Abstract 4794: Mitochondrial matrix protease ClpP agonists inhibit cell growth and cancer stem cell function in breast cancer cells
Authors: Yoshimi E. Greer, Lidia Hernandez, Voeller Donna, Raj Chari, Sam Gilbert, Binwu Tang, Christina Annunziata, Lalage Wakefield, Edwin Iwanowicz, Lee M. Graves, Stanley Lipkowitz
Journal: Cancer Research (2020)

Abstract Background: We previously reported that ONC201, a novel anti-tumor drug currently being tested in early phase clinical trials, impairs mitochondrial structure and function in breast cancer cells (Greer et al., 2018). Recent studies demonstrated that ONC201 and the related TR compounds are agonists of mitochondrial caseinolytic protease P (ClpP), an essential protein to mitochondrial protein homeostasis (Graves et al. 2019). Cancer stem cells (CSCs) preferentially use mitochondrial oxidative metabolism for energy production. Here, we tested the impact of ClpP agonists-mediated mitochondrial dysregulation on breast CSCs. Methods: Mitochondrial DNA (mtDNA)-depleted (rho0) MDA-MB231 (MB231) cells were generated by ethidium bromide treatment. MtDNA copy numbers were measured by qPCR. Mitochondrial respiration was monitored by Seahorse XF analyzer. Cell viability was analyzed by CellTiter-Glo 2.0 and RealTime-Glo assay. Protein expression was examined by Western blotting. ClpP knockout (KO) cells were generated by CRISPR/Cas9 technology. Mammosphere formation assay was performed to evaluate CSC function. MB231 cells stably expressing SORE6 (OCT4/SOX2 response element)-mCherry reporter gene were used to monitor CSC numbers. In vivo limiting dilution analysis was performed to examine the effect of ClpP agonists on MB231 mammary fat pad tumor initiation capability in athymic nude female mice. Results: We observed that mammospheres formed by MB231were enriched in mtDNA compared to cells grown in 2D culture while rho0 cells lacking functional mitochondria failed to form mammospheres. This suggested that mitochondria are required for CSC function. The effects of ONC201 and TR compounds in breast cancer cells were tested. First, we confirmed that TR compounds (TR-65, TR-57) had ~60-250 fold higher potency compared to ONC201 in cell viability assays. Both ONC201 and TR-57 impaired mitochondrial structure, function, inhibited cell growth in 2D, and significantly inhibited mammosphere formation in multiple breast cancer cell lines. Importantly, ONC201 and TR-57 did not affect mitochondrial function, cell growth in 2D, nor mammosphere formation in CLPP KO cell lines, confirming ClpP is the target of these drugs. Flow cytometry analysis of MB231-SORE6-mCherry expressing cells demonstrated that ONC201 treatment significantly reduced the fraction of cells that were positive for the SORE6-mCherry activity, consistent with loss of CSCs. Finally, using in vivo limiting dilution experiments, CSC frequency was significantly lower (p&amp;lt;0.05) in the ONC201-treated group compared with the control group, indicating that ONC201 inhibits tumor initiation capability in breast cancers. Conclusion: Our studies suggest that targeting mitochondria by ClpP agonists inhibit cell growth and tumor initiation in breast cancer cells. Citation Format: Yoshimi E. Greer, Lidia Hernandez, Voeller Donna, Raj Chari, Sam Gilbert, Binwu Tang, Christina Annunziata, Lalage Wakefield, Edwin Iwanowicz, Lee M. Graves, Stanley Lipkowitz. Mitochondrial matrix protease ClpP agonists inhibit cell growth and cancer stem cell function in breast cancer cells [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4794.