Deciphering LonP1's role in glioblastoma: an alternative mechanism of treatment resistance.

Temozolomide (TMZ) remains the standard-of-care chemotherapy for glioblastoma, yet resistance severely limits its clinical efficacy. To identify alternative pathways, we developed TMZ-resistant (TR) and MGMT inhibitor O6-benzylguanine (O6-BG)-resistant (OTR) glioblastoma models that differ in MGMT status, but both show elevated expression compared with parental cells, implicating Lon protease (LonP1) in the resistant phenotype. Functional analyses showed that LonP1 drives metabolic reprogramming toward oxidative phosphorylation (OXPHOS) and supports survival under therapeutic stress. To establish LonP1's causal role, we genetically overexpressed LonP1 in glioma lines, which conferred robust TMZ resistance, whereas LonP1 downregulation via inducible shRNA or pharmacologic inhibition restored TMZ sensitivity, reduced cell viability, and compromised mitochondrial integrity and OXPHOS capacity. Remarkably, our findings confirm LonP1 as a strong contributor to de novo TMZ resistance in treatment-naïve tumor cells and to maintain/enhance resistance in established resistant models. However, the initial rescue experiment partially supports the specificity of the LonP1-dependent phenotype. Together, our data identify LonP1 as a potential therapeutic target to overcome TMZ resistance and provide a rationale for developing LonP1-directed interventions as adjuncts to standard TMZ therapy with the potential to improve glioblastoma. Additional in vivo orthotopic or PDX-based models will be required to define the full translational relevance of LonP1 in glioblastoma outcomes and delay or reverse chemoresistance.NEW & NOTEWORTHY Temozolomide (TMZ) is the standard therapy for glioblastoma, but resistance is common and often linked to MGMT-mediated DNA repair. We found that LonP1, a mitochondrial protease, also drives MGMT-independent resistance by maintaining oxidative phosphorylation. Cells chronically exposed to TMZ upregulate LonP1, increase mitochondrial mass, and adopt a more oxidative phosphorylation (OXPHOS)-dependent metabolic phenotype accompanied by altered extracellular acidification. Targeting LonP1 reverses this adaptation and sensitizes glioblastoma cells to TMZ.