Small Molecule CLPP Activators Impair Mitochondrial Functions and Induce Senescence in Triple-Negative Breast Cancer

ClpP activators are an emerging field of pharmacology, lead by ONC201 (Dordaviprone) which was FDA approved for the treatment of H3K27M mutant diffuse midline gliomas in 2025. More potent and selective ClpP activators, such as the TR compounds, are in pre-clinical testing for a variety of cancers including triple-negative breast cancer (TNBC). ClpP activators significantly impair mitochondrial function and metabolism across cancer cell lines and inhibit cancer cell proliferation. Chapters 2 and 3 discovered that the ClpP activator, TR-57, inhibits mitochondrial transcription, likely via degradation of the mitochondrial RNA polymerase (POLRMT). In TNBC, ClpP activation also decreased mitochondrial transcription factor A (TFAM) and depleted mtDNA content. Overexpressing POLRMT slightly increased the IC50 of TR-57, indicating that POLRMT might be important for the mechanism of action of ClpP activators. The response to ClpP activation varies from cancer to cancer, with some cancers undergoing apoptosis, while others only have halted proliferation. Chapter 4 details the discovery that the TR compounds induce senescence in TNBC cells, as evidenced by increased -galactosidase activity and cytokines, a reduction in Lamin B1, Myc, and p-Rb, as well as impaired mitochondrial metabolism and sustained proliferation arrest. TR-57 induced nuclear DNA damage and S-phase arrest. When TNBC cells were treated with TR-57 and drug was washed out, cellular proliferation did not resume and mitochondrial impairment and senescence was perpetuated. These observations were reproduced in vivo where TR-57-induced senescent mouse TNBC cells failed to form tumors in female mice. These results indicate that ClpP activation irreversibly induces senescence and impairs mitochondrial health. A synergistic combination therapy was identified with ClpP activators and the Bcl-2 inhibitor, venetoclax. TR-57 and venetoclax induced significant cell death in immortalized TNBC and patient-derived cells. These studies demonstrate that TNBC cells are sensitized to targeting survival pathways following ClpP hyperactivation, informing further studies that can progress ClpP activators such as the TR compounds into the clinic.