Abstract
O(6)-Methylguanine-DNA methyltransferase (MGMT) acts as a genomic custodian, reversing alkylation damage to preserve DNA integrity. However, when its regulatory balance tips via promoter methylation, polymorphisms, or epigenetic silencing, MGMT can become a liability, fuelling cancer progression, treatment resistance, and poor outcomes across malignancies. This review uncovers the nuanced control of MGMT, revealing how its genetic and epigenetic shifts shape tumor behavior, therapeutic response, and risk stratification. We aim to transform molecular insights into actionable clinical strategies, reimagining MGMT as both a biomarker and therapeutic lever. We curated high-impact studies (up to 2025) from PubMed, Scopus, and Web of Science, focusing on MGMT modulation, synthetic lethality, CRISPR-based restoration, and epigenetic therapies. Emerging multi-omics and translational frameworks were prioritized. MGMT's activity is choreographed by an intricate interplay of promoter methylation, histone marks, transcriptional regulation, and microRNA influence. These dynamics critically affect sensitivity to alkylating agents like temozolomide. Intriguingly, MGMT also engages with the immune landscape modulating response to immunotherapies. Innovations in multi-omics, single-cell analytics, and AI-based biomarker profiling are unveiling previously hidden regulatory layers. Decoding MGMT's regulation unlocks new therapeutic frontiers. Cutting-edge strategies from CRISPR to liquid biopsy promise more personalized, resistance-proof cancer care.