The MGMT gene is crucial for DNA repair. It protects cells by reversing DNA damage caused by alkylating agents chemicals that add harmful methyl or ethyl groups to DNA. MGMT specifically removes the alkyl group from the O⁶ position of guanine, a site that, if left unrepaired, can cause incorrect base pairing, mutations, and eventually cancer [7]. This reaction occurs in a single step where MGMT transfers the alkyl group to its own cysteine residue, resulting in its inactivation. For this reason, MGMT is often referred to as a “suicide” repair enzyme [2].

The MGMT gene is located on chromosome 10q26 and consists of 5 exons and 4 introns and spans greater than 300 kb [7]. MGMT protein has 207 amino acids and has many conserved regions throughout. Structurally, MGMT has a conserved DNA-binding domain and an active site that includes a cysteine at position 145, essential for its repair function [7]. Although MGMT is expressed in most normal tissues, the level of expression can vary significantly, influencing how cells respond to DNA damage.

It has repetitive GC-rich sequences comprising a CpG island. The promoter region of MGMT spans 1.2 kb and includes the first exon and part of the first intron [7]. Expression of MGMT can be induced mainly by DNA damage, glucocorticoids, cyclic AMP, protein kinase C and interaction of several transcriptional factors like SP1, activator proteins 1 and 2 (AP-1 and AP-2) with its promoter region. MGMT functions as a transferase and an alkyl-group acceptor (Figure 1). MGMT recognizes alkyl DNA adducts at the O⁶ position of guanine and transfers the alkyl moiety to a cysteine residue within its own structure, effectively repairing lesions in a stoichiometric and irreversible manner [8]. While its primary substrate is O⁶-MeG, MGMT is also capable of repairing larger alkyl adducts such as O⁶-ethylguanine and O⁴-methylthymine, indicating its versatility beyond standard substrates [9]. Interestingly, the base excision repair (BER) mechanism addresses other types of alkyl damage, specifically N7-methylguanine and N3-methyladenine, whereas MGMT focuses predominantly on O⁶ and O⁴ adducts [10].

The repair of O⁶-MeG lesions can lead to G:T mismatches, which can subsequently be rectified through mismatch repair (MMR) mechanisms [11]. It is well documented that downregulation of the MGMT gene is reported in a variety of tumor types, including gliomas, colorectal cancers, and lung cancers. This loss significantly contributes to tumorigenesis by facilitating the persistence of mutations resulting from mispairing during replication [12, 13]. Therefore, MGMT plays a crucial role in protecting cells from mutagenic DNA adducts, and its diminished expression is associatively linked to various malignancies. This underscores its importance as a biomarker in cancer prognosis and therapy [13, 14].

The expression of MGMT is tightly regulated at multiple levels, which influences both cancer development and treatment response. Abnormal MGMT regulation is often observed in cancers like glioblastoma, colorectal, lung, and cervical cancer, where it affects sensitivity to alkylating chemotherapies [15, 16].

MGMT expression is orchestrated by promoter activity, anchored in a CpG island, an epigenetic hotspot dense with cytosine-guanine sequences that acts as a regulatory epicenter (Figure 1). In many cancers, this region becomes hypermethylated, leading to transcriptional silencing of MGMT and making tumor cells more responsive to alkylating agents like TMZ [4, 17]. Histone modifications, particularly trimethylation of histone H3 at lysine 9 (H3K9me3), also suppress MGMT expression by condensing the chromatin structure and making it inaccessible for transcription [18].

Transcription factors such as AP-1 and SP1 usually enhance MGMT expression by binding to its promoter. In contrast, p53 may either repress or activate MGMT depending on the context, highlighting its complex regulatory role [19, 20]. Non-coding RNAs (ncRNAs), especially microRNAs (miRNAs), regulate MGMT after transcription by binding to its mRNA and blocking translation or promoting degradation, further fine-tuning its protein levels in cells [5].

Gene variants in the MGMT gene can significantly affect its DNA repair efficiency, contributing to individual cancer risk. These single-nucleotide polymorphisms (SNPs) may influence protein expression, structure, or function and are often investigated as biomarkers of cancer susceptibility.

One well-studied variant is rs12917 (C>T), located in exon 5 of the MGMT gene. This polymorphism has been associated with reduced MGMT activity, which limits the cell’s ability to repair O⁶-MeG lesions effectively. The resulting accumulation of DNA damage increases the risk of tumor initiation, particularly in tissues frequently exposed to genotoxic agents, such as the colon and lungs [6].

Another important SNP is rs2308321 (T>C), a nonsynonymous change that leads to an amino acid substitution. This variant has been linked to increased tumor progression in several cancers. It may alter the three-dimensional structure of the MGMT protein, affecting its stability or ability to interact with damaged DNA substrates [2]. Evidence suggests that individuals carrying the C allele may exhibit poorer outcomes when treated with alkylating agents due to altered MGMT activity.

Additionally, the rs16906222 (A>G) variant, found in the promoter region, is believed to impact chromatin accessibility. This alteration can reduce transcription factor binding efficiency and thus lower MGMT gene expression. A decrease in expression can sensitize tumors to alkylating drugs but may also lead to increased baseline mutation rates [5].

Beyond inherited polymorphisms, somatic mutations in the MGMT gene are increasingly observed in several malignancies including glioblastoma and colorectal carcinoma. These mutations are often acquired during tumor evolution and lead to loss-of-function changes in the MGMT protein [2]. Without functional MGMT, cells lose a critical mechanism for repairing alkylating DNA damage, resulting in genomic instability.

Such instability promotes further mutations in tumor suppressors and oncogenes, accelerating tumor progression. Interestingly, tumors harboring inactivating MGMT mutations often become more sensitive to chemotherapy, particularly alkylating agents, since they lack an effective repair mechanism [3, 19]. However, this also increases the risk of developing secondary malignancies or drug resistance through alternative repair pathways.

Promoter hypermethylation is one of the most studied epigenetic silencing mechanisms of MGMT. This process involves the addition of methyl groups to CpG islands in the gene’s promoter region, leading to chromatin condensation and transcriptional repression. MGMT promoter methylation has been observed across several cancers and is often linked to enhanced treatment response due to impaired DNA repair capacity [16, 17].

In glioblastoma multiforme (GBM), MGMT promoter methylation is a well-established biomarker for predicting the effectiveness of the alkylating agent TMZ. Patients whose tumors have methylated MGMT promoters often show better responses to TMZ and improved overall survival, since the absence of MGMT prevents repair of TMZ-induced DNA lesions [2, 16].

In colorectal cancer, MGMT promoter methylation is frequently associated with microsatellite instability (MSI), a condition where DNA MMR is impaired. MGMT silencing leads to the accumulation of mutations, contributing to tumorigenesis and resistance to DNA-damaging treatments [15].

In cervical cancer, MGMT promoter methylation has been correlated with radiation sensitivity. The loss of MGMT-mediated DNA repair may make tumor cells more vulnerable to DNA strand breaks induced by radiotherapy, offering a possible explanation for better treatment outcomes in methylation-positive patients [6].

Apart from DNA methylation, MGMT expression is also regulated by post-translational modifications of histone proteins. These modifications alter chromatin structure, influencing gene accessibility.

Two key repressive marks, H3K9me3 and histone H3 lysine 27 trimethylation (H3K27me3) are particularly involved in MGMT silencing. High levels of these marks around the MGMT promoter region led to tightly packed chromatin, which blocks RNA polymerase from accessing the gene, thereby halting transcription [2, 18].

These histone modifications often work in conjunction with DNA methylation, creating a robust silencing environment that further diminishes MGMT expression. The loss of MGMT reduces the cell’s capacity to repair O⁶-alkylguanine lesions, increasing susceptibility to mutagenesis and, paradoxically, to chemotherapy agents that rely on such damage for their cytotoxic effects.

Emerging evidence highlights the role of ncRNAs, particularly miRNAs in the post-transcriptional regulation of MGMT. These small RNAs bind to the 3′ untranslated region (3′UTR) of MGMT mRNA, blocking its translation or leading to degradation.

Two miRNAs, miR-181c and miR-648, have been shown to directly target MGMT mRNA, decreasing protein production. This downregulation exacerbates DNA repair deficiencies in cancer cells and enhances sensitivity to DNA-damaging agents like TMZ and radiotherapy [2, 5]. High expression levels of these miRNAs are often observed in tumors with low MGMT protein, suggesting a potential feedback loop between ncRNA activity and chemotherapeutic response.

In addition to miR-181c and miR-648, other miRNAs have also been implicated in MGMT regulation. For instance, miR-370-3p has been shown to suppress MGMT expression in glioma by targeting its mRNA, contributing to TMZ resistance and malignant progression [21]. Similarly, miR-221-3p downregulates MGMT in hepatocellular carcinoma, thereby promoting tumorigenesis [22]. These findings highlight the tissue-specific regulatory roles of ncRNAs and reinforce their potential as both therapeutic targets and biomarkers across different cancer types.

Targeting these miRNAs or their interaction with MGMT mRNA may open new therapeutic avenues for overcoming drug resistance, particularly in glioblastoma and colorectal cancer, where MGMT activity critically determines treatment outcome.

Resistance to alkylating agents such as TMZ and BCNU is closely linked to the DNA repair activity of MGMT. Tumors with high MGMT expression can effectively reverse therapy-induced DNA damage, limiting drug efficacy and promoting cell survival [16, 53, 54]. Elevated MGMT activity in tumors has been recognized as a significant mechanism behind resistance to alkylating therapies, allowing malignant cells to efficiently repair alkylation damage before it induces lethal replication errors [55].

The prognostic significance of MGMT in GBM is well-documented, particularly regarding its promoter methylation status. Methylation of the MGMT promoter leads to gene silencing and decreased expression of the MGMT protein, which correlates with diminished DNA repair capability and increased sensitivity to TMZ treatment. Patients with GBM exhibiting a methylated MGMT promoter tend to have better treatment outcomes, as they are less able to proficiently repair the damage caused by alkylating agents, compared with those possessing unmethylated promoters who often show poor responses [16, 55, 56].

Gupta et al. [6] demonstrated that tumor-specific MGMT promoter methylation serves both as a prognostic biomarker and as a predictive factor for therapeutic response. For instance, the EORTC-NCIC trial highlighted the correlation between MGMT promoter methylation and favourable treatment outcomes following TMZ and radiotherapy, reinforcing the utility of this biomarker in guiding clinical treatment decisions [55, 57]. Additionally, studies indicate that higher levels of methylation within the MGMT promoter region are associated with improved overall survival rates in GBM patients, underscoring the need for routine assessment of MGMT status in neuro-oncology [16, 58, 59].

In summary, alkylating agents such as TMZ and BCNU induce DNA damage through O⁶-alkylation of guanine. The MGMT enzyme mitigates this damage via DNA repair; thus, high expression levels of MGMT can lead to treatment resistance. Conversely, MGMT promoter methylation silences the expression of this enzyme, enhancing sensitivity to TMZ and correlating with improved patient outcomes. This dynamic positions MGMT methylation as both a prognostic and predictive biomarker essential for patient stratification in glioblastoma therapies [60–63] (Table 1).

Recent reports highlight the synthetic lethality, a strategy that targets vulnerabilities in tumor cells that lack functional MGMT. It plays a crucial role in the repair of DNA damage caused by alkylating agents such as TMZ and BCNU, which modify the O⁶ position of guanine and can lead to replication errors and cell death if unrepaired [64, 65]. In tumors where MGMT is overexpressed, these repair mechanisms can confer resistance to overcome this; one promising strategy involves the combination of MGMT inhibitors (e.g., O⁶-BG) with poly(ADP-ribose) polymerase (PARP) inhibitors (e.g., olaparib, niraparib), which disrupt parallel DNA repair pathways. This dual inhibition causes the accumulation of DNA damage and enhances tumor cell death, particularly in MGMT-deficient settings [66–68]. For instance, studies reveal that inhibiting PARP activity in conjunction with the removal of MGMT allows for heightened sensitivity to TMZ by diminishing the tumor’s ability to repair alkylated DNA. Specifically, PARP inhibitors can reduce the ability of MGMT to repair O⁶-MeG lesions, effectively enhancing the cytotoxic effects of alkylating agents [69]. Additionally, the use of ATR (ataxia telangiectasia and Rad3-related protein) inhibitors alongside PARP inhibitors has been shown to further intensify replication stress and drive tumor cell apoptosis in certain cancer models [66, 70].

Furthermore, the strategic combination of PARP and MGMT inhibitors is being explored in various cancers that exhibit similar resistance mechanisms [71, 72]. Preclinical data suggest that the disruption of both DNA repair pathways exploits the vulnerabilities of certain tumors and holds potential for improving patient outcomes in challenging clinical contexts, including those with mutant or hyperactive MGMT profiles [64, 68].

In summary, the strategy of utilizing synthetic lethality through the combination of MGMT inhibitors and PARP inhibitors represents a promising avenue for circumventing drug resistance in MGMT-overexpressing tumors, facilitating more effective treatment regimens and potentially leading to better clinical outcomes in patients afflicted with these malignancies.

The methylation status of the MGMT promoter has emerged as a critical biomarker in the management of GBM and other malignancies. Numerous studies underscore its dual role as both a predictive marker for treatment response and a prognostic marker for overall survival. This understanding is integral to the concept of precision medicine, where therapeutic strategies are tailored based on the molecular characteristics of a patient’s tumor.

Research has consistently demonstrated that MGMT promoter methylation is associated with enhanced sensitivity to alkylating agents, particularly TMZ in GBM. In the article by Hegi et al. [16], they conducted seminal work showing that patients with methylated MGMT promoters exhibited significantly better survival outcomes when treated with TMZ compared to those with unmethylated MGMT, who often showed rapid tumor progression and limited response to therapy. This study has laid the groundwork for incorporating MGMT methylation testing into clinical decision-making [16].

Esteller et al. [15] also established the significance of MGMT promoter methylation as a marker of cancer-specific prognosis, demonstrating that gene silencing through methylation leads to reduced MGMT expression and an augmented response to alkylating chemotherapy. The ability of MGMT to counteract the effects of alkylating agents underlines its importance in therapeutic resistance.

Beyond predicting treatment response, MGMT promoter methylation serves as a prognostic marker. Studies indicate that patients with methylated MGMT have longer overall survival and progression-free survival rates compared to their counterparts with active MGMT expression. In their analysis, Hegi et al. [16] reported that MGMT methylation status significantly correlated with improved overall survival in patients receiving TMZ and radiation therapy.

More recent investigations have reinforced these findings. For instance, a meta-analysis by Brandner et al. [73] confirmed that methylated MGMT is linked to better clinical outcomes across various studies, asserting that MGMT promoter methylation influences both the efficacy of chemotherapy and the overall survival of GBM patients.

The implications of these findings for precision medicine cannot be overstated. By utilizing MGMT methylation status, clinicians can stratify patients for more personalized treatment approaches. For instance, patients with unmethylated MGMT may require alternative therapies or more aggressive treatment strategies, while those with methylated MGMT can potentially benefit from alkylating chemotherapeutics such as TMZ.

The methylation status of the MGMT promoter is not only predictive of response to treatment but also serves as a valuable prognostic marker in GBM and other malignancies. Its dual role underscores the importance of integrating molecular features into treatment algorithms, aligning with the principles of precision medicine.

Gene therapy represents a promising strategy to reintroduce MGMT activity in tumors where it has been silenced. It was demonstrated that delivering MGMT via lentiviral vectors into glioblastoma cells restored repair capacity, increasing resistance to alkylating agents like TMZ [16]. Although reactivation could aid in preventing further genomic instability, it might also affect chemotherapy sensitivity, highlighting the importance of precise therapeutic design.

MGMT silencing often occurs through promoter methylation, a key epigenetic event. DNA methyltransferase (DNMT) inhibitors such as 5-azacytidine and decitabine have shown potential in reversing this silencing and restoring gene expression [53, 54]. These agents demethylate CpG islands, allowing transcriptional machinery to access the MGMT gene, though re-expression could paradoxically increase drug resistance, necessitating tailored use (Table 1).

Histone deacetylase inhibitors (HDACi), like vorinostat, also enhance chromatin accessibility by maintaining histone acetylation [55, 57]. Combining DNMT and HDACi has demonstrated synergistic effects, promoting MGMT reactivation and impacting tumor suppressor gene expression [5]. These epigenetic strategies hold promise but require careful clinical evaluation to balance therapy efficacy and resistance risk.

RNA interference and small molecule inhibitors: In tumors where MGMT contributes to chemotherapy resistance, downregulating its expression is a potential therapeutic route. Small molecules and RNA-based tools, including siRNAs and antisense oligonucleotides, have been used to reduce MGMT levels in cancer cells, enhancing TMZ sensitivity [55]. By specifically targeting MGMT mRNA, these strategies can achieve tumor-specific effects while minimizing harm to normal tissues.

Combining therapies to overcome resistance: Using MGMT inhibitors alongside chemotherapy can enhance treatment outcomes. Bai et al. [2] showed that pairing O⁶-BG with TMZ improved efficacy in GBM models by inhibiting MGMT’s repair function [56]. Such combination approaches exploit DNA damage accumulation and repair inhibition, making tumors more susceptible to chemotherapy.

Integrating immunotherapy with MGMT modulation: Emerging evidence suggests that MGMT status influences tumor immune profiles. Knocking down MGMT might boost tumor mutational burden and enhance immune system recognition, potentially improving immune checkpoint inhibitor responses [57]. Integrating MGMT modulation with immunotherapy could pave the way for more personalized and effective cancer treatments.

Epigenetic therapy is a promising avenue in GBM, where MGMT promoter methylation often mediates chemotherapy resistance. By reversing silencing, these therapies could restore DNA repair capacity and modify treatment response.

DNMT inhibitors: Agents such as decitabine and azacitidine inhibit DNMTs, preventing DNA methylation and reactivating silenced genes such as MGMT [16, 53]. Decitabine has been shown to restore MGMT expression in previously methylated tumor cells [54]. However, this reactivation may decrease TMZ sensitivity, necessitating personalized treatment planning (Table 1).

HDACi: HDACi like vorinostat maintain acetylation of histone tails, leading to an open chromatin structure that allows transcription of silenced genes. Combining DNMT and HDACi can reactivate MGMT synergistically and enhance chemotherapy sensitivity [5]. These findings support the exploration of combination therapies targeting multiple epigenetic pathways (Table 1).

Recent advancements in epigenome-editing tools such as CRISPR-dCas9 systems offer a precise approach to modulate MGMT expression by targeting its promoter methylation or enhancer elements. This opens new avenues for reactivating silenced MGMT in normal tissue or silencing overexpressed MGMT in chemoresistant tumors [74]. Such programmable systems, especially when coupled with epigenetic effectors like DNMT3A or TET1 fusions, have demonstrated efficacy in preclinical models of glioblastoma and colorectal cancer [75].

Demethylation of the MGMT promoter: CRISPR/Cas9 can target the methylated MGMT promoter to reactivate its expression, increasing DNA repair and potentially sensitizing tumors to chemotherapy [2, 3]. This approach could benefit tumors with epigenetically silenced MGMT [16, 53].

Repairing MGMT mutations: CRISPR also allows precise correction of MGMT gene mutations, potentially restoring normal function and reducing chemotherapy resistance [54, 55]. Correcting dysfunctional sequences can support overall DNA repair pathways and limit mutation accumulation.

Understanding regulatory networks: CRISPR can dissect MGMT regulatory pathways by editing transcription factors like SP1 and NF-κB [57]. This capability enhances understanding of MGMT modulation and could reveal combinatorial targets for therapy [16, 56].

The integration of multi-omics approaches combining methylome, transcriptome, proteome, and chromatin accessibility data is transforming our understanding of MGMT regulation and its context-dependent impact on therapy resistance. Single-cell and spatial omics further unravel the intratumoral heterogeneity of MGMT expression and its epigenetic determinants across cancer types [76] (Table 1).

Genomic profiling: Assessing MGMT mutations and other key oncogenic alterations like IDH1 can refine therapy choices [16]. Broader sequencing informs the overall mutational landscape, enabling tailored interventions.

Epigenomic assessment: MGMT promoter methylation profiling informs therapy decisions, as methylated tumors are more responsive to alkylating agents like TMZ [53]. Incorporating methylation data with other molecular profiles enhances therapy selection.

Transcriptomic insights: Examining MGMT and DNA repair gene expression helps predict therapy response and guides combination treatments [54]. Transcriptomics aids in identifying patients who may benefit from alkylating agents or need alternative strategies.

Customized treatment plans: Integrating multi-omics data allows clinicians to design precise therapies. Patients with methylated MGMT may benefit from alkylating agents, while those with high MGMT activity might need MGMT inhibitors or other approaches [55]. This ensures that therapy matches the tumor’s unique profile.

Balancing efficacy and toxicity: Multi-omics integration optimizes drug selection and reduces side effects. Evidence shows that patients with methylated MGMT fare better on alkylating agents, while others may require different strategies [56]. This personalized strategy aligns therapy with tumor biology, improving outcomes.

Thanks to advances in multi-omics technologies, scientists are now able to paint a much clearer picture of MGMT’s role in cancer well beyond what single methylation tests can offer. For instance, a 2016 study by Ceccarelli et al. [77] utilized integrative multi-omics analysis to identify MGMT promoter methylation and IDH mutation subtypes in glioblastoma, which correlated with therapy response and survival outcomes. The GLASS consortium showed that shifts in MGMT methylation and expression directly tie into drug resistance and tumor evolution in glioblastoma, revealing layers of complexity that routine biopsies miss [78]. Building on that, clinical trials like NCT04555577 and projects like PCAWG are using multi-omics data combining epigenetic, genomic, and immune profiles to tailor more precise, combination-based cancer therapies [79]. It’s a new era of biomarker-driven personalization, powered by integrated biology. Similarly, The Cancer Genome Atlas (TCGA) project incorporated genomic, transcriptomic, and methylation profiles to guide patient stratification and therapeutic development based on MGMT status. These cases highlight the translational potential of multi-omics approaches in personalizing MGMT-related cancer therapy [77, 80]. MGMT status varies across different cancers, influencing therapeutic outcomes and survival (Table 2).

Above mentioned innovative approaches highlight the dynamic interplay between MGMT expression, chemotherapy sensitivity, and treatment resistance. Integrating gene editing, epigenetic therapies, and multi-omics data can refine precision oncology and improve care for patients with MGMT-driven treatment challenges.