Targeting USP7 Identiﬁes a Metastasis- Competent State ... · Targeting USP7 Identiﬁes a Metastasis-Competent State within Bone Marrow–Resident Melanoma CTCs Monika Vishnoi1,

Translational Science

Targeting USP7 Identifies a Metastasis-Competent State within Bone Marrow–ResidentMelanoma CTCsMonika Vishnoi1, Debasish Boral1, Haowen Liu1, Marc L. Sprouse1,Wei Yin1,Debalina Goswami-Sewell1, Michael T. Tetzlaff2, Michael A. Davies2,Isabella C. Glitza Oliva2, and Dario Marchetti1

Abstract

Systemic metastasis is the majorcause of death from melanoma,themost lethal formof skin cancer.Although most patients with mel-anoma exhibit a substantial gapbetweenonset of primary andmet-astatic tumors, signaling mechan-isms implicated in the period ofmetastatic latency remain unclear.We hypothesized that melanomacirculating tumor cells (CTC)home to and reside in the bonemarrow during the asymptomaticphase of disease progression.Using a strategy to deplete normalcell lineages (Lin�), we isolatedCTC-enriched cell populationsfrom the blood of patients withmetastatic melanoma, verified bythe presence of putative CTCs characterized by melanoma-specific biomarkers and upregulated gene transcripts involved in cellsurvival and prodevelopment functions. Implantation of Lin� population in NSG mice (CTC-derived xenografts, i.e., CDX), andsubsequent transcriptomic analysis of ex vivo bone marrow–resident tumor cells (BMRTC) versus CTC identified proteinubiquitination as a significant regulatory pathway of BMRTC signaling. Selective inhibition of USP7, a key deubiquinatingenzyme, arrested BMRTCs in bonemarrow locales anddecreased systemicmicrometastasis. This study provides first-time evidencethat the asymptomatic progression ofmetastaticmelanoma can be recapitulated in vivo using patient-isolated CTCs. Furthermore,these results suggest that USP7 inhibitors warrant further investigation as a strategy to prevent progression to overt clinicalmetastasis.

Significance: These findings provide insights into mechanism of melanoma recurrence and propose a novel approach toinhibit systematic metastatic disease by targeting bone marrow-resident tumor cells through pharmacological inhibition ofUSP7.

Graphical Abstract: http://cancerres.aacrjournals.org/content/canres/78/18/5349/F1.large.jpg. Cancer Res; 78(18); 5349–62.�2018 AACR.

IntroductionMelanoma, the most aggressive skin cancer, frequently

metastasizes to distant organs such as bone, lung, liver, andbrain (1). Unlike localized melanoma, which can be sur-gically resected, metastatic melanoma with extranodalinvolvement is typically treated with systemic therapiesincluding targeted and immune therapy (2). Although out-comes are improving due to new therapies, the majorityof patients with stage IV melanoma will die from theirdisease (1, 2).

© 2018 American Association for Cancer Research

Liquid biopsy Asymptomaticprogression

CTCs BMRTCs

Distant organ micrometastasis

USP7

Melanoma progresses asymptomatically in patient-isolated CTC-derived xenografts and distantorgan micrometastasis can be inhibited by targeting USP7.

1Biomarker Research Program Center, Houston Methodist Research Institute,Houston, Texas. 2Department of Melanoma Medical Oncology, The University ofTexas MD Anderson Cancer Center, Houston, Texas.

Note: Supplementary data for this article are available at Cancer ResearchOnline (http://cancerres.aacrjournals.org/).

Corresponding Author: Dario Marchetti, Houston Methodist Research Institute,Suite R7-111, MS R7-414, 6670 Bertner Avenue, Houston, TX 77030. Phone: 713-363-7769; Fax: 713-363-7717; E-mail: [email protected]

doi: 10.1158/0008-5472.CAN-18-0644

�2018 American Association for Cancer Research.

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Metastasis is a multistep process that is initiated when tumorcells leave the primary tumor and traverse through the circu-lation to reach distinct organs (3, 4). While transiting throughthe peripheral blood, a subset of CTCs can home to the bonemarrow where they reside in a state of sustained quiescence(parallel progression model of metastatic melanoma; ref. 5).The unique bone marrow microenvironment exerts temporaland spatial selection pressures that are conducive for thesurvival of cell clones retaining long-term, self-renewal abilitywhile acquiring the necessary traits for successful organ colo-nization, even in the absence of overt local bone invasion(3, 5, 6). These bone marrow–resident tumor cells (BMRTC)are considered to be the "seeds" of future metastasis; targetingthem for elimination or promoting the prolongation of theirquiescent/growth-arrested state can therefore be a promisingstrategy to overcome or delay metastatic onset (5, 7). Accord-ingly, it is imperative to identify novel biomarkers for BMRTCdetection and to develop new therapeutic strategies that caneliminate BMRTCs before they progress to overt metastasis.

Successful surgical resection of primary melanoma (with clearmargins) along with satisfactory lymph node dissection doesnot always prevent the incidence of late metastatic recurrence(5, 8). This suggests that metastatic dissemination is an earlyevent wherein melanoma cells remain dormant but viable indistant organs while retaining abilities to generate overt meta-stasis at a later time (3, 8–11). For example, using the RET.AADmelanoma mouse models, Eyles and colleagues demonstratedthat the process of tumor cell dissemination preceded the onsetof symptomatic metastasis (4). Furthermore, Ghossein andcolleagues found that the presence of melanoma-specific tran-scripts in clinical blood and bone marrow samples is associatedwith shorter median survival of patients (12). Despite thesereports pointing to bone marrow as the likely reservoir fordisseminated CTCs, the precise role of BMRTCs in the patho-genesis of metastatic melanoma remains unclear and no signif-icant progress has been made to target melanoma CTCs/BMRTCs for clinical metastasis prevention (4, 10–13).

We hypothesized that melanoma CTCs migrate to and residein the bone marrow during asymptomatic progression of thedisease. We report here the isolation of CTC-enriched popula-tions from the peripheral blood of patients with metastaticmelanoma, their expansion in immunocompromised mice,followed by their harvesting and subsequent immunopheno-typing and molecular characterization. Using these approaches,we identified elevated USP7/PTEN expression as a distinct genesignature of BMRTCs. Furthermore, we demonstrated that inhi-bition of USP7 reduces the metastatic potential of BMRTCs byprolonging their arrest in bone marrow. Our investigationsprovide novel insights into the identity of BMRTCs that maybe detectable in patients with melanoma during asymptomaticmetastasis, and present data to support the evaluation of USP7inhibitors in patients with melanoma at risk of developingmetastasis.

Materials and MethodsPatient blood collection

Patients with melanoma with stage III or stage IV diseasewere accrued according to protocols approved by the Institu-tional Ethical Review Boards at the University of Texas MDAnderson Cancer Center (Houston, TX) and Houston Method-

ist Research Institute (HMRI, Houston, TX). All patient bloodsamples were collected after receiving informed written consentand according to the principles of Declaration of Helsinki.Clinical details of each patient and PTEN H-score, includedin the study, are provided in Supplementary Table S1 andSupplementary Fig. S1. Peripheral blood (18–20 mL) wasobtained at the middle of vein puncture and was collected inCellSave tubes (Menarini Silicon Biosystems, Inc.), or EDTAtubes under aseptic conditions. Samples were provided imme-diately to the laboratory for CTC isolation and analysis.

Antibodies and inhibitors used for the studyFor multiparametric flow cytometry and DEPArray, primary

antibodies were obtained from the following sources: FITC-CD45(#304054; 1:200), FITC-CD34 (#343504; 1:200), FITC-CD105(#323204; 1:200), FITC-CD90 (#328108; 1:200), FITC-CD73(#344016; 1:200), FITC HLA-A/B/C antibody (#311404;1:200), PerCP/Cy5.5-CD146 (#342014; 1:100), PE-HumanNG2/MCSP (#FAB2585P, 1:100), BV421-Ki67 (#350506;1:100) were obtained from BioLegend, anti–Melan-A antibody(# AC12-0297-03; 1:200) from Abcore, and FITC-Anti-S100(#ab76749; 1:50) was purchased from Abcam.

For IHC, anti-human, anti–Melan-A antibody (#ab51061;1:100), anti–tenascin-C (#ab108930, 1:100), and anti-p21(#ab188224, 1:100) were purchased from Abcam; HLA-ABC(#565292; 1:100) from BD Biosciences; USP7 (#GTX125894;1:200) from Genetex. PTEN antibody (#sc-7974; 1:20) waspurchased from Santa Cruz Biotechnology. Anti-p53 anti-body (#SAB4503021, 1:100) was purchased from Sigma, andCCP110 (#12780-I-AP, 1:100) antibody was obtained fromProteintech. Anti-mouse, USP7 (#26948-1-AP, 1:200), and PTEN(#603000-1-Ig, 1:200) antibodies were purchased from Protein-tech. Alexa Fluor-conjugated anti-mouse, anti-rabbit secondaryIgG antibodies used for immunofluorescence staining (1:500dilution) were obtained from Cell Signaling Technology. USP7inhibitors P5091 (#SML0770) and P22077 (#2301) were pur-chased from BioVision.

PBMC isolation and CTC enrichment by multiparametricflow cytometry

Peripheral blood mononuclear cells (PBMC) were isolatedby established procedures (14). Briefly, whole blood was trea-ted with red blood cell lysis buffer (154 mmol/L NH4Cl,10 mmol/L KHCO3, 0.1 mmol/L EDTA) at 1:25 ratio, followedby incubation at room temperature (25�C) for 5 minutes, thenpelleting the remaining blood cells at 300 � g for 10 minutes.Mononucleated cell pellet was then washed twice with 1� PBS(with 5 mmol/L EDTA) and used for fluorescence labelingfollowed by multiparametric flow sorting (FACSAria II; BDBiosciences). Data recorded during cell sorting were analyzedby DIVA acquisition software version 8.0.1 (BD Biosciences).Antibodies and reagents described above were used. Datagenerated by FACS were analyzed by FlowJo V10.

Experimental xenograftsAll animal study was approved by our Institutional Animal

Care and Use Committee protocol. Immunodeficient animalexperiments were performed using 4- to 8-week-old NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ (NSG) mice (The JacksonLaboratory). Flow-sorted Lin� PBMCs (�50,000 cells) deriv-ed from patient blood (8 mL volume) were injected in

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anesthetized NSG mice through intracardiac injection underaseptic conditions. Previous reports show that injection ofmelanoma cell lines (1 � 106 cells) with different metastaticabilities in immunodeficient mice typically develop overtmacrometastases in 10 to 12 weeks (poorly metastatic) and3 to 4 weeks (highly metastatic melanoma cell lines; ref. 15).Based upon this reference time-frame and considering thatmelanoma CTCs are rare cells with low transcriptional activity(Supplementary Fig. S2A and S2B; Supplementary Tables S2and S3), xenograft mice were euthanized 6 months after injec-tion with patient-isolated Lin� cells (13, 16–19). This wasbased upon the reasoning that this period should suffice forin vivo selection and elimination of nontumor cells and suc-cessful establishment of a niche for resident single cells insystemic viscera and bone marrow. Peripheral blood, bonemarrow, and organs were harvested for downstream analyses.Approximately 800 to 900 mL blood was collected in EDTAtubes by cardiac puncture of anesthetized mice. Animals weresacrificed soon afterwards and bone marrow/organ tissues wereharvested. In particular, bone marrow was obtained from femurand tibia by flushing them out with 1� PBS with EDTA(5 mmol/L) using a 28 G � 1/2 needle, followed by centrifu-gation at 300 � g for 10 minutes. Blood and bone marrow wereprocessed immediately for PBMC isolation and FACS analyses.

To study the effect of USP7 inhibitors on BMRTCs/CTCs, 2 to3 immunodeficient mice were injected with Lin� population(50,000 cells/mouse) selected from the same individual patientwith melanoma (16 mL blood volume). One animal of eachpatient group was then treated with USP7 inhibitor (eitherP5091 or P22077). CTC-derived xenografts (CDX) were treatedtwice a week subcutaneously with USP7 inhibitors at an MTD[P22077 (15 mg/kg); P5091 (10 mg/kg); ref. 20]. Controlvehicle consisted of 1� PBS (100 mL) injected in untreatedCDXs. After 11 weeks of treatment, mice were sacrificed andnecropsy performed.

Eight to 10-week-old, syngeneic mice C57BL/6J (n ¼ 30)were purchased from The Jackson Laboratory. Xenografts weredeveloped by intracardiac injection of aggressive B16F10melanoma cells (ATCC; 50,000 cells/mouse) and treatedwith or without USP7 inhibitors (P5091 and P22077) at theirMTD as described previously. After 3 weeks, each group ofanimals (n ¼ 10) was euthanized and organs (bone marrow,lung, brain, liver, and lymph node) were harvested to per-form hematoxylin and eosin (H&E) staining analyses. Allmelanoma cells were obtained from ATCC, DNA fingerprinted,and routinely validated for Mycoplasma-free testing at Charac-terized Cell Line Core Facility, MD Anderson, Houston, Texas.

Immunofluorescence and IHCFACS-isolated CTCs were subjected to quick air-dry on

Millennia 2000 adhesive glass slides (StatLab), and fixed with4% paraformaldehyde (21). Cells were permeabilized (0.05%Triton X-100 in 1� PBS) for 30 minutes, followed by 30-minuteincubation in blocking buffer (1% BSA þ 1% normal goatserum in 1� PBS). Next, immunofluorescent cell staining wasemployed using selected primary and secondary antibodies.Magnified (�100) images were captured using Zeiss AxioObserver microscope Z1 (Carl Zeiss), and data were analyzedusing Zeiss ZEN2 software. Harvested tissue was processedand stained for H&E and other IHC markers by the researchpathology core at HMRI. Images were captured by using EVOS

XL Cell Imaging System (Thermo Fisher Scientific). IHC imageswere taken and quantified by free-access ImageJ software.Reciprocal intensity (250 � y) was measured by subtractingthe mean intensity of the stained area (y) from the maximum(250) unstained white area intensity as described previously(22). Student t test, type 2, paired 2 was used to calculate thedifference in reciprocal intensity between two groups (USP7inhibitors treated vs. untreated) for each protein.

DEPArray and CellSearch CTC interrogationFlow-sorted CDX-derived HLAþ/Melan-Aþ were fixed with

2% paraformaldehyde for 20 minutes at 25�C followed bywashing with 1� PBS to visualize cells at single-cell level. CTCswere then stained with appropriate antibodies, rewashed withthe SB115 buffer, and loaded onto the DEPArray platform(Menarini Silicon Biosystems, Inc.). Cells were then detectedand analyzed by using Cell Browser software v3.0 (MenariniSilicon Biosystems, Inc.), as reported previously (14, 21). ForCellSearch CTC enumeration, harvested murine blood wasspiked with blood from healthy donors and was processedusing CellTracks circulating Melanoma Cell Kit and CellSearchplatform (Menarini Silicon Biosystems, Inc.), following themanufacturer's guidelines.

RNA isolation and gene expression profilingTotal RNA isolation was performed using NucleoSpin RNA XS

Isolation Kit (Macherey-Nagel, Inc.), then immediately providedto the sequencing/noncoding RNA core facility (MD AndersonCancer Center). RNA and cDNA amplifications, quality controls,and gene expression arrays were performed using the humanmicroarray platform (Clariom D, Affymetrix, Inc.). BMRTC/CTCsamples were RNA-normalized using Affymetrix Powertool1.18.0, and annotations were taken from Affymetrix version36. Gene expression data analysis from each of BMRTC/CTCsubsets (derived from asymptomatic CDX mice, with absence ofhistopathologic confirmed macrometastasis) was performedusing Transcriptome Analysis Console 3.0.0.466 (Affymetrix,Inc.). Two-way ANOVA was performed to calculate fold change(FC) and P value. Pathway enrichment analyses were subsequent-ly performed using the Ingenuity Pathway Analysis softwareversion 01-07 (Qiagen, Inc.).

WTA amplification and qRT-PCRmRNA and cDNA amplification were carried out from isolated

RNA using REPLI-g single-cell WTA Amplification Kit, accordingto the manufacturer's instructions (Qiagen, Inc.). AmplifiedcDNA was purified using the ExoSAP method (Affymetrix;#78202.4X.1.ML), and subjected to qRT-PCR using the SensiFASTSYBR Hi-ROX Kit (Bioline, #BIO-92020). CT values were normal-ized with housekeeping gene b-actin and log2 (�DDCT) werecalculated. Student t test, type 2, paired 2was performed to obtaintheP valueof each gene. Primer sequences for each gene are shownin Supplementary Table S4.

Whole-genome amplification and next-generation sequencingWhole-genome amplification (WGA) was performed by

REPLI-g Single-Cell WGA Kit, according to the manufacturer'sinstructions (Qiagen, Inc.). Briefly, the pool of CTC subsets waslysed and denatured followed by amplification to obtain intactamplified DNA of 10 kb. Ion Torrent next-generation sequenc-ing (NGS) was performed by using Ion AmpliSeq Cancer

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Hotspot Panel v2 (Thermo Fisher Scientific, #4475346).Libraries were constructed using amplified DNA and loadedon Ion Torrent Pmt sequencer. Sequences were assembled withhuman reference hg19 assembly and variant analyses wereperformed by Advaita iVariantGuide (http://www.advaitabio.com/ipathwayguide). Analyses were based on NGS for thedetection of somatic mutations in the coding sequence of aminimum of 46 genes (range 46–128), which were performedon DNAs extracted from samples in the CLIA-certified molec-ular diagnostic laboratory (University of Texas MD AndersonCancer Center).

Mitochondrial DNA assessment and cell surface andintranuclear flow cytometry

The ratio of mitochondrial DNA (mtDNA) to nuclear DNA(nDNA) copies was used to verify cell proliferative status (MTNindex; ref. 23). The mitochondrial target was strategically selectedwithin the noncoding displacement loop (MTDL) of themtGenome because of the rare occurrence of large-scale deletionsthat are common to other areas, for example, the major arc55.Single-copy and low variability b2-microglobulin (B2M) genewas selected as the nuclear target. Relative quantitative PCR(qPCR) was performed and MTN index was calculated [MTNindex ¼ 2 � 2DCT, where DCT ¼ (CTB2M � CTMTDL)].

We used the Permiflow method (US patent #US7326577 B2)of cell fixation for concomitant cell surface (e.g., HLAþ/Mel-Aþ), and intranuclear staining of target proteins (e.g.,Ki67) in CDX-derived cell populations, as described previously(14). Briefly, xenograft-isolated bone marrow and bloodPBMCs were first stained with the live/dead fixable ZombieNIR dye (BioLegend, #423105, 1:100) for 15 minutes, followedby two sequential washes with 1� PBS. All samples were thenfixed and permeabilized with the Permiflow solution by incu-bating at 42�C for 1 hour, followed by washing with 1� PBS.Cells were then resuspended in staining buffer and stained withappropriate antibodies for flow cytometric analyses.

ResultsIsolation and validation of melanoma CTC-enriched cellpopulations isolated from patients' blood

CTCs derived from patients display significant heterogeneityin cell surface biomarkers, epitomized by the coexistence oftumor-initiating and stem cell populations within a singleblood draw (16, 17). A consequence of this diversity is thatmultiple marker-based platforms (including CellSearch, theonly FDA-cleared platform for clinical CTC testing), and cellsize-based microfiltration devices are unable to capture theentire spectrum of CTC subtypes that may be biologicallyand/or clinically relevant in cancer progression (18, 24, 25).Because there is no universal melanoma CTC marker capable ofidentifying the heterogeneous CTC subgroups, particularlyclones that may transit to- and from the bone marrow, weused immunocompromised mice for the in vivo selection ofcells capable of bone marrow homing and residence. To imple-ment this strategy, we first employedmultiparametric flow cyto-metry to deplete normal cell lineages present in the peripheralblood of patients with melanoma (patients enrolled in thisstudy are listed in Supplementary Table S1), thereby enrichingcell populations containing putative as well as uncharacterizedabnormal cell populations (here referred as Lin� cells). Flow

cytometric gating consisted of doublet discrimination anddead cell (DAPIþ) elimination, followed by depletion of leu-kocytes (CD45þ), circulating endothelial cells (CD34þ), andmesenchymal stem cells (CD73þ/CD90þ/CD105þ; Fig. 1A).Using this strategy, we successfully captured putative melano-ma CTCs identified by the presence of established melanomamarkers (Melan-A and S100) from the peripheral blood of8 patients with melanoma validated through immunofluo-rescence (Fig. 1B; ref. 16). Second, we performed Ion Torrentnext-generation DNA sequencing and analyzed the geneticprofiles of 409 oncogenes and tumor suppressor genes of CTCs(Lin� and Melan-Aþ cells) derived from two independentpatients with melanoma (patients #9 and #10). We identified176 and 231 clinicopathologic mutations in respective patients(#9 and #10), present on NF1, KRAS, TP53, RB1, ATM, andEGFR genes (Fig. 1C; Supplementary Table S5A and S5B),confirming the neoplastic identity of these cells. Of note, CTCsfrom one of the analyzed patients (patient #10) harbored amelanoma-associated mutation in CDKN2A (p.Ala148Thr;COSM3736958;https://www.ncbi.nlm.nih.gov/clinvar/variation).We also confirmed the presence of skin cancer–associated muta-tions in XPA (c.-4A>G), ERCC1 (p.Asn118Asn), and ERCC5(p.Gly1534Arg; https://www.ncbi.nlm.nih.gov/clinvar/variation).Third, to validate that melanoma patient–isolated Lin� cells(patients #2, #3, #4, and #7) were distinct from normal PBMCs,we performed whole-genome microarray analyses on these cells(n ¼ 4), and compared them with the gene expression profiles ofPBMCs derived from healthy donors (n ¼ 9). PBMC dataset(GSE100054) was specifically chosen because it was obtainedusing the same human microarray platform (Clariom D; Affyme-trix, Inc.). A total of 15,056 genes were upregulated and 23,430genes were downregulated (fold change <�2 or >2, P ¼ 0.05) inLin� cell populations compared with healthy donor PBMCs,suggesting low transcriptional activity (Fig. 1D and E; Supple-mentary Table S2). Unsupervised hierarchical clusteringshowed distinct gene expression patterns in Lin� and PBMCcell populations. (Fig. 1F). Lin� populations possessed signi-ficantly higher expression of BAGE, MAGEA1, B4GALNT1,DCD, and S100A3 confirming the presence of specific mela-noma-associated markers within this population (Supplemen-tary Table S3; refs. 16, 19, 26). Downstream pathway enrich-ment analyses in Lin� cell pools predicted increased activationof nuclear receptor andoncogenic signaling, alongwith inhibitionof immune regulation and progrowth signaling pathways(Supplementary Fig. S3; Supplementary Tables S6 and S7). Thesefindings were reflected in cellular functions, for example, upre-gulated transcripts implicated in cell survival and prodevelop-ment functionswith a concomitant decrease in pathways involvedin cell proliferation and inflammation. Collectively, these resultsasserted the presence of putative and uncharacterized melanomaCTCs in Lin� cell populations isolated by multiparametric flowcytometry.

Expansion of melanoma patient–isolated Lin� cells in vivoTo evaluate whether Lin� cells isolated from patients with

melanoma could recapitulate the metastatic cascade, we firstinjected Lin� cell populations (n ¼ 8) into the left ventricle ofimmunodeficient mice (patients #1–8). Mice were euthanizedat the predesignated study endpoint (6 months), after collect-ing blood by cardiac puncture. Bone marrow from long bones(femur, tibia, and humerus) and organs typically affected by

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Figure 1.

Isolation, validation, and molecular characterization of de novo Lin� cell population. A, Enrichment of viable Lin� cell population (CD45�/CD34�/CD90�/CD73�/CD105� cells) from the blood of 8 patients with melanoma through multiparametric flow sorting (patients #1–8). B, Validation of FACS procedures bymelanoma markers (S100 and Melan-A) expression in Lin� cell population through immunofluorescence staining. Representative images are shown. Scale bar,10 mmol/L. C, Genetic mutational profiling of Lin�/Melan-Aþ cell populations derived from two representative patients with melanoma (patients #9 and #10) byAmpliSeq Ion Torrent Cancer Hotspot Panel V2. Number of missense, nonsense, and silent mutations of each patient is indicated. D, Differential geneexpression profiling of Lin� cell population from patients with melanoma (n ¼ 4; patients #2, #3, #4, and #7), compared with dataset of PBMCs isolated fromblood of normal healthy donors (GSE100054; n¼ 9). Two-wayANOVAwere performed to calculate FC and P value. E, Scatter plots showing global gene expressionchanges of Lin� cell population. F, Hierarchical clustering showing distinct transcriptomic signatures of Lin� cell population versus PBMCs.






metastatic melanoma (lung, brain, and liver) were collected fordownstream analyses. Interestingly, although routine histo-pathologic inspection did not identify any macrometastaticdisease in these organs, extensive IHC analyses employinghuman (HLA-ABC) and melanoma (Melan-A) biomarkersdetected discrete resident human melanoma cells in multiplemurine organs (CDXs; Supplementary Fig. S4A and S4B;ref. 27). Second, to assess the presence of human melanomaCTCs in blood of CDX models (derived from patient #4), wespiked CDX blood with healthy donor blood and performedanalyses using the CellSearch platform to visualize and enu-merate CTCs. We identified eight DAPIþ/CD45�/Melan-Aþ

cells from 500 mL of murine blood by CellSearch (Fig. 2A).Next, we harvested blood and bone marrow for PBMC iso-lation followed by multiparametric flow sorting, and isolatedHLAþ/Melan-Aþ ex vivo CTCs and BMRTCs (Fig. 2B). Wevalidated the veracity of flow procedures by immunofluores-cence analyses, detecting the presence of human (HLA) andmelanoma (Melan-A) markers in HLAþ/Melan-Aþ FACS-sortedpopulations (Fig. 2C). Third, we confirmed the heterogeneityof a pool of melanoma markers (Melan-A/CD146/NG2/S100)in CDX-derived BMRTC/CTC populations (patient #3) by theDEPArray (Fig. 2D; refs. 16, 17). The DEPArray is an antigen-agnostic CTC capturing technology that separates viable rarecells at a single-cell level. An additional important aspect of thissystem is the absence of shear force stress on cells duringrecovery (14). We identified, analyzed, and quantitated het-erogeneous melanoma cell populations displaying specificmelanoma markers in FACS-sorted HLAþ/Melan-Aþ cell popu-lation derived from BMRTCs/CTCs of the same CDX mice:Melan-Aþ/S100þ/CD146þ/NG2þ (BMRTCs ¼ 27; CTCs ¼ 8),Melan-Aþ/S100�/CD146þ/NG2� (BMRTCs ¼ 11; CTCs ¼ 10),Melan-Aþ/S100þ/CD146þ/NG2� (BMRTCs ¼ 11; CTCs ¼ 14),and Melan-Aþ/S100�/CD146�/NG2� (BMRTCs ¼ 0; CTCs ¼

14; Fig. 2D). Fourth, we performed genetic profiling for a familyof 50 key cancer genes by Ion Torrent NGS on ex vivo BMRTCsand CTCs (patients #3 and 4). We discovered novel mutationsin PIK3CA, NRAS, KRAS, ERBB4, PTEN, and APC genes alongwith two hotspot cosmic mutations (COSM21451 andCOSM25085) in the PIK3CA gene using hg19 human genomeassembly as reference (Fig. 2B; Supplementary Table S8). Final-ly, we performed differential gene expression analysis of ex vivoversus de novo CTCs (Lin�/CTC-enriched population) frompaired patient samples (patients #2, #3, #4, and #7) by humanmicroarray profiling (ClariomD; Affymetrix, Inc.). We observed3,777 significantly upregulated and 3,879 significantly down-regulated transcripts (FC ¼ 2.5; P ¼ 0.05) when CTCs versusLin�/CTC-enriched population-derived microarray data werecompared. We also observed 12,431 significantly overlappinggenes between arrays of these population (P ¼ 0.05; Supple-mentary Fig. S5). Collectively, these results demonstratethat CTC-enriched cell populations isolated from melanomapatients and expanded in CDX models could be successfullyharvested from blood and bone marrow of these mice.

Divergent transcriptomic signatures between ex vivo BMRTCsand CTCs

To determine functional differences between BMRTCs andCTCs, we performed transcriptome profiling of HLAþ/Melan-Aþ

cell populations obtained from CDX (derived from patients#1–8) bone marrow, and blood. We used paired BMRTC/CTCpopulations derived from eight CDX models (showing absenceof macrometastatic disease) generated by injecting Lin� cellpopulation from individual patients with metastatic melanoma(n ¼ 8), and performed human microarray profiling (ClariomD; Affymetrix, Inc.). Analyses identified 3,645 differentiallyexpressed genes; 2,439 genes were upregulated, whereas 1,206genes were downregulated (FC¼ 2.5, P-value¼ 0.05) in BMRTCs

Figure 2.

Isolation and validation of melanoma patient CTCs/BMRTCs from CDXs. A, Capture, visualization, and enumeration of human melanoma CTCs byCellSearch (CD45�/CD34�/DAPIþ/Melan-Aþ cells; patient #4). B, Multiparametric flow cytometry gating used to isolate human melanoma cells frombone marrow and blood of CDXs models (DAPI�/HLAþ/Melan-Aþ cells). Table showing number of genomic mutations (50 cancer gene sequencingpanel) analyzed by AmpliSeq Ion Torrent in representative patients (see Materials and Methods for details). C, Immunofluorescence visualization ofpatient-derived CTCs and BMRTCs from CDXs. Scale bar, 10 mm. D, Representative images of ex vivo CTCs and BMRTCs isolated by the DEPArray (patient #3).

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versus CTCs (Fig. 3A and B). Divergent transcriptomic signaturesof BMRTCs/CTCs in CDXmodels reflected the differential behav-ior of Lin� cells owing to their spatial niche (Fig. 3A and B;Supplementary Fig. S6). Subsequent pathway enrichment analy-ses showed that the top five upregulated canonical pathways wereEIF2 signaling, actin cytoskeleton, systemic lupus erythematosus,hypoxia signaling, and, notably, the protein ubiquitination path-way (Fig. 3B; Supplementary Table S9). Molecular and cellularfunctional annotations also indicated that BMRTCs upregulatedgenes involved in gene expression, RNA posttranslational mod-ifications, protein synthesis, cell cycle, and cell survival with aconcomitant decrease in cell death and apoptosis. Second,because the distinct gene expression pattern of ex vivo BMRTCsmay have been conferred by the osteogenic niche during theirresidence in bone marrow and could be a reflection of theirdependence on these unique signaling mechanisms, we focusedon individual genes in the protein ubiquitination pathwaythat were most strikingly elevated in BMRTCs. We found thatPTEN, a known tumor suppressor gene, and Ubiquitin SpecificPeptidase 7 (USP7), a deubiquitinating enzyme involved inposttranslational modifications, were significantly upregulated(PTEN, FC ¼ 219, P ¼ 0.0028; USP7, FC ¼ 5, P ¼ 0.0072)in ex vivo BMRTCs (Fig. 3B; refs. 28, 29). To confirm thesefindings, we assessed USP7 and PTEN expression on FACS-sortedHLAþ/Melan-Aþ ex vivo BMRTCs/CTCs by immunofluorescence,and observed elevated expression of USP7 and PTEN in BMRTCs(Fig. 3C). Next, we performed survival analyses of USP7/PTEN

gene expression in patients (n ¼ 114) with cutaneous melanomawhose data were obtained from The Cancer Genome Atlas data-base by employing Oncolnc software tool (http://www.oncolnc.org/; ref. 30). Kaplan–Meier survival analyses show that highexpression of USP7 correlated with decreased patient survival,whereas PTEN expression was not significantly associated (log-rank P values of 0.0167 vs. 0.112; HR, 1.598 vs. 0.7937 with 95%confidence interval; Fig. 3D). In addition, we did not observe anycorrelation between patient's PTEN mutational status and itsexpression on CDX-derived BMRTCs population. Collectively,these findings revealed that USP7 is uniquely upregulated in exvivo BMRTCs and correlates with shorter patient survival.

USP7 inhibition modulates metastatic competence andproliferative properties of CDX-isolated BMRTCs/CTCs

We investigated the effects of USP7 inhibitors on metastaticcompetency of BMRTCs as melanoma-derived, bone marrow–

associated CTC population. We performed drug susceptibilityassays in CDXs using two clinically relevant USP7 inhibitors:P5091 and P22077. We injected Lin� cells (50,000 cells/mouse) from 6 patients with melanoma (patients #11–16)simultaneously into 13 NSG mice and treated mice in parallelwith: P5091, P22077, or vehicle (PBS). Mice were euthanizedafter 11 weeks of treatment and visceral organs and CTCsfrom blood and bone marrow were harvested for downstreamanalysis. After euthanization, vehicle and USP7 inhibitor-treated CDX mice (11 weeks of USP7 inhibitor treatment)

Figure 3.

Transcriptional profiling of patients with melanoma BMRTCs versus CTCs in CDXs. A, Hierarchical clustering (FC ¼ 2.5; P ¼ 0.05). B, Volcano plotshowing differentially expressed genes in ex vivo BMRTCs versus CTCs by human array (Clariom D; Affymetrix, Inc.); two-way ANOVA was performedto calculate FC and P value [patients #1–8; BMRTCs ¼ 2 and CTCs ¼ 1 (shown below)]. Table displaying significantly altered top canonical pathwaysin ex vivo BMRTC versus CTC population. C, Increased expression of USP7-PTEN axis in BMRTs versus CTCs by immunofluorescence analyses (scale bar,10 mm). Graph shows the number of USP7 and PTENþ cells in BMRTC/CTC populations. One-sample t test was performed to compare the P value.D, Kaplan–Meier plot analyses of USP7 and PTEN performed using cutaneous melanoma patients' The Cancer Genome Atlas database (n ¼ 114; http://www.oncolnc.org/). Blue/red lines indicate USP7 and PTEN low/high expression, respectively. Log rank P value and HR (USP7 ¼ 1.599 and PTEN ¼ 0.7937) werecalculated by GraphPad Prism ver7 (95% confidence interval).





http://www.oncolnc.org/





injected with Lin� cell population from the same individualpatient, we performed IHC to assess USP7 and PTEN expressionin bone marrow and lung as most common metastatic mela-noma sites (1). We found a significant reduction of USP7,PTEN, and Melan-Aþ cells in bone marrow and lung tissues(micrometastatic disease) in USP7 inhibitor-treated versusPBS-treated CDXs, revealing that USP7 targeting leads to asignificant reduction of metastatic competency in patient-derived, bone marrow–associated CTCs (Fig. 4A–E; Supple-mentary Fig. S7A–S7E).

We also investigated the effects of the USP7 inhibitors onmetastatic competency in a syngeneic mice model. We injectedmetastasis-competent B16F10 melanoma cells through intra-cardiac procedure in syngeneic C57BL/6J mice. We groupedmice into three categories (n ¼ 10 for each group): (i)PBS-treated control group and groups treated with USP7 inhi-bitors (ii) P5091 and (iii) P22077, respectively. Mice wereeuthanized after 21 days, and bone marrow, lung, liver, lymphnode, and brain tissues were harvested for analysis. Histo-pathologic evaluation showed a significant reduction of mela-noma metastasis in with both USP7 inhibitors versus untreatedcontrol mice groups (Fig. 5A and B). Third, we investigatedthe effect of USP7 inhibitors on nuclear-cytoplasmic localiza-tion of PTEN (Supplementary Fig. S8; ref. 28). We observedsignificant reduction of nuclear:cytoplasmic PTEN ratio onBMRTC population derived from USP7 inhibitor treated versusuntreated CDXs (Supplementary Fig. S8A and S8B). However,we observed restoration of nuclear PTEN in lung mets of micetreated with USP7 inhibitors versus untreated (SupplementaryFig. S8B and S8C). This suggests that distinct microenvironmentproperties influence the USP7 regulation of PTEN expression.

Fourth, we investigated the effects of USP7 inhibitors on themetastatic competency of ex vivo BMRTCs, considering thatroles of USP7 in the pathogenesis of metastatic melanomaremain unclear. We assessed USP7 inhibitors' effect on theproliferative capacity of ex vivo BMRTCs and CTCs (derived frompatients #13 and #14) by performing flow cytometric analyses forKi67, a marker of cellular proliferation (14). We detected greaterpercentage of Ki67-high cells in CTCs versus BMRTCs irrespectiveof USP7 inhibitor treatment [P5091: 89.1% (CTCs) vs. 68.4%(BMRTCs); P22077: 91.5% (CTCs) vs. 42.4% (BMRTCs);Fig. 6A]. We observed increased percentage of Ki67high amongex vivo BMRTCs isolated from USP7 inhibitor-treated versusuntreated CDXs. Conversely, we did not observe any differencein Ki67 status among ex vivo CTCs isolated from USP7 inhibitors-treated versus untreated CDXs. Furthermore, because mtDNAcopy number is an indicator of mitochondrial biogenesis andproliferative potency (31), we compared mtDNA versus nuclearDNA content ratios in ex vivo BMRTCs versus CTCs of six CDXmice (derived from patients #12, #13, and #14). QuantitativePCR showed significantly higher MTN index in BMRTCs derivedfrom USP7 inhibitor-treated versus untreated CDX mice(Fig. 6B). In addition, CellSearch analyses of ex vivo BMRTCs/CTCs from CDXs (derived from patient #12) treated with USP7inhibitors showed higher number of melanoma CTCs (CD45�/CD34�/DAPIþ/Melan-Aþ cells) in BMRTCs versus correspondingCTCs (Fig. 6C). Cumulatively, these findings suggest that USP7inhibition causes a significant reduction of metastatic compe-tency of BMRTCs by restricting melanoma cell populationwithin bone marrow locales (Figs. 4A–E, 5A–B, and 6A–C;Supplementary Fig. S7A–S7E).

Effects of USP7 inhibitors on ex vivo BMRTCs/CTCsTo delineate genes involved in the metastatic potency of

BMRTCs and affected by USP7 inhibition, we performed com-parative transcriptional profiling of ex vivo BMRTCs from pairedvehicle versus USP7 inhibitor-treated CDX mice (derived frompatients #11, #12, and #16). Differential gene expression pro-filing revealed 321 significantly altered genes (138 upregulated,183 downregulated) in BMRTCs treated with USP7 inhibitors(Fig. 6D and E). CDX-derived BMRTCs clustered together intwo groups when hierarchical clustering was performed (FC ¼2, P ¼ 0.05), suggesting that USP7 affected BMRTCs by adistinct gene signature (Fig. 6F). We also evaluated USP7-altered, metastasis-competent targets in distinct CDX-derivedBMRTCs and found significantly four upregulated (POLE,CCP110, SMAD13, and MTND4LP12) and nine downregulated(TNC, FBXO25, RGS22, PDE1A, GPCPD1, ABSB4, SERINC3,EIF3F, and UBXN4) genes associated with metastatic coloni-zation. Furthermore, miRNA analyses revealed upregulation ofMIR885-5p, MIR9-1, MIR6870, and MIR3657 and downregu-lation of MIR4719, MIR 548A2, MIR548AE1, MIR548AJ1, andMIR548AX2 transcripts in USP7-modulated BMRTCs. Quanti-tative PCR revealed significant downregulation of USP7, PTEN,TNC, and GPCPD1 transcripts in HLAþ/Melan-Aþ ex vivoBMRTCs derived from CDXs treated with USP7 inhibitors(P5091 and P22077) versus vehicle (Fig. 6G). RGS22 tran-scripts were significantly downregulated in CDX BMRTCs whentreated with inhibitor P22077 only, but not with inhibitorP5091 versus vehicle (Fig. 6G). Furthermore, expression ofgenes involved in cell invasion (TNC, SERINC3, ABSB4, andSMAD13), cell migration (TNC, ASB4, RGS22, and GPCPD1),cell proliferation (MIR885-p and CCP110), and cell apoptosis(TNC, FBXO25, PDE1A, EIF3F) were decreased by USP7 inhib-itor treatment, possibly highlighting regulatory roles of USP7at various metastatic steps during melanoma progression(Fig. 7; refs. 32–39). Next, to determine the role of USP7inhibition in BMRTC-driven lung colonization, we performedIHC for two USP7-modulated markers (TNC and CCP110) inlung tissue of CDX mice treated versus untreated with USP7inhibitors (derived from patients #11, #15, and #16). IHCquantification shows significant upregulation of CCP110 anddownregulation of TNC in CDX-derived lung tissue treatedversus untreated with USP7 inhibitors (P ¼ 0.05; Supplemen-tary Fig. S9A–S9D). Of relevance, USP7 inhibition has beenshown to play a role in MDM2 degradation, which leads toreactivation of tumor suppressor genes p53 and p21, resultingin tumor growth inhibition (40). Accordingly, we quantifiedp53 and p21 expression in lung and bone marrow tissues(derived from patients #13 and #15) of CDXs treated versusuntreated with USP7 inhibitors (Supplementary Fig. S10A–S10D). IHC quantification shows significantly elevated expres-sion of p53 and p21 on lung and bone marrow tissue derivedfrom CDX mice treated with versus without USP7 inhibitors (P¼ 0.05).

DiscussionThis study provides first-time evidence that asymptomatic

progression of metastatic melanoma can be recapitulated in vivousing patient-isolated CTCs, and demonstrates that melanomaCTCs at advanced disease stages contain heterogeneous cellpools bearing distinct characteristics associated with bone

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Figure 4.

USP7 inhibitors affect the metastatic potency of BMRTCs population in CDXs. A–E, Histopathologic Melan-A evaluation with colocalization of USP7 andPTEN staining in lung and bone marrow (BM) tissues from USP7 inhibitor-treated versus untreated CDXs (immunodeficient mice; patients #11–15).Black arrows, positive staining for respective markers. HLA and H&E staining of the same region are shown in extended Fig. 7 of "Supplementaryinformation" (Supplementary Fig. S7A–S7E). Graph showing semiquantitative estimation of USP7/PTEN staining by using ImageJ software. Student t test,type 2, paired 2 were performed to calculate the P value. Scale bar, 100 mm.






marrow versus blood locales. In addition, we show that USP7plays critical roles in BMRTC metastatic potential by regulatinggenes related to cellular proliferation, apoptosis, and cell migra-tion. Complementing previous investigations that have charac-terized the genetic landscape of primary and metastatic mela-noma lesions, we demonstrate that transcriptional subtyping ofmelanoma CTCs provides key insights into the molecularmechanisms that regulate metastatic potency by proposing anovel rationale-based approach for targeting BMRTCs (5, 7, 8,10, 11, 13, 16, 17). In contrast to the majority of CTC studiesthat have largely elucidated the spatial attributes of peripheralblood-based CTCs in the context of metastasis, we provideinsights into the primordial stages of CTC dissemination andsurvival in bonemarrowwithout progression to overt metastasis.

Employing a negative selection strategy to deplete normal cellpopulations of blood, we implanted patient Lin� cells in immu-nodeficient mice to generate CDX models that can faithfullyrecapitulate the early events of melanoma metastasis. Lin� cellpopulations not only had overexpression of melanoma markers,but also harbored mutations on multiple melanoma oncogenes,demonstrating their atypical nature (Fig. 1A–F; SupplementaryTable S5; refs. 14, 41). Furthermore, combining the use of anantigen-agnostic platform (DEPArray) with genomic sequencing,we show that melanoma CTCs home to and reside in the bonemarrowofCDXmicewhen injectedwithpatient-isolated Lin� cellpopulation (Figs. 2A–D and 4A–E). We consider this approachuseful for establishing patient-derived xenograft models particu-larly when patient tumor tissue may not be available.

Whole-genome transcriptomic arrays and qPCR for individualtarget genes identified distinct transcriptional signatures of ex vivomelanoma BMRTCs, potentially reflecting effects of bonemarrowmicroenvironment on BMRTC gene expression. In addition, theproportion of Ki67-low cells was approximately 50% higher inex vivo BMRTCs versus CTCs, suggesting that BMRTCs containa distinct cell subpopulation that may be in a state of subduedproliferation (Fig. 6A). However, we did not observe any corre-lation between high Ki67 index present in USP7 inhibitors–treated BMRTCs population and metastatic burden in visceralorgans or bone. Of note, Ki67 as a good indicator of proliferationand present in G1–M and S phases of cell cycle while absent inthe G0 phase (42). Therefore, this is likely due to the effect ofUSP7 inhibitors, which arrests the Ki67þ BMRTC population atG1–S phase of cell cycle and inhibits their metastatic potential(43, 44). This suggests that USP7 inhibitors, although augment-ing the proliferative potency of BMRTCs, do not augment theirdissemination from bone marrow to generate lethal metastasis.Interestingly, we detected BMRTC-specific mutations in SMAD4and APC genes, suggesting that besides affecting gene expression,bone marrow microenvironment may impart temporal selectivepressures on BMRTCs, resulting in their acquiring unique muta-tions (Fig. 2B; Supplementary Table S8; ref. 5).

The deubiquitinase enzyme USP7 is involved in the posttrans-lational modification of PTEN altering its tumor-suppressiveactivity. Loss of PTEN function in patients with melanomahas been linked to significantly faster progression to overtbrain metastasis along with poorer overall survival (45). In

Figure 5.

USP7 inhibitors affect the metastatic potency of BMRTC population in syngeneic mice. A, Representative H&E staining of lung, bone marrow (BM), liver, brain,and lymph node tissue section derived from syngeneic mice treated versus untreated with USP7 inhibitors. B, Five to eight images of each mousewere quantified for melanoma cells in distant tissue organs. Student t test, type 2, paired 2 were performed to calculate the P value. Scale bar, 100 mm.

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our CDX models, we found significantly higher USP7 expres-sion that colocalized with PTEN in BMRTCs when comparedwith ex vivo CTCs (Figs. 3A–D and 4A–E). It is likely that thehigh expression of USP7 transcripts detected in BMRTCsreflects the intrinsic behavior of heterogeneous CTC poolas well as properties of the bone marrow microenvironment.

This is because: (i) patient-derived CTCs contain a heteroge-neous pool of melanoma cells and CTC subsets have thetendency to seed the bone marrow and (ii) USP7 modulatesthe osteogenic differentiation of mesenchymal stem cells inbone marrow (5, 46). Kaplan–Meier survival analyses indicat-ed that high USP7 (but not PTEN) expression is significantly

Figure 6.

Effects of USP7 inhibitors on the proliferative ability of patient BMRTCs/CTCs and BMRTC gene expression isolated from CDXs. A, Effects of USP7 inhibitorson Ki67 HLAþ/Melan-Aþ BMRTC/CTC populations. Mean fluorescence intensity (MFI) of Ki-67 via intracellular multiparametric flow cytometry(patients #13 and #14). Details of procedures and reagents used are described in the Materials and Methods. B, MTN index of FACS-sorted HLAþ/Melan-Aþ

population derived from CDXs (n ¼ 3) paired bone marrow and blood (patients #12, #13, and #14). Total gDNA was extracted and purified, then subjectedto MTN determination using qPCR. C, Capture, visualization, and enumeration of CTCs by CellSearch analyses of melanoma patient BMRTCs/CTCsisolated from CDXs treated with or without USP7 inhibitors (patient #12). A total of 500 mL of CDX blood was collected and analyzed according toCellSearch specifications (CD45�/CD34�/DAPIþ/Melan-Aþ cells). Displayed are BMRTC/CTC numbers/500 mL blood per treatment condition. D,Differential gene expression profiling of BMRTC population isolated from CDXs treated with and without USP7 inhibitors. E, Scatter plot showingdifferentially expressed genes in BMRTCs by human array (Clariom D; Affymetrix, Inc.). Two-way ANOVA was performed to calculate FC and P value(FC ¼ 2, P ¼ 0.05). F, Hierarchical clustering (FC ¼ 2.5, P ¼ 0.05) showing differential expressed genes in BMTRCs isolated from CDXs treatedversus untreated USP7 inhibitors P5091 (patients #11 and 12) and P22077 (patients #12 and 16). Upregulated/downregulated genes are highlighted inred/black, respectively. G, Validation of microarray data by qPCR analyses. Relative mRNA expression and SD were calculated for each gene. Student ttest, type 2, paired 2 were performed to calculate respective P values.






associated with decreased overall survival of patients withmelanoma (Fig. 3D), while targeting USP7 with specific inhi-bitors reduced ex vivo CTC numbers at metastatic organ sitesalong with a concomitant decrease in USP7 and PTEN expres-sion (Figs. 4A–E and 5A and B). Our immunohistopathologicevaluation shows a significant upregulation of p53 and p21expression in lung and bone marrow tissues derived from CDXmice treated with USP7 inhibitors versus PBS-treated group(Supplementary Fig. S10A–S10D); however, we did notobserve a significant difference in p53 transcript expressionbetween ex vivo BMRTCs versus CTCs (28, 40). However, geneexpression microarray analysis of USP7 inhibitor treated versusuntreated ex vivo BMRTCs revealed several candidate genessignificantly affected by USP7 inhibition (Fig. 6D–F). Amongthem, TNC and FBXO25 are genes implicated in the develop-ment of metastatic melanoma, breast and non–small cell lungcancers (32, 33, 47). Furthermore, downregulation of genesinvolved in cell migration (RGS22, GPCPD1 and ASB4) andinvasion (TNC, SERINC3, ABSB4 and SMAD13) was alsodetected, suggesting that USP7 inhibition may impede BMRTCreshedding from bone marrow, arresting them in bone marrowlocales (35–37, 48). USP7 inhibition also decreased the expres-sion of proliferation-related biomarkers (Ki67 and MTNindices; Fig. 6A–F), possibly by affecting genes (e.g., CCP110,POLE, p21 and p53; Supplementary Figs. S9A–S9D and S10A–S10D) that arrest BMRTC cell-cycle progression and mayinduce senescent phenotype (38, 39). Collectively, these find-ings suggest that USP7 plays a central role in mediatingmelanoma CTC residence in bone marrow (Figs. 6A–G and 7).

Our study has some limitations. First, analyses were perform-ed on a low number of patients; therefore, we cannot concludethat all patients with melanoma follow these models and path-ways. Second, we profiled a limited number of cells in eachpatient, which, although done stochastically, may have someinherent sampling bias. Third, although we suggest that USP7inhibition–mediated reduction of micrometastasis is not a directeffect on cells already disseminated to distant organs, we cannotexclude a direct effect of USP7 inhibition on these cells. However,our results demonstrate that melanoma patient–isolated CTCshome to and reside in bonemarrow, and emphasize the relevanceof determining the prognostic value of bone marrow–associated,CTC-derived cell populations. The systematic interrogation of

these BMRTCs identified USP7 as an important mediator ofBMRTC-specific signaling and provided evidence that applyingUSP7 inhibitors in clinical settings can be relevant to eliminateresidual melanoma cells in bone marrow and thereby preventfurther metastatic spread.

Disclosure of Potential Conflicts of InterestM.T. Tetzlaff is a consultant/advisory board member of Seattle Genetics,

Novartis, and Myriad Genetics. M.A. Davies reports receiving commercialresearch grants from GSK, Astrazeneca, Roche/Genentech, Oncothyreon, andSanofi-Aventis and is consultant/advisory board member of GSK, Novartis,BMS, Sanofi-Aventis, Syndax, and Nanostring. No potential conflicts of interestwere disclosed by the other authors.

Authors' ContributionsConception and design: M. Vishnoi, D. Boral, D. MarchettiDevelopment of methodology: M. Vishnoi, D. BoralAcquisition of data (provided animals, acquired and managed pati-ents, provided facilities, etc.): M. Vishnoi, H. Liu, W. Yin, M.L. Sprouse,D. Goswami-Sewell, M.T. Tetzlaff, M.A. Davies, I.C. Glitza OlivaAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): M. Vishnoi, D. Boral, M.L. Sprouse, W. Yin,D. Goswami-Sewell, M.T. TetzlaffWriting, review, and/or revision of the manuscript: M. Vishnoi, D. Boral,M.T. Tetzlaff, M.A. Davies, I.C. Glitza Oliva, D. MarchettiAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): M. Vishnoi, H. Liu, W. YinStudy supervision: D. Marchetti

AcknowledgmentsThis studywas supported by theNIH grants (1 R01CA 216991 and 1 R01 CA

160335 to D. Marchetti); the Dr. Miriam and Sheldon G. Adelson MedicalResearch Foundation (to M.A. Davies); the AIM at Melanoma Foundation(to M.A. Davies); and by philanthropic support from the MD AndersonMelanoma Shot Program (to M.A. Davies). We are thankful to Dr. DavidHaviland, Director of the Flow Cytometry Core at HoustonMethodist ResearchInstitute (HMRI), Dr. Zhubo Wei of the Biostatistics core at HMRI, and toDr. Chang-Gong Liu, Director of the sequencing and ncRNA core at MDAnderson Cancer Center for their respective expertise.

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 27, 2018; revised June 12, 2018; accepted July 13, 2018;published first July 19, 2018.

Figure 7.

Model representing the asymptomaticprogression of melanoma in patient-derived xenografts and effects ofUSP7 inhibition, either as a potentialdirect action on cells alreadydisseminated to distant organs or as aresult of USP7 targeting BMRTCs.

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References1. Howlader N, Noone AM, Krapcho M, Miller D, Bishop K, Kosary CL, et al.

SEER cancer statistics review, 1975-2014. Bethesda, MD: NCI.2. Luke JJ, Flaherty KT, Ribas A, Long GV. Targeted agents and immunothera-

pies: optimizing outcomes in melanoma. Nat Rev Clin Oncol 2017;14:463–82.

3. Friberg S, Nystrom A. Cancer metastases: early dissemination and laterecurrences. Cancer Growth Metastasis 2015;8:43–9.

4. Eyles J, Puaux AL, Wang X, Toh B, Prakash C, Hong M, et al. Tumorcells disseminate early, but immunosurveillance limits metastaticoutgrowth, in a mouse model of melanoma. J Clin Invest 2010;120:2030–39.

5. Werner-Klein M, Scheitler S, Hoffmann M, Hodak I, Dietz K, Lehnert P,et al. Genetic alterations driving metastatic colony formation areacquired outside of the primary tumour in melanoma. Nat Commun2018;9:595.

6. Rocken M. Early tumor dissemination, but late metastasis: insights intotumor dormancy. J Clin Invest 2010;120:1800–3.

7. Wong JH. Early diagnosis ofmetastatic disease inmelanoma: does itmake adifference?: Comment on "Long-term follow-up and survival of patientsfollowing a recurrence of melanoma after a negative sentinel lymph nodebiopsy result". JAMA Surg 2013;148:462.

8. Jones EL, Jones TS, Pearlman NW, Gao D, Stovall R, Gajdos C, et al. Long-term follow-up and survival of patients following a recurrence of mela-noma after a negative sentinel lymph node biopsy result. JAMA Surg2013;148:456–61.

9. Olmeda D, Cerezo-Wallis D, Riveiro-Falkenbach E, Pennacchi PC,Contreras-Alcalde M, Ibarz N, et al. Whole-body imaging of lympho-vascular niches identifies pre-metastatic roles of midkine. Nature2017;546:676–80.

10. Bourgault-Villada I, Hong M, Khoo K, Tham M, Toh B, Wai L-E, et al.Current insight into the metastatic process and melanoma cell dis-semination. In: Murph M, editor. Research on melanoma—aglimpse into current directions and future trends. Rijeka, Croatia:IntechOpen; 2011.

11. TsengWW, Doyle JA, Maguiness S, Horvai AE, Kashani-Sabet M, Leong SP.Giant cutaneous melanomas: evidence for primary tumour induced dor-mancy in metastatic sites? BMJ Case Rep 2009;2009:2073.

12. Ghossein RA, Carusone L, Bhattacharya S. Molecular detection of micro-metastases and circulating tumor cells in melanoma prostatic and breastcarcinomas. In Vivo 2000;14:237–50.

13. Klinac D, Gray ES, Freeman JB, Reid A, Bowyer S, Millward M, et al.Monitoring changes in circulating tumour cells as a prognostic indicator ofoverall survival and treatment response in patients with metastatic mel-anoma. BMC Cancer 2014;14:423.

14. Boral D, Vishnoi M, Liu HN, Yin W, Sprouse ML, Scamardo A, et al.Molecular characterization of breast cancer CTCs associated with brainmetastasis. Nat Commun 2017;8:196.

15. Thies A, Mauer S, Fodstad O, Schumacher U. Clinically proven markers ofmetastasis predictmetastatic spread of humanmelanoma cells engrafted inscid mice. Br J Cancer 2007;96:609–16.

16. Khoja L, Shenjere P, Hodgson C, Hodgetts J, Clack G, Hughes A, et al.Prevalence and heterogeneity of circulating tumour cells in metastaticcutaneous melanoma. Melanoma Res 2014;24:40–6.

17. Gray ES, Reid AL, Bowyer S, Calapre L, Siew K, Pearce R, et al. Circulatingmelanoma cell subpopulations: their heterogeneity and differentialresponses to treatment. J Invest Dermatol 2015;135:2040–48.

18. Luo X, Mitra D, Sullivan RJ, Wittner BS, Kimura AM, Pan S, et al. Isolationand molecular characterization of circulating melanoma cells. Cell Rep2014;7:645–53.

19. Kiyohara E, Hata K, Lam S, Hoon DS. Circulating tumor cells asprognostic biomarkers in cutaneous melanoma patients. Methods MolBiol 2014;1102.

20. Chauhan D, Tian Z, Nicholson B, Kumar KG, Zhou B, Carrasco R, et al. Asmall molecule inhibitor of ubiquitin-specific protease-7 induces apopto-sis in multiple myeloma cells and overcomes bortezomib resistance.Cancer Cell 2012;22:345–58.

21. VishnoiM, Peddibhotla S, YinW, ScamardoAT,GeorgeGC,HongDS, et al.The isolation and characterization of CTC subsets related to breast cancerdormancy. Sci Rep 2015;5:17533.

22. Nguyen D. Quantifying chromogen intensity in immunohistochem-istry via reciprocal intensity. Cancer InCytes 2013;2:e. doi: 10.5281/zenodo.13670.

23. Rooney JP, Ryde IT, Sanders LH, Howlett EH, Colton MD, Germ KE, et al.PCR based determination ofmitochondrial DNA copy number inmultiplespecies. Methods Mol Biol 2015;1241:23–38.

24. Attard G, Crespo M, Lim AC, Pope L, Zivi A, de Bono JS. Reporting thecapture efficiency of a filter-basedmicrodevice: a CTC is not a CTC unless itis CD45 negative–letter. Clin Cancer Res 2011;17:3048–49.

25. Mazzini C, Pinzani P, Salvianti F, Scatena C, Paglierani M, Ucci F, et al.Circulating tumor cells detection and counting in uveal melanomas by afiltration-based method. Cancers 2014;6:323–32.

26. Ortega-Martinez I, Gardeazabal J, Erramuzpe A, Sanchez-Diez A, Cortes J,Garcia-Vazquez MD, et al. Vitronectin and dermcidin serum levels predictthe metastatic progression of AJCC I-II early-stage melanoma. Int J Cancer2016;139:1598–607.

27. Jung IH, Chung YY, Jung DE, Kim YJ, Kim do H, Kim KS, et al. Impairedlymphocytes development and xenotransplantation of gastrointestinaltumor cells in prkdc-null SCID zebrafish model. Neoplasia 2016;18:468–79.

28. Carra G, Panuzzo C, Torti D, Parvis G, Crivellaro S, Familiari U, et al.Therapeutic inhibition of USP7-PTEN network in chronic lymphocyticleukemia: a strategy to overcome TP53mutated/deleted clones.Oncotarget2017;8:35508–22.

29. Zhou J, Wang J, Chen C, Yuan H, Wen X, Sun H. USP7: Target validationand drug discovery for cancer therapy. Med Chem 2018;14:3–18.

30. Anaya J. OncoLnc: linking TCGA survival data to mRNAs, miRNAs,lncRNAs. PeerJ Computer Sci 2016;2:e1780v1.

31. Clay Montier LL, Deng JJ, Bai Y. Number matters: control of mammalianmitochondrial DNA copy number. J Genet Genomics 2009;36:125–31.

32. Shao H, Kirkwood JM, Wells A. Tenascin-C signaling in melanoma. CellAdh Migr 2015;9:125–30.

33. Jiang GY, Zhang XP, Wang L, Lin XY, Yu JH, Wang EH, et al. FBXO25promotes cell proliferation, invasion, and migration of NSCLC. TumourBiol 2016;37:14311–9.

34. HuY, Xing J,Wang L,HuangM,GuoX,Chen L, et al. RGS22, a novel cancer/testis antigen, inhibits epithelial cell invasion and metastasis. Clin ExpMetastasis 2011;28:541–9.

35. Marchan R, Buttner B, Lambert J, Edlund K, Glaeser I, Blaszkewicz M, et al.Glycerol-3-phosphate acyltransferase 1 promotes tumor cell migrationand poor survival in ovarian carcinoma. Cancer Res 2017;77:4589–601.

36. Margue C, Philippidou D, Reinsbach SE, Schmitt M, Behrmann I, Kreis S.New target genes ofMITF-inducedmicroRNA-211 contribute tomelanomacell invasion. PLoS One 2013;8:e73473.

37. Javelaud D, Alexaki VI, Dennler S, Mohammad KS, Guise TA, Mauviel A.TGF-beta/SMAD/GLI2 signaling axis in cancer progression andmetastasis.Cancer Res 2011;71:5606–10.

38. Barenz F, Hoffmann I. Cell biology: DUBing CP110 controls centrosomenumbers. Curr Biol 2013;23:R459–60.

39. Aoude LG, Heitzer E, Johansson P, Gartside M, Wadt K, Pritchard AL,et al. POLE mutations in families predisposed to cutaneous melanoma.Fam Cancer 2015;14:621–8.

40. Turnbull AP, Ioannidis S, Krajewski WW, Pinto-Fernandez A, Heride C,Martin ACL, et al. Molecular basis of USP7 inhibition by selectivesmall-molecule inhibitors. Nature 2017;550:481–6.

41. Liu Z, Fusi A, Klopocki E, Schmittel A, Tinhofer I, Nonnenmacher A, et al.Negative enrichment by immunomagnetic nanobeads for unbiased char-acterization of circulating tumor cells from peripheral blood of cancerpatients. J Transl Med 2011;9:70.

42. Bruno S, Darzynkiewicz Z. Cell cycle dependent expression and stability ofthe nuclear protein detected by Ki-67 antibody in HL-60 cells. Cell Prolif1992;25:31–40.

43. Reverdy C, Conrath S, Lopez R, Planquette C, Atmanene C, Collura V, et al.Discovery of specific inhibitors of human USP7/HAUSP deubiquitinatingenzyme. Chem Biol 2012;19:467–77.

44. JagannathanM,Nguyen T, GalloD, LuthraN, BrownGW, Saridakis V, et al.A role for USP7 in DNA replication. Mol Cell Biol 2014;34:132–45.

45. Bucheit AD, Chen G, Siroy A, Tetzlaff M, Broaddus R, Milton D, et al.Complete loss of PTEN protein expression correlates with shorter time to






brain metastasis and survival in stage IIIB/C melanoma patients withBRAFV600 mutations. Clin Cancer Res 2014;20:5527–36.

46. Tang Y, Lv L, Li W, Zhang X, Jiang Y, Ge W, et al. Protein deubiquitinaseUSP7 is required for osteogenic differentiation of human adipose-derivedstem cells. Stem Cell Res Ther 2017;8:186.

47. Vanharanta S, Massague J. Origins of metastatic traits. Cancer Cell 2013;24:410–21.

48. Hu Y, Xing J, Chen L, Zheng Y, Zhou Z. RGS22 inhibits pancreaticadenocarcinoma cell migration through the G12/13 alpha subunit/F-actinpathway. Oncol Rep 2015;34:2507–14.


Vishnoi et al.




2018;78:5349-5362. Published OnlineFirst July 19, 2018.Cancer Res Monika Vishnoi, Debasish Boral, Haowen Liu, et al.

Resident Melanoma CTCs−Bone Marrow Targeting USP7 Identifies a Metastasis-Competent State within

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Targeting USP7 Identiﬁes a Metastasis- Competent State ... · Targeting USP7 Identiﬁes a Metastasis-Competent State within Bone Marrow–Resident Melanoma CTCs Monika Vishnoi1,