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
Previous studies have suggested that statins can be repurposed for cancer treatment. However, the therapeutic efficacy of statins in chronic myeloid leukemia (CML) has not yet been demonstrated. In this study, we retrospectively evaluated the outcomes of 408 CML patients who underwent imatinib therapy. The deep molecular response rates in patients treated with the statin/TKI combination were significantly higher than those in patients treated with TKI alone (p = 0.0016). The statin/TKI combination exerted potent cytotoxic effects against wild-type and ABL1 mutant CML, BaF3, and K562/T315I mutant cells. Furthermore, the statin/TKI combination additively inhibited the colony-forming capacity of murine CML-KLS+ cells in vitro. In addition, we examined the additive growth-inhibitory effects of the statin/tyrosine kinase inhibitor (TKI) combination against CML patient-derived CD34+ cells. The growth-inhibitory effects of the statin/imatinib combination against CD34+/CML primary cells were higher than those against CD34+/Norm cells (p = 0.005), suggesting that the combination of rosuvastatin and imatinib exerted growth-inhibitory effects against CML CD34+ cells, but not against normal CD34+ cells. Furthermore, results from RNA sequencing of control and statin-treated cells suggested that statins inhibited c-Myc-mediated and hematopoietic cell differentiation pathways. Thus, statins can be potentially repurposed to improve treatment outcomes in CML patients when combined with TKI therapy.
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.
Cellular stress response has dual aspects; cell-protective or lethal. Mitochondria have their unique organellar response termed "mitochondrial unfolded protein response (UPRmt)" induced by damaged mitochondrial (mt) matrix proteins. While recent discoveries have successfully targeted BCL2, a regulator of mt integrity in acute myeloid leukemia (AML), the significance of UPRmt is unknown. We hypothesized that priming UPRmt towards cell death would be a novel therapeutic strategy for AML. UPRmt is generally induced by dysregulation of mt protein pools. Therefore, to test if UPRmt signaling is also operational in AML cells, we selected classical or putative UPRmt inducers; the mt translation inhibitors tetracycline and tigecycline, the mt protein transport inhibitor MitoBlock6, and the mtDNA damaging agent ethidium bromide. In OCI-AML3 and HL60 cells, these agents indeed induced the transcription factor ATF5, which was reported as a central inducer of UPRmt, and its targets (e.g., LonP, HSPA9), triggering apoptosis in AML cells. In addition, we here report imipridones (ONC201 and ONC212), the activators of mt protein degradation, as novel UPRmt inducers. We recently reported that imipridones non-covalently bind the mt protease ClpP and allosterically activate it. They induced prominent apoptosis in primary AML progenitor and leukemia initiating cells (LICs) in vitro and in vivo, but not in normal bone marrow cells, following "mitochondrial proteolysis" with reduction of selective mt matrix proteins (e.g., SDHB, NDUFA12) and resultant inhibition of oxidative phosphorylation (Oxphos) (Ishizawa, Zarabi et al, Cancer Cell 2019). We then postulated that dysregulation of mt protein pools by mitochondrial proteolysis can also induce UPRmt. Indeed, our gene expression profiles of ONC201-treated Z138 and Jeko-1 cells were highly enriched for previously published UPRmt gene signatures, and UPRmt effectors were induced also in AML cells. Of potentially high clinical significance is the finding of synergistic anti-leukemia effects of imipridones when combined with the selective BCL2 inhibitor venetoclax, in vitro and in vivo (Ishizawa et al. Science Signaling 2016, and Nii et al. Blood 2019). However, its underlying molecular mechanism is unclear. Since BCL2 is reported to be induced by UPRmt, we hypothesized that BCL2 is critical for the ClpP-mediated UPRmt to have the cell protective effects, contrary to lethal effects, as dual aspects of stress response. We utilized the tetracycline-inducible system of an activated mutant (Y118A) form of ClpP in OCI-AML3 cells, and demonstrated that venetoclax treatment sensitizes OCI-AML3 cells to genetic activation of ClpP towards apoptosis. Furthermore, other UPRmt inducers (tetracycline, tigecycline, and MitoBlock6) in combination with venetoclax also synergistically induced apoptosis in AML cells, suggesting that BCL2 inhibition and UPRmt induction generally exerts synergistic anti-leukemia effects. We next focused on the enhanced effect observed for the combination of imipridones with venetoclax as compared to other UPRmt inducers, searching for other targets that could further enhance the synergy. We then hypothesized that the synergism between ClpP activation and BCL2 inhibition involves SDHB, a respiratory chain complex II subunit degraded by activated ClpP but not targeted by any of other UPRmt inducers. Consistently, SDHB knockdown sensitized OCI-AML3 cells to venetoclax-induced apoptosis, indicating that SDHB reduction and UPRmt by ClpP activation concomitantly enhance the cell lethality by BCL2 inhibition. Collectively, UPRmt is a new potential therapeutic target for AML, which significantly enhances the cell death effects of BCL2 inhibition on AML cells. In particular, ClpP activation induces UPRmt and, concomitantly, downregulates SDHB, thus targeting the respiratory chain complex II, which results in improved synergistic leukemia cell apoptosis when combined with BCL2 inhibition. Oxphos is also a hallmark of drug resistant AML stem cells, which supports the notion that Oxphos inhibition by this combination targets LICs. Based on promising preclinical anti-tumor efficacy, ONC201 as a single agent is being evaluated in early phase clinical trials, showing clinical responses in AML and midline gliomas. A clinical trial testing the combinatorial strategy of targeting ClpP and Bcl-2 is under development. Disclosures Borthakur: Novartis: Research Funding; NKarta: Consultancy; Eisai: Research Funding; Oncoceutics: Research Funding; BioLine Rx: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Cyclacel: Research Funding; Strategia Therapeutics: Research Funding; Eli Lilly and Co.: Research Funding; Arvinas: Research Funding; Merck: Research Funding; AstraZeneca: Research Funding; PTC Therapeutics: Consultancy; Agensys: Research Funding; Argenx: Membership on an entity's Board of Directors or advisory committees; FTC Therapeutics: Membership on an entity's Board of Directors or advisory committees; GSK: Research Funding; Incyte: Research Funding; Janssen: Research Funding; AbbVie: Research Funding; BMS: Research Funding; Oncoceutics, Inc.: Research Funding; Bayer Healthcare AG: Research Funding; BioTheryX: Membership on an entity's Board of Directors or advisory committees; Tetralogic Pharmaceuticals: Research Funding; Cantargia AB: Research Funding; Polaris: Research Funding; Xbiotech USA: Research Funding. Stogniew:Oncoceutics, Inc.: Employment. Oster:Oncoceutics, Inc.: Employment. Kantarjian:BMS: Research Funding; AbbVie: Honoraria, Research Funding; Ariad: Research Funding; Amgen: Honoraria, Research Funding; Jazz Pharma: Research Funding; Pfizer: Honoraria, Research Funding; Cyclacel: Research Funding; Immunogen: Research Funding; Agios: Honoraria, Research Funding; Actinium: Honoraria, Membership on an entity's Board of Directors or advisory committees; Novartis: Research Funding; Takeda: Honoraria; Astex: Research Funding; Daiichi-Sankyo: Research Funding. Schimmer:Novartis Pharmaceuticals: Consultancy; Medivir Pharmaceuticals: Research Funding; Jazz Pharmaceuticals: Consultancy; Otsuka Pharmaceuticals: Consultancy. Andreeff:Eutropics: Equity Ownership; Daiichi Sankyo, Inc.: Consultancy, Patents & Royalties: Patents licensed, royalty bearing, Research Funding; Jazz Pharmaceuticals: Consultancy; Celgene: Consultancy; Aptose: Equity Ownership; Reata: Equity Ownership; 6 Dimensions Capital: Consultancy; AstaZeneca: Consultancy; Amgen: Consultancy; Breast Cancer Research Foundation: Research Funding; CPRIT: Research Funding; NIH/NCI: Research Funding; Center for Drug Research & Development: Membership on an entity's Board of Directors or advisory committees; Cancer UK: Membership on an entity's Board of Directors or advisory committees; NCI-CTEP: Membership on an entity's Board of Directors or advisory committees; German Research Council: Membership on an entity's Board of Directors or advisory committees; Leukemia Lymphoma Society: Membership on an entity's Board of Directors or advisory committees; NCI-RDCRN (Rare Disease Cliln Network): Membership on an entity's Board of Directors or advisory committees; CLL Foundation: Membership on an entity's Board of Directors or advisory committees; BiolineRx: Membership on an entity's Board of Directors or advisory committees; Oncolyze: Equity Ownership; Oncoceutics: Equity Ownership; Senti Bio: Equity Ownership, Membership on an entity's Board of Directors or advisory committees. Ishizawa:Daiichi Sankyo: Patents & Royalties: Joint submission with Daiichi Sankyo for a PTC patent titled "Predictive Gene Signature in Acute Myeloid Leukemia for Therapy with the MDM2 Inhibitor DS-3032b," United States, 62/245667, 10/23/2015, Filed.
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