The Role of m6A-Mediated DNA Damage Repair in Tumor Development and Chemoradiotherapy Resistance

Abstract

Among the post-transcriptional modifications, m6A RNA methylation has gained significant research interest due to its critical role in regulating transcriptional expression. This modification affects RNA metabolism in several ways, including processing, nuclear export, translation, and decay, making it one of the most abundant transcriptional modifications and a crucial regulator of gene expression. The dysregulation of m6A RNA methylation-related proteins in many tumors has been shown to lead to the upregulation of oncoprotein expression, tumor initiation, proliferation, cancer cell progression, and metastasis.Although the impact of m6A RNA methylation on cancer cell growth and proliferation has been extensively studied, its role in DNA repair processes, which are crucial to the pathogenesis of various diseases, including cancer, remains unclear. However, recent studies have shown accumulating evidence that m6A RNA methylation significantly affects DNA repair processes and may play a role in cancer drug resistance. Therefore, a comprehensive literature review is necessary to explore the potential biological role of m6A-modified DNA repair processes in human cancer and cancer drug resistance.In conclusion, m6A RNA methylation is a crucial regulator of gene expression and a potential player in cancer development and drug resistance. Its dysregulation in many tumors leads to the upregulation of oncoprotein expression and tumor progression. Furthermore, the impact of m6A RNA methylation on DNA repair processes, although unclear, may play a crucial role in cancer drug resistance. Therefore, further studies are warranted to better understand the potential biological role of m6A-modified DNA repair processes in human cancer and cancer drug resistance.

Introduction

The N6-methyladenosine (m6A) transcriptional marker has recently emerged as a pivotal regulator of gene expression and diverse cellular pathways in eukaryotic messenger RNA. ¹ m6A modification is indispensable for the proper functioning of numerous cellular processes, including circadian rhythm, cell regeneration, differentiation, neurogenesis, and immunity. ² Disruptions in the regulation of m6A have been implicated in various pathological conditions, such as cancer, obesity, and immune disorders. In addition to its well-known roles in gene expression regulation, m6A has been found to be an important player in preserving genomic stability and integrity by regulating DNA repair processes, particularly in response to oxidative stress and DNA damage. ³ Thus, a comprehensive understanding of the complex regulation of m6A modification in health and disease can pave the way for the development of innovative therapeutic strategies.

The pathogenesis of cancer is commonly associated with genomic instability, which arises from a combination of various factors, including DNA damage, tumor-specific DNA repair defects, and impaired regulation of the cell cycle. ⁴ This genomic instability is often manifested by chromosomal aberrations, such as changes in chromosome number and structure, and by structural changes in DNA, including nucleotide substitution, insertion, and deletion. In particular, the occurrence of driver mutations in a small number of genes can have a profound impact on cellular behavior, providing a selective advantage for cancer cell growth and promoting disease development ⁵ Such mutations also determine the tumor’s response to treatment, which further highlights the significance of genomic instability in cancer. By investigating the cellular response to DNA damage, we can gain deeper insights into the development of cancer and improve disease classification and treatment strategies. A comprehensive understanding of the genomic instability mechanisms in cancer is therefore crucial for advancing our knowledge of cancer biology and developing effective therapies.

DNA repair is a complex and essential process that maintains genomic integrity by efficiently repairing various forms of DNA damage, including base mismatches, methylation events, oxidized bases, intrastrand and interstrand DNA crosslinks, and protein-DNA adducts. ⁶ To achieve this task, cells utilize a diverse range of specialized pathways, such as mismatch repair, base excision repair, nucleotide excision repair (NER), and homologous directed repair of Fanconi anemia. However, mutations in the genes that encode proteins involved in these pathways can lead to genomic instability, which may promote cancer development.^7-9 Therefore, the identification and targeting of DNA repair pathways and proteins in the development of novel anticancer therapies have become increasingly relevant. By enhancing the sensitivity of cancer cells to conventional chemotherapy, such an approach may ultimately lead to improved treatment efficacy, particularly given the critical role of DNA repair capacity in determining the cellular response to genotoxic stress.

The Function of m6A

RNA methylation refers to a process wherein methyltransferase enzymes add methyl groups to the Nmur6 site of RNA molecules, including both messenger RNA (mRNA) and noncoding RNA (ncRNA). This phenomenon is extensively involved in the internal modification of eukaryotic mRNAs and represents a crucial mode of epigenetic regulation.^9,10 At the molecular level, m6A has been shown to regulate a diverse array of processes undergone by mRNA molecules, encompassing variable splicing, nucleation, translation, and degradation (Figure 1).^11-13 At the physiological level, m6A can regulate stem cell differentiation, ¹⁴ animal growth and development, ¹⁵ Drosophila sex determination, ¹⁶ DNA damage repair, ¹⁷ heat shock response, ¹⁸ immune response, and cancer development.^19-21 Other mRNA modifications, such as N1-methyladenosine (M1A), 5-methylcytosine, and pseudouridine (Y), form epitope-coding groups alongside m6A, which together form a complex that encodes and regulates protein synthesis.^1,22,23

Figure 1.

Molecular composition of the M6A modifications. The M6A modification are managed by “writers”, “readers” and “erasers”. The m6A “writer” complex, consisting of METTL13, METTL14, WTAP, RBM15/15B, ZC3H13, CBLL1, VIRMA, and METTL16, catalyzes the modification of M6A. M6A “erasers” proteins FTO and ALKBH5,facilitate demethylation on M6A site. M6A “reader” proteins perform their biological functions by binding to M6A modifications. YTHDC1 is involved in pre-mRNA splicing and RNA exportation, while YTHDC2 and YTHDF2/3 induce intracellular RNA degradation. IGF2BP family proteins sustain RNA stability.YTHDF1/3, YTHDC2 and eIF3 promote RNA translation. hnRNPC and hnRNPG are involved in pre-mRNA processing and structure switching, while hnRNPA2B1 promotes primary miRNA processing. fMRP binds to YTHDF2 to stabilize mRNA.

m6A Methylase (Writers)

The m6A RNA modification is facilitated by various methyltransferases known as m6A “writers”. These writers include Methyltransferase 3 (METTL3), Methyltransferase 14 (METTL14), Wilms Tumor 1-Associated Protein (WTAP), RNA Binding Motif Protein 15 (RBM15), Vir-like m6A Methyltransferase Associated (VIRMA) - also called KIAA1429, Zinc Finger CCCH-Type Containing 13 (ZC3H13), Zinc Finger CCHC-Type Containing 4 (ZCCHC4), Methyltransferase 16 (METTL16), and Methyltransferase 5 (METTL5).^21,24,25

METTL3 was the first human RNA m6A methyltransferase to be identified. METTL14 is another active component of the m6A methyltransferase complex (MTC). Together, they play a crucial role in substrate recognition and form stable hybrids at a 1:1 proportion. While METTL3 is the main catalytic core, METTL14 acts as the supporting structure of RNA binding. WTAP is the third key component of the MTC.^24,26 Though WTAP lacks a conservative catalytic methylation domain, it interacts with METTL3 and METTL14 as a connecting protein to ensure the localization of the METTL3-METTL14 heterodimer on nuclear spots and promote catalytic activity. ²⁷

RBM15 and RBM15 B have no catalytic function; however, they can bind to METTL3 and WTAP to direct the two proteins to specific RNA sites for m6A modification. ²⁸ KIAA1429 is the largest known component of the methyltransferase complex. It guides regioselective methylation of specific m6A near the 3′UTR and stop codon by recruiting the m6A methyltransferase component METTL3/METTL14/WTAP. ²⁹ ZC3H13 is a newly discovered regulatory protein for RNA m6A, which can bridge WTAP and mRNA binding factor Nito, and regulate the complex to be anchored in the nucleus. ³⁰ ZCCHC4, the main methyltransferase responsible for m6A methylation of AAC in 28SrRNA, affects translation by binding to a part of mRNA and plays a role in cell proliferation and tumorigenesis. ³¹ METTL16, an independent mRNA methyltransferase, can act alone and catalyze the m6A of U6snRNA. It can also target pre-mRNAs and ncRNAs to regulate tumorigenesis. ³² METTL5 is a newly discovered methyltransferase responsible for the m6A modification of 18SrRNA.^25,33

m6A Demethylase (Erasers)

m6A RNA demethylation, carried out by a group of demethylases known as “erasers,” is a crucial process in ensuring the dynamic and reversible nature of m6A methylation. The members of this group include fat mass and obesity-associated protein (FTO), α-ketoglutarate-dependent dioxygenase alkB homolog 5 (ALKBH5), and α-ketoglutarate-dependent dioxygenase alkB homolog 3 (ALKBH3).^34,35 FTO, also known as the gene related to fat weight and obesity, was the first m6A demethylase to be discovered. FTO exhibits high oxidative demethylation activity on m6A-rich RNA in vitro and regulates the m6A content of RNA in vivo. ³⁶ ALKBH5, the second m6A demethylase to be identified, possesses a catalytic domain capable of demethylating single-stranded RNA (ssRNA) and single-stranded DNA (ssDNA), with a particular preference for m6A demethylation in ssRNA. This ability renders m6A methylation reversible in RNA. ³⁷ Finally, ALKBH3, primarily located in the cytoplasm with a strong binding affinity to tRNA, can demethylate N1-methyladenosine (M1A) and 3-methylcytosine (M3C) in RNA and N6-meA in tRNA. Modification of tRNA by ALKBH3 improves protein translation efficiency. ³⁸

m6A Binding Protein (Readers)

m6A modification plays a crucial role in producing diverse downstream biological functions, but the recognition and binding of m6A modification by different “reader” proteins are necessary to decode m6A markers and produce functional signals, ultimately resulting in the specific destination of target RNA. ³⁹ These reader proteins include YT521-B homologous (YTH) domain family proteins 1, 2, and 3 (YTHDF1, YTHDF2, and YTHDF3), YTH domain-containing protein 1 and 2 (YTHDC1 and YTHDC2), insulin-like growth factor 2 mRNA binding protein (IGF2BP; comprising IGF2BP1/2/3), eukaryotic translation initiation factor 3 (Eif3), heterogeneous ribonucleoproteins (HNRNPs; including HNRNPA2/B1 and HNRNPC/G), ⁴⁰ leucine-rich pentapeptide repeat sequence (PPR) motif protein (LRPPRC), and YTH family proteins. The YTH family proteins specifically recognize m6A modification in a methylation-dependent manner through a particular YTH domain. ⁴¹ Among the five YTH family proteins, YTHDC1 is the only nuclear protein involved in transcription, mRNA splicing, and transcription, while YTHDF1, YTHDF2, YTHDF3, and YTHDC2 are cytoplasmic m6A readers mainly involved in mRNA translation and degradation.^16,42,43 IGF2BPs, including IGF2BP1-3, can bind to m6A and enhance RNA expression by stabilizing RNA,^44,45 while EIF3 promotes cap-independent translation. ⁴⁰ The HNRNP family, including HNRNPA2/B1, facilitates alternative splicing of target RNA and improves primary miRNA processing through interaction with miRNA microprocessor complex protein DGCR8. ⁴⁶ After recognizing m6A, HNRNPC/G can regulate mRNA abundance and splicing, known as the “m6A switch.” LRPPRC is a member of the PPR motif protein family that binds to RNA and regulates transcription, RNA processing, splicing, stability, editing, and translation. ⁴⁷

DNA Repair of m6A Regulation Affecting Genomic Stability

1m6A Regulates DNA Repair to Regulate Genomic Stability

m6A and UV Damage Repair

The profound role of m6A modification in facilitating the response and repair of UV-induced DNA damage has been comprehensively explored. ⁴⁸ Compelling research indicates that m6A is not only induced by UV irradiation, but also selectively recruited to UV-damaged sites to accelerate DNA repair and heighten cell resistance to UV damage. Remarkably, m6A produced by METTL3 emerges as a vital contributor to the response and repair of UV damage, potentially mediated by cross-lesion DNA polymeraseκ. ⁴⁸ Additionally, RNA encoding m6A in the cytoplasm undergoes translocation back to the nucleus after UV-induced damage, where it accrues at the location of the UV-induced DNA damage, potentially leading to Nucleotide excision repair (NER) to mitigate damage through ncRNA. ⁴⁹ In essence, these discoveries underscore the fundamental significance of m6A in promoting the response and repair of UV damage, mandating a continued exploration into the intricate mechanisms that underpin its profound influence.

m6A-mediated dsDNA break repair to keep genomic stability

The repair of DNA damage is a critical process for the maintenance of genomic stability and the prevention of cancer. ¹⁸ Recent studies have highlighted the potential of N6-methyladenosine (m6A), a prevalent RNA modification, in repairing double-stranded DNA (dsDNA) breaks through a complex mechanism involving the activation of METTL3 via ATMATM phosphorylation, recruitment of YTHDC1 to the RNA encoded by m6A, and subsequent recruitment of and BRCA1 for dsDNA break repair via homologous recombination (HR) (Figure 2). This process not only facilitates the repair of DNA damage but also averts the risk of genomic instability. ¹⁸ The discovery of this intricate repair mechanism sheds new light on the significance of RNA modifications in maintaining genomic integrity and holds promising implications for the development of novel cancer therapies.

Figure 2.

M6A regulates DNA repair through the R-loop to maintain genomic stability. DNA damage agents induce strand breaks to activate DNA damage response and ATM, thereby phosphorylating and activating METTL3. DNA damage response leads to gene transcription and M6A production by activating METTL3 on mRNA transcripts. Subsequently, the mRNAs wrapped in M6A hybridize with the DNA template to form an R ring. Then, M6A readers, such as YTHDF2 and YTHDC1, combine with M6A on the R ring to promote DNA repair and R loop resolution through RNaseH1, thereby maintaining genome stability.

Direct repair of m6A and DNA

Recent research has shed light on the crucial role of m6A modification in regulating direct DNA repair and preventing genomic instability in mice. ⁵⁰ Recognition of m6A modification by YTHDF1 promotes cap-dependent translation, leading to the translation of O6 methylguanine-DNA methyltransferase, a direct DNA repair enzyme that prolongs the lifespan of mice. Additionally, altering levels of METTL3/14, ALKBH5, and FTO can also influence lifespan, further supporting the role of m6A modification in preventing genomic instability and promoting longevity. ⁵⁰ These findings suggest that targeting m6A modification may have therapeutic potential for preventing genomic instability and age-related diseases.

The m6a Modification and Genomic Instability

DNA translesion synthesis (TLS) is a DNA damage tolerance pathway that allows the cell to overcome replication barriers. ⁵¹ In TLS, specialized low-fidelity polymerases utilize the damaged template to restart DNA synthesis.Eukaryotic TLS involves polymerases of the Y-family ⁵² — including REV1, Polη, Polιand Poκ—and the B-family (Polζ). ⁵³ These Y-family and B-family TLS polymerases lack 3′-to-5′nucleotide proofreading and exhibit a decreased capacity to distinguish between incoming nucleotides relative to replicative polymerases. ⁵⁴ The replication stress response that occurs during treatment, cancer cells activate various DNA damage tolerance (DDT) pathways, ⁵⁵ DDT pathways broadly include translesion DNA synthesis.TLS involves specialized polymerases that can replicate through a damaged DNA template ⁵⁴ and is generally regarded as a lower-fidelity form of DDT because the TLS polymerases recruited to stalled replication forks have a high potential for mutagenesis. ⁵⁶ Mutagenic events induced by TLS polymerases can contribute to tumorigenesis36 and impact the response of cancer cells to DNA-damaging chemotherapies, ⁵⁷ highlighting the importance of these mechanisms in the context of tumour treatment.Several studies have found that METTL3-mediated m6 A modification plays a critical role in the DNA damage response (DDR) to regulate the DNA repair pathway. RNA m6 A modification rapidly occurs in ultraviolet (UV)-irradiated chromatin, indicating that METTL3 is specifically recruited to the UV-damaged chromatin region. ⁴⁸ This extensive recruitment of METTL3 depends on ADP-ribose polymerase 1 (PARP1). This DNA repair pathway may be mediated by trans-lesion DNA polymerase κ (Pol κ), which has been implicated in both nucleotide excision repair (NER) and trans-lesion synthesis (TLS).^58,59

m6A Processing Protein and DNA Damage and Repair

m6A’s Regulatory Role in Anticancer Drug Resistance through DNA Repair

The regulatory function of N6-methyladenosine (m6A) has emerged as a crucial research area in the fight against cancer. m6A plays a regulatory role in modulating drug resistance in cancer cells by regulating DNA repair. ⁶⁰ Investigating m6A regulatory proteins is essential to develop effective anticancer drug targets.m6A has been shown to increase the stability of transcription factor activation enhancer binding protein 2C (Tfap2c) mRNA, leading to the survival of seminoma cells treated with cisplatin and resistance to drug-induced DNA damage. Mechanistically, m6A upregulates the DNA repair gene via Tfap2c, rendering cells resistant to drug-induced DNA damage. Conversely, the downregulation of m6A in β-catenin mRNA triggers drug resistance in cervical squamous cell carcinoma, potentially by upregulating excision repair cross-complementation group 1 (ERCC1). ⁶¹ The multifaceted role of m6A in cancer, including its dual role in tumor type and treatment through DNA damage repair, plays a critical role in regulating drug resistance in anticancer therapy. Elucidating the cellular pathways underlying m6A-mediated anticancer drug resistance may pave the way for the development of innovative therapeutic strategies to overcome drug resistance and improve cancer patient outcomes. ⁶² Therefore, understanding the mechanisms underlying m6A regulation in cancer cells is essential for the development of targeted therapeutic strategies to combat drug resistance in cancer treatment.

m6A-related regulators and DNA damage

The involvement of m6A in DNA damage and repair is a complex and pivotal area of investigation that holds great promise for identifying potential therapeutic targets. ⁶³ By scrutinizing the effects of m6A regulators on cell sensitivity to DNA damage, valuable insights into DNA repair mechanisms can be gained. Studies have revealed that the knockout of the FTO gene in mouse osteoblasts leads to increased cell sensitivity to ultraviolet light and hydrogen peroxide, ultimately resulting in cell death. However, erasure of m6A by FTO can enhance the stability of mRNAs of Hspa1a and DNA repair genes, thereby protecting cells from DNA damage.^64,65 The competing activity of ALKBH1 in m6A demethylation and AP lyase activity potentially results in the accumulation of AP sites in the genome. Further investigation is vital for a complete understanding of the synergistic effect between ALKBH1 m6A demethylation and DNA damage repair, which will enable us to unravel the role of m6A in DNA damage response and repair, and potentially pave the way for the development of new therapeutic strategies.

m6A’s Role in Regulating Bone Development

Bone development is a complex process governed by intricate molecular mechanisms. Recent studies have demonstrated the crucial role of m6A, an RNA modification, in regulating bone development through its regulation of DNA repair.FTO, an essential protein for mammalian osteoblast survival, differentiation, and bone development, has been shown to be severely compromised in mice with FTO global gene knockout or osteoblast selective gene knockout. This impairment is characterized by age-related bone volume reduction, which can be attributed to the accumulation of DNA double-strand breaks in osteoblasts of FTOKO mice. The accumulation of DNA double-strand breaks leads to metabolic stress and downregulation of DNA repair proteins Hspa1a and Kdm2a, promoting apoptosis and osteoblast death. ⁶³ These findings underscore the vital importance of FTO and m6A in the proper orchestration of bone development and suggest potential therapeutic targets for bone-related disorders (Figure 3).

Figure 3.

The role of M6a-modified DNA repair in cancer. The increased phosphorylation of CHK1 inhibited by ALKBH5 siRNA makes CHK1 inactive, resulting in the inhibition of DNA repair and other processes; thus, the maintenance of glioma stem cells is reduced, and the radiosensitivity of glioma stem cells is promoted. Mettl14 promotes genome-wide repair by mediating the mRNA methylation of DDB2 and affecting its translation. It also inhibits skin cancer formation induced by UVB radiation. IGF2BP2/3 increases the M6A level of VANGL1 after irradiation by regulating the miR-29b-3p-VANGL1 axis. VANGL1 increases BRAF by inhibiting protein degradation leading to the increase in BRAF-related downstream effect factors TP53BP1 and RAD51. These effectors participate in the repair of DNA damage and protect DNA from damage, thereby reducing the harmful effects of radiation on LUAD. YTHDF1 acts on the target molecule E2F8. It regulates the stability of E2F8 mRNA and the repair of DNA damage in an METTL14-dependent manner, thereby promoting cell proliferation, inhibiting apoptosis, and promoting the growth of breast tumor cells. METTL16 interacts with Mre11 in an RNA-dependent manner, thereby inhibiting DNA terminal excision. During DNA damage, ATM phosphorylates METTL16 at its C-terminal Ser419, resulting in conformational changes and self-inhibition of the RNA binding of METTL16. Thus, the METTL16-RNA-Mre11 complex is dissociated, and the inhibition of Mre11 is released.

The Role of m6A-Modified DNA Repair in Cancer Treatment and Drug Resistance

Skin Cancer

Skin cancer is a significant global public health challenge, with chronic ultraviolet B (UVB) radiation exposure being a prominent risk factor. ⁶⁶ UVB radiation exposure triggers DNA damage and somatic mutations in skin cells, leading to the development of skin cancer. ⁶⁷ Recent studies have revealed that METTL14, a critical constituent of the m6A RNA methyltransferase complex, mediates mRNA methylation of damage-specific DNA-binding protein 2 (DDB2), a protein involved in genome-wide repair and UVB-induced skin cancer inhibition. Additionally, YTHDF1 knockdown regulates the level of DDB2 mRNA methylation and reduces DDB2 protein levels, leading to genome-wide repair impairment. ⁶⁸ These results indicate that METTL14 serves as a crucial transcription mechanism governing global genome repair and impeding UVB-induced skin cancer, representing a potential therapeutic target.

Osteosarcoma

Osteosarcoma is a highly malignant and heterogeneous bone tumor that primarily affects children and adolescents, with a high mortality rate. ⁶⁹ Despite extensive research efforts, the survival rate of osteosarcoma patients has remained stagnant for the past three decades, mainly due to the rarity of somatic mutations that hamper the development of effective treatments. Recent studies have revealed that ALKBH5, an RNA demethylase, is frequently amplified in osteosarcoma cells and plays a pivotal role in promoting tumor growth and progression. ⁷⁰ Mechanistic analysis has further demonstrated that knockdown of ALKBH5 induces cell cycle arrest by destabilizing ubiquitin-specific peptidase (USP22) and ubiquitin ligase ring finger protein 40 (RNF40), resulting in the upregulation of p27Kip1 and Wee1, which are cyclin-dependent kinase inhibitory proteins, and downregulation of cell cycle and DNA damage and repair-related genes in osteosarcoma cells. ⁷¹ Thus, ALKBH5 exerts its tumorigenic effects by downregulating genes involved in cell cycle progression and DNA repair in osteosarcoma cells.

Lung Adenocarcinoma

Lung adenocarcinoma(LUAD) is a significant contributor to global morbidity and mortality, with advanced-stage lung cancer commonly treated using a range of modalities such as radiotherapy, chemotherapy, cotherapy adjuvant immunotherapy, or targeted therapy.^72,73 Recent studies have elucidated the crucial role of IGF2BP2/3 in upregulating m6A levels and downregulating miR-29b-3p expression in VANGL1 post-irradiation by modulating the miR-29b-3p-VANGL1 axis. Furthermore, the activation of multiple signaling pathways, including cell cycle, and basic transcription factors.^74,75 These findings highlight the utmost importance of comprehensively investigating the molecular mechanisms underlying LUAD and devising targeted interventions that can effectively counteract the deleterious effects of radiation on LUAD.

Breast Cancer (BC)

BC is a significant global public health challenge, with the highest incidence rate among all cancers and the second leading cause of cancer-related deaths in women. ⁷⁶ Despite notable progress in the clinical management of BC, Triple-negative breast cancer (TNBC) poses a significant hurdle due to its poor prognosis and resistance to conventional therapies. ⁷⁷ Recent studies have illuminated the roles of m6A binding proteins YTHDF1 and METTL3 in BC, providing new insights into their mechanisms of action. ⁷⁸ Specifically, YTHDF1 has emerged as a potent tumor promoter in BC, exerting its effects by modulating E2F8mRNA stability and DNA damage repair through METTL14. By stimulating cell proliferation, suppressing apoptosis, and promoting tumor growth, YTHDF1 represents a promising therapeutic target for the treatment of BC. ⁷⁹ Conversely, METTL3 participates in diverse biological and pathological processes, including DNA repair, and enhances the resistance of BC cells to chemotherapeutic drugs. These findings suggest that the targeting of YTHDF1 and METTL3 may hold promise as a novel approach to overcoming chemotherapy resistance and improving the outcomes of BC treatment.

Glioblastoma(GBM)

GBM is a highly aggressive brain tumor characterized by a high incidence of recurrence and poor prognosis. ⁸⁰ The resistance of glioblastoma stem cells (GBMSCs) to radiotherapy and chemotherapy is a significant obstacle to effective treatment. To overcome this resistance, the DNA damage response (DDR) pathway is an attractive target, as it plays a vital role in GBM progression. Specifically, checkpoint kinases CHK1 and CHK2 are critical components of the DDR pathway, which are activated by effector kinases ATR and ATM, resulting in cell cycle checkpoint activation, DNA repair initiation, and cell cycle arrest. ⁸¹ However, the m6A RNA demethylase ALKBH5 has been shown to inhibit CHK1 activation, thereby promoting the maintenance of glioma stem cells and increasing radioresistance. ⁷¹ In light of these findings, we propose that downregulating ALKBH5 gene expression represents a promising strategy for enhancing the radiosensitivity of GBMSCs and improving the efficacy of GBM treatment.

Pancreatic Ductal Adenocarcinoma (PDAC)

PDAC is a prevalent subtype of pancreatic cancer and ranks among the most invasive and malignant cancers, exhibiting an overall 5-year survival rate of approximately 10%. ⁸² Despite the use of conventional chemotherapy and radiotherapy, the 5-year survival rates remain unimproved ⁸³ In PDAC, upregulated METTL16 expression, one of the RNA N6-methyladenosine writers, may offer high efficacy in treating multiple (ADP ribose)-polymerase inhibitor (PARPI) and increasing chemotherapeutic drug sensitivity. When DNA suffers damage, ATM initiates METTL16 phosphorylation, leading to conformational changes, and auto-inhibition of RNA binding. Consequently, the METTL16-RNA-Mre11 complex disassembles, releasing Mre11 inhibition, and reducing the inhibitory function of METTL16 in HR repair. ⁸⁴ In METTL16-expressing PDAC, there exists a synergistic effect between gemcitabine and PARPI, and the therapeutic potential of gemcitabine is further potentiated when combined with oxalapril. ⁸⁴ Fisetin has shown potential as an antitumor drug by inducing DNA damage. ⁸⁵ It targets PHF10, a subunit of the m6A writer ZC3H13 and PBAF chromatin remodeling complex. Loss of PHF10 function enhances γ-H_2AX, RAD51, and 53BP1 recruitment at the DSB site, thereby decreasing HR repair efficiency. Moreover, the ZC3H13 gene downregulates PHF10 m6A methylation in a YTHDF1-dependent manner and reduces PHF10 translation. ⁸⁵ In conclusion, fisetin enhances DSB through ZC3H13-mediated m6A modification of PHF10, providing a novel perspective for PDAC treatment (Table.1).

Table 1.

Role of m6a-Mediated Regulation of DNA Repair in Tumor Drug Resistance. Targeting m6a.

Cancer Type	Therapeutic Agents	Effect of m6A	Critical m6A Regulators	Targeted mRNA/Pathways	Refs
BC	Chemotherapy (cisplatin/PARP inhibitor)	Resistant	YTHDF1	E2F8 mRNA	[75]
		Resistant	METTL3	EGF-RAD51	[79]
				Enhanced HR activity, DNA damage
Cervical squamous cell carcinoma	Irradiation	Antiresistant	FTO	β-catenin-ERCC1	[104]
Lung adenocarcinoma	Irradiation	Antiresistant	IGF2BP2/3	BRAF/DNA repair	[74]
Glioblastoma	Irradiation	Antiresistant	ALKBH5	CHK1/cell cycle checkpoint, cell cycle arrest, and DNA repair	[82]
PDAC	chemotherapy (gemcitabine)	Antiresistant	METTL16	ATM-mediated phosphorylation of METTL16	[85]

FU, 5-fluorouracil; ALKBH5, AlkB homolog 5; E2F8, E2F transcription factor 8; EGFR, epidermal growth factor receptor; ERK, extracellular signal-regulated kinase; FTO, fat mass and obesity-associated protein; ERCC1, excision repair cross-complementation; METTL3, methyltransferase-like protein 3; PARP, poly(adenosine diphosphate) ribose polymerase; IGF2BP2, insulin-like growth factor 2 mRNA-binding protein 2; YTHDF, YT521-B homolog family; β-catenin, β-linked protein; ZC3H13, zinc finger CCCH-type containing 13.

The Role of m6A-Modified DNA Repair in Cancer Drug Resistance

The development of targeted therapies has revolutionized cancer treatment; however, drug resistance remains a major impediment to effective cancer management. Excitingly, recent research has highlighted the critical role of m6A, an epigenetic RNA modification, in regulating the resistance of cancer cells to chemotherapy through DNA repair. ² The identification and investigation of m6A regulatory proteins as potential anticancer drug targets represents a key avenue to overcome drug resistance in cancer patients. Moreover, targeting m6A RNA modification through regulation represents a promising therapeutic strategy for various types of cancer cells. ⁶² Understanding the mechanisms by which m6A regulates RNA splicing, degradation, and translation, as well as cell proliferation, metabolism, and metastasis, is essential for the development of novel therapeutic approaches. ⁸⁶ Therefore, given that m6A is one of the most prevalent epigenetic RNA modifications, extensive investigation into its underlying mechanisms of action is critical for the advancement of cancer treatment.

The Role of m6a Modified DNA Repair in Chemotherapy Resistance

Chemotherapeutic drugs are known to trigger apoptosis, which is caused by DNA damage.^87-89 When exposed to cytotoxic agents, eukaryotic cells typically halt their cell cycle to repair DNA damage or undergo apoptosis. The failure to halt the cell cycle or repair DNA damage can lead to drug resistance in cancer cells destroyed by DNA-damaging agents.^89,90 RNA m6A modification’s regulatory effect on TP53 suggests that targeting m6A may be an effective therapeutic strategy to prevent or overcome drug resistance by controlling downstream pathways that induce cell cycle arrest and DNA damage repair.^91,92 A comprehensive elucidation of the correlation between M6A-modified DNA repair and tumor resistance is as follows:

Platinum drugs

The first generation of platinum-based drug cisplatin, which was approved by the FDA in 1965, demonstrated successful efficacy in treating testicular cancer. Subsequently, the second generation carboplatin and third generation oxaliplatin were developed consecutively and are now extensively utilized worldwide for various tumor types. However, there is a growing concern regarding the emergence of resistance to platinum-based drugs among cancer cells. Studies have indicated that m6A modified DNA damage repair plays a crucial role in regulating resistance to cisplatin (DDP) and oxaliplatin (OXA).Study have demonstrated that PARP1 plays a crucial role in repairing DNA damage induced by oxaliplatin through mediating base excision repair, thereby contributing to the emergence of drug resistance. In gastric cancer CD133+ stem cells, METTTL3 can enhance the stability of PARP1 by recruiting YTHDF1 to the 3′-untranslated region (3′-UTR) of PARP1 mRNA, thus facilitating the development of oxaliplatin resistance in gastric cancer CD133+ stem cells. ⁹³ In seminoma, METTL3 enhances the stability of IGF2BP2 mRNA through m6A methylation, thereby upregulating the expression of transcription factor activated enhancer binding protein 2C (TFAP1C). TFAP2C further activates DNA repair genes WEE1 G2 checkpoint kinase (WEE1) and breast cancer type 1 to promote resistance against cisplatin. ⁶⁰ The studies have demonstrated that tumor cells exhibit an elevated occurrence of DNA damage upon VIRMA knockdown, as evidenced by increased expression of γH2AX and GADD45 A/B, which are key players in DNA damage repair. Additionally, the downregulation of XRCC4-like factor (XLF) and meiotic recombination 11 homolog 1 (MRE11) was observed, and the occurrence of DNA damage is correlated with the development of resistance to cisplatin. ⁹⁴ The m6a modification mediated by YTHDF1 in breast cancer cells enhances the expression of FOXM1, thereby facilitating DNA damage repair and promoting resistance to cisplatin treatment.^95,96 Moreover, the inhibition of METTL3 can enhance ADR-induced DNA damage by mediating m6A modification of the EGF/RAD51 axis, thereby increasing the sensitivity of breast cancer cells to Adriamycin. ⁷⁹ The METTL3-mediated upregulation of YAP also enhances NER by increasing the expression of ERCC1 in the middle and lower regions of non-small cell lung cancer. ⁹⁷

Temozolomide and Gemcitabine

Temozolomide (TMZ), an alkylating agent in addition to platinum-based drugs, is the primary first-line chemotherapy agent for advanced gliomas. However, the overall clinical efficacy of this regimen in glioma (GBM) remains suboptimal due to inherent or acquired resistance to TMZ therapy. Gemcitabine (GEM) is a widely utilized pyrimidine analogue in the treatment of various tumors, particularly pancreatic cancer (PC). The emergence of chemical resistance to GEM has become a significant concern and recent studies have revealed its regulation by m6A modification.The METTL3 enzyme in glioma stem cells enhances the stability of MGMT and ANPG mRNA, thereby up-regulating the expression of MGMT and ANPG through M6A modification, consequently facilitating TMZ.^98,99 The M6A modification in pancreatic cancer cells enhances the expression of ANRIL-L, thereby promoting resistance to gemcitabine. Additionally, ANRIL-L facilitates DNA damage repair by forming complexes with Ring1b and EZH2, thus enhancing the DNA HR repair ability of cells and ultimately contributing to gemcitabine resistance. ¹⁰⁰

Adriamycin/Doxorubicin and 5-FU

Doxorubicin (DOX), one of the anthracyclines, exerts its anti-tumor effects by intercalating into DNA and subsequently disrupting DNA repair mechanisms, or by inducing oxidative stress. The uracil analogue 5-FU, which functions by incorporating into nucleic acids to disrupt nucleotide metabolism, is a key component in various commonly used chemotherapy regimens, such as bimodal PF regimens, that are extensively employed in cancer treatment.The protein arginine methyltransferase 5 (PRMT5) impedes RNA m6A modification by facilitating the nuclear translocation of the RNA demethylase ALKBH5, which has been previously reported to be exclusively localized in the nucleus. Through m6A demethylation, ALKBH5 augments BRCA6 mRNA stability and further enhances DNA repair, thereby promoting doxorubicin resistance in breast cancer cells. ¹⁰¹ In BC cells, the YTHDF1-dependent regulation of E2F8 mRNA stability, which is METTL14-dependent, promotes S-phase entry, DNA replication, and DNA damage repair. This renders YTHDF1-deficient BC cells sensitive to drugs such as Adriamycin, cisplatin, and the PARP inhibitor orapanil.^48,78 Moreover, METTL3 upregulates the expression of the ubiquitin-binding enzyme E2B (RAD6B), a critical enzyme that triggers DNA damage repair through FANCD2 homogenization, leading to resistance to drugs like 5-fluorouracil (5-FU), cisplatin, and gemcitabine by promoting cell nuclear antigen and γ-H2AX.^102-104

Furthermore, METTL3 promotes the stability of PARP1 mRNA, increasing NER activity and promoting the resistance of CD133+ gastric cancer stem cells to oxaliplatin. ⁹³ The m6A modification also upregulates the expression of ankyrin repeat and LEM domain-containing protein 1, a collaborator of the fundamental HR factor BRC-1/BRCA1 in colorectal cancer cells, indicating its role in promoting DNA repair.^105,106

RNA modification through N6-methyladenosine (m6A) has a complex and dynamic impact on the DNA damage repair pathway in various cancer types. ¹⁰⁶ The impact of m6A modification on DNA damage repair can be both positive and negative, depending on the type of cancer. For example, in bladder cancer cells, CircMORC3-mediated m6A modification can lead to cisplatin resistance, whereas in other tumors, m6A modification can have the opposite effect on DNA damage repair. The upregulation of β-catenin by FTO-mediated mechanisms can also contribute to the resistance of cervical squamous cell carcinoma to cisplatin and radiotherapy. ⁶¹ Conversely, m6A modification can increase the stability of Tfap2c mRNA, resulting in the upregulation of DNA repair genes and the conferment of drug resistance to seminoma cells treated with cisplatin. These findings demonstrate that m6A plays a dual role in regulating DNA damage repair, depending on the tumor type and treatment modality. m6A modification can also modulate the drug resistance of anticancer therapy.^60,107 Further investigation of the determinants of m6A function in DNA damage repair may help develop innovative strategies to sensitize cancer cells to DNA damage agents. The complex nature of m6A modification suggests that a better understanding of its impact on DNA damage repair could lead to more effective cancer treatments.

Role of m6a Modified DNA Repair in Radiotherapy Resistance

Radiation therapy remains one of the primary modalities for cancer treatment, inducing DNA damage in cancer cells to achieve cell death. Therefore, it is imperative to develop dependable strategies for overcoming tumor radioresistance. ⁷¹ The inhibition of ALKBH5 demethylase in GBM resulted in the downregulation of CHK1 mRNA expression, leading to impaired DNA damage repair in GBMSCs and consequently enhancing radiosensitivity. ⁷¹ And The ALKBH5 protein can also enhance the expression of FOXM1 through m6a demethylation modification, thereby facilitating DNA damage repair and consequently promoting the radiotherapy resistance of GBMSC. ¹⁰⁸ In addition METTL3 increases SOX mRNA stability through M6A modification, resulting in decreased sensitivity to gamma irradiation and increased DNA repair in GSC cells, which is marked by downregulation of γ-H2AX expression. ¹⁰⁴ The depletion of METTL3 in pancreatic cancer cells has been demonstrated to enhance their susceptibility to radiation. ⁶¹ The current research on radiation resistance and M6A-modified DNA damage repair is not comprehensive; therefore, further exploration of the role of M6A-modified DNA damage in radiation resistance is necessary.

Challenges and Perspectives

This review examines M6A-modified DNA damage repair associated with chemotherapy resistance and radiation therapy. Although M6A regulation impacts drug resistance to targeted therapy, it does not influence DNA damage repair; hence, the aforementioned two types are primarily introduced. The complexity of m6A modification in drug resistance is highlighted by the diverse functions played by m6A RNA-modified DNA damage repair in different types of tumors. The swift progress in m6A RNA methylation research has facilitated the identification of potential mechanisms underlying cancer occurrence and progression. The regulation of DNA damage repair via m6A methylation, the most common RNA modification, exhibits a dual nature. Specifically, an increase in m6A methylation in certain genes promotes DNA damage repair, while a decrease in the m6A methylation level in other genes also stimulates DNA damage repair.

The function of DNA damage repair plays a crucial role in the onset and progression of cancer, as well as in cancer drug resistance. Recent studies have revealed the involvement of various proteins in mediating mRNA methylation and modulating DNA repair processes, thereby affecting cancer development. For instance, methylation protein METTL14 has been found to mediate mRNA methylation of DDB2, which promotes genome-wide repair and inhibits UVB-induced skin cancer. ⁶⁸ On the other hand, a high expression of demethylase ALKBH5 in osteosarcoma has been shown to decrease the m6A level, resulting in increased gene expression related to cell cycle and DNA repair, thus promoting the growth of osteosarcoma. ⁷⁰ Furthermore, METTL3 has been observed to enhance PARP1-mediated DNA damage repair by promoting the stability of the DNA repair enzyme PARP1 mRNA. This leads to increased activity of NER and promotes the resistance of CD133+ gastric cancer stem cells to oxaliplatin. ⁹³ These studies highlight the role of m6A RNA-modified DNA damage repair in different tumor types, indicating the complexity of m6A modification in drug resistance.

Targeting m6A regulation or inhibition may offer a novel strategy for overcoming chemotherapy resistance. The m6A modification facilitated by METTL3 stimulates the expression of EGF, which in turn elevates the expression of RAD51, resulting in increased HR activity and reduced DNA damage brought on by ADR (doxorubicin). Consequently, it reinforces the resistance of MDA-MB-231 breast cancer cells against chemotherapeutic drugs. ⁷⁹ Given METTL3’s critical role in mediating HR and chemotherapeutic drug responses, it is plausible that it could prove beneficial in cancer treatment.

The multifaceted role of N6-methyladenosine (m6A) in the regulation of DNA repair and genomic stability is increasingly appreciated, yet the underlying molecular mechanisms remain incompletely understood. A key challenge is that the expression and activity of m6A-modifying enzymes in diverse tissue contexts are not yet well-characterized, which critically impacts cancer biology. The m6A methyltransferase METTL3 exhibits divergent expression and activity profiles in different tumor tissues, with consequential effects on the progression and prognosis of various cancers. For instance, the transcriptional coactivator HBXIP, which inhibits miRNAlet-7g, has been linked to the upregulation of METTL3 and the acceleration of breast cancer development. Nonetheless, the regulatory mechanisms governing METTL3 in other cancer types are yet to be fully elucidated. Further research on m6A and its regulatory factors is essential to identify novel therapeutic targets and improve our understanding of cancer pathophysiology.

Recent studies have suggested that m6A modifications on mRNA can influence the translation of certain proteins, including those involved in DNA repair and replication. Therefore, it is plausible that m6A modifications could indirectly affect the regulation of low-fidelity polymerases and TLS by influencing the expression of relevant genes.how M6A modification could influence DNA repair mechanisms and potentially contribute to treatment resistance in various therapeutic settings. However, the details of these interactions and their functional significance are likely to be specific to the cellular context, the particular RNA molecules involved, and the specific proteins affected.

Conclusion

This article presents an overview of the recent advances in research on m6A RNA-modified DNA damage repair and its role in regulating tumor development and drug resistance. In recent years, there has been an increasing focus on the role of m6A modification in regulating drug resistance in tumor therapy. Targeted m6A modification systems hold significant importance in enhancing the efficacy of chemotherapy and radiotherapy for cancer treatment, while further advancing the clinical significance of m6A in preventing drug resistance and developing novel treatment strategies.

ORCID iD

Wen Jun Yin https://orcid.org/0000-0002-6264-9353

Data Availability Statement

Data sharing does not apply to this article as no new data were created or analyzed in this study.*