ONC201 kills breast cancer cellsin vitroby targeting mitochondria

Abstract

We report a novel mechanism of action of ONC201 as a mitochondria-targeting drug in cancer cells. ONC201 was originally identified as a small molecule that induces transcription of TNF-related apoptosis-inducing ligand (TRAIL) and subsequently kills cancer cells by activating TRAIL death receptors. In this study, we examined ONC201 toxicity on multiple human breast and endometrial cancer cell lines. ONC201 attenuated cell viability in all cancer cell lines tested. Unexpectedly, ONC201 toxicity was not dependent on either TRAIL receptors nor caspases. Time-lapse live cell imaging revealed that ONC201 induces cell membrane ballooning followed by rupture, distinct from the morphology of cells undergoing apoptosis. Further investigation found that ONC201 induces phosphorylation of AMP-dependent kinase and ATP loss. Cytotoxicity and ATP depletion were significantly enhanced in the absence of glucose, suggesting that ONC201 targets mitochondrial respiration. Further analysis indicated that ONC201 indirectly inhibits mitochondrial respiration. Confocal and electron microscopic analysis demonstrated that ONC201 triggers mitochondrial structural damage and functional impairment. Moreover, ONC201 decreased mitochondrial DNA (mtDNA). RNAseq analysis revealed that ONC201 suppresses expression of multiple mtDNA-encoded genes and nuclear-encoded mitochondrial genes involved in oxidative phosphorylation and other mitochondrial functions. Importantly, fumarate hydratase deficient cancer cells and multiple cancer cell lines with reduced amounts of mtDNA were resistant to ONC201. These results indicate that cells not dependent on mitochondrial respiration are ONC201-resistant. Our data demonstrate that ONC201 kills cancer cells by disrupting mitochondrial function and further suggests that cancer cells that are dependent on glycolysis will be resistant to ONC201.

Imipridone Anticancer Compounds Ectopically Activate the ClpP Protease and Represent a New Scaffold for Antibiotic Development

“…TFAM itself was also depleted in response to ONC212, in agreement with previous studies in other cell lines (Greer et al 2018;Graves et al 2019). Consistent with previous reports of mitochondrial defects caused by ONC201 treatment in various cell lines (Greer et al 2018;Ishizawa et al 2019), we found that both ONC201 and ONC212, but not an inactive isomer of ONC201, caused a collapse of mitochondrial membrane potential and an increase in mitochondrial reactive oxygen species levels in NALM-6 cells ( Figure S3). The broad impact of ectopic CLPP activation on the mitochondrial proteome indicates that the primary defect elicited by ONC212 is a collapse of mitochondrial integrity and function.…”

“…CLPX also performs chaperone functions independently of CLPP, as it promotes heme biosynthesis and enhances the DNA-binding activity of TFAM, a mitochondrial transcription factor (Kasashima et al 2012;Yien et al 2017). TFAM itself was also depleted in response to ONC212, in agreement with previous studies in other cell lines (Greer et al 2018;Graves et al 2019). Consistent with previous reports of mitochondrial defects caused by ONC201 treatment in various cell lines (Greer et al 2018;Ishizawa et al 2019), we found that both ONC201 and ONC212, but not an inactive isomer of ONC201, caused a collapse of mitochondrial membrane potential and an increase in mitochondrial reactive oxygen species levels in NALM-6 cells ( Figure S3).…”

“…Although it is possible that cell type-specific gene expression may explain the absence of these genetic hits, the most parsimonious explanation is that CLPP is the primary target of the imipridones. The proximal effect of unregulated CLPP activation on mitochondrial integrity is consistent with the established correlation between ONC201 sensitivity and reliance on mitochondrial respiration (Greer et al 2018), as well as the depletion of proteins involved in oxidative phosphorylation (OXPHOS), as shown here and elsewhere (Greer et al 2018;Graves et al 2019;Ishizawa et al 2019). The activation of CLPP by the imipridones represents a new avenue in the emerging anticancer strategy of targeting mitochondrial functions (Cole et al 2015;Zong et al 2016;Baccelli et al 2019).…”

“…Several mechanisms of action have been ascribed to ONC201 and its more potent derivate ONC212 (Wagner et al 2017), including: FOXO3A-mediated induction of TRAIL (Allen et al 2013) upon inhibition of the prosurvival AKT/ERK pathway (Allen et al 2013); activation of the integrated stress response (ISR) via the eIF2a kinases HRI and PKR, resulting in ATF4/CHOP-mediated upregulation of the TRAIL DR5 receptor (Kline et al 2016); activation of the ISR independent of eIF2a (Ishizawa et al 2016); and antagonism of dopaminergic G protein-coupled receptors (GPCRs) or activation of other GPCRs (Kline et al 2018;Madhukar et al 2019;Nii et al 2019;Prabhu et al 2019). Cell biological characterization suggests that ONC201 disrupts mitochondrial function and that cancer cells that do not depend on mitochondrial respiration are ONC201-insensitive (Greer et al 2018). It has recently been shown that ONC201 and other imipridones directly bind and activate CLPP in the absence of CLPX, resulting in the degradation of selected mitochondrial proteins and loss of mitochondrial integrity (Graves et al 2019;Ishizawa et al 2019).…”

Impact of pharmacological agents on mitochondrial function: a growing opportunity?

“…ONC201 was identified from a screen for compounds that induced the transcription of tumour necrosis-related apoptosis-inducing ligand (TRAIL), an inducer of apoptosis [74]. Follow-up studies discovered that ONC201 did not directly activate TRAIL in all of the cancer cells in which it was effective but was impairing mitochondrial function [75]. Recent data from two studies identified the target of ONC201 as being the mitochondrial CLPP protease which is activated by ONC201 and results in increased cancer cell killing [76,77].…”