Abstract B023: ONC201 kills breast cancer cells by targeting mitochondria

• Published in: Molecular cancer therapeutics 2018-01, Vol.17 (1_Supplement), p.B023-B023
• Main Authors: Greer, Yoshimi Endo, Gilbert, Samuel, Islam, Celia, Ubaldini, Ashley, Stuelten, Christina, Porat-Shliom, Natalie, Weigert, Roberto, Ji, Yun, Gattinoni, Luca, Soheilian, Ferri, Nagashima, Kunio, Wang, Xiantao, Hafner, Markus, Shetty, Jyoti, Tran, Bao, Koparde, Vishal, Jailwala, Parthav, Cam, Maggie, Crooks, Dan, Linehan, W. Marston, Voeller, Donna, Reinhold, William, Rajapakse, Vinodh, Pommier, Yves, Lipkowitz, Stanley
• Format: Article
• Language: English
• ISSN: 1535-7163; 1538-8514
• DOI: 10.1158/1535-7163.TARG-17-B023

ONC201 is a small molecule originally identified as a TRAIL (TNF-related apoptosis-inducing ligand) 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. In this study, we tested ONC201 toxicity in breast cancer cell lines. ONC201 was obtained from Oncoceutics, Inc. Cell viability was tested with MTS assay and CytoTox-Glo assay. ATP level was measured with CellTiter-Glo 2.0 assay. RNA-seq 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 and electron microscopy analysis were performed to study mitochondrial morphology. Mitochondrial DNA (mtDNA) copy number was analyzed by Quantitative PCR (qPCR). Mitochondrial defective (rho0) cell lines were generated by ethidium bromide treatment. We tested the effects of ONC201 on 18 human breast cancer cell lines that represent ER+, HER2-amplified, basal A triple-negative (TNBC), and basal B TNBC 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. 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 suggested that ONC201 kills breast cancer cells via a caspase-independent, TRAIL-receptor-independent mechanism. Western blots confimed that ONC201 induces ATF4 and CHOP, consistent with reported observations. ONC201 also induced phosphorylation of AMP-dependent kinase (AMPK) and depletion of cellular ATP in multiple breast cancer cell lines. Cytotoxicity and ATP depletion induced by ONC201 were significantly enhanced in nonglucose (galactose) medium compared with glucose-containing medium, suggesting that ONC201 targets mitochondrial respiration. Supplementing glucose to cells grown in galactose medium rescued ONC201-dependent ATP depletion, cytotoxicity, and induction of p-AMPK, ATF4, and CHOP. Seahorse XF analyzer indicated that ONC201 inhibits mitochondrial respiration via an indirect mechanism. Confocal imaging revealed tha