Abstract 991: ONC201 & TR57 reversibly depletes mtDNA content & regulates mitochondrial biogenesis in BT-474, human breast cancer cells

Abstract

Abstract Recent demonstration that mechanism of action of small molecule imipridone ONC201 could be mediated through mitochondrial targeting (Graves et al., 2019; Greer et al., Ishizawa et al., 2019), was confirmed by identification of mitochondrial caseinolythic protease (ClpP), as the only target of ONC201 and its chemical analog TR57 (Graves et al., 2019). We further studied direct effect of these drugs on mitochondrial functions and mtDNA distribution in human breast cancer cells. Using respirometry and confocal microscopy, we demonstrated dose- and time dependent suppression of mitochondria oxygen consumption and degradation of mtDNA in cultured human BT-474 cell lines, treated with ONC201 and TR57. Both drugs, used in this study, induced significant and reversible decrease in the rate of proliferation as well as the content of mtDNA within the mitochondria (> by 50%), which was released into the cytosol. Semi-quantitative analysis of confocal images of mitochondrial matrix (MTDR, red fluorescence) and mtDNA (SYBR Green-1, green fluorescence) demonstrated 30-35% release of mtDNA from mitochondria/mitochondrial fragments. Exposure to ONC201 (10µM) or TR57 (50nM) for 48 h decreased mtDNA content by 60% and 85%, respectively. Effect of these drugs was reversible and washout of drugs restored the rate of proliferation as well as mtDNA content in time-dependent manner. Within 24h of washout, the total cellular mtDNA reached almost 90% of initial level, demonstrating fast activation of mtDNA synthesis upon removal of toxins. RT-PCR approach to monitor mtDNA in the cytosol and mitochondria confirmed release of mtDNA into the cytosol and associated decrease of mtDNA within mitochondria/mitochondrial fragments, following ONC201 or TR57 treatment of BT-474 cells. Taken together, our data demonstrate that these drugs reversibly modulate and regulate intracellular mtDNA content, through ONC201 and TR57 dependent regulation of mitochondrial biogenesis in human breast cancer cells. In conclusion, ONC201 & TR57 dependent reversible inhibition of cell proliferation and decrease in mtDNA content allow suggesting drug-dependent regulation of mitochondrial biogenesis in these BCC. Our observation warrants new direction in the elucidation of the mechanism of action of these drugs and search for novel efficient anti-cancer drugs. Support of Funding Information: Russian Science Foundation № 19-75-20145 Citation Format: Margarita I. Kobyakova, Serazhutdin Abdullaev, Yana V. Evstratova, Artem Mishukov, Irina Odinokova, Lee M. Graves, Ekhson Holmuhamedov. ONC201 & TR57 reversibly depletes mtDNA content & regulates mitochondrial biogenesis in BT-474, human breast cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 991.