LAUSR

Background: Advanced and metastatic melanoma carries significant mortality despite recent advances. Metabolic plasticity is a hallmark of cancer and an important resistance mechanism in the face of anti-neoplastic therapies. We sought to understand the effect of chemotherapy, Temozolomide (TMZ), on OXPHOS and TCA cycle metabolism in melanoma cell lines. By understanding these metabolic adaptations to chemotherapy, we hope to identify novel combination therapies. Methods: A375 and SK-MEL-28 cell lines were used for all experiments. Cell viability assays were performed with 1500 cells per well, treated with TMZ and oligomycin or phenformin, and viability was assessed using PicoGreen dsDNA assay. Western blot was used with primary antibodies against TOM20 and Β-actin. LCMS metabolites were assessed by QTRAP triple quadrupole mass spectrometry coupled to a Prominence UFLC HPLC system. This assessed approximately 299 metabolites. Seahorse XFp miniextracellular analyzer was used to obtain OCR with and without TMZ exposure, and after sequential injections of oligomycin, FCCP, and rotenone with antimycin A. Mitochondrial assays included TMRE staining (membrane potential) and Mitotracker Green (mitochondrial mass). Results: Metabolic profiles of melanoma cell lines change with exposure to chemotherapy. After melanoma cells are exposed to IC30 dose of TMZ, LCMS metabolomics revealed statistically significant increase in TCA cycle metabolites such as succinate, succinyl CoA, malate, oxaloacetate, isocitrate, alpha-ketoglutarate, and fumarate. Melanoma increases OXPHOS in the presence of chemotherapy. Mitochondrial mass, measured by Mitotracker, increased in response to TMZ exposure. We also found increased mitochondrial membrane potential and increased TOM20 expression after cells were treated with TMZ. Seahorse XF Cell Mito Stress Assay demonstrated increased oxygen consumption rate after exposure to TMZ. We then combined TMZ and two mitochondrial inhibitors, phenformin (complex I) and oligomycin (complex V). Combination of phenformin and TMZ was synergistic in cell viability assays as determined by Bliss equation. Oligomycin sensitized both cell lines to TMZ. Discussion: We have shown that melanoma cells rely on mitochondrial function when exposed to TMZ by increasing levels of TCA cycle intermediates, increasing mitochondrial tropism, and increased expression of mitochondrial enzymes. This demonstrates a metabolic resistance mechanism in response to chemotherapy to promote melanoma cell survival. Finally, we exploit this adaptation by pharmacologic inhibition of mitochondrial electron transport chain (ETC) with phenformin and oligomycin A. This treatment was synergistic with conventional chemotherapy. Our findings point out that the ETC compartment of mitochondria could be a novel and readily available combination treatment strategy for patients with advanced and refractory melanoma. Citation Format: Alexander W. Loftus, Mehrdad Zarei, Omid Hajihassani, Johnathan J. Hue, Hallie J. Graor, Ali Vaziri-Gohar, Jordan M. Winter, Luke D. Rothermel. Chemotherapy alters mitochondrial metabolism in melanoma. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4839.