ONC201 and Its Potent Analogues Disrupt Mitochondrial Metabolic Function in Triple Negative Breast Cancer

Abstract

BackgroundONC201 is an imipridone compound in clinical trials for multiple cancer types, including triple negative breast cancer (TNBC). ONC201 is a promising anticancer therapy whose pharmacological target and function are not clearly defined. We have demonstrated that the target of ONC201 and its potent analogues (TR compounds) is the mitochondrial protease ClpP, consistent with previous reports of ONC201‐induced mitochondrial stress and depolarization in TNBC cells.Study ObjectiveThe objective of this study is to determine if ONC201 and TR compound treatment result in a loss of mitochondrial protein through activation of ClpP, leading to metabolic disruption in glutamine utilization and mitochondrial function.MethodsTo determine if ONC201 and TR compounds disrupt aerobic respiration, galactose was substituted for glucose in cell culture media. We used galactose‐containing cell culture media to shift cells from anaerobic to aerobic respiration, as galactose is utilized through aerobic respiration. The IC50 of ONC201 and TR compounds was measured with glucose‐ or galactose‐containing media by MTS. MitoPlate assays (Biolog) were performed to identify perturbations in the utilization of 30 different substrates in TNBC cells treated with ONC201 or TR compounds. Proteomic analysis of ONC201 or TR compound‐treated cells was conducted using LC‐MS.ResultsProteomic analysis revealed a large decline in mitochondrial peptides following ONC201 or TR compound treatment. We also demonstrated that substitution of glucose with galactose reduced the IC50 of ONC201 and TR compounds in TNBC cells. In addition, MitoPlate assays suggest a reduced utilization of TCA cycle substrates and L‐glutamine.ConclusionsAddition of galactose to cell culture media improved efficacy of ONC201 and TR compounds in TNBC cells. Sensitization and loss of mitochondrial peptides suggest that ONC201 and TR compounds cause loss of mitochondrial protein and reduction metabolic activity. Consistent with sensitization results, MitoPlate data demonstrates a loss of TCA cycle substrate and L‐glutamine utilization following ONC201 or TR compound treatment. Glutamine is known to facilitate both cell proliferation and energy production in cancer cells through protein biosynthesis and the TCA cycle. Proteomic analysis is being used to identify ClpP‐dependent protein loss. Uniformly 13C‐labeled substrates will be used to identify perturbations in energetic and biosynthetic pathways following ONC201 and TR compound treatment. Loss of mitochondrial metabolic activity and glutamine utilization may play a role in ONC201 and TR compound anticancer activity.Support or Funding InformationNIH Pharmacological Sciences T32 (GM007040), University Cancer Research Fund