The interplay between RNA m⁶A modification and radiation biology of cancerous and non-cancerous tissues: a narrative review

Abstract

The diverse radiation types in medical treatments and the natural environment elicit complex biological effects on both cancerous and non-cancerous tissues. Radiation therapy (RT) induces oncological responses, from molecular to phenotypic alterations, while simultaneously exerting toxic effects on healthy tissue. N⁶-methyladenosine (m⁶A), a prevalent modification on coding and non-coding RNAs, is a key epigenetic mark established by a set of evolutionarily conserved enzymes. The interplay between m⁶A modification and radiobiology of cancerous and non-cancerous tissues merits in-depth investigation. This review summarizes the roles of m⁶A in the biological effects induced by ionizing radiation and ultraviolet (UV) radiation. It begins with an overview of m⁶A modification and its detection methods, followed by a detailed examination of how m⁶A dynamically regulates the sensitivity of cancerous tissues to RT, the injury response in non-cancerous tissues, and the toxicological effects of UV exposure. Notably, this review underscores the importance of novel regulatory mechanisms of m⁶A and their potential clinical applications in identifying epigenetically modulated radiation-associated biomarkers for cancer therapy and estimation of radiation dosages. In conclusion, enzyme-mediated m⁶A-modification triggers alterations in target gene expression by affecting the metabolism of the modified RNAs, thus modulating progression and radiosensitivity in cancerous tissues, as well as radiation effects on normal tissues. Several promising avenues for future research are further discussed. This review highlights the importance of m⁶A modification in the context of radiation biology. Targeting epi-transcriptomic molecules might potentially provide a novel strategy for enhancing the radiosensitivity of cancerous tissues and mitigating radiation-induced injury to normal tissues.

Introduction

RNA modifications have garnered significant attention over an extensive historical period since their initial identification¹. The N⁶-methyladenosine (m⁶A) base was first reported in bacteria and yeast in 1958^2,3, and the mRNA m⁶A sequence motif was discovered in the 1970s^4–6. However, the methyltransferase METTL3 was not identified until the 1990s⁷. A major breakthrough came in 2012, with the development of a novel method combining RNA immunoprecipitation and next-generation sequencing⁸, which enabled comprehensive mapping of this post-transcriptional modification. To date, more than 150 chemical modifications have been identified across various cellular RNAs, including N¹ (or N⁶)-methyladenosine (m¹A or m⁶A), N⁶, 2′-O-dimethyladenosine (m⁶Am), inosine, 5-methylcytidine (m⁵C), N⁴-acetylcytidine (ac⁴C), 2′-O-methylated nucleotide (Nm), pseudouridine (Ψ), and internal N⁷-methylguanosine (m⁷G). These modifications, particularly m⁶A, have introduced a new paradigm for genetic regulation that has enriched the framework of the central dogma in molecular biology.

Substantial research has revealed the interplay between RNA m⁶A modification and diverse physiological and pathological processes, including cancer. Aberrant m⁶A modification, mediated by specific enzymes, has been shown to have tumor promoting or suppressing activity. Given the role of radiation therapy (RT) as a classical treatment method used alongside surgery, chemotherapy, and immunotherapy, the link between m⁶A and tumorigenesis makes this modification relevant to cancer radiation biology⁹. Radiation-responsive m⁶A-modified genes might serve as potential diagnostic or therapeutic targets facilitating the development of strategies to sensitize tumors to radiation. In cancer irradiation, normal tissues inevitably experience iatrogenic radiation exposure during RT, medical workers face occupational radiation exposure, and organisms in the environment are exposed to radioactivity after nuclear accidents. Radiation’s adverse effects encompass a wide range of injuries, including those affecting the skin¹⁰, hematopoietic system¹¹, gastrointestinal system¹², lungs¹³, neurological system¹⁴, and reproductive system¹⁵. These effects are closely associated with molecular, cellular and tissue responses. Genetic transformation after radiation exposure, often associated with epigenetics, has been extensively studied^16,17. The epigenetic modifications that occur under low-dose irradiation, because of the sensitivity and dynamic nature of epigenetic components, are particularly noteworthy. In this regard, illustrating the interplay between m⁶A modification and radiation effects on normal tissues is important in the context of the ongoing Fukushima nuclear contaminated water discharge plan.

Emerging evidence implicates RNA m⁶A modification in radiation biology. Cancer occurrence and development are accompanied by a series of biological events. RT aimed at curbing cancer growth can restore the aberrant expression status of m⁶A regulators, including “writer”, “eraser”, and “reader” enzymes, as well as their target genes, which are involved in multiple cellular events such as apoptosis¹⁸, ferroptosis¹⁹, proliferation²⁰, stem cell self-renewal²¹, and the tumor microenvironment (TME)²², in an m⁶A-dependent manner. Thus, RNA m⁶A modification plays crucial roles in tumor radiation resistance and the efficacy of sensitization approaches. Furthermore, m⁶A modification participates in radiation-induced biological effects, such as DNA damage repair²³, apoptosis²⁴, pyroptosis²⁵, inflammation²⁶, and epithelial-to-mesenchymal transition (EMT)²⁷ of normal tissues, and consequently influences radioactive injury effects.

Despite the importance of RNA m⁶A modification across various life domains, the interplay between RNA m⁶A modification and radiation biology in the context of cancerous and non-cancerous tissues remains understudied. This review provides a comprehensive overview of the regulatory roles of RNA m⁶A modification in ionizing radiation (IR)- and ultraviolet (UV) radiation-induced biological effects. It includes a brief introduction to this modification and its detection methods, as well as an exploration of the regulatory mechanisms of m⁶A on diverse RNAs involved in cancer sensitivity to RT, non-cancer radiation injury, and UV-related toxicity. This review emphasizes the importance of novel regulatory mechanisms involving m⁶A and their potential applications in estimating radiation dosage, screening for epigenetic radiation-associated biomarkers, or serving as therapeutic targets in precision therapy. Additionally, potential directions for future research are proposed, to shed light on the importance of RNA m⁶A modification in cancer and non-cancer radiation biology, and expand the research field of epigenetic modifications.

RNA m⁶A modification

Epigenetics refers to the study of heritable changes in gene expression that do not involve alterations in the DNA sequence; examples include DNA methylation, RNA editing, histone modification, maternal effects, and genomic imprinting²⁸. All RNA modifications collectively form a new level of gene expression regulation known as the epi-transcriptome. Among the numerous RNA modifications, m⁶A is the most prevalent post-transcriptional modification in eukaryotic messenger RNAs (mRNAs) and non-coding RNAs—including long noncoding RNAs (lncRNAs), circular RNAs (circRNAs), ribosomal RNAs (rRNAs), transfer RNAs (tRNAs), and small nuclear RNAs (snRNAs)—accounting for more than 60% of the total RNA modifications^29–32.

m⁶A is formed by the addition of a methyl group to the sixth nitrogen atom of adenosine in RNA and is observed across diverse organisms including bacteria and mammals, thus demonstrating evolutionary conservation^33,34. Accumulating evidence indicates that m⁶A modification, governed by specific enzymes, affects virtually every step in the RNA lifecycle, including transcription, splicing, polyadenylation, translation, nuclear export, RNA localization, stability, turnover, the DNA damage response, apoptosis, and chromatin assembly^35,36.

Abnormal m⁶A modification is closely associated with the activation and inhibition of signaling pathways involved in various physiological and pathological processes^37,38. According to previous reports, m⁶A modification plays roles in gametogenesis and pregnancy²⁸, embryonic development³⁹, stem cell renewal and differentiation⁴⁰, neurogenesis⁴¹, and immunity^42,43. Moreover, m⁶A modification has been implicated in a variety of human diseases, including obesity^44,45, cardiovascular disease⁴⁶, metabolic disease⁴⁷, neuropsychiatric disorders⁴⁸, viral infection^49,50, preeclampsia⁵¹, kidney disease⁵², steatotic liver disease⁵³, rheumatoid arthritis⁵⁴, and a wide variety of cancers^55,56.

Thus, RNA m⁶A modification is a versatile player across biological kingdoms that influences a wide range of cellular functions and disease states.

Writers, erasers, and readers of RNA m⁶A modifications

The conserved m⁶A modification motif DRACH (D = G/A/U, R = G/A, H = A/U/C), frequently identified around the stop codon or in 3′-untranslated regions (3′-UTRs) by whole-transcriptome m⁶A mapping, is maintained by 3 classes of regulators: writers, erasers, and readers³⁴. Writers and erasers add or remove the methyl group in the motif, respectively, and consequently determine RNA methylation level, whereas the readers recognize m⁶A modification and mediate the recruitment of downstream functional complexes, which influence the fate of modified RNAs^57–60.

The m⁶A methyltransferase complex adds methyl groups to the adenine nucleotides of RNA transcripts. Within this complex, methyltransferase-like 3 (METTL3) binds S-adenosylmethionine (SAM) and forms a stable dimer with methyltransferase-like 14 (METTL14), which has a substrate recognition function and is a core component of the methyltransferase complex⁶¹. Wilms’ tumor 1-associated protein (WTAP) is a crucial bridging protein that interacts with METTL3 and METTL14 in the complex, combines with other components, and forms a functional unit⁶². Vir-like m⁶A methyltransferase-associated protein (VIRMA) (KIAA1429) recruits core components and regulates region-specific methylation⁶³. Other essential components of the complex include zinc finger CCCH-type containing 13 (ZC3H13) and RNA binding motif protein 15/15B (RBM15/15B)^32,62.

Substantial progress has recently been made in identifying enzymes mediating N⁶-adenosine methylation on non-coding RNAs (ncRNAs) and mRNAs. Zinc finger CCHC domain-containing protein 4 (ZCCHC4), methyltransferase-like 5 (METTL5), methyltransferase-like 16 (METTL16), and methyltransferase-like 4 (METTL4) are involved in the methylation of ncRNAs^64–68. METTL5 and ZCCHC4 are the methyltransferases catalyzing m⁶A formation on 18S and 28S rRNA, respectively. Adenosines of the U2 and U6 spliceosomal snRNAs are m⁶Am or m⁶A methylated by METTL4 and METTL16, respectively^69–72. METTL16 also catalyzes m⁶A modification of the SAM synthetase methionine adenosyltransferase 2A (MAT2A) mRNA⁷¹. Another evolutionarily conserved RNA methyltransferase, phosphorylated RNA polymerase II CTD interacting factor 1 (PCIF1), catalyzes further methylation at the N⁶ position if the first nucleoside of the nascent mRNA is adenosine, and subsequently generates the m⁶Am modification^73–76.

m⁶A demethylases remove the methyl group from adenine nucleotides bearing m⁶A substrates, thereby providing insight into the dynamic potential of m⁶A. Two m⁶A demethylases have been identified in eukaryotes: the fat mass and obesity-associated protein (FTO)⁴⁴ and α-ketoglutarate-dependent dioxygenase homolog 5 (ALKBH5)⁷⁷. Both demethylases contain a similar catalytic center using ferrous iron as a cofactor and α-ketoglutarate as a co-substrate.

The m⁶A modification recognition enzymes recognize and ensure the diverse biological functions of various m⁶A motifs, thus influencing the binding between RNA and RNA-binding proteins⁷⁸. The YT521-B homology (YTH) domain family is the most conventional reader in eukaryotes. Each member of this family contains a conserved C-terminal domain that recognizes RNA and a variable N-terminal domain that binds RNA⁷⁹. This family is present in both the cytoplasm (YTH N⁶-methyladenosine RNA binding protein 1/2/3, YTHDF1/2/3) and the nucleus (YTH domain-containing protein 1/2, YTHDC1/2)⁸⁰. Specifically, YTHDF2 shows selective binding to mRNAs carrying m⁶A and delivers them to cellular RNA decay sites⁶⁰. YTHDF3 forms complexes with YTHDF1 that increase RNA translation, as well as complexes with YTHDF2 that accelerate RNA degradation^81,82. YTHDC1 is responsible for RNA splicing and export from the nucleus^83,84, whereas YTHDC2 is involved in the translation of specific target RNAs⁸⁵. Other common readers include heterogeneous nuclear ribonucleoproteins⁸⁶, fragile X messenger ribonucleoprotein (FMRP)⁸⁷, and insulin-like growth factor 2 mRNA-binding proteins (IGF2BPs)⁸⁸.

An outline of the identified m⁶A writers, erasers, and readers, and their corresponding functions is depicted in Figure 1.

Figure 1.

Outline of the identified writers, erasers, and readers and their corresponding functions in the dynamic and reversible m⁶A modification process (created in Figdraw). m⁶A modification is governed by a series of enzymes. Writers are m⁶A methyltransferases including METTL3, METTL14, WTAP, KIAA1429, RBM15/RBM15B, ZC3H13, METTL16 [catalyzes m⁶A methylation of the S-adenosylmethionine (SAM) synthetase methionine adenosyltransferase 2A (MAT2A) mRNA], and PCIF1 (catalyzes further methylation at the N⁶ position if the first nucleoside of the nascent mRNA is adenosine, thus generating the m⁶Am modification), which add methyl groups from the SAM substrate to adenine nucleotides of mRNAs in the nuclear speckles. In addition, ZCCHC4 (28S rRNA), METTL5 (18S rRNA), METTL4 (U2 spliceosomal snRNAs), and METTL16 (U6 spliceosomal snRNAs) are involved in the methylation of ncRNAs. The m⁶A erasers are demethylases including FTO and ALKBH5; these enzymes use ferrous iron as a cofactor and α-ketoglutarate as a co-substrate. They remove the methyl group from adenine, thus providing a dynamic counterbalance of m⁶A on the transcripts. Reader proteins recognize the m⁶A site, and consequently ensure the binding of RNA and RNA-binding proteins, and coordinate the diverse biological functions of distinct methylated transcripts. Readers have been implicated in directing the fate of RNA metabolism—such as splicing (YTHDC1 and HNRNPC), nuclear export (YTHDC1), decay (YTHDF2/3 and YTHDC2), stability (IGF2BP1/2/3), translation (YTHDF1/3, YTHDC2 and FMRP), and miRNA processing (HNRNPA2B1)—and typically have multiple roles.

RNA m⁶A modification detection methods

Although research on RNA modification began in the 1970s, no substantial progress was made until the development of a novel approach combining RNA immunoprecipitation and next-generation sequencing in 2012⁸. With the continued advancement of existing techniques and the advent of new technologies, the study of m⁶A modification has great potential in uncovering novel biological regulatory mechanisms and providing therapeutic targets. Here, we briefly introduce the currently available, mature, or exploratory methods for total m⁶A quantification and identification of m⁶A within genes.

For determination of overall m⁶A levels, mass spectrometry, colorimetry, and dot blot assays are frequently used. Among these methods, mass spectrometry is a rapid, low-cost, and relatively accurate approach with high sensitivity and good resolution; in addition, high-performance liquid chromatography can locate specific m⁶A residues^89–92. A colorimetric method based on similar principles to classical enzyme-linked immunosorbent assays can easily detect global RNA m⁶A levels⁹³. Dot blot assays can be used as a semi-quantitative method to identify m⁶A alterations in samples^90,94. In addition, several novel m⁶A quantitative technologies have been developed, such as RNA photoactivatable-ribonucleoside-enhanced crosslinking and immunoprecipitation (PAR-CLIP), based on the incorporation of photoactivatable nucleoside analogs into nascent RNA^89,95, and electrochemical immunosensors, a simple, sensitive and selective electrochemical method using gold nanoparticle modified glassy carbon electrodes (AuNP/GCE)⁸⁹.

The development of a series of m⁶A identification technologies that are antibody-dependent or -independent has been a milestone in the m⁶A research field. For example, methylated RNA immunoprecipitation sequencing (MeRIP-seq), m⁶A-seq2, PA-m⁶A-seq, and m⁶A individual-nucleotide-resolution cross-linking and immunoprecipitation (miCLIP)-seq have been reported to detect m⁶A modifications with improved resolution from 100 nt to 200 nt, and eventually to single-base resolution ^68,96–99. MeRIP-seq, the primary technology, combines methylated RNA fragments and high-throughput sequencing to detect m⁶A peaks with 100 nt to 200 nt resolution but is unable to precisely detect m⁶A sites. The m⁶A-seq2 method enhances m⁶A quantification at modification site, gene, or sample resolution. The PA-m⁶A-seq technique increases the resolution to 30 nt via 4SU strengthening cross-links. This method can access only m⁶A residues around 4SU operating sites, whereas miCLIP ultimately enhances the detection precision to single-base resolution. m⁶A-REF-seq can detect m⁶A sites in the whole transcriptome in an antibody-dependent manner¹⁰⁰. DART-seq induces C-to-U deamination at sites adjacent to m⁶A bases¹⁰¹. m⁶A-label-seq marks methylation sites to achieve detection at a single-base resolution¹⁰². m⁶A-SEAL uses streptavidin beads to enrich biotin-labeled m⁶A-modified sites for subsequent sequencing and analysis¹⁰³. m⁶A-LAIC-seq uses the spike-in External RNA Controls Consortium to quantitatively detect m⁶A presence¹⁰⁴. SELECT precisely distinguishes m⁶A modified residues at single-base resolution, independently of antibody immunoprecipitation or isotope labeling¹⁰⁵. SCARLET is a digestion-based method used to determine the percentage of m⁶A at single-nucleotide resolution for mRNAs and lncRNAs¹⁰⁶.

Insights into RNA m⁶A modification in cancer radiation biology

Links between m⁶A modification and cancer RT

As a major treatment modality for tumors, RT is used in the early, late, or postoperative adjuvant stages of tumor treatment. Multiple cancer types are suitable for RT, primarily head and neck tumors, such as nasopharyngeal carcinoma (NPC), oral cancer, and throat cancer¹⁰⁷; thoracic tumors, such as esophageal cancer, lung cancer, and breast cancer¹⁰⁸; and pelvic and abdominal tumors, such as locally advanced colorectal cancer (CRC), gastric cancer, cervical cancer, and endometrial cancer in females, or penile and testicular cancer in males, and kidney cancer and bladder cancer of the urinary system¹⁰⁹; certain bone metastases or brain metastases¹⁰⁹; and soft tissue sarcomas of the extremities¹¹⁰. RNA m⁶A modification has emerged as an important regulator in radiation oncology, particularly in modulating the sensitivity of tumor cells to RT. The tumor-inhibitory effects of RT can restore the aberrant expression of m⁶A enzymes including writers, erasers, and readers, as well as the RNA m⁶A modification levels of target genes. The expression changes in various target RNAs in tumor tissues are reversed via the modulation of their stability, translation, splicing, and localization in an m⁶A-dependent manner. Consequently, RNA m⁶A modification contributes to radiation-mediated inhibitory or promoting effects on oncogenes or tumor suppressors, thereby modifying the responses of cancer cells to radiation, and potentially leading to resistance or sensitivity to RT treatment.

m⁶A modification of diverse RNAs in IR biology of cancer tissues

Importantly, the close relationship between m⁶A modification and neoplasms has implicated these modifications in cancer RT. Distinct types of RNAs, such as mRNAs, lncRNAs, circRNAs, and microRNAs (miRNAs), are modulated by m⁶A modification. Elucidating RNA m⁶A-mediated mechanisms in cancer radiation biology would facilitate improved therapeutic outcomes in clinical practice.

mRNAs

As the primary carriers of genetic information, mRNAs account for most m⁶A-annotated RNAs. The biological functions of many mRNAs regulated by m⁶A have been best elucidated in cancer. The particular role of m⁶A in cancer is exemplified by the regulation of 2 main classes of tumor-related genes: oncogenic genes and oncosuppressive genes³². m⁶A and its mediators collectively regulate all transcriptional and post-transcriptional regulatory processes, including processing, export, translation, and decay of mRNAs throughout their lifecycle¹¹¹. Consequently, the expression levels of oncogenic genes or oncosuppressive genes are influenced, thereby determining the fate of cancer development. mRNA m⁶A modification is involved in cancer radiation sensitivity as well as the efficacy of radiotherapy. The m⁶A enzymes play major roles, which differ across different tumor types and mechanisms; these roles include affecting DNA damage and repair, regulating tumor cell stemness, and controlling the cell cycle, thus modulating the radioresistance of diverse tumors¹¹².

First, the importance of m⁶A writers in the radiation sensitivity of various cancers has been reported. In pancreatic cancer cells, METTL3 promotes chemo- and radio-resistance, and METTL3-depleted cells show enhanced sensitivity to anticancer agents and irradiation¹¹³. Carbon-ion radiotherapy is a radical nonsurgical treatment with high local control rates and minimal serious adverse events¹¹⁴. Xu et al.¹¹⁴ have reported that METTL3 levels and mediation of m⁶A modification are elevated in non-small cell lung cancer (NSCLC) cells after carbon-ion radiotherapy. METTL3-mediated m⁶A modification of mRNA inhibits the decay of H2A histone family member X (H2AX) mRNA, enhances its expression, and consequently enhances DNA damage repair and cell survival¹¹⁴. Thus, METTL3-mediated m⁶A modification of mRNA contributes to the resistance to carbon-ion radiotherapy in NSCLC. Han and colleagues²¹ have shown that the recently identified methyltransferase METTL16 is aberrantly overexpressed in human acute myeloid leukemia (AML) cells, particularly in leukemia stem cells and leukemia-initiating cells. Mechanistically, METTL16 exerts its oncogenic role by promoting the expression of branched-chain amino acid (BCAA) transaminase 1 (BCAT1) and BCAT2 in an m⁶A-dependent manner and reprogramming BCAA metabolism in AML²¹. In endometrial carcinoma, protein arginine methyltransferase (PRMT) interacts with METTL14 and mediates its arginine methylation. Blocking PRMT enhances tumor suppression in response to cisplatin and RT, through a process involving ferroptosis [promoting methylation modification via an m⁶A-YTHDF2-dependent mechanism, and consequently decreasing glutathione peroxidase 4 (GPX4) mRNA stability]¹¹⁵. In breast cancer, neuropilin-1 (NRP1) enhances the stem cell properties and confers radioresistance to breast cancer cells. Mechanistically, NRP1 decreases IR-induced apoptosis by downregulating B-cell lymphoma-2 (Bcl-2) via WTAP-mediated m⁶A modification of Bcl-2 mRNA¹¹⁶.

Second, the roles of the m⁶A erasers FTO and ALKBH5 have been examined in the radiation sensitivity of NPC and hepatocellular carcinoma (HCC). Huang et al.¹⁹ have reported that the demethylase FTO erases the m⁶A modification of transcripts of OTUB1, a member of the ovarian tumor domain protease (OTU) subfamily of deubiquitinases (DUBs), thus promoting its expression, inhibiting the ferroptosis of cells induced by radiation, and ultimately triggering radiotherapy resistance in NPC. Chen et al.¹¹⁷ have revealed that irradiation upregulates ALKBH5 in hepatic stellate cells and consequently mediates monocyte recruitment; M2 polarization; and secretion of cysteine-cysteine motif chemokine ligand 20 (CCL20), which in turn upregulates ALKBH5 expression, thereby forming a positive feedback loop that promotes radiation-induced liver fibrosis (RILF) and decreases HCC radiosensitivity. This process involves the demethylation of Toll-interleukin 1 receptor domain-containing adaptor protein (TIRAP) mRNA and activation of its downstream NF-κB and JNK/Smad2 pathways¹¹⁷. The dual roles of ALKBH5 as a radiosensitization target and microenvironmental regulator provide new insights for RILF prevention and radiosensitization of HCC¹¹⁷.

Finally, m⁶A readers also play crucial roles in the radiation tolerance of myeloid-derived suppressor cells, cervical cancer, and lung adenocarcinoma (LUAD). A recent study has revealed the roles of YTHDF2 in tumor radiotherapy and immunotherapy²². YTHDF2 contributes to myeloid-derived suppressor cell (MDSC)-induced immunosuppression and treatment resistance to combined IR and/or anti-PD-L1²². Specifically, IR induces immunosuppressive MDSC expansion and YTHDF2 expression in both humans and murine models. IR-induced YTHDF2 expression relies on NF-κB signaling, and YTHDF2 in turn leads to NF-κB activation, thus forming an IR-YTHDF2-NF-κB circuit²². Therefore, YTHDF2 is a promising target for improving radiotherapy and radiotherapy/immunotherapy combinations²². Du et al.¹¹⁸ have reported that YTHDF3 mediates regulation of cervical cancer radio-resistance by hepatocyte nuclear factor 1-alpha (HNF1-α) through promoting the translation of DNA repair protein RAD51 homologue 4 (RAD51D) in an m⁶A-dependent manner. In LUAD, Hao et al.¹¹⁹ have demonstrated that irradiation causes overexpression of Van Gogh-like protein 1 (VANGL1) through increasing its mRNA m⁶A levels; consequently, the detrimental effects of irradiation on LUAD are alleviated by protecting DNA from damage, probably through the activation of v-raf murine sarcoma viral oncogene homolog B1 (BRAF)/tumor protein p53 binding protein 1 (TP53BP1)/RAD51 cascades. IGF2BP2/3 facilitates VANGL1 mRNA stability and expression¹¹⁹. Elevated m⁶A levels of VANGL1 and diminished miR-29b-3p have been found to be responsible for VANGL1 overexpression after irradiation¹¹⁹.

Notably, deciphering novel mechanisms underlying classical molecular processes from a new perspective has contributed to illustrating key biological events. Targeting nuclear factor erythroid 2-related factor 2 (Nrf2), a key transcription factor well known to be responsible for tumor radio-resistance, is a promising strategy to enhance the radiation sensitivity of cancer¹²⁰. The epi-transcriptomic regulation of Nrf2 in carcinogenesis has gained increasing attention in recent years. m⁶A-mediated Nrf2 RNA methylation has been reported to be associated with the upregulation of YTHDC2 as well as YTHDF1-3, and the downregulation of FTO and ALKBH5 in HepG2 human hepatoma cells¹²¹. Targeting Nrf2 by regulating m⁶A modification might therefore provide a new strategy for promoting cancer radiotherapy.

lncRNAs

LncRNAs are arbitrarily defined as non-coding transcripts longer than 200 nucleotides, encompassing RNAs transcribed by RNA polymerase I (Pol I), Pol II, and Pol III, as well as RNAs derived from processed introns^122,123. LncRNA-annotated genes occupy a substantial portion of the genomes of complex organisms¹²². One important way in which lncRNAs function is as scaffolds or bridges for protein interactions, thus affecting the formation of protein complexes and regulating protein activity. They recruit chromatin-modifying factors that alter the levels of chromatin modification, and consequently influence gene transcription and expression¹²⁴. LncRNA LINC00839, modified by METTL3-mediated m⁶A, maintains glioma stem cells and exerts radiation resistance by acting as a scaffold lncRNA; it promotes c-Src-mediated phosphorylation of β-catenin and subsequently activates the Wnt/β-catenin signaling pathway²⁰. Additionally, Yin et al.¹²⁵ have identified a lncRNA-RNA component of mitochondrial RNA processing endoribonuclease (RMRP) in NSCLC. The highly methylated RMRP promotes cancer stem cell properties and EMT, and enhances resistance to radiotherapy and cisplatin. Mechanistically, RMRP recruits Y-box binding protein 1 (YBX1) to the transforming growth factor beta receptor 1 (TGFBR1) promoter region, thereby resulting in upregulation of TGFBR1 transcription. The TGFBR1/SMAD2/SMAD3 pathway is also modulated by RMRP¹²⁵. m⁶A RNA methylation-mediated RMRP stability has been found to drive the progression of NSCLC through regulating the TGFBR1/SMAD2/SMAD3 signaling pathway¹²⁵.

circRNAs

Endogenous circRNAs are covalently closed RNA molecules that are more stable than linear RNAs, such as mRNA, miRNA, and lncRNA¹²⁶. CircRNAs participate in gene expression regulation and perform various functions in plasma, exosomes, and urine¹²⁶. Therefore, circRNAs are expected to serve as biomarkers and therapeutic targets for many tumors¹²⁷. The mechanisms of circRNAs in human cancer are diverse, including acting as miRNA sponges, interacting with proteins, regulating gene splicing or transcription, being translated into proteins or peptides, and being involved in epigenetic regulation. m⁶A modification participates in the diverse functions of circRNAs, although current evidence remains limited¹²⁷.

Shao et al.¹²⁸ have identified and characterized circAFF2 as a novel m⁶A-modified circRNA that binds Cullin-associated NEDD8-dissociated protein 1 (CAND1), promotes the binding of CAND1 to Cullin1 and inhibits the neddylation of Cullin1 and subsequently enhances the radiosensitivity of CRC. Importantly, the regulation of circAFF2 involves ALKBH5-mediated m⁶A demethylation, followed by recognition and degradation via YTHDF2¹²⁸. Thus, the ALKBH5/YTHDF2/circAFF2/Cullin-NEDD8 axis might serve as a potential radiotherapy target for CRC¹²⁸. Zhong et al.¹²⁹ have revealed that the expression of circ_0124554 is upregulated in CRC tissues and cells, and miR-1184 has been identified as a target of circ_0124554 and targeted LIM and SH3 protein 1 (LASP1). METTL3 mediates m⁶A modification of circ_0124554, promoting CRC progression and radio-resistance through the miR-1184/LASP1 pathway¹²⁹. Moreover, m⁶A modification and its related enzymes affect the resistance of cancer to radiotherapy by regulating circRNAs, and METTL3 promotes the stabilization of circCUX1 through mediating its m⁶A modification¹³⁰. Additionally, circCUX1 binds caspase-1 and inhibits its expression, thus decreasing inflammatory factor release and enhancing tolerance to radiotherapy in hypopharyngeal squamous cell carcinoma (HPSCC)¹³⁰.

The roles of m⁶A modification and corresponding enzymes involving diverse RNAs in the IR sensitivity of cancer are listed in Table 1.

Table 1.

m⁶A modification of diverse RNAs involved in ionizing radiation biology in cancer tissues

RNA type	Cancer	Enzyme	Target	Function
mRNAs	Pancreatic cancer	METTL3	Ubiquitin-dependent process↓	Promote chemo- and radio-resistance113
	NSCLC	METTL3	H2AX↑	Enhance DNA damage repair and cell survival, promoting radio-resistance114
	AML	METTL16	BCAT↑	Drive leukemogenesis and leukemia stem cell self-renewal21
	Endometrial carcinoma	METTL14 YTHDF2	GPX4↓	Enhance tumor suppression in response to cisplatin and RT by blocking PRMT3115
	Breast cancer	WTAP	Bcl-2↓	Contribute to stemness and potentiate radioresistance via downregulation of Bcl-2 by NRP1116
	NPC	FTO	OTUB1↑	Inhibit ferroptosis and trigger radiotherapy resistance19
	HCC	ALKBH5	TIRAP↑	Promote liver fibrosis (RILF) and decrease HCC radiosensitivity117
	MDSC	YTHDF2	Adrb2, Metrnl, smpdl3b↓	Contribute to MDSC-induced immunosuppression and resistance to combined IR and/or anti-PD-L122
	Cervical cancer	YTHDF3	RAD51D↑	Promote radioresistance of cervical cancer118
	LUAD	IGF2BP2/3	VANGL1↑	Attenuate detrimental effects of irradiation on LUAD119
lncRNAs	Glioma	METTL3 YTHDF2	LINC00839↑	Promote tumor progression and radiation resistance20
	NSCLC	METTL3	RMRP (lncRNA-RNA component)↑	Promote cancer stem cell properties and EMT, enhancing resistance to radiotherapy and cisplatin125
circRNAs	CRC	ALKBH5 YTHDF2	circAFF2↓	Decrease radiosensitivity128
	CRC	METTL3	circ_0124554↑	Promote progression and radioresistance129
	HPSCC	METTL3	circCUX1↑	Enhance tolerance to RT130

↑: promotes, ↓: inhibits.

m⁶A modification with UV-induced radiation in cancer biology

UV radiation, a major carcinogen in the occurrence and development of all cutaneous cancer types¹³¹, is a common form of non-IR received by terrestrial organisms. UV can induce melanogenesis and even melanoma. Guo et al.¹³² have reported that UVB irradiation promotes global m⁶A modification in melanocytes and upregulates METTL3, thereby increasing the expression of YAP1 via m⁶A modification, activating the co-transcription factor TEA domain transcription factor 1 (TEAD1), and promoting melanogenesis. An early study on the importance of m⁶A modification in UV radiation focused on its function in the UV-induced DNA damage response¹³³. Xiang et al.¹³³, in 2017, reported that m⁶A modification in RNA is rapidly (within 2 min) and transiently induced at DNA damage sites in response to UV irradiation. This modification occurs on numerous poly(A)+ transcripts and is regulated by the methyltransferase METTL3 and the demethylase FTO¹³³. Multiple DNA polymerases participate in the UV response, some of which resynthesize DNA after the lesion has been excised by the nucleotide excision repair pathway, whereas others are involved in trans-lesion synthesis and allow replication past damaged lesions during S phase¹³³. DNA polymerase κ (Pol κ), which has been implicated in both nucleotide excision repair and trans-lesion synthesis, requires METTL3 for immediate localization to UV-induced DNA damage sites¹³³. This notable finding indicates that m⁶A RNA serves as a beacon for the selective, rapid recruitment of Pol κ to damage sites, and consequently facilitates repair and cell survival¹³³. Recently, the m⁶A reader protein YTHDC2 has been found to function in regulating UVB-induced DNA damage repair and histone modification H3K27me3; therefore, it may potentially serve as a biomarker in cutaneous squamous cell carcinomas¹³¹.

Insights into RNA m⁶A modification in non-cancer radiation biology

m⁶A modification participates in a wide variety of biological events. However, studies on the role of this modification in non-cancer radiation biology are relatively scarce. As a dynamic and reversible process, m⁶A modification responds sensitively to external stimuli^133,134. Traditional viewpoints regarding the mechanisms underlying IR-induced injury are centered on the direct or indirect (via oxidative stress) effects causing breakage of DNA strands to varying degrees^134,135. With the rapid advancement of epi-transcriptome research in recent years, unraveling novel epigenetic mechanisms from a post-transcriptional perspective is an important mission in radiobiology. To date, studies examining the roles of m⁶A modification in non-tumor radiation biology have focused on radiation-induced lung injury (RILI), radiation-induced liver disease (RILD), and radiation-induced hematopoietic injury (RIHI).

RILI

Occupational exposure to IR, such as exposure during radiological medical activities, can cause lung injury, denoted RILI^136,137. Clinically, RILI is a common complication in some patients undergoing thoracic radiotherapy^138,139. The biological roles of EMT in the pathogenesis of RILI have been widely documented. Feng et al.²⁷ have detected markedly increased m⁶A levels, accompanied by upregulated METTL3 and downregulated ALKBH5, after IR-induced EMT. Forkhead box O1 (FOXO1) has been identified as a key target of METTL3, and its expression is downregulated by METTL3-mediated mRNA m⁶A modification in a YTHDF2-dependent manner, thus activating the AKT and ERK signaling pathways²⁷. This study has shed light on the novel epigenetic mechanism involved in the pathogenesis of RILI. In parallel, Zhao et al.²⁶ have revealed that zinc finger and BTB domain-containing protein 7B (Zbtb7b) suppresses aseptic inflammation in RILI. Mechanistically, Zbtb7b recruits the RNA demethylase ALKBH5 to interleukin-6 (IL-6) mRNA, and subsequently demethylates m⁶A modification of IL-6 mRNA and inhibits its nuclear export²⁶. Thus, Zbtb7b epigenetically suppresses irradiation-induced IL-6 production through inhibiting the m⁶A modification and nucleocytoplasmic transport of IL-6 mRNA in the lungs²⁶.

RILD

RILD is a complication of HCC radiotherapy. Chen et al.¹⁴⁰ have found that ALKBH5-modified human high-mobility group box-1 (HMGB1)/stimulator of interferon genes (STING) activation contributes to RILD via the innate immune response. Specifically, hepatic stellate cells show significant differences in methylation patterns after X-ray irradiation at 8 Gy¹⁴⁰. Irradiation recruits ALKBH5, which in turn demethylates m⁶A residues in the 3′-UTR of HMGB1, and results in the activation of STING-interferon regulatory factor 3 (IRF3) signaling. YTHDF2 directly binds m⁶A-modified sites in HMGB1 and promotes its degradation¹⁴⁰. Wang et al.²⁵ have investigated the function of m⁶A modification in Kupffer cells during RILD and observed significantly increased m⁶A modification levels after irradiation. Their findings have confirmed that METTL3 is a primary modulator that promotes the methylation and gene expression of TEAD1, and consequently leads to STING/nucleotide-binding oligomerization domain (NOD)-like receptor pyrin domain-containing 3 (NLRP3) signaling activation²⁵. IGF2BP2 recognizes methylated TEAD1 mRNA and promotes its stability²⁵. As previously described, after irradiation, upregulated ALKBH5 mediates m⁶A demethylation of TIRAP mRNA and promotes RILF in hepatic stellate cells through inducing monocyte recruitment and M2 polarization¹¹⁷.

RIHI

Hematopoiesis is a developmental or physiological process that generates all blood lineages¹⁴¹. Total body irradiation triggers a series of adverse health effects, collectively termed acute radiation syndrome (ARS)¹⁴². The hematopoietic system is recognized as the organ with the highest radiation sensitivity, and the hematopoietic subsyndrome of ARS (H-ARS) occurs in humans exposed to total body irradiation of 4 to 6 Gy^143,144. Mortality from H-ARS is known to be due to hematopoietic insufficiency (both leukopenia and anemia), opportunistic infection from immune suppression, and coagulation dysfunction^144–148. Investigations of the function of m⁶A modification in hematopoietic injury and recovery under radiation stress are extremely rare. Hematopoietic stem cells (HSCs) are central to the hematopoiesis process, because they are responsible for self-renewal and generation of all hematopoietic lineages, which are essential for various stress conditions, including IR^149,150. IR damages the hematopoietic system via direct effects on the viability and/or function of hematopoietic stem/progenitor cells¹⁵¹. Wang et al.¹⁵² have generated hematopoietic-specific Ythdf2 knockout mice by using 3 Cre/LoxP systems, and identified a unique role of YTHDF2-mediated m⁶A recognition in adult mouse HSCs. Mechanistically, the expression of Ythdf2 in HSCs facilitates the decay of the m⁶A-modified mRNAs of Wnt target genes and consequently contributes to the repression of Wnt signaling at steady state. After deletion of YTHDF2, the number of HSCs expands, and the regenerative ability of HSCs also increases under stress conditions such as radiation¹⁵². YTHDF2 deficiency simultaneously prevents the degradation of mRNAs of both Wnt target genes and survival-related genes during hematopoietic stresses, and this combined effect synergistically enhances the regenerative ability of HSCs¹⁵². These findings have established the critical role of YTHDF2 in modulating HSC self-renewal, regeneration, and protection, thereby providing preliminary results supporting translation to clinical applications¹⁵².

The bone marrow is the primary site for hematopoiesis¹⁵³, and severe myelosuppression is a common adverse effect of radiotherapy or chemotherapy in clinical settings¹⁵⁴. Our group previously revealed the time-varying epi-transcriptome-wide m⁶A methylome and transcriptome alterations in γ-ray-exposed mouse bone marrow¹³⁴. We have observed that 4 Gy γ-irradiation rapidly (5 min and 2 h) and severely impairs the mouse hematopoietic system, including spleen and thymus atrophy, alterations in blood cell proportions, tissue inflammation, and malondialdehyde elevation¹³⁴. The m⁶A content and expression of m⁶A-related enzymes are altered, and radiation triggers dynamic and reversible m⁶A modification profiles and alters mRNA expression, wherein both m⁶A fold-enrichment and mRNA expression first increase and subsequently decrease¹³⁴. The affected pathways are closely related to those of classical radiation response and metabolism¹³⁴. Interestingly, blocking the expression of FTO and ALKBH5 mitigates radiation hematopoietic toxicity; therefore, inhibiting the expression of m⁶A demethylases might enhance recovery from radiation exposure¹³⁴. This study indicates the importance of m⁶A modification in hematopoietic radiobiology and suggests that modulation of the epi-transcriptome might be exploited as a strategy against radiation damage.

The roles of m⁶A modification and corresponding enzymes in the IR biology of normal tissues are summarized in Table 2.

Table 2.

RNA m⁶A methylation in ionizing radiation biology in normal tissues

Organ	Enzyme	Target	Function
Lung	METTL3 ALKBH5 YTHDF2	FOXO1 mRNA↓	Facilitate IR-induced EMT during RILI27
	ALKBH5	IL-6 mRNA↓	Suppress aseptic inflammation in RILI26
Liver	ALKBH5 YTHDF2	HMGB1 mRNA↓	Activate STING-IRF3 and contribute to RILD via the innate immune response140
	METTL3 IGF2BP2	TEAD1 mRNA↑	Activate STING-NLRP3 and contribute to RILD via pyroptosis25
	ALKBH5	TIRAP mRNA↑	Promote RILF through monocyte recruitment and M2 polarization117
Hematopoietic organs	YTHDF2	Wnt target genes↓	Decrease the regenerative ability of HSC152
	FTO ALKBH5	Radiation response and metabolism pathways	Inhibit recovery from radiation exposure134

↑: promotes, ↓: inhibits.

m⁶A modification with UV radiation in the biology of normal tissues

Several studies have focused on the roles of RNA m⁶A modification in non-IR effects on non-cancer tissues. A recent article has revealed that deficiency in ALKBH5 alleviates UVB radiation-induced chronic actinic dermatitis via regulating pyroptosis, in a manner involving alteration of levels of cell proliferation and apoptosis, inflammatory cytokines, and pyroptosis-associated proteins, such as gasdermin D (GSDMD), caspase-1, and caspase-4²⁴. Long-term exposure to UV radiation from sunlight facilitates the progression of skin photoaging. A recent study has reported that microRNA m⁶A modification regulates this process. A single UVB dose of 30 mJ/cm² elicits a photoaging phenotype in skin fibroblasts, and is accompanied by a decrease in total m⁶A levels and METTL14 expression. The m⁶A modification level of pri-miR-100, identified as the target of METTL14, is decreased by photoaging. The modification of pri-miR-100 influences the binding of DiGeorge syndrome critical region 8 (DGCR8) on this pri-miRNA, thereby obstructing the maturation of miR-100-3p. The restrained miR-100-3p maturation eventually results in elevated expression of the target gene epidermal growth factor receptor feedback inhibitor 1 (ERRFI1). Therefore, UVB exposure triggers dermal photoaging by affecting the METTL14/m⁶A-miR-100-3p/ERRFI1 axis. This finding clarifies the miRNA m⁶A modification-mediated mechanism underlying skin photoaging, and enriches evidence of the important roles of RNA m⁶A modification in UVB-induced skin injury¹⁵⁵. Supplementing the findings of Xiang et al.¹³³, Liu and colleagues²³ have revealed the altered transcriptome and epigenetic signatures in the UV-induced DNA damage response through multi-omic analysis, including the DNA methylation (5 mC) and RNA m⁶A methylome. They have defined the temporally specific m⁶A RNA methylation that appears to affect DNA repair by modulating translation²³. Evidence of the functions of RNA m⁶A modification in non-ionizing radiobiology has verified its spatiotemporally sensitive characteristics, which contribute to the precise regulation of genetic information.

Notably, investigations of the roles of m⁶A modification in radiation-induced normal tissue injuries are far from comprehensive. How this modification functions in other typical radiation effects, such as radiation-induced gastrointestinal injury, radiation-induced neurobehavioral injury, and radiation-induced reproductive injury, remains unclear and requires urgent elucidation (Figure 2).

Figure 2.

Outline of the interplay between m⁶A modification and radiation-induced injury effects on distinct normal organ tissues (created in Figdraw). Investigated effects included those on the lungs, liver, and hematopoietic system. A. In radiation-induced lung injury (RILI), the METTL3/m⁶A-FOXO1 mRNA/YTHDF2/AKT-ERK signaling pathway contributes to epithelial-to-mesenchymal transition (EMT). BTB domain-containing protein 7B (Zbtb7b) recruits ALKBH5 to demethylate of IL-6 mRNA, thereby inhibiting its nucleocytoplasmic transport and production, and alleviating aseptic inflammation in RILI. B. In radiation-induced liver disease (RILD), the ALKBH5/m⁶A-HMGB1 mRNA/STING-IRF3 signaling pathway contributes to RILD via the innate immune response. In Kupffer cells, irradiation increases m⁶A modification levels, and the METTL3/m⁶A-TEAD1 mRNA/IGF2BP2/STING-NLRP3 signaling pathway contributes to the injury effects via pyroptosis. In hepatic stellate cells, irradiation upregulates ALKBH5, and the ALKBH5/m⁶A-TIRAP mRNA pathway facilitates radiation-induced liver fibrosis (RILF) progression by inducing monocyte recruitment and M2 polarization. C. The hematopoietic process is crucial for recovery and survival after radiation exposure, whereas hematopoiesis is vulnerable during RT. In hematopoietic Ythdf2 knockout mice, the mRNA degradation of both Wnt target genes and survival-associated genes under radiation stress is suppressed, and the regenerative ability of hematopoietic stem cells (HSCs) is therefore restrained. Our previous study has revealed that inhibiting the expression of FTO and ALKBH5 ameliorates radiation-induced hematopoietic injury (RIHI); therefore, suppression of m⁶A demethylase might improve recovery from radiation exposure. D. MicroRNA m⁶A modification regulates UV-induced skin photoaging. UVB exposure elicits a photoaging phenotype in skin fibroblasts and is accompanied by a decrease in total m⁶A level and METTL14 expression. After METTL14 overexpression, the downregulated m⁶A modification level of pri-miR-100, identified as the target of METTL14, is rescued, thus enhancing binding of DiGeorge syndrome critical region 8 (DGCR8) to this pri-miRNA and restoring miR-100-3p maturation. The resurgent miR-100-3p eventually decreases expression of its target gene, epidermal growth factor receptor feedback inhibitor 1 (ERRFI1). Therefore, UVB exposure triggers dermal photoaging by affecting the METTL14/m⁶A-miR-100-3p/ERRFI1 axis. Of note, how the m⁶A modification functions in other typical radiation effects, such as radiation-induced neurobehavioral injury (RINI, E), radiation-induced gastrointestinal injury (RIGI, F), and radiation-induced reproductive injury (RIRI, G), requires further study.

Novel regulatory mechanisms of RNA m⁶A modification

With the continued advancement of m⁶A-related research, multiple novel regulatory mechanisms are being elucidated (Figure 3). One novel mechanism of m⁶A modification in radiobiology affects m⁶A enzymes’ chromatin remodeling—a critical regulatory layer, alongside m⁶A modification, in controlling gene expression and DNA damage signaling^156,157. Sun et al.¹⁵⁷ have reported that poly[adenosine diphosphate (ADP)-ribose] polymerase 1 (PARP1), interacting with the transcription factors nuclear factor I-C (NFIC) and TATA binding protein (TBP), modulates the chromatin accessibility of the METTL3 promoter, and consequently activates METTL3 transcription and influences the m⁶A modification of poly(A)+ RNA, such as lysophosphatidic acid receptor 5 (LPAR5) mRNA. However, irradiation causes disassociation of PARP1 from the promoter of METTL3, and impairs the accessibility of the transcription factors NFIC and TBP to the promoter region. Subsequently, chromatin condensation and repressed transcription of METTL3 decrease the m⁶A levels and consequently accelerate the degradation of LPAR5 mRNA¹⁵⁷. These findings advance the conventional understanding of PARP’s roles, and may have important implications in exploring valuable therapeutic strategies for PARP1 inhibitors in oncology¹⁵⁷.

Figure 3.

The newly elucidated regulatory mechanisms relevant to RNA m⁶A modification (created in Figdraw). As shown, the novel m⁶A-associated mechanisms in radiobiology involve the chromatin remolding of m⁶A regulatory enzymes and the function of m⁶A-modified enhancer RNAs (eRNAs). A. Irradiation causes disassociation of poly(ADP-ribose) polymerase 1 (PARP1) from the promoter of METTL3 and impairs the accessibility of the transcription factors nuclear factor I-C (NFIC) and TATA binding protein (TBP) to the promoter region. Subsequently, chromatin condensation and repressed transcription of METTL3 occur. Decreased expression of METTL3 abates the m⁶A levels of its target genes, such as Lysophosphatidic Acid Receptor 5 (LPAR5), whose degradation is consequently accelerated after radiation. B. Top, in nasopharyngeal carcinoma (NPC), METTL3 methylates the super enhancer (SE) RNA lncRNA succinate-CoA ligase GDP-forming beta subunit-antisense 1 (SUCLG2-AS1), located in the SE region of sex-determining region Y-box 2 (SOX2). The stability of SUCLG2-AS1 is enhanced by the recognition of the reader protein insulin-like growth factor 2 mRNA-binding protein 3 (IGF2BP3), and the expression is elevated in an m⁶A-dependent fashion, thereby facilitating the formation of a complex comprising CCCTC binding factor (CTCF), SUCLG2-AS1, SOX2 promoter, and the SE region through a long chromatin loop; increasing SOX2 expression; and conferring radioresistance to NPC cells. Bottom, the bone-specific enhancer RNA MLXIPe, with an m⁶A site, along with the mRNA of its target gene PSMD9, with m⁶Am site on the 5′-UTR, is recognized by, and interacts with, RNA-binding protein KH-type splicing regulator protein (KHSRP). The interaction protects against PSMD9 mRNA degradation by 5′-3′ exoribonuclease 2 (XRN2). Thus, the m⁶A-methylated eRNA MLXIPe facilitates radioresistance in metastatic prostate cancer (mPa). C. In lung cancer, the reader protein IGF2BP2 is upregulated and augments radioresistance. Mechanistically, the m⁶A-modified mRNA of solute carrier family 7 member 5 or LAT1 (SLC7A5), encoding a sodium-independent transporter of large neutral amino acids across the cell membrane, is stabilized by IGF2BP2, which is elevated in lung cancer. The upregulation of SLC7A5 promotes the transport of Met, the substrate for SAM synthesis, into cells. Increased intracellular SAM further serves as a substrate for the histone lysine methyltransferase SETD1A, thus leading to formation of trimethylated lysine 4 of histone H3 (H3K4me3) at the promoter region of the IGF2BP2 gene, and enhancing IGF2BP2 transcription through formation of a positive feedback loop between IGF2BP2 and SLC7A5. Consequently, IGF2BP2 enhances lung cancer radioresistance via the AKT/mTOR pathway.

Some studies have focused on m⁶A modification on enhancer RNAs. METTL3 mediates the methylation of super enhancer (SE) RNA, the lncRNA succinate-CoA ligase GDP-forming beta subunit-antisense 1 (SUCLG2-AS1). SUCLG2-AS1 is recognized and stabilized by the reader protein insulin-like growth factor 2 mRNA-binding protein 3 (IGF2BP3), and the expression of SUCLG2-AS1 increases in an m⁶A-dependent fashion in NPC¹⁸. SUCLG2-AS1, located in the SE region of sex-determining region Y-box 2 (SOX2), regulates the expression of SOX2 through long-range chromatin loop formation, via mediating the occupancy of the CCCTC binding factor CTCF at the SE and promoter regions of SOX2, thus modulating the metastasis and radiosensitivity of NPC¹⁸. Zhao et al.¹⁵⁸ have screened and identified bone-specific m⁶A modifications on enhancer RNA (eRNA) that play a post-transcriptional functional role in bone metastatic prostate cancer and correlate with RT resistance. The enhancer RNA MLXIPe with an m⁶A site, along with the mRNA of its target gene PSMD9, with an m⁶Am site at the 5′-UTR, are recognized and interact with the RNA-binding protein KH-type splicing regulator protein (KHSRP), thereby protecting against PSMD9 mRNA degradation by 5′-3′ exoribonuclease 2 (XRN2) and increasing the radioresistance of metastatic prostate cancer¹⁵⁸.

Novel functions of m⁶A modification in radiotherapy efficacy are also reflected in regulation of the TME. This epigenetic modification modulates the immune milieu of the radiation-remodeled TME and has a nexus role in both anti-tumor immunity and the radiation response: it influences both tumor cell intrinsic radioresistance and MDSC-mediated extrinsic radioresistance¹⁵⁹.

With continued deep examination of the molecular mechanisms underlying cancer radiosensitivity, researchers are delineating the cross-talk between RNA m⁶A modification and other epigenetic modifications, such as histone modification and DNA methylation. In lung cancer, the reader protein IGF2BP2 is upregulated and enhances radioresistance. Mechanistically, the m⁶A-modified mRNA of solute carrier family 7 member 5 or LAT1 (SLC7A5), a sodium-independent transporter of large neutral amino acids across the cell membrane, is stabilized by IGF2BP2. The upregulation of SLC7A5 promotes the transportation of Met, the substrate for SAM synthesis in cells. Intracellular SAM enrichment provides a substrate for SETD1A, a histone lysine methyltransferase, thus leading to trimethylation of lysine 4 of histone H3 (H3K4me3) in the promoter of the IGF2BP2 gene, enhancing IGF2BP2 transcription, and forming a positive feedback loop between IGF2BP2 and SLC7A5¹⁶⁰. This finding emphasizes the importance of the interactions among multiple regulatory layers underlying the RT sensitivity of cancer. These studies have provided new insights into new types of RNA m⁶A modification and indicated multiple regulatory paradigms mediated by this modification.

Exploration of the application of RNA m⁶A modification in radiation medicine

Accurate quantification of IR, particularly low-dose radiation, is important in protecting organisms against damage caused by environmental radioactive incidents or clinical radiotherapy¹⁶¹. As described previously, the abundant m⁶A modification responds sensitively and rapidly to external stimulation¹³⁴. By exploiting this feature of m⁶A modification, Jin et al.¹⁶¹ have reported a Cas13a-microdroplet platform that enables sensitive detection of ultra-low doses of radiation (0.5 Gy vs. 1 Gy traditionally) within 1 h. The micro-platform uses an ideal, specific radiation-sensitive marker, m⁶A on the nuclear receptor coactivator 4 (NCOA4) gene (NCOA4-m⁶A)^161,162. The microfluidics of the platform generates uniform microdroplets that encapsulate a detection system consisting of CRISPR/Cas13a and NCOA4-m⁶A targets derived from total RNA extraction, thereby achieving a 10-fold enhancement in sensitivity and significantly decreasing the limit of detection¹⁶¹. Clinical patient samples and systematic mouse models have indicated a limit of detection (0.5 Gy) and superior sensitivity to that of traditional quantitative PCR¹⁶¹. Generally, through screening and identification of radiation-sensitive m⁶A sites on specific mRNAs or non-coding RNAs and the m⁶A-correlative enzymes involved in certain cancer types, m⁶A-related biomarkers can be used for tumor diagnosis in early stages, prediction of RT response before treatment, and evaluation of prognosis after treatment, and can also serve as a target in combination therapy to enhance the efficacy of RT. Thus, strategic and rational utilization of m⁶A-related biomarkers may enhance the effects of cancer RT in clinical settings.

m⁶A modification-based biomarkers have also been explored for absorbed IR dose estimation¹⁶². A genome-wide screen of radiation-responding mRNAs, whose m⁶A levels show significant alteration after acute irradiation, has been conducted¹⁶². The NCOA4 m⁶A level shows highly responsive specificity to radiation, which is conserved across mice, monkeys, and humans, and complies with the dose-response relationship observed in peripheral blood mononuclear cells from patients with cancer receiving RT¹⁶². In the screening of m⁶A-based biomarkers for radiation dose estimation, the specifically sensitive genes should be identified, and a dose-m⁶A level curve should be established for dose derivation. Subsequently, dose-prediction models based on the biomarker of m⁶A modifications might be established, through which the radiation doses absorbed into the body are prospectively and accurately predicted, thereby maximally alleviating the adverse effect toxicity during RT, as well as injury in the event of accidental nuclear exposure.

Identification of epigenetic biomarkers of radiation sensitivity is beneficial in precision medicine¹⁶³. Using multi-omics analysis, Zeng et al.¹⁶³ have identified SETD2, a factor important in repairing DNA double-strand breaks and maintaining chromatin integrity, as a radio-sensitivity signature in LUAD. Moreover, SETD2 is a favorable prognostic factor whose effects are antagonized by RBM15 and YTHDF3; therefore, SETD2 is a promising epigenetic biomarker in patients with LUAD¹⁶³. These studies have indicated promising prospects of m⁶A modification in radiation monitoring for radioactive injury management and screening radiosensitivity-related epigenetic biomarkers^161,162.

Given that multiple m⁶A-associated modulators are dysregulated in various patterns of malignancy, targeting overexpressed m⁶A enzymes is considered a promising strategy for cancer therapy. Small-molecule inhibitors targeting the catalytic domains of writers, erasers, and readers have been designed to diminish their enzymatic activities. For instance, METTL3 or METTL3-14 complex inhibitors such as UZH1a/2 and CDIBA derivative have been found to exhibit anti-tumor efficacy in a glioblastoma PDX mouse model or AML^164,165. The most widely investigated METTL3 inhibitor STM2457 impairs stem cell viability in leukemia, by suppressing the cell proliferation of prostate cancer and intrahepatic cholangiocarcinoma, and notably enhancing the anti-PD-1 treatment effects in CRC^166–169. Delicaflavone, a natural product from Selaginella doederleinii Hieron, prevents expression of METTL3/14 and promotes the anti-tumor immune response in lung cancer¹⁷⁰. Additionally, CS1/CS2 and Dac51, inhibitors of FTO, hinder CSCs self-renewal and facilitate anti-tumor immunity^171,172. Cucurbitacin B, targeting IGF2BP1, induces HCC cell apoptosis and suppress PD-L1 expression¹⁷³. Furthermore, on the basis of the primary effects of small-molecule inhibitors, the nanoparticle delivery system can achieve targeted delivery and controllable release of molecules. A co-delivery nanoplatform carrying the FTO inhibitor (FB23-2) and tumor-associated antigens has been designed to target tumor-infiltrating dendritic cells in HCC, and consequently restrain HCC progression through accelerating dendritic cell maturation as well as effector T cell infiltration¹⁷⁴. These studies have indicated that the integrative utility of inhibitors targeting m⁶A-related regulators or specific m⁶A-modified oncogenes may achieve radiosensitizing effects in cancer RT in the clinical practice of radiation medicine.

Conclusions and prospects

In the past several years, knowledge of RNA m⁶A modifications in distinct biological processes has rapidly increased, and the intricate interplay between this modification and radiation biology in both cancer and non-cancer contexts has profound implications for elucidating the molecular mechanisms underlying radiation responses and devising novel strategies for RT or radiation protection. Accumulating evidence suggests that m⁶A modifications play critical roles in tumor responsiveness to RT and the propagation of radiation injury in normal tissues. This modification is also a potential biomarker and therapeutic target, and modulating the activity of associated enzymes to alter m⁶A levels might facilitate RT efficacy while mitigating toxic effects on normal tissues. Accordingly, this feature of m⁶A modification might be used to develop dual-benefit strategies in cancer treatment in the future.

Despite the current exploration of the relationship between RNA m⁶A modification and radiation biology, studies in this area are far from sufficient. More in-depth efforts are crucial. A goal of molecular biologists is to further decipher the precise novel molecular pathways through which RNA m⁶A modification governs tumor radio-sensitivity and the radiation injury effects on normal tissues. Subsequently, designing novel radio-sensitizers or radio-protectors based on m⁶A modification-related pathways could pave the way to personalized RT and radiation protection approaches. Furthermore, clinical trials to validate the potential of m⁶A modification as a predictive biomarker for cancer prognosis and RT efficacy would help assess the clinical utility of targeted therapies against the m⁶A pathway in patients with cancer. By monitoring the expression of m⁶A-based biomarkers, clinicians could tailor RT regimens to individual patients, including optimization of doses and schedules for maximum efficacy and minimal toxicity. This personalized approach might achieve better outcomes and diminished adverse effects for RT. Collectively, m⁶A-related regulator-mediated m⁶A-modification triggers the expression alterations of oncogenes or tumor suppressors by affecting the metabolism of modified-RNAs, thus modulating the progression and RT outcomes for cancer tissues. Similarly, radiation-induced aberrantly expressed m⁶A-related enzymes and the abnormal modification of key target RNAs contribute to the toxic injury effects to normal tissues. Delving deeper into the intrinsic connection between RNA m⁶A modification and radiation biology promises to enhance RT outcomes, decrease adverse reactions, and mitigate the toxic effects derived from environmental radiation stress, thereby driving advancements in precision medicine.

Funding Statement

This work was supported by grants from the National Natural Science Foundation of China (Grant No. 82173467 and 82273577) and CAMS Innovation Fund for Medical Sciences (Grant Nos. CIFMS, 2022-I2M-2-003 and 2021-I2M-1-042).

Conflict of interest statement

No potential conflicts of interest are disclosed.

Author contributions

Conceived and designed the analysis: Saijun Fan and Shuqin Zhang.

Collected the data: Yajia Cheng and Shuqin Zhang.

Contributed data or analysis tools: Yajia Cheng and Yue Shang.

Wrote the paper: Shuqin Zhang and Yajia Cheng.

Revised the manuscript and figures: Yajia Cheng, Yue Shang and Saijun Fan.