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