ONC201-Induced Mitochondrial Dysfunction, Senescence-like Phenotype, and Sensitization of Cultured BT474 Human Breast Cancer Cells to TRAIL

Abstract

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.

Targeting Mitochondria with ClpP Agonists as a Novel Therapeutic Opportunity in Breast Cancer

“…ClpP agonists induce degradation of mitochondrial proteins (e.g., TFAM, mitochondrial elongation factor Tu [TUFM], and ETC complex proteins), depletion of mtDNA copy number, downregulation of OxPhos, loss of ATP, mitochondrial structural damage, and elevation of ROS level in multiple breast cancer cell lines [ 58 , 269 , 270 , 304 ]. Moreover, ClpP agonists dysregulate metabolic pathways not affected by other mitochondrial drugs such as metformin, oligomycin, CPI-613 [ 58 ].…”

“…Studies testing ONC201 and/or TR compounds in breast cancer models collectively show that ClpP agonists exert a significant anti-proliferative effect accompanied with ISR and cell cycle arrest, rather than inducing apoptosis [ 58 , 268 , 270 , 298 , 299 , 301 , 302 , 304 , 306 ]. These observations are distinct from what has been reported as apoptosis observed in other malignancies, such as colon cancers and hematological malignancies [ 307 , 308 , 309 ].…”

“…These observations are distinct from what has been reported as apoptosis observed in other malignancies, such as colon cancers and hematological malignancies [ 307 , 308 , 309 ]. Recent studies revealed that the anti-proliferative effect of ClpP agonists observed in breast cancers represent a senescence-like phenotype [ 304 , 310 ]. These findings suggests that the impact of ClpP targeting in cancers is context dependent.…”

“…Peritumoral abundance of NK cells is shown to correlate with better pathological complete response (pCR) rate after neoadjuvant chemotherapy in large and locally advanced breast cancer [ 337 ]. ClpP agonists recruit and activate NK cells in breast and other cancer models [ 300 , 301 ], and the cytotoxicity of ClpP-agonist was enhanced by NK cells in breast cancer cell line [ 304 ].…”

In vitro vascular differentiation system efficiently produces natural killer cells for cancer immunotherapies

“…Additionally, antibody-dependent cellular cytotoxicity, mediated by the Fc receptor CD16, can trigger NK cell degranulation against antibody-coated target cells 25–30 . Another advantage of NK cell versatility is the ability to recognize cancer cells despite their continuous mutations and to recognize stress and senescence markers induced by chemotherapy during cancer treatment 31 . Moreover, NKs avoid graft-vs-host disease, commonly associated with T cell therapy, and were recently shown to be more effective than T cells in killing DIPG 32 .…”