Abstract P127: Mitochondrial matrix protease ClpP agonists suppress breast cancer stem cell function by downregulating multiple stem cell regulatory mechanisms
Authors: Greer Yoshimi E. , Hernandez Lidia , Voeller Donna , Chari Raj , Tang Binwu , Annunziata Christina M. , Gilbert Sam , Wakefield Lalage , Iwanowicz Edwin , Graves Lee M. , Lipkowitz Stanley 
Published: December 2021

Abstract
Background: We previously reported that small molecule ONC201 induces mitochondrial structural and functional damage, leading to death in breast cancer cells. Subsequent studies demonstrated that ONC201 and the related analogs TR compounds are agonists of mitochondrial caseinolytic protease P (ClpP), an essential protein for maintenance of mitochondrial protein homeostasis. Recent studies have shown that cancer stem cells (CSCs) preferentially use mitochondrial oxidative metabolism for energy production. Here, we report that ClpP agonists inhibit breast CSCs by unique mechanisms targeting pathways vital to maintain CSC function. Methods: ONC201 and other ClpP agonists (TR-57, 65), other mitochondria-targeting drugs, such as oligomycin, metformin, CPI-613 were used. MDA-MB231 cell line was used as a primary in vitro model system. CLPP knockout (KO) cells were generated by CRISPR/Cas9 technology. Seahorse XF analyzer was used for cellular respiration analysis. Luminescence-based assays were used for cell viability and metabolism assays. Protein expression was examined by Western blotting. Aldefluor assay and SORE6 (OCT4/SOX2 response element)-reporter gene were used to monitor CSC fraction. Mammosphere formation assay was used to evaluate CSC function in vitro. In vivo limiting dilution analysis was used to evaluate tumor initiation capability of cells injected into mammary fat pad of athymic nude female mice. Results: Seahorse XF analyzer showed that mammospheres are more dependent on OxPhos than glycolysis compared with cells grown in 2D, supporting the hypothesis that breast CSCs rely on OxPhos. ClpP agonists reduced the CSC fraction in both Aldefluor and SORE6 reporter assays. ClpP agonists inhibited mammosphere formation in CLPP WT, but not in CLPP KO cells, demonstrating the on-target effects on CSC function. In in vivo assays, tumor formation was significantly (p&lt;0.001) inhibited in the ClpP agonist-treated group compared with the control group, and the effect was CLPP-dependent. Altogether, these findings support that ClpP agonists inhibit CSC in breast cancers. We found that ClpP agonists downregulate multiple pathways and proteins critical for CSC maintenance including mevalonate pathway, HIF1a, EPAS1, YAP, and Myc. We also observed that other mitochondria targeting drugs such as oligomycin, metformin downregulate these signaling pathways and proteins to some extent. Importantly, however, ClpP agonists showed significantly greater impact in mammosphere formation and cell growth assays, compared with other mitochondria-targeting drugs. Further studies revealed that ClpP agonists uniquely deplete NAD+/NADH and promote reactive oxygen species, both of which are shown as key factors to maintain pluripotency of stem cells. Moreover, ClpP agonists uniquely inhibited enzymes involved with glutamine catabolism and proline biosynthesis, vital to amino acids and nucleotide synthesis. Conclusion: ClpP agonists inhibit cell growth and tumor initiation in breast cancer cells by targeting multiple pathways essential to maintain CSC function.
Citation Format: Yoshimi E. Greer, Lidia Hernandez, Donna Voeller, Raj Chari, Binwu Tang, Christina M. Annunziata, Sam Gilbert, Lalage Wakefield, Edwin Iwanowicz, Lee M. Graves, Stanley Lipkowitz. Mitochondrial matrix protease ClpP agonists suppress breast cancer stem cell function by downregulating multiple stem cell regulatory mechanisms [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2021 Oct 7-10. Philadelphia (PA): AACR; Mol Cancer Ther 2021;20(12 Suppl):Abstract nr P127.