Biology subjects

Fennell, E. M. J.

Publications and source records attributed to Fennell, E. M. J..

3 recordsLinked to original sources

bioRxiv2022

SMALL MOLECULE CLPP AGONISTS INDUCE SENESCENCE AND ALTER TRAIL-MEDIATED APOPTOTIC RESPONSE OF TRIPLE-NEGATIVE BREAST CANCER CELLS

Imipridones are a novel class of anticancer drugs with promising antiproliferative effects in several cancer cell types, including breast cancer. Recent studies identified the mitochondrial ATP-dependent caseinolytic peptidase P (ClpP) as the target for imipridones and related analogs. Despite these findings, the specific processes by which ClpP activators inhibit cancer cell growth remain poorly understood. Here we report that two structurally distinct ClpP activators, ONC201 and TR-57, promote senescence in SUM159 and MDA-MB-231 triple-negative breast cancer (TNBC) cell lines. Induction of senescence was measured through {beta}-galactosidase assays and confirmed by the increase of H2A.X phosphorylation, hypophosphorylation of retinoblastoma protein (Rb), upregulation of multiple interleukin mRNAs and other markers. The level of senescence induced by these compounds was equivalent to that observed with the CDK4/6 inhibitor and positive control abemaciclib. To confirm the crucial role of ClpP activation in senescence induction, we generated ClpP null TNBC cell lines using CRISPR interference (CRISPRi). Neither ONC201 nor TR-57 induced senescence in the ClpP null models. Incubation of WT cells with ClpP activators led to a reduction in the levels of apoptosis-related proteins like XIAP, SMAC/DIABLO, Survivin, DR4 and DR5, which correlated with the lack of apoptosis observed in these cells. Interestingly, treatment with TR-57 strongly reduced apoptosis induced by staurosporine but increased sensitivity to tumor necrosis factor-related apoptosis-inducing ligand (TRAIL). To investigate the enhanced effects of TRAIL, we examined the expression of Wee1 in senescent cells and found that both TR-57 and abemaciclib down-regulated Wee1. Addition of a Wee1 inhibitor partially sensitized cells to TRAIL suggesting the importance of Wee1 in this process. In summary, we show that ClpP activators induce senescence in a ClpP-dependent manner and that combined treatment of ClpP activators with TRAIL provides an effective approach to eliminate malignant senescent cells in vitro. HIGHLIGHTSO_LITreatment of TNBC cells with ClpP activators induces senescence in vitro C_LIO_LIInduction of senescence is ClpP dependent C_LIO_LIActivation of ClpP leads to changes in mRNA levels of senescence associated cytokines C_LIO_LISenescent TNBC cells are sensitized to TRAIL mediated apoptosis C_LI

cancer biology↗

bioRxiv2022

Mitochondrial Matrix Protease ClpP Agonists inhibit Cancer Stem Cell Function in Breast Cancer Cells by Disrupting Mitochondrial Homeostasis

Mitochondria are multifaceted organelles which are important for bioenergetics, biosynthesis and signaling in metazoans. Mitochondrial functions are frequently altered in cancer to promote both the energy and the necessary metabolic intermediates for biosynthesis required for tumor growth. Cancer stem cells (CSCs) contribute to chemotherapy resistance, relapse, and metastasis. Recent studies have shown that while non-stem, bulk cancer cells utilize glycolysis, breast CSCs are more dependent on oxidative phosphorylation (OxPhos) and therefore targeting mitochondria may inhibit CSC function. We previously reported that small molecule ONC201, which is an agonist for the mitochondrial caseinolytic protease (ClpP), induces mitochondrial dysfunction in breast cancer cells. In this study, we report that ClpP agonists inhibit breast cancer CSC function in vitro and in vivo. Mechanistically, we found that OxPhos inhibition downregulates multiple pathways required for CSC function, such as the mevalonate pathway, YAP, Myc, and the HIF pathway. ClpP agonists showed significantly greater inhibitory effect on CSC functions compared with other mitochondria-targeting drugs. Further studies showed that ClpP agonists deplete NAD(P)+ and NAD(P)H and induce redox imbalance, and dysregulate one-carbon metabolism and proline biosynthesis. Downregulation of these pathways by ClpP agonists further contribute to the inhibition of CSC function. In conclusion, ClpP agonists inhibit breast CSC functions by disrupting mitochondrial homeostasis in breast cancer cells and inhibiting multiple pathways critical to CSC function.

cancer biology↗

bioRxiv2022

Characterization of TR-107, a Novel Chemical Activator of the Human Mitochondrial Protease ClpP

We recently described the identification of a new class of small molecule activators of the mitochondrial protease ClpP. These compounds synthesized by Madera Therapeutics showed increased potency of cancer growth inhibition over the related compound ONC201. In this study, we describe chemical optimization and characterization of the next generation of highly potent and selective small molecule ClpP activators (TR compounds) and demonstrate their efficacy against breast cancer models in vitro and in vivo. One of these compounds (TR-107) with excellent potency, specificity and drug-like properties was selected for further evaluation. Examination of TR-107 effects in triple-negative breast cancer (TNBC) cell models showed growth inhibition in the low nanomolar range, equipotent to paclitaxel, in a ClpP-dependent manner. TR-107 reduced specific mitochondrial proteins including OXPHOS and TCA cycle components, in a time, dose and ClpP-dependent manner. Seahorse XF analysis and glucose deprivation experiments confirmed inactivation of OXPHOS and demonstrated an increased dependence on glycolysis following TR-107 exposure. The pharmacokinetic properties of TR-107 were compared to other known ClpP activators including ONC201 and ONC212. TR-107 displayed excellent exposure and serum t1/2 after oral administration. The antitumor response to TR-107 was investigated using human TNBC MDA-MB-231 cell line-induced xenograft tumors. Oral administration of TR-107 resulted in reduction in tumor volume and extension of survival in the treated compared with vehicle control mice. In summary, we describe the identification of highly potent new ClpP agonists with improved efficacy against TNBC, through targeted inactivation of OXPHOS and disruption of mitochondrial metabolism.

pharmacology and toxicology↗