Highlights d Activation of the mitochondrial ClpP induces p53independent cancer cell lethality d Imipridones are allosteric agonists of ClpP, being tested in human clinical trials d ClpP activation increases proteolysis of mitochondrial proteins d ClpP-mediated mitochondrial proteolysis impairs mitochondrial respiratory function

Imipridones constitute a novel class of anti-tumor agents. Here, we report that a second-generation imipridone, ONC212, possesses highly increased anti-tumor activity compared to the first-generation compound ONC201. In vitro studies using human acute myeloid leukemia (AML) cell lines, primary AML, and normal bone marrow (BM) samples demonstrate that ONC212 exerts prominent apoptogenic effects in AML, but not in normal BM cells, suggesting potential clinical utility. Imipridones putatively engage G protein-coupled receptors (GPCRs) and/or trigger an integrated stress response in hematopoietic tumor cells. Comprehensive GPCR screening identified ONC212 as activator of an orphan GPCR GPR132 and Gαq signaling, which functions as a tumor suppressor. Heterozygous knock-out of GPR132 decreased the anti-leukemic effects of ONC212. ONC212 induced apoptogenic effects through the induction of an integrated stress response, and reduced MCL-1 expression, a known resistance factor for BCL-2 inhibition by ABT-199. Oral administration of ONC212 inhibited AML growth in vivo and improved overall survival in xenografted mice. Moreover, ONC212 abrogated the engraftment capacity of patient-derived AML cells in an NSG PDX model, suggesting potential eradication of AML initiating cells, and was highly synergistic in combination with ABT-199. Collectively, our results suggest ONC212 as a novel therapeutic agent for AML.

Early clinical trials using murine double minute 2 (MDM2) inhibitors demonstrated proof-of-concept of p53-induced apoptosis by MDM2 inhibition in cancer cells; however, not all wild-type tumors are sensitive to MDM2 inhibition. Therefore, more potent inhibitors and biomarkers predictive of tumor sensitivity are needed. The novel MDM2 inhibitor DS-3032b is 10-fold more potent than the first-generation inhibitor nutlin-3a. mutations were predictive of resistance to DS-3032b, and allele frequencies of mutations were negatively correlated with sensitivity to DS-3032b. However, sensitivity to DS-3032b of wild-type tumors varied greatly. We thus used two methods to create predictive gene signatures. First, by comparing sensitivity to MDM2 inhibition with basal mRNA expression profiles in 240 cancer cell lines, a 175-gene signature was defined and validated in patient-derived tumor xenograft models and human acute myeloid leukemia (AML) cells. Second, an AML-specific 1,532-gene signature was defined by performing random forest analysis with cross-validation using gene expression profiles of 41 primary AML samples. The combination of mutation status with the two gene signatures provided the best positive predictive values (81% and 82%, compared with 62% for mutation status alone). In addition, the top-ranked 50 genes selected from the AML-specific 1,532-gene signature conserved high predictive performance, suggesting that a more feasible size of gene signature can be generated through this method for clinical implementation. Our model is being tested in ongoing clinical trials of MDM2 inhibitors. This study demonstrates that gene expression profiling combined with mutational status predicts antitumor effects of MDM2 inhibitors and .

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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.