Abstract 1487: ONC201 kills breast cancer cells by inhibiting mitochondrial respiration
Authors: Yoshimi Greer, Samuel Gilbert, Celia Islam, Yun Ji, Luca Gattinoni, Christina Stuelten, Natalie Porat-Shliom, Roberto Weigert, Xiantao Wang, Markus Hafner, Kunio Nagashima, Donna Voeller, Stanley Lipkowitz
Journal: Cancer Research (2017)

Abstract Background: ONC201 is a small molecule originally identified as a TRAIL inducing compound currently being tested in phase1/2 clinical trials in multiple cancer types. Two recent studies reported that ONC201 also induces an atypical stress response mediated in part by ATF4 and CHOP. Methods: ONC201 was obtained from Oncoceutics, Inc. Recombinant GST-TRAIL was prepared in the laboratory. Cell viability was tested with MTS assay and CellTiter-Glo luminescent cell viability assay. ATP level was measured with CellTiter-Glo 2.0 assay. RNAseq and western blotting were performed to investigate change of gene expression. Mitochondrial respiration was monitored by Seahorse XF analyzer. Live cell imaging was performed to examine the mode of cell death. Confocal microscopy and electron microscopy analysis were performed to study mitochondrial morphology. Results: We tested the effects of ONC201 on 18 human breast cancer cell lines that represent ER+, HER2 amplified, TNBC basal A and TNBC basal B breast cancer. ONC201 reduced cell viability in breast cancer cell lines in all subtypes tested with IC50s ranging from 0.8-5 μM, similar to what has been reported for other cancer cell types. Unexpectedly, ONC201 toxicity was not dependent on TRAIL receptors or caspases and live cell imaging revealed ONC201 induces cell membrane ballooning followed by rupture. By contrast, GST-TRAIL induced TRAIL-receptor dependent caspase mediated death and classic apoptosis morphology. These results suggest that ONC201 kills breast cancer cells via a caspase-independent, TRAIL-receptor-independent mechanism distinct from TRAIL-induced apoptosis. Western blots revealed that ONC201 induces the stress pathway proteins ATF4 and CHOP, consistent with the recently published observations. ONC201 also induced phosphorylation of AMP-dependent kinase (AMPK) and depletion of cellular ATP in multiple breast cancer cell lines. Seahorse XF analysis found that ONC201 inhibited mitochondrial oxygen consumption rate but did not inhibit glycolysis as measured by the extracellular acidification rate. Both ONC201-induced toxicity and ATP depletion were enhanced when cells were cultured in non-glucose (galactose) medium. Supplementing glucose to cells grown in galactose medium partially prevented ONC201-dependent ATP depletion, induction of phospho-AMPK, ATF4 and CHOP, and cell death. These data are consistent with an inhibition of oxidative phosphorylation (OxPhos) by ONC201. RNAseq revealed ONC201 inhibits expression of multiple mitochondrial genes involved in OxPhos and other mitochondrial functions, and western blot confirmed those findings. Confocal and electron microscopic evaluation revealed abnormal mitochondrial morphology. Conclusion: Our data demonstrate that ONC201 can kill breast cancer cells by a novel mechanism involving disruption of mitochondrial morphology and inhibition of mitochondrial respiration. Citation Format: Yoshimi Greer, Samuel Gilbert, Celia Islam, Yun Ji, Luca Gattinoni, Christina Stuelten, Natalie Porat-Shliom, Roberto Weigert, Xiantao Wang, Markus Hafner, Kunio Nagashima, Donna Voeller, Stanley Lipkowitz. ONC201 kills breast cancer cells by inhibiting mitochondrial respiration [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1487. doi:10.1158/1538-7445.AM2017-1487