Abstract B52: Bioenergetic switch confers acquired resistance to BEZ235 in EGFR T790M-mutant non-small cell lung cancer
Authors: Bhattacharya Bhaskar , Koh King Xin , Mohamed Omar Mohamed Feroz , Low Sarah , Tan Juleen , Sapari Nur Sabrina , Iacopetta Barry , Soo Ross , Beloueche-Babari Mounia , Soong Richie 
Published: May 2016

Abstract
Introduction:
Acquired resistance (AQR) continues to be source of treatment failure in EGFR inhibitor therapy and subsequent second-line strategies. In this study, AQR to the PI3K/mTOR inhibitor BEZ235 was generated in EGFR T790M non-small cell lung cancer (NSCLC) cells refractory to EGFR inhibitors to better understand AQR in this setting.
Methods:
Acquired resistant clones of H1975 were generated by continual exposure of H1975 cells to parental IC50 concentrations of BEZ235, followed by monoclonal selection. Phenotypic and protein analysis were carried out using standard techniques. Differentially expressed genes were determined using Affymetrix Gene 1.0ST analysis. Combination analysis was carried out using Chou and Talalay's median-effect equation. Oxygen consumption rates (OCR) were measured using the Seahorse XFp analyzer. Metabolomic analysis was carried out with 1H NMR. Depletion of mitochondrial DNA (mtDNA) was achieved by ethidium bromide treatment.
Results:
Two resistant clones were generated with stably higher IC50 values (Clone 5.2 μM, Clone 6 7.8μM) to parental H1975 cells (0.32μM) after 6 months. The clones were resistant to other PI3K and mTOR inhibitors but not chemotherapeutic drugs, indicating class-specific resistance. Compared to parental cells, the clones exhibited a reduced G1 block, increased migratory capability, reduced PTEN and increased p-AKT. Gene array analysis revealed monocarboxylate transporters as top differentially expressed genes, prompting an investigation of glycolytic status. Proliferation was inhibited in the absence of glucose for the AQR clones but not parental cells. Compared to parental cells, AQR cells were more sensitive to the glycolytic inhibitor, 3BP. Combination treatment with 3BP and BEZ235 reversed the resistance of AQR cells, while in parental cells the combination was antagonistic. Resistant clones had higher extracellular lactate, and lower reactive oxygen species and ATP levels. Metabolomic analysis indicated that the clones had increased intracellular glucose, lactate, NADH and glutamine levels, consistent with enhanced glycolysis. The AQR clones also had lower OCR, reflecting impaired mitochondrial respiratory function. Depletion of mitochondrial DNA in parental cells led to resistance to PI3K and mTOR inhibitors, but not cisplatin.
Conclusion:
These results highlight that cells could acquire drug resistance through a bioenergetics switch from mitochondrial respiration to glycolysis.
Citation Format: Bhaskar Bhattacharya, King Xin Koh, Mohamed Feroz Mohamed Omar, Sarah Low, Juleen Tan, Nur Sabrina Sapari, Barry Iacopetta, Ross Soo, Mounia Beloueche-Babari, Richie Soong. Bioenergetic switch confers acquired resistance to BEZ235 in EGFR T790M-mutant non-small cell lung cancer. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr B52.