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

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

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.

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 70C 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. For studies evaluating cellular effects of compound treatment, 250 to 2,000 cells in 40 µl of recommended medium were seeded per well in 384-well plates (Corning 3764). DMSO-solubilized compounds were added in duplicate in a two-fold dilution series using a D300e Digital Dispenser (Hewlett-Packard). Following a 5-day incubation, cell viability was measured using CellTiter-Glo (Promega) following the manufacturer’s instructions. Luminescence was measured using a CLARIOstar plate reader (BMG LABTECH) and normalized to that of DMSO-treated cells. CC50 values were determined by non-linear regression using a 4-parameter fit in Prism (GraphPad Software). Inhibitor profiling of the 430-cell line panel was conducted and analyzed similarly at Crown Bio (Beijing, China). RNASeq analysis. RNA was extracted from approximately 1 million H526 cells treated with DMSO or 0.5 µM USP7-797 using the Allprep DNA/RNA Mini Kit (Qiagen). Whole transcriptome non-stranded libraries were prepared using the TruSeq RNA Library Prep Kit v2 (Illumina). Sequencing was performed on Illumina NovaSeq using paired-end mode, 200 cycles, targeting 40 million reads per sample, by MedGenome. Sequencing results were processed on the Seven Bridges Genomics platform through Kallisto Quant (version 0.43.1) aligning to the gencode.v27 transcript reference. Aligned reads were imported by tximport (version 1.10.1) and Treatment fold change and significance were

calculated with DESeq2 (version 1.22.2) with an experimental design of “~ Time + Treatment + Time:Treatment” on genes with at least 10 counts. Shrunk log2 fold changes (lfcShrink function, type=”apeglm”) were used for significance calculation. For gene set enrichment analysis, genes with at least 2-fold up or down change and adjusted p < 0.01 were considered significantly differentially expressed. Gene sets were downloaded from MSigDB v6.2 (http://software.broadinstitute.org/gsea/msigdb) and those with at least 4 overlapping genes with the significantly regulated genes were used for hypergeometric test calculation. The RNASeq data is accessible at NCBI Gene Expression Omnibus (GEO): accession number GSE151286. Statistical analysis. Cancer Cell Line Encyclopedia (CCLE) mutation data was downloaded from https://data.broadinstitute.org/ccle/CCLE_DepMap_18Q2_maf_20180502.txt.gz. Genes were considered mutated if they were annotated in CCLE as containing variants that were either a TCGA Hotspot, a COSMIC Hotspot, or a deleterious variant. Genes with at least 6 mutated cell lines out of the 397 cell lines that overlapped with the cell line screening panel were tested one gene at a time in linear models or coordinately in multivariate models where the fraction cell signal at 1 µM compound was the dependent variable. For compound synergy analysis, a Bliss-independence model was used. Briefly, the products of the fraction of maximal cell signal for each of the tested compounds alone were calculated as the expected signal in the absence of any compound interaction. The log2 of the ratio of expected:observed signal was calculated to indicate synergy (positive) or interference (negative) interactions. For in vivo xenograft studies, a two-way ANOVA with repeated measures was performed for statistical analysis of differences in tumor volumes between treatment groups. Western blotting, caspase activation, propidium iodide, -H2AX and phospho-S6 assays. For Western blotting of cell lines, compound-treated cells were lysed in RIPA lysis buffer (ThermoScientific) containing Halt Protease and Phosphatase Inhibitor Cocktail (Life Technologies). For Western blotting of tumors, tumors were harvested 24 hours post treatment, homogenized under liquid nitrogen and lysed in a buffer containing 150 mM NaCl, 20 mM Tris, pH 7.5, 1 mM EDTA, 1 mM EGTA, 1% Triton X- 100, 1% NP-40, and Halt Protease and Phosphatase Inhibitor Cocktail (Life Technologies). Lysates were quantified using the BCA protein assay (Pierce) and equivalent protein was separated by SDS-PAGE (4-12%, ThermoScientific). Following electrophoresis, proteins were transferred to PVDF membranes. Membranes were blocked with 5% milk or BSA in TBS + 0.05% Tween-20 (TBST) and subjected to Western blot analysis using the following primary antibodies, diluted in blocking solution and incubated overnight at 4C: GAPDH (14C10, Cell Signaling, 1:1000), p53 (DO1, Santa Cruz, 1:200), p21 (12D1, Cell Signaling, 1:500), UHRF1 (H-8, Santa Cruz, 1:100), MYCN (D4B2Y, Cell Signaling, 1:1000), -Actin (13E5, Cell Signaling, 1:10,000). Blots of cell line samples were then incubated for 1 hour at room temperature in secondary antibodies, diluted 1:10,000, and imaged using SuperSignal West chemiluminescent reagents (Life Technologies). Blots of tumor samples were

incubated for 1 hour at room temperature in IR dye-labeled secondary antibodies (Li- Cor), diluted 1:15,000, and imaged using Odyssey CLx (Li-Cor). Band intensity was quantitated using Image Studio Lite (Li-Cor). Caspase activation assays were performed on compound-treated cells using the Caspase-Glo 3/7 Assay Kit (Promega) according to the manufacturer’s protocol, and luminescence was measured using a CLARIOstar plate reader (BMG LABTECH). Propidium iodide staining was performed on compound-treated cels using FxCycle™ PI/RNase Staining Solution (ThermoFisher) according to the manufacturer’s protocol, and data were collected using the Attune (Thermo Fisher Scientific) flow cytometer and analyzed with FlowJo software (FlowJo LLC). For -H2AX measurement, compound-treated cells were harvested, fixed and stained using the H2A.X Phosphorylation Assay Kit (Sigma) according to the manufacturer’s protocol. Data were collected using FACSCanto (Becton Dickinson) and Attune (Thermo Fisher Scientific) flow cytometers and analyzed with FlowJo software (FlowJo LLC). For phospho-S6 ELISA assays, compound-treated cells were lysed in PathScan Sandwich ELISA Lysis Buffer (Cell Signaling) containing Halt Protease and Phosphatase Inhibitor Cocktail (Life Technologies). Lysates were quantified using the BCA protein assay (Pierce) and equivalent protein was used in the PathScan Phospho- S6 Ribosomal Protein (Ser235/236) Sandwich ELISA Kit (Cell Signaling) according to the manufacturer’s protocol. Absorbance was measured using the Spectramax 300 plate reader (Molecular Devices). Animal experiments. MM.1S and H526 xenograft studies were conducted at Crown Bio (Beijing, China) and RAPT Therapeutics, Inc., respectively, according to the guidelines approved by the respective Institutional Animal Care and Use Committees (IACUC), protocol numbers 001243 (Crown Bio) and FL0002 (RAPT). MM.1S cells were inoculated in PBS into irradiated female NOD/SCID mice (Beijing Anikeeper Biotech, Beijing, China), while H526 cells were inoculated in PBS/Matrigel (Corning) into female Nu/Nu mice (Jackson Laboratories). At the start of the study, NOD/SCID mice were 9-10 weeks old and 17.2-22.3 g in weight, while Nu/Nu mice were 7 weeks old and 18.1-25.4 g in weight. Mice were randomized into groups when mean tumor size reached 150 mm3 (MM.1S) or 50-100 mm3 (H526), respectively, and drug administration by oral gavage was started on day of randomization. Tumor volumes and body weights were subsequently measured twice per week. Tumor volumes were calculated using the formula: V = 0.5(A x B2), where A and B are the long and short diameters of the tumor, respectively.

Results

Discovery of potent and selective USP7 inhibitors. We employed structure-based drug design and other medicinal chemistry techniques to enable the design of novel potent and selective USP7 inhibitors. To guide simultaneous optimization of inhibitor potency and selectivity we developed a high-throughput screening cascade. Inhibitor potency was evaluated using a biochemical assay of USP7 activity employing

rhodamine-labeled ubiquitin, and a cellular activity assay using a p53 response element-driven luciferase reporter engineered into the TP53 wild-type RKO cell line. Inhibitor selectivity was guided by counter-screening in a similar biochemical assay measuring activity of the most closely structurally related deubiquitinase, USP47. A strong correlation was observed between p53 EC50 values and USP7 IC50 values, suggesting that p53 elevation was occurring due to USP7 inhibitor-mediated MDM2 destabilization rather than non-specific cellular cytotoxicity (Supplementary Figure S1A). In addition, we measured cytotoxicity in the RKO cell line and eliminated compounds that sharply reduced ATP levels, suggestive of physicochemical toxicity. Using this cascade, we optimized several series of USP7 inhibitors to sub-nM biochemical potency, sub-100 nM cellular potency, and >5,000-fold selectivity over USP47. Representative compounds used in this paper are listed in Table 1; chemical structures are shown in Figure 1A and Supplementary Figure S1B and are discussed in more detail elsewhere (30). We further tested the biochemical activity of an early tool compound, USP7-443, in a panel of 41 deubiquitinases and demonstrated that while it inhibited USP7 at an IC50 of 9 nM, it was inactive against all other deubiquitinases at up to 50 µM, a >5,000-fold selectivity (Figure 1C and Supplementary Table S1). The USP7 catalytic domain forms a hand-like structure with thumb, fingers and palm sub-domains, and ubiquitin binding induces a change from an inactive conformation to an active conformation in which the catalytic triad residues are aligned for catalysis (31). X-ray diffraction analysis of a USP7/USP7-443 co-crystal structure showed binding of the inhibitor to the allosteric site of the palm region, distal to the catalytic cysteine-223 (Figure 1B). USP7-443 represents a novel pharmacophore and binds to a similar pocket as the previously described 4-hydroxy-piperidine USP7 inhibitors, forming some of the same contacts in addition to some new ones (7,11,12). A detailed description of the binding of these novel inhibitors to USP7 is provided elsewhere (30). Our inhibitors, like other reported allosteric USP7 inhibitors, hinder binding of ubiquitin, preventing transition of the USP7 catalytic domain into its active conformation and resulting in a misaligned catalytic triad that prevents enzymatic ubiquitination (7,8,10,11). The USP7/USP7-443 co-crystal structure enabled structure-guided development of the more potent in vitro tool compound USP7-866, the active and inactive enantiomer pair USP7- 414 and USP7-777, and the in vivo tool compounds USP7-055 and USP7-797 (Table 1 and Supplementary Figure S1B). As predicted by the binding mode, experiments performed on USP7-797 and other closely related compounds indicated that binding was reversible (Supplementary Figure S1C). This contrasts with published nitro- thiophene-containing compounds which irreversibly bind to catalytic cysteine-223 (9,14,32). Notably, these covalent inhibitors are not selective against USP47, the closest homologue to USP7 (14,15), whereas USP7-443 is highly selective against USP47 and other deubiquitinases (Supplementary Table S1). Computational docking studies with a docking grid generated using the USP7/USP7-443 co-crystal structure showed that all the USP7 inhibitors used in this study bind in the same allosteric pocket of USP7 and in the same manner (Supplementary Figure S2). Given their similarities

in binding and structure (Supplementary Figure S1B), they are expected to have the same mode of action and similarly high selectivity for USP7 over other deubiquitinases.

USP7 inhibitors inhibit growth of TP53 wild-type tumor cell lines in vitro and in vivo. Since multiple pro-tumor functions of USP7 have been reported (7-13,18,22-24), we assessed the inhibitory effects of our potent and selective USP7 inhibitor USP7-866 on cell growth of a panel of 430 cancer cell lines. Using gene annotation data from the Cancer Cell Line Encyclopedia (CCLE) (33) to identify mutations that were significantly associated with USP7 inhibitor sensitivity, we found that TP53 mutational status was most significant, with wild-type TP53 status associated with greater sensitivity to USP7 inhibition (Figure 2A and Supplementary Table S2). As a comparison we used idasanutlin (RG7388), which elevates p53 by inhibiting the interaction between p53 and its E3 ubiquitin ligase MDM2 (34), and observed a similar but more significant association of drug sensitivity with wild-type TP53 status (Supplementary Figure S3A). Hematologic cancers, which are predominantly TP53 wild-type, seemed to be particularly sensitive to both USP7 inhibitor and idasanutlin (Supplementary Figure S3B, C). Following up on this observation, we tested the effect of our specific USP7 inhibitors on viability of the TP53 wild-type multiple myeloma cell line, MM.1S. Treatment of MM.1S for 5 days with the USP7 inhibitors USP7-866, -055 and -797 significantly inhibited viability in vitro with a CC50 of 100 nM as determined by the CellTiter-Glo assay (Figure 2B and Table 1). Testing of the paired active and inactive enantiomers, USP7-414 and USP7-777, suggested that the inhibitory effect was on- target (Figure 2B and Table 1). USP7-866 was able to engage USP7 and prevent its binding to and conjugation with the active-site probe, ubiquitin propargylamide (Ub-PA) (Figure 2C). USP7 inhibition increased levels of p53 and p21, whose transcription is dependent on p53 induction (Figure 2D). USP7-866, -055 and -797, which are structurally very similar, showed similar activities across several assays in vitro (Supplementary Figures S1B and S2, Tables 1, 2 and 3). However, USP7-055 and USP7-797 had significantly improved in vivo pharmacokinetic properties over USP7-866 and the enantiomeric pair, USP7-414 and USP7-777, and were therefore used for in vivo studies. We assessed the effects of USP7 inhibition in vivo in the MM.1S xenograft model in NOD-SCID mice via daily oral dosing of USP7-055 at 50 mg/kg and 100 mg/kg, and twice-daily oral dosing of USP7-797 at 50 mg/kg. These doses were well tolerated in preliminary experiments in tumor-free NOD-SCID mice (Supplementary Figure S4A, B). In the longer-term xenograft study in tumor-bearing mice we observed dose-dependent body weight loss in a fraction of mice. However, brief dosing holidays allowed the mice to recover and dosing to continue. USP7-055 effectively inhibited MM.1S tumor growth and prolonged survival in a dose-dependent manner, and USP7- 797 was highly efficacious as well (Figure 2E). Similar to results in vitro, USP7 inhibition resulted in an increase in p21 levels in tumors (Supplementary Figure S4C) suggesting that this reduction in tumor growth was mediated at least in part through activation of the p53 pathway. USP7 inhibition also reduced the viability of several

additional TP53 wild-type blood cancer cell lines, including M07e, OCI-AML5 and MOLM13, in vitro (Table 2). Since USP7 has been reported to stabilize MYCN (18), and since MYCN amplification is an established driver mutation in neuroblastoma, we also tested the effect of USP7 inhibition on a panel of TP53 wild-type, MYCN- or MYC-amplified neuroblastoma cell lines. We found that our USP7 inhibitors reduced viability of all three MYCN-amplified and two MYC-amplified neuroblastoma cell lines tested (Table 3). We used idasanutlin as a positive control, and as expected these cell lines were also sensitive to this compound (Tables 2 and 3). It is possible that MYCN amplification and wild-type TP53 status both contribute to USP7 inhibitor sensitivity.

USP7 inhibitors inhibit growth of a subset of TP53 mutant tumor cell lines in vitro and in vivo. In addition to the many TP53 wild-type cell lines in the cell line panel that were sensitive to USP7 inhibition, we found a subset of TP53 mutant cell lines that were inhibited by our USP7 inhibitors (Table 4). These included two MYCN-amplified neuroblastoma cell lines and one MYCN-amplified small cell lung cancer cell line, along with other cell lines of differing genetic status. We further evaluated the effect of pharmacological inhibition of USP7 on viability of the TP53 mutant, MYCN-amplified small cell lung cancer cell line, H526. The USP7 inhibitors USP7-055 and USP7-797 significantly inhibited viability of H526 in vitro with a CC50 of 500 nM using the CellTiter- Glo assay. Testing of paired active and inactive enantiomers indicated that this effect was on-target (Figure 3A and Table 1). In contrast, idasanutlin, whose inhibitory effect is dependent on a functional p53 pathway, had no effect (Figure 3A). USP7-797 did not induce any change in p21 level after 2, 8 or 24 hours of treatment, confirming that the p53 pathway is non-functional in this cell line. Similarly, USP7 inhibition did not reduce MYCN protein levels after 2, 8 or 24 hours of treatment, indicating that although MYCN is amplified in H526 cells, the killing of these cells by USP7 inhibitor was not due to destabilization of MYCN. In vivo, USP7-797 inhibited tumor growth and prolonged survival in the H526 xenograft model in athymic nude mice, when administered via well- tolerated twice-daily oral dosing at 50 mg/kg (Figure 3B). Since multiple reports point to a role for USP7 in stabilizing proteins involved in DNA damage repair, we tested whether USP7-797 could potentiate the activity of the DNA- damaging agents commonly used for SCLC chemotherapy, carboplatin and doxorubicin, or the PARP inhibitor olaparib. We found that USP7-797 potentiates the activity of these agents in H526 cells and, as expected, does not synergize with a second USP7 inhibitor, USP7-866 (Figure 3C and Supplementary Figure S5A). The level of phosphorylated histone H2AX (-H2AX), a marker of DNA damage (35), was increased to a much greater extent in cells treated with a combination of USP7-797 and carboplatin compared to carboplatin alone, and only slightly increased by USP7-797 alone, suggesting that USP7 inhibition may hinder the repair of DNA damage induced by carboplatin (Supplementary Figure S5B).

USP7 inhibitors have also been shown to potentiate the activity of proviral integration site for moloney murine leukemia virus (PIM) kinase and phosphoinositide-3 kinase (PI3K) inhibitors in the TP53 wild-type EOL-1 AML cell line (8), so we investigated the effect of combining USP7-797 with the PIM kinase inhibitor AZD1208 or the PI3K inhibitors pictilisib or taselisib in the TP53 mutant H526 cell line. We found that USP7- 797 strongly synergized with AZD1208, pictilisib and taselisib, suggesting that USP7 functions in a related pathway in these cells (Figure 3D and Supplementary Figure S5C). As expected, no synergy was observed when USP7-797 was dosed with itself. Both PIM and PI3K promote the activity of mammalian target of rapamycin (mTOR), which regulates cellular protein translation and controls phosphorylation of S6 at the Ser235/236 site (36-39). Treatment with USP7-797 alone decreased levels of phosphorylated S6 (Ser235/236) suggesting that USP7 inhibition might directly affect this pathway (36), (Supplementary Figure S5D). In order to identify pathways impacted by USP7 inhibition, we used RNASeq to profile transcriptomic changes in H526 cells following treatment with USP7-797 for 24 or 48 hours. These time points were informed by our observation that USP7-797 induced apoptosis in H526 cells in a dose-dependent manner starting 72 hours post treatment, as measured by caspase-3/7 induction (Supplementary Figure S6A) and an increase in the sub-G1 population upon propidium iodide labeling (Supplementary Figure S6B). Based on target engagement experiments showing that our inhibitors fully engage USP7 by 4 hours of treatment (Figure 2C)), we reasoned that reduction in USP7 substrate protein levels would lead to changes in downstream gene expression at the later time points of 24 and 48 hours. Measurement of transcriptional changes prior to 72 hours should thus uncover programs triggered by USP7 inhibition without being confounded by apoptosis pathways themselves. USP7 inhibitor treatment resulted in upregulation of a large number of genes normally silenced by the polycomb repressive complex 2 (PRC2) (Table 5, Supplementary Figure S6C, and Supplementary Tables S3, S4). PRC2, whose components include the proteins EZH2, SUZ12 and EED, trimethylates histone H3 at lysine 27 (H3K27me3) (40). Gene set enrichment analysis showed that the top 10 significantly enriched gene sets are all regulated by this pathway (Table 5). To more broadly discover pathways activated in USP7 inhibitor-sensitive cell lines, we analyzed the data from our USP7-866-treated 430-cancer cell line panel using gene expression data from CCLE (33) to identify repressed or overexpressed gene sets that were significantly associated with sensitivity to USP7 inhibition. We found that PRC2- regulated gene sets were generally significantly lower expressed in USP7 inhibitor- sensitive cell lines (Supplementary Table S5) although the differential regulation of some genes in related gene sets (eg. EZH2 and SUZ12) complicates the analysis. In addition, gene sets belonging to DNA damage repair and translation-related pathways were significantly overexpressed in USP7 inhibitor-sensitive cell lines (Supplementary Table S6). These repression and overexpression patterns occurred in sensitive cell lines of both TP53 wild-type and mutant status (Supplementary Tables S5 and S6).

Interestingly, the top 4 significantly overexpressed gene sets in TP53 mutant USP7 inhibitor-sensitive cell lines belong to DNA damage repair pathways.

Discussion USP7 has attracted much attention as a novel target for the treatment of cancer. USP7 has been reported to function in several pro-tumor pathways by indirectly reducing the levels of the tumor suppressor protein p53, protecting oncogenes and DNA damage repair proteins from proteasomal degradation, and promoting the function of regulatory T cells. While the role of USP7 in limiting p53 levels is well established, the dearth of potent and specific USP7 inhibitors with drug-like in vivo pharmacokinetic properties has hindered further exploration and validation of the other reported roles of USP7 in tumors of differing TP53 status (Supplementary Table S7). Many previous reports utilized USP7 inhibitors that have been reported to inhibit a wide range of other deubiquitinases including USP1, USP2, USP4, USP5, USP6, USP9x, USP10, USP15, USP16, USP20, USP21, USP25, USP28, USP47, OTUB2, OTUD5, A20, vOTU and VCPIP, in addition to the unrelated protein MMP13 (13-15,29). Here we report the discovery of novel, potent, highly selective, reversible, and orally bioavailable small-molecule inhibitors of USP7 with sub-nanomolar biochemical potency and >5,000-fold selectivity for USP7 over its closest homologue, USP47, and all 40 other deubiquitinases profiled. This high potency and selectivity allowed us to investigate the role of USP7 in cellular pathways important for tumor growth. An assessment of the cytotoxicity of our USP7 inhibitor in a large cell line panel showed that TP53 status is an important predictor of USP7i activity, validating the role of USP7 in the p53 pathway. We also confirmed previous reports that inhibition of USP7 is highly deleterious to leukemia cell lines (7-9). Our reversible, USP7-specific inhibitors were more potent against MM.1S cells in vitro than an irreversible, USP7/USP47- targeting inhibitor (CC50 of 100 nM vs. 6-14 µM) (9) and significantly reduced tumor growth in vivo. USP7 inhibition also inhibited the viability of several additional hematologic cancer cell lines and neuroblastoma cell lines with both MYCN and MYC amplifications, genetic modifications that are associated with worse prognoses (41,42). Our mouse xenograft experiments demonstrated that oral delivery of our USP7 inhibitors impacted tumor growth in vivo at generally well-tolerated doses. In cases where short dosing holidays were given, the rapid recovery of body weight indicated the reversibility of these short-term adverse effects. USP7 modulates the levels of proteins other than MDM2, and previous studies have shown that USP7 inhibition can also reduce the viability of TP53 mutant cell lines (4,5,17,18,22-24). USP7 has been reported to stabilize MYCN, a driver of some aggressive neuroblastomas; DNMT1 and UHRF1, which maintain DNA methylation, as well as proteins involved in DNA damage repair, both pathways that are often dysregulated in cancers (18-20, 22-24). For example, USP7 inhibition disrupts DNA

damage repair in TP53 mutant chronic lymphocytic leukemia via destabilization of Rad18 (22), and in TP53 mutant lung neuroendocrine tumor cells via destabilization of CCDC6 (23). Since p53 is inactivated in over 90% of small cell lung cancers (SCLC), an aggressive tumor type with poor prognosis and limited treatment options, we investigated the effect of USP7 inhibition in the SCLC cell line H526 (43,44). The p53 pathway is expected to be non-functional in H526 cells, as one TP53 allele is deleted and the other lost by a splice site mutation (33). Our reversible, USP7-specific inhibitor reduced viability of H526 cells with a lower CC50 than previously reported with an irreversible, USP7/USP47-targeting inhibitor (500 nM vs. 7.41 µM) (23) and significantly reduced tumor growth in vivo. USP7 inhibition strongly synergized with inhibition of proviral integration site for moloney murine leukemia virus (PIM) kinase and phosphoinositide-3 kinase (PI3K). Both PIM and PI3K promote the activity of mTOR, a master regulator of cellular protein translation that is often hyper-activated in cancers, via PRAS40 and TSC2 phosphorylation (36-39). mTOR regulates cellular protein translation partly via activation of p70 ribosomal S6 kinase 1 (S6K1), leading to phosphorylation of S6 at the Ser235/236 site (36). Phosphorylation of S6 has been reported to correlate with increased translation of transcripts encoding proteins involved in cell cycle progression, ribosomal proteins, and transcription elongation factors (45,46). Our observation that USP7 inhibition alone reduced levels of phosphorylated ribosomal protein S6 suggests that USP7 functions in a related pathway in these cells and perhaps explains the synergy between USP7 inhibitors and PIM or PI3 kinase inhibitors. In addition, our USP7 inhibitor potentiated the activity of DNA-damaging chemotherapeutic agents carboplatin and doxorubicin, and the PARP inhibitor olaparib. USP7 inhibition could thus increase the efficacy of certain standard of care chemotherapeutics. Profiling of transcriptomic changes in H526 cells upon short-term USP7 inhibitor treatment revealed upregulation of a large number of genes normally silenced by the polycomb repressive complex 2 (PRC2), suggesting that USP7 may stabilize or activate this complex or its components. PRC2 initiates transcriptional silencing by trimethylating histone H3 at Lys27 (H3K27me3), which is then bound by the maintenance complex PRC1 (40). PRC1 monoubiquitinates histone H2A, leading to chromatin compaction and stabilizing transcriptional silencing. The catalytic subunit of PRC2 is the histone methylase enhancer of zeste 2 (EZH2). EZH2 is upregulated in many cancers, leading to epigenetic repression of genes encoding tumor suppressors, proteins involved in DNA damage response pathways, and negative regulators of the mTOR pathway (47,48). Inhibition of EZH2 is actively being pursued clinically and preclinically as an anti-tumor strategy (40). The observation that FKBP11 and RGS16, two mTOR-inhibitory proteins reported to be epigenetically silenced by EZH2, are among those upregulated by USP7 inhibition in H526 cells, dovetails with the USP7/PIM/PI3K inhibitor synergy we observe. However, given the many pro-tumor functions of EZH2, other pathways altered as a consequence of its reduced activity likely also contribute to the observed tumor growth inhibition. USP7 has previously

been reported to modulate the activity of PRC1 by stabilizing its component proteins RING1B and BMI1 (21,49,50) and USP7 ablation abolishes PRC1 binding at the INK4a tumor suppressor locus, derepressing it and causing proliferative arrest (50). USP7 may promote PRC2 activity similarly by stabilizing its component proteins and this will be explored in future studies. Intriguingly, an investigation into gene expression patterns significantly associated with USP7 inhibitor sensitivity in our large cell line panel revealed that PRC2-regulated gene sets were significantly lower expressed in USP7 inhibitor-sensitive cell lines, while gene sets belonging to DNA damage repair and translation-related pathways were significantly overexpressed in these cell lines. These expression patterns occurred in sensitive cell lines of both TP53 wild-type and mutant status, indicating that the mechanisms of action uncovered in the H526 cell line generalize to other cell lines in which USP7 inhibitors are effective and that activation of these pathways are markers of sensitivity to USP7 inhibition. Our discovery of highly potent, specific, reversible, orally bioavailable USP7 inhibitors confirms that USP7 is a pharmacologically tractable oncology drug target. Inhibition of USP7 reduces the viability of both TP53 wild-type and TP53 mutant tumors in vitro and in vivo by multiple mechanisms. Our findings extend the utility of USP7 inhibitors beyond TP53 wild-type tumors and suggest novel avenues for combination therapies to improve outcomes in less tractable tumor types. The ability to specifically inhibit the activity of deubiquitinases such as USP7 opens a path to target otherwise undruggable targets by modulating their protein stability. Further work elucidating how USP7- dependent cell death is induced will aid in ultimately defining a clinical development path for these inhibitors.

Acknowledgments. We would like to acknowledge Lavanya Adusumilli, Deepika Kaveri, Oezcan Talay and Andrea Kim for helpful discussions related to our USP7 program.

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Tables and Table Legends Table 1. Biochemical and cellular potency of USP7 inhibitors and selectivity over USP47 (nd = not determined, PK = pharmacokinetic properties).

Compound USP7 USP47 p53 MM.1S H526 Other properties

IC50 IC50 EC50 CC50 CC50 (nM) (nM) (µM) (µM) (µM) USP7-443 9 > 50,000 0.7 nd nd Early tool compound, less potent in vitro, unsuitable in vivo PK USP7-866 0.2 10,000 0.07 0.1 0.4 Potent in vitro but unsuitable in vivo PK USP7-055 0.3 > 10,000 0.03 0.1 0.5 Potent in vitro with suitable in vivo PK USP7-797 0.5 > 10,000 0.03 0.1 0.5 Potent in vitro with suitable in vivo PK USP7-414 0.2 > 10,000 0.03 0.3 1.3 Active enantiomer, potent in vitro but unsuitable in vivo PK USP7-777 67 > 10,000 > 1.0 > 5 27 Inactive enantiomer, not potent in vitro and unsuitable in vivo PK

Table 2. Cytotoxicity of USP7 inhibitors and idasanutlin in TP53 wild-type blood cancer cell lines (acute myeloid leukemia, AML; multiple myeloma, MM; not determined, nd). Cell Line Cancer Idasanutlin USP7-055 USP7-797 Type CC50 (µM) CC50 (µM) CC50 (µM) M07e AML 0.03 0.1 0.2 OCI-AML5 AML 0.04 0.2 0.2 MOLM13 AML 0.02 0.1 0.4 EOL-1 AML 0.05 0.04 nd MOLP8 MM 0.3 0.2 nd NCI-H929 MM 0.2 0.4 nd MM.1S MM 0.02 0.1 0.1

Table 3. Cytotoxicity of USP7 inhibitors and idasanutlin in TP53 wild-type neuroblastoma cell lines (not determined, nd). Cell Line MYC/MYCN Idasanutlin USP7-866 USP7-055 USP7-797 Amplification CC50 (µM) CC50 (µM) CC50 (µM) CC50 (µM) Status SH-SY5Y MYC amplified 0.1-0.5 1.7 1.8 1.9 SK-N-SH MYC amplified 0.2 1.2 nd nd IMR-32 MYCN amplified 0.24 2.1 nd nd CHP-134 MYCN amplified 0.01 nd 0.5 0.6 NB-1 MYCN amplified 0.1 nd 0.6 0.5

Table 4. Cytotoxicity of USP7 inhibitors and idasanutlin in TP53 mutant cancer cell lines (not determined, nd). Cell Line Cancer Type Idasanutlin USP7-866 USP7-055 USP7-797 CC50 (µM) CC50 (µM) CC50 (µM) CC50 (µM) H526 lung, small cell, 6.5 0.4 0.5 0.5 MYCN-amplified LA-N-2 neuroblastoma, 5.3 0.2 0.6 0.2 MYCN-amplified SK-N-DZ neuroblastoma, > 12.5 nd 0.2 0.2 MYCN-amplified EBC-1 lung, squamous 16.2 2.5 nd nd cell carcinoma NCI-H820 lung, papillary 9.7 2.1 nd nd

adenocarcinoma NCI-H2009 lung, > 25 1.8 nd nd adenocarcinoma HUH-7 hepatocellular > 25 2.8 nd nd carcinoma MHCC97-H hepatocellular > 25 1.9 nd nd carcinoma NCI-N87 gastric 8.6 2.8 nd nd JJN-3 multiple > 25 2.6 nd nd myeloma HCC1806 breast, triple- 7.5 2.9 nd nd negative HCC1937 breast, triple- 7.1 0.1 0.1 nd negative, BRCA1 mutant

Table 5. Top ten gene sets significantly upregulated upon USP7 inhibitor treatment of H526 cells. Pathway FDR p- Number of value differentially expressed genes found within gene set MEISSNER_BRAIN_HCP_WITH_H3K4ME3_AND_H3K2 1.04E-54 226 7ME3 BENPORATH_ES_WITH_H3K27ME3 1.75E-47 213 BENPORATH_SUZ12_TARGETS 2.82E-46 201 BENPORATH_EED_TARGETS 2.30E-35 185 BENPORATH_PRC2_TARGETS 1.72E-31 131 MEISSNER_NPC_HCP_WITH_H3K4ME2 2.40E-28 113 MIKKELSEN_MCV6_HCP_WITH_H3K27ME3 3.72E-28 102 MIKKELSEN_MEF_HCP_WITH_H3K27ME3 9.98E-24 110 MEISSNER_NPC_HCP_WITH_H3_UNMETHYLATED 1.46E-23 105 MEISSNER_NPC_HCP_WITH_H3K4ME2_AND_H3K27 3.68E-23 83 ME3

Figure Legends

Figure 1. Structure of USP7 inhibitor in complex with USP7. (A) Chemical structure of USP7 inhibitor USP7-443. (B) Co-crystal structure of USP7-443 and USP7. (C) Biochemical IC50 of USP7-443 for 41 deubiquitinases tested. Figure 2. USP7 inhibitors inhibit TP53 wild-type tumors in vitro and in vivo. (A) Association of TP53 status with USP7 inhibitor sensitivity in 430-cell line panel. (B) MM.1S cells were treated with USP7 inhibitors for 5 days, after which viability was measured by CellTiter Glo assay. (C) MM.1S cells were treated with USP7 inhibitor for 4h, after which USP7 engagement was measured using Ub-PA probe. (D) MM.1S cells were treated with USP7 inhibitor or idasanutlin for 6h, after which levels of the indicated proteins were measured. (E) NOD-SCID mice engrafted with MM.1S tumors were treated with USP7 inhibitor for the indicated time, and tumor volume and survival were monitored. Figure 3. USP7 inhibitors inhibit TP53 mutant H526 tumors in vitro and in vivo. (A) H526 cells were treated with USP7 inhibitors or idasanutlin for 5 days, after which viability was measured by CellTiter Glo assay. (B) Nu/Nu mice engrafted with H526 tumors were treated with USP7 inhibitor for the indicated time, and tumor volume and survival were monitored. (C, D) H526 cells were treated with indicated drug (y-axis) and USP7 inhibitor (x-axis) at indicated dose (µM) for 5 days, after which viability was measured by CellTiter Glo assay and synergy was calculated by Bliss analysis.

OH N HO NH S Cl O N

Cl O

Thumb Fingers

Palm Cys223 USP7-443 C 5.0E-09

5.0E-08

(M) 5.0E-07 50 IC 5.0E-06

5.0E-05 BAP1 USP1 USP2 USP4 USP5 USP6 USP7 USP8 CYLD OTU1 VCPIP USP9x USP11 USP14 USP15 USP16 USP19 USP20 USP21 USP25 USP28 USP30 USP35 USP36 USP45 JOSD1 JOSD2 UCHL1 UCHL3 UCHL5 OTUB2 Ataxin3 OTUD1 OTUD3 USP27x Ataxin3L Cezanne OTUD6A OTUD6B AMSH-LP Deubiquitinase OTUD5 (p177S)

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 Figure 2Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.

A 2.3 e-7 B

µM 120

1 ta langis dezilamroN langis ta 1 100 100 1.0 - 80 80 60 60 40 0.5 - 40 20 20 Active enantiomer USP7-055 Inactive enantiomer

0 % Luminescence (ATP) 0 -8 -7 -6 % Luminescence (ATP) -8 -7 -6 -5 10 10 10 0.0 - 10 10 10 10 Mutant Wild-type [Compound] (M) [Compound] (M) TP53 status

C D probe: - + + + + + + + + + + + + USP7-055 (nM): - 100 500 - Idasanutlin (nM): 125 USP7-866 (nM): 0 0 500 200 85 35 15 6 2 1 0.4 0.2 0.06 --- p53 USP7-Ub - USP7- p21 GAPDH β−actin

E Tumor Volume Survival **** 3

) ***

3 Vehicle control 2500 100 USP7-055 50 mpk QD 2000 USP7-055 100 mpk QD *** 80 USP7-797 50 mpk BID **** 1500 **** 60 1000 40 Vehicle control USP7-055 50 mpk QD 500 20 USP7-055 100 mpk QD Percent Animals With

Tumor Volume (mm USP7-797 50 mpk BID

0 Tumor Volume < 1500 mm 0 0 5 10 15 20 25 0 5 10 15 20 25 Days of Treatment Days of Treatment

Downloaded from mct.aacrjournals.org on October 3, 2021. © 2020 American Association for Cancer Research. Figure 3 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. A

120 120 120 100 100 100 80 80 80 60 60 60 40 40 40 20 20 Active enantiomer 20 USP7-797 Inactive enantiomer Idasanutlin 0 0 % Luminescence (ATP) % Luminescence (ATP) 0 -8 -7 -6 -5 -8 -7 -6 -5 -8 -7 -6 -5 10 10 10 10 10 10 10 10 % Luminescence (ATP) 10 10 10 10 [Compound] M [Compound] M [Compound] M

B Tumor Volume Survival TmT ) **** 3

) 2000 Vehicle control 100 3 USP7-797 1500 80 15 00 mm **** 60 1000 40

500 Percent Survival 20 Vehicle control Tumor Volume (mm USP7-797

0 (Tumor Reaching 0 0 5 10 15 20 25 0 5 10 15 20 25 Days of Treatment Days of Treatment C carboplatin doxorubicin olaparib USP7-866 25 0.25 75 1 synergy 12.5 0.12 37.5 0.5 4 6.25 18.8 0.06 0.25 2 3.1 9.4 0.03 0 1.6 0.12 0.02 4.1 -2 0.8 0.06 0.01 2.3 -4 0.4 1.2 interference 0.004 0 0.2 0.6 1 2 0 0.5 0.1 0.002 0.3 0.06 0.12 0.25 0.05 0.001 0.2 0 0 0 0 1 2 1 2 1 2 0.5 0 0.5 0 0.5 0.06 0.12 0.25 0.06 0.12 0.25 0.06 0.12 0.25 D AZD1208 pictilisib taselisib USP7-797 10 2 1.2 2 synergy 5 1 0.6 1 3 0.5 2 2.5 0.3 0.5 1.2 0.25 0.16 1 0.6 0.12 0.08 0.25 0 0.3 0.06 0.04 0.12 -1 0.16 0.03 0.02 0.06 interference 0.02 0.08 0.01 0 0.04 0.01 0.005 1 2 0.004 0 0.5 0.02 0.002 0.06 0.12 0.25 0 0 0 0 1 2 1 2 0.5 0 1 2 0 0.5 0.06 0.12 0.25 0.5 0.06 0.12 0.25 0.06 0.12 0.25

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

Yamini M. Ohol, Michael T. Sun, Gene Cutler, et al.

Mol Cancer Ther Published OnlineFirst August 11, 2020.

Updated version Access the most recent version of this article at: doi:10.1158/1535-7163.MCT-20-0184

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