Abstract 2720: Mitochondrial ClpP-mediated proteolysis induces selective cancer cell lethality
Authors: Ishizawa Jo , Zarabi Sarah F. , Davis R Eric , Halgas Ondrej , Nii Takenobu , Jitkova Yulia , Zhao Ran , St-Germain Jonathan , Heese Lauren E. , Egan Grace , Ruvolo Vivian R. , Barghout Samir H. , Nishida Yuki , Hurren Rose , Ma Wencai , Gronda Marcela , Link Todd , Wong Keith , Mabanglo Mark , Kojima Kensuke , Borthakur Gautam , MacLean Neil , Ma John Man Chun , Leber Andrew B. , Minden Mark D. , Houry Walid , Kantarjian Hagop , Stogniew Martin , Raught Brian , Pai Emil F. , Schimmer Aaron D. , Andreeff Michael 
Published: August 2019

Abstract
ClpP is a mitochondrial protease and a major protein quality control mediator that primarily interacts with metabolic enzymes in mitochondria. Here, we demonstrate that activation of this protease results in prominent anti-cancer activity, and propose ClpP activation as a novel therapeutic strategy for cancer and hematologic malignancies. We used genetic and chemical tools to activate ClpP. In a genetic approach, we tested the anti-cancer effects of ClpP activation by expressing a constitutively active ClpP mutant. Indeed, induction of the active ClpP mutant induced apoptosis in vitro and inhibited tumor progression in vivo. To further explore the antineoplastic effects of ClpP activation, we then performed a chemical screen of an in-house library of on-patent and off-patent drugs and identified imipridones (ONC201 and ONC212) as potent ClpP agonists. Imipridones are first-in-class antineoplastic agents and have shown preclinical efficacy in various malignancies in vitro and in vivo and are currently being evaluated in clinical trials in a diverse spectrum of cancers. Importantly, we and others have shown that their activity is agnostic to TP53 mutational status. Of note, molecular targets of imipridones that bind the drugs and are functionally important for their cytotoxicity have never been identified. Through extensive chemical investigations, including analysis of binding mechanism of the compounds to ClpP in cell free (ITC) and cell based assays (CETSA) as well as molecular analysis of the crystal structure, we demonstrate that these molecules bind ClpP non-covalently, and activate the protease by stabilizing the ClpP 14-mer, enlarging the axial pores of the complex, and inducing structural changes in the residues surrounding and including the catalytic triad. In leukemia, lymphoma and colon cancer cells including primary acute myeloid leukemia (AML) cells, both compounds displayed potent ClpP-dependent cytotoxicity with IC50s in low micro- or nanomolar ranges. Importantly, in primary AML samples, pretreatment ClpP levels correlated with response to imipridones. In lymphoma and AML xenograft models, both genetic and chemical activation of ClpP resulted in antitumor effects, while expression of inactive D190A ClpP mutant induced resistance. Mechanistically, ClpP activation leads to increased degradation of substrates of the enzyme including respiratory chain complex subunits and mitochondrial translation system. The resultant impaired mitochondrial structure and reduction in oxygen consumption is selectively cytotoxic to malignant cells that rely highly on mitochondrial energy production for their survival, whereas normal cells are not affected. In conclusion, ClpP activation is an entirely novel therapeutic strategy for malignant tumors. Our findings also suggest a general concept of inducing TP53-independent cancer cell lethality through activation of mitochondrial proteolysis.
Citation Format: Jo Ishizawa, Sarah F. Zarabi, R Eric Davis, Ondrej Halgas, Takenobu Nii, Yulia Jitkova, Ran Zhao, Jonathan St-Germain, Lauren E. Heese, Grace Egan, Vivian R. Ruvolo, Samir H. Barghout, Yuki Nishida, Rose Hurren, Wencai Ma, Marcela Gronda, Todd Link, Keith Wong, Mark Mabanglo, Kensuke Kojima, Gautam Borthakur, Neil MacLean, John Man Chun Ma, Andrew B. Leber, Mark D. Minden, Walid Houry, Hagop Kantarjian, Martin Stogniew, Brian Raught, Emil F. Pai, Aaron D. Schimmer, Michael Andreeff. Mitochondrial ClpP-mediated proteolysis induces selective cancer cell lethality [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2720.