Onc201 Kills Breast Cancer Cells By Inhibiting Mitochondrial Respiration

Background: ONC201 is a small molecule originally identified as a TRAIL inducing compound (Allen et al., Sci. Trans. Med 2013). Two recent studies reported that ONC201 also induces an atypical stress response mediated in part by ATF4 and CHOP (Klein et al., Sci. Signal 2016 and Ishizawa et al., Sci. Signal 2016). ONC201 is currently being tested in phase1/2 clinical trials in multiple cancer types. In this study, we tested the effects of ONC201 on human breast cancer cells. Methods: We tested ONC201 in 18 human breast cancer cell lines that represent ER+, HER2 amplified, TNBC basal A and TNBC basal B breast cancer. Cell death was analyzed by MTS assay after 5 days of treatment. Cells were treated with GST-TRAIL in parallel for comparison. Z-VAD-FMK was used as a pan-caspase inhibitor. To verify the mechanism of action of ONC201, siRNA against death receptors (DR) 4 and 5 were transfected to cells and tested in MTS assay and Western blotting. Seahorse XF analyzer and live cell imaging were used to further characterize the effect of ONC201. Results: ONC201 reduced cell viability in breast cancer cell lines in all subtypes tested with IC50s ranging from 0.8-5 uM, similar to what has been reported for other cancer cell types. Unexpectedly, ONC201 did not induce caspase 3 or PARP cleavage, and its toxicity was not inhibited by Z-VAD-FMK, nor by siRNA knockdown of DR4 or DR5. By contrast GST-TRAIL induced caspase 3 and PARP cleavage and GST-TRAIL-induced cell death was inhibited by Z-VAD-FMK and by siRNA knockdown of DR5. Live cell imaging revealed ONC201 induces cell membrane ballooning followed by rupture, whereas GST-TRAIL induced classic apoptosis morphology. Together these results suggest that ONC201 kills breast cancer cells via a caspase-independent, DR4/5-independent mechanism distinct from TRAIL-induced apoptosis. Western blots revealed that ONC201 induces ATF4 and CHOP, consistent with the recently published observations. ONC201 also induced phosphorylation of AMP-dependent kinase (AMPK) in multiple breast cancer cell lines, suggesting that cellular ATP level is decreased by ONC201. ATP depletion by ONC201 was confirmed by direct measurement of cellular ATP. Seahorse XF analysis found that ONC201 inhibited mitochondrial oxygen consumption rate (OCR) but did not inhibit glycolysis as measured by the extracellular acidification rate. Long exposure to ONC201 significantly attenuated OCR, while acute treatment did not inhibit OCR. These data suggest that ONC201 inhibits mitochondrial oxidative phosphorylation via an indirect mechanism. Western blots demonstrated that ONC201 decreases expression of multiple mitochondrial proteins involved in oxidative phosphorylation. Both ONC201-induced toxicity and ATP depletion were enhanced when cells were cultured in non-glucose (galactose) medium. This is consistent with ONC201-induced inhibition of mitochondrial respiration. Supplementing glucose to cells grown in galactose medium partially rescued ONC201-dependent ATP depletion and cell death, and reversed ONC201-induced phospho-eIF2, ATF4 and CHOP induction. Conclusion: Together, these data demonstrate that ONC201 can kill breast cancer cells by a novel mechanism involving inhibition of mitochondrial respiration. Citation Format: Greer YE, Gilbert SF, Islam C, Ji Y, Gattinoni L, Stuelten C, Voeller D, Lipkowitz S. ONC201 kills breast cancer cells by inhibiting mitochondrial respiration [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P3-06-02.