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
Acute myeloid leukemia (AML) is an aggressive hematologic malignancy for which new therapeutic approaches are required. One such potential therapeutic strategy is to target the ubiquitin-like modifier-activating enzyme 1 (UBA1), the initiating enzyme in the ubiquitylation cascade in which proteins are tagged with ubiquitin moieties to regulate their degradation or function. Here, we evaluated TAK-243, a first-in-class UBA1 inhibitor, in preclinical models of AML. In AML cell lines and primary AML samples, TAK-243 induced cell death and inhibited clonogenic growth. In contrast, normal hematopoietic progenitor cells were more resistant. TAK-243 preferentially bound to UBA1 over the related E1 enzymes UBA2, UBA3, and UBA6 in intact AML cells. Inhibition of UBA1 with TAK-243 decreased levels of ubiquitylated proteins, increased markers of proteotoxic stress and DNA damage stress. In vivo, TAK-243 reduced leukemic burden and targeted leukemic stem cells without evidence of toxicity. Finally, we selected populations of AML cells resistant to TAK-243 and identified missense mutations in the adenylation domain of UBA1. Thus, our data demonstrate that TAK-243 targets AML cells and stem cells and support a clinical trial of TAK-243 in this patient population. Moreover, we provide insight into potential mechanisms of acquired resistance to UBA1 inhibitors.
Edited by Ursula Jakob Prions are infectious protein aggregates that cause several fatal neurodegenerative diseases. Prion research has been hindered by a lack of cellular paradigms for studying the replication of prions from different species. Although hamster prions have been widely used to study prion replication in animals and within in vitro amplification systems, they have proved challenging to propagate in cultured cells. Because the murine catecholaminergic cell line CAD5 is susceptible to a diverse range of mouse prion strains, we hypothesized that it might also be capable of propagating nonmouse prions. Here, using CRISPR/ Cas9-mediated genome engineering, we demonstrate that CAD5 cells lacking endogenous mouse PrP expression (CAD5-PrP ؊/؊ cells) can be chronically infected with hamster prions following stable expression of hamster PrP. When exposed to the 263K, HY, or 139H hamster prion strains, these cells stably propagated high levels of protease-resistant PrP. Hamster prion replication required absence of mouse PrP, and hamster PrP inhibited the propagation of mouse prions. Cellular homogenates from 263K-infected cells exhibited prion seeding activity in the RT-QuIC assay and were infectious to naïve cells expressing hamster PrP. Interestingly, murine N2a neuroblastoma cells ablated for endogenous PrP expression were susceptible to mouse prions, but not hamster prions upon expression of cognate PrP, suggesting that CAD5 cells either possess cellular factors that enhance or lack factors that restrict the diversity of prion strains that can be propagated. We conclude that transfected CAD5-PrP ؊/؊ cells may be a useful tool for assessing the biology of prion strains and dissecting the mechanism of prion replication.
At times, it can be difficult to discern if a lack of overlap in reported interactions for a protein-of-interest reflects differences in methodology or biology. In such instances, systematic analyses of protein-protein networks across diverse paradigms can provide valuable insights. Here, we interrogated the interactome of the prion protein (PrP), best known for its central role in prion diseases, in four mouse cell lines. Analyses made use of identical affinity capture and sample processing workflows. Negative controls were generated from PrP knockout lines of the respective cell models, and the relative levels of peptides were quantified using isobaric labels. The study uncovered 26 proteins that reside in proximity to PrP. All of these proteins are predicted to have access to the outer face of the plasma membrane, and approximately half of them were not reported to interact with PrP before. Strikingly, although several proteins exhibited profound co-enrichment with PrP in a given model, except for the neural cell adhesion molecule 1, no protein was highly enriched in all PrP-specific interactomes. However, Gene Ontology analyses revealed a shared association of the majority of PrP candidate interactors with cellular events at the intersection of transforming growth factor β and integrin signaling.
Trypanothione reductase (TR) plays a key role in the unique redox metabolism of trypanosomatids, the causative agents of human African trypanosomiasis (HAT), Chagas’ disease, and leishmaniases. Introduction of a new, lean propargylic vector to a known class of TR inhibitors resulted in the strongest reported competitive inhibitor of Trypanosoma (T.) brucei TR, with an inhibition constant Ki of 73 nm, which is fully selective against human glutathione reductase (hGR). The best ligands exhibited in vitro IC50 values (half‐maximal inhibitory concentration) against the HAT pathogen, T. brucei rhodesiense, in the mid‐nanomolar range, reaching down to 50 nm. X‐Ray co‐crystal structures confirmed the binding mode of the ligands and revealed the presence of a HEPES buffer molecule in the large active site. Extension of the propargylic vector, guided by structure‐based design, to replace the HEPES buffer molecule should give inhibitors with low nanomolar Ki and IC50 values for in vivo studies.
Dedicated to the memory of an exceptional scientist, Professor François Diederich l-2-Haloacid dehalogenases, industrially and environmentally important enzymes that catalyse cleavage of the carbonhalogen bond in S-2-halocarboxylic acids, were known to hydrolyse chlorinated, brominated and iodinated substrates but no activity towards fluorinated compounds had been reported. A screen for novel dehalogenase activities revealed four l-2haloacid dehalogenases capable of defluorination. We now report crystal structures for two of these enzymes, Bpro0530 and Rha0230, as well as for the related proteins PA0810 and RSc1362, which hydrolyse chloroacetate but not fluoroacetate, all at ~2.2 Å resolution. Overall structure and active sites of these enzymes are highly similar. In molecular dynamics (MD) calculations, only the defluorinating enzymes sample more compact conformations, which in turn allow more effective interactions with the small fluorine atom. Structural constraints, based on X-ray structures and MD calculations, correctly predict the defluorination activity of the homologous enzyme ST2570.
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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