RNA N ⁶‐methyladenosine modification in the lethal teamwork of cancer stem cells and the tumor immune microenvironment: Current landscape and therapeutic potential

Abstract

N⁶‐methyladenosine (m⁶A), the newest and most prevalent layer of internal epigenetic modification in eukaryotic mRNA, has been demonstrated to play a critical role in cancer biology. Increasing evidence has highlighted that the interaction between cancer stem cells (CSCs) and the tumor immune microenvironment (TIME) is the root cause of tumorigenesis, metastasis, therapy resistance, and recurrence. In recent studies, the m⁶A modification has been tightly linked to this CSC‐TIME interplay, participating in the regulation of CSCs and TIME remolding. Interestingly, the m⁶A modification has also been identified as a novel decisive factor in the efficacy of immunotherapies—particularly anti‐PD‐1/PD‐L1 monotherapies—by changing the plasticity of the TIME. Given the functional importance of the m⁶A modification in the crosstalk between CSCs and the TIME, targeting m⁶A regulators will open new avenues to overcome therapeutic resistance, especially for immune checkpoint‐based immunotherapy. In the present review, we summarize the current landscape of m⁶A modifications in CSCs and the TIME, and also prospect the underling role of m⁶A modifications at the crossroads of CSCs and the TIME for the first time. Additionally, to provide the possibility of modulating m⁶A modifications as an emerging therapeutic strategy, we also explore the burgeoning inhibitors and technologies targeting m⁶A regulators. Lastly, considering recent advances in m⁶A‐seq technologies and cancer drug development, we propose the future directions of m⁶A modification in clinical applications, which may not only help to improve individualized monitoring and therapy but also provide enhanced and durable responses in patients with insensitive tumors.

In this review, we evaluate the current landscape of m6A modification in the lethal teamwork of CSCs and the TIME for the first time. In addition, we summarize the existing inhibitors targeting m⁶A regulators and discuss the potential of modulating m⁶A modification as a therapeutic strategy. Moreover, we propose the future directions of using m⁶A modification in cancer diagnosis and treatment, as well as providing an overview of the emerging approaches to m⁶A‐targeted immunotherapy. We believe that this review will provide novel insight into the role of m⁶A modification in cancer‐specific basic and translational medicine.

1. BACKGROUND

Since RNA modifications were first reported in 1951,¹, ² more than 150 types have been discovered owing to advances in high‐throughput sequencing technology.³ Of these, N ⁶‐methyladenosine (m⁶A) is recognized as the most essential and widespread type of modification in eukaryotic mRNAs and noncoding RNAs.⁴ A lack of advanced sequencing technologies has prevented any major breakthroughs in this field over past decades; however, with the successive discoveries of m⁶A regulatory components, the importance and function of the m⁶A modification has gradually been revealed. High‐throughput sequencing technologies have identified that m⁶A modification sites are often enriched in the coding sequence, 3’ untranslated region (3’UTR), and in the vicinity of stop codons.¹, ⁵, ⁶ As with DNA and protein modifications, RNA m⁶A modification is a dynamic, reversible, and multilayered process that alters target gene expression based on the three types of m⁶A regulators (methyltransferases, demethylases, and binding proteins).⁷, ⁸ To date, numerous studies have demonstrated its involvement in various physiological and pathological processes, most notably in tumorigenesis.⁹

Increasing evidence suggests that the m⁶A modification plays a nonnegligible role in the evolution and progression of multiple tumors. It is noteworthy that a number of studies have found that m⁶A is engaged in the maintenance and modulation of the stemness property of cancer stem cells (CSCs).¹⁰, ¹¹, ¹² CSCs are a small subpopulation of tumor cells that possess self‐renewal and clonal tumor initiation potential, and are often regarded as one of the sources of tumor relapse, metastasis, and therapeutic resistance.¹³ Meanwhile, the tumor immune microenvironment (TIME), the lethal synthesis partner of CSCs, unites CSCs to form an ecological system for the acceleration of tumor progression.¹⁴, ¹⁵, ¹⁶ From the perspective of the seed and soil hypothesis, CSCs can be likened to the most tenacious intrinsic seed, while the TIME tends to represent the fertile soil conductive to the growth and survival of CSCs, which collude to promote a more malignant tumor phenotype with stronger metastatic and invasive capacity.¹⁷, ¹⁸ Theoretically, CSCs are able to remodel the TIME but are also inversely affected by signals originating from it.¹⁵, ¹⁹ Notably, it has also been reported that both CSCs and the TIME are intimately associated with resistance to the most promising immunotherapy, in particular immune checkpoint blockade therapy, and disruption of the CSC‐TIME interplay is a critical step in reducing resistance propensity and enhancing antitumor activity.²⁰, ²¹, ²², ²³ Intriguingly, m⁶A modification not only actively participates in the remodeling of TIME processes but also appears to be involved in the crosstalk between CSCs and the TIME during various immune responses and hypoxia‐related reactions.²⁴, ²⁵, ²⁶, ²⁷ It is reasonable to infer that m⁶A may be a pivotal factor in this deadly teamwork.

To date, the function of the m⁶A modification in the interaction between CSCs and the TIME remains under active investigation. In the present review, we provide a novel perspective on the role of the m⁶A regulatory network with respect to CSCs and the TIME. Additionally, we discuss the relevant molecular mechanisms and potential therapeutic strategies based on m⁶A modification, providing an overview of targeting the crosstalk between CSCs and the TIME for cancer therapies.

2. THE REGULATORY COMPONENTS OF RNA m⁶A MODIFICATION

Similar to DNA and protein modifications, RNA m⁶A modification is a dynamic, reversible, and multilayered process that is determined by m⁶A‐specialized methyltransferases (writers), demethylases (erasers), and binding proteins (readers)²⁸ (Figure 1). Owing to significant developments in the field of epitranscriptomics, a large number of writers, erasers, and readers have been identified.

FIGURE 1.

The cellular m⁶A machinery. m⁶A modification is a dynamic, reversible, and multilayered process. The writer complex has been identified as METTL3, METTL14, WATP, RBM15/15B, KIAA1429, and ZC3H13, which adds m⁶A methylation on target RNAs. The two erasers, FTO and ALKBH5, remove the m⁶A methylation from target RNAs. m⁶A is recognized by diverse readers, mainly HNRNPC, HNRNPG, HNRNBPA2B1, YTHDC1/2, and YTHDF1/2/3, which mediate various posttranscriptional processes including mRNA export, splicing, stabilization, decay, and translation, in addition to miRNA processing.

2.1. m⁶A methyltransferases/writers

m⁶A writers are capable of adding m⁶A to RNA, which is regarded as the installation of the m⁶A modification, and often form a multisubunit complex to exert their effects, which includes methyltransferase‐like 3 (METTL13), methyltransferase‐like 14 (METTL14), wilms tumor 1‐associated protein (WTAP), vir‐like m⁶A methyltransferase associated (VIRMA/KIAA1429), RNA‐binding motif protein 15/15B (RBM15/15B), and zinc finger CCCH‐type containing 13 (ZC3H13). METTL3 and METTL14 are the core subunits of this methyltransferase complex, with METTL3 playing a leading enzyme‐catalyzing role and METTL14 being less enzymatically efficient. In addition, METTL14 also functions to stabilize METTL3 and recognize the target RNA.²⁹ Despite lacking catalytic function, WTAP facilitates the localization of the METTL3/14‐METTL14 heterodimer to nuclear speckles.³⁰ The function of ZC3H13 is to maintain and enhance the nuclear localization of the writer complex.³¹ KIAA1429 and RBM15/15B are responsible for ensuring that this complex is recruited in a specific region to exert catalytic action.³², ³³, ³⁴ In addition to this classical writer complex, other m⁶A methyltransferases have been consecutively discovered: methyltransferase‐like 5 (METTL5), methyltransferase‐like 16 (METTL16), zinc finger CCHC‐type containing 4 (ZCCHC4), phosphorylated CTD interacting factor 1 (PCIF1), and NOP2/Sun RNA methyltransferase 2 (NSun2). PCIF1 performs m⁶A methylation on 2‐O‐methylated adenine located at the 5’ end of mRNAs.³⁵ With the exception of PCIF1, these writers are involved in the m⁶A modification of noncoding RNAs. In particular, METTL5 and ZCCHC4 are responsible for adding m⁶A on 18S and 28S ribosomal RNAs.³⁶, ³⁷ METTL16 is specifically engaged in the m⁶A modification of U6 small nuclear RNAs and also regulates the expression of MAT2A.³⁸ Nsun2 actively participates in the regulation of m⁶A modification of tRNAs.³⁹ Further research will reveal the more detailed functions of these writers.

HIGHLIGHTS

1. The teamwork of cancer stem cells (CSCs) and the tumor immune microenvironment (TIME) is the root cause of tumor progression;

2. The m⁶A modification is participated in the CSC−TIME interplay, and the interaction between m⁶A and CSC−TIME was overviewed for the first time;

3. This will provide novel insight into the role of m⁶A in cancer‐specific basic and translational medicine.

2.2. m⁶A demethylases/erasers

Erasers ensure that the m⁶A modification is a dynamic and reversible process. Currently, there are two predominant m⁶A demethylases, fat mass and obesity associated protein (FTO) and AlkB homolog 5 (ALKBH5), which belong to the ALKB family of dependent dioxygenases and collaborate to balance the m⁶A levels in the transcriptome by abrogating m⁶A modification of RNAs.⁴⁰, ⁴¹, ⁴² However, these two erasers work in different ways, with FTO sequentially converting m⁶A to N ⁶‐hydroxymethyladenosine (hm⁶A), N ⁶‐formyladenosine (f⁶A), and adenosine, while ALKBH5 removes m⁶A in a straightforward manner.⁴², ⁴³ To a large extent, these erasers select target RNAs to perform demethylation based on the structure and conformation elicited by m⁶A.⁴⁴ Additionally, recent studies have shown that AlkB homolog 3 (ALKBH3) is also an emerging eraser of m⁶A modifications but acts preferentially on tRNAs.⁴⁵

2.3. m⁶A binding proteins/readers

The mechanism by which m⁶A truly exerts its biological effects is through the recruitment of relevant binding proteins (readers). At present, the YT521‐B homology (YTH) domain family proteins (YTH domain‐containing proteins 1 [YTHDC1], YTH domain‐containing proteins 2 [YTHDC2], YTH N6‐methyladenosine RNA binding protein 1 [YTHDF1], YTH N6‐methyladenosine RNA binding protein 2 [YTHDF2], and YTH N6‐methyladenosine RNA binding protein 3 [YTHDF3]) are the most well‐studied m⁶A readers and can be classified into three types based on location: nuclear YTHDC1, nucleocytoplasmic YTHDC2, and cytoplasmic YTHDF1, YTHDF2, and YTHDF3.⁴⁶, ⁴⁷, ⁴⁸ The main functions of YTHDC1 include RNA splicing, X‐chromosome silencing mediation, and facilitation of m⁶A‐modified RNA export from the nucleus to the cytoplasm.⁴⁶, ⁴⁹ YTHDC2 is tightly linked to both translation and RNA degradation.⁵⁰ YTHDF1 directly promotes the translation of m⁶A‐modified RNAs in cooperation with the translation machinery.⁵¹ The ability of YTHDF2 to trigger the decay of target RNAs is achieved by the direct recruitment of CCR4‐NOT adenosine complexes.⁵² Interestingly, YTHDF3 serves as a synergistic role, assisting YTHDF1 in enhancing translation and YTHDF2 in causing RNA degradation.⁵³ However, recent study revealed that YTHDF2 may play a double‐faceted role in RNA stabilization, not only inducing RNA degradation, but also stabilizing some RNA transcripts.⁵⁴ In addition to the YTH domain family, other m⁶A‐binding protein families have also been identified, such as the insulin‐like growth factor 2 mRNA‐binding proteins (IGF2BP1, IGF2BP2, and IGF2BP3) and the heterogeneous nuclear ribonucleoprotein (HNRNP) family (HNRNPC, HNRNPG, and HNRPA2B1). IGF2BP1‐3 are involved in the maintenance and enhancement of target RNA stability and storage by interacting with the typical consensus GG(m⁶A)CU sequence.⁵⁵ Unlike the YTH domain family, HNRNPC and HNRNPG are recognized as indirect readers since they do not bind to m⁶A modification sites, but preferentially bind to emerging sites generated by changes in RNA structure caused by m⁶A modification. This process, in which m⁶A modification regulates RNA‐structure‐based accessibility of m⁶A readers to alter target RNA biological function, is known as the m⁶A switch mechanism. It is through this m⁶A switch mechanisms that HNRNPC and HNRNPG affect the abundance and splicing of target RNAs.⁵⁶, ⁵⁷ In addition to being an indirect m⁶A reader, HNRNPA2B1 also actively modulates microRNA (miRNA) processing.⁴⁸ Furthermore, other novel m⁶A readers have been discovered, including leucine‐rich pentatricopeptide repeat containing (LRPPRC), fragile X mental retardation protein (FMRP), eukaryotic initiation factor 3 (eIF3), and ATP binding cassette subfamily F member 1 (ABCF1).⁵⁸, ⁵⁹, ⁶⁰, ⁶¹ These novel readers can also recognize m⁶A modifications to influence the fate of RNAs.

2.4. m⁶A modification in CSCs

A growing body of evidence shows that CSCs are inextricably linked to tumor initiation and development⁶², ⁶³; therefore, it is believed that targeting CSC‐relevant mechanisms is the key foundation of anticancer treatment. Recently, it has been demonstrated that m⁶A modification contributes greatly to the pluripotency and differentiation of mammalian stem cells.⁶⁴, ⁶⁵ In particular, many representative pluripotent genes, such as nanog homeobox (NANOG), octamer binding transcription factor 3/4 (OCT3/4), kruppel like factor 4 (KLF4), and SRY‐Box transcription factor 2 (SOX2), have been found to possess a large number of m⁶A modifications in their corresponding RNA transcripts.⁶⁶, ⁶⁷ Moreover, recent studies have shown that aberrant m⁶A deposition is also closely related to CSCs, which has been confirmed in a variety of malignancies.¹⁰, ¹¹, ⁶⁸ In the subsequent sections, we elaborate on the role of m⁶A modification in CSCs in different organ systems (Figure 2, Table 1).

FIGURE 2.

The relationship between m⁶A‐related regulators and various CSCs in different organ systems. The m⁶A‐related regulators are involved in various CSCs of different organ systems, including the nervous, digestive, reproductive, and hematologic systems. These regulators play a dual role in the modulation of various CSCs, promoting or inhibiting the stemness characteristics of CSCs by regulating the decay, splicing, stabilization, and translation of target RNAs.

TABLE 1.

The relationship between m⁶A regulators and various CSCs

Cancer types	M6A regulators	Molecular axis	Function	Model system	Reference
GBM	METTL3	METTL3/SOX2	Promoting stemness of GSCs	In vivo	71
	METTL14	METTL3/METTL14/ADAM19	Inhibiting stemness of GSCs	In vitro and in vivo	12
	ALKBH5	ALKBH5/FOXM1	Promoting stemness of GSCs	In vitro and in vivo	11
	FTO		Promoting stemness of GSCs	In vivo	12
	IGF2BP2	IGF2BP2/Let‐7family	Promoting stemness of GSCs	In vitro and in vivo	73
		HMGA1	Promoting stemness of GSCs	In vivo	74
	YTHDF2	YTHDF2/MYC/IGFBP2	Promoting stemness of GSCs	In vitro and in vivo	75
PC	HNRNPA2B1	HNRNPA2B1/UCA1/KRAS	Promoting stemness of pancreatic CSCs	In vitro	76
	IGF2BP2	IGF2BP2/DANCR	Promoting stemness of pancreatic CSCs	In vitro and in vivo	77
CRC	METTL3	METTL3/SOX2	Promoting stemness of colorectal CSCs	In vitro and in vivo	78
	YTHDF1	YTHDF1/FZD9/WNT6/Wnt//β‐cantenin pathway	Promoting stemness of colorectal CSCs	In vitro and in vivo	79
HCC	YTHDF2	YTHDF2/OCT4	Promoting stemness of liver CSCs	In vitro and in vivo	80
	IGF2BP2	IGF2BP2/ROS production			81
BC	ALKBH5	ALKBH5/NANOG	Promoting stemness of BCSCs	In vitro and in vivo	10
OC	FTO	FTO/PDEC1/PDE4B/cAMP pathway	Inhibiting stemness of OCSCs	In vitro and in vivo	85
EC	ALKBH5	ALKBH5/SOX2	Promoting stemness of ECSCs	In vitro and in vivo	25
AML	METTL14	SPI1/METTL14/MYB/MYC	Promoting stemness of LSCs	In vitro and in vivo	68
	ALKBH5	ALKBH5/TACC3	Promoting stemness of LSCs	In vitro and in vivo	88
	FTO		Promoting stemness of LSCs	In vitro and in vivo	89, 90
	IGF2BP1	IGF2BP1/ALDH1A1/HOXB4/MYB	Promoting stemness of LSCs	In vitro and in vivo	91
	YTHDF2		Promoting stemness of LSCs	In vitro and in vivo	92
OS	METTL3	Pluripotency of stem cells/Wnt pathway	Promoting stemness of OSCs	In vitro	94
BCa	METTL3	METTL3/AFF4/SOX2/MYC	Promoting stemness of BCa stem cells	In vitro	95
cSCC	METTL3	METTL3/ΔNp6	Promoting stemness of cSCC stem cells	In vitro	96

m6A, N6‐methyladenosine; GBM, glioblastoma; PC, pancreatic cancer; CRC, colorectal cancer; HCC, hepatocellular carcinoma; BC, breast cancer; OC, ovarian cancer; EC, endometrial cancer; AML, acute myeloid leukemia; OS, osteosarcoma; BCa, bladder cancer; cSCC, cutaneous squamous cell carcinoma; GSCs, glioblastoma stem cells; CSCs, caner stem cells; BCSCs, breast cancer stem cells; OCSCs, ovarian cancer stem cells; ECSCs, endometrial cancer stem cells; LSCs, leukemia stem cells; OSCs, osteosarcoma cancer stem cells.

2.5. Nervous system

Glioblastoma (GBM) is the most prevalent and aggressive type of malignant nervous system tumors, with a high relapse potential and unfavorable prognosis.⁶⁹, ⁷⁰ The presence of glioblastoma stem cells (GSCs) is one of the root causes of this phenomenon and the primary concern related to GBM treatment strategies. Recent studies have demonstrated that m⁶A regulatory components, such as METTL3, ALKBH5, FTO, YTHDF2, and IGF2BP2, are involved in the modulation of GSCs.¹¹, ¹², ⁷¹, ⁷², ⁷³, ⁷⁴, ⁷⁵ First, the m⁶A reader METTL3, a key mediator of GSCs, promotes the growth and self‐renewal of GSCs by stabilizing SOX2 mRNA.⁷¹ Downregulation of METTL3 has been shown to suppress the stemness features of GSCs and attenuate GBM invasiveness. Furthermore, high m⁶A modification levels enable reprogramming of GBM cells and transform non‐GSCs into GSCs. An in‐depth study uncovered that METTL3 protects serine and arginine rich splicing factors (SRSFs) mRNAs from nonsense‐mediated mRNA decay, which facilitates GBM development and progression⁷²; however, with regard to the function of METTL3 in GBM, the opposite notion has been proposed.¹² It was noted that overexpression of METTL3 inhibits the growth, self‐renewal, and frequency of GSCs by increasing the m⁶A abundance in the target mRNAs (eg, ADAM metallopeptidase domain 19 [ADAM19]), subsequently decreasing their expression. Further research is needed to explore these contradictory conclusions.

In addition, the eraser ALKBH5 is highly overexpressed in GSCs, predicting an unfavorable prognosis in patients with GBM. ALKBH5‐mediated demethylation of the transcription factor forkhead box M1 (FOXM1) results in elevated corresponding nascent transcripts and the subsequently detectable increased expression of mRNA and protein, which ultimately boosts GSC function and GBM germination. Moreover, knockdown of ALKBH5 has been shown to be effective in reducing the proliferative capacity of GSCs and weakening their stemness features.¹¹ Furthermore, pharmacological inhibition of eraser FTO impedes GBM growth and tumor initiation while also prolonging the life expectancy of GSC‐engrafted mice.¹²

Moreover, two m⁶A binding proteins have been reported to participate in the regulation of GSCs. IGF2BP2 specifically binds to the let‐7 miRNA recognition sites of target transcripts to protect against let‐7 miRNA‐based splicing and silencing of these target genes, which is thought to increase the expression levels of corresponding mRNA and protein levels and subsequently induce and preserve GSCs specificity.⁷³ Another study revealed the relationship between IGF2BP2 and GSCs in mesenchymal GBM, where IGF2BP2, DExH‐Box helicase 9 (DHX9), and HIF1A antisense RNA 2 (HIF1A‐AS2) can directly interact to stimulate the expression of target genes (high mobility group A1 [HMGA1]), eventually driving the GBM phenotype and enabling GSCs to acclimatize to hypoxic conditions.⁷⁴ Similarly, YTHDF2 serves as an oncogenic trigger in GBM hierarchy, being upregulated in GSCs and supporting their stemness. This reader stabilizes transcripts of MYC and VEGFA in an m⁶A modification‐dependent manner, both of which subsequently interact with the downstream effector IGF2BP2 to establish a strong axis in GBM. The YTHDF2‐MYC‐IGF2BP2 axis may be a potential novel therapeutic target for GBM treatment.⁷⁵

2.6. Digestive system

At present, it is understood that m⁶A regulators act as essential modulators of CSCs in three digestive malignancies: pancreatic cancer (PC), colorectal cancer (CRC), and hepatocellular carcinoma (HCC). First, HNRNPA2B1 has been reported to synergize with a long noncoding RNA urothelial cancer associated 1 (UCA1) to enhance KRAS expression and activity, ultimately facilitating CSCs properties and tumor growth in PC.⁷⁶ In addition, IGF2BP2 stabilizes the long noncoding differentiation antagonizing nonprotein coding RNA (DANCR) in an m⁶A modification‐based manner, which work in concert to contribute to the stemness properties and progression in PC.⁷⁷ Moreover, METTL3 collaborates with IGF2BP2 to increase m⁶A enrichment of SOX2 transcripts as well as extend their lifespan, maintaining and motivating the stemness of CRC cells.⁷⁸ Researchers have also found that a high expression level of YTHDF1 promotes the tumorigenicity of CRC cells both in vivo and in vitro, while silencing YTHDF1 gives rise to a corresponding downregulation of classical CSC markers (CD44, CD133, OCT4, aldehyde dehydrogenase 1 [ALDH1], and leucine‐rich repeat‐containing G‐protein coupled receptor 5 [LGR5]), a smaller clonosphere, and slower tumor formation. Theoretically, suppression of YTHDF1 decreases frizzled class receptor 9 (FZD9) and Wnt family member 6 (WNT6) levels, ultimately limiting the stem cell‐related Wnt/β‐catenin signaling pathway.⁷⁹ Several m⁶A regulators have been identified as playing pivotal roles in HCC CSCs regulation. YTHDF2 functions to regulate m⁶A methylation levels in the 5’UTR of OCT4 mRNA and influence the protein translation of OCT4 mRNA; therefore, the activity of CSCs can be enhanced or weakened by altering the expression level of YTHDF2. In vitro experiments have demonstrated that the loss of YTHDF2 lowers tumor burden and the likelihood of lung metastasis in HCC.⁸⁰ Furthermore, another m⁶A binding protein IGF2BP2 enables to improve reactive oxygen species (ROS) production and induce genomic instability in HCC CSCs.⁸¹

2.7. Reproductive system

Breast cancer (BC) is the leading killer in women, with unparalleled morbidity and mortality rates.⁸² Similar to other tumors, aberrant m⁶A modification also promotes the strength of breast cancer stem cells (BCSCs).¹⁰ Upregulation of ALKBH5 increases m⁶A demethylation of NANOG, NANOG mRNA stability, and protein expression, ultimately boosting the BCSCs phenotype in BC. In vitro experiments have demonstrated that silencing ALKBH5 suppresses tumor formation and dramatically diminishes the proportion and function of BCSCs.¹⁰ Moreover, both ovarian and endometrial cancer (OC and EC) are serious diseases in women.⁸³, ⁸⁴ In OC, FTO plays a tumor suppressor role and impairs ovarian cancer stem cells (OCSCs) function when overexpressed. Owing to its demethylase activity, FTO destabilizes the mRNAs of two phosphodiesterase genes (phosphodiesterase 1C/4B [PDEC1 and PDE4B]) to strengthen cyclic adenosine monophosphate (cAMP) signaling and dampen the stemness characteristics of OCSCs.⁸⁵ Similarly, endometrial cancer stem cells (ECSCs) are also controlled by ALKBH5, the high expression of which mediates the SOX2 level through its enhanced demethylation capacity, triggering ECSCs initiation and stemness states.²⁵

2.8. Hematologic system

Leukemia stem cells (LSCs), featured by an unparalleled self‐renewal and growth capacity, are believed to be the initial trigger of the emergence and development of this hematologic malignancy, as well as problems related to treatment resistance and recurrence.⁸⁶, ⁸⁷ Researchers have successively unraveled the connection between m⁶A modification and LSCs, elucidating that m⁶A regulation is indispensable for LSCs growth.

METTL14, an essential element of the m⁶A writer complex, has been found to be highly expressed in both normal hematopoietic stem cells (HSCs) and acute myeloid leukemia (AML) cells; however, its expression decreases when these cells begin to differentiate. MYB and MYC, playing significant roles in the differentiation and self‐renewal of AML cells, are the direct targets of METTL14. This m⁶A writer targets the posttranscriptional regulation of MYB and MYC based on m⁶A modification, while its own expression is controlled by SFI1 centrin binding protein (SFI1). This biological process constructed a novel signaling axis (SPI1‐METTL14‐MYB/MYC) in the AML that determines the fate and activity of LSCs.⁶⁸ Notably, the small molecule inhibitor of METTL3 was effective to decrease the AML stem cells growth and propagating, also enabled to prolong the survival of multiple AML patients‐derived‐xenografts mouses models.⁸⁸ ALKBH5 is also aberrantly elevated in AML, which often predicts poor survival in AML patients. Inhibition of ALKBH5 impairs the growth and self‐renewal of LSCs yet has little effect on normal HSCs. Mechanistically, it has been discovered that ALKBH5 directly modifies Transforming acidic coiled‐coil 3 (TACC3) in an m⁶A posttranscriptional manner to ensure its transcripts half‐life and high expression. Moreover, it was highlighted that ALKBH5 is a specific target for eradicating LSCs in AML.⁸⁹ With respect to another identified m⁶A eraser FTO, both its pharmacological inhibition and knockdown can inhibit LSCs population growth and function, which also increases the toxic effect of T cells on LSCs.⁹⁰, ⁹¹ The m⁶A binding proteins, IGF2BP1 and YTHDF2, have also been associated with LSCs. IGF2BP1 orchestrates LSCs phenotype and tumor‐initial property. Suppression of IGF2BP1, genetically and pharmacologically, decreases LSCs proliferation, increases LSCs differentiation, induces programmed death of LSCs, and enhances the sensitivity of LSCs to chemotherapy. The potential and underlying cause may be the ability of IGF2BP1 to directly modulate crucial regulators and stemness markers of HSCs, including Aldehyde dehydrogenase 1 family member A1 (ALDH1A1), Homeobox B4 (HOXB4), and MYB.⁹² Similarly, YTHDF2 is also an active participant in the transformation of AML, and inactivation of YTHDF2 prolongs the half‐life of transcripts with m⁶A modifications, which are mostly associated with LSCs. The absence of YTHDF2 renders LSCs more sensitive to TNF‐induced apoptotic signals. Interestingly, silencing of YTHDF2 promotes HSCs to a certain extent, but does not allow hematopoiesis to become out of control.⁹³

3. OTHER SYSTEMS

Unsurprisingly, in other systems, recent literature has also uncovered the presence of m⁶A modifications in the regulation of various CSCs. Since related studies are still at a relatively early stage, we have included these different systems into the same section for elaboration.

METTL3 is perceived as an essential performer of m⁶A modifications in osteosarcoma (OS), bladder cancer (BCa), and cutaneous squamous cell carcinoma (cSCC), mediating the stemness properties of CSCs and tumor progression.⁹⁴, ⁹⁵, ⁹⁶, ⁹⁷ The expression of METTL3 is elevated in osteosarcoma stem cells (OSCs), causing the increased m⁶A modifications in OSCs as compared with non‐OSCs, which may be one of the principal reasons why OS is prone to chemoresistance and metastasis. Moreover, bioinformatics analysis has suggested that these variations in m⁶A enrichment are likely related to pluripotency of stem cells and the Wnt pathway.⁹⁵ In BCa, the emergence and self‐renewal of CSCs is limited when METTL3 is depleted. Mechanistically, METTL3 controls the expression of AF4/FMR2 family member 4 (AFF4), and AFF4 upregulates the essential stemness genes SOX2 and MYC by binding to their promoter regions.⁹⁶ METTL3 deficiency has been proven to alter the expression of typical differentiation and undifferentiation markers (K10 and K14) in cSCC stem cells. Depletion of METTL3 dramatically restricts ΔNp63 expression, which consequently leads to poorer growth and tumorigenicity of cSCC stem cells.⁹⁷ In addition, YTHDF1 has also been demonstrated to help tumor cells adapt to hypoxic conditions in lung cancer, while hypoxia‐related molecular events are known to facilitate and support CSCs development. This phenomenon suggests that YTHDF1 may govern CSCs in an indirect manner in lung cancer.²⁴, ⁹⁸

In summary, it can be clearly observed that m⁶A modification plays a key role in the emergence and development of CSCs in a variety of malignancies. The presence of CSCs is often considered the root source of tumor propagation and relapse, which in turn are also responsible for chemoradiotherapy resistance. Furthermore, chemotherapy drug transport and metabolism are also mediated by m⁶A modification, which is the first step in determining drug effectiveness against CSCs.⁹⁹ Therefore, it is reasonable to assume that the biological process of m⁶A modification may be pivotal in eliminating CSCs and destroying tumors, providing a novel and potential therapeutic avenue for several cancers.

3.1. m⁶A modification in the TIME

With the recognition of cancer as a heterogeneous disease, the TIME can no longer be separated from tumorigenesis and progression.¹⁰⁰ The TIME is a complex and systematic structure, analogous to fertile soil for tumor growth, featured by various immune cellular compositions, regulatory‐protein expression, and inflammatory cytokines.¹⁸, ²¹ Most importantly, the TIME status is the leading cause of the differential responses and outcomes in cancer patients receiving the same treatment, especially for multiple immunotherapies.²¹, ¹⁰¹ Therefore, explaining the diversity and complexity of the TIME is an indispensable step in enhancing the predictive capacity and clinical guidance of immunotherapy, which will benefit countless patients. It is becoming apparent that TIME remodeling includes various biological processes, such as immune cell infiltration, immune checkpoint protein expression, and cytokine production, in which the m⁶A modifications function as a critical mediator (Figure 3, Table 2).

FIGURE 3.

The m⁶A‐mediated TIME remodeling includes multiple aspects. m⁶A‐related regulators actively reprogram the TIME in various ways. For immune cells, these regulators are able to influence MDSC and Treg infiltration, M1 and M2 polarization, DC migration and function, and naïve T cell proliferation and differentiation. For immune checkpoints, PD‐L1, LIRB4, CD40, and CD80 are upregulated by certain m⁶A regulators, while MICB and ULPB2 are downregulated. For cytokine production, the secretion of IL6, IL12, TNF‐α, and TGF‐β is increased, but that of CXCL9, CXCL10, and IFN‐γ production is decreased.

TABLE 2.

m6A‐mediated TIME remodeling in various aspects

TIME compositions		M6A regulators	Molecular mechanisms	Functions	Reference
Immune cells	DC	METTL3, HNRNPC	Regulating CD40 and CD80 expression	DC migration and function	27, 102
		YTHDF1, YTHDF2	Regulating lysosomal protease expression	CCR7‐mediated DC migration and DC‐based immunec responses	103
	Macrophage	FTO	Regulating STAT1 and PPAR‐γ mRNA degradation	M1 and M2 macrophages polarization	105
	Naïve T cell	METTL3	Regulating SOCS family mRNA degradation	naïve T cell proliferation and differentiation	106
	Treg	METTL3	Regulating SOCS family mRNA degradation	Tregs stability and suppressive function	107
		ALKBH5	Regulating MCT4 expression	Tregs infiltration	26
	MDSC	METTL3		MDSCs infiltration	108
		ALKBH5	Regulating MCT4 expression	MDSCs infiltration	26
Immune checkpoints	PD‐L1	FTO	Regulating IFN‐γ pathway	PD‐L1 expression	110
	LIRB4	FTO	Regulating mRNA degradation	LIRB4 expression	88
	ULPB2, MICB	IGF2BP3		ULPB2 and MICB expression and migration	111
	CD40, CD80	METTL3, HNRNPC		CD40 and CD80 expression	27, 102
Cytokines	IL6, IL12, and TNF‐α	METTL3	Regulating NF‐κB signaling	Production of IL6, IL12, and TNF‐α	27
	IFN‐γ, CXCL9, and CXCL10	METTL3, METTL14	Regulating IFN‐γ‐STAT1‐IRF1 signal pathway	Production of IFN‐γ, CXCL9, and CXCL10	113
	TGF‐β1	METTL3	Regulating TGF‐β1 mRNAs degradation and translation elongation	Production and activation of TGF‐β1	115

m⁶A, N ⁶‐methyladenosine; TIME, tumor immune microenvironment; DC, dendritic cell; CCR7, C‐C motif chemokine receptor 7; STAT1, signal transducer and activator of transcription 1; PPAR, peroxisome proliferators‐activated receptor; SOCS, suppressor of cytokine signaling; Treg, regulatory T cells; MCT4, monocarboxylate transporter 4; MDSC, myeloid‐derived suppressor cell; IFN‐γ, interferon gamma; LIRB4, leukocyte immunoglobulin like receptor B4; LIRB4, leukocyte immunoglobulin like receptor B4; MICB, MHC class I polypeptide‐related sequence B; IL‐6, interleukin 6; IL‐12, interleukin 12; TNF‐α, tumor necrosis factor alpha; NK‐κB, nuclear factor kappa B; CXCL9, C‐X‐C motif chemokine ligand 9; CXCL10, C‐X‐C motif chemokine ligand 10; TFG‐β1, transforming growth factor beta 1.

3.2. Immune cells

The TIME is broadly populated with various immune cells, and their composition, distribution, and function largely determine the TIME characteristics.²¹ Dendritic cell (DC)‐directed antigen presentation is the initial step in the activation of antitumor effects of specific immunity, while m⁶A modification influences DCs in many ways. METTL3, HNRNPC, YTHDF1, and YTHDF2 affect the maturation and phenotype of DCs, further disrupting their key immune functions. On one hand, both METTL3 and HNRNPC regulate DC maturation and function by altering the costimulatory molecular (CD40 and CD80) expression.²⁷, ¹⁰² METTL3 has been found to promote translation of CD40 and CD80 mRNAs. In addition, loss of METTL3 in DCs decreases the transcription of TLR4 signaling adaptor Tirap, resulting in a diminished ability to activate T cells.²⁷ On the other hand, YTHDF1 and YTHDF2 manipulate DC migration and function through completely different mechanisms. In DCs, YTHDF1 directly binds to lysosomal protease transcripts marked by m⁶A and promotes the translation of lysosomal cathepsins, rendering DCs incapable of neoantigen‐presentation and cross‐priming.¹⁰³ CCR7‐mediated DC migration and DC‐based immune response pathways can also involve the presence of m⁶A modifications. The lnc‐RNA double PHD fingers 3 (Dpf3), which plays a pivotal role in these pathways, directly impedes hypoxia‐inducible factor 1‐alpha (HIF‐1α) activity and HIF‐1α‐dependent glycolytic metabolism, culminating in the inhibition of DC migration and inflammatory responses. Silencing of YTHDF2 further exacerbates this CCR7‐induced DC migration process and completely disables DCs by alleviating m⁶A modification‐based RNA degradation of lnc‐Dpf3.¹⁰⁴ In addition to DCs, macrophage polarization is also fine‐tuned by m⁶A modification. Depletion of the m⁶A demethylase FTO not only inactivates the NF‐κB signaling pathway but also restricts the polarization of M1 and M2 macrophages owing to the accelerated degradation of signal transducer and activator of transcription 1 (STAT1) and peroxisome proliferators‐activated receptor‐γ (PPAR‐γ) mRNA marked by m⁶A.¹⁰⁵ Furthermore, m⁶A methylation‐mediated mRNA decay disturbs homeostasis and activates naïve T cells and Tregs.¹⁰⁶, ¹⁰⁷ Downregulation of METTL3 results in a considerable decline in m⁶A‐dependent mRNA degradation of the suppressor of cytokine signaling (SOCS) family, which encode many STAT family repressor proteins. Consequently, these inhibitor proteins, such as rate‐limiting enzymes, restrain naïve T cell proliferation and differentiation mediated by IL‐7 signaling and also decrease IL‐2 signaling, disturbing the stability and suppressive function of Tregs.¹⁰⁶, ¹⁰⁷ There is no doubt that CD8+ T cells are also influenced by m⁶A regulators. In vivo experiments, both METTL3 and METTL14‐deficient tumors render higher infiltration of CD8+ T cells and enhanced secretion ability of cytokines than the controls.¹⁰⁸ Meanwhile, YTHDF1‐knock out mice exhibit increased cross‐priming of CD8+ T cells by DCs as compared with WT mice.¹⁰³ Interestingly, patients with low YTHDF1 expression have higher proportion of CD8+ T cells in the TIME.¹⁰³

Moreover, the expression level of METTL3 is positively correlated with myeloid‐derived suppressor cells (MDSCs) infiltration in the TIME, which work together to create an immunosuppressive environment. Both METTL3 and MDSCs are independent factors for reduced survival in cervical cancer patients.¹⁰⁹ Similarly, the m⁶A eraser ALKBH5 indirectly manipulates the splicing and expression of the target gene monocarboxylate transporter 4 (MCT4), a crucial lactate transporter, the expression of which is subject to m⁶A demethylation by ALKBH5. Inhibition of ALKBH5 by CRISPR or pharmacological molecules reduces MCT4 expression, leading to a dramatic decrease in lactate content of the TIME. A lower lactate concentration is accompanied by a smaller proportion of suppressive immune cells (Tregs and MDSCs) in the TIME, which notably harbor ALKBH5 deletions or mutations and typically predict sensitive responses and favorable efficacy of anti‐PD‐1 treatment in melanoma patients.²⁶ Moreover, Li et al constructed a comprehensive m⁶A regulator‐based risk signature that implicates a strong relationship between m⁶A regulators and immune cell infiltration in the TIME. Based on bioinformatics analysis, they also prompted m⁶A may collaborate with the PI3K‐AKT‐mTOR signaling pathway to reprogram the TIME.¹¹⁰

3.3. Immune checkpoints

Conceivably, some critical immune checkpoints are also supervised by m⁶A regulators. In colon cancer cells, FTO overexpression causes a corresponding boost in PD‐L1 protein expression in an IFN‐γ‐dependent manner.¹¹¹ In addition, FTO actively upregulates immune checkpoint LIRB4 in AML cells by reducing YTHDF2‐induced mRNA degradation. Blockade of FTO is an effective way to sensitize AML cells to T‐cell cytotoxicity by targeting leukocyte immunoglobulin like receptor B4 (LIRB4), which alleviates tumor immune evasion in AML to some degree.⁹⁰ Further, the m⁶A binding protein IGF2BP3 also takes part in the modulation of tumor immune evasion, showing a remarkably higher expression level in tumor cells than in normal tissue. Powerfully oncogenic IGF2BP3 not only downregulates the expression of stress‐induced ligands (UL16 binding protein 2 [ULPB2] and MHC class I polypeptide‐related sequence B [MICB]) but also prohibits these proteins from trafficking to the cell surface. Moreover, NK cells fail to recognize and annihilate these ingenious tumor cells via the natural killer group 2 member D (NKG2D) receptor.¹¹² Furthermore, both METTL3 and HNRNPC mediate the expression of the costimulatory molecules CD40 and CD80 in DCs. METTL3‐deficient DCs demonstrate lower CD40 and CD80 translation levels than METTL3‐wild type DCs. It has also been shown that METLL3 promotes CD40 and CD80 expression in DCs by increasing the translation efficiency of the corresponding mRNAs.²⁷

Anti‐PD‐1/L1 treatment is perceived to be the most promising method of annihilating and eradicating malignancies,¹¹³ and researchers have gradually unveiled the roles of certain m⁶A regulators in anti‐PD‐1/L1 therapy. Intriguingly, the efficacy of anti‐PD‐1 is considerably augmented by simultaneous depletion of METTL3 and METTL14 in both colorectal cancer and melanoma. Mechanistically, the depletion of METTL3 and METTL14 may increase the infiltration and function of CD8+T cells.¹⁰⁸ Similarly, the m⁶A eraser ALKBH5 indirectly affects responses to anti‐PD‐1 immunotherapy by creating a suppressive TIME. Conceivably, harboring ALKBH5 deletions or mutations typically predicts sensitive responses and a favorable efficacy of anti‐PD‐1 treatment in melanoma patients.²⁶ In parallel, knockdown of FTO results in increased PD‐1 expression, which reverses melanoma resistance to anti‐PD‐1 therapy in preclinical experiments.¹¹⁴ Moreover, YTHDF1‐deficient mice are featured by more CD8+ T cells in TIME, tending to display preferable efficacy profiles and outcomes for PD‐L1 checkpoint blockade.¹⁰³ Taken together, this evidence demonstrates that all aspects of m⁶A regulators are competent in modulating the immune responses to anti‐PD‐1/L1 treatment.

3.4. Cytokines

It has been reported that the cytokine production process is also controlled by m⁶A regulators‐mediated signaling pathways. METLL3 enhances NF‐κB signaling by controlling the levels of downstream effector molecules. MELLT3‐knockout DCs exhibit a dramatic decrease in production of the cytokines IL6, IL12, and TNF‐α following lipopolysaccharides (LPS) stimulation. Mechanistically, METTL3 deficiency causes a lower translation level of these cytokine mRNAs.²⁷ Also, the type IFN‐β production raised by human cytomegalovirus or double‐stranded DNA was influenced by METTL14 and ALKBH5. Inhibiting the METTL14 genetic expression decreased the production and accumulation of type IFN‐β in the above process; however, inhibition of ALKBH5 obtained the opposite result.¹¹⁵ Previous studies have uncovered that these cytokines are positively involved in the accumulation and response of T cells and the subsequent antitumor effect.¹¹⁶, ¹¹⁷, ¹¹⁸, ¹¹⁹ Importantly, the combined targeting of IL6 with PD‐1/PD‐L1 and blocking of TNF and PD‐1 enables to increase the efficacy of PD‐1/PD‐L1 treatment, which significantly reduce tumor progression.¹¹⁷, ¹¹⁸, ¹²⁰ Moreover, it has also been found that FTO is engaged in NF‐κB signaling, and inhibition of FTO leads to inactivation of this pathway.¹⁰⁵ In colorectal cancer and melanoma, simultaneous depletion of METTL3 and METTL14 induces mass production of cytokines, including IFN‐γ, CXCL9, and CXCL10. The underlying mechanism is that suppression of METTL3 and METLL14 contributes to the stabilization of STAT1 and interferon regulatory factor 1 (IRF1) mRNAs, which orchestrate the IFN‐γ‐STAT1‐IRF1 signaling pathway in the TIME.¹⁰⁸ Most strikingly, METTL3 has also been found to mediate the expression and secretion of TGF‐β1, which further modulates the TGF‐β‐induced epithelial‐mesenchymal transition (EMT) process of tumors. It has been suggested that the METTL3 expression level is negatively correlated with TGF‐β1 mRNA decay and translation elongation. In METTL3‐knockdown cancer cells, the half‐life, secretion, and activation of TGF‐β are inhibited, which contributes to the suppression of the biological process of EMT.¹²¹

To summarize, the m⁶A modification has shown tremendous potential in remodeling the TIME in various dimensions, including the function of multiple immune cells, cytokine secretion, and expression of immune checkpoint proteins. Therefore, it is believed that only the tip of the iceberg related to the role and mechanisms of the m⁶A modification in the reprograming of the TIME has been uncovered to date. Elucidation of the manner by which m⁶A modification remodels the TIME is not only essential for perceiving tumor biology but also provides a novel opportunity to explore the potential of this RNA regulation‐based therapy in cancer.

3.5. m⁶A modification: an underlying bridging between CSCs and the TIME

CSCs are notorious for being the root cause of tumor recurrence and resistance, which render current treatments ineffective in a vast number of patients. Moreover, the TIME is also a primary determinant of the efficacy of various therapies, especially immunotherapy. It has become apparent that CSCs and the TIME are never mutually exclusive in tumors but establish a deadly teamwork to facilitate tumor development and progression, serving as putative catalysts for each other. In the crosstalk between the CSC‐TIME, both intercellular contact and noncontact interactions occur. Taking the noncontact interactions as an example, tumor cells produce various cytokines to stimulate the expansion of suppressive immune cells, while these immune cells also secrete soluble factors to enhance the CSCs plasticity and the EMT process.¹²²

Due to the rapid and unrestrained proliferation of tumor cells, hypoxia is a pervasive and prominent feature of the TIME. Hypoxia is indispensable for CSCs maintenance but also supports the acquisition of stemness characteristics in tumors. Strikingly, it has been reported that certain m⁶A regulators collaborate with HIF‐1α and HIF‐2α to promote the CSCs phenotype in various tumors.¹⁰, ²⁵ Exposure to a hypoxic microenvironment profoundly stimulates the expression of HIFs and ALKBH5 in BC cells, which ultimately advances BCSCs stemness features and enrichment. During this biological process, HIFs may act as upstream regulators of ALKBH5‐mediated demethylation targeting the pluripotent gene NANOG, since changing the expression level of HIFs alters the activity of ALKBH5 accordingly. Dual regulation by HIFs and ALKBH5 gives rise to higher expression and lower degradation levels of target gene NANOG, ultimately increasing the percentage of BCSCs in BC.¹⁰ The same applies to ECSCs, in which suppression of HIFs also markedly decreases the protein expression level of ALKBH5 and thus diminishes its demethylation capacity. HIFs and ALKBH5 form a subtle collaboration to mediate the level of SOX2, which is the trigger for ECSCs initiation and development. This HIFs‐ALKBH5‐SOX2 axis endeavors to maintain the ECSCs phenotype and function in the TIME.²⁵ These findings indicate that the m⁶A modification functions as a connector in the process of hypoxia‐induced stemness in ECSCs.

Meanwhile, the costimulatory and adhesion molecules also actively participate in the contact interaction in the CSC‐TIME connection, for instance, PD‐L1 signaling acts as an essential role to by promoting immune evasion and CSC growth. They work together to create a hypoxic and immunosuppressive environment that inhibits antitumor effects and promotes tumor progression and metastasis.¹²² The interaction between CSCs and the TIME remains under active investigation; however, some clues have been found that m⁶A modifications may play an important role in this interaction. m⁶A modification not only influences CSCs and the TIME but also mediates communication between them, and dissecting the detailed role of m⁶A modification will have profound implications in the discovery of novel related targets for tumor therapy.

PD‐L1 signaling is also an essential bridge between CSCs and the TIME. Immune cells produce cytokines to promote PD‐L1 expression in CSCs, while CSCs with a higher PD‐L1 expression induce immune evasion in the TIME.¹²³ The m⁶A regulator FTO was identified to participate in this process. FTO enables to upregulate the PD‐L1 expression in colon cancer cells and subsequently promotes immune escape in the TIME.¹¹¹

Integrins family members are known to participate in the signaling transduction between intracellular and extracellular matrix within the TIME and also play a significant role in cancer stemness, progression, and drug resistance.¹²⁴, ¹²⁵ Previous studies have elucidated that integrin‐α₆ is concentrated on CSCs and maintains the stemness characteristics of CSCs.¹²⁶ Moreover, recent studies have suggested that the m⁶A writer MELLT3 upregulates the expression level of integrin‐α₆ in BCa cells, facilitating a more malignant phenotype with greater migration and invasion abilities.¹²⁷ Therefore, it is reasonable to speculate that m⁶A modification may also determine the expression level of integrins, which are involved in prosurvival signaling and TIME reprogramming, thus facilitating the stemness features and function of cancer cells.

Taken together, m⁶A regulators are frequently present during the interaction between CSCs and the TIME. Even though the direct regulatory function of m⁶A modifications in this interaction has not yet been uncovered, there are many clues indicating that it plays an important role in regulating a variety of key molecules in this process. The specific communication mechanisms remain elusive, warranting further research to explore the role of m⁶A modification in the CSC‐TIME interplay.

4. THERAPEUTIC POTENTIAL OF TARGETING m⁶A REGULATORS

Based on existing research, it is clear that m⁶A regulators play pivotal roles in carcinogenesis, demonstrating great potential in cancer treatment. To date, several relevant studies have explored the therapeutic value of certain m⁶A regulators. FTO inhibitors are the most well‐researched candidates targeting m⁶A modification in cancer therapy, with several FTO inhibitors that increase the m⁶A abundance on RNAs having already been successfully identified. First, FB23 and FE23‐2, other types of small‐molecule FTO inhibitors, are able to bind directly to the FTO active pocket, dealing a fatal blow to the proliferation of AML cells.¹²⁸ In addition, another two FTO inhibitors, CS1 and CS2, have been found to limit the growth and function of LSCs, sensitize them to T‐cell cytotoxicity, and decrease their immune evasion ability.⁹⁰ Another MA2 has also been shown to successfully suppress the phenotype of GSCs and impair tumor progression.¹² Noticeably, entacapone has been identified as a potential novel FTO inhibitor in metabolic diseases based on the structure virtual screening FDA‐approved drugs. In addition, IGF2BP1 inhibitors have also demonstrated favorable antitumor effects in several malignancies including leukemia, melanoma, and ovarian cancer.⁹², ¹²⁹ Moreover, a small‐molecule ALKBH5 inhibitor, ALK‐04, has been shown to cause a considerable increase in the efficacy of anti‐PD‐1 therapy both in vivo and in vitro.²⁶ Further, the simultaneous suppression of METTL3 and METTL14 has also been demonstrated to augment the efficacy of anti‐PD‐1 therapy in colorectal cancer and melanoma, owing to the higher infiltration of CD8+ T cells and massive cytokine release in the TIME.¹⁰⁸ Additionally, the METTL3 pharmacological inhibitor (STM2457) was also capable of effectively preventing AML growth and improving survival in AML models in preclinical experiments.⁸⁸ Meanwhile, in NSCLC, the resistance to gefitinib caused by METTL3‐mediated autophagy process was reversed by β‐elemene.¹³⁰ Together, these studies indicate that m⁶A regulator inhibitors will be useful in cancer treatment.

On the other hand, a bidirectional dCasRx m⁶A modification editing platform has been constructed, which is composed of nucleus dCasRx and either a reader (METTL3) or an eraser (ALKBH5). This editing platform is capable of regulating the methylation status at specific m⁶A sites in HEK293T and GBS cells, ultimately affecting the expression of target genes and cancer cell proliferation.¹³¹ This remarkable emerging technology provides a solid foundation for the clinical targeting of m⁶A modification in cancer treatment. Furthermore, some preclinical research also found that depletion of some m⁶A regulators can sensitize tumor cells to chemotherapies, including breast cancer and NK/T cell lymphoma.¹³², ¹³³ Nevertheless, further detailed studies are required to realize its potential. Finally, it is notable that m⁶A expression profiles have great potential to differentiate the immune characteristics of patients with tumors, which is likely to accurately guide the application of immunotherapy in clinical.¹³⁴, ¹³⁵

5. DISCUSSION AND PERSPECTIVE

Despite still being in the immature stage of exploration, numerous studies have indicated that m⁶A modification and the corresponding regulators orchestrate a range of critical pathological processes in tumorigenesis and development by regulating the epitranscriptome. Notably, m⁶A modification actively participates in the development of CSCs in various tumors, determining their fate and functions to influence tumor progression. Both genetic and pharmacological inhibition of m⁶A regulators enable the suppression of CSCs self‐renewal and growth, thus limiting tumor formation and progression. Moreover, m⁶A modification also remodels the TIME in various aspects, including immune cell regulation, cytokine production, and immune checkpoint expression. Targeting m⁶A regulators is an effective method for sensitizing immune responses in anti‐PD‐1/L1 immunotherapy both in vitro and in vivo, which further profoundly indicates a novel strategy to compensate for the limitations of immunotherapy. The m⁶A modification is a dual regulator in both CSCs and TIME, but its direct function in this interaction has not yet been proposed. However, some clues indicate that it is involved in the deadly teamwork of the CSCs‐TIME. m⁶A regulators collaborate with hypoxic factors, integrins, and PD‐L1 to influence interactions between the CSC and TIME, ultimately promoting the tumor development and progression. It is clear that the CSC‐TIME interplay has an essential function in the therapeutic resistance and unfavorable survival. We believe more relevant research will further reveal the regulate function of m⁶A modification in such crosstalk, which will also be informative for cancer eradication and therapy.

As rapid advancements are made in m⁶A sequencing and detection methods and continuous refinement of m⁶A‐based drugs development is performed, it is promising that m⁶A modification will open a new opportunity for tumor diagnosis and treatment.¹³⁶, ¹³⁷, ¹³⁸, ¹³⁹ Given the central role of m⁶A modification in tumorigenesis and development, it is reasonable to speculate that m⁶A modification possesses significant value in the clinical diagnosis and treatment of cancer. First, m⁶A regulators are promising biomarkers for distinguishing benign from malignant tumors and predicting metastasis, therapy resistance, and recurrence, which may be helpful in early diagnosis and individualized monitoring. In addition to m⁶A regulators, the global m⁶A profile based on blood or tissue may also be a reliable choice as a cancer biomarker for diagnosis, classification, and prognosis, warranting more advanced m⁶A‐seq technologies. Furthermore, it is noteworthy that m⁶A modification is closely related to the most promising immunotherapies, in particular anti‐PD‐1/PD‐L1 monotherapies, and its associated regulators hold great promise for the screening of appropriate immunotherapy candidates to achieve precision medicine. Moreover, the combination of m⁶A‐related biomarkers and other classical biomarkers is likely to spark new ideas for better clinical guidelines.

With regard to cancer treatment, m⁶A modification has demonstrated tremendous potential in various aspects. First, as more studies explore and validate the specificity and side effects of m⁶A regulatory inhibitors and editing platforms, the two m⁶A‐related therapies may function as emerging targeted treatments for tumor eradication. Second, combining m⁶A‐based and mainstream treatments is also an attractive and promising blueprint for the future. These m⁶A‐targeted regimens may compensate for the limitations and deficiencies of other current therapies to some extent. Nevertheless, the timing and sequence of combined regimens are critical during cancer treatment, and more research is needed to investigate and validate optimal decisions with a view to maximizing patient benefit. In addition, it is clear that aberrant m⁶A deposition level and m⁶A regulators expression play pivotal roles in therapeutic resistance mechanisms, such as chemoradiotherapy resistance and immunotherapy unresponsiveness. This may provide a new opportunity to patients with advanced drug‐resistant cancer for whom no medication is currently available, filling the significant gap still remaining in the current field of cancer therapeutics.

6. CONCLUSION

In conclusion, we have overviewed the landscape of m⁶A modifications in the cross‐linkage between CSC and TME for the first time, which may bring the possibility of m⁶A modifications as a new therapeutic target for tumor treatment. In addition, we also attempted to point out the direction of m⁶A modifications in the future clinical applications, which can be suggestive for individual therapy and improvement in efficacy of current treatments. Lastly, only the tip of the iceberg has been uncovered regarding the mechanisms related to m⁶A modification in the crosstalk between CSCs and the TIME, and deeper large‐scale studies are warranted for further exploration with a view to opening a new therapeutic avenue in cancer.

AUTHOR CONTRIBUTIONS

NS and JH designed this study and provided funding support. CQZ, YJL, and ZHZ drafted the manuscript and completed the figures. YJL, GCZ, and PW collected the references and completed the tables. All the authors reviewed and approved the final manuscript.

CONSENT FOR PUBLICATION

Consent for publication of this paper has been obtained from the authors.

COMPETING INTERESTS

The authors declare that they have no competing interests.

Zhang Z, Zhang C, Luo Y, et al. RNA N ⁶‐methyladenosine modification in the lethal teamwork of cancer stem cells and the tumor immune microenvironment: Current landscape and therapeutic potential. Clin Transl Med. 2021;11:e525. 10.1002/ctm2.525