Exploring the Role of Mitochondrial ClpP Activators on Immune Signaling in Breast Cancer

Abstract

Approximately 20% of breast cancer cases diagnosed yearly are of the triple‐negative breast cancer (TNBC) type. Current treatments for TNBC patients are limited to surgical intervention and traditional chemotherapies. ONC201 is a recently discovered orally‐active imipridone molecule in phase I/II clinical trials for a large variety of aggressive cancers including glioblastoma, breast, endometrial, and others. We have been studying highly potent analogs of ONC201, known as the TR compounds (TRs), for their efficacy in TNBC. To better understand the mechanism of action, we coupled a TR compound to agarose beads and applied proteomics to identify potential targets. Our studies identified the mitochondrial caseinolytic peptidase (ClpP), as a major ONC201 and TRs binding protein. We further demonstrated that these compounds activated the proteolytic activity of ClpP and that this mitochondrial signal was required for subsequent activation of the integrated stress response as determined by increased CHOP and ATF4 proteins. In addition to activation of the integrated stress response, other recent studies have suggested that the antitumor effects of ONC201 may in part be dependent on increased immune cell recruitment and cancer growth inhibition through this mechanism. Thus, one of our major objectives is to understand how activation of ClpP by ONC201 or TRs impacts cellular signaling events that lead to enhanced immune cell recruitment. We hypothesize that activation of ClpP promotes activation of key immune signaling proteins, like NF‐kB, which subsequently leads to an increase in proteins required for immune cell recruitment. Accordingly, our preliminary data shows activation or up‐regulation of proteins connected to innate immune signaling including MAVS, NF‐kB, and TBK1 in response to ONC201 or TRs treatment. Additionally, our qPCR data shows that treatment of TNBC cells with ONC201 or TRs leads to increased mRNA levels of specific Interleukins (IL‐12, IL‐10). Collectively, these results suggest that activation of ClpP by ONC201 or TRs increases innate immune signaling and expression of cytokines, which are crucial events for successful recruitment of immune cells. Future experiments will evaluate if activation of NF‐κB by ONC201 or TRs treatment is ClpP dependent using a ClpP knockout in TNBC cell lines. Lastly, we will also measure changes in levels of known NF‐κB regulated cytokines (TNFα, IL‐6, IL‐18 and IL‐1β) after treating TNBC cells with ONC201 or TRs using flow cytometry. Overall, this work provides valuable new insight into potential mechanisms by which ClpP activators mediate their anti‐cancer effects through immune signaling and recruitment.Support or Funding InformationL.J.A.‐C was supported by the NIH 5 T32 GM007040