Abstract Temozolomide (TMZ) continues to serve as the frontline chemotherapy for glioblastoma (GBM), yet its long-term efficacy remains critically compromised due to the emergence of treatment resistance. While the DNA repair enzyme O6-methylguanine-DNA methyltransferase (MGMT) is the well-known contributor to TMZ resistance, clinical observations have shown that even tumors with minimal or silenced MGMT expression eventually acquire resistance, indicating additional, MGMT-independent pathways are involved. To investigate these alternative resistance mechanisms, we established two distinct glioblastoma models: TMZ-resistant (TR) cells characterized by high MGMT expression, and O6-Benzylguanine and TMZ-resistant (OTR) cells exhibiting low MGMT levels relative to parental controls. Our extensive analyses reveal a key function of the mitochondrial protease LonP1 in promoting TMZ resistance through metabolic adaptation. Both TR and OTR cell lines demonstrated significantly upregulated LonP1 expressions compared to parent glioblastoma cells, implicating elevated LonP1 as a central mediator in resistance development. Functional studies underscore that increased LonP1 expression contributes to a metabolic shift within resistant glioblastoma cells, transitioning from glycolytic metabolism towards enhanced oxidative phosphorylation (OXPHOS). This metabolic reprogramming equips resistant cells with improved capacity to sustain energetic and biosynthetic demands under TMZ-induced therapeutic stress. To validate LonP1’s causal role in resistance, we genetically overexpressed LonP1 in established glioma and patient-derived glioblastoma cell lines, resulting in robust acquisition of TMZ resistance. Conversely, downregulating LonP1 via targeted knockdown or pharmacologic inhibition restored sensitivity to TMZ, reducing cell viability and disrupting mitochondrial integrity. It is noteworthy that while our data firmly establish the necessity of LonP1 in maintaining TMZ resistance, the sufficiency of LonP1 overexpression to initiate resistance de novo in naïve tumor cells remains untested, as LonP1 knockout models were not leveraged for resistance induction. Collectively, our findings identify mitochondrial LonP1 protease as a promising target to overcome TMZ resistance in glioblastoma therapy. Inhibition of LonP1 activity could potentially reverse metabolic adaptations, thereby resensitizing resistant tumor cells to TMZ and improving treatment efficacy. This study provides a strong rationale for developing LonP1-targeted therapeutics as adjunctive agents in standard TMZ chemotherapy regimens, with the hope of delaying or reversing chemoresistance to improve clinical outcomes for glioblastoma patients. Citation Format: Shashi Jain, Dahlia A. Ordaz, Javier Lepe, Naomi Lomeli, James Pham, Bhaskar Das, Daniela A. Bota. Exploring the role of LONP1 in lon-traditional mechanisms of glioblastoma resistance abstract. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 382.
Jain et al. (Fri,) studied this question.