Overcoming temozolomide resistance in glioma: recent advances and mechanistic insights

Abstract

Temozolomide (TMZ) remains the cornerstone chemotherapy for glioma, yet intrinsic and acquired resistance mechanisms significantly limit its clinical effectiveness. This review summarizes the multifaceted molecular pathways contributing to TMZ resistance, including enhanced DNA repair mechanisms such as O⁶-methylguanine-DNA methyltransferase (MGMT), mismatch repair (MMR), and base excision repair (BER). Additional resistance factors include genetic mutations that affect the drug response, dysregulated non-coding RNAs (miRNAs, lncRNAs, and circRNAs), glioma stem cells (GSCs), cytoprotective autophagy, an immunosuppressive tumor microenvironment (TME), altered signaling pathways, and active drug efflux transporters. Recent advancements to overcome these resistance mechanisms, including enhancing TMZ bioavailability through nanoparticle-based delivery systems and the inhibition of efflux transporters, have been explored. Novel therapeutic approaches that target DNA repair pathways and manipulate autophagy are highlighted. Immunotherapeutic interventions reversing immune suppression and metabolic strategies targeting tumor metabolism offer additional avenues. Emerging therapies such as CRISPR-based gene editing, phytochemical combinations, repurposed drugs, and novel TMZ analogs designed to bypass MGMT-mediated resistance are also discussed. This review highlights current developments and identifies emerging areas, with the goals of enhancing clinical outcomes and prolonging survival for glioma patients.

Keywords: Chemoresistance; Glioma; Temozolomide; Treatment strategies.

Fig. 1

Mechanism of action of TMZ. TMZ is an orally administered imidazotetrazine prodrug that undergoes pH-dependent conversion under physiological conditions into its active metabolite MTIC. MTIC subsequently reacts with water, generating 5-aminoimidazole-4-carboxamide (AIC) and a highly reactive methyldiazonium cation. This methyldiazonium cation preferentially methylates DNA at the N⁷ position of guanine (N⁷-MeG; approximately 70%), predominantly in guanine-rich regions but also at adenine residues (N³-MeA; approximately 9%) and guanine residues at the O⁶ position (O⁶-MeG; approximately 6%). The cytotoxic effect of TMZ primarily results from the formation of O⁶-MeG lesions, which are carcinogenic, mutagenic, and toxic. These lesions are repaired directly by the suicide enzyme MGMT, which removes the methyl group from O⁶-MeG, restoring the original guanine residue. If left unrepaired, O⁶-MeG mispairs specifically with thymine during DNA replication, activating DNA MMR. MMR recognizes and excises the mispaired thymine on the daughter strand; however, the persistent O⁶-MeG lesion in the template strand results in futile cycles of thymine reinsertion and excision. These continuous futile repair cycles generate persistent DNA strand breaks, leading to G2/M cell cycle arrest and eventually cell death. The more abundant DNA adducts, N⁷-MeG and N³-MeA, are rapidly repaired via DNA BER. Therefore, the most important DNA repair systems affecting the mechanism of action and cytotoxicity of TMZ are MGMT, MMR, and BER

Fig. 2

Mechanisms of TMZ resistance in GBM. Resistance arises through enhanced DNA damage repair pathways, including the overexpression of the MGMT and BER proteins and the inactivation of MMR. Drug efflux transporters promote TMZ extrusion, reducing intracellular drug levels. Genetic mutations and non-coding RNAs contribute to metabolic reprogramming, immune escape, and the activation of survival signaling pathways such as the PI3K/AKT/mTOR and RAS/RAF/MEK/ERK pathways. GSCs play crucial roles in tumor formation, treatment resistance, and recurrence, largely due to their self-renewal ability and adaptability. Autophagy is regulated through the RAS/RAF/MEK/ERK, ATM/AMPK/ULK1, and PI3K/AKT/mTOR pathways, further supporting cell survival under TMZ treatment. GSCs play a central role in maintaining therapeutic resistance via pathways such as the PI3K/AKT, Wnt/β-catenin, and JAK/STAT pathways, which sustain stemness, promote immune evasion, and modulate inflammation. Additionally, the tumor immune microenvironment, shaped by glioma-associated microglia and macrophages (GAMs), microglia, and secreted factors, inhibits cytotoxic T-cell activity and enhances regulatory T-cell function, facilitating tumor progression and metastasis

Fig. 3

Autophagy in TMZ-treated cells. Autophagy is a multistep process consisting of initiation, nucleation, elongation, maturation, and fusion. In glioma cells treated with TMZ, autophagy is activated through multiple signaling cascades. (1) TMZ induces DNA damage, which activates the ATM/AMPK/ULK1 signaling axis, subsequently promoting the assembly of the class III PI3K (Vps34) complex, which initiates autophagosome formation. (2) TMZ-induced oxidative stress results in the accumulation of ROS, which stimulates receptor tyrosine kinases (RTKs). Activated RTKs trigger the RAS/RAF/MEK/ERK and PI3K/AKT pathways, leading to the activation of downstream transcription factors that modulate autophagy. Specifically, ERK1/2 activation facilitates autophagy by enhancing the formation of the Vps34 complex, whereas AKT activation inhibits autophagy by promoting mTORC1 activity, which suppresses the ULK1 complex. Notably, elevated intracellular ROS levels also activate PTEN, a negative regulator of the PI3K/AKT pathway. This PTEN-mediated inhibition is more pronounced than the autophagy-promoting effect of RTKs, resulting in overall suppression of the PI3K/AKT pathway under TMZ treatment. (3) The Vps34 complex is essential for the nucleation of autophagic vesicles, whereas vesicle elongation and maturation into autophagosomes require additional autophagy-related proteins (ATG) and LC3. Mature autophagosomes subsequently fuse with lysosomes to form autolysosomes, where autophagic substrates are degraded. Cytoprotective autophagy supports protein synthesis, energy production, and cell survival, thereby contributing to TMZ resistance in glioma cells

Fig. 4

Strategies for overcoming drug resistance in GBM. Strategies include improving the bioavailability of TMZ by enhancing its delivery across the blood-brain barrier and reducing efflux via P-glycoprotein inhibition. Targeting DNA damage repair pathways, such as the MGMT, PARP, and BER pathways, can increase TMZ-induced cytotoxicity. Modulation of key signaling pathways (JAK2/STAT3, MAPK, and Wnt/β-catenin) through targeted inhibitors offers another route to sensitize tumor cells. Autophagy manipulation, through the inhibition of cytoprotective autophagy or activation of cytotoxic autophagy, synergistically enhances the TMZ response. Metabolic interventions aim to disrupt glycolysis, lipid metabolism, and amino acid utilization by targeting enzymes such as LDH, FASN, and BCAT1. Immunotherapeutic strategies, including immune checkpoint inhibitors, tumor vaccines, and oncolytic viruses (OVs), are employed to boost anti-tumor immune responses. Additional treatments, such as tumor-treating fields, gene editing, nano-red light therapy, and plant-derived compounds, represent emerging modalities with the potential to overcome resistance and improve therapeutic outcomes in GBM

Fig. 5

Strategies to improve TMZ delivery and reduce drug efflux in GBM. Research has focused on two main strategies: enhancing drug delivery to bypass biological barriers and reducing drug efflux to maintain higher intracellular TMZ concentrations. (1) Cationic liposomes and transferrin-modified nanoparticles facilitate BBB crossing via adsorption-mediated endocytosis and receptor targeting, respectively. POSS-based nanocarriers and tFNA nanoparticles enhance nuclear localization and tumor cell apoptosis. Encapsulation of TMZ in Calix nanocapsules increases early uptake and cytotoxicity. Additional strategies, including folate receptor-targeted exosomes co-loaded with quercetin, siRNA micelles targeting STAT3, MTIC prodrug micelles, and UiO-66-NH₂ nanocomposites activated by ultrasound, further improve BBB penetration and therapeutic efficiency while minimizing toxicity. (2) To counteract TMZ efflux, inhibitors such as Reversan block P-gp-mediated drug expulsion, and EPIC-1042 reduces the release of sEVs by disrupting PTRF/Cavin1-caveolin-1 interactions. Ultra-small, large-pore silica nanoparticles (USLPs) help evade efflux pump recognition and enhance cytotoxicity. Additional strategies include quadruple therapy using targeted exosome systems to downregulate TMZ-resistance genes such as RASGRP1 and VPS28, and approaches that reduce cerebrospinal fluid (CSF) clearance by modulating ependymal cilia activity, increasing TMZ accumulation at tumor sites