small-molecule inhibitors of usp1 target id1 degradation in ......small molecule therapeutics...

Small Molecule Therapeutics

Small-Molecule Inhibitors of USP1 Target ID1 Degradationin Leukemic Cells

HelenaMistry1, Grace Hsieh1, Sara J. Buhrlage2, Min Huang1, Eunmi Park1, Gregory D. Cuny4, Ilene Galinsky3,Richard M. Stone3, Nathanael S. Gray2, Alan D. D'Andrea1, and Kalindi Parmar1

AbstractInhibitor of DNA binding 1 (ID1) transcription factor is essential for the proliferation and progression of

many cancer types, including leukemia. However, the ID1 protein has not yet been therapeutically targeted in

leukemia. ID1 is normally polyubiquitinated anddegradedby theproteasome. Recently, it has been shown that

USP1, a ubiquitin-specific protease, deubiquitinates ID1 and rescues it from proteasome degradation.

Inhibition of USP1 therefore offers a new avenue to target ID1 in cancer. Here, using a ubiquitin-rhoda-

mine–based high-throughput screening, we identified small-molecule inhibitors of USP1 and investigated

their therapeutic potential for leukemia. These inhibitors blocked thedeubiquitinating enzymeactivity ofUSP1

in vitro in a dose-dependent manner with an IC50 in the high nanomolar range. USP1 inhibitors promoted the

degradation of ID1 and, concurrently, inhibited the growth of leukemic cell lines in a dose-dependent manner.

A knownUSP1 inhibitor, pimozide, also promoted ID1 degradation and inhibited growth of leukemic cells. In

addition, the growth of primary acute myelogenous leukemia (AML) patient-derived leukemic cells was

inhibited by a USP1 inhibitor. Collectively, these results indicate that the novel small-molecule inhibitors of

USP1 promote ID1 degradation and are cytotoxic to leukemic cells. The identification of USP1 inhibitors

therefore opens up a new approach for leukemia therapy. Mol Cancer Ther; 12(12); 2651–62. �2013 AACR.

IntroductionID1 (inhibitor of DNAbinding 1) protein is amember of

the helix-loop-helix (HLH) family of transcriptional reg-ulatory proteins,which consists of 4members, ID1–ID4 (1,2). Basic HLH (bHLH) family of transcription factors arekey regulators of cellular development and differentia-tion. The bHLH proteins (e.g., E2A, E2-2, and HEB) formhomodimers or heterodimers with other tissue-specificbHLH proteins (e.g., MyoD and SCL/tal1), bind DNAupon dimerization, and activate transcription of differen-tiation-associatedproteins. The ID familyofHLHproteinscan also form dimers with bHLH proteins (3). However,the ID-bHLH heterodimers are transcriptionally inactiveand are unable to bind to the DNA because ID proteins

lack a basic DNA binding domain (2). Accordingly, IDproteins antagonize the functions of bHLH proteins andinhibit the bHLH protein–mediated transactivation ofgenes that promote cellular differentiation. ID proteins,therefore, are also termed inhibitors of differentiation. Inaddition to binding to the bHLHproteins, ID proteins canalso interact with non-bHLH proteins and antagonizetheir function (4).

ID1, one of the ID proteins, is known to play a role incellular transformation. It is overexpressed in a variety ofhuman cancer types, including pancreatic, cervical, ovar-ian, prostate, breast, colon, and brain cancer (5–11). Inmost of these cancer types, high ID1 expression correlateswith poor prognosis and increased chemoresistance(7, 9, 10, 12, 13). Moreover, functional studies have dem-onstrated that ID1 can promote cell proliferation, inhibitdifferentiation, delay cellular senescence in primaryhuman cells, and promote cell migration and the meta-static phenotype of cancers (14). ID1 function is alsorequired for maintaining the "stemness" of many normaland malignant tissues (10, 11, 15).

Recent studies indicate that ID1 is also an activator ofleukemic cell growth. First, Tang and colleagues (16)observed high levels of ID1 expression in primary acutemyelogenous leukemia (AML) samples, and high expres-sion correlated with poor prognosis. Second, ID1 wasidentified as a common downstream target of constitu-tively activated oncogenic tyrosine kinases, such as BCR-ABL and TEL-ABL (17). Third, ID1 can immortalizehematopoietic progenitors in vitro and promote a

Authors' Affiliations: Departments of 1Radiation Oncology, 2CancerBiology, and 3Medical Oncology, Dana-Farber Cancer Institute, HarvardMedical School, Boston; and 4Laboratory for Drug Discovery in Neurode-generation, Harvard Center for Neurodegeneration and Repair, Brighamand Women's Hospital and Harvard Medical School, Cambridge,Massachusetts

Note: Supplementary data for this article are available at Molecular CancerTherapeutics Online (http://mct.aacrjournals.org/).

CorrespondingAuthors:Kalindi Parmar, Division ofGenomicStability andDNA Repair, Department of Radiation Oncology, Dana-Farber CancerInstitute, Harvard Medical School, 450 Brookline Avenue, Boston, MA02215. Phone: 617-632-4114; Fax: 617-582-8213; E-mail:[email protected]; and Alan D. D'Andrea. Phone: 617-632-2112; Fax: 617-632-5757; E-mail: [email protected]

doi: 10.1158/1535-7163.MCT-13-0103-T

�2013 American Association for Cancer Research.

MolecularCancer

Therapeutics

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Figure 1. High-throughput screening and identification of USP1/UAF1 inhibitors. A, schematic of the USP1/UAF1 constructs. B, Coomassie Bluestaining of the purified USP1/UAF1 complex. C, a schematic of the Ub-rhodamine based screening assay. D, chemical structure of a parental USP1 inhibitorcompound527 (C527). E, C527 inhibitsUSP1activity in a time-dependentmanner. PurifiedUSP1/UAF1 complexwas incubatedwith 1mmol/LC527orDMSOfor the indicated time, followed by the addition of Ub-AMC at a 0.5 mmol/L final concentration. Relative fluorescence units (RFU) at 535 nanometers wasmeasured to indicate the enzymatic activity of USP1. F, Dose-dependent inhibition of USP1 enzymatic activity by USP1 inhibitor C527. Purified USP1/UAF1complex was incubated with C527 or DMSO for 3 hours followed by the addition of Ub-AMC at 0.5 mmol/L concentration. (Continued on the following page.)

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Mol Cancer Ther; 12(12) December 2013 Molecular Cancer Therapeutics2652




myeloproliferative disease in mice in vivo (18). Moreover,the knockdown of ID1 expression inhibited leukemic cellgrowth (18). Collectively, these observations suggest thatID1 is a prime therapeutic target for leukemia and othercancer types. However, suitable drugs to therapeuticallytarget ID1 have not been developed to date (14). Protein–protein interactions in the nucleus, such as the interactionof ID1withHLHfactors, are notoriouslydifficult to inhibitwith small molecules (19).A recent report offers an alternative strategy for knock-

ing down the ID1 protein, namely, through inhibition ofthe ubiquitin-specific protease, USP1 (20). USP1 is a deu-biquitinating (DUB) enzyme, which removes polyubiqui-tin chains from the ID1 protein (20). ID1 is normallypolyubiquitinated and rapidly degraded by the protea-some (21–23). USP1 removes the polyubiquitin chains andrescues ID1 fromdegradation. Selectively knocking downUSP1 using short hairpin RNA (shRNA) results in therapid degradation of ID1 in osteosarcoma cells. Impor-tantly, USP1 knockdown results in decreased mesenchy-mal cell proliferation and enhanced differentiation ofosteosarcoma cells that overexpress USP1 and ID1 (20),providing a rationale for differentiation therapy of manycancer types including leukemia (e.g., AML), chronicmyelogenous leukemia (CML). We, therefore, reasonedthat pharmacologic inhibition of USP1 would promoteubiquitin-mediateddegradationof the ID1protein, result-ing in differentiation and growth inhibition of immatureleukemic cells. Our laboratory has previously shown thathuman USP1 forms a stable complex with its bindingpartner, USP1-associated factor 1 (UAF1; ref. 24). USP1by itself exhibits low DUB activity; however, this activityis significantly enhanced when bound as a USP1/UAF1complex. Using high-throughput screening, we identifieda small-molecule inhibitor of the USP1/UAF1 complex.We describe here a novel small molecule (C527), andmultiple derivatives, that inhibit USP1 catalytic activity,promote ID1 degradation, and inhibit leukemic cellgrowth.

Materials and MethodsHigh-throughput screeningThe USP1/UAF1 complex was prepared as described

previously (ref. 24; Fig. 1) and the protein complex wasused for high-throughput screening. The fluorogenic ubi-quitin-rhodamine (Ub-rhodamine)-based enzyme assaywas established in a 384-well format for high-throughputscreening. The reaction buffer containing free ubiquitinandUSP1/UAF1 enzyme complexwas added in 384-wellplates using automated liquid handling robot-BioTekMicrofill (BioTek Instruments Inc.), followed by the addi-

tion of the compounds (in dimethyl sulfoxide, DMSO)from the compound library plates to wells using apin transfer robotic system at a final concentration of10 mmol/L. The reactions were then incubated for 30minutes at room temperature, followed by the additionof Ub-rhodamine to initiate the reactions. The enzymeactivity of the USP1/UAF1 complex was determined bymeasuring the fluorescence of Ub-rhodamine. Of note,150,000 compounds were screened from the library platesat the Partners Center for Drug Discovery (Cambridge,MA). Details of the screen are provided in the Supple-mentary Methods.

In vitro deubiquitination assaysPurified USP5 enzyme was purchased from Boston

Biochem. UCH-L1 and UCH-L3 were as reported previ-ously (25). USP12/46 was prepared in our laboratory asdescribed previously (26, 27). The in vitro enzymaticassayswere performed as described previously (24) usingubiquitin-AMC (Ub-7-amido-4methylcoumarin; BostonBiochem) as a substrate in a reaction buffer containing20 mmol/L HEPES-KOH (pH 7.8), 20 mmol/L NaCl, 0.1mg/mL ovalbumin, 0.5 mmol/L EDTA, and 10 mmol/Ldithiothreitol. The fluorescencewasmeasured by FLUOs-tar Galaxy Fluorometer (BMG LABTECH). For the Ub-vinylsulfone (VS) assay, the proteins were incubatedwithUb-VS (BostonBiochem) at 0.5mmol/Lfinal concentrationfor 45 minutes at 30�C, followed by the immunoblotting.

Cells and drug treatmentsLeukemic cell lines were grown in RPMI-1640 medium

(Invitogen) supplemented with 10% FBS (Invitrogen) andpenicillin/streptomycin (Invitrogen).HeLacells andU2OScells were grown in Dulbecco’s Modified Eagle Medium(Invitogen) supplemented with 10% FBS (Invitrogen) andpenicillin/streptomycin (Invitrogen). K562 cells were pro-vided by Dr. Dipanjan Chowdhury (Dana-Farber CancerInstitute, DFCI, Boston, MA). MOLM14 cells were provid-ed by Dr. A. Thomas Look (DFCI). Luciferase-expressingAMLcell lineswereprovidedbyDr.AndrewKung (DFCI).Mouse osteosarcoma cells were provided by Dr. StuartOrkin (DFCI). The cells were provided within the last 24months. No authentication of the cell lineswas done by theauthors. USP1 inhibitor C527 and its derivatives weresynthesized and the purity was validated by high-perfor-mance liquid chromatography. Pimozide was purchasedfrom Sigma. Primary human patient samples with AMLwere collected from DFCI leukemia program under theapproval of the appropriate protocols. Cells were treatedwith DMSO or USP1 inhibitors in appropriate medium for24 to 72 hours. The viable cell counts were determined

(Continued.) The RFU at 535 nanometers was measured to indicate the enzymatic activity of USP1. G, IC50 plot of C527 against the USP1/UAF1 complexin an enzymatic reaction, as described in E and F. H and I, USP1 inhibitor SJB2-043 inhibits the activity of native USP1/UAF1. K562 cells were treated withDMSO or SJB2-043 (1 mmol/L) for 24 hours and cell extracts were incubated with 0.5 mmol/L Ub-Vs (see structure in H) for 45 minutes. An aliquot ofuntreated cell extract was treated with Ub-aldehyde (5 mmol/L) before adding Ub-Vs. The reactions were then subjected to immunoblotting with anti-HA oranti-USP1 antibodies.

Targeting ID1 in Leukemia by USP1 Inhibitor

www.aacrjournals.org Mol Cancer Ther; 12(12) December 2013 2653




using Trypan Blue staining, CellTiter-Glo reagent (Pro-mega) or MTT assay. The apoptotic cells were detectedusing Annexin V and 7-amino-actinomycin D (7-AAD; BDBiosciences) staining according to the manufacturer’sinstructions using flow cytometry. For benzidine staining,the cells were washed twice with PBS and resuspended in45 mL of PBSþ 5 mL of benzidine stain solution (0.2% in 0.5M glacial acetic acid, 3% H2O2). After 45 minutes incuba-tion at room temperature, the benzidine-positive cellsweredetected by light microscopy.

Antibodies and shRNAsWestern blotting was performed with the following

antibodies: anti-USP1 (A301-699A; Bethyl lab), anti-ID1(B-8; Santa Cruz Biotechnology), antivinculin (H-10;Santa Cruz Biotechnology), anti-a tubulin (DM1A; SantaCruz Biotechnology), anti-ID2 (C-20; Santa Cruz Biotech-nology), anti-ID3 (C-20; Santa Cruz Biotechnology), anti-FANCD2 (H-300; Santa Cruz Biotechnology), and anti-FANCI (28). Doxycycline-inducible shRNAs in TRIPZLentivirus vectors against human USP1 were purchasedfrom Open Biosystems (Thermo Scientific). Mature anti-sense sequence for the shRNAs were (i) V2THS_171887:TTTATCTTCTCCTACAATC, (ii) V2THS_218649: TTAA-GATAGCAAGTATTGC, and (iii) V2THS_171886:TAA-ACTTTCACATTCCAAG.

Mouse xenograft experimentsSix- to 8-week-old athymic nude mice were used for

xenograft studies. Themouse experimentswere reviewedand approved by DFCI’s Animal Care and Use Commit-tee. A total of 5� 106 K562 cells weremixedwithMatrigel(BD Biosciences; 1:1 ratio) and injected subcutaneouslyinto the flanks of nude mice. When the tumors reached 2mm3 size, 15 mg/kg pimozide (dissolved in 2% DMSO þ0.3% tartaric acid) or vehicle was administered daily. Thetumor growth was monitored by measuring the size.

Homologous recombination analysis andimmunofluorescence

Homologous recombination activity was analyzed byDRGFP reporter as previously described (29). U2OS-DRGFP cells carrying a chromosomally integrated singlecopy of homologous recombination repair substrate wereused. Double-strand break (DSB)–induced homologousrecombination in these cells results in restoration andexpression of GFP. Briefly, 24 hours after induction ofchromosomal DSBs through the expression of I-SceI,U2OS-DRGFP cells were treated with USP1 inhibitorC527 for 24 hours. Cells were then subjected to fluores-cence-activated cell sorting analysis to quantify the per-centage of viableGFP-positive cells. TheRad51 foci in cellswere detected by immunofluoroscence as described pre-viously (30) using anti-RAD51 (Santa Cruz Biotechnolo-gy) and Alexa Fluor 488–conjugated secondary antibo-dies. The quantification of cells with RAD51 foci wasperformed by counting the number of cells with RAD51foci.

ResultsIdentification of a novel USP1 inhibitor using high-throughput screening

We have previously reported that USP1 by itself exhi-bits poor DUB activity. However, when bound to itsinteracting partner, UAF1, the catalytic activity of USP1is significantly enhanced (24). We therefore used a puri-fied USP1/UAF1 complex and performed a high-throughput screening to identify a USP1 inhibitor (Fig.1). We used the cDNA encoding a full-length USP1polypeptide, which had a substitution of GG to AA atamino acid residues 670 to 671 (Fig. 1A). We havepreviously shown that this amino acid change disruptsan autocleavage site in USP1 but does not alter theinteraction of USP1 with UAF1 or the DUB activity ofthe complex (24, 31). Human USP1 (with GG to AAmutation) and UAF1 proteins were coexpressed in SF9insect cells to generate a protein complex with elevatedDUB activity. The USP1/UAF1 protein complex waspurified and the purity was confirmed by coomassiestaining (Fig. 1B). The stable USP1/UAF1 enzyme com-plex was then subjected to a high-throughput inhibitorscreen, described schematically in Fig. 1C. A small mol-ecule, C527 (heterocyclic tricyclic 1,4-dihydro-1,4-dioxo-1H-naphthalene; Fig. 1D) was identified, which inhib-ited the USP1/UAF1 complex.

USP1 inhibitor C527 has a high affinity for USP1We next confirmed the ability of C527 to inhibit the

USP1/UAF1 complex in a dose-dependent and time-dependent manner, using Ub-AMC as a substrate (Fig.1E–G). Pretreatment ofUSP1/UAF1withC527 resulted ininhibition of its enzyme activitywith an IC50 of 0.88� 0.03mmol/L. To determine the specificity of C527 in USP1/UAF1 inhibition, we next examined its ability to inhibitother DUB enzymes in vitro. UAF1 stimulates not onlyUSP1, but also twootherDUBenzymes,USP12 andUSP46(26). We therefore tested C527 for its ability to inhibit thepurified USP12/USP46 complex or other DUBs. C527inhibited the DUB activity of the USP12/USP46 complexand other DUB enzymes in vitro. However, the IC50 ofC527 for these DUB enzymes was higher in comparisonwith USP1/UAF1 complex (Table 1). C527 had consider-ably less inhibitory effect on UCH-L1 and UCH-L3, adifferent subclass of DUB enzymes, referred to as the

Table 1. IC50 values of USP1 inhibitor C527 forthe indicated DUB enzymes

DUBs IC50 (mmol/L)

USP1 0.88 � 0.07USP12/46 5.97 � 0.37USP5 1.65 � 0.42UCH-L1 >10UCH-L3 2.18 � 0.69

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ubiquitin C-terminal hydrolases (25), even though theyare also cysteine proteases. Taken together, C527 is a pan-DUBenzyme inhibitor in vitro,with ahighnanomolar IC50

for the USP1/UAF1 complex.To generate a USP1 inhibitor with improved cellular

potency, we chemically synthesized several analogs/derivatives of C527 and determined their IC50 for USP1/UAF1 inhibition (data not shown). We then selected aderivative, SJB2-043 (IC50 ¼ 0.544 mmol/L), for furtherstudies due to the feasibility of its large-scale chemicalsynthesis.

USP1 inhibitor SJB2-043 inhibits the enzyme activityof native USP1Wenext determinedwhether SJB2-043 inhibits theDUB

activity of native USP1 complexes isolated from humancells (Fig. 1H and I). For this purpose, we performedcompetition assays between SJB2-043 and the ubiquitinactive site probe HA-Ub-vinyl sulfone (HA-Ub-VS)reagent (Fig. 1H). This affinity reagent covalentlymodifiesand traps active DUB enzymes in cell extracts (32). Cellswere treated with SJB2-043 for 24 hours and cell extractswere incubated with HA-Ub-VS, followed by immuno-blottingwith anti-USP1 or anti-HA antibodies. Consistentwith our previous studies and the most recent studies byPiatkov and colleagues (24, 31, 33), USP1 in the untreatedcells was detected on Western blot analysis as a doublet;full-length USP1 and an autocleaved USP1 with only N-terminal (Fig. 1I, lane 1). The Ub–USP1 conjugates weregenerated in the untreated cell lysates and were detectedbyan increase inmolecularweight ofUSP1 (Fig. 1I, lane 2).However, the formation of the Ub–USP1 conjugate wasinhibited by the addition of SJB2-043 (lane 4). Ubiquitin-aldehyde (Ub-aldehyde), a potent known nonspecificDUB inhibitor, caused a complete loss of USP1–Ub-VSconjugates in this competition assay (lane 3). SJB2-043inhibited the Ub-VS labeling of a limited number ofendogenous DUB enzymes (Fig. 1I; anti-HA blot). Inaddition, SJB2-043 inhibited the labeling of USP1 withUb-VS in a dose-dependent manner (Supplementary Fig.S1A). Taken together, these data demonstrate that SJB2-043 is an inhibitor of the native USP1/UAF1 complex.

Novel USP1 inhibitors promote degradation of ID1in leukemic cellsshRNA knockdown of USP1 results in enhanced pro-

teasome-mediated degradation of ID1 in osteosarcomacells (20). ID1 degradation in osteosarcoma cells results inmesenchymal differentiation and decreased cell prolifer-ation. We therefore tested whether small molecule USP1inhibitors enhance ID1 degradation in cells. Indeed, USP1inhibitor C527 promoted the dose-dependent degrada-tion of ID1 in human U20S osteosarcoma cells (data notshown). We next tested USP1 inhibitors for their ability toinhibit USP1 in various cell lines. The K562 cell line (theerythroleukemia cell line derived from a patient withCML) was treated with SJB2-043 (Fig. 2A) for 24 hours,and USP1 and ID1 protein levels were determined by

Western blotting. Interestingly, the SJB2-043 caused adose-dependent decrease in USP1 levels and a concom-itant degradation of the ID1 protein in the K562 cells at amicromolar drug concentration (Fig. 2B). The decrease inUSP1 and ID1 levels by SJB2-043 exposure resulted fromproteasomal degradation because the proteasome inhib-itor, MG-132, restored the levels of both proteins (Fig. 2Cand Supplementary Fig. S1B).

SJB2-043 also caused a decrease in the levels of other IDproteins, namely ID2 and ID3 in K562 cells (Supplemen-tary Fig. S1C). To determine whether the decrease in ID1levels was secondary to a decrease inUSP1 levels, we nextgenerated a K562 stably transfected with a cDNA encod-ing doxycycline-induced shRNA to USP1. As expected,doxycycline exposure of these cells resulted in a dose-dependent decrease in USP1 expression (SupplementaryFig. S2A). Moreover, depletion of USP1 by siRNA pro-moted ID1 degradation in K562 cells (Supplementary Fig.S2B). Importantly, siRNA-mediated knockdown of USP1in leukemic K562 cells resulted in growth inhibition,increased apoptosis and cell-cycle arrest (SupplementaryFig. S2C–S2F). Collectively, these results indicated thatalthough SJB2-043 may have off-target effects, it exhibitsits activity in K562 cells at least in part by promotingdegradation of USP1, which results in ID1–ID3degradation.

USP1 inhibitors are cytotoxic to leukemic cellsWe next determined whether the USP1 inhibitors are

cytotoxic to leukemic cells and whether the cytotoxicityresults from ID1 degradation. K562 leukemic cells weretreated with SJB2-043, and survival and differentiationwere assessed. SJB2-043 caused a dose-dependentdecrease in the number of viable K562 cells, with anEC50 of approximately 1.07 mmol/L � 0.08 (95% confi-dence interval; Fig. 2D). Moreover, SJB2-043 inducedapoptosis of K562 cells in a dose-dependent manner (Fig.2E). Cytotoxic drug concentrations correlated with theconcentrations required for ID1 degradation. In addition,low-dose treatments with SJB2-043 activated differentia-tion of K562 cells into hemoglobin-expressing erythroidcells, as detected by benzidine staining (Fig. 2F).

In addition to the CML cells (K562 cells), we alsoevaluated the cytotoxicity of SJB2-043 in multiple AMLcell lines.We choseAMLcell lines thatwere engineered toexpress the luciferase enzyme so that these cell lines canbeused in future for in vivo optical imaging experiments. TheAML cell lines, namely,MOLM14, OCI-AML3, U937, andSK-NO1 (Fig. 3) expressed ID1 proteins. Upon treatmentwith SJB2-043, a dose-dependent cytotoxicity and con-comitant ID1 degradation was observed in all of the AMLcell lines (Fig. 3) further suggesting the role of ID1 inleukemic cell survival.

To determine whether the cytotoxicity of the USP1inhibitor was due to USP1/ID1 degradation, wenext evaluated analogs of SJB2-043 in leukemic cells(Supplementary Fig. S3). The USP1 inhibitor analogs(e.g., SJB2-127) with no significant activity on USP1 (IC50






> 10 mmol/L) or ID1 degradation at concentrations up to10 mmol/L, did not exhibit cytotoxic effects on leukemiccells (Supplementary Fig. S3A). The analog SJB2-109 hadcomparable biochemical inhibitory activity (IC50 ¼ 0.416mmol/L) to our lead inhibitor SJB2-043 and exhibits com-parable effects on ID1 degradation and leukemic cellsurvival. Importantly, a more potent analog SJB3-019A(IC50 ¼ 0.0781 mmol/L) was five times more potent thanSJB2-043 in promoting ID1 degradation and cytoxicity inK562 cells.

To determine whether ID1 degradation resulted fromUSP1 inhibition and was not secondary to apoptosis, wenext analyzed USP1/ID1 levels and apoptosis aftertreating K562 cells with SJB2-043 along with a caspaseinhibitor in a time-course manner. USP1/ID1 degrada-tion was observed as early as 7 hours after the 2 mmol/Lof SJB2-043 exposure when apoptosis was not significant

(Supplementary Fig. S4A). At 18 or 26 hours (Supple-mentary Fig. S4B and S4C) after the SJB2-043 exposure(2 mmol/L), complete degradation of USP1/ID1 wasobserved with concurrent robust increase in apoptosis.As expected, caspase inhibitor treatment resulted indecreased apoptosis and inhibition of caspase activity(Supplementary Fig. S4). However, ID1 degradationwas not affected by caspase inhibition (SupplementaryFig. S4, left). Even when the caspase activity was sig-nificantly abrogated with caspase inhibitor, the apopto-sis was not completely blocked. These results suggestthat ID1 degradation by SJB2-043 may not be occurringas a result of induction of apoptosis, although apoptosismay play a role in destabilizing ID1 independent ofUSP1 inhibition.

Collectively, these results indicated that novel USP1inhibitors promote ID1 degradation by specifically

Figure 2. USP1 inhibitor promotesID1 degradation and inhibitsgrowth of the leukemic cell lineK562. A, chemical structure ofUSP1 inhibitor SJB2-043. B,Western blot analysis of lysatesfromK562 cells treatedwith DMSOor USP1 inhibitor SJB2-043 for 24hours. C, degradation of USP1 andID1 in leukemic cells by USP1inhibitor is proteasome mediated.Western blot analysis of the lysatesfrom K562 cells treated with SJB2-043 for 24 hours followed by thetreatment with MG-132 are shown.D, dose-dependent growthinhibition of K562 cells by USP1inhibitor SJB2-043. Cells weregrown in triplicate with DMSO orSJB2-043 for 24 hours, andcytotoxicity was determined. E,induction of apoptosis in K562cells treated with USP1 inhibitor.Cells were grown in triplicate withDMSO or SJB2-043 for 24 hours,and apoptosis was determined.F, differentiation of K562 cells aftertreatments with low doses of theUSP1 inhibitor. Benzidine stainingof the cells treated with SJB2-043for 5 days is shown.

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targeting native USP1 and cause cytotoxicity in multipleleukemic cell lines.

Pimozide, a known USP1 inhibitor, promotes ID1degradation and inhibits leukemic cell growthA known drug, pimozide, is a weak USP1 noncom-

petitive inhibitor (34). Pimozide may also inhibitSTAT5 activation, thereby reducing the survival ofBCR/ABL–driven CML cells (35). We examined pimo-zide for its possible cytotoxic effects on K562 cells(Fig. 4) via destabilization of ID1. Interestingly, pimo-zide promoted ID1 degradation, decreased USP1 levels,and caused a dose-dependent inhibition of K562 cellgrowth in vitro with induction of apoptosis (Fig. 4B–D),though at a higher drug concentrations as comparedwith SJB2-043. The inhibitory drug concentrations cor-related with the concentrations required for ID1 deg-radation. We next examined the effect of pimozide in amouse xenograft model of leukemia. K562 cells wereinjected into nude mice and xenografts were estab-lished followed by the treatment with pimozide orvehicle. As shown in Fig. 4E, pimozide treatmentcaused a modest inhibition of tumor growth. Takentogether, pimozide, a commercially available weakUSP1 inhibitor, promotes ID1 degradation and inhibitsleukemic cell growth in vitro and in vivo, suggesting

that our more potent USP1 inhibitors may have astronger antileukemic effect.

USP1 inhibitor promotes ID1 degradation andinhibits growth of primary AML cells from patients

Human AML precursor cells arise from leukemic stemcells (LSC), and LSC differentiation is controlled throughcoordinate transcriptional regulation including the bHLHproteins. We therefore, tested whether the USP1 inhibitoralso promotes ID1 degradation and cytoxicity in primaryhuman leukemic cells. Primary fresh bone marrow andperipheral blood samples were acquired from patientswith AML and mononuclear cells were harvested. Thecells were then exposed to the USP1 inhibitor for 48 to 72hours in culture and ID1 levels aswell as cell viability wasdetermined. USP1 inhibitor SJB2-043 exhibited dose-dependent cytotoxicity on the primary bone marrow aswell as peripheral blood from patients with AML (Fig. 5Aand B). Moreover, SJB2-043 treatment caused inactivationof USP1 and promoted ID 1 degradation in primary AMLcells (Fig. 5C). The ID1 protein was detected as a doublet,which is likely due to the posttranslationalmodification ofthe protein. These results demonstrate thatUSP1 inhibitorSJB2-043 inactivates native USP1 in primary AML cellsand exhibits cytotoxicity. The selectivity of SJB2-043against normal human hematopoietic cells was then

Figure 3. USP1 inhibitor promotes ID1degradation andcauses cytotoxicity inmultiple AMLcell lines. A, dose-dependent growth inhibition ofMOLM14cells bySJB2-043. Cells were grown in triplicate in presence of DMSO or SJB2-043 for 24 hours, and cytotoxicity was determined. B, Western blot analysisof the lysates from MOLM14 cells treated with DMSO or SJB2-043 for 24 hours. C, dose-dependent growth inhibition of luciferase-expressing AMLcell lines by SJB2-043. Cells were grown in triplicate in the presence of DMSO or SJB2-043 for 24 hours, and cytotoxicity was determined. D, Western blotanalysis of the lysates from luciferase-expressing AML cells treated with DMSO or SJB2-043 for 24 hours.






examined in comparison with pimozide using cord bloodCD34þ cells. Although pimozide was tolerated at highmicromolar doses, low micromolar levels of SJB2-043 didresult in growth inhibition of primary human cord bloodCD34þ cells (Supplementary Fig. S5).

USP1 inhibitor increases the levels of Ub-FANCD2and disrupts the homologous recombination

Previous studies indicate thatUSP1has other substratesbesides ID1 (36). For instance, the USP1/UAF1 complexdeubiquitinates the cellular monoubiquitinated substrates

Figure 5. USP1 inhibitors promoteID1 degradation and growthinhibition of primary AML cells frompatients. Primary bone marrowcells or peripheral bloodmononuclear cells from patientswith AMLwere cultured in triplicatecultures in the presence of DMSOor USP1 inhibitor, and cytotoxicitywas determined. Survival plots ofprimary AML bone marrow cells (A)or peripheral blood cells (B) treatedwith DMSO or SJB2-043 for 48 to72 hours are shown. The data arefrom three different patientsamples (n¼ 3�SEM). C,Westernblot analysis of the lysates fromAML peripheral blood cells treatedwith SJB2-043 for 48 hours.

Figure 4. Treatment of K562 cells with a known USP1 inhibitor, pimozide, leads to ID1 degradation and growth inhibition. A, chemical structure of a knownUSP1 inhibitor pimozide. B, dose-dependent growth inhibition of K562 cells by pimozide. Cells were grown in triplicate with DMSO or pimozide for48 hours, and cytotoxicity was determined. C, induction of apoptosis in K562 cells treated with pimozide. Cells were grown in triplicate cultures, as describedin B, and apoptosis was determined. D, Western blot analysis of the lysates from cells treated with DMSO or pimozide for 48 hours. E, in vivoefficacyof pimozide inK562 xenograft. Nudemice (n¼10/group) bearing xenografts of K562cellswere injected intraperitoneallywith pimozide or vehicle daily

and tumor volumes ½tumorvolume ¼ ðwidthÞ2 � length=2� were measured. P ¼ 0.0042 for pimozide versus vehicle groups, using paired t test.

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Ub-FANCD2andUb-PCNA (31, 37).Deubiquitination ofFANCD2 is a critical step in the Fanconi anemia (FA)/BRCADNA repair pathway (38, 39). Knockout or knock-down of USP1/UAF1 results in cellular hypersensitivityto DNA cross-linking chemotherapy agents such asmitomycin C (40, 41). Murine knockout of USP1 resultsin cellular increase in Ub-FANCD2 and a mouse pheno-

type resembling Fanconi anemia (41). Knockout of USP1also results in elevated levels of Ub-PCNA, leading to anincrease in DNA translesion synthesis (31).

We next determined whether the USP1 inhibitors canalter cellular levels of Ub-FANCD2 and Ub-FANCI, abinding partner of FANCD2 (Fig. 6). HeLa cells weretreated with USP1 inhibitor C527 and levels of FANCD2

Figure 6. USP1 inhibitor increases the levels of Ub-FANCD2, decreases the homologous recombination activity, and sensitizes the cells tochemotherapy agents. A,Western blot analysis of the lysates fromHeLa cells treatedwith DMSOorUSP1 inhibitor C527 for 8 hours. B, increased cytotoxicityof C527 on HeLa cells in presence of chemotherapy agents. Cells were treated with C527 (1 mmol/L) for 24 hours, followed by the treatment with mitomycinC (MMC; 0.25 mmol/L), camptothecin (CPT; 0.1 mmol/L), or etoposide (0.5 mmol/L) for 4 days, and cell survival was determined. C, C527 inhibits DRGFPreporter for homologous recombination repair activity. U2OS-DRGFP cells were transfected with I-SCE-I reporter plasmid and then exposed to C527 at theindicated concentration for 24 hours. Cells were then subjected to flow cytometry analysis. The percentage of GFP-positive cells was normalized bysolvent vehicle treated group are shown. D, C527 inhibits camptothecin (CPT)-inducedRAD51 foci formation. HeLa cells were pretreatedwith DMSOorC527at the indicated concentration and then exposed to camptothecin for 1 hour. RAD51 foci were detected using immunofluorescence. Data are representedas mean � SD from three independent experiments.






and FANCI were determined by Western blotting of thecell lysates. C527 treatments caused an increase in thelevels of Ub-FANCD2 and Ub-FANCI (Fig. 6A). SJB2-043(another USP1 inhibitor) also increased the level of Ub-FANCD2 (Supplementary Fig. S6A and S6B). An increasein Ub-PCNA was also observed in cells exposed to SJB2-043 (Supplementary Fig. S6C), suggesting that besidesFANCD2, PCNA is also affected byUSP1 inhibition.HeLacells were next pretreated with C527 followed by thetreatment with DNA damaging agents including mito-mycin C, a DNA cross-linking agent. Pretreatment of cellswith the USP1 inhibitor caused an enhancement in thecytoxicity of mitomycin C and camptothecin (Fig. 6B).

BecausedisruptionofUSP1activity results indecreasedhomologous recombination (41), we next used a geneconversion assay to examine the effect of the USP1 inhib-itor on cellular homologous recombination activity.Interestingly, C527 treatments caused a dose-dependentdecrease in gene conversion (Fig. 6C), based on the mea-surement of cellular GFP in this assay. We also confirmedhomologous recombination defect by evaluating Rad51foci formation, another surrogate marker of cellularhomologous recombination activity (Fig. 6D). C527-inhib-ited camptothecin induced the Rad51 foci in HeLa cells.The more active USP1 inhibitor SJB3-019A also increasedthe levels of Ub-FANCD2 and Ub-PCNA, and decreasedthe homologous recombination activity (SupplementaryFig. S6D–S6F). Taken together, these results suggest thatUSP1 inhibitor treatments lead to an increase in ubiqui-tinated forms of FANCD2 and FANCI, cause a decrease inhomologous recombination activity, and sensitize cells toDNA damaging agents.

DiscussionID1 is essential for proliferation and maintenance of

many cancer types (14, 42). However, it has not yet beentherapeutically targeted with a small molecule in leuke-mia. Here, we demonstrate that targeting USP1 with asmall molecule can promote ID1 degradation and celldeath in multiple leukemic cell lines. Our data demon-strate that ID1 can be targeted even in primary humanleukemic cells using novel inhibitors of USP1.

The drug of choice for CML is imitanib, although drugresistance remains a problem. Novel therapies are alsoneeded for the cure of AML, the most common acuteleukemias of adults. USP1 inhibitors represent a novelclass of drugs for these cancers. USP1 inhibitors candegrade ID1, promote differentiation, and induce apopto-sis in these types of leukemic cells and can be potentialtherapeutic agents. ID1 blocks cellular differentiation atleast in part, through its repressive effect on the expres-sion of cell-cycle inhibitor p21 (20). Consistent with thisprevious report (20), our USP1 inhibitor C527 promotesthe degradation of ID1 and the concurrent upregulation ofp21 in mouse osteosarcoma cells (Supplementary Fig.S6B). Cell-cycle arrest through p21 upregulation providesa mechanistic explanation for the increase in erythroiddifferentiation of leukemic cells. ID proteins have central

role in keeping cells in an immature state. Various reportssuggest that ID proteins including ID1 play an importantrole in maintaining cancer-initiating cells in a variety ofsolid tumors (10, 19, 43, 44). Therefore, therapeuticallytargeting ID1 may also eradicate leukemic cancer stemcells.

An inhibitor of USP1, pimozide, was recently identified(34). Pimozide, an antipsychotic drug, is currently U.S.Food andDrugAdministration approved for treatment ofTourette syndrome. However, its efficacy in promotingID1 degradation has not been previously reported. In ourstudies, pimozide promotes ID1 degradation and causescytoxicity in leukemic cells. Our in vivo studies usingpimozide showed a modest decrease in tumor volume inxenograftmodels ofK562 leukemic cells. This is consistentwith the recent report by Nelson and colleagues (45), whoobserved a modest effect of pimozide in FLT3-AML–driven tumor models. Of note, the EC50 of pimozide onleukemic cells is significantly higher than our novel USP1inhibitors. Even though pimozide is currently used inhumans for the treatment of Tourette syndrome, its effi-cacy in mouse tumor models is limited because of itstoxicity. Accordingly, at the maximum tolerated doses,pimozidemay not be an effective anticancer agent, at leastin leukemic mouse models. Moreover, in addition toexhibiting an inhibitory activity to USP1, it also abrogatesSTAT5 activity in leukemic cells (35, 45).

ID1 plays an important role in invasiveness of manycancer types including breast and brain tumors(11, 46, 47). Specifically, ID1 depletion by antisense oligosto ID1 in BCR-ABL–bearing 32D3 cells significantlyreduces its invasiveness in vitro and in vivo severe com-bined immunodeficient mouse models via MMP9 axis(48). It remains to be investigated whether our small-molecule inhibitors of USP1 can reduce the invasivenessof leukemic cells. Although ID1 has been previouslytargeted using peptide-conjugated antisense oligomers(49), peptide aptamers (50), or cannabidiol (11) as poten-tial ways to inhibit ID1 function in solid tumors, none ofthese applications have been explored for leukemia.

The USP1 inhibitors reported in this study may haveother advantages, because USP1 has other known sub-strates (31, 37). We have previously shown that USP1, incomplex with its stimulatory binding partner, UAF1,deubiquitinates the Fanconi anemia proteins, FANCD2and FANCI (24). Knockdown of USP1 activity results inelevated cellular levels of Ub-FANCD2, resulting in dis-ruption of the Fanconi anemia pathway (40, 41). Function-al Fanconi anemia DNA repair pathway is required forcellular resistance to DNA cross-linking agents and Fan-coni anemia pathway deficient cells including USP1�/�

cells are hypersensitive to DNA cross-linking agents (39–41). USP1 inhibitors may therefore block the Fanconianemia pathway, thereby promoting cellular hypersensi-tivity toDNAcross-linking agents and chromosome insta-bility. In the short run,USP1 inhibitorsmay thereforehavean additional useful function as anticancer agents, bysensitizing tumors to conventional chemotherapeutic

Mistry et al.





agents, for example, cisplatin (34). In our study, USP1inhibitors increased the levels of Ub-FANCD2, decreasedthe homologous recombination activity, and sensitizedcells to chemotherapy agents’ mitomycin C and camp-tothecin. The increase in Ub-FANCD2may result directlyfromUSP1 inhibition or indirectly from the DNAdamageincurred by USP1 inhibitor exposure. In either case, USP1inhibition may have additional anticancer functions thatextend beyond the promotion of cellular differentiation.Accordingly, the inhibitors may be useful as radiation orcisplatin sensitizers, based on their ability to disrupt theFA/BRCA or translesion synthesis pathways.In summary, we have identified novel small-molecule

inhibitors of USP1 that exhibit nanomolar IC50 for USP1activity. These inhibitors also promote ID1 degradationand cause cytotoxicity in a variety of leukemic cell linesand primary leukemic cells. Further preclinical assess-ment of these small molecules in mouse models willdetermine their efficacy as novel anticancer agents. Ifsuccessful, these newly identified USP1 inhibitors canhave broader implications and can be valuable therapeu-tic agents for not only leukemia but also for other cancertypes (e.g., breast cancer and glioblastoma) in which ID1may be a prime therapeutic target.

Disclosure of Potential Conflicts of InterestNo potential conflicts of interest were disclosed.

Authors' ContributionsConception anddesign:H.Mistry, G.Hsieh, S.J. Buhrlage,N.S. Gray,A.D.D’Andrea, K. ParmarDevelopment of methodology: H. Mistry, G. Hsieh, S.J. Buhrlage, M.Huang, E. Park, K. ParmarAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.):H. Mistry, G. Hsieh, M. Huang, I. Galinsky, N.S.Gray, K. ParmarAnalysis and interpretation of data (e.g., statistical analysis, biostatis-tics, computational analysis): H. Mistry, G. Hsieh, S.J. Buhrlage,M. Huang, G.D. Cuny, N.S. Gray, K. ParmarWriting, review, and/or revision of the manuscript:H. Mistry, G. Hsieh,S.J. Buhrlage, G.D. Cuny, R.M Stone, A.D. D’Andrea, K. ParmarAdministrative, technical, or material support (i.e., reporting or orga-nizing data, constructing databases): E. Park, R.M Stone, K. ParmarStudy supervision: N.S. Gray, A.D. D’Andrea, K. Parmar

AcknowledgmentsThe authors thank Anna Blachman, Kevin O’Connor, and Benjamin

Primack for their technical assistance. The authors also thank Jung MinKim for her help in generating the data on p21 expression.

Grant SupportThis research was supported by a Translational Research Grant from

the Leukemia & Lymphoma Society (6237-13) and the NIH grants (1RC4DK090913-01, 2P01HL048546, 2RO1 DK43889, and 5R37HL52725-18; toA.D. D’Andrea).

The costs of publication of this article were defrayed in part by thepayment of page charges. This article must therefore be hereby markedadvertisement in accordance with 18 U.S.C. Section 1734 solely to indicatethis fact.

Received February 19, 2013; revisedOctober 4, 2013; acceptedOctober 7,2013; published OnlineFirst October 15, 2013.

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2013;12:2651-2662. Published OnlineFirst October 15, 2013.Mol Cancer Ther Helena Mistry, Grace Hsieh, Sara J. Buhrlage, et al. Leukemic CellsSmall-Molecule Inhibitors of USP1 Target ID1 Degradation in

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