The Temozolomide Mutational Signature: Mechanisms, Clinical Implications, and Therapeutic Opportunities in Primary Brain Tumor Management

Temozolomide (TMZ) remains foundational in the management of adult-type diffuse gliomas in general, and glioblastoma specifically. However, its efficacy harbors an evolutionary trade-off. TMZ drives its cytotoxicity through generating O⁶-methylguanine lesions, especially active in MGMT-silenced, mismatch repair (MMR)-proficient tumors. By selecting for acquired MMR-deficient subclones, often via MSH6 inactivation, this process escalates into a hypermutator phenotype, generating thousands of de novo alterations. This is a hallmark of the mutational signature known as SBS11, characterized by C>T transitions, which is associated with TMZ treatment. The hypermutator phenotype drives heterogeneity, therapeutic resistance, spatial diversification, and distant recurrence. Despite harboring a mutational burden comparable to melanoma and lung cancer, TMZ-induced hypermutation does not sensitize gliomas to immune checkpoint blockade. This resistance reflects the profoundly immunosuppressive brain microenvironment, impaired antigen presentation, marked transcriptional plasticity, and perhaps also the frequent use of corticosteroids. Emerging strategies aim to exploit vulnerabilities created by TMZ-mediated genomic instability, including PARP, ATR, WEE1, and AURKA inhibition; alternative alkylators; metabolic rewiring; and G-quadruplex stabilization. Notably, the real-time detection of evolving mutational signatures via CSF-based liquid biopsies may enable adaptive therapy before radiographic progression. By reframing TMZ as a potent evolutionary agent rather than a conventional chemotherapy, this review synthesizes recent mechanistic insights and translational opportunities to guide a next-generation, evolution-informed treatment paradigm for glioma.