USP7 Cooperates with NOTCH1 to Drive the Oncogenic Transcriptional Program in T-Cell Leukemia Qi Jin1, Carlos A

Published OnlineFirst September 17, 2018; DOI: 10.1158/1078-0432.CCR-18-1740

Translational Cancer Mechanisms and Therapy Clinical Cancer Research USP7 Cooperates with NOTCH1 to Drive the Oncogenic Transcriptional Program in T-Cell Leukemia Qi Jin1, Carlos A. Martinez1, Kelly M. Arcipowski1, Yixing Zhu1, Blanca T. Gutierrez-Diaz1, Kenneth K. Wang2, Megan R. Johnson1, Andrew G. Volk3, Feng Wang4, Jian Wu4, Charles Grove4, Hui Wang4, Ivan Sokirniy4, Paul M. Thomas5, Young Ah Goo1,5, Nebiyu A. Abshiru5, Nobuko Hijiya6,Soﬁe Peirs7,8, Niels Vandamme8,9, Geert Berx8,9, Steven Goosens7,8,9, Stacy A. Marshall1, Emily J. Rendleman1,Yoh-hei Takahashi1, Lu Wang1, Radhika Rawat1, Elizabeth T. Bartom1, Clayton K. Collings1, Pieter Van Vlierberghe7,8, Alexandros Strikoudis10, Stephen Kelly10, Beatrix Ueberheide11, Christine Mantis12, Irawati Kandela12, Jean-Pierre Bourquin13, Beat Bornhauser13, Valentina Seraﬁn14, Silvia Bresolin14, Maddalena Paganin14, Benedetta Accordi14, Giuseppe Basso14, Neil L. Kelleher1,5,15, Joseph Weinstock4, Suresh Kumar4, John D. Crispino1,3,16, Ali Shilatifard1,16, and Panagiotis Ntziachristos1,3,16

Abstract

Purpose: T-cell acute lymphoblastic leukemia (T-ALL) is an demethylase. USP7 is highly expressed in T-ALL and is tran- aggressive disease, affecting children and adults. Chemother- scriptionally regulated by NOTCH1. In turn, USP7 controls apy treatments show high response rates but have debilitating NOTCH1 levels through deubiquitination. USP7 binds onco- effects and carry risk of relapse. Previous work implicated genic targets and controls gene expression through stabiliza- NOTCH1 and other oncogenes. However, direct inhibition tion of NOTCH1 and JMJD3 and ultimately H3K27me3 of these pathways affects healthy tissues and cancer alike. Our changes. We also show that USP7 and NOTCH1 bind T-ALL goal in this work has been to identify enzymes active in T-ALL superenhancers, and inhibition of USP7 leads to a decrease of whose activity could be targeted for therapeutic purposes. the transcriptional levels of NOTCH1 targets and signiﬁcantly Experimental Design: To identify and characterize new blocks T-ALL cell growth in vitro and in vivo. NOTCH1 druggable partners in T-ALL, we coupled studies of Conclusions: These results provide a new model for USP7 the NOTCH1 interactome to expression analysis and a series of deubiquitinase activity through recruitment to oncogenic functional analyses in cell lines, patient samples, and xenograft chromatin loci and regulation of both oncogenic transcription models. factors and chromatin marks to promote leukemia. Our Results: We demonstrate that ubiquitin-speciﬁc protease 7 studies also show that targeting USP7 inhibition could be (USP7) interacts with NOTCH1 and controls leukemia growth a therapeutic strategy in aggressive leukemia. Clin Cancer Res; by stabilizing the levels of NOTCH1 and JMJD3 histone 1–18. 2018 AACR.

Introduction risk of recurrence due to tumor cells that are refractory to che- Acute lymphoblastic leukemia (ALL) represents 25% of child- motherapy, a common characteristic of high-risk T-ALL. In hood tumors (1, 2). T-cell ALL (T-ALL; refs. 1–7) is the most this regard, 20% of pediatric, and more than 50% of adult aggressive subtype of ALL and these tumors exhibit a signiﬁcant patients, with T-ALL fail to achieve a complete remission after

1Department of Biochemistry and Molecular Genetics, Northwestern University, Woman's and Child's Health, University of Padova, Padova, Italy. 15Department Chicago, Illinois. 2Master of Science in Biotechnology Graduate Program, North- of Chemistry, Northwestern University, Chicago, Illinois. 16Robert H. Lurie western University, Evanston, Illinois. 3Division of Hematology/Oncology, Comprehensive Cancer Center, Northwestern University, Chicago, Illinois. Department of Medicine, Northwestern University, Chicago, Illinois. 4Progenra Note: Supplementary data for this article are available at Clinical Cancer Inc., Malvern, Pennsylvania. 5Proteomics Center of Excellence, Northwestern Research Online (http://clincancerres.aacrjournals.org/). University, Evanston, Illinois. 6Ann & Robert H. Lurie Children's Hospital, North- western University, Chicago, Illinois. 7Center for Medical Genetics, Ghent Uni- Q. Jin, C.A. Martinez, and K.M. Arcipowski contributed equally for this article. versity Hospital, Ghent, Belgium. 8Cancer Research Institute Ghent (CRIG), Ghent, Belgium. 9Molecular Cellular Oncology Lab, Department for Biomedical Corresponding Author: Panagiotis Ntziachristos, Northwestern University, Molecular Biology, Ghent University, Ghent, Belgium. 10Department of Pathol- Feinberg School of Medicine, 320 East Superior Street, Searle 6-523, Chicago, ogy, New York University, New York, New York. 11Department of Biochemistry IL 60611. Phone: 347-703-0048; E-mail: [email protected]; Website: and Molecular Pharmacology, New York University, New York, New York. Ntziachristoslab.com 12 Center for Developmental Therapeutics, Northwestern University, Evanston, doi: 10.1158/1078-0432.CCR-18-1740 Illinois. 13University Children's Hospital, Division of Pediatric Oncology, Univer- sity of Zurich, Switzerland. 14Oncohematology Laboratory, Department of 2018 American Association for Cancer Research.

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number of patient samples and cell lines of T-ALL, as well as Translational Relevance preclinical models, including patient-derived xenografts. We Current therapeutic options and drugs in clinical trials for identified a novel member of the NOTCH1 transcriptional com- T-cell acute lymphoblastic leukemia (T-ALL) are rather limited plex in T-ALL, the first deubiquitinase (DUB) protein with critical and mainly restricted to chemotherapy, unlike other hemato- role in leukemia, referred to as ubiquitin-specific peptidase 7 logic malignancies, such as B-ALL. In addition, T-ALL presents (USP7; refs. 32–43). We have shown that: (i) the NOTCH1 with higher rates of relapse compared with B-ALL, rendering complex interacts with and is stabilized by USP7; (ii) USP7 levels this disease an unmet clinical need. Here, we report a novel are significantly increased in T-ALL tumor samples compared to targeted therapy in T-ALL, against a protein (USP7) that is normal T cells via a positive feedback loop with NOTCH1; (iii) a upregulated and transcriptionally regulated by oncogenic small chemical molecule that inhibits USP7 (USP7i), leads to a NOTCH1 in T-ALL. Inhibition of USP7 significantly blocks blockade in T-ALL cell growth, without associated toxicity in disease progression and extends survival, without associated healthy animals. Thus, it is proposed to determine the mechan- toxicities, in preclinical models of T-ALL. We speculate that isms employed by USP7/NOTCH1 that leads to a T-ALL pheno- this targeted therapy (USP7 inhibition), could be used in type and most importantly, can targeting USP7 reverse this combination with systemic chemoradiation, to increase front- phenotype in patients. line response rates, especially in patients with high risk of relapse/nonresponse, or potentially as a replacement for sys- Materials and Methods temic cytotoxics in frail patients, likely to suffer from extensive Cell lines and primary cells adverse events associated with frontline intensive chemother- The human T-ALL cell lines CUTLL1 (gift from Iannis Aifantis, apy. USP inhibition could be also used as an addition to New York University, New York, NY), LOUCY (gift from Pieter immunotherapy approaches. Van Vlierberghe, Cancer Research Institute, Belgium), CCRF-CEM (ATCC; #CCL-119), JURKAT (ATCC), HPB (HPB-ALL), and KOPTK1 (Iannis Aifantis' group) were cultured in RPMI1640 medium supplemented with 10% heat-inactivated FBS (Sigma- chemotherapy, making resistance to therapy the most substantial Aldrich), 2% penicillin/streptomycin (Gibco, Fisher Scientific), challenge in treatment (5, 8, 9). Immunotherapy using engi- and 1% GlutaMAX (Gibco, Fisher Scientific). 293T cells (ATCC, neered T cells appears promising for some patients, but these #CRL-11268) were maintained in DMEM medium supplemented experimental therapies also appear fraught with unacceptable with 10% heat-inactivated FBS, 2% penicillin/streptomycin, and toxicities. ALL, thus, represents a high unmet need. To this end, 1% GlutaMAX. Human pan T cells were purchased from AllCells. improvement in therapy, as well as personalized cancer medicine, com. Primary human samples were collected by collaborating should identify subgroups of patients, based on the molecular institutions with informed consent and analyzed under the mechanism of resistance, which will benefit patients through the supervision of the Institutional Review Board of Ghent University. development of novel therapeutic strategies, including targeted Informed consent to use leftover material for research purposes agents in patients with high-risk T-ALL. was obtained from all of the patients at trial entry in accordance The NOTCH1 (1, 2, 10–26) signaling pathway is activated in with the Declaration of Helsinki. more than 50% of T-ALL cases. NOTCH1 mutations were initially identified in the form of chromosomal translocation t(7;9)(q34; q34.3), which leads to the expression of a truncated and consti- Antibodies and reagents tutively active form of NOTCH1 (10). In most T-ALL cases, The following antibodies were used for Western blot analysis: NOTCH1 is activated via mutations that disrupt specificdomains mouse anti-actin (Millipore; clone C4), rabbit anti-JMJD3 (Cell responsible for the nuclear levels of NOTCH1 protein. NOTCH1- Signaling Technology; #3457), rabbit anti-USP7 (Bethyl Labora- positive leukemia presents with infiltration of bone marrow, tories, Montgomery, TX; #A300-033A-7), rabbit anti-cleaved spleen, and liver with leukemic blasts and lead to a fulminant NOTCH1 (Val1744; Cell Signaling Technology; #4147), rabbit terminal disease that, if untreated, can lead to death over the course anti-K48 specific ubiquitin (Millipore; 05-1307), rabbit anti- of months. Seminal work has described the major oncogenic Lamin B1 (Proteintech; 12987-1-AP), rabbit anti-USP9X (Bethyl pathways (i.e., NOTCH1) in T-ALL (1, 2, 10–21). Chemical inhi- Laboratories; #A301-351A), anti-Flag (Sigma; M2 clone), anti-HA bitors or antibodies against NOTCH1 have been unsuccessful in the (Abcam; ab18181), and rabbit anti-USP24 (Bethyl Laboratories; clinic, due to high levels of on-target toxicity, given the involvement #A300-938A). of this protein in multiple physiologic processes (11, 27). The following antibodies were used for ChIP: rabbit anti- Others and we have previously shown that loss of the repressive H3K27Ac (Cell Signaling Technology; #8173S), rabbit anti- mark trimethylation of lysine 27 on histone H3 (H3K27me3) is H3K27me3 (Cell Signaling Technology; #9733S), rabbit important for leukemia initiation and progression (15), and anti-H2BK120ub (Cell Signaling Technology; #5546S), rabbit NOTCH1 recruitment to target genes and loss of H3K27me3 anti-H2AK119ub (Cell Signaling Technology; #8240S), rabbit correlate during leukemogenesis (15). We also identified and anti-H3K79me2 (made by Ali Shilatifard's laboratory, Northwest- characterized the pro-oncogenic role of the epigenetic modulator ern University), rabbit anti-H3K4me3 (Ali Shilatifard's laborato- Jumonji D3 (JMJD3; refs. 28–31), which mediates NOTCH1 ry), normal rabbit IgG (Millipore; #12-370), anti-Flag (Sigma; M2 oncogenic function in T-ALL (16). JMJD3 is a NOTCH1 interactor clone), and rabbit anti-USP7 (Bethyl Laboratories; #A300-033A-7). and is recruited by NOTCH1 to oncogenic targets (16). The rabbit anti-C-MYC:(N-262; Santa Cruz Biotechnology) and Here we sought out to identify additional NOTCH1 cofactor goat anti-RUNX1 (Santa Cruz Biotech; SC- 8563) antibodies were proteins with pro-oncogenic functions that can be targeted in used in published ChIP-seq studies we use in our analysis. A T-ALL contexts without causing toxicity. In this study, we used a RUNX1 antibody from Cell Signaling Technology was used for

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our Western blot and ChIP-qPCR studies (AML1 antibody #4334). human database, using Sequest within Proteome Discoverer. The Secondary antibodies for Western blots were HRP-conjugated anti- results were filtered with a 1% FDR searched against a decoy rabbit and anti-mouse IgG (GE Healthcare). database and for proteins with at least 2 unique peptides. Word Quick Start Bovine Gamma Globulin (BGG) Standard Set clouds were generated using the statistical program R, and gene (protein standards) were purchased from Bio-Rad; benzonase, ontology was performed using the Gene Ontology Consortium RNase A, dithiothreitol (DTT), EZview Red Anti-HA Affinity Gel (http://www.geneontology.org/). (HA beads), and Influenza Hemagglutinin (HA) peptide were purchased from Sigma-Aldrich; NaV and NaF were purchased In more detail from New England BioLabs; Protein G Dynabeads were purchased As previously described (47), the affinity purified proteins were from Life Technologies; IgG-free BSA was purchased from Jackson resuspended in NuPAGE LDS Sample Buffer (Novex). The sam- ImmunoResearch Laboratories (West Grove, PA); phenol chlo- ples were reduced with 2 mL of 0.2M dithiothreitol (Sigma) for roform was purchased from ThermoScientific; and proteinase K, 1 hour at 57 C at pH 7.5. Next the samples were alkylated with 2 Tousimis formaldehyde, and Peptide International Z-Leu-Leu- mL of 0.5M iodoacetamide (Sigma) for 45 minutes at room Leu-H aldehyde (MG132) were purchased from Fisher Scientific. temperature in the dark. The samples were loaded on a NuPAGE The latest generation of Progenra inhibitors (USP7i), P217564, 4% to 12% Bis-Tris Gel 1.0 mm (Life Technologies) and run for 8 was a kind gift from Progenra (Malvern, PA). Several additional minutes at 200 V. The gel was stained with GelCode Blue Stain inhibitors from Progenra were used (P5091 and P22077) yielding Reagent (Thermo). The gel plugs were excised and destained for results similar to P217564. All USP7 inhibitors used in the 15 minutes in a 1:1 (v/v) solution of methanol and 100 mmol/L manuscript have been published previously as referred to in ammonium bicarbonate. The buffer was exchanged and the the manuscript. GSKJ4 was purchased from Cayman Chemical; samples were destained for another 15 minutes. This was repeated #12073. for another 3 cycles. The gel plugs were dehydrated by washing with acetonitrile, and further dried by placing them in a SpeedVac Immunoprecipitation for 20 minutes. Three hundred nanograms of sequencing grade One hundred million T-ALL cells were collected and washed modified trypsin (Promega) was added directly to the dried gel with chilled PBS. Cells were resuspended in 5 volumes of Buffer plugs followed by enough 100 mmol/L ammonium bicarbonate A [10 mmol/L HEPES, 1.5 mmol/L MgCl , 10 mmol/L KCl, 1:100 2 to cover the gel pieces. The gel plugs were allowed to shake at room protease inhibitor (Sigma-Aldrich; P8340), 1 mmol/L NaV, temperature and digestion proceeded overnight. The digestion 1 mmol/L NaF, and 0.5 mmol/L DTT in H O], incubated on ice 2 was halted by adding a slurry of R2 50 mm Poros beads (Applied for 10 minutes, and lysed using a Dounce homogenizer. Nuclear Biosystems) in 5% formic acid and 0.2% trifluoroacetic acid (TFA) pellets were resuspended in 1.5 mL of TENT buffer [50 mmol/L to each sample at a volume equal to that of the ammonium Tris pH 7.5, 5 mmol/L EDTA, 150 mmol/L NaCl, 0.05% (v/v) bicarbonate added for digestion. The samples were allowed to Tween 20, 1:100 protease inhibitor (Sigma-Aldrich, P8340), shake at 4C for 120 minutes. The beads were loaded onto C18 1 mmol/L NaV, 1 mmol/L NaF, and 0.5 mmol/L DTT in H O] 2 ziptips (Millipore), equilibrated with 0.1% TFA, using a micro- containing 5 mmol/L MgCl and 100 units benzonase, and 2 centrifuge for 30 s at 6,000 rpm. The beads were washed with 0.5% incubated at 4C for 30 minutes, rotating. Lysates were passed acetic acid. Peptides were eluted with 40% acetonitrile in 0.5% through a 251/2G needle/syringe 5 times, and spun down at 4C, acetic acid followed by 80% acetonitrile in 0.5% acetic acid. The 2000 RPM, for 7 minutes to remove debris. Protein G magnetic organic solvent was removed using a SpeedVac concentrator and beads were added to the lysates to decrease nonspecific binding the sample reconstituted in 0.5% acetic acid. and incubated at 4C for 30 minutes, rotating. Precleared lysates were then incubated with the appropriate antibody-conjugated beads (5 mg antibody per 100 million cells) at 4C overnight, Analysis–mass spectrometry rotating. Beads were washed 4 times in TENT buffer at 4C An aliquot of each sample was loaded onto an Acclaim Pep- for 3 minutes, and protein complexes were eluted in 200-mL Map100 C18 75-mm 15-cm column with 3-mm bead size 0.1 mol/L glycine pH 2.5 for 10 minutes at 25C, shaking. Twenty coupled to an EASY-Spray 75-mm 50-cm PepMap C18 analytical microliters of 1 mol/L Tris pH 8.0 was then added to the super- HPLC column with a 2-mm bead size using the auto sampler of an natants. For IP of HA-JMJD3, precleared lysates were incubated EASY-nLC 1000 HPLC (ThermoFisher) and solvent A (2% ace- with HA-coated beads overnight, and protein complexes were tonitrile, 0.5% acetic acid). The peptides were eluted into a eluted in 200-mL TENT buffer containing 400 mg/mL HA peptide Thermo Fisher Scientific Orbitrap Q Exactive Mass Spectrometer (Sigma-Aldrich) at 4C overnight, rotating. increasing from 2% to 30% solvent B (90% acetonitrile, 0.5% acetic acid) in 60 minutes, followed by an increase from 30% to Mass spectrometry 40% solvent B in 10 minutes and 40% to 100% solvent B in Histone epiproteomics analysis was performed as previously another 10 minutes. described (44, 45). For the JMJD3 mass spectrometry, the affinity- High-resolution full MS spectra were obtained with a resolu- purified proteins were reduced, alkylated, and loaded onto an tion of 70,000, an AGC target of 1e6, with a maximum ion time of SDS-PAGE gel to remove any detergents and LC/MS incompatible 120 milliseconds, and a scan range from 400 to 1,500 m/z. reagents. The gel plugs were excised, destained, and subjected to Following each full MS scan, 20 data-dependent MS-MS spectra proteolytic digestion with trypsin. The resulting peptides were were acquired. The MS-MS spectra were collected with a resolution extracted and desalted as previously described (46). An aliquot of of 17,500 an AGC target of 5e4, maximum ion time of 120 the peptides was analyzed with LC/MS coupled to a ThermoFisher milliseconds, 1 microscan, 2 m/z isolation window, fixed first Scientific Orbitrap QExactive Mass Spectrometer operated in data mass of 150 m/z, dynamic exclusion of 30 seconds, and normal- dependent mode. The data were searched against a UniProt ized collision energy (NCE) of 27.

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Data processing—mass spectrometry added for 5 minutes. Pelleted cells were then lysed according to All acquired MS2 spectra were searched against a UniProt the type of ChIP performed. human database using Sequest within Proteome Discoverer For histone ChIPs, cells were lysed in 375 mL of nuclei incu- (ThermoScientific). The search parameters were as follows: pre- bation buffer [15 mmol/L Tris pH 7.5, 60 mmol/L KCl, 150 cursor mass tolerance 10 ppm, fragment mass tolerance 0.02 mmol/L NaCl, 15 mmol/L MgCl2, 1 mmol/L CaCl2, 250 Da, digestion parameters trypsin allowing 2 missed cleavages, mmol/L Sucrose, 0.3% NP-40, 1 mmol/L NaV, 1 mmol/L NaF, fixed modification of carbamidomethyl on cysteine, variable and 1 EDTA-free protease inhibitor tablet (Roche)/10 mL in H2O] modification of oxidation on methionine, and variable modifi- for 10 min on ice. Nuclei were washed once with digest buffer cation of deamidation on glutamine and asparagine. The results [10 mmol/L NaCl, 10 mmol/L Tris pH 7.5, 3 mmol/L MgCl2,1 were filtered with a 1% FDR searched against a decoy database and mmol/L CaCl2, 1 mmol/L NaV, 1 mmol/L NaF, and 1 EDTA-free for proteins with at least 2 unique peptides. Proteins enriched in protease inhibitor tablet (Roche)/10 mL in H2O] and resus- the sample affinity purification over the empty vector control were pended in 57-mL Digest Buffer containing 4.5 units MNase (USB) further interrogated. for 1 hour at 37C. MNase activity was quenched for 10 minutes on ice upon the addition of EDTA to a final concentration of 20 Reverse phase protein array mmol/L. Pelleted nuclei were lysed in 300-mL Nuclei Lysis Buffer Reverse phase protein array (RPPA) was performed as described [50 mmol/L Tris-HCl pH 8.0, 10 mmol/L EDTA pH 8.0, 1% SDS, 1 in Milani G. and colleagues, Oncotarget 2014, and Serafin V. and mmol/L NaV, 1 mmol/L NaF, and 1 EDTA-free protease inhibitor colleagues, Leukemia 2017. Briefly, cells were lysed in an appro- tablet (Roche)/10 mL in H2O] using a Bioruptor Pico (Diage- priate lysis buffer with proteases and phosphatases inhibitors, node) for 5 minutes (30 seconds on, 30 seconds off). Lysate was serially diluted into 4-point dilution curves and printed on centrifuged at max speed for 5 minutes to remove debris, and 9 nitrocellulose-coated glass slides with the 2470 Aushon Arrayer volumes of IP Dilution Buffer [0.01% SDS, 1.1% Triton X-100, (Aushon Biosystems). 1.2 mmol/L EDTA pH 8.0, 16.7 mmol/L Tris-HCl pH 8.0, 167 mmol/L NaCl, 1 mmol/L NaV, 1 mmol/L NaF, and 1 EDTA-free Western blot analysis protease inhibitor tablet (Roche)/10 mL in H2O] were added to To make total cell extracts, up to 10 million cells were collected the supernatant. Fifty microliters of protein G magnetic beads, and resuspended in 20-mL RIPA buffer [50 mmol/L Tris HCl pH blocked with IgG-free BSA, were added to the sample and incu- 8.0, 150 mmol/L NaCl, 1% NP-40/IGEPAL, 0.5% sodium deox- bated at 4C for 30 minutes, rotating. 1% of the precleared sample ycholate, 0.1% SDS, 1:100 protease inhibitor (Sigma-Aldrich, was kept out as input, and the remaining sample was split into 3 P8340), 1 mmol/L NaV, and 1 mmol/L NaF in H2O] per 1 million tubes. Fifty microliters of protein G magnetic beads conjugated to cells. Cells were lysed on ice for 20 minutes, and spun down at 15 mL of the appropriate antibody were added to each tube, and 4C, max speed, for 10 minutes to remove debris. incubated at 4C overnight, rotating. Bead-bound complexes Protein concentrations were determined via Bradford assay. were washed for 5 minutes each in 1 mL of low salt buffer [20 Samples and buffer were diluted 1:10 in H2O. Two microliters of mmol/L Tris-HCl pH 8.0, 150 mmol/L NaCl, 2 mmol/L EDTA, 1% protein standards, H2O, or diluted sample were added to wells of (w/v) Triton X-100, and 0.1% w/v SDS in H2O], high salt buffer a 96-well plate in duplicate. Then, 2 mL of diluted buffer and [20 mmol/L Tris-HCl pH 8.0, 500 mmol/L NaCl, 2 mmol/L EDTA, 100-mL Quick Start Bradford 1 Dye Reagent (Bio-Rad) were 1% (w/v) Triton X-100, and 0.1% w/v SDS in H2O], LiCl buffer added to each well, and absorbance was measured at 600 nm [10 mmol/L Tris-HCl pH8.0, 250 mmol/L LiCl, 1 mmol/L EDTA, using the GloMax-Multi Detection System (Promega). 1% (w/v) NP-40, and 1% (w/v) deoxycholic acid in H2O], and TE. Up to 50-mg sample was boiled in 1 SDS loading dye (Bio- For epigenetic regulator and transcription factor ChIPs, cells Rad) at 95C for 10 minutes prior to loading into 4% to 15% were lysed in 1-mL LB1 buffer (50 mmol/L HEPES-KOH pH 7.5, Tris-glycine polyacrylamide gels (Bio-Rad). Eight microliters of 140 mmol/L NaCl, 1 mmol/L EDTA, 10% glycerol, 0.5% NP-40, PageRuler Plus Prestained Protein Ladder (10–250 kD; Fisher 0.25% Triton X-100, 1:100 protease inhibitor [Sigma-Aldrich, Scientific) was also loaded. Gels were run at 100 V until P8340), 1 mmol/L NaV, and 1 mmol/L NaF in H2O] for 10 samples reached the separating part of the gel, and then were minutes at 4C, rotating. Nuclei pellets were resuspended in LB2 run at 130 V. Gels were transferred for 1.5 hours at 80 V or Buffer [10 mmol/L Tris-HCl pH 8.0, 200 mmol/L NaCl, 1 mmol/L overnight at 35 to 40 V, and membranes were blocked in 5% EDTA, 0.5 mmol/L EGTA, 1:100 protease inhibitor (Sigma- milk in TBST (0.1% Tween 20 in 1 TBS) for 1 hour. Mem- Aldrich, P8340), 1 mmol/L NaV, and 1 mmol/L NaF in H2O] branes were incubated at 4C overnight with the appropriate and incubated for 10 minutes at 25C, rotating. Finally, nuclei antibody in TBST. Then, the membranes were washed 3 times pellets were lysed in 300 mL LB3 buffer [10 mmol/L Tris-HCl pH for 10 minutes with TBST, incubated for 2 hours at 4Cwith 8.0, 100 mmol/L NaCl, 1 mmol/L EDTA pH 8.0, 0.5 mmol/L the appropriate secondary antibody, washed 3 times for 10 EGTA pH 8.0, 0.1% sodium deoxycholate, 0.5% N-lauroylsarco- minutes with TBST, and developed using Clarity Western ECL sine, 1:100 protease inhibitor (Sigma-Aldrich, P8340), 1 mmol/L Substrate (Bio-Rad), or SuperSignal West Femto Maximum NaV, and 1 mmol/L NaF in H2O] using a Bioruptor Pico (Diag- Sensitivity Substrate (ThermoScientific) as needed, on a Bio- enode) for 5 minutes (30 seconds on, 30 seconds off). 1% Triton Rad ChemiDoc Touch Imaging System. Analysis was performed X-100 was added, and samples were centrifuged at max speed to using Image Lab software (Bio-Rad). remove debris. Fifty microliters of protein G magnetic beads, blocked with IgG-free BSA, were added to the sample and incu- Chromatin immunoprecipitation bated at 4C for 30 minutes, rotating. One percent of the pre- Ten million T-ALL cells were cross-linked in 1 mL/million cells cleared sample was kept out as input, and the remaining sample fixation buffer (1% formaldehyde, 1 PBS, and 1% FBS in H2O) was used for IP. Fifty microliters of protein G magnetic beads for 10 minutes at 25C. Then, 1:12.5 glycine [2.5 mol/L] was conjugated to 5 to 10 mg of the appropriate antibody were added

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to each tube, and incubated at 4C overnight, rotating. Bead- Peak calling bound complexes were washed for 5 minutes each in 1 mL Peaks were called using MACS (51) version 1.4.2 with the of RIPA wash buffer (50 mmol/L HEPES-KOH pH 7.6, 300 default parameters, using the specific aligned ChIP-Seq data for mmol/L LiCl, 1 mmol/L EDTA, 1% NP-40, and 0.7% sodium a particular antibody as the treatment sample and an aligned bam deoxycholate in H2O), 5 times, and 1 time in 1-mL TE wash file of the unprecipitated input as a control sample. Only the top buffer (10 mmol/L Tris-HCl pH 8.0, 1 mmol/L EDTA, and 50 10,000 peaks ordered by peak score (which is proportional to the mmol/L NaCl in H2O). FDR of the peak) were chosen for further analysis. To elute bead-bound complexes, 50 mL of elution buffer (for 10 mL total volume, 7.5-mL H2O, 0.5 mL 20% SDS, and 2-mL 0.5M Calculation of overlaps and statistical significance sodium bicarbonate) was added to each sample, and samples Overlap between 2 ChIP-Seq peak datasets were calculated were incubated at 65C for 15 minutes, shaking at 1,000 RPM on a using the mergePeaks tool in the HOMER (52) tools. In brief, thermomixer (ThermoScientific). Elution was repeated a second the intersection between 2 ChIP-Seq peak datasets was deter- time, and then 100-mL RNase Buffer (12-mL of 5M NaCl, 0.2-mL mined by calculating the number of peaks in 1 set that 30 mg/mL RNase, and 88-mL TE) was added to each ChIP and overlap with the peaks in the second set. Statistical significance input sample. Samples were incubated at 37C for 20 minutes, of the overlaps between ChIP-Seq datasets was done by followed by the addition of PK Buffer (2.5-mL 20 mg/mL pro- using reservoir sampling (53) with a set of 161 transcription teinase K, 5-mL 20% SDS, and 92.5-mL TE) overnight at 65C. An factor peak binding sites downloaded from http://hgdownload. equal volume of phenol chloroform solution was added to cse.ucsc.edu/goldenpath/hg19/encodeDCC/wgEncodeRegTfb the samples, which were vortexed for 1 minute and transferred sClustered/ (wgEncodeRegTfbsClusteredV3.bed.gz, ENCODE to MaXtract High Density tubes (Qiagen). Samples were centri- data). The tool for implementing the sampling schema is called fuged for 8 minutes at max speed, and the upper phase was poverlaps and can be downloaded from github at https://github. transferred to new tubes containing 1.5-mL 20 mg/mL glycogen. com/brentp/poverlap. A total of 100 permutations per pair-wise Then, 30-mL sodium acetate and 800-mL 100% ethanol were combination of transcription factor overlaps was used. Venn added, and tubes were incubated on dry ice to 30 to 60 minutes. diagrams of overlaps were generated using an online Venn dia- DNA pellets were washed in 70% ethanol, air-dried, and resus- gram generator (http://jura.wi.mit.edu/bioc/tools/venn.php). pended in 30-mLH2O. Motif analysis and pathway enrichment analysis SRM method development and mass spectral data analysis HOMER, with the standard default parameters, was used to SRMs capable of discriminating 19 acetylation sites from determine the enrichment for known and unknown motifs in the 13 different histone peptides were developed with 2 to 4 fragment USP7 ChIP-Seq peaks. Pathway enrichment analysis of USP7- ions for each species, as described previously (44). Various his- bound genes were determined using GREAT (Genomic Regions tone peptides with different modifications were purchased or Enrichment of Annotations Tool; ref. 54). custom synthesized from Anaspec or Genescript to assist the method development. Data were analyzed in Skyline with Generation of heatmaps Savitzky–Golay smoothing (34). All labeled and unlabeled peaks Heatmap representation of log2 fold-changes in epigenetic were grouped as a whole for manual peak determination to avoid marks between DMSO and USP7 inhibition were visualized using bias. Total peak areas from SRMs were used for quantification (as ngs.plot (55). In brief, log2 fold-changes of H3K27me3, H2Aub, listed in ftp://PASS01134:[email protected]/). To H2Bub, and H3K79me2 upon USP7 inhibition versus DMSO access files via FTP, use credentials: treatment at the top 10,000 USP7 peaks were visualized. A k- Servername: ftp.peptideatlas.org means clustering algorithm option was used with the number of Username: PASS01134 clusters set to 5. Correlated gene expression changes were calcu- Password: HG6634ca lated by determining the log2 fold-change in gene expression upon USP7 or JMJD3 inhibition of the nearest protein-coding ChIP-Seq gene to the USP7 peak. Gene expression changes were visualized Libraries were prepared using Agencourt AMPure XP beads using Java TreeView (56). (Beckman Coulter) and the KAPA HTP Library Preparation Kit (KAPA Biosystems), according to the manufacturer's protocol Quantitative PCR (v4.15). DNA fragment size was determined using High Sensi- RNA was isolated from T-ALL cells using the Aurum Total tivity DNA Chips read on a 2100 Bioanalyzer (Agilent Technol- RNA Mini Kit (Bio-Rad), and was quantified on a Qubit 3.0 ogies). Libraries were sequenced on the Illumina NextSeq 500 Fluorometer (Life Technologies) using the Qubit RNA HS (50bp single reads). FASTQ reads were first trimmed from the 30 (High Sensitivity) Assay Kit (Life Technologies) according to the end using Trimmomatic (48) version 0.33, such that the Phred33 manufacturers' instructions. cDNA was made using the High score of all the nucleotides was above 30 and all reads shorter than Capacity cDNA RT Kit (Applied Biosystems), according to the 20bp were discarded. The resulting reads were then aligned to the manufacturer's protocol. qPCR reactions were carried out using hg19 genome using bowtie version 1.1.2 with the following iTaq Universal SYBR Green Supermix (Bio-Rad) and the following parameters: bowtie -p 5 -m 1 -v 2 -S (49). Next, the bam file was primers: USP7:50-CGGTGTTGTGTCCATCACTC-30 (forward) sorted and bigwig tracks were created by extending each read by and 50-AGTTGAGCGAGCCCGAG-30 (reverse); NOTCH3:50- 150 bp and using the GenomicAlignments (50) package in R in GTAGAGGGCATGGTGGAAGA-30 (forward) and 50-AAGTGGTC- order to calculate the coverage of reads in counts per million CAACAGCAGCTT-30 (reverse); DTX1:50-CTCGCCACTGCTATC- (CPM) normalized to the total number of reads for each sample in TACCC-30 (forward) and 50-CGTGCCGATAGTGAAGATGA-30 the library. (reverse); and HES1:50-GCAGATGACGGCTGCGCTGA-30

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(forward) and 50-AAGCGGGTCACCTCGTTCATGC-30 (reverse). Annexin V Binding Buffer (BD Biosciences), according to the Samples were run on a CFX Connect Real-Time System (Bio-Rad) manufacturers' instructions. To measure cell cycle, cells were fixed under the following conditions: 95C for 10 seconds (denatur- in 100-mL Fix and Perm Medium A (Life Technologies) for 15 ation), 60C for 30 seconds (annealing), and 72C for 30 seconds minutes, washed with PBS, and incubated with 100-mL Fix and (elongation), for 40 cycles. Analyses were performed using Graph- Perm Medium B (Life Technologies) þ 1 mg/mL DAPI (Invitro- Pad Prism software (GraphPad Software). Statistical comparisons gen) for 1 hour at 4C. Flow cytometry was performed on an LSR II were made using the Student unpaired, 2-sided t test. P values (BD Biosciences) and analyses were performed using FlowJo <0.05 were considered statistically significant. Relative mRNA software (Tree Star). Statistical analyses were performed using abundance refers to the levels of the gene of interest normalized GraphPad Prism software (GraphPad Software). Comparisons to housekeeping genes GAPDH or G6PD. All values were then were made using the Student unpaired, 2-sided t test. P values normalized to the control sample. <0.05 were considered statistically significant.

Analysis of data from publically available databases TUBE and ubiquitination assays Analysis of microarray data from GEO was done using the NCBI A total of 200 million JURKAT/CUTLL1 T-ALL cells were treated GEO2R online tool for microarray analysis. Adjusted P value in duplicate with either DMSO or 5 mmol/L USP7i for 8 hours. calculations were done using Benjamini and Hochberg (False Cell pellets were collected, washed with PBS, and frozen. Ubiqui- < discovery rate or FDR) option. An FDR of 0.05 was considered to tinated substrates were pulled down using agarose-TUBE beads, fi be statistically signi cant. eluted, and treated with the DUB USP2 to remove ubiquitin prior to Western blotting as previously described (57). The wild-type Ubiquitin competition assay and mutant USP7-expressing plasmids are purchased by addgene This assay was performed as previously described, using recom- (#46751 and #46752 correspondingly, from the Maertens and binant enzymes and a panel of USP7 inhibitors, including Peters labs). P217564 (57). In brief, in vitro recombinant enzymes in 20 mmol/L Tris-HCl (pH 8.0), 2 mmol/L CaCl2, and 2 mmol/L b- mercaptoethanol were incubated with dose ranges of P5091 for RNA-Seq 30 minutes in a 96-well plate before the addition of Ub-PLA2 and RNA was extracted from up to 10 million T-ALL cells using the NBD C6-HPC (Invitrogen) or Ub-EKL and EKL substrate, as Aurum Total RNA Mini Kit (Bio-Rad) according to the manufac- fi previously described (58). The liberation of a fluorescent product turer's instructions. RNA was quanti ed on a Qubit 3.0 Fluorom- within the linear range of the assay was monitored using a Perkin eter (Life Technologies) using the Qubit RNA HS (High Sensitiv- Elmer Envision fluorescence plate reader. Activity-based DUB ity) Assay Kit (Life Technologies) according to the manufacturer's probes were used for the evaluation of the in vivo activity of DUBs. instructions. RNA integrity and DNA fragment size were deter- These are based on the sequence of Ub as the DUB-targeting motif mined using RNA Nano Chips and High Sensitivity DNA Chips and comprise a reactive C-terminal warhead such as vinyl methyl read on a 2100 Bioanalyzer (Agilent Technologies). Libraries were ester (VME), and an N-terminal epitope tag (59). prepared using Agencourt AMPure XP beads (Beckman Coulter) and the TruSeq RNA Library Prep Kit v2 (Illumina), according to Cell transfection and virus production the manufacturer's low sample (LS) protocol, and sequenced on 293T cells that reach up to 70% confluency were used for the Illumina NextSeq 500 (50 bp single reads). FASTQ reads were transfection. 48 hours after transfection, 293T cells were collected aligned to the hg19 genome using tophat version 2.1.0 (60) using — — — for the subsequent experiment as required. The following USP7- the following options no-novel-juncs read-mismatches 2 — fi specific shRNA (Sigma-Aldrich, MISSION system) was used: read-edit-dist 2 max-multihits 5. The generated bam les were shUSP7.1:50-CCGGCCTGGATTTGTGGTTACGTTACTCGAGTA- then used to count the reads only at the exons of genes using ACGTAACCACAAATCCAGGTTTTT-30 and shUSP7.2:50-C- htseq-count (61) with the following parameters -q -m intersec- CGGCCAGCTAAGTATCAAAGGAAACTCGAGTTTCCTTTGATA- tion-nonempty -s reverse -t exon. Differential expression analysis CTTAGCTGGTTTTT-30. Retroviral NOTCH1 plasmid (gift from was done using the R package edgeR (62). Bigwig tracks of RNA- Iannis Aifantis, New York University) and retroviral JMJD3 Seq expression were generated by using the GenomicAlignments plasmids (from addgene and Paul Khavari's group, wild-type package in R in order to calculate the coverage of reads in CPM #21212 and mutant #21214) were used for retrovirus produc- normalized to the total number of uniquely mapped reads for tion. Retrovirus was used to infect T-ALL cells as previously each sample in the library. described (16). Superenhancer analysis Viability assays, apoptosis, and cell-cycle analysis Superenhancers (SEs) were determined using a H3K27Ac ChIP- A total of 500,000 cells were plated in wells of a 24-well plate, Seq dataset in JURKAT cells treated with DMSO or USP7 inhibitor. and treated with DMSO or USP7i as indicated in the Figure Aligned bam files of H3K27Ac ChIP-Seq signal and input control Legends. Cells were counted each day via trypan blue, and were analyzed using the Rank Ordering of Super-Enhancers inhibitor/medium was changed. When cells reached a confluency algorithm (ROSE; refs. 63, 64). A stitching distance of 12.5Kb of >1 million cells/mL, cells were diluted 1:2, and this dilution was were used to stitch together enhancer regions, and regions within factored into the cell numbers for viability assays. For apoptosis 2.5Kb of a transcriptional start site were ignored in order to analysis, cells were stained with LIVE/DEAD Fixable Near-IR Dead prevent promoter bias. The initial set of enhancer regions used Cell Stain (Life Technologies) according to the manufacturer's in the analysis was determined by merging the peaks of H3K4me1 protocol, except that cells were stained for 20 minutes at 4C, prior and H3K27Ac in CUTLL1 cells (ChIP-Seq dataset for H3K4me1 to staining with PE-conjugated Annexin V (Life Technologies) in signal and H3K27Ac was downloaded from GEO with the

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accession numbers GSM732910 and GSM1252938 for H3K4me1 was administered on days 1 to 5 and 8 to 11 intravenously, and on and H3K27Ac, respectively). days 12 and 15 to 22 intraperitoneally. Blood was collected via tail nick on days 17 and 22 to measure tumor burden by staining for Gene set enrichment analysis hCD45. Gene set enrichment analysis (GSEA) was done using the For toxicity studies, 8-week-old NOD.Cg-Prkdcscid male Broad Institute GSEA software (http://software.broadinstitute. mice (#005557; Jackson Laboratories) were randomly assigned org/gsea/index.jsp; refs. 65, 66). RNA-Seq gene expression data to treatment groups (3 mice/group). Mice were treated with were used to create a preranked order of genes, where genes either vehicle (10% DMSO, 2% Tween-80, and 10% captisol in were sorted using the P value of the differential expression PBS), 10 mg/kg USP7i, 10 mg/kg USP7i þ 10 mg/kg GSKJ4, between USP7 inhibition and DMSO treatment, and gene up- 10 mg/kg USP7i þ 20 mg/kg GSKJ4, or 10 mg/kg USP7i þ 50 or downregulation upon USP7 inhibition was determined. In mg/kg GSKJ4 i.v. daily for 5 days to evaluate treatment toxicity. brief, the genes were ranked by calculating –log(P value)sign For combination treatment studies, 1 million JURKAT T-ALL (logFC), and a PreRanked GSEA analysis was done using the cells were injected subcutaneously into the right flank of 8- Hallmark database and the standard weighted enrichment week-old NOD.Cg-Prkdcscid male mice (#005557; Jackson statistic with 5,000 permutations. Enriched datasets with a Laboratories) with an equal volume of BD Matrigel, at 50 mL FDR < 0.05 were considered statistically significant. of cells to 50-mL Matrigel. After tumors reached 150 to 200 mm3, mice were randomly assigned to treatment groups and Intravenous and subcutaneous xenograft studies treated intravenously with vehicle (10% DMSO, 2% Tween 80, All mice were housed in a barrier facility, and procedures were and 10% captisol in PBS; n ¼ 10), 10 mg/kg USP7i (n ¼ 6per performed as approved by the Northwestern University Institu- inhibitor), or 10 mg/kg USP7i þ 50 mg/kg GSKJ4 (n ¼ 10) tional Animal Care and Use Committee (protocol Ntziachristos 5timesthefirst week, and then 3 times per week until tumors #IS00002058 and Mazar #IS00000556) and the Ghent University reached 1.5 cm3. Animal Ethics Committee (protocol #ECD16/56). Analyses were performed using GraphPad Prism software For CUTLL1 T-ALL subcutaneous studies, 0.7 million (GraphPad Software). Statistical comparisons were made using (PLKO.1 and shUSP7.1) cells were injected subcutaneously the Student unpaired, 2-sided t test. P values <0.05 were consid- into the right flank of 8-week-old NOD.Cg-Prkdcscid male ered statistically significant. mice (#005557; Jackson Laboratories, Portage, MI) with an m m equal volume of BD Matrigel, at 50 L of cells to 50- L Complete blood cell count analysis Matrigel. Body weight and tumor size (via calipers) were Mouse blood samples were collected via cardiac puncture and measured 3 times per week. run on a Hemavet 950 FS (Drew Scientific, Inc.) to obtain blood For JURKAT T-ALL subcutaneous studies, 0.7 million cells were cell counts. injected subcutaneously into the right flank of 8-week-old NOD. Cg-Prkdcscid male mice (#005557; Jackson Laboratories) with an Mouse histology equal volume of BD Matrigel, at 50 mL of cells to 50-mL Matrigel. This was performed as described in the past by Milano and The following day, mice were randomly assigned to treatment colleagues (67), and Real and colleagues (11). groups (7 mice/group), and were treated with either DMSO or 10 mg/kg USP7i 3 times per week intraperitoneally for 8 doses. One week later, mice were treated 5 times per week intraperito- Drug synergism neally for 17 days. Body weight and tumor size (via calipers) were A total of 3,000 cells per well were seeded using a microplate measured 3 times per week. Dispenser (MultiFlo, BioTek) in 384-well clear bottom, black wall For JURKAT T-ALL intravenous transplantation studies, 1 mil- plates (Corning). Drugs were added using the Tecan D300e digital lion cells were injected via tail vein into 8-week-old NOD.Cg- dispenser (Tecan). After 72-hour incubation, alamarBlue cell Prkdcscid male mice (#005557; Jackson Laboratories) in 100-mL viability reagent (ThermoFisher) was added and viability was fi fl PBS. Animals were monitored by IVIS every 3 days until luciferase quanti ed by measuring uorescence in a plate reader (Tecan fi signal was detected, and then animals were randomly assigned In nite m1000 pro, Ex: 530 nm; El: 590). Synergy analysis was to treatment groups (9 mice/group). Mice were treated 3 times per conducted using Compusyn software (68). week intraperitoneally with DMSO or 10 mg/kg USP7i (in 10% DMSO þ 2% Tween 80) for up to 10 doses. IVIS images were taken Ub-VME based DUB activity assay twice per week (Perkin Elmer) and animal weight was measured The studies were performed as previously described (69). 3 times per week to ensure accurate dosing. JURKAT cells were treated with compound or DMSO for 5 hours. For primagraft studies, 1.2 million primary human T-ALL cells Cells were washed once with PBS, harvested, and lysed in 1% (from the spleen of leukemic primary xenograft engrafted with NP40 lysis buffer (50 mmol/L Tris-HCl, pH 7.5, 150 mmol/L cells of T-ALL patient FV, N16/0267) in 150-mL PBS were injected NaCl, 1% NP40, 10% Glycerol, 1 mmol/L PMSF, 2 mmol/L via tail vein into 1.5-month-old female NGS mice (Jackson b-ME). Twenty micrograms of lysate per sample was incubated Laboratories). Two weeks after transplantation, blood was col- at 37 C for 30 minutes with Ub-VME at a final concentration of lected via tail nick for analysis of hCD45 expression (engraftment) 400 nmol/L in a total reaction volume of 10 mL. The reaction was using PE-conjugated mouse anti-human CD45 antibody (Milte- stopped by adding 2 mLof6 SDS sample buffer and boiling for 5 nyi Biotec; clone 5B1). Flow cytometry data were analyzed using minutes, followed by SDS-PAGE electrophoresis, and transfer to Flowjo software. One week later, mice were randomly divided PVDF membranes. The blots were probed with antibodies against into 2 groups (7 mice/group) and treated with either DMSO or USP7 and USP24 to determine the formation of DUB-Ub-VME 10 mg/kg USP7i in 2% Tween 80 þ 10% DMSO in PBS. Treatment complex.

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Results Mov34/Mpr1 Pad1 N-terminalþ (MPNþ) (JAMM, 14) domain To identify potential NOTCH1/JMJD3-associated proteins and superfamily proteins that binds zinc (metalloproteases). understand NOTCH1 complex stability in leukemia, we retro- USP7 (32–43) was a top interacting partner of JMJD3. USP7 virally expressed hemaglutinin (HA) tagged-JMJD3 in CCRF-CEM might also interact with NOTCH1 (75) and has a demonstrated T-ALL cells. We performed LC/MS-MS following immunoprecip- role in other hematologic malignancies, in multiple myeloma in itation (IP) of HA-JMJD3 (Fig. 1A). One hundred and one particular (76). We confirmed the interaction of NOTCH1 and proteins were uniquely associated with HA-JMJD3, and these JMJD3 with USP7 in reciprocal IP experiments (Fig. 1C and D). proteins were enriched in members of the ubiquitin-specific USP24 and USP9x (77), in contrast, have not been found to protease (USP) family of DUBs, including USP7, USP9X, and interact with NOTCH1 (75). Indeed, our pull-down studies for USP24 (Fig. 1B; Supplementary Table S1; Supplementary Fig. NOTCH1, followed by detection of USP24 and USP9x in Western S1A). Indeed, gene ontology analysis of the JMJD3 interactome blot experiments, showed that neither USP9x nor USP24 interact showed enrichment in proteins involved in various metabolic with NOTCH1 (Supplementary Fig. S1C and S1D). Furthermore, processes, cell cycle progression and members of the proteasome our studies show that, in contrast to USP7 and NOTCH1, both complex (Supplementary Fig. S1B). USP9x and USP24 are mainly localized in the cytoplasm (Sup- Dynamic NOTCH1 (de)ubiquitination plays a pivotal role in plementary Fig. S1E). the regulation of NOTCH1 protein levels in leukemia, and Given the potential participation of USP7 in NOTCH1 protein ubiquitin ligases such as FBXW7 have been demonstrated to complexes, we studied the levels of USP7 mRNA in cancer using regulate NOTCH1 degradation and are tumor suppressors in data from 176 cancer cell lines (cancer cell line encyclopedia). Our T-ALL (70, 71). To this end, we hypothesized that USPs might analysis showed that USP7,likeNOTCH1, is significantly over- be acting as oncogenic co-factors for the NOTCH1 complex, expressed in T-ALL compared with other hematologic and solid controlling complex stability and, ultimately, transcriptional tumors (Fig. 2A). We then used reverse phase protein array (RPPA) potency. Ubiquitination occurs through a sequence of enzy- analysis (78, 79) to quantify NOTCH1 and USP7 protein levels in a matic reactions involving the activity of 2 E1 ubiquitin- panel of 64 pediatric leukemia patient samples. We noticed that activating enzymes, multiple E2 enzymes and hundreds USP7 and intracellular or total NOTCH1 levels significantly cor- (700) of E3 enzymes in humans. E3 ligases determine com- relate in T-ALL (Fig. 2B; Supplementary Fig. S2A). We then plex specificity (72). In contrast, DUBs (73, 74) mainly include hypothesized that NOTCH1 might directly bind and control the the ubiquitin-specificproteasesuperfamily(USP/UBP,58 USP7 gene locus. Analysis of ChIP-Seq data in CUTLL1 T-ALL cells members), the ovarian tumor (OTU, 14) superfamily, the showed that NOTCH1 directly binds the promoter of the USP7 Machado-Josephin domain (MJD, 5) superfamily, the ubiqui- gene (Fig. 2C). We also demonstrated that inhibition of NOTCH1, tin C-terminal hydrolase (UCH, 4) superfamily (all the afore- using an established method for NOTCH1 inhibition, using mentioned categories are cysteine proteases), and the Jab1/ gamma secretase inhibitors (GSI; refs. 11, 15), decreased NOTCH1

A B JMJD3 mass spectrometry JMJD3-1 Avg. peptide Accession Protein Figure 1. spectral matches 48 USP7 is a member of the JMJD3/ O15054 KDM6B 184 NOTCH1 complex in T-ALL. A, HA Q9UPU5 USP24 43 immunoprecipitation (for HA-tagged 13 JMJD3) followed by mass B7Z815 USP7 33 31 spectrometry in CCRF-CEM T-ALL Q6KC79-2 NIPBL 22 cells. Shown is the overlap of 101 P31689 DNJA1 20 HA-JMJD3-associated proteins across Q93008-1 USP9X 19 3 biological replicates, revealing 46 101 common proteins associated with Q6ZMT4 KDM7A 17 HA-JMJD3. B, Top interactors for 29 9 JMJD3, showing the average peptide JMJD3-2 JMJD3-3 spectral matches. Members of the USP family are among these top interacting proteins. C, Representative western blot studies following immunoprecipitation of HA-JMJD3 in C IP Inputs D CCRF-CEM T-ALL cells. Control cells IP express the HA-tagged vector. USP7 inputcon D, Western blot studies following

Control JMJD3 Control JMJD3 immunoprecipitation of USP7 in JMJD3 CUTLL1 T-ALL cells, showing JMJD3 interactions of JMJD3 and NOTCH1 USP7 with USP7. For all immune-blots USP7 presented here, 1 of 3 representative NOTCH1 biological replicates is shown.

ACTIN Actin-β

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binding enrichment on the USP7 gene locus as well as other well- As USP7 has a characterized enzymatic activity as deubiquiti- characterized NOTCH1 targets, DTX1 and NOTCH3 (Supplemen- nating enzyme, we hypothesized that it might control NOTCH1 tary Fig. S2B). Furthermore, GSI treatment or NOTCH1 silencing protein levels through active deubiquitination. We silenced USP7 using short hairpins against NOTCH1 (shNOTCH1), led to down- in T-ALL using short hairpin RNAs (shUSP7) and examined regulation of USP7 mRNA and protein levels and other well- NOTCH1 protein levels, to observe a decrease in the levels of characterized NOTCH1 targets (Fig. 2D; Supplementary Fig. NOTCH1, suggesting that USP7 might control NOTCH1 protein S2C–S2E). In contrast, NOTCH1 overexpression leads to a tran- stability (Fig. 3A). We also noticed a marked inhibition of T-ALL scriptional upregulation of USP7 (Supplementary Fig. S2F). growth upon shUSP7 compared with the control (Fig. 3A;

A B 15,000 R 2 = 0.61

) USP7 mRNA P < 0.0001 100 * 80 10,000 60 5,000 RPKM 40 N1IC protein levels (RPPA) 20

eads per kilobase 0

milion mapped reads 0 (r 0 25,00050,000 75,000100,000125,000150,000 T-ALL (17) USP7 protein levels (RPPA) Non T-ALL (159) NOTCH1 mRNA C 80 ) NOTCH1 150 **** 100 1 80 3.3 USP7 60 0

RPKM 40 2.6 H3K27ac 20 0 0 eads per kilobase

milion mapped reads 1.8 (r H3K4me3 0 T-ALL (17)

Non T-ALL (159) USP7

D 4 CUTLL1 DMSO vs. GSI

0 USP7 logFC −2 MYC

−4 DTX1 NOTCH3

−6 0 5 10 15 20 logCPM

Figure 2. USP7 is highly expressed in T-ALL and is transcriptionally regulated by NOTCH1. A, RPKM values for NOTCH1 and USP7 in 176 blood cancer cell lines were obtained from https://software.broadinstitute.org/morpheus/, using the CCLE RNAseq data. These data are available for download at https://portals.broadinstitute.org/ ccle_legacy/. These include several T-ALL cell lines, ALLSIL, DND41, HPBALL, HUT78, JURKAT, KE37, LOUCY, MOLT13, MOLT16, MOLT3, P12ICHIKAWA, PEER, PF382, RPMI8402, SUPT1, SUPT11, and TALL1. All other cell lines were analyzed against these T-ALL cell lines. Two-tailed unpaired t test was conducted using the RPKM values (, P < 0.05; , P < 0.0001). B, USP7 protein levels correlate with NOTCH1 intracellular (N1IC) levels in primary T-ALL (n ¼ 64). C, Tracks showing NOTCH1, USP7, and the activating histone marks H3K27Ac and H3K4me3 ChIP-Seq signal enrichment in T-ALL cells (CUTLL1), at the USP7 locus. D, Gene expression analysis from CUTLL1 RNA-Seq showing up- and downregulated genes (red and blue dots, respectively) upon GSI treatment, as well as genes with no statistically signiﬁcant expression changes (black dots, P < 0.05). NOTCH1 target genes showing statistically signiﬁcant down regulation upon GSI treatment are indicated in the plot. One of 3 representative biological replicates is shown.

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Figure 3. USP7 plays a critical role in T-ALL maintenance via regulation of NOTCH1 protein levels. A, CUTLL1 T-ALL cells were transduced with a control retroviral vector and 2 different shUSP7 (shUSP7.1 and shUSP7.2). Left: Protein levels of USP7 and NOTCH1 in CUTLL1 T-ALL cells (control, shUSP7.1,and shUSP7.2). Right: Growth study was performed after knocking down of USP7 in CUTLL1 cells. Data are shown as mean SD derived from 3 independent biological replicates; , P 0.05. B, IC50 curve of USP7i in several T-ALL cell lines (JURKAT, CUTLL1, HPB, and KOPTK). C, Growth curve of CUTLL1 T-ALL cells upon daily treatment with increasing concentrations of USP7i. Data are shown as mean SD derived from 3 independent experiments; , P 0.05. D, Representative Western blot studies following immunoprecipitation of Flag-NOTCH1 in 293T cells at denaturing condition. 293T cells were transfected with Flag- NOTCH1, ubiquitin, and FBXW7 associated with USP7 WT or USP7 CS as indicated. E, A representative Western blot analysis following immunoprecipitation of Flag- NOTCH1, conducted in 3 biological replicates, is shown. Flag-NOTCH1 overexpressed CUTLL1 T-ALL cells were treated with DMSO (control) or USP7i (5 mmol/L) for 8 hours, whole cell extracts were isolated under denaturing conditions to disrupt protein–protein interactions, followed by Flag-NOTCH1 immunoprecipitation. F, Western blot analysis of JMJD3, NOTCH1, and USP7 protein levels upon treatment of CUTLL1 T-ALL cells with increasing concentrations of USP7i for 24 hours. For all immune-blots presented, 1 of 3 representative biological replicates is shown.

Supplementary Fig. S3A and S3B). USP7 silencing led to increased icantly inhibited the growth of various T-ALL lines, including apoptosis and a decrease in S phase of T-ALL cells, as demon- CUTLL1, JURKAT, and CEM, in the micromolar (mmol/L) range strated using shUSP7 (Supplementary Fig. S3C and S3D). We then of concentrations, similarly to shUSP7 (Fig. 3B and C; Supple- took advantage of established USP7-small molecule inhibitors mentary Fig. S4A and 5A). The compound inhibits USP7 (USP7i, see also Materials and Methods), developed by Progenra activity in a dose-dependent manner from 1 to 5 mmol/L (Sup- Inc., and successfully used in preclinical models of multiple plementary Fig. 4B), without significantly blocking total DUB myeloma and other types of cancer and in immunologic contexts activity in T-ALL, even at 5 mmol/L concentrations (Supplemen- (57, 76, 80–84), to block USP7 activity in leukemia cells. The tary Fig. S4C). Treatment of T-ALL cells with USP7i for 1 and 3 efficacy and specificity of those compounds has been previously days coupled to apoptosis and cell-cycle analysis, using Annexin V demonstrated in several systems (57, 76, 80–84). USP7i signif- and DAPI staining respectively, led to a significant increase in

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apoptosis and a decrease in S phase (Supplementary Fig. S5B and We and others have shown that NOTCH1 activity leads to S5C), similarly to the shUSP7. eviction of the repressive mark H3K27me3 from the promoters of To confirm the role of USP7 in NOTCH1 stabilization, we oncogenic targets (15, 16, 87). We hypothesized that inhibition of expressed wild-type or a catalytic mutant of USP7 together with USP7 would lead to an increase in H3K27me3 levels through NOTCH1, and measured NOTCH1 expression in 293T cells. reduction of JMJD3 protein levels. We assessed the levels of Indeed, NOTCH1 was stabilized in the presence of wild-type H3K27me3 on USP7 targets upon USP7i treatment for 24 hours. USP7 (Fig. 3D, right). Similarly, we show that only the wild-type Indeed, USP7 and NOTCH1 target loci presented a significant but not the catalytically inactive USP7 can efficiently deubiqui- gain of H3K27me3 upon USP7i treatment (Fig. 4E; Supplemen- tinate NOTCH1 (Fig. 3D, left). These results show that USP7 and tary Fig. S8C). As USP7 has been shown to control histone its catalytic activity are critical for NOTCH1 deubiquitination and ubiquitination (88, 89), we also examined histone H2B lysine stabilization. To evaluate the role of USP7 in NOTCH1 ubiqui- 120 ubiquitination (H2Bub) levels following USP7 inhibition. tination in T-ALL, we inhibited USP7 catalytic activity, pulled We showed that USP7 colocalized with genes exhibiting a gain of down ubiquitinated proteins using tandem ubiquitin binding H2B ubiquitination upon USP7 inhibition (Fig. 4E; Supplemen- entity (TUBE) technology (85), and measured NOTCH1 levels tary Figs. S8C and S9; refs. 90, 91). These changes in H2Bub and (Supplementary Fig. S6A). We then performed similar cell treat- H3K27ac/me3 seemed to localize to USP7 targets and not a ments followed by Flag-tagged NOTCH1 pull-down and detec- genome-wide phenomenon, as global levels of these marks were tion of ubiquitination using antibodies against lysine 48 (K48)- not significantly altered following treatment with USP7i as we linked poly-ubiquitin chains, a type of polyubiquitination cou- demonstrated using global bottom-up proteomics (Supplemen- pled to protein degradation through proteasomal activity (86). tary Fig. S10; ref. 92). Changes in H2A lysine 119 ubiquitination We demonstrated an increase of NOTCH1 poly-ubiquitination (H2Aub) on downregulated loci were minimal but detectable, upon USP7i treatment (Fig. 3E). NOTCH1 is a critical target of potentially associated to H3K27me3 changes (Fig. 4E; Supple- USP7, as NOTCH1 overexpression partially rescues USP7i-asso- mentary Figs. S8C and S9; ref. 93). ciated blockade in T-ALL growth and expression of NOTCH1 Our data suggest that USP7 regulates gene expression of critical targets in T-ALL cells (Supplementary Fig. S7A–S7C). Similar to NOTCH1 targets. To delineate the molecular effects of USP7 NOTCH1, we demonstrated a significant decrease in the levels of activity, we performed global expression analysis using RNA-Seq JMJD3 upon silencing of USP7 (Supplementary Fig. S6B). Treat- upon treatment with USP7i, and comparison with GSI (NOTCH1 ment with USP7i led to a similar decrease in NOTCH1 and JMJD3 inhibitor), and GSKJ4 (JMJD3 inhibitor). The transcriptional protein levels, starting at 1 mmol/L USP7i (Fig. 3F). Likewise, signatures of NOTCH1, USP7, and JMJD3 chemical inhibition inhibition of USP7 in T-ALL cells leads to an increasing of K48- overlapped significantly, both with regards to downregulated as linked ubiquitination of JMJD3 (Supplementary Fig. S6C). JMJD3 well as upregulated genes (Fig. 5A and B, top; Supplementary Fig. overexpression using a retroviral system could not rescue the effect S11A–S11C). Specifically, inhibition of USP7 led to downregula- of the drug on leukemic cells (Supplementary Fig. S7D), poten- tion of NOTCH1 targets, including NOTCH3 and DTX1 (Fig. 5B tially due to the simultaneous requirement for high levels of and C; Supplementary Fig. S11C). Similar to enzymatic inhibi- NOTCH1 expression for leukemia maintenance. tion, USP7 silencing led to downregulation of NOTCH1 targets Because of the activity of intracellular NOTCH1 (N1-IC) on (Fig. 5A and B; bottom) and significant overlap with USP7i chromatin, we evaluated genome-wide USP7 binding via chro- treatment (Supplementary Fig. S11D). Gene set enrichment anal- matin immunoprecipitation combined with sequencing (ChIP- ysis upon USP7 inhibition showed that major oncogenic path- Seq). We identified 10,000 high-confidence peaks for USP7 that ways (NOTCH1, MYC, DNA damage response, and metabolism) localized near promoters (Fig. 4A) and had a strong overlap with were downregulated (Fig. 5D; Supplementary Fig. S12). Together, NOTCH1 binding sites (Fig. 4B). Both USP7 and NOTCH1 bound these data show that USP7 enzymatic activity is important for to classical NOTCH1 targets, such as DTX1 and NOTCH3, as well sustaining oncogenic activity in T-ALL. as NOTCH1 itself (Supplementary Fig. S8A). Pathway analysis of It is well known that NOTCH1 regulates oncogenic enhancers the ChIP-Seq data showed a striking enrichment for NOTCH1 (94, 95). Analysis of USP7 ChIP-Seq data showed that leukemia- signaling (Fig. 4C). Furthermore, NOTCH1 recruited USP7 to specific SEs (65, 96), as determined by H3K27 acetylation signal chromatin, as treatment with GSI for depletion of intracellular intensity (Supplementary Fig. S13A), were enriched for binding of NOTCH1 levels significantly decreased chromatin-bound USP7 USP7 and NOTCH1: 192 out of 254 leukemia SEs were cobound levels (Fig. 4D). USP7 controls NOTCH1 stability and inhibition by USP7 and NOTCH1. In agreement with the association of SEs of USP7 led to a reduction of global NOTCH1 levels and deple- with transcriptional activation, we found that the USP7-bound tion of NOTCH1 from chromatin (Fig. 4D). We further performed genes were more highly expressed compared to the rest of the USP7 and NOTCH1 ChIP studies focusing on USP7 targets upon transcriptome (Supplementary Fig. S13B). We hypothesized that USP7 inhibition. Our results show that NOTCH1 and USP7 combinatorial inhibition of USP7 and the SE component BRD4 binding to chromatin is significantly affected upon USP7i treat- using JQ1 inhibitors might be an additive or synergistic effect in ment whereas binding of another T-ALL transcription factor, inhibiting T-ALL growth. Indeed, our studies using combinatorial RUNX1 (used as control) remained largely unaffected (Supple- inhibition of the bromodomain proteins that recognize acetylat- mentary Fig. S8B). Global cellular levels of USP7 did not change ed histones (JQ1; refs. 97, 98) and USP7 showed a synergistic over the period of treatment (Fig. 4D). These findings suggest that effect (Supplementary Fig. S13C) on T-ALL inhibition. This was USP7 and NOTCH1 form a positive feedback loop (see also our further underlined by our targeted and genome-wide analysis of discussion), where NOTCH1 induces USP7 gene expression and USP7 interactions showing that USP7 interacts with Mediator 1, a then recruits USP7 to chromatin, resulting in increased expression main component of SEs (Ntziachristos group, unpublished), of NOTCH1 targets, through stabilization of the NOTCH1 suggesting that USP7 may regulate gene activation via interactions complex. with the T-ALL transcriptional machinery in SE areas.

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These findings show that USP7 inhibition is a valid therapeutic icant finding led us to assess the potential of chemical inhibition tool in high-risk leukemia, where other treatments have failed to of USP7 alone or in combination with JMJD3, using the GSKJ4 lead to significant disease regression. Silencing of USP7 in human: compound, to block leukemia growth in vivo. Because of the role mouse xenograft models, using CUTLL1 T-ALL cells, led to a of the H3K27me3 epigenetic mark in the regulation of NOTCH1 significant inhibition of tumor growth in animals (Fig. 6A) targets in T-ALL progression, we and others have successfully used coupled to an extension of mouse survival (Fig. 6B). This signif- GSKJ4 to target JMJD3 in T-ALL in the past with decent effect on

A USP7 B USP7 NOTCH1 ChIP-Seq 0 10 Peaks Peaks

4 Gene 6 body 5 3 4 56124388 4580 2 3 2 1

mapped reads 1 0 0 P < 0.01 Reads count per million -3000 TSS 33% 66% TES +3000 −3000 NOTCH1 +3000 USP7 peaks Peaks C Pathway enrichment analysis −log10 (Hypergeometric P) 012345678910 Notch signaling 10.81 CXCR3 signaling 6.19 NOTCH3 signaling 4.69 NOTCH1 signaling 3.86 Thrombin signaling 3.54

D USP7i GSI E HES1 Control 21 1 (μmol/L) DMSO 6 USP7i 2 μmol/L USP7 5 4 Chromatin NOTCH1 3 bound 1.5 1.0 ** ** *** LAMIN 0.5

P ercentage of input 0 USP7 b b c IgG H2Au H2Bu NOTCH1 Whole cell H3K27aH3k4me1 extracts H3K27me3 ACTIN MYC enhancer 8 7 6 1.5 USP7 5 * Control 4 GSI 3 1.0 1.5 * ** ** 1.0 0.5 0.5

P ercentage of input 0 0 3

Relative protein levels IgG Chromatin-bound Whole cell H2AubH2Bub extracts extracts H3K27acH3K4me1 H3K27me

Figure 4. USP7 and NOTCH1 co-bind oncogenic targets. A, Heatmap of USP7 peak intensity (left) and metaplots showing the average counts per million of USP7 ChIP-Seq signal across all genes (middle), and NOTCH1 peaks (right). One of 3 representative biological replicates is shown.B,Analysis of ChIP-Seq data showing the overlap of USP7 peaks with NOTCH1 peaks. C, GREAT (Genomic Regions Enrichment of Annotations Tool) analysis showing enrichment for speciﬁc gene functions amongst the USP7-bound peaks used in B. D, Top: representative protein levels of USP7 and NOTCH1 in the chromatin-bound fraction and whole cell extracts of JURKAT T-ALL cells upon treatment with DMSO (control), USP7i, or GSI. One of 3 biological replicates is shown. Bottom: quantiﬁcation of USP7 protein levels in top. E, CUTLL1 T-ALL cells were treated with DMSO or USP7i 2 mmol/L for 2 days. ChIP of indicative histone marker coupled to detection by quantitative real-time PCR at indicative loci were presented. Data are shown as mean SD derived from 3 independent experiments: , P 0.05; , P 0.01; , P 0.001.

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A Down Down B DMSO USP7i GSI 1.5 USP7i 5 μmol/L

1.0

379 124 1611 0.5

P < 0.001 Relative mRNA level 0

DTX1 C-MYC HES1 USP7 NRARP NOTCH1NOTCH3 Down Down shUSP7.1 GSI 1.5 PLKO.1 shUSP7.1

1.0

447 298 1437 0.5 Relative mRNA level 0 P < 0.001

DTX1 C-MYC HES1 USP7 NRARP NOTCH1 NOTCH3

C D FDR = 0.021 CUTLL1 DMSO vs. USP7i NOTCH signaling

2 logFC

0 MYC

−2 NOTCH3 DTX1 −4 Rank in ordered dataset 0 5 10 15 20 logCPM

Figure 5. USP7 and NOTCH1 control the expression of oncogenic targets. A, Analysis of RNA-Seq data showing the overlap of downregulated genes upon USP7 inhibition (1 mmol/L) and GSI (1 mmol/L) treatment in CUTLL1 T-ALL cells (top). Analysis of RNA-Seq data showing the overlap of downregulated genes upon GSI (1 mmol/L) and knockdown of USP7 in CUTLL1 T-ALL cells (bottom). RNA-Seq data are the average of 3 biological replicates. B, Top: CUTLL1 T-ALL cells were treated with DMSO or 5 mmol/L of USP7i for 3 days. NOTCH1 targets were determined by RT-PCR at mRNA level. Bottom: RT-PCR of NOTCH1 targets upon treatment of CUTLL1 T-ALL cells with shUSP7.1 and a control retroviral vector. Data are shown as mean SD derived from 3 independent experiments. C, Gene expression data from CUTLL1 RNA-Seq showing up- and downregulated genes (red and blue dots, respectively) upon USP7i treatment, as well as genes with no statistically signiﬁcant expression changes (black dots, P < 0.05). NOTCH1 target genes are indicated in the plot. One of 3 representative biological replicates is shown.D,Gene set enrichment analysis for downregulated genes upon USP7 inhibition. Shown is the NOTCH1 signaling pathway that is signiﬁcantly downregulated in USP7i versus control cells (false discovery rate [FDR] ¼ 0.021). leukemia inhibition (16, 99). We initially evaluated inhibitor mined by complete blood cell count, body weight, and analysis of toxicity by injecting mice intravenously with vehicle, USP7i alone spleen and liver (Supplementary Fig. S14A and data not shown). (10 mg/kg body mass), or USP7i with increasing concentrations We also examined USP7i-treated animals for gastrointestinal (GI) of GSKJ4 (up to 50 mg/kg body mass). The GSKJ4 concentrations toxicity as GSI treatment for direct NOTCH1inhibition leads to used have been previously shown to successfully inhibit tumor goblet cell metaplasia in animals. Our studies show no signs of growth (99, 100). Immunocompromised mice treated daily for 5 toxicity associated with USP7i inhibition in the GI track (Supple- days showed no signs of treatment-associated toxicity, as deter- mentary Fig. S14B). We then transplanted luciferase-expressing

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JURKAT T-ALL cells either intravenously or subcutaneously into (Fig. 6C; Supplementary Fig. S15A). Similar results were obtained immunocompromised mice (15, 16). Upon tumor detection using a primagraft intravenous model using a patient sample with using bioluminescence imaging (IVIS), mice were randomized mutations affecting the gene of the main ubiquitin ligase for into different groups that were treated with vehicle or USP7i (10 NOTCH1, FBXW7, and high NOTCH1 expression levels as well mg/kg). In both models, tumors in USP7i-treated animals showed as CCND3 mutation (Supplementary Table S2), where T-ALL a signiﬁcant growth disadvantage compared to the control group progression was detected in the peripheral blood using the marker

PLKO.1 20 * ) 1,500

3 shUSP7.1

15 1,000 * 10 500

total ﬂux [p/s] 5 Tumor volume (mm 0 0 Fold change (day 7 to day 21), 0101520255 PLKO.1 shUSP7.1 Days post inoculation

Radiance B PLKO.1 (n = 6) C 2 Vehicle USP7i (p/sec/cm /sr) shUSP7.1 (n = 6) 4×105 80 P = 0.031

5 100 3×10 60

P = 0.0015 Day 7 2×10 40 50 1×105

total ﬂux [p/s] 20 8×106 0

6 Fold change (day 7 to day 21), Percent mouse survival 0 6×10 02030405010 Vehicle USP7i Days post inoculation 4×106 Day 21 2×106

D Vehicle 100 400 ** ** USP7i *** 300 USP7i/GSKJ4 , day 28) 3

50 200

*** * 100 Percent mouse survival 0 * 0

040608020 Tumor volume (mm Vehicle USP-7i USP-7i/GSKJ4 Days posttransplantation

Figure 6. USP7 inhibition blocks leukemia growth in preclinical models of T-ALL. A, Luciferase-expressing CUTLL1 cells were transduced with a control or shUSP7.1 retroviral vectors. CUTLL1 T-ALL cells (PLKO.1 and shUSP7.1) were injected subcutaneously into immunocompromised mice. Left: Fold change in total ﬂux from day 7 to day 21 is shown (PLKO.1, n ¼ 6; shUSP7.1, n ¼ 6). Right: tumor volume for a period of 21 days is shown; , P 0.05. B, Mouse survival for the PLKO.1 and shUSP7.1 groups. C, JURKAT T-ALL cells were injected intravenously into immunocompromised mice. Upon detection of leukemic blasts using bioluminescence (with IVIS), mice were treated intraperitoneally with 10 mg/kg USP7i 3 days/week for 3 weeks. Relative luminescence intensity is shown for 2 representative mice per treatment group on days 7 and 21 of treatment. The fold change in total ﬂux from day 7 to day 21 is shown on the right (vehicle, n ¼ 8; USP7i, n ¼ 9). D, Immunocompromised mice were injected subcutaneously with JURKAT T-ALL cells. Once tumors became visible by IVIS, mice were administered USP7i (10 mg/kg body mass), alone or in combination with GSKJ4 (50 mg/kg body mass), intravenously, 3 times/week till termination of the study. Shown is mouse survival (bottom left) and the mean tumor volume SD (right) for the different groups (vehicle, n ¼ 7; USP7i, n ¼ 5; and USP7i/GSKJ4, n ¼ 8). , P 0.05; , P 0.01; , P 0.001.

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human CD45 (Supplementary Fig. S15B and S15C). To further note, there are no hitherto identified ankyrin mutants for potentiate the therapeutic impact of USP7i in T-ALL, we investi- NOTCH1 in T-ALL (10). We mined 264 T-ALL patient data gated the effect of combinatorial USP7 and JMJD3 inhibition on available through the pediatric cancer genome project (PeCan) leukemia growth by treating mice with USP7i (10 mg/kg body data portal (https://pecan.stjude.cloud/home), in our search for mass) and GSKJ4 (50 mg/Kg body mass) in a subcutaneous model NOTCH1 mutations. No mutations in the ankyrin domain of of T-ALL and monitored animal survival (Fig. 6D). Similar to our NOTCH1 were identified, in agreement with published evidence. previous studies (Fig. 6C; Supplementary Fig. S15B), we found A better understanding of H3K27me3 demethylation and H2B that USP7i as a single therapy significantly inhibits tumor growth deubiquitination changes and whether levels of those 2 marks are (Fig. 6D, right) and extends mouse survival (Fig. 6D, left). More- intertwined provide us with information useful in designing over, combinatorial inhibition significantly decreased tumor epigenetic therapies. Intriguingly, and in contrast to the previ- growth, more effectively than vehicle or USP7 inhibition alone ously characterized pro-oncogenic role of USP7 in T-ALL and (Fig. 6D, right) without any associated toxicity based on our multiple myeloma (76), neuroblastoma (81), chronic lympho- analysis of mouse weight (Supplementary Fig. S15D) and histol- cytic leukemia (101), and other solid tumors (102–104), there are ogy studies (not shown). Moreover, we demonstrated significant mutations affecting USP7 in pediatric cancers, TAL1-positive survival differences between mice injected with vehicle or those leukemias in particular (105–107). Moreover, it is known that injected with a combination of USP7 and JMJD3 inhibitors (Fig. USP7 can have different substrates, including PTEN (35), and the 6D, left). USP7i/GSKJ4 treatment yielded a prolonged mouse p53–HDM2 axis (36–38, 108, 109). Thus, similarly to what we survival compared to vehicle or USP7i therapy (Fig. 6D, left and and others have shown for another epigenetic modulator, the data not shown), lending rationale to the use of combinatorial histone demethylase UTX, which can play dual roles (oncogene or drug treatments against epigenetic regulators in T-cell leukemia. tumor suppressor) in NOTCH1- and TAL1-postitive contexts of T-ALL (16, 99, 69), the role of USP7 might also be context-specific. These context-specific roles of the epigenetic modulators are Discussion another intriguing aspect of their biology that we can exploit to Therapeutic targeting of high-risk ALL has been challenging, develop elegant targeted therapeutic approaches in cancer. Fur- rendering this disease an unmet clinical need. In this study, we ther research is needed to study the role of USP7 in leukemia performed a series of epigenetic, biochemical, functional and contexts other than NOTCH1-positive T-ALL, such as TAL1- pharmacologic studies to show that the DUB USP7 functionally positive leukemia. and physically interacts with and controls the NOTCH1 pathway Our findings strongly suggest that USP proteins, and USP7, in and ultimately the oncogenic transcriptional circuitry in T-cell particular, may be exploited for pharmacological inhibition in ALL. USP7 inhibition of its enzymatic activity could be a valid certain T-ALL patients. Previous studies using the same USP7 therapeutic target in this disease. inhibitor backbone in mouse models showed that the drug Given the previously unrecognized importance of deubiquiti- inhibits USP7 in specific physiological mouse systems and in nation in acute leukemia progression, we characterized a novel mouse tumor cells (57, 81, 110). This is not surprising as the role for USP7 in controlling NOTCH1 stability T-ALL, providing USP7 gene/protein is significantly conserved between human and the first evidence that the ubiquitination–methylation axis can mouse. Although further studies are needed to directly compare serve as an oncogenic switch and therapeutic target in T-ALL. USP7 the effect of the drug on human and mouse tissues, these pub- is the first DUB identified and thoroughly characterized to regu- lished and our findings suggest that USP7 compounds might have late the NOTCH oncogenic pathway. Our studies showed that significant efficacy with minimal toxicity in clinical trials against USP7, NOTCH1, and JMJD3 act in a positive feedback loop patients with T-ALL. The therapeutic window for USP7 inhibition (Supplementary Fig. S16), where the NOTCH1/JMJD3 complex in NOTCH1þ T-ALL might be explained by the significantly higher induces expression of USP7, and it subsequently results in USP7 levels of USP7 in this disease compared to physiologic tissues. recruitment to target genes. Ultimately, USP7 recruitment leads to Combinations of targeted therapies are hailed as the future of stabilization of the oncogenic complex and activation of targets cancer therapy, with hematological malignancies leading the way through demethylation of H3K27 and deubiquitination of H2B, (i.e. bortezomib, lenalidomide, and dexamethasone for multiple as well as changes in H3K27Ac at SEs. Past and ongoing studies in myeloma, and rituximab and ibrutinib for chronic lymphocytic our group aim at mapping NOTCH1 areas that interact with leukemia). As epigenetic modulators are 1 of the main gene USP7. NOTCH1 molecules interact with and are controlled by families controlling tumor biology, we feel our study can provide the E3 ubiquitin ligase FBXW7 via their PEST domain and engi- insights in the use of DUBs inhibitors and combination with neered PEST mutants (theonine 2512 to alanine, T2512A) for chemotherapy or other epigenetics inhibitors for therapy in NOTCH1, are impervious to FBXW7 regulation (70, 71). In leukemia. Three recent studies characterized small molecule inhi- contrast, we demonstrate that this NOTCH1 mutant interacts bitors with efficacy against USP7 in different cancer contexts, with USP7, suggesting that other parts of the NOTCH1 intracel- including colorectal carcinoma, osteosarcoma and prostate cancer lular fragment might control its binding to and regulation via (111–113). Thus, combined inhibition of USP7 and JMJD3 may USP7 (Supplementary Fig. S17A). To this end, we generated a represent an attractive therapeutic approach for T-ALL. series of NOTCH1 truncations to demonstrate that the ankyrin domain of NOTCH1 (amino acids 1850–2123) mediates inter- fl action with USP7 (Supplementary Fig. S17B). Although further Disclosure of Potential Con icts of Interest studies might be required to further map the exact interacting N. Hijiya reports receiving speakers bureau honoraria from Novartis. fi N. L. Kelleher is an employee of Quest Diagnostics and Thermo Fisher, and amino acid regions on USP7 and NOTCH1, our ndings poten- is a consultant/advisory board member for Quest Diagnostics. J. Weinstock is an tially suggest a novel mode of post-translational regulation of employee of Progenra. No potential conflicts of interest were disclosed by the NOTCH1 independent of the PEST domain and FBXW7 ligase. Of other authors.

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Jin et al.

Data availability Study supervision: Q. Jin, N.L. Kelleher, S. Kumar, P. Ntziachristos The raw files and the Proteome discoverer results are now accessible at Other (synthesize compounds): C. Grove MassIVE accession: MSV000081771 and ProteomeXchange accession: Other (performed experiments and contributed ideas): R. Rawat fi PXD008330. For now, the log in information for reviewers at MassIVE is: Other (performed experiments): V. Sera n Username: MSV000081771_reviewer Other (clinical data): G. Basso Password: reviewersonly Other (design some of the compounds which were used): J. Weinstock The raw MS and skyline files for the epiproteomics analysis are deposited to PeptideAtlas (http://www.peptideatlas.org/) Official URL for this dataset: ftp://PASS01134:[email protected]. org/ To access files via FTP, use credentials: Acknowledgments Servername: ftp.peptideatlas.org This work was supported by the NIH T32CA080621-11 Oncogenesis and Username: PASS01134 Developmental Biology Training Grant and Cancer Smashers Postdoctoral Password: HG6634ca Fellowship (to K.M. Arcipowski), and by the National Cancer Institute (R00CA188293-02), the American Society of Hematology, the Leukemia Authors' Contributions Research Foundation, the St. Baldrick's Foundation, the H Foundation, the Gabrielle's Angel Foundation, and the Zell Foundation (to P. Ntziachristos). Conception and design: Q. Jin, K.M. Arcipowski, Y. Zhu, B.T. Gutierrez Diaz, This work is also supported by AIRC IG 19186 grant to G. Berx. High- M.R. Johnson, A.G. Volk, S. Kumar, J.D. Crispino, P. Ntziachristos throughput sequencing data have been deposited into Gene Expression Development of methodology: Q. Jin, Y. Zhu, B.T. Gutierrez Diaz, K.K. Wang, Omnibus, accession number GSE97435. Proteomics services were per- A.G. Volk, I. Kandela, N.L. Kelleher, S. Kumar, , P. Ntziachristos formed by the Northwestern proteomics core, generously supported by NCI Acquisition of data (provided animals, acquired and managed patients, CCSG P30 CA060553 awarded to the Robert H Lurie Comprehensive Cancer provided facilities, etc.): Q. Jin, K.M. Arcipowski, Y. Zhu, K.K. Wang, Center and the National Resource for Translational and Developmental M.R. Johnson, A.G. Volk, F. Wang, C. Grove, H. Wang, I. Sokirniy, P.M. Thomas, Proteomics supported by P41 GM108569. For some of the in vivo studies Y. Ah Goo, N.A. Abshiru, N. Hijiya, S. Peirs, N. Vandamme, G. Berx, S. Goosens, we collaborated with the Developmental Therapeutics Core (DTC), also S.A. Marshall, E.J. Rendleman, Y.-H. Takahashi, R. Rawat, B. Ueberheide, supported by Cancer Center Support Grant P30 CA060553 from the Nation- C. Mantis, I. Kandela, J.-P. Bourquin, B. Bornhauser, S. Bresolin, M. Paganin, al Cancer Institute. B. Accordi, G. Basso, , P. Ntziachristos We want to thank all members of the Ntziachristos laboratory for critical Analysis and interpretation of data (e.g., statistical analysis, biostatistics, review of the manuscript and their comments. We want to thank Miklos Bekes computational analysis): Q. Jin, C.A. Martinez, K.M. Arcipowski, B.T. Gutierrez and Tony Huang (New York University) for providing USP7-expressing Diaz, K.K. Wang, A.G. Volk, F. Wang, I. Sokirniy, P.M. Thomas, Y. Ah Goo, plasmids. N. Hijiya, S. Peirs, S.A. Marshall, E.T. Bartom, C.K. Collings, S. Kelly, B. Ueberheide, I. Kandela, S. Bresolin, B. Accordi, G. Basso, N.L. Kelleher, S. Kumar, J.D. Crispino, , P. Ntziachristos The costs of publication of this article were defrayed in part by the Writing, review, and/or revision of the manuscript: Q. Jin, C.A. Martinez, payment of page charges. This article must therefore be hereby marked K.M. Arcipowski, B.T. Gutierrez Diaz, M.R. Johnson, A.G. Volk, Y. Ah Goo, advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate N. Hijiya, S. Peirs, R. Rawat, B. Accordi, S. Kumar, J.D. Crispino, , P. Ntziachristos this fact. Administrative, technical, or material support (i.e., reporting or organizing data, constructing databases): Q. Jin, Y. Zhu, B.T. Gutierrez Diaz, K.K. Wang, M. Received June 5, 2018; revised July 18, 2018; accepted September 11, 2018; R.Johnson, J.Wu, C. Grove,L. Wang, A. Strikoudis, A. Shilatifard, , P.Ntziachristos published first September 17, 2018.

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OF18 Clin Cancer Res; 2018 Clinical Cancer Research

USP7 Cooperates with NOTCH1 to Drive the Oncogenic Transcriptional Program in T-Cell Leukemia

Qi Jin, Carlos A. Martinez, Kelly M. Arcipowski, et al.

Clin Cancer Res Published OnlineFirst September 17, 2018.

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