Targeting USP7 Identifies a Metastasis- Competent State Within

Published OnlineFirst July 19, 2018; DOI: 10.1158/0008-5472.CAN-18-0644

Cancer Translational Science Research

Targeting USP7 Identiﬁes a Metastasis- Competent State within Bone Marrow–Resident Melanoma CTCs Monika 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 major Asymptomatic cause of death from melanoma, Liquid biopsy Distant organ micrometastasis the most lethal form of skin cancer. progression Although most patients with melanoma exhibit a substantial gap between onset of primary and metastatic tumors, signaling mechanisms implicated in the period of USP7 metastatic latency remain unclear. We hypothesized that melanoma circulating tumor cells (CTC) home to and reside in the bone marrow during the asymptomatic CTCs BMRTCs phase of disease progression. Melanoma progresses asymptomatically in patient-isolated CTC-derived xenografts and distant Using a strategy to deplete normal organ micrometastasis can be inhibited by targeting USP7. cell lineages (Lin ), we isolated © 2018 American Association for Cancer Research CTC-enriched cell populations from the blood of patients with metastatic melanoma, verified by the presence of putative CTCs characterized by melanoma-specific biomarkers and upregulated gene transcripts involved in cell survival and prodevelopment functions. Implantation of Lin population in NSG mice (CTC-derived xenografts, i.e., CDX), and subsequent transcriptomic analysis of ex vivo bone marrow–resident tumor cells (BMRTC) versus CTC identified protein ubiquitination as a significant regulatory pathway of BMRTC signaling. Selective inhibition of USP7, a key deubiquinating enzyme, arrested BMRTCs in bone marrow locales and decreased systemic micrometastasis. This study provides first-time evidence that the asymptomatic progression of metastatic melanoma 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 clinical metastasis.

Signiﬁcance: These ﬁndings provide insights into mechanism of melanoma recurrence and propose a novel approach to inhibit systematic metastatic disease by targeting bone marrow-resident tumor cells through pharmacological inhibition of USP7. Graphical Abstract: http://cancerres.aacrjournals.org/content/canres/78/18/5349/F1.large.jpg. Cancer Res; 78(18); 5349–62. 2018 AACR.

1Biomarker Research Program Center, Houston Methodist Research Institute, Introduction 2 Houston, Texas. Department of Melanoma Medical Oncology, The University of Melanoma, the most aggressive skin cancer, frequently Texas MD Anderson Cancer Center, Houston, Texas. metastasizes to distant organs such as bone, lung, liver, and Note: Supplementary data for this article are available at Cancer Research brain (1). Unlike localized melanoma, which can be sur- Online (http://cancerres.aacrjournals.org/). gically resected, metastatic melanoma with extranodal Corresponding Author: Dario Marchetti, Houston Methodist Research Institute, involvement is typically treated with systemic therapies Suite R7-111, MS R7-414, 6670 Bertner Avenue, Houston, TX 77030. Phone: 713- including targeted and immune therapy (2). Although out- 363-7769; Fax: 713-363-7717; E-mail: [email protected] comes are improving due to new therapies, the majority doi: 10.1158/0008-5472.CAN-18-0644 of patients with stage IV melanoma will die from their 2018 American Association for Cancer Research. disease (1, 2).

www.aacrjournals.org 5349

Vishnoi et al.

Metastasis is a multistep process that is initiated when tumor ist Research Institute (HMRI, Houston, TX). All patient blood cells leave the primary tumor and traverse through the circu- samples were collected after receiving informed written consent lation to reach distinct organs (3, 4). While transiting through and according to the principles of Declaration of Helsinki. the peripheral blood, a subset of CTCs can home to the bone Clinical details of each patient and PTEN H-score, included marrow where they reside in a state of sustained quiescence in the study, are provided in Supplementary Table S1 and (parallel progression model of metastatic melanoma; ref. 5). Supplementary Fig. S1. Peripheral blood (18–20 mL) was The unique bone marrow microenvironment exerts temporal obtained at the middle of vein puncture and was collected in and spatial selection pressures that are conducive for the CellSave tubes (Menarini Silicon Biosystems, Inc.), or EDTA survival of cell clones retaining long-term, self-renewal ability tubes under aseptic conditions. Samples were provided imme- while acquiring the necessary traits for successful organ colo- diately to the laboratory for CTC isolation and analysis. nization, even in the absence of overt local bone invasion (3,5,6).Thesebonemarrow–resident tumor cells (BMRTC) Antibodies and inhibitors used for the study are considered to be the "seeds" of future metastasis; targeting For multiparametric flow cytometry and DEPArray, primary them for elimination or promoting the prolongation of their antibodies were obtained from the following sources: FITC-CD45 quiescent/growth-arrested state can therefore be a promising (#304054; 1:200), FITC-CD34 (#343504; 1:200), FITC-CD105 strategy to overcome or delay metastatic onset (5, 7). Accord- (#323204; 1:200), FITC-CD90 (#328108; 1:200), FITC-CD73 ingly, it is imperative to identify novel biomarkers for BMRTC (#344016; 1:200), FITC HLA-A/B/C antibody (#311404; detection and to develop new therapeutic strategies that can 1:200), PerCP/Cy5.5-CD146 (#342014; 1:100), PE-Human eliminate BMRTCs before they progress to overt metastasis. NG2/MCSP (#FAB2585P, 1:100), BV421-Ki67 (#350506; Successful surgical resection of primary melanoma (with clear 1:100) were obtained from BioLegend, anti–Melan-A antibody margins) along with satisfactory lymph node dissection does (# AC12-0297-03; 1:200) from Abcore, and FITC-Anti-S100 not always prevent the incidence of late metastatic recurrence (#ab76749; 1:50) was purchased from Abcam. (5, 8). This suggests that metastatic dissemination is an early For IHC, anti-human, anti–Melan-A antibody (#ab51061; event wherein melanoma cells remain dormant but viable in 1:100), anti–tenascin-C (#ab108930, 1:100), and anti-p21 distant organs while retaining abilities to generate overt meta- (#ab188224, 1:100) were purchased from Abcam; HLA-ABC stasis at a later time (3, 8–11). For example, using the RET.AAD (#565292; 1:100) from BD Biosciences; USP7 (#GTX125894; melanoma mouse models, Eyles and colleagues demonstrated 1:200) from Genetex. PTEN antibody (#sc-7974; 1:20) was that the process of tumor cell dissemination preceded the onset purchased from Santa Cruz Biotechnology. Anti-p53 anti- of symptomatic metastasis (4). Furthermore, Ghossein and body (#SAB4503021, 1:100) was purchased from Sigma, and colleagues found that the presence of melanoma-specific tran- CCP110 (#12780-I-AP, 1:100) antibody was obtained from scripts in clinical blood and bone marrow samples is associated Proteintech. Anti-mouse, USP7 (#26948-1-AP, 1:200), and PTEN with shorter median survival of patients (12). Despite these (#603000-1-Ig, 1:200) antibodies were purchased from Protein- reports pointing to bone marrow as the likely reservoir for tech. Alexa Fluor-conjugated anti-mouse, anti-rabbit secondary disseminated CTCs, the precise role of BMRTCs in the patho- IgG antibodies used for immunofluorescence staining (1:500 genesis of metastatic melanoma remains unclear and no signif- dilution) were obtained from Cell Signaling Technology. USP7 icant progress has been made to target melanoma CTCs/ inhibitors P5091 (#SML0770) and P22077 (#2301) were pur- BMRTCs for clinical metastasis prevention (4, 10–13). chased from BioVision. We hypothesized that melanoma CTCs migrate to and reside in the bone marrow during asymptomatic progression of the PBMC isolation and CTC enrichment by multiparametric disease. We report here the isolation of CTC-enriched popula- flow cytometry tions from the peripheral blood of patients with metastatic Peripheral blood mononuclear cells (PBMC) were isolated melanoma, their expansion in immunocompromised mice, by established procedures (14). Briefly, whole blood was trea- followed by their harvesting and subsequent immunopheno- ted with red blood cell lysis buffer (154 mmol/L NH4Cl, typing and molecular characterization. Using these approaches, 10 mmol/L KHCO3, 0.1 mmol/L EDTA) at 1:25 ratio, followed we identified elevated USP7/PTEN expression as a distinct gene by incubation at room temperature (25C) for 5 minutes, then signature of BMRTCs. Furthermore, we demonstrated that inhi- pelleting the remaining blood cells at 300 g for 10 minutes. bition of USP7 reduces the metastatic potential of BMRTCs by Mononucleated cell pellet was then washed twice with 1 PBS prolonging their arrest in bone marrow. Our investigations (with 5 mmol/L EDTA) and used for fluorescence labeling provide novel insights into the identity of BMRTCs that may followed by multiparametric flow sorting (FACSAria II; BD be detectable in patients with melanoma during asymptomatic Biosciences). Data recorded during cell sorting were analyzed metastasis, and present data to support the evaluation of USP7 by DIVA acquisition software version 8.0.1 (BD Biosciences). inhibitors in patients with melanoma at risk of developing Antibodies and reagents described above were used. Data metastasis. generated by FACS were analyzed by FlowJo V10.

Experimental xenografts Materials and Methods All animal study was approved by our Institutional Animal Patient blood collection Care and Use Committee protocol. Immunodeﬁcient animal Patients with melanoma with stage III or stage IV disease experiments were performed using 4- to 8-week-old NOD. were accrued according to protocols approved by the Institu- Cg-Prkdcscid Il2rgtm1Wjl/SzJ (NSG) mice (The Jackson tional Ethical Review Boards at the University of Texas MD Laboratory). Flow-sorted Lin PBMCs (50,000 cells) deriv- Anderson Cancer Center (Houston, TX) and Houston Method- ed from patient blood (8 mL volume) were injected in

5350 Cancer Res; 78(18) September 15, 2018 Cancer Research

Molecular Insights of BM-Resident Melanoma Cells

anesthetized NSG mice through intracardiac injection under XL Cell Imaging System (Thermo Fisher Scientific). IHC images aseptic conditions. Previous reports show that injection of were taken and quantified by free-access ImageJ software. melanoma cell lines (1 106 cells) with different metastatic Reciprocal intensity (250 y) was measured by subtracting abilities in immunodeficient mice typically develop overt the mean intensity of the stained area (y) from the maximum macrometastases in 10 to 12 weeks (poorly metastatic) and (250) unstained white area intensity as described previously 3 to 4 weeks (highly metastatic melanoma cell lines; ref. 15). (22). Student t test,type2,paired2wasusedtocalculatethe Based upon this reference time-frame and considering that difference in reciprocal intensity between two groups (USP7 melanoma CTCs are rare cells with low transcriptional activity inhibitors treated vs. untreated) for each protein. (Supplementary Fig. S2A and S2B; Supplementary Tables S2 and S3), xenograft mice were euthanized 6 months after injec- DEPArray and CellSearch CTC interrogation þ þ tion with patient-isolated Lin cells (13, 16–19). This was Flow-sorted CDX-derived HLA /Melan-A were fixed with based upon the reasoning that this period should suffice for 2% paraformaldehyde for 20 minutes at 25Cfollowedby in vivo selection and elimination of nontumor cells and suc- washing with 1 PBS to visualize cells at single-cell level. CTCs cessful establishment of a niche for resident single cells in were then stained with appropriate antibodies, rewashed with systemic viscera and bone marrow. Peripheral blood, bone the SB115 buffer, and loaded onto the DEPArray platform marrow, and organs were harvested for downstream analyses. (Menarini Silicon Biosystems, Inc.). Cells were then detected Approximately 800 to 900 mL blood was collected in EDTA and analyzed by using Cell Browser software v3.0 (Menarini tubes by cardiac puncture of anesthetized mice. Animals were Silicon Biosystems, Inc.), as reported previously (14, 21). For sacrificed soon afterwards and bone marrow/organ tissues were CellSearch CTC enumeration, harvested murine blood was harvested. In particular, bone marrow was obtained from femur spiked with blood from healthy donors and was processed and tibia by flushing them out with 1 PBS with EDTA using CellTracks circulating Melanoma Cell Kit and CellSearch 1 (5 mmol/L) using a 28 G /2 needle, followed by centrifu- platform (Menarini Silicon Biosystems, Inc.), following the gation at 300 g for 10 minutes. Blood and bone marrow were manufacturer's guidelines. processed immediately for PBMC isolation and FACS analyses. To study the effect of USP7 inhibitors on BMRTCs/CTCs, 2 to RNA isolation and gene expression profiling 3 immunodeficient mice were injected with Lin population Total RNA isolation was performed using NucleoSpin RNA XS (50,000 cells/mouse) selected from the same individual patient Isolation Kit (Macherey-Nagel, Inc.), then immediately provided with melanoma (16 mL blood volume). One animal of each to the sequencing/noncoding RNA core facility (MD Anderson patient group was then treated with USP7 inhibitor (either Cancer Center). RNA and cDNA amplifications, quality controls, P5091 or P22077). CTC-derived xenografts (CDX) were treated and gene expression arrays were performed using the human twice a week subcutaneously with USP7 inhibitors at an MTD microarray platform (Clariom D, Affymetrix, Inc.). BMRTC/CTC [P22077 (15 mg/kg); P5091 (10 mg/kg); ref. 20]. Control samples were RNA-normalized using Affymetrix Powertool vehicle consisted of 1 PBS (100 mL) injected in untreated 1.18.0, and annotations were taken from Affymetrix version CDXs. After 11 weeks of treatment, mice were sacrificed and 36. Gene expression data analysis from each of BMRTC/CTC necropsy performed. subsets (derived from asymptomatic CDX mice, with absence of Eight to 10-week-old, syngeneic mice C57BL/6J (n ¼ 30) histopathologic confirmed macrometastasis) was performed were purchased from The Jackson Laboratory. Xenografts were using Transcriptome Analysis Console 3.0.0.466 (Affymetrix, developed by intracardiac injection of aggressive B16F10 Inc.). Two-way ANOVA was performed to calculate fold change melanoma cells (ATCC; 50,000 cells/mouse) and treated (FC) and P value. Pathway enrichment analyses were subsequent- with or without USP7 inhibitors (P5091 and P22077) at their ly performed using the Ingenuity Pathway Analysis software MTD as described previously. After 3 weeks, each group of version 01-07 (Qiagen, Inc.). animals (n ¼ 10) was euthanized and organs (bone marrow, lung, brain, liver, and lymph node) were harvested to per- WTA amplification and qRT-PCR form hematoxylin and eosin (H&E) staining analyses. All mRNA and cDNA amplification were carried out from isolated melanoma cells were obtained from ATCC, DNA fingerprinted, RNA using REPLI-g single-cell WTA Amplification Kit, according and routinely validated for Mycoplasma-free testing at Charac- to the manufacturer's instructions (Qiagen, Inc.). Amplified terized Cell Line Core Facility, MD Anderson, Houston, Texas. cDNA was purified using the ExoSAP method (Affymetrix; #78202.4X.1.ML), and subjected to qRT-PCR using the SensiFAST Immunofluorescence and IHC SYBR Hi-ROX Kit (Bioline, #BIO-92020). CT values were normal- FACS-isolatedCTCsweresubjectedtoquickair-dryon ized with housekeeping gene b-actin and log2 (DDCT) were Millennia 2000 adhesive glass slides (StatLab), and fixed with calculated. Student t test, type 2, paired 2 was performed to obtain 4% paraformaldehyde (21). Cells were permeabilized (0.05% the P value of each gene. Primer sequences for each gene are shown Triton X-100 in 1 PBS) for 30 minutes, followed by 30-minute in Supplementary Table S4. incubation in blocking buffer (1% BSA þ 1% normal goat serum in 1 PBS). Next, immunofluorescent cell staining was Whole-genome amplification and next-generation sequencing employed using selected primary and secondary antibodies. Whole-genome amplification (WGA) was performed by Magnified (100) images were captured using Zeiss Axio REPLI-g Single-Cell WGA Kit, according to the manufacturer's Observer microscope Z1 (Carl Zeiss), and data were analyzed instructions (Qiagen, Inc.). Briefly, the pool of CTC subsets was using Zeiss ZEN2 software. Harvested tissue was processed lysed and denatured followed by amplification to obtain intact and stained for H&E and other IHC markers by the research amplified DNA of 10 kb. Ion Torrent next-generation sequenc- pathology core at HMRI. Images were captured by using EVOS ing (NGS) was performed by using Ion AmpliSeq Cancer

www.aacrjournals.org Cancer Res; 78(18) September 15, 2018 5351

Vishnoi et al.

Hotspot Panel v2 (Thermo Fisher Scientific, #4475346). cytometric gating consisted of doublet discrimination and þ Libraries were constructed using amplified DNA and loaded dead cell (DAPI ) elimination, followed by depletion of leu- þ þ on Ion Torrent Pmt sequencer. Sequences were assembled with kocytes (CD45 ), circulating endothelial cells (CD34 ), and þ þ þ human reference hg19 assembly and variant analyses were mesenchymal stem cells (CD73 /CD90 /CD105 ;Fig.1A). performed by Advaita iVariantGuide (http://www.advaitabio. Using this strategy, we successfully captured putative melano- com/ipathwayguide). Analyses were based on NGS for the ma CTCs identified by the presence of established melanoma detection of somatic mutations in the coding sequence of a markers (Melan-A and S100) from the peripheral blood of minimum of 46 genes (range 46–128), which were performed 8 patients with melanoma validated through immunofluo- on DNAs extracted from samples in the CLIA-certified molec- rescence (Fig. 1B; ref. 16). Second, we performed Ion Torrent ular diagnostic laboratory (University of Texas MD Anderson next-generation DNA sequencing and analyzed the genetic Cancer Center). profiles of 409 oncogenes and tumor suppressor genes of CTCs þ (Lin and Melan-A cells) derived from two independent Mitochondrial DNA assessment and cell surface and patients with melanoma (patients #9 and #10). We identified intranuclear flow cytometry 176 and 231 clinicopathologic mutations in respective patients The ratio of mitochondrial DNA (mtDNA) to nuclear DNA (#9 and #10), present on NF1, KRAS, TP53, RB1, ATM, and (nDNA) copies was used to verify cell proliferative status (MTN EGFR genes (Fig. 1C; Supplementary Table S5A and S5B), index; ref. 23). The mitochondrial target was strategically selected confirming the neoplastic identity of these cells. Of note, CTCs within the noncoding displacement loop (MTDL) of the from one of the analyzed patients (patient #10) harbored a mtGenome because of the rare occurrence of large-scale deletions melanoma-associated mutation in CDKN2A (p.Ala148Thr; that are common to other areas, for example, the major arc55. COSM3736958; https://www.ncbi.nlm.nih.gov/clinvar/variation). Single-copy and low variability b2-microglobulin (B2M) gene We also confirmed the presence of skin cancer–associated muta- was selected as the nuclear target. Relative quantitative PCR tions in XPA (c.-4A>G), ERCC1 (p.Asn118Asn), and ERCC5 (qPCR) was performed and MTN index was calculated [MTN (p.Gly1534Arg; https://www.ncbi.nlm.nih.gov/clinvar/variation). – index ¼ 2 2DCT, where DCT ¼ (CTB2M CTMTDL)]. Third, to validate that melanoma patient isolated Lin cells We used the Permiflow method (US patent #US7326577 B2) (patients #2, #3, #4, and #7) were distinct from normal PBMCs, þ of cell fixation for concomitant cell surface (e.g., HLA / we performed whole-genome microarray analyses on these cells þ Mel-A ), and intranuclear staining of target proteins (e.g., (n ¼ 4), and compared them with the gene expression profiles of Ki67) in CDX-derived cell populations, as described previously PBMCs derived from healthy donors (n ¼ 9). PBMC dataset (14). Briefly, xenograft-isolated bone marrow and blood (GSE100054) was specifically chosen because it was obtained PBMCs were first stained with the live/dead fixable Zombie using the same human microarray platform (Clariom D; Affyme- NIR dye (BioLegend, #423105, 1:100) for 15 minutes, followed trix, Inc.). A total of 15,056 genes were upregulated and 23,430 by two sequential washes with 1 PBS. All samples were then genes were downregulated (fold change <2or>2, P ¼ 0.05) in fixed and permeabilized with the Permiflow solution by incu- Lin cell populations compared with healthy donor PBMCs, bating at 42C for 1 hour, followed by washing with 1 PBS. suggesting low transcriptional activity (Fig. 1D and E; Supple- Cells were then resuspended in staining buffer and stained with mentary Table S2). Unsupervised hierarchical clustering appropriate antibodies for flow cytometric analyses. showed distinct gene expression patterns in Lin and PBMC cell populations. (Fig. 1F). Lin populations possessed significantly higher expression of BAGE, MAGEA1, B4GALNT1, Results DCD, and S100A3 confirming the presence of specificmela- Isolation and validation of melanoma CTC-enriched cell noma-associated markers within this population (Supplemen- populations isolated from patients' blood tary Table S3; refs. 16, 19, 26). Downstream pathway enrich- CTCs derived from patients display significant heterogeneity ment analyses in Lin cell pools predicted increased activation in cell surface biomarkers, epitomized by the coexistence of of nuclear receptor and oncogenic signaling, along with inhibition tumor-initiating and stem cell populations within a single of immune regulation and progrowth signaling pathways blood draw (16, 17). A consequence of this diversity is that (Supplementary Fig. S3; Supplementary Tables S6 and S7). These multiple marker-based platforms (including CellSearch, the findings were reflected in cellular functions, for example, upre- only FDA-cleared platform for clinical CTC testing), and cell gulated transcripts implicated in cell survival and prodevelop- size-based microfiltration devices are unable to capture the ment functions with a concomitant decrease in pathways involved entire spectrum of CTC subtypes that may be biologically in cell proliferation and inflammation. Collectively, these results and/or clinically relevant in cancer progression (18, 24, 25). asserted the presence of putative and uncharacterized melanoma Because there is no universal melanoma CTC marker capable of CTCs in Lin cell populations isolated by multiparametric flow identifying the heterogeneous CTC subgroups, particularly cytometry. clones that may transit to- and from the bone marrow, we used immunocompromised mice for the in vivo selection of Expansion of melanoma patient–isolated Lin cells in vivo cells capable of bone marrow homing and residence. To imple- To evaluate whether Lin cells isolated from patients with ment this strategy, we first employed multiparametric flow cyto- melanoma could recapitulate the metastatic cascade, we first metry to deplete normal cell lineages present in the peripheral injected Lin cell populations (n ¼ 8) into the left ventricle of blood of patients with melanoma (patients enrolled in this immunodeficient mice (patients #1–8). Mice were euthanized study are listed in Supplementary Table S1), thereby enriching at the predesignated study endpoint (6 months), after collect- cell populations containing putative as well as uncharacterized ing blood by cardiac puncture. Bone marrow from long bones abnormal cell populations (here referred as Lin cells). Flow (femur, tibia, and humerus) and organs typically affected by

5352 Cancer Res; 78(18) September 15, 2018 Cancer Research

Molecular Insights of BM-Resident Melanoma Cells

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 by melanoma 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) by AmpliSeq Ion Torrent Cancer Hotspot Panel V2. Number of missense, nonsense, and silent mutations of each patient is indicated. D, Differential gene expression profiling of Lin cell population from patients with melanoma (n ¼ 4; patients #2, #3, #4, and #7), compared with dataset of PBMCs isolated from blood of normal healthy donors (GSE100054; n ¼ 9). Two-way ANOVA were performed to calculate FC and P value. E, Scatter plots showing global gene expression changes of Lin cell population. F, Hierarchical clustering showing distinct transcriptomic signatures of Lin cell population versus PBMCs.

www.aacrjournals.org Cancer Res; 78(18) September 15, 2018 5353

Vishnoi et al.

Figure 2. Isolation and validation of melanoma patient CTCs/BMRTCs from CDXs. A, Capture, visualization, and enumeration of human melanoma CTCs by CellSearch (CD45/CD34/DAPIþ/Melan-Aþ cells; patient #4). B, Multiparametric ﬂow cytometry gating used to isolate human melanoma cells from bone marrow and blood of CDXs models (DAPI/HLAþ/Melan-Aþ cells). Table showing number of genomic mutations (50 cancer gene sequencing panel) analyzed by AmpliSeq Ion Torrent in representative patients (see Materials and Methods for details). C, Immunoﬂuorescence visualization of patient-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).

metastatic melanoma (lung, brain, and liver) were collected for 14; Fig. 2D). Fourth, we performed genetic profiling for a family downstream analyses. Interestingly, although routine histo- of 50 key cancer genes by Ion Torrent NGS on ex vivo BMRTCs pathologic inspection did not identify any macrometastatic and CTCs (patients #3 and 4). We discovered novel mutations disease in these organs, extensive IHC analyses employing in PIK3CA, NRAS, KRAS, ERBB4, PTEN, and APC genes along human (HLA-ABC) and melanoma (Melan-A) biomarkers with two hotspot cosmic mutations (COSM21451 and detected discrete resident human melanoma cells in multiple COSM25085) in the PIK3CA gene using hg19 human genome murine organs (CDXs; Supplementary Fig. S4A and S4B; assembly as reference (Fig. 2B; Supplementary Table S8). Final- ref. 27). Second, to assess the presence of human melanoma ly, we performed differential gene expression analysis of ex vivo CTCs in blood of CDX models (derived from patient #4), we versus de novo CTCs (Lin /CTC-enriched population) from spiked CDX blood with healthy donor blood and performed paired patient samples (patients #2, #3, #4, and #7) by human analyses using the CellSearch platform to visualize and enu- microarray profiling (Clariom D; Affymetrix, Inc.). We observed þ þ merate CTCs. We identified eight DAPI /CD45 /Melan-A 3,777 significantly upregulated and 3,879 significantly down- cells from 500 mL of murine blood by CellSearch (Fig. 2A). regulated transcripts (FC ¼ 2.5; P ¼ 0.05) when CTCs versus Next, we harvested blood and bone marrow for PBMC iso- Lin /CTC-enriched population-derived microarray data were lation followed by multiparametric flow sorting, and isolated compared. We also observed 12,431 significantly overlapping þ þ HLA /Melan-A ex vivo CTCs and BMRTCs (Fig. 2B). We genes between arrays of these population (P ¼ 0.05; Supple- validated the veracity of flow procedures by immunofluores- mentary Fig. S5). Collectively, these results demonstrate cence analyses, detecting the presence of human (HLA) and that CTC-enriched cell populations isolated from melanoma þ þ melanoma (Melan-A) markers in HLA /Melan-A FACS-sorted patientsandexpandedinCDXmodelscouldbesuccessfully populations (Fig. 2C). Third, we confirmed the heterogeneity harvested from blood and bone marrow of these mice. of a pool of melanoma markers (Melan-A/CD146/NG2/S100) in CDX-derived BMRTC/CTC populations (patient #3) by the Divergent transcriptomic signatures between ex vivo BMRTCs DEPArray (Fig. 2D; refs. 16, 17). The DEPArray is an antigen- and CTCs agnostic CTC capturing technology that separates viable rare To determine functional differences between BMRTCs and þ þ cells at a single-cell level. An additional important aspect of this CTCs, we performed transcriptome profiling of HLA /Melan-A system is the absence of shear force stress on cells during cell populations obtained from CDX (derived from patients recovery (14). We identified, analyzed, and quantitated het- #1–8) bone marrow, and blood. We used paired BMRTC/CTC erogeneous melanoma cell populations displaying specific populations derived from eight CDX models (showing absence þ þ melanoma markers in FACS-sorted HLA /Melan-A cell popu- of macrometastatic disease) generated by injecting Lin cell lation derived from BMRTCs/CTCs of the same CDX mice: population from individual patients with metastatic melanoma þ þ þ þ Melan-A /S100 /CD146 /NG2 (BMRTCs ¼ 27; CTCs ¼ 8), (n ¼ 8), and performed human microarray profiling (Clariom þ þ Melan-A /S100 /CD146 /NG2 (BMRTCs ¼ 11; CTCs ¼ 10), D; Affymetrix, Inc.). Analyses identified 3,645 differentially þ þ þ Melan-A /S100 /CD146 /NG2 (BMRTCs ¼ 11; CTCs ¼ 14), expressed genes; 2,439 genes were upregulated, whereas 1,206 þ and Melan-A /S100 /CD146 /NG2 (BMRTCs ¼ 0; CTCs ¼ genes were downregulated (FC ¼ 2.5, P-value ¼ 0.05) in BMRTCs

5354 Cancer Res; 78(18) September 15, 2018 Cancer Research

Molecular Insights of BM-Resident Melanoma Cells

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 plot showing differentially expressed genes in ex vivo BMRTCs versus CTCs by human array (Clariom D; Affymetrix, Inc.); two-way ANOVA was performed to calculate FC and P value [patients #1–8; BMRTCs ¼ 2 and CTCs ¼ 1 (shown below)]. Table displaying significantly altered top canonical pathways in 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) were calculated by GraphPad Prism ver7(95%confidence interval). versus CTCs (Fig. 3A and B). Divergent transcriptomic signatures gene expression in patients (n ¼ 114) with cutaneous melanoma of BMRTCs/CTCs in CDX models reflected the differential behav- whose data were obtained from The Cancer Genome Atlas data- ior of Lin cells owing to their spatial niche (Fig. 3A and B; base by employing Oncolnc software tool (http://www.oncolnc. Supplementary Fig. S6). Subsequent pathway enrichment analy- org/; ref. 30). Kaplan–Meier survival analyses show that high ses showed that the top five upregulated canonical pathways were expression of USP7 correlated with decreased patient survival, EIF2 signaling, actin cytoskeleton, systemic lupus erythematosus, whereas PTEN expression was not significantly associated (log- hypoxia signaling, and, notably, the protein ubiquitination path- rank P values of 0.0167 vs. 0.112; HR, 1.598 vs. 0.7937 with 95% way (Fig. 3B; Supplementary Table S9). Molecular and cellular confidence interval; Fig. 3D). In addition, we did not observe any functional annotations also indicated that BMRTCs upregulated correlation between patient's PTEN mutational status and its genes involved in gene expression, RNA posttranslational mod- expression on CDX-derived BMRTCs population. Collectively, ifications, protein synthesis, cell cycle, and cell survival with a these findings revealed that USP7 is uniquely upregulated in ex concomitant decrease in cell death and apoptosis. Second, vivo BMRTCs and correlates with shorter patient survival. because the distinct gene expression pattern of ex vivo BMRTCs may have been conferred by the osteogenic niche during their USP7 inhibition modulates metastatic competence and residence in bone marrow and could be a reflection of their proliferative properties of CDX-isolated BMRTCs/CTCs dependence on these unique signaling mechanisms, we focused We investigated the effects of USP7 inhibitors on metastatic on individual genes in the protein ubiquitination pathway competency of BMRTCs as melanoma-derived, bone marrow– that were most strikingly elevated in BMRTCs. We found that associated CTC population. We performed drug susceptibility PTEN, a known tumor suppressor gene, and Ubiquitin Specific assays in CDXs using two clinically relevant USP7 inhibitors: Peptidase 7 (USP7), a deubiquitinating enzyme involved in P5091 and P22077. We injected Lin cells (50,000 cells/ posttranslational modifications, were significantly upregulated mouse) from 6 patients with melanoma (patients #11–16) (PTEN, FC ¼ 219, P ¼ 0.0028; USP7, FC ¼ 5, P ¼ 0.0072) simultaneously into 13 NSG mice and treated mice in parallel in ex vivo BMRTCs (Fig. 3B; refs. 28, 29). To confirm these with: P5091, P22077, or vehicle (PBS). Mice were euthanized findings, we assessed USP7 and PTEN expression on FACS-sorted after 11 weeks of treatment and visceral organs and CTCs þ þ HLA /Melan-A ex vivo BMRTCs/CTCs by immunofluorescence, from blood and bone marrow were harvested for downstream and observed elevated expression of USP7 and PTEN in BMRTCs analysis. After euthanization, vehicle and USP7 inhibitor- (Fig. 3C). Next, we performed survival analyses of USP7/PTEN treated CDX mice (11 weeks of USP7 inhibitor treatment)

www.aacrjournals.org Cancer Res; 78(18) September 15, 2018 5355

Vishnoi et al.

injected with Lin cell population from the same individual Effects of USP7 inhibitors on ex vivo BMRTCs/CTCs patient, we performed IHC to assess USP7 and PTEN expression To delineate genes involved in the metastatic potency of in bone marrow and lung as most common metastatic mela- BMRTCs and affected by USP7 inhibition, we performed com- noma sites (1). We found a significant reduction of USP7, parative transcriptional profiling of ex vivo BMRTCs from paired þ PTEN, and Melan-A cells in bone marrow and lung tissues vehicle versus USP7 inhibitor-treated CDX mice (derived from (micrometastatic disease) in USP7 inhibitor-treated versus patients #11, #12, and #16). Differential gene expression pro- PBS-treated CDXs, revealing that USP7 targeting leads to a filing revealed 321 significantly altered genes (138 upregulated, significant reduction of metastatic competency in patient- 183 downregulated) in BMRTCs treated with USP7 inhibitors derived, bone marrow–associated CTCs (Fig. 4A–E; Supple- (Fig.6DandE).CDX-derivedBMRTCs clustered together in mentary Fig. S7A–S7E). two groups when hierarchical clustering was performed (FC ¼ We also investigated the effects of the USP7 inhibitors on 2, P ¼ 0.05), suggesting that USP7 affected BMRTCs by a metastatic competency in a syngeneic mice model. We injected distinct gene signature (Fig. 6F). We also evaluated USP7- metastasis-competent B16F10 melanoma cells through intra- altered, metastasis-competent targets in distinct CDX-derived cardiac procedure in syngeneic C57BL/6J mice. We grouped BMRTCs and found significantly four upregulated (POLE, mice into three categories (n ¼ 10 for each group): (i) CCP110, SMAD13, and MTND4LP12) and nine downregulated PBS-treated control group and groups treated with USP7 inhi- (TNC, FBXO25, RGS22, PDE1A, GPCPD1, ABSB4, SERINC3, bitors (ii) P5091 and (iii) P22077, respectively. Mice were EIF3F, and UBXN4) genes associated with metastatic coloni- euthanized after 21 days, and bone marrow, lung, liver, lymph zation. Furthermore, miRNA analyses revealed upregulation of node, and brain tissues were harvested for analysis. Histo- MIR885-5p, MIR9-1, MIR6870, and MIR3657 and downregu- pathologic evaluation showed a significant reduction of mela- lation of MIR4719, MIR 548A2, MIR548AE1, MIR548AJ1, and noma metastasis in with both USP7 inhibitors versus untreated MIR548AX2 transcripts in USP7-modulated BMRTCs. Quanti- control mice groups (Fig. 5A and B). Third, we investigated tative PCR revealed significant downregulation of USP7, PTEN, þ þ the effect of USP7 inhibitors on nuclear-cytoplasmic localiza- TNC, and GPCPD1 transcripts in HLA /Melan-A ex vivo tion of PTEN (Supplementary Fig. S8; ref. 28). We observed BMRTCs derived from CDXs treated with USP7 inhibitors significant reduction of nuclear:cytoplasmic PTEN ratio on (P5091 and P22077) versus vehicle (Fig. 6G). RGS22 tran- BMRTC population derived from USP7 inhibitor treated versus scripts were significantly downregulated in CDX BMRTCs when untreated CDXs (Supplementary Fig. S8A and S8B). However, treated with inhibitor P22077 only, but not with inhibitor we observed restoration of nuclear PTEN in lung mets of mice P5091 versus vehicle (Fig. 6G). Furthermore, expression of treated with USP7 inhibitors versus untreated (Supplementary genes involved in cell invasion (TNC, SERINC3, ABSB4, and Fig. S8B and S8C). This suggests that distinct microenvironment SMAD13), cell migration (TNC, ASB4, RGS22, and GPCPD1), properties influence the USP7 regulation of PTEN expression. cell proliferation (MIR885-p and CCP110), and cell apoptosis Fourth, we investigated the effects of USP7 inhibitors on the (TNC, FBXO25, PDE1A, EIF3F) were decreased by USP7 inhib- metastatic competency of ex vivo BMRTCs, considering that itor treatment, possibly highlighting regulatory roles of USP7 roles of USP7 in the pathogenesis of metastatic melanoma at various metastatic steps during melanoma progression remain unclear. We assessed USP7 inhibitors' effect on the (Fig. 7; refs. 32–39). Next, to determine the role of USP7 proliferative capacity of ex vivo BMRTCs and CTCs (derived from inhibition in BMRTC-driven lung colonization, we performed patients #13 and #14) by performing flow cytometric analyses for IHC for two USP7-modulated markers (TNC and CCP110) in Ki67, a marker of cellular proliferation (14). We detected greater lung tissue of CDX mice treated versus untreated with USP7 percentage of Ki67-high cells in CTCs versus BMRTCs irrespective inhibitors (derived from patients #11, #15, and #16). IHC of USP7 inhibitor treatment [P5091: 89.1% (CTCs) vs. 68.4% quantification shows significant upregulation of CCP110 and (BMRTCs); P22077: 91.5% (CTCs) vs. 42.4% (BMRTCs); downregulation of TNC in CDX-derived lung tissue treated Fig. 6A]. We observed increased percentage of Ki67high among versus untreated with USP7 inhibitors (P ¼ 0.05; Supplemen- ex vivo BMRTCs isolated from USP7 inhibitor-treated versus tary Fig. S9A–S9D). Of relevance, USP7 inhibition has been untreated CDXs. Conversely, we did not observe any difference shown to play a role in MDM2 degradation, which leads to in Ki67 status among ex vivo CTCs isolated from USP7 inhibitors- reactivation of tumor suppressor genes p53 and p21, resulting treated versus untreated CDXs. Furthermore, because mtDNA in tumor growth inhibition (40). Accordingly, we quantified copy number is an indicator of mitochondrial biogenesis and p53 and p21 expression in lung and bone marrow tissues proliferative potency (31), we compared mtDNA versus nuclear (derived from patients #13 and #15) of CDXs treated versus DNA content ratios in ex vivo BMRTCs versus CTCs of six CDX untreated with USP7 inhibitors (Supplementary Fig. S10A– mice (derived from patients #12, #13, and #14). Quantitative S10D). IHC quantification shows significantly elevated expres- PCR showed significantly higher MTN index in BMRTCs derived sion of p53 and p21 on lung and bone marrow tissue derived from USP7 inhibitor-treated versus untreated CDX mice from CDX mice treated with versus without USP7 inhibitors (P (Fig. 6B). In addition, CellSearch analyses of ex vivo BMRTCs/ ¼ 0.05). CTCs from CDXs (derived from patient #12) treated with USP7 inhibitors showed higher number of melanoma CTCs (CD45 / þ þ CD34 /DAPI /Melan-A cells) in BMRTCs versus corresponding Discussion CTCs (Fig. 6C). Cumulatively, these findings suggest that USP7 This study provides first-time evidence that asymptomatic inhibition causes a significant reduction of metastatic compe- progression of metastatic melanoma can be recapitulated in vivo tency of BMRTCs by restricting melanoma cell population using patient-isolated CTCs, and demonstrates that melanoma within bone marrow locales (Figs. 4A–E, 5A–B, and 6A–C; CTCs at advanced disease stages contain heterogeneous cell Supplementary Fig. S7A–S7E). pools bearing distinct characteristics associated with bone

5356 Cancer Res; 78(18) September 15, 2018 Cancer Research

Molecular Insights of BM-Resident Melanoma Cells

Figure 4. USP7 inhibitors affect the metastatic potency of BMRTCs population in CDXs. A–E, Histopathologic Melan-A evaluation with colocalization of USP7 and PTEN 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 "Supplementary information" (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.

www.aacrjournals.org Cancer Res; 78(18) September 15, 2018 5357

Vishnoi et al.

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 mouse were 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.

marrow versus blood locales. In addition, we show that USP7 Whole-genome transcriptomic arrays and qPCR for individual plays critical roles in BMRTC metastatic potential by regulating target genes identified distinct transcriptional signatures of ex vivo genes related to cellular proliferation, apoptosis, and cell migra- melanoma BMRTCs, potentially reflecting effects of bone marrow tion. Complementing previous investigations that have charac- microenvironment on BMRTC gene expression. In addition, the terized the genetic landscape of primary and metastatic mela- proportion of Ki67-low cells was approximately 50% higher in noma lesions, we demonstrate that transcriptional subtyping of ex vivo BMRTCs versus CTCs, suggesting that BMRTCs contain melanoma CTCs provides key insights into the molecular a distinct cell subpopulation that may be in a state of subdued mechanisms that regulate metastatic potency by proposing a proliferation (Fig. 6A). However, we did not observe any corre- novel rationale-based approach for targeting BMRTCs (5, 7, 8, lation between high Ki67 index present in USP7 inhibitors– 10, 11, 13, 16, 17). In contrast to the majority of CTC studies treated BMRTCs population and metastatic burden in visceral that have largely elucidated the spatial attributes of peripheral organs or bone. Of note, Ki67 as a good indicator of proliferation blood-based CTCs in the context of metastasis, we provide and present in G1–M and S phases of cell cycle while absent in insights into the primordial stages of CTC dissemination and the G0 phase (42). Therefore, this is likely due to the effect of þ survival in bone marrow without progression to overt metastasis. USP7 inhibitors, which arrests the Ki67 BMRTC population at Employing a negative selection strategy to deplete normal cell G1–S phase of cell cycle and inhibits their metastatic potential populations of blood, we implanted patient Lin cells in immu- (43, 44). This suggests that USP7 inhibitors, although augment- nodeficient mice to generate CDX models that can faithfully ing the proliferative potency of BMRTCs, do not augment their recapitulate the early events of melanoma metastasis. Lin cell dissemination from bone marrow to generate lethal metastasis. populations not only had overexpression of melanoma markers, Interestingly, we detected BMRTC-specific mutations in SMAD4 but also harbored mutations on multiple melanoma oncogenes, and APC genes, suggesting that besides affecting gene expression, demonstrating their atypical nature (Fig. 1A–F; Supplementary bone marrow microenvironment may impart temporal selective Table S5; refs. 14, 41). Furthermore, combining the use of an pressures on BMRTCs, resulting in their acquiring unique muta- antigen-agnostic platform (DEPArray) with genomic sequencing, tions (Fig. 2B; Supplementary Table S8; ref. 5). we show that melanoma CTCs home to and reside in the bone The deubiquitinase enzyme USP7 is involved in the posttrans- marrow of CDX mice when injected with patient-isolated Lin cell lational modification of PTEN altering its tumor-suppressive population (Figs. 2A–D and 4A–E). We consider this approach activity. Loss of PTEN function in patients with melanoma useful for establishing patient-derived xenograft models particu- hasbeenlinkedtosignificantly faster progression to overt larly when patient tumor tissue may not be available. brain metastasis along with poorer overall survival (45). In

5358 Cancer Res; 78(18) September 15, 2018 Cancer Research

Molecular Insights of BM-Resident Melanoma Cells

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 inhibitors on 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 subjected to MTN determination using qPCR. C, Capture, visualization, and enumeration of CTCs by CellSearch analyses of melanoma patient BMRTCs/CTCs isolated from CDXs treated with or without USP7 inhibitors (patient #12). A total of 500 mL of CDX blood was collected and analyzed according to CellSearch 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, Scatterplotshowing differentially 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 treated versus untreated USP7 inhibitors P5091 (patients #11 and 12) and P22077 (patients #12 and 16). Upregulated/downregulated genes are highlighted in red/black, respectively. G, Validation of microarray data by qPCR analyses. Relative mRNA expression and SD were calculated for each gene. Student t test, type 2, paired 2 were performed to calculate respective P values. our CDX models, we found significantly higher USP7 expres- This is because: (i) patient-derived CTCs contain a heteroge- sion that colocalized with PTEN in BMRTCs when compared neous pool of melanoma cells and CTC subsets have the with ex vivo CTCs (Figs. 3A–Dand4A–E).Itislikelythatthe tendency to seed the bone marrow and (ii) USP7 modulates high expression of USP7 transcripts detected in BMRTCs the osteogenic differentiation of mesenchymal stem cells in reflects the intrinsic behavior of heterogeneous CTC pool bone marrow (5, 46). Kaplan–Meier survival analyses indicat- as well as properties of the bone marrow microenvironment. ed that high USP7 (but not PTEN) expression is significantly

www.aacrjournals.org Cancer Res; 78(18) September 15, 2018 5359

Vishnoi et al.

Figure 7. Model representing the asymptomatic progression of melanoma in patient- derived xenografts and effects of USP7 inhibition, either as a potential direct action on cells already disseminated to distant organs or as a result of USP7 targeting BMRTCs.

associated with decreased overall survival of patients with these BMRTCs identified USP7 as an important mediator of melanoma (Fig. 3D), while targeting USP7 with specificinhi- BMRTC-specific signaling and provided evidence that applying bitors reduced ex vivo CTC numbers at metastatic organ sites USP7 inhibitors in clinical settings can be relevant to eliminate along with a concomitant decrease in USP7 and PTEN expres- residual melanoma cells in bone marrow and thereby prevent sion (Figs. 4A–E and 5A and B). Our immunohistopathologic further metastatic spread. evaluation shows a significant upregulation of p53 and p21 expression in lung and bone marrow tissues derived from CDX Disclosure of Potential Conflicts of Interest mice treated with USP7 inhibitors versus PBS-treated group M.T. Tetzlaff is a consultant/advisory board member of Seattle Genetics, (Supplementary Fig. S10A–S10D); however, we did not Novartis, and Myriad Genetics. M.A. Davies reports receiving commercial fi research grants from GSK, Astrazeneca, Roche/Genentech, Oncothyreon, and observe a signi cant difference in p53 transcript expression Sanofi-Aventis and is consultant/advisory board member of GSK, Novartis, between ex vivo BMRTCs versus CTCs (28, 40). However, gene BMS, Sanofi-Aventis, Syndax, and Nanostring. No potential conflicts of interest expression microarray analysis of USP7 inhibitor treated versus were disclosed by the other authors. untreated ex vivo BMRTCs revealed several candidate genes significantly affected by USP7 inhibition (Fig. 6D–F). Among Authors' Contributions them, TNC and FBXO25 are genes implicated in the develop- Conception and design: M. Vishnoi, D. Boral, D. Marchetti ment of metastatic melanoma, breast and non–smallcelllung Development of methodology: M. Vishnoi, D. Boral Acquisition of data (provided animals, acquired and managed pati- cancers (32, 33, 47). Furthermore, downregulation of genes ents, provided facilities, etc.): M. Vishnoi, H. Liu, W. Yin, M.L. Sprouse, involved in cell migration (RGS22, GPCPD1 and ASB4) and D. Goswami-Sewell, M.T. Tetzlaff, M.A. Davies, I.C. Glitza Oliva invasion (TNC, SERINC3, ABSB4 and SMAD13) was also Analysis and interpretation of data (e.g., statistical analysis, biostatistics, detected, suggesting that USP7 inhibition may impede BMRTC computational analysis): M. Vishnoi, D. Boral, M.L. Sprouse, W. Yin, reshedding from bone marrow, arresting them in bone marrow D. Goswami-Sewell, M.T. Tetzlaff locales (35–37, 48). USP7 inhibition also decreased the expres- Writing, review, and/or revision of the manuscript: M. Vishnoi, D. Boral, M.T. Tetzlaff, M.A. Davies, I.C. Glitza Oliva, D. Marchetti sion of proliferation-related biomarkers (Ki67 and MTN – Administrative, technical, or material support (i.e., reporting or organizing indices; Fig. 6A F), possibly by affecting genes (e.g., CCP110, data, constructing databases): M. Vishnoi, H. Liu, W. Yin POLE, p21 and p53; Supplementary Figs. S9A–S9D and S10A– Study supervision: D. Marchetti S10D) that arrest BMRTC cell-cycle progression and may induce senescent phenotype (38, 39). Collectively, these find- Acknowledgments ings suggest that USP7 plays a central role in mediating This study was supported by the NIH grants (1 R01 CA 216991 and 1 R01 CA melanoma CTC residence in bone marrow (Figs. 6A–Gand7). 160335 to D. Marchetti); the Dr. Miriam and Sheldon G. Adelson Medical Our study has some limitations. First, analyses were perform- Research Foundation (to M.A. Davies); the AIM at Melanoma Foundation (to M.A. Davies); and by philanthropic support from the MD Anderson ed on a low number of patients; therefore, we cannot conclude Melanoma Shot Program (to M.A. Davies). We are thankful to Dr. David that all patients with melanoma follow these models and path- Haviland, Director of the Flow Cytometry Core at Houston Methodist Research ways. Second, we profiled a limited number of cells in each Institute (HMRI), Dr. Zhubo Wei of the Biostatistics core at HMRI, and to patient, which, although done stochastically, may have some Dr. Chang-Gong Liu, Director of the sequencing and ncRNA core at MD inherent sampling bias. Third, although we suggest that USP7 Anderson Cancer Center for their respective expertise. inhibition–mediated reduction of micrometastasis is not a direct effect on cells already disseminated to distant organs, we cannot The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked exclude a direct effect of USP7 inhibition on these cells. However, – advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate our results demonstrate that melanoma patient isolated CTCs this fact. home to and reside in bone marrow, and emphasize the relevance of determining the prognostic value of bone marrow–associated, Received February 27, 2018; revised June 12, 2018; accepted July 13, 2018; CTC-derived cell populations. The systematic interrogation of published first July 19, 2018.

5360 Cancer Res; 78(18) September 15, 2018 Cancer Research

Molecular Insights of BM-Resident Melanoma Cells

References 1. Howlader N, Noone AM, Krapcho M, Miller D, Bishop K, Kosary CL, et al. 22. Nguyen D. Quantifying chromogen intensity in immunohistochem- SEER cancer statistics review, 1975-2014. Bethesda, MD: NCI. istry via reciprocal intensity. Cancer InCytes 2013;2:e. doi: 10.5281/ 2. Luke JJ, Flaherty KT, Ribas A, Long GV. Targeted agents and immunothera- zenodo.13670. pies: optimizing outcomes in melanoma. Nat Rev Clin Oncol 2017; 23. Rooney JP, Ryde IT, Sanders LH, Howlett EH, Colton MD, Germ KE, et al. 14:463–82. PCR based determination of mitochondrial DNA copy number in multiple 3. Friberg S, Nystrom A. Cancer metastases: early dissemination and late species. Methods Mol Biol 2015;1241:23–38. recurrences. Cancer Growth Metastasis 2015;8:43–9. 24. Attard G, Crespo M, Lim AC, Pope L, Zivi A, de Bono JS. Reporting the 4. Eyles J, Puaux AL, Wang X, Toh B, Prakash C, Hong M, et al. Tumor capture efficiency of a filter-based microdevice: a CTC is not a CTC unless it cells disseminate early, but immunosurveillance limits metastatic is CD45 negative–letter. Clin Cancer Res 2011;17:3048–49. outgrowth, in a mouse model of melanoma. J Clin Invest 2010; 25. Mazzini C, Pinzani P, Salvianti F, Scatena C, Paglierani M, Ucci F, et al. 120:2030–39. Circulating tumor cells detection and counting in uveal melanomas by a 5. Werner-Klein M, Scheitler S, Hoffmann M, Hodak I, Dietz K, Lehnert P, filtration-based method. Cancers 2014;6:323–32. et al. Genetic alterations driving metastatic colony formation are 26. Ortega-Martinez I, Gardeazabal J, Erramuzpe A, Sanchez-Diez A, Cortes J, acquired outside of the primary tumour in melanoma. Nat Commun Garcia-Vazquez MD, et al. Vitronectin and dermcidin serum levels predict 2018;9:595. the metastatic progression of AJCC I-II early-stage melanoma. Int J Cancer 6. Rocken M. Early tumor dissemination, but late metastasis: insights into 2016;139:1598–607. tumor dormancy. J Clin Invest 2010;120:1800–3. 27. Jung IH, Chung YY, Jung DE, Kim YJ, Kim do H, Kim KS, et al. Impaired 7. Wong JH. Early diagnosis of metastatic disease in melanoma: does it make a lymphocytes development and xenotransplantation of gastrointestinal difference?: Comment on "Long-term follow-up and survival of patients tumor cells in prkdc-null SCID zebrafish model. Neoplasia 2016;18: following a recurrence of melanoma after a negative sentinel lymph node 468–79. biopsy result". JAMA Surg 2013;148:462. 28. Carra G, Panuzzo C, Torti D, Parvis G, Crivellaro S, Familiari U, et al. 8. Jones EL, Jones TS, Pearlman NW, Gao D, Stovall R, Gajdos C, et al. Long- Therapeutic inhibition of USP7-PTEN network in chronic lymphocytic term follow-up and survival of patients following a recurrence of mela- leukemia: a strategy to overcome TP53 mutated/deleted clones. Oncotarget noma after a negative sentinel lymph node biopsy result. JAMA Surg 2017;8:35508–22. 2013;148:456–61. 29. Zhou J, Wang J, Chen C, Yuan H, Wen X, Sun H. USP7: Target validation 9. Olmeda D, Cerezo-Wallis D, Riveiro-Falkenbach E, Pennacchi PC, and drug discovery for cancer therapy. Med Chem 2018;14:3–18. Contreras-Alcalde M, Ibarz N, et al. Whole-body imaging of lympho- 30. Anaya J. OncoLnc: linking TCGA survival data to mRNAs, miRNAs, vascular niches identifies pre-metastatic roles of midkine. Nature lncRNAs. PeerJ Computer Sci 2016;2:e1780v1. 2017;546:676–80. 31. Clay Montier LL, Deng JJ, Bai Y. Number matters: control of mammalian 10. Bourgault-Villada I, Hong M, Khoo K, Tham M, Toh B, Wai L-E, et al. mitochondrial DNA copy number. J Genet Genomics 2009;36:125–31. Current insight into the metastatic process and melanoma cell dis- 32. Shao H, Kirkwood JM, Wells A. Tenascin-C signaling in melanoma. Cell semination. In: Murph M, editor. Research on melanoma—a Adh Migr 2015;9:125–30. glimpse into current directions and future trends. Rijeka, Croatia: 33. Jiang GY, Zhang XP, Wang L, Lin XY, Yu JH, Wang EH, et al. FBXO25 IntechOpen; 2011. promotes cell proliferation, invasion, and migration of NSCLC. Tumour 11. Tseng WW, Doyle JA, Maguiness S, Horvai AE, Kashani-Sabet M, Leong SP. Biol 2016;37:14311–9. Giant cutaneous melanomas: evidence for primary tumour induced dor- 34. Hu Y, Xing J, Wang L, Huang M, Guo X, Chen L, et al. RGS22, a novel cancer/ mancy in metastatic sites? BMJ Case Rep 2009;2009:2073. testis antigen, inhibits epithelial cell invasion and metastasis. Clin Exp 12. Ghossein RA, Carusone L, Bhattacharya S. Molecular detection of micro- Metastasis 2011;28:541–9. metastases and circulating tumor cells in melanoma prostatic and breast 35. Marchan R, Buttner B, Lambert J, Edlund K, Glaeser I, Blaszkewicz M, et al. carcinomas. In Vivo 2000;14:237–50. Glycerol-3-phosphate acyltransferase 1 promotes tumor cell migration 13. Klinac D, Gray ES, Freeman JB, Reid A, Bowyer S, Millward M, et al. and poor survival in ovarian carcinoma. Cancer Res 2017;77:4589–601. Monitoring changes in circulating tumour cells as a prognostic indicator of 36. Margue C, Philippidou D, Reinsbach SE, Schmitt M, Behrmann I, Kreis S. overall survival and treatment response in patients with metastatic mel- New target genes of MITF-induced microRNA-211 contribute to melanoma anoma. BMC Cancer 2014;14:423. cell invasion. PLoS One 2013;8:e73473. 14. Boral D, Vishnoi M, Liu HN, Yin W, Sprouse ML, Scamardo A, et al. 37. Javelaud D, Alexaki VI, Dennler S, Mohammad KS, Guise TA, Mauviel A. Molecular characterization of breast cancer CTCs associated with brain TGF-beta/SMAD/GLI2 signaling axis in cancer progression and metastasis. metastasis. Nat Commun 2017;8:196. Cancer Res 2011;71:5606–10. 15. Thies A, Mauer S, Fodstad O, Schumacher U. Clinically proven markers of 38. Barenz F, Hoffmann I. Cell biology: DUBing CP110 controls centrosome metastasis predict metastatic spread of human melanoma cells engrafted in numbers. Curr Biol 2013;23:R459–60. scid mice. Br J Cancer 2007;96:609–16. 39. Aoude LG, Heitzer E, Johansson P, Gartside M, Wadt K, Pritchard AL, 16. Khoja L, Shenjere P, Hodgson C, Hodgetts J, Clack G, Hughes A, et al. et al. POLE mutations in families predisposed to cutaneous melanoma. Prevalence and heterogeneity of circulating tumour cells in metastatic Fam Cancer 2015;14:621–8. cutaneous melanoma. Melanoma Res 2014;24:40–6. 40. Turnbull AP, Ioannidis S, Krajewski WW, Pinto-Fernandez A, Heride C, 17. Gray ES, Reid AL, Bowyer S, Calapre L, Siew K, Pearce R, et al. Circulating Martin ACL, et al. Molecular basis of USP7 inhibition by selective melanoma cell subpopulations: their heterogeneity and differential small-molecule inhibitors. Nature 2017;550:481–6. responses to treatment. J Invest Dermatol 2015;135:2040–48. 41. Liu Z, Fusi A, Klopocki E, Schmittel A, Tinhofer I, Nonnenmacher A, et al. 18. Luo X, Mitra D, Sullivan RJ, Wittner BS, Kimura AM, Pan S, et al. Isolation Negative enrichment by immunomagnetic nanobeads for unbiased char- and molecular characterization of circulating melanoma cells. Cell Rep acterization of circulating tumor cells from peripheral blood of cancer 2014;7:645–53. patients. J Transl Med 2011;9:70. 19. Kiyohara E, Hata K, Lam S, Hoon DS. Circulating tumor cells as 42. Bruno S, Darzynkiewicz Z. Cell cycle dependent expression and stability of prognostic biomarkers in cutaneous melanoma patients. Methods Mol the nuclear protein detected by Ki-67 antibody in HL-60 cells. Cell Prolif Biol 2014;1102. 1992;25:31–40. 20. Chauhan D, Tian Z, Nicholson B, Kumar KG, Zhou B, Carrasco R, et al. A 43. Reverdy C, Conrath S, Lopez R, Planquette C, Atmanene C, Collura V, et al. small molecule inhibitor of ubiquitin-specific protease-7 induces apopto- Discovery of specific inhibitors of human USP7/HAUSP deubiquitinating sis in multiple myeloma cells and overcomes bortezomib resistance. enzyme. Chem Biol 2012;19:467–77. Cancer Cell 2012;22:345–58. 44. Jagannathan M, Nguyen T, Gallo D, Luthra N, Brown GW, Saridakis V, et al. 21. Vishnoi M, Peddibhotla S, Yin W, Scamardo AT, George GC, Hong DS, et al. A role for USP7 in DNA replication. Mol Cell Biol 2014;34:132–45. The isolation and characterization of CTC subsets related to breast cancer 45. Bucheit AD, Chen G, Siroy A, Tetzlaff M, Broaddus R, Milton D, et al. dormancy. Sci Rep 2015;5:17533. Complete loss of PTEN protein expression correlates with shorter time to

www.aacrjournals.org Cancer Res; 78(18) September 15, 2018 5361

Vishnoi et al.

brain metastasis and survival in stage IIIB/C melanoma patients with 47. Vanharanta S, Massague J. Origins of metastatic traits. Cancer Cell 2013; BRAFV600 mutations. Clin Cancer Res 2014;20:5527–36. 24:410–21. 46. Tang Y, Lv L, Li W, Zhang X, Jiang Y, Ge W, et al. Protein deubiquitinase 48. Hu Y, Xing J, Chen L, Zheng Y, Zhou Z. RGS22 inhibits pancreatic USP7 is required for osteogenic differentiation of human adipose-derived adenocarcinoma cell migration through the G12/13 alpha subunit/F-actin stem cells. Stem Cell Res Ther 2017;8:186. pathway. Oncol Rep 2015;34:2507–14.

5362 Cancer Res; 78(18) September 15, 2018 Cancer Research

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

Monika Vishnoi, Debasish Boral, Haowen Liu, et al.

Cancer Res 2018;78:5349-5362. Published OnlineFirst July 19, 2018.

Updated version Access the most recent version of this article at: doi:10.1158/0008-5472.CAN-18-0644

Supplementary Access the most recent supplemental material at: Material http://cancerres.aacrjournals.org/content/suppl/2018/07/19/0008-5472.CAN-18-0644.DC1

Visual A diagrammatic summary of the major findings and biological implications: Overview http://cancerres.aacrjournals.org/content/78/18/5349/F1.large.jpg

Cited articles This article cites 45 articles, 5 of which you can access for free at: http://cancerres.aacrjournals.org/content/78/18/5349.full#ref-list-1

E-mail alerts Sign up to receive free email-alerts related to this article or journal.

Reprints and To order reprints of this article or to subscribe to the journal, contact the AACR Publications Department at Subscriptions [email protected].

Permissions To request permission to re-use all or part of this article, use this link http://cancerres.aacrjournals.org/content/78/18/5349. Click on "Request Permissions" which will take you to the Copyright Clearance Center's (CCC) Rightslink site.