JunB and CCAAT/enhancer binding protein-b(C/EBPb) ) in the m⁶A/YTHDF2 pathway in B16-OVA cells, which reduces gluconeogenic activity and facilitates the infiltration and function of CD8⁺ T cells by removing the metabolic barrier to their activation.¹¹⁹ YTHDF1 also potentiates glycolysis. circRHBDD1 is a novel circRNA that enhances the glycolysis rate and glycolysis capacity via the phosphatidylinositol 3-kinase (PI3K)/AKT pathway, and its inhibition enhances the anti-PD-L1 therapeutic effect.¹²⁰ Mechanistically, circRHBDD1 promotes YTHDF1 binding to PIK3R1 mRNA, thereby facilitating PIK3R1 translation and activating downstream signaling pathways.¹²⁰ At present, studies of how m⁶A modification affects metabolic reprogramming to alter immune responses are focused mainly on glucose metabolism, and the regulation of other tumor metabolism pathways needs to be investigated further (Figure 7B; Table 1).

Secretion of immune-related factors and other mechanisms

The immune response can be regulated by immune-related factors, including chemokines, cytokines, and IFNs, in the tumor microenvironment. An increasing number of studies have proven the connection between m⁶A and these immune-related factors. For example, METTL3 or METTL14 deficiency in colorectal carcinoma cells increases recruitment of CD8⁺ T cells and the levels of IFN-γ, CXCL9, and CXCL10 in the tumor microenvironment.¹²¹ Mechanistically, STAT1 and INF1 mRNA stability is negatively regulated in an m⁶A-Ythdf2-dependent manner, and IFN-γ-STAT1-IRF1 signaling affect the immune response.¹²¹ In another study, METTLE3 facilitated MDSC accumulation in colorectal cancer by promoting CXCL1 expression and thereby compromised the function of CD4⁺ and CD8⁺ T cells.¹²² Notably, m⁶A regulates CXCL1 expression not by directly regulating CXCL1 mRNA but by promoting the translation efficiency of BHLHE41, a transcription factor that binds the promoter region of CXCL1.¹²² RBM-15 has been identified as an essential component of writer complexes. Similarly, RBM-15 has been shown to promote CXCL11 expression, although it accomplishes this through its direct regulation of CXCL11 mRNA stability, thereby increasing macrophage infiltration and M2 polarization.¹²³ ALKBH5 has been identified as another regulator that fosters immunosuppression. It promotes secretion of CXCL8/IL-8 and recruitment of TAMs under anoxic conditions in a glioblastoma multiforme (GBM) model.¹²⁴ ALKBH5 regulates CXCL8/IL-8 not by directly removing m⁶A modifications on CXCL8 mRNA but by stabilizing the lncRNA NEAT1; hence, the transcriptional repressor splicing factor proline and glutamine rich (SFPQ) is removed from the CXCL8 promoter and transferred to paraspeckles.¹²⁴ Similarly, another study showed that ALKBH5 upregulates IL-8 expression in HCC, thereby increasing PD-L1⁺ macrophage recruitment and facilitating immunosuppressive microenvironment formation.¹²⁵ Mechanistically, ALKBH5 destabilizes MAP3K8 mRNA in a YTHDF2-mediated manner and activates the JNK/extracellular signal-regulated kinase (ERK) pathway.¹²⁵ In head and neck squamous cell carcinoma (HNSCC), knocking down ALKBH5 inhibits tumor progression. Depletion of ALKBH5 increases m⁶A abundance on DDX58 mRNA and increases retinoic acid-inducible gene I (RIG-I) expression by increasing DDX58 mRNA maturation in a manner dependent on the function of the m⁶A reader HNRNPC.¹²⁶ RIG-I positively regulates secretion of IFNα through the inhibitor of kappa B kinase ε (IKKε)/TBK1/IRF3 pathway (Figure 7C; Table 1).

In addition, tumor cells regulate antigen expression and the IFN response through modulation of m⁶A modifications. The YY1-CDK9 transcription elongation complex affects the expression levels of METTL3 and YTHDF2, thus inhibiting the IFN response and antigen presentation in glioblastoma stem cells (GSCs).¹²⁷ Mechanistically, IFN-related genes (IFNB1, STAT1, and IRF1) are stabilized in an m⁶A-dependent manner, and IFN signaling is enhanced when the complex is inhibited.¹²⁷

In summary, the roles played by m⁶A regulators in tumor cells are more consistent than the complex roles they play in immune cells. As an immune evasion regulator in tumors, m⁶A upregulates the expression of immune checkpoints and promotes recruitment of immunosuppressive cells such as Tregs, MDSCs, and M2 macrophages, thus contributing to an immunosuppressive tumor microenvironment. Therefore, targeting the m⁶A pathway may be a new strategy for tumor immunotherapy.

The m¹A RNA modification in tumor immunity

Overview of the m¹A RNA modification

The m¹A modification, which was first discovered decades ago, was initially recognized as a reliable way to regulate the function and stability of transfer RNA (tRNA) and rRNA.^128,129,130 More recently, m¹A modifications have been shown to mark eukaryotic mRNAs.¹³¹ However, in contrast to the m⁶A modification, which preferentially decorates stop codons and 3′ UTRs, the m¹A modification is enriched mainly on start codons upstream of the first splice site and 5′ UTRs of mRNA transcripts.^131,132 M1ARegpred¹³³ and RAMPred¹³⁴ are powerful tools for predicting m¹A modification sites (Table 3).

Similar to the m⁶A modification, the m¹A modification is also regulated by specific regulators. The methyl group in the m¹A modification is mainly added to mitochondrial and cytoplasmic RNAs by specific writers. tRNA methyltransferase 6 noncatalytic subunit (TRMT6) and RNA methyltransferase 61A (TRMT61A) form the α2β2 heterotetramer TRMT6/61A,¹³⁵ which is a methyltransferase targeting cytoplasmic tRNAs, while TRMT61B¹³⁶ and TRMT10C¹³⁷ are tRNA methyltransferases that act mainly in mitochondria.¹³⁸ Moreover, TRMT6/TRMT61A and TRMT10C catalyze the m¹A modification of mRNAs in the cytoplasm and mitochondria.¹³⁸ In addition, nucleomethylin (NML) has been discovered to be a writer for 28S rRNA m¹A modification.¹³⁹ Some readers have also been identified. YTHDF proteins, first identified as m⁶A readers, have been shown to recognize and bind to the m¹A modification.¹⁴⁰ Their roles as m¹A-binding proteins remain to be further elucidated. In terms of erasers, ALKBH1,¹⁴¹ ALKBH3,¹⁴² ALKBH7,¹⁴³ and FTO¹⁴⁴ can remove m¹A on tRNA, while ALKBH3 can also target modifications on mRNA.¹³²

Generally, m¹A is crucial for the structure and function of tRNAs and mRNAs. m¹A modifications were first identified on tRNAs, and the positive electrostatic charge carried by m¹A has been shown to be essential for maintaining the structure and function of tRNAs.¹⁴⁵ In a subsequent study, ALKBH1 was found to act as a demethylase that removes methyl groups from m¹A modifications on tRNAs and tRNA^iMet, thereby reducing translation initiation and translation elongation.¹⁴¹ Recently, m¹A modifications were found on mRNAs and disrupted Watson-Crick base pairing.¹³¹ Their function on mRNAs is still being explored. Li et al.¹³² found that m¹A on 5′ UTRs is associated with higher translational efficiency, whereas m¹A on mitochondrial mRNAs, located mainly in the coding sequence (CDS), inhibits translation. Consistent with these findings, ALKBH3 knockdown results in the downregulation of ErbB2 and AKT1S1 expression.¹⁴⁶ However, Safra et al.¹³⁸ found that only low m¹A abundance on mRNA in the cytosol results in translational repression. In addition to affecting the translation efficiency of the target mRNA, ALKBH3, a targeted mRNA m¹A demethylase, reduces the stabilization of macrophage colony-stimulating factor (CSF-1) mRNA.¹⁴⁷ RNA-binding proteins may play essential roles in the function of m¹A. YTHDF1 binds to m¹A on ATP5D mRNA and subsequently forms a complex with eRF1 to terminate translation.¹⁴⁸ In addition, YTHDF2 and YTHDF3 have been demonstrated to reduce the stability of target transcripts^149,150 (Figure 1).

Aberrant m¹A RNA modification in tumor immunity

Previous studies have revealed a relationship between m¹A modification and tumorigenesis; for example, m¹A modification of tRNAs drives liver tumorigenesis through activation of cholesterol synthesis.¹⁵¹ However, the relationship between m¹A methylation and tumor immunity remains largely unknown. A recent study reported that tRNA-m¹A modification was essential for rapid T cell proliferation.¹⁵² TRMT61A, which has been recognized as a typical m¹A writer, is crucial for CD4⁺ T cell immune function, as indicated by an experiment in which its absence led to arrest of activated CD4⁺ T cells.¹⁵² Further studies revealed that this effect is mediated by tRNA-m¹A enhancement of the translation efficiency of Myc mRNA, mainly mediated by an improved elongation process, which enhances expression of Myc and ultimately regulates cell cycling and metabolism.¹⁵² In addition, the analysis showed that m¹A regulatory genes are positively correlated with immune cell infiltration and that YTHDF3, which had been identified previously as an m¹A reader, may regulate macrophage activation.¹⁵³ Bioinformatics studies have largely aimed to study m¹A regulators, including three writers (TRMT6, TRMT61A, and TRMT10C), two erasers (ALKBH1 and ALKBH3), and four readers (YTHDF1, YTHDF2, YTHDF3, and YTHDC1). In oral squamous cell carcinoma,¹⁵⁴ colon cancer,¹⁵⁵ and HCC,¹⁵⁶ the m¹A score was found to be negatively correlated with patient prognosis and immune cell infiltration. In contrast, in ovarian cancer¹⁵⁷ and lung adenocarcinoma,¹⁵⁸ the m¹A score was found to be positively correlated with prognosis and immunotherapy outcome. In ovarian cancer, innate immune cells were increased in the high m¹A score subpopulation, whereas adaptive immune cells were increased in the low m¹A score subpopulation.¹⁵⁷ The m1A score showed a positive correlation with almost all immune cells in lung adenocarcinoma.¹⁵⁸ The differences in these outcomes are rarely studied, and further research regarding recent theories is urgently needed.

The m⁵C RNA modification in tumor immunity

Introduction to the m⁵C RNA modification

The m⁵C RNA modification involves methylation of cytidine residues at position 5. It is widespread on rRNAs, tRNAs, other ncRNAs, and mRNAs. The m⁵C modification is conserved in tRNAs and rRNAs, but in mRNAs, the m⁵C modification is located primarily in 5′ UTRs and 3′ UTRs.¹⁵⁹ Various m⁵C prediction models based on different machine learning algorithms have emerged; for example, Chen et al.¹⁶⁰ predicted other modification sites with various machine learning models based on known m⁵C modification sites on mRNAs in humans and mice. Further predictive models are described in Table 3.

Similar to m⁶A and m¹A modifications, m⁵C modifications are regulated by writers, readers, and erasers. m⁵C modification relies mainly on the NOL1/NOP2/sun (NSUN) family, which includes NSUN1,¹⁶¹ NSUN2,¹⁶² NSUN3,¹⁶³ NSUN4,¹⁶⁴ NSUN5, NSUN6,¹⁶⁵ NSUN7, and the DNA methyltransferase homologue DNMT2.¹⁶⁶ These writers mainly catalyze methylation on tRNAs (NSUN2, NSUN3, NSUN6, and DNMT2), rRNAs (NSUN1, NSUN4, and NSUN5), and other ncRNAs (NSUN2 and NSUN7) in the nucleus and mitochondria, while NSUN2¹⁶⁷ and NSUN6¹⁶⁸ mediate m⁵C modification on mRNAs. m⁵C function partially depends on RNA-binding proteins, and research in this area is still in an early stage. RNA and export factor-binding protein 2 (ALYREF), Y-box-binding protein 1 (YBX1), ypsilon schachtel (YPS), and YTHDF2 are the currently known m⁵C-binding proteins,^{167,169,170,171} and their functions are described below. Recently, ten-eleven translocation (TET) family members and ALKBH1 were identified as m⁵C demethylases that dynamically regulate m⁵C content.¹⁷²

m⁵C controls the fate of modified RNA. Research on m⁵C initially focused on rRNAs and tRNAs. Regarding its effect on rRNAs, m⁵A regulates rRNA maturation,¹⁷¹ rRNA stability,¹⁶¹ and protein translation.^161,173 In tRNAs, m⁵A is involved in stabilizing tRNA structure¹⁷⁴ and regulating selective translation during oxidative stress.¹⁷⁵ NSUN2 mediates methylation of tRNAs, reducing tRNA cleavage by nucleic acid endonucleases and thereby increasing tRNA stability, ultimately affecting translation efficiency.¹⁷⁶ In addition, TET mediates oxidation of m⁵C in tRNAs and promotes translation.¹⁷⁷ In recent years, with advances in detection methods, studies on m⁵C modification of mRNAs have gradually increased. NSUN2 has been found to also mediate m⁵C modification of mRNAs; ALYREF is an mRNA export adaptor that specifically binds to the m⁵C site after mRNA m⁵C modification to regulate targeted mRNA export.¹⁶⁷ In addition, ALYREF enhances the stability of pyruvate kinase M2 (PKM2) mRNA.¹⁷⁸ Similarly, YBX1 has been shown to stabilize mRNA by recruiting ELAVL1, which is another important m⁵C reader protein.¹⁷⁹ In addition to affecting stability and export, YBX1 regulates translation of mRNAs. m⁵C methylation of IL-17A mRNA and intercellular adhesion molecule 1 (ICAM-1) mRNA promotes their translation.^180,181 Moreover, NSUN2-mediated m⁵C methylation has also been shown to synergize with METTL3/METTL14-mediated m⁶A methylation to promote translation of p21 mRNA.¹⁸² However, m⁵C methylation catalyzed by NSUN2 had the opposite effect on p27 mRNA translation, suppressing its gene expression.¹⁸³ Furthermore, m⁵C modifications are involved in regulation of mitochondrial function, stem cell development and differentiation, neurological development, cellular senescence, and other processes^184,185,186 (Figure 1).

Aberrant m⁵C RNA modifications in tumor immunity

m⁵C modification is associated with development of GC,¹⁸⁷ bladder cancer,¹⁷⁹ HCC,¹⁸⁸ esophageal squamous cell carcinoma,¹⁸⁹ and other tumors. In addition, in previous studies, the m⁵C demethylase TET has been proven to affect the function of immune cells.^190,191,192 However, research on m⁵C in tumor immunity is still in its infancy. Previous studies have shown that NSUN2 methylates IL-17A mRNA and thereby increases IL-17 expression in T lymphocytes,¹⁸⁰ and it can also increase leukocyte adhesion by methylating ICAM-1 mRNA.¹⁸¹ Regarding tumor immunity, the m⁵C reader YBX1 has been identified as a positive regulator of PD-L1 that induces immune evasion.¹⁹³ Recent studies have shown that m⁵C RNA methylation regulators are prognostic predictors in certain cancers and can regulate immune cells in the tumor microenvironment. NSUN2 and NSUN6 have been identified as risk and protective factors, respectively, in triple-negative breast cancer (TNBC) and have been associated with six major immune cells, with the highest correlation between NSUN6 and CD4⁺ T cells.¹⁹⁴ In another study, NSUN2 was found to negatively regulate immune cell infiltration, while high NSUN2 expression was associated with downregulation of multiple immune checkpoints.¹⁹⁵ NSUN3 and NSUN4 have also been found to be related to immune cells in lung squamous cell carcinoma (LUSC); notably, NSUN3 was closely associated with CD8⁺ T cell infiltration, and NSUN4 was closely associated with neutrophils.¹⁹⁶ m⁵C modifications may enhance humoral immunity, and in HNSCC, DNMT1 expression levels promote peptide cross-linking.¹⁹⁷ In addition, the m⁵C eraser TET2 has also been shown to be significantly associated with infiltration of many immune cells. In papillary thyroid carcinoma,¹⁹⁸ oral squamous cell carcinoma,¹⁹⁹ and renal cell carcinoma,²⁰⁰ m⁵C scores were found to correlate with different immune phenotypes. A high m⁵C score (associated with an immunosuppressive phenotype) predicted a poor prognosis, while a low m⁵C score (associated with an immune-activating phenotype) predicted a good prognosis. Furthermore, in a study in prostate cancer, naive B cells, CD8⁺ T cells, M1 macrophages, and M2 macrophages were identified as key immune cells involved in distinguishing m⁵C-related immune phenotypes, and CTLA4 was differentially expressed between the two phenotypes.²⁰¹

Discussion

In this review, we focused on the relationship between m⁶A, m¹A, and m⁵C modifications and tumor immunity and outlined different mechanisms by which RNA modifications affect antitumor immunity. Although RNA has over 100 modifications, the majority of studies have focused on m⁶A. In this review, we not only discussed m⁶A extensively but also focused on m¹A and m⁵C, which have differences and similarities with m⁶A. On the one hand, these RNA modifications are all dependent on regulation of writers, erasers, and readers. In the presence of these regulators, RNA modifications are reversible and are precisely regulated to perform biological functions. All modifications are able to regulate the stability and translational efficiency of target RNAs. On the other hand, most of the more than 170 RNA modifications identified so far have been identified to occur on ncRNAs.²⁰² m⁶A modifications are mainly found on mRNAs, whereas m¹A and m⁵C modifications are mainly found on tRNAs and rRNAs²⁰³ and are present at much lower levels on mRNAs.²⁰⁴ While numerous studies have revealed the role played by m⁶A in tumor immunity, studies of m¹A and m⁵C are still in the initial stages, and more investigation is needed. Recent studies have demonstrated the crucial roles played by m¹A and m⁵C in tumorigenesis and immunity. For instance, the m⁵C modification at position 34 on mitochondrial tRNA^Met promotes metastasis by driving mitochondrial mRNA translation, increasing mitochondrial function, and affecting metabolic plasticity in tumors,²⁰⁵ indicating a pivotal function of m⁵C in tumorigenesis. TRMT61A mediates tRNA-m¹A58 modification and is crucial for rapid T cell activation because it enhances the translation efficiency of Myc mRNA,¹⁵² indicating an essential role of m¹A in regulating immunity. Therefore, it has been speculated that m¹A modification is a vital regulator of tumor immunity. More studies should be carried out to determine the exact function of m¹A and m⁵C in tumor immunity.

In addition, other types of RNA modifications have also been found to play a key role in regulation of tumor immunity. For example, the immunomodulatory function of A-to-I editing, which is catalyzed by adenosine deaminases acting on RNA (ADARs), has been described in previous studies.²⁰⁶ Recently, a new mechanism underlying the cytotoxicity of 6-thioguanine (6TG), a widely used chemotherapeutic agent, was found.²⁰⁷ 6TG increased A-to-I editing in bladder cancer-associated protein (BLCAP) mRNA, eventually decreasing cell viability.²⁰⁷ Moreover, knockdown of ADAR1 in tumor cells was found to enhance the response to ICIs by overcoming inactivation of antigen presentation.²⁰⁸ In addition to A-to-I editing, m⁷G modification of tRNA has recently been found to promote formation of an immunosuppressive microenvironment in HCC tumors mediated via the METTL1-transforming growth factor β2 (TGF-β2)-MDSC axis.²⁰⁹ Further exploration focusing on other types of RNA modifications is also needed.

Notably, increasing evidence suggests the potential for cross-talk between different RNA modifications. m⁶A, m¹A, m⁵C, and other RNA modifications, including A-to-I editing and m⁷G²¹⁰ and pseudouridine (Ψ)²¹¹ modification, have been found to modify mRNA. The cross-talk of mRNA modifications includes interactions of content and function. First, the abundance of RNA modifications on mRNA is modulated by other modifications. For instance, there is a negative correlation between m6A and A-to-I editing, two of the most abundant RNA modifications affecting mRNA, and both occur at adenosine.²¹² Second, different modifications on mRNA cooperate in regulating the fate of mRNA. For example, NSUN2-mediated m⁵C methylation promotes METTL3/METTL14-mediated m⁶A methylation in p21 mRNA and enhances p21 expression and vice versa.¹⁸² HRSP12 binds to m¹A and promotes the interaction between m⁶A modifications and YTHDF2, thus cooperatively promoting rapid mRNA degradation.²¹³ Additionally, modifications of ncRNAs can also cross-talk with modifications of mRNAs. For example, the tRNA methyltransferase TRMT10A not only installs N1-methylguanosine (m¹G) on tRNA but also facilitates the m⁶A demethylase activity of FTO and potentially accelerates degradation of mRNA in a YTHDF2-dependent manner.²¹⁴ However, the precise coregulation networks involving different RNA modifications and the impact of these interactions on tumor immunity remain largely unclear; these topics are promising areas for further research.

Furthermore, studies have revealed that different epigenetic modifications, including DNA methylation, chromatin remodeling, histone modification, and RNA modification, do not function independent of each other and share interaction networks.²¹⁵ For example, m⁶A modification of CBX1 (a histone methylation regulator) mRNA destabilized the transcript via the m⁶A reader YTHDF3.¹¹³ In addition, METTL3/METTL14 downregulates H3K9me2 modification by YTHDC1-mediated recruitment of the H3K9me2 demethylase KDM3B to m⁶A-associated chromatin regions, which indicates that there is direct information flow from RNA to chromatin.⁷¹ On the other hand, m⁶A modification of mRNA can be regulated by histone modifications.²¹⁶ For example, histone H3 trimethylation at Lys36 (H3K36me3) can be directly recognized by METTL14 and promote m⁶A modification of nascent RNAs to coregulate transcription, with MTC interacting with adjacent RNA polymerase II molecules.²¹⁷ Recent studies have shown a similar regulatory mechanism between DNA methylation and RNA modifications. For instance, RNA m⁶A guides DNA 5-methylcytosine (5mC) demethylation via the m⁶A reader FXR1, recruiting TET1 to genomic loci and reprogramming chromatin accessibility.²¹⁸ However, the precise cross-talk between different RNA modifications that regulate the antitumor immune response needs to be further explored and verified.

In summary, RNA modifications are closely associated with tumor immunity and may be novel targets for preventing immune escape. The regulation of oncogenic signaling pathways by RNA modifications revealed in previous studies has identified potential drug targets, such as the Wnt/β-catenin pathway, PI3K-Akt-mTOR pathway, and JAK-STAT pathway.²¹⁹ In this review, we examined RNA modifications and tumor immunity, offering ideas for therapy for many patients who have shown poor responses to existing immunotherapies. Inhibitors of ALKBH5/FTO and targeted deletion of METTL3/14 have been shown to enhance the effects of PD-L1 inhibitor immunotherapy by enhancing the expression of immune checkpoints and secretion of cytokines.^112118121 In addition, RNA-modified regulators hold promise for development as biological indicators of cancer prognosis and response to immune checkpoint blockade. Further clinical studies are urgently needed. However, current applications generally remain limited by the lack of relevant small-molecule inhibitors and unknown mechanisms of action. A deeper understanding of the biological mechanisms is conducive to selection of more efficient targets and drugs. These outcomes might further improve the survival time of cancer patients and their quality of life.

Published Online: May 29, 2023

Lead contact website

https://www.shsmu.edu.cn/info/1026/5530.htm

http://daoshi.shsmu.edu.cn/Pages/TeacherInformationView.aspx?uid=3823333C-AD6F-44C4-9118-C632E793BCBD&from=s&pId=100212&tId=