Mounting evidence has shown that m6A modification is always misregulated in various types of cancers and that m6A regulatory factor expression is significantly related to the expression of PD-1. Yang et al. revealed that highly expressed fat mass and obesity-associated (FTO) gene plays a carcinogenic role in melanoma growth via m6A RNA modification, and FTO knockdown increased m6A methylation of PD-1 through the m6A reader YTHDF2, leading to the promotion of cell growth and proliferation. Simultaneously, FTO inhibition enhances the response to anti-PD-1 therapy in mice (130). The IGF2BP family, a regulatory subunit of m6A readers, is positively correlated with PD-1 expression, demonstrating that m6A modification regulated by the IGF2BP family may have potential benefits for patients with lung adenocarcinoma treated with ICIs (133). In recent years, integrative and comprehensive genomic and molecular analyses have been established to explore potential prognostic targets predicting the response of patients with pancreatic cancer to immunotherapy. A low m6A score, based on the expression of some m6A regulators, revealed a low abundance of PD-1 and CTLA-4, suggesting the key role of m6A in clinical application in predicting the response to ICIs in patients with pancreatic cancer (137).

PD-L1, as a PD-1 ligand and another critical immune checkpoint protein, binds to PD-1, contributing to the immune escape of cancer cells (138). Emerging studies have shown that dysregulation of m6A-related regulators, such as methylases, demethylases, and m6A binding proteins, is pivotal in affecting PD-L1 expression. Depletion of Mettl3 and Mettl14 significantly upregulated PD-L1, and Mettl3 or Mettl14 deficient enhanced the response to anti-PD-1 treatment in pMMR-MSI-L CRC and melanoma. Notably, the growth of tumors, loss of Mettl3 or Mettl14, was inhibited analogous to multiple combinatorial immunotherapy regimens (anti-PD-1+anti-CTLA-4) (128). Except for the direct action of m6A regulators on PD-L1, the upstream genes of PD-L1 were also extensively referred to as the modification of m6A RNA methylation and then increasing or reducing PD-L1 expression. In non-small cell lung cancer (NSCLC), METTL3 mediates m6A modification of the circIGF2BP3 (hsa_circ_0079587) gene in a YTHDC1-m6A-dependent manner, which competitively upregulates PKP3 expression. PKP3 stabilizes PD-L1 protein by facilitating its deubiquitination, eventually contributing to the immune escape of NSCLC cells (122). Similar to NSCLC, in cholangiocarcinoma (CCA), METTL14 binds Siah2 mRNA in the 3′-UTR region and triggers m6A modification, promoting its degradation in a YTHDF2-dependent manner. Upon the depletion of Siah2, it increased the protein stability of PD-L1 and then inhibited T cells expansion and T-cell–mediated anti-tumor activity in CCA cells, indicating the clinical potential of the METTL14-Siah2-PD-L1–regulating axis for CCA immunotherapy (139). Furthermore, lncRNA is also known as the target of METTL14. METTL14 enhanced the m6A methylation of MIR155HG relying on the “reader” protein HuR-dependent pathway and then modulated PD-L1 expression, contributing to the immune escape of HCC cells (127). In intrahepatic cholangiocarcinoma (ICC), the loss of ALKBH5 enhanced m6A abundances of PD-L1 transcripts in the 3′-UTR region in a YTHDF2-dependent manner (132), downregulating its expression. A recent study demonstrated that PD-L1 expression is positively correlated with the expression of METTL3 and IGF2BP3 in breast cancer. Mechanistically, METTL3 upregulated PD-L1 expression by facilitating its mRNA stability in an m6A-IGF2BP3-dependent manner (123), which may guide new directions for breast cancer immunotherapy. Recently, PD-L1 expression has also been found to be regulated by some other m6A regulators in other types of cancer, such as mediated by METTL3 in OSCC (124), by FTO in colon cancer cells (131), and by YTHDF1 and YTHDF2 in NSCLC (136). Additionally, Liu found that the knockdown of IGF2BP1 downregulated PD-L1 expression and activated immune cell infiltration, thus leading to the inhibition of HCC progression (134).

However apart from PD-1 and PD-L1, m6A regulates other checkpoints, such as CD80, ICOS, and VISTA. Consistent with PD-1 and PD-L1, METTL3 was found to promote the translational expression of CD80, which was identified as another novel checkpoint that promotes DCs activation and maturation in an m6A-dependent manner (125). Moreover, METTL3 has been reported to promote T follicular helper (TFH) cell differentiation through m6A modification. Meanwhile, the m6A level and expression of inducible co-stimulatory (ICOS) were decreased in METTL3-deficient TFH cells, indicating that m6A modification regulated ICOS expression (126). A similar study from Yangyang Zhu’s group reported that the decreased expression of ICOS, caused by glyceraldehyde 3-phosphate dehydrogenase protein weakened TFH cell initiation via METTL3/METTL14-mediated m6A modification (129). In CRC, YTHDF1 has been observed to enhance the protein levels of PD-L1 and VISTA (also known as PD-1 homolog (PD-1H), DD1α, Gi24), and the IgV domain of which shares a sequence homology with both CD28 and B7 families (140), in an m6A dependent manner (135). In addition to the aforementioned regulatory pathways validated experimentally, researchers have analyzed the relationship between m6A methylation modification and the expression of immunotherapeutic targets, such as PD-1, PD-L1, CTLA4, TIGIT, and LAG3 by bioinformatics, revealing the important roles of m6A modification patterns in the anti-tumor immunotherapy strategy for various cancers. For instance, the m6A score or m6A-related high/low-risk subgroup, based on the expression of m6A methylation-related genes, was characterized by differential expression of several immune checkpoints, including PD-1, PD-L1, and CTLA-4 in head and neck squamous cell carcinoma (141), bladder cancer (142–144), lung adenocarcinoma (145), clear cell renal cell carcinoma (146, 147), glioblastoma multiforme (148), HCC (149), gastric cancer (150), and small cell lung cancer (151). The aberrant expression of these immune checkpoints plays an important role in the efficacy of anti-PD-L1 immune checkpoint therapy, considered a predictor for the prognosis in patients treated with ICIs. However, these conclusions were assessed based on the dataset extracted from public databases and bioinformatics analysis, and clinical samples and cellular experiments are required to verify our findings.

In addition to the effect on immune checkpoints, the response to anti-tumor immune therapy has been reported to be affected via reshaping the TME and regulating various signal pathways by m6A regulators. For example, depletion of Mettl3/14 strengthens the response to anti‐PD‐1 therapy by promoting interferon-gamma (IFN‐γ)‐Stat1‐Irf1 signaling in CRC and melanoma (152). Similarly, in melanoma, FTO knockdown increased tumor cell sensitivity to anti-PD-1 treatment by IFNγ in mice (153). Likewise, a recent study found that knockout of Alkbh5 inhibited tumor growth during anti–PD-1 treatment by regulating Mct4/Slc16a3 expression, lactate content, and suppressive immune cell accumulation in TME (37).

Furthermore, m6A modification was also reported to active and mature DC (125), promote more antigen presentation and CTL responses (154), increase infiltration of M1/M2-like TAMs and Treg cells (155), and suppress Tfh differentiation and maturation (126), all of which protect the homeostasis and tumor immunosurveillance function. For example, Yin’s group has reported that deletion of METTL3 in myeloid cells attenuated the effect of PD-1 blockade in B16 melanoma, and METTL3 KO mice showed increased infiltration of M1/M2-like TAMs and Treg cells in tumor sites compared to control mice (155). In a CRC-bearing mouse model, YTHDF1-deficient classical DCs presented a better therapeutic effect along with anti-PD-L1 treatment compared with that of the control mice by enhancing the ability of cross-presentation of tumor antigens of DCs and promoting the activation of T cells (154). Recently, Chen et al. demonstrated that METTL3 inhibited anti-tumor immunity by promoting the m6A-BHLHE41-CXCL1/CXCR2 axis to recruit immunosuppressive MDSCs suppressing CD8 + T cells, thereby facilitating CRC progression (156). Except for solid tumors, in AML, FTO inhibition has been reported to inhibit the expression of LILRB4, identified as an immune checkpoint gene that plays a vital role in the immunotherapy of AML, enabling AML cells to be more sensitive to T-cell toxicity and preventing immune escape, thus serving as a potential enhancer for immunotherapy of AML (157).

Aside from the remodeled TME described above, m6A modifcation mediated the anti-tumor immunity by affecting some tranditional pathways. Song et al. found that the deletion of METTL3 in tumor-infltrating NK cells is associated with malignant progression and shorter survival time in mouse models by downregulating SHP-2. Then, reduced SHP-2 made NK cells less responsive to IL-15, which is related to the inhibition of AKT-mTOR and MAPK-ERK signaling pathways (158). Recently, an analogous result has shown that METTL3-deficiency in macrophages destroyed the translation of SPRED2 mediated by YTHDF1, which promoted tumor growth and metastasis through the activation of ERK and NF-κB/STAT3 pathways (155).

Accumulating evidences have demonstrated that out-of-balance between host microbiota and immune system played a role in tumorigenesis and progression (159). For example, the composition of gut microbiome differed significantly between responders and nonresponders in melanoma patients receiving anti-PD1 therapy (46, 160). According to transcriptome map, the microbiome is a significant influence factor on host m6A modification (161). As a result, the link between gut microbes and the host m6A modification may open up new avenues for ICIs against cancer. This data identifies a correlation between m6A and synergistic effects on immunotherapy outcomes, making it a promising target for combination cancer immunotherapy.

Among the m6A regulators, FTO is the most promising target for the development of inhibitors, and to date, over 10 FTO inhibitors have been discovered, and their treatment efficiency has been verified in different models (168). Rhein, identified as the first FTO inhibitor, reversibly binds to the catalytic domain of FTO and inhibits the demethylation of m6A catalyzed by FTO with low cytotoxicity. Yan et al. demonstrated that in combination with Rhein and tyrosine kinase inhibitor (TKI), resistant cells were sensitive to TKIs and inhibited colony formation compared with that of the single-agent treatment in leukemia cells (169). Unfortunately, no clinical studies have reported the effect of the combination of rhein and immunotherapy on tumors.

Recent studies have shown that the combination strategy of antigen-capturing nanodrug and FTO inhibitor (FB23-2) showed tremendous potential to drive immune checkpoint blockade (ICB)-based immunotherapy, thereby assisting ICB in preventing distant tumor growth and metastasis after the primary tumor ablation therapy of HCC. FB23-2 was released intracellularly from the nanodrug and triggered a significant anti-tumor immune response by promoting antigen presentation and maturation of TIDC by increasing the m6A modification level (165). Meclofenamic acid, a highly selective FTO inhibitor, has been loaded into the γ-cyclodextrin cavity, which enhanced photothermal immunotherapy against PCa when combined with near-infrared radiation -II-mediated photothermal therapy based on gold nanorods. In vivo, the mice with the synergistic therapy showed greater tumor growth inhibition and significantly fewer pulmonary metastatic nodules than that of the control mice by promoting the proliferation and infiltration of CD8+ T cells in the TME and suppressing the transcript of PD-L1 (166).

Recently, a study reported that loss of FTO inhibited tumor growth and blocked FTO-mediated immune evasion by enhancing CD8+ T cell infiltration in tumors. Furthermore, a novel FTO inhibitor, Dac51, facilitated the effect of PD-L1 blockade therapy for better tumor control, showing slower growth and longer overall survival by increasing T cell infiltration, indicating a potent strategy to synergistically improve the immune response in melanoma and NSCLC (167). Consistently, Yang et al. reported that inhibiting FTO enabled the sensitivity of the response to anti-PD-1 therapy in vivo by mediating the regulation of PD-1, CXCR4, and SOX10 in melanoma (130), revealing a critical role of FTO inhibition in accelerating melanoma cell resistance to anti-PD-1 blockade (170).

Subsequently, Su et al. screened two highly effective and selective FTO inhibitors, CS1 and CS2, showing a better effect in suppressing leukemia cell activity than FB23-2 and MO-I-500 (two other previously reported FTO inhibitors). CS1/CS2 treatment restricted AML stem cell self-renewal and suspended immune escape by decreasing the expression of the immune checkpoint gene LILRB4 (157). And the combinatorial treatment of MA2, another inhibitor of FTO, plus the chemotherapy drug temozolomide(TMZ), exhibited a synergistic effect in suppressing glioma cells (171). Thus, developing m6A-based targeted drugs to improve cancer immunotherapy will occur in the near future.

In another case, ALK-04, a specific inhibitor of ALKBH5, functioned synchronously to accelerate anti-PD-1 treatment in melanoma, significantly decreasing tumor growth compared to control, by regulating the recruitment of immunosuppressive regulatory T cells and myeloid-derived suppressor cells, and lactate accumulation in the TME (37). It was also demonstrated that downregulation or mutation of ALKBH5 was correlated with a positive response to PD-1 therapy in patients with melanoma treated with pembrolizumab or nivolumab (37). In ICC, studies on specimens have revealed that patients with stronger nuclear enrichment of ALKBH5 are more sensitive to anti-PD1 immunotherapy (132). Therefore, ALKBH5 inhibitors/activators are potential therapeutic targets for improving the efficacy of immunotherapy.

METTL3/METTL14, two core catalytic subunits of MTC, have been reported to play a crucial role in tumorigenesis and the maintenance of CRC. A recent study identified that in combination with STM2457, a highly potent and selective catalytic inhibitor of METTL3, anti-PD1 treatment resulted in a remarkable anti-tumor effect compared with that of the individual treatments in CRC mouse models through the increased infiltration of activated T cells (156). As mentioned before, METTL3/14 knockout in CRC enhanced the response to anti–PD-1 therapy in constructed Patient-Derived Xenograft (PDX) mice by increasing the infiltration of cytotoxic CD8+ T cells into the tumor cell and a marked increase in the concentration of IFN-c, Cxcl9, and Cxcl10 in the tumor microenvironment (128). Similar to immunotherapies listed above, Li et al. showed that METTL3 was upregulated in glioblastoma patients with TMZ treatment, and silencing METTL3 inhibited the tumor growth in TMZ-resistant xenograft mouse in combination with TMZ (172).

Several different inhibitors/activators have also been reported to exhibit anti-tumor effects. For example, STM2457 targeted key stem cell populations of AML to reverse the malignant phenotype of AML, revealing significant anti-leukemic effects (173). A synergistic anti-cancer effect through the combination of STM2457 and immunotherapy is looking forward to going into clinical trials.

In line with the inhibitors of writer/eraser, a similar synergistic effect was observed when combined with anti-PD-L1 therapy. Han et. reported that when WT and Ythdf1 −/− tumor-bearing melanoma cells mice were treated with an anti-PD-L1 antibody, all Ythdf1−/− mice showed complete tumor suppression, whereas only 40% of the control group showed regression (154). Similar results were observed in CRC, mice-bearing YTHDF1-knockdown CT26 and MC38 tumor cells showed significantly reduced tumor growth rate and weight, and the overall survival of mice receiving the combination therapy was dramatically prolonged compared with those in either monotherapy group (135). MDNP/siYTH polyplexes, employed for the non-viral delivery of YTHDF1 siRNA into DCs, displayed the best tumor growth inhibition in the MDNP/siYTH polyplexes + aPD-L1 combinational treatment mice group (174). It uncovered YTHDF1 depletion that could potentiate the efficacy of anti-PD-L1 immunotherapy. Unfortunately, no corresponding inhibitor of YTHDF1 has been developed and applied to practice so far.

Accumulating evidence has demonstrated that the connection between the dysregulation of m6A regulators and the efficiency of ICIs treatment,which played an essential role in tumor progression. Thus, inhibitors/agonists of the m6A regulator protein can be applied as adjuvant therapies for immunotherapy to make ICI therapy safer and more effective. However, inhibitors/activators of m6A regulators have not yet been used for the treatment of cancer, and future clinical trials are required to prove the synergistic anti-cancer effect of m6A inhibitors combined with ICIs.