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Novel, Selective Inhibitors of USP7 Uncover Multiple Mechanisms of Antitumor Activity in Vitro and in Vivo

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Novel, Selective Inhibitors of USP7 Uncover Multiple Mechanisms of Antitumor Activity in Vitro and in Vivo Author Manuscript Published OnlineFirst on August 11, 2020; DOI: 10.1158/1535-7163.MCT-20-0184 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Title Novel, Selective Inhibitors of USP7 Uncover Multiple Mechanisms of Antitumor Activity in Vitro and in Vivo Authors and affiliations Yamini M. Ohol1, Michael T. Sun1, Gene Cutler1, Paul R. Leger1, Dennis X. Hu1, Berenger Biannic1, Payal Rana1, Cynthia Cho1, Scott Jacobson1, Steve T. Wong1, Jerick Sanchez1, Niket Shah1, Deepa Pookot1, Betty Abraham1, Kyle Young1, Silpa Suthram1, Lisa A. Marshall1, Delia Bradford1, Nathan Kozon1, Xinping Han1, Akinori Okano1, Jack Maung1, Christophe Colas1, Jacob Schwarz1, David Wustrow1, Dirk G. Brockstedt1, Paul D. Kassner1 1 RAPT Therapeutics, Inc., South San Francisco, California, USA Running title Mechanisms of Antitumor Activity of Novel, Selective USP7 Inhibitors Corresponding author information Yamini M. Ohol: 561 Eccles Avenue, South San Francisco, CA 94080, USA; [email protected] Paul D. Kassner: 561 Eccles Avenue, South San Francisco, CA 94080, USA; [email protected] Conflict of interest statement All authors are current or former employees of RAPT Therapeutics, Inc., where this research was conducted. Keywords Drug targets, Drug mechanisms, Small molecule agents, USP7, p53 Abstract The deubiquitinase USP7 regulates the levels of multiple proteins with roles in cancer progression and immune response. USP7 inhibition may thus decrease oncogene 1 Downloaded from mct.aacrjournals.org on October 3, 2021. © 2020 American Association for Cancer Research. Author Manuscript Published OnlineFirst on August 11, 2020; DOI: 10.1158/1535-7163.MCT-20-0184 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. function, increase tumor suppressor function and sensitize tumors to DNA-damaging agents. We have discovered a novel chemical series that potently and selectively inhibits USP7 in biochemical and cellular assays. Our inhibitors reduce the viability of multiple TP53 wild-type cell lines, including several hematologic cancer and MYCN- amplified neuroblastoma cell lines, as well as a subset of TP53 mutant cell lines in vitro. Our work suggests that USP7 inhibitors upregulate transcription of genes normally silenced by the epigenetic repressor complex PRC2 and potentiate the activity of PIM and PI3K inhibitors as well as DNA-damaging agents. Further, oral administration of USP7 inhibitors inhibits MM.1S (multiple myeloma; TP53 wild-type) and H526 (small cell lung cancer; TP53 mutant) tumor growth in vivo. Our work confirms that USP7 is a promising, pharmacologically tractable target for the treatment of cancer. Introduction USP7 is a deubiquitinase that regulates the levels of several proteins with roles in cancer development and antitumor immunity. USP7 stabilizes MDM2, the oncogenic E3 ubiquitin ligase that promotes proteasomal degradation of the tumor suppressor p53 (encoded by TP53) (1). Genetic depletion or pharmacological inhibition of USP7 reduces cellular MDM2 levels and subsequently elevates levels of p53 (2-8). Small- molecule inhibitors of USP7 with varying potency and selectivity have been developed and are cytotoxic to TP53 wild-type tumor cells in vitro and in vivo (6-16). Multiple other substrates of USP7 have also been reported (17). USP7 has been reported to stabilize the pro-survival PIM2 kinase, the oncogenic protein MYCN, and the DNA methyltransferases DNMT1 and UHRF1 (8,18-20). USP7 may also promote the activity of the DNA methylation complex PRC1 (21). In addition, USP7 may stabilize multiple proteins involved in DNA damage repair, thus imparting resistance to DNA- damaging chemotherapy, PARP inhibitors, and radiotherapy (17,22-24). Via these mechanisms of action, cell lines of both wild-type and mutant TP53 status have been shown to be directly sensitive to chemical inhibition of USP7. In addition to these tumor intrinsic mechanisms, USP7 has also been reported to suppress immune responses in the tumor microenvironment by stabilizing the transcription factor Foxp3, which is essential for the development and function of regulatory T cells (Treg), or by stabilizing the Foxp3 activator Tip60, thereby enhancing the suppressive function of Treg (25-28). However, many of the USP7 inhibitors that have previously been used to explore USP7 biology have been reported to inhibit a wide range of other deubiquitinases and unrelated proteins (13-15,29), (Supplementary Table S7). Hence, the role of highly specific pharmacologic inhibition of USP7 and its impact on tumor growth is less well understood. In this report, we describe the discovery of a series of novel, potent and highly selective small-molecule inhibitors of USP7. These inhibitors bind non-covalently to USP7 and 2 Downloaded from mct.aacrjournals.org on October 3, 2021. © 2020 American Association for Cancer Research. Author Manuscript Published OnlineFirst on August 11, 2020; DOI: 10.1158/1535-7163.MCT-20-0184 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. impede binding of USP7 to ubiquitin, preventing cleavage of ubiquitinated substrates. We show that selective chemical inhibition of USP7 potently reduces growth of multiple TP53 wild-type and a subset of TP53 mutant cell lines in vitro. We demonstrate in vivo tumor growth inhibition in the TP53 wild-type MM.1S (multiple myeloma) and the TP53 mutant H526 (small cell lung cancer) xenograft models. We observe an elevation of p53 and p21 in USP7 inhibitor-treated MM.1S cells, indicating that p53-mediated growth arrest may contribute to growth inhibition. In the TP53 mutant H526 tumor cell line we observe synergy between USP7 inhibitors and DNA-damaging agents, as well as PIM and PI3 kinase inhibitors, suggesting novel approaches to combination therapies. USP7 inhibitor treatment leads to transcriptional changes that reflect suppression of the epigenetic repressor complex PRC2. Analysis of gene expression patterns in a 430- tumor cell line panel suggests that activation of PRC2, translation-related pathways and DNA damage repair pathways are markers of sensitivity to USP7 inhibition. USP7 is thus an attractive target for the treatment of both TP53 wild type and TP53 mutant tumors. Materials and Methods Compounds. USP7 inhibitors were synthesized as described (30). In that paper, USP7-443 is referred to as compound 14, USP7-866 as compound 30, and USP7-797 as compound 41. USP7-055 was synthesized in an analogous fashion to compound 37, while USP7-414 and USP7-877 were synthesized in an analogous fashion to compound 18, following the same general procedures as outlined in the paper. The USP7/USP7-443 co-crystal structure is accessible at the RCSB Protein Data Bank (code: 6VN6). Deubiquitinase biochemical assays. For the USP7 biochemical assay, a 25 µl reaction volume containing recombinant full-length USP7 (62 pM) in 20 mM HEPES pH 7.3, 150 mM NaCl, 1 mM TCEP, and 125 µg/ml BSA was assembled in wells of 384 well plates. Compounds were dispensed with a Hewlett Packard D300 digital dispenser (1% final DMSO). Following a 30-minute incubation at room temperature, ubiquitin- rhodamine (BostonBiochem) was added with the D300 to a final concentration of 100 nM and the reaction was allowed to proceed for 1 hour at room temperature protected from light. The reaction was stopped by the addition of 5 µl 1M acetic acid. Rhodamine fluorescence was measured using an Envision plate reader (Perkin Elmer) and IC50 values were determined by non-linear regression using a 4-parameter fit in the Dotmatics software package. USP47 biochemical assays were conducted using recombinant full-length USP47 (1 nM) and the same protocol. Deubiquitinase selectivity profiling was conducted by Ubiquigent (Dundee, UK) using a similar assay protocol with the following changes: assay buffer was 40 mM Tris/HCl pH 7.4, 5% glycerol, 0.005% Tween-20, 1 mM DTT, 0.05 mg/ml ovalbumin, and the reaction was stopped by the addition of 5 µl 100 mM N-Ethylmaleimide. 3 Downloaded from mct.aacrjournals.org on October 3, 2021. © 2020 American Association for Cancer Research. Author Manuscript Published OnlineFirst on August 11, 2020; DOI: 10.1158/1535-7163.MCT-20-0184 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. p53 cellular assay. RKO cells stably transfected with a p53 luciferase reporter vector (Signosis) were seeded at 2500 cells per well in 25µl of recommended media in 384- well black-walled tissue plates (Greiner). Compounds were added with a D300 digital dispenser (0.5% final DMSO). Following an 18-hour incubation, p53-dependent luciferase levels were measured via Bright-Glo Luciferase (Promega), following the manufacturer’s instructions, using a CLARIOstar plate reader (BMG LABTECH). IC50 values were determined by non-linear regression using a 4-parameter fit in the Dotmatics software package. Target engagement assay. Cells were seeded at 1 million per well in recommended media and treated for 4 hours with compounds added with a D300 digital dispenser. Cells were lysed in IP Lysis Buffer (Pierce) and lysates were quantified using the BCA assay (Pierce). 20 µg lysate was treated with 2 µM Ubiquitin-propargylamide (Ub-PA) probe (UbiQ-057) for 15 minutes at room temperature, following which probe activity was stopped by the addition of 1× LDS Sample Buffer (ThermoFisher). Samples were heated at 70C for 10 minutes, separated by SDS-PAGE, and analyzed by Western blotting using anti-USP7 (Millipore 05-1946, 1:2000), anti--actin (Invitrogen MA515739, 1:5000), and goat anti-mouse HRP (Invitrogen 31430, 1:5000) antibodies. Cell culture, cell treatments, and viability assays. CHP-134 was purchased from Sigma Aldrich, MOLM-13 was purchased from AddexBio, and NB-1 was purchased from Sekisui XenoTech. All other cell lines were purchased from American Type Culture Collection. For all cell lines, short tandem repeat profiling and Mycoplasma testing were conducted (IDEXX Bioresearch, August 6, 2018), culturing was performed in recommended growth medium, and passages 2-10 from thawing were used for experiments.
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