Abstract Glioblastoma multiforme (GBM) is the most aggressive primary brain tumor and remains difficult to treat. Temozolomide (TMZ) is a cornerstone chemotherapeutic agent for GBM; however, the early development of TMZ resistance significantly limits its therapeutic benefit. ATP-binding cassette transporters such as ABCB1 reduce intracellular drug levels through active efflux. We hypothesized that ABCB1 mediates resistance to TMZ through metabolic rewiring. We investigated whether enhanced mitochondrial bioenergetics fuels ABCB1 function and promotes TMZ resistance. To test this, we generated LN-229 cells stably overexpressing ABCB1 and examined metabolic and bioenergetic changes using gas chromatography–mass spectrometry based metabolomics. We also conducted ATP and lactate assays, mitochondrial mass measurements, ROS profiling, and apoptosis analyses. ABCB1 overexpression caused marked metabolic shifts, including increased tricarboxylic acid (TCA) cycle intermediates, glycolytic activity, higher ATP levels, and increased mitochondrial mass. These findings indicate that metabolic alterations potentially fueled ABCB1drug efflux, as evidenced by reduced doxorubicin retention. We next inhibited mitochondrial complex I with metformin, which decreased ATP production, impaired efflux activity, and restored TMZ sensitivity. Combined metformin and TMZ treatment induced increased apoptotis, demonstrated by caspase-3 activation, PARP cleavage, and increased Annexin V-positive cells. Our findings identify mitochondrial metabolism as a key driver of ABCB1-mediated chemoresistance in GBM and suggest that targeting bioenergetic pathways may be an effective strategy to overcome TMZ resistance. Citation Format: Ashutosh Shrivastava, Manendra Singh Tomar, Chirag Kulkarni, Sreyanko Sadhukhan, Priyanka Prajapati, Amit Lahiri, Naibedya Chattopadhyay. Mitochondrial bioenergetics sustain ABCB1-driven temozolomide resistance in glioblastoma multiforme 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 373.
Shrivastava et al. (Fri,) studied this question.