ONC201, the anticancer drug, targets and activates mitochondrial ATP-dependent caseinolytic peptidase P (ClpP), a serine protease located in the mitochondrial matrix. Given the promise of ONC201 in cancer treatment, we evaluated its effects on the breast ductal carcinoma cell line (BT474). We showed that the transient single-dose treatment of BT474 cells by 10 µM ONC201 for a period of less than 48 h induced a reversible growth arrest and a transient activation of an integrated stress response indicated by an increased expression of CHOP, ATF4, and GDF-15, and a reduced number of mtDNA nucleoids. A prolonged exposure to the drug (>48 h), however, initiated an irreversible loss of mtDNA, persistent activation of integrated stress response proteins, as well as cell cycle arrest, inhibition of proliferation, and suppression of the intrinsic apoptosis pathway. Since Natural Killer (NK) cells are quickly gaining momentum in cellular anti-cancer therapies, we evaluated the effect of ONC201 on the activity of the peripheral blood derived NK cells. We showed that following the ONC 201 exposure BT474 cells demonstrated enhanced sensitivity toward human NK cells that mediated killing. Together our data revealed that the effects of a single dose of ONC201 are dependent on the duration of exposure, specifically, while short-term exposure led to reversible changes; long-term exposure resulted in irreversible transformation of cells associated with the senescent phenotype. Our data further demonstrated that when used in combination with NK cells, ONC201 created a synergistic anti-cancer effect, thus suggesting its possible benefit in NK-cell based cellular immunotherapies for cancer treatment.

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Background and goals: ONC201 an experimental anticancer drug, targeting mitochondrial caseinolytic peptidase, ClpP is currently under investigation. Current data on the effect of ONC201 along with observations that ONC201 causes cytotoxic effect, there are data demonstrating that ONC201 is rather cytostatic and does not induce cell death. Recently, we demonstrated ONC201-induced arrest of proliferation and a decrease in the number of mitochondrial nucleoids. Here we extended our study to describe the effect of short-and long-term exposure of BT474 cells to ONC201. Observations: Proliferation and cell cycle arrest. ONC201 in a dose-dependent manner (0-50 µM) inhibited the proliferation of BT474 cells without induction of apoptotic cell death. Cells exposed to 10 µM ONC201 accumulated in G0/G1 phase (49.4 ± 11.5% vs 78.7 ± 5.1%) and declined in the S-phase of the cell cycle (39.2 ± 6.6% vs 9.0 ± 2.1%). In parallel, ONC201 exposure induced a decline in Cyclin E and Cdk2 protein levels. Nucleoids and mtDNA. Exposure of BT474 cells, to 10 µM ONC 201, decreased the number of mitochondrial nucleoids from 249 ± 52 to 155 ± 38 per cell after 24 h. Longer 72 h exposure further reduced the number of nucleoids to 84 ± 36 per cell. ONC201 exposure also induced reduction of mitochondrial size from 2.8 ± 0.5 µm2 to 1.5 ± 0.2 µm2 and depletion of mtDNA. Short-term and long-term consequences of cell exposure to ONC201. Short-term treatment (24 h exposure) demonstrated the reversible effect of ONC201 on the expression of stress proteins (ATF4, CHOP, and GDF-15), cell cycle regulatory proteins (cyclin E, Cdk2), and partial restoration of mitochondrial nucleoids (to 214 ± 46 nucleoids per cell). On the contrary, long-term treatment (72 h) resulted in the irreversible and persistent arrest of proliferation, decline of cell cycle regulatory proteins, and activation of stress proteins. Respiration. ONC201 exposure suppressed State 3, State 4, and uncoupled respiration in digitonin-permeabilized BT474 cells. Complex I activity was decreased from 3.3 ± 0.4 ng-atoms O/min/106 cells to 0.5 ± 0.1 and 0.4 ± 0.3 ng-atoms O/min/106 cells, in cells exposed to ONC201 for 24h and 72h, respectively. Similarly, ONC201 decreased the activity of Complex II from 3.5 ± 0.5 ng-atoms O/min/106 cells to 1.5 ± 0.6 and 0.3 ± 0.1 ng-atoms O/min/106. Conclusions: The consequences of ONC201 treatment were dependent on the duration of exposure to the drug. Short-term (24 h) exposure of BT474 cells resulted in reversible induction of stress proteins (ATF4, CHOP, and GDF-15), which were reversed following 120 h incubation in drug-free media. On the contrary, long-term (72 h) exposure of cells to ONC201 induced irreversible and persistent arrest of proliferation and elevation of expression of stress proteins. Thus, ONC201 can induce prolonged and irreversible elevation of stress proteins and persistent G1/S cell cycle arrest. Citation Format: Artem A. Mishukov, Irina V. Odinokova, Serazhutdin A. Abdullaev, Vitaly K. Zhalimov, Emily M. Fennel, Lucas J. Aponte-Collazo, Paul R. Graves, Lee M. Graves, Ekhson L. Holmuhamedov. Anticancer compound ONC201 induces prolonged stress and arrest of proliferation in BT474 human breast cancer cells without induction of cell death [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5380.