il2 inducible t-cell kinase, a novel therapeutic target in ...biology of human tumors il2 inducible...

Biology of Human Tumors

IL2 Inducible T-cell Kinase, a Novel TherapeuticTarget in MelanomaCraig C. Carson1, Stergios J. Moschos2,3, Sharon N. Edmiston3, David B. Darr3, NanaNikolaishvili-Feinberg3, Pamela A. Groben4, Xin Zhou5, Pei Fen Kuan3,5, Shaily Pandey1,Keefe T. Chan3,6, Jamie L. Jordan3, Honglin Hao1, Jill S. Frank7, Dennis A. Hopkinson1,David C. Gibbs1, Virginia D. Alldredge1, Eloise Parrish3, Sara C. Hanna3, Paula Berkowitz1,David S. Rubenstein1,3, C. Ryan Miller3,4,8,9, James E. Bear3,6, David W. Ollila3,7,Norman E. Sharpless2,3, Kathleen Conway3,10, and Nancy E. Thomas1,3

Abstract

Purpose: IL2 inducible T-cell kinase (ITK) promoter CpG sitesare hypomethylated in melanomas compared with nevi. Theexpression of ITK in melanomas, however, has not been estab-lished and requires elucidation.

Experimental Design: An ITK-specific monoclonal antibodywas used to probe sections from deidentified, formalin-fixedparaffin-embedded tumor blocks or cell line arrays and ITK wasvisualized by IHC. Levels of ITK protein differed among melano-ma cell lines and representative lines were transduced with fourdifferent lentiviral constructs that each contained an shRNAdesigned to knockdown ITK mRNA levels. The effects of theselective ITK inhibitor BI 10N on cell lines and mouse modelswere also determined.

Results: ITK protein expression increased with nevus tometastatic melanoma progression. In melanoma cell lines,

genetic or pharmacologic inhibition of ITK decreased prolif-eration and migration and increased the percentage of cellsin the G0–G1 phase. Treatment of melanoma-bearing micewith BI 10N reduced growth of ITK-expressing xenograftsor established autochthonous (Tyr-Cre/Ptennull/BrafV600E)melanomas.

Conclusions: We conclude that ITK, formerly considered animmune cell–specific protein, is aberrantly expressed in mela-noma and promotes tumor development and progression. Ourfinding that ITK is aberrantly expressed in most metastaticmelanomas suggests that inhibitors of ITK may be efficaciousfor melanoma treatment. The efficacy of a small-molecule ITKinhibitor in the Tyr-Cre/Ptennull/BrafV600E mouse melanomamodel supports this possibility. Clin Cancer Res; 21(9); 2167–76.�2015 AACR.

IntroductionAdvances in understanding the genetic aberrations associated

with melanoma initiation and progression have led to the recentU.S. Food and Drug Administration approval of three small-

molecule inhibitors against components in the BRAF–MEKpathway (1–3). However, these systemic therapies rarely leadto melanoma cures, even if used in combination (4). Next-generation sequencing analyses of melanoma specimens iden-tified a handful of genetic aberrations that may have "driver"roles in melanoma development and progression but somedistinct melanoma subtypes bear none of the identified "driver"genetic aberrations (5). Current immunotherapies have admit-tedly led to cures, albeit in a small group of patients (6–8).Although early clinical trials testing combinations of antibodiesagainst immune checkpoint proteins have shown great promise,the toxicities and long-term efficacy are unknown and underinvestigation (9).

Using DNA-methylation profiling to discriminate primarycutaneous melanomas from benign moles, we determined thatthepromoter for IL2 inducible T-cell kinase (ITK)was significantlyhypomethylated in primary melanomas compared with nevi(10). This finding was unexpected because the expression of ITK,a member of the TEC family of tyrosine kinases that includes theBruton's tyrosine kinase (BTK), is normally restricted to distinctimmune cell subsets. In T cells, ITK functions downstream of theT-cell receptor and is important for T-cell activation, develop-ment, differentiation, and production of many proinflammatorycytokines (11). Given previous reports about the potential forlineage reprogramming of melanoma cells with acquisition ofneural or angiogenic properties (12, 13), we hypothesized that

1Department ofDermatology,TheUniversity ofNorthCarolina,ChapelHill, North Carolina. 2Department of Medicine,The University of NorthCarolina, Chapel Hill, North Carolina. 3Lineberger ComprehensiveCancer Center, The University of North Carolina, Chapel Hill, NorthCarolina. 4Department of Pathology and Laboratory Medicine, TheUniversity of North Carolina, Chapel Hill, North Carolina. 5Departmentof Biostatistics, The University of North Carolina, Chapel Hill, NorthCarolina. 6Department of Cell Biology and Physiology, The UniversityofNorthCarolina,Chapel Hill, North Carolina. 7Departmentof Surgery,The University of North Carolina, Chapel Hill, North Carolina. 8Depart-ment ofNeurology,TheUniversityofNorthCarolina,Chapel Hill, NorthCarolina. 9Neuroscience Center, The University of North Carolina,Chapel Hill, North Carolina. 10Department of Epidemiology, The Uni-versity of North Carolina, Chapel Hill, North Carolina.

Note: Supplementary data for this article are available at Clinical CancerResearch Online (http://clincancerres.aacrjournals.org/).

Corresponding Author: Nancy E. Thomas, Department of Dermatology, Uni-versity ofNorthCarolina, 413Mary Ellen JonesBuilding, CB#7287, ChapelHill, NC27599. Phone: 919-966-0785; Fax: 919-966-6460; E-mail:[email protected]

doi: 10.1158/1078-0432.CCR-14-1826

�2015 American Association for Cancer Research.

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aberrant ITK expression in melanoma cells might foster tumorgrowth.

On the basis of the finding that ITK CpG sites are hypo-methylated in melanoma compared with nevi, we conductedexperiments to determine whether ITK was expressed in neviand melanomas. The availability of potent ITK-selective inhi-bitors makes ITK an attractive target, and the TEC kinase BTKhas been successfully targeted in hematologic malignancies(14). We therefore examined ITK expression in primary andmetastatic melanomas and tested the effects of genetic andpharmacologic inhibition of ITK in cultured cells and murinemelanoma models.

Materials and MethodsTissue procurement

Human tissues analyzed (Supplementary Table S1) wereobtained as deidentified, formalin-fixed paraffin-embeddedtumor blocks from the University of North Carolina (UNC;Chapel Hill, NC)Health Care archives under Institutional ReviewBoard (IRB)-approved protocols.

Tissue sections and tissue microarray constructionFourmicron-thick whole tissue sections (WTS) of nevi (n¼ 30)

and primary invasive melanomas (n ¼ 20) were used for tumortissue analyses. Tissue microarrays (TMA) of metastatic melano-mas (n ¼ 82) containing triplicate cores (0.6 mm) from eachspecimen were also constructed. A pathologist (P.A. Groben)reviewed hematoxylin and eosin (H&E; hematoxylin 7211, eosin7111, Richard-Allan)-stained tissue sections to confirm the speci-mens' diagnoses and marked representative tumor areas on theH&Es for cores to be included in TMAs. TMA blocks were cut into4-mm thick sections. A pathologist examined H&E slides of theTMAs to confirm tumor presence.

Cell culture and Western blot analysesCells were grown in 10% FBS (S12450, Atlanta Biologicals) in

base media without antibiotic unless noted. Human melanomacell lines A375, VMM 39, SKMEL 103, SKMEL 131, SKMEL 153,SKMEL 147, RPMI-8322, and PMWK were used for the cellbiology experiments. A375, VMM 39, SKMEL 103, and SKMEL

147 were passaged in DMEM (Gibco). SKMEL 131, SKMEL 153,and RPMI-8322 were grown in RPMI-1640 media. PMWK cellswere passaged in Alpha Minimum Essential Medium (MEM)media (Gibco) with 0.5� minimal essential amino acids. Thesources and base media for the cell lines have been reported (15)except for SKMEL 235 and WM 1232 (obtained from Wistar, M.Herlyn) and grown in RPMI-1640 (11875-093, Gibco) and TU2% [80%MCDB 153 (M-7403, Sigma), 20% L-15media (41300-070, Gibco)], 2% FBS (S12450, Atlanta Biologicals), 5 mg/mLbovine insulin (I-5500, Sigma), 1.68 mmol/L Calcium Chloride(C-5670, Sigma). Cell lines have been tested and authenticatedand verified as mycoplasma free. Normal human melanocytes(NHM; Clonetech) were cultured andWestern blot analyses wereperformed as described previously (16).

Melanocyte-melanoma cell line array constructionCell line arrays (CLA) were constructed from 38melanoma cell

line and three NHM pellets that were fixed in 10% bufferedformalin (SF98-4, Fisher) for 16 to 24 hours, washed twice in70% ethanol, clotted in 2% low-melting agarose (BP165-25,Fisher), processed, and embedded in paraffin wax (23-021-400,Fisher). Cell pellet blocks were sectioned and H&E-stained toverify the quality of the cell clots and guide CLA construction.Three 1-mm diameter cores were removed from each cell pelletblock and randomly embedded into recipient CLA blocks, whichwere cut into 5-mm thick sections.

Specimen pathology reviewMelanomas were reviewed by a dermatopathologist. Demo-

graphic characteristics were extracted from the clinical record.

Antibodies, tissue staining protocols, imaging, and analysisITK (rabbitmonoclonal Y401; Abcamab32039)was utilized to

probe human ITK. GAPDH (Abcam ab9484mousemonoclonal),cyclin D2 (Santa Cruz Biotechnology sc-563056), and LC3(Novus Biologicals NB 100-2220) were used for Western blotanalyses. Other antibodies and tissue staining protocols for theIHC experiments are summarized in Supplementary Table S2.Single or dual IF on CLAs, melanoma TMAs, andWTS of nevi andmelanomas were performed in the Bond fully-automated slidestaining system as previously described (17).

H&E stained WTS, CLA, and melanoma TMA slides weredigitally imaged at�20magnificationusing theAperio ScanScopeXT (Aperio Technologies). High-resolution acquisition (�20objective) of fluorescently stained slides in the DAPI, Cy3, andCy5 channels was performed in the Aperio-FL (Aperio Technol-ogies) and fluorescently stained CLAs in PM2000 (HistoRx).Fluorescently stained WTS and TMAs (ITK-S100) were submittedfor analysis through Spectrum using HistoRx automated quanti-tative analysis (AQUA) software version 2.2. Expression of ITKprotein labeled by Cy5 (red) was measured in an S100-specifictumormask labeled by Alexa555 (green; ref. 6). ITK expression inCLAs was measured in the autofluorescent (Cy3) mask. Samespecimen array cores were averaged. AQUA scores were normal-ized across the TMAs and WTS using cell line standards run inevery batch. The Aperio Area Quantification FL algorithm wasused to quantify ITK (Cy5) colocalization with CD3, CD19,CD56, CD57, CD68, MPO, and MCT markers (Cy3) in threeprimary melanomas and two melanomas metastatic to lymphnodes from 5 different patients.

Translational Relevance

Emerging systemic therapies for metastatic melanomaextend life, but are rarely curative for the majority of patients.Ourfinding that IL2 inducible T-cell kinase (ITK), a TEC familytyrosine kinase, is aberrantly expressed in most metastaticmelanomas suggests that inhibitors of ITK might be usefulfor melanoma treatment. The efficacy of a small-molecule ITKinhibitor in the Tyr-Cre/Ptennull/BrafV600E mouse melanomamodel suggests that ITK is a driver of melanoma progressionand a potential therapeutic target. Ibrutinib, an inhibitor of theTEC family Bruton's tyrosine kinase, also inhibits ITK and wasrecently FDA approved for treatment of hematologic malig-nancies. Therefore, considering the clinical success of targetingtyrosine kinases for treatment of other malignancies, trialsusing ITK inhibitors for treatment of humanmelanoma, aloneor in combination with existing therapies, are warranted.

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Clin Cancer Res; 21(9) May 1, 2015 Clinical Cancer Research2168



Tyr-Cre/Ptennull/BrafV600E mouse melanomas were stained asdescribed (ref. 18; Supplementary Table S2). Slides were blockedwith DakoCytomation, X0909 (KEY 2006) and incubated withAbcamab32113 for 1 hour at room temperature or 4�Covernight.The slides were blocked with Dako X0590 biotin blocking solu-tion (X059030-2, Agilent). Chromogenic and fluorescentlystained images were stored within the Aperio SpectrumDatabase.

Melanoma mutational statusThe BRAF (exons 11 and 15) and NRAS (exons 2 and 3)

mutational status of primary melanomas and cell lines wasdetermined as reported (19). Metastatic melanoma TMA coreswere stained with BRAF VE1 antibody (20) and scored forVE1 cytoplasmic staining by a pathologist as 0 (no staining),1þ (weak background), 2þ (moderate staining), or 3þ (strongstaining). VE1 scores of 2þ and 3þ were considered positive forBRAFV600E.

shRNA lentivirus production and useLentiviral shRNA constructs TRCN0000010020 (ITK4),

TRCN0000010021 (ITK5), TRCN0000010022 (ITK6), andTRCN0000010023 (ITK7) from the ThermoScientific TRC shRNAlibrary TRC-Hs1.0 (Human) were supplied by the UNC-CH'sLenti-shRNA Core Facility. Lentivirus was produced according tothe ViraPower Lentiviral Packaging Mix instructions (#44-2050,Invitrogen). Approximately 1�106 lentiviral particleswere addedto transduce approximately 50% of the cells in a 7.5 cm dish. Onday 2,media were removed and fresh completemedia added thenon day 3media were replacedwith fresh completemedia contain-ing puromycin (final concentration 10 mg/mL). Cells wereallowed to grow for 4 days before use.

BI 10N, a small-molecule ITK inhibitorBI 10N (21) was from Changchun Discovery Sciences Ltd.

Aliquots of a 1,000� stock solution in DMSO were prepared andstored at �20�C. Carna Biosciences performed selectivity assays.

Two-dimensional gel electrophoresisTwo-dimensional gel electrophoresis was performed using the

Immobiline DryStrip 7 cm, pH 6 to 11 gel system according to themanufacturer's specifications (17-6001-94, GE Healthcare). Gelswere then transferred and Western blot analyses were performedusing the Y401 antibody.

Proliferation and migration assaysHuman melanoma cell lines were added to 10 cm2 6-well

dishes at a density of 50,000 cells per well. BI 10N was added inDMSO; DMSO was used as a drug vehicle control. Cells wereharvested using Trypsin (0.025%) in PBS solution (R-001-100,Gibco) containing 0.01% EDTA for approximately 5 minutes.Cells were counted using the Countess Automated Cell Counter(C10227, Life Technologies). Graphs were generated usingGraphPad Prism version 5 (GraphPad Software).

Single-cell tracking was performed to calculate the averagemotility rate, as described previously (22). Cells were incubatedfor 24 hours with BI 10N before tracking. At least 50 cells weretracked at each BI 10N concentration.

EdU – FxCycle violet staining of melanoma cellsMelanoma cells were grown to approximately 60% confluence

in T25 tissue cultureflasks (CorningProduct #430639).Cellswerelabeled with Click-iT EdU Alexa fluor 488 (C10425, Invitrogen)followed by detection using FxCycle Violet (F-10347, Invitrogen),as per manufacturer's recommendations. Data acquisition wasaccomplished using CyanADP from Bechman Coulter, and cell-cycle analysis accomplished using Summit (version 4.3) software(DAKO).

Caspase glo 3/7 assayThe Promega Caspase-GLO 3/7 Assay Kit (G8090, Promega)

was utilized as per the manufacture's protocols. Ten thousandmelanoma cells were plated in 96-well dishes in quadruplicatewith the indicated drug concentrations. Of note, 100 nmol/Lstaurosporine was used as the positive control.

Reverse Phase Protein ArrayPMWKs andRPMI-8322 cells were treatedwith increasing BI 10

concentrations, lysateswere produced, and theMDAnderson corefacility performed Reverse Phase Protein Array (RPPA) analyses(23).

In vivo studiesAll mice were housed and followed in the UNC LCCC mouse

phase I unit (MP1U) under UNC-CH Institute for Animal Careand Use Committee approved protocols. Ten-week-old malenude athymic mice (Jackson Labs 000819) were subcutaneouslyinjected into the flank with 500,000 cells, which were previouslysuspended in a 50:50 mixture of Matrigel Basement MembraneMatrix (356234, BD Biosciences) and Hank's Solution with 2%FBS. The Tyr-Cre/Ptennull/BrafV600E GEMM was induced asdescribed (24). Treatment beganwhen tumors reached anapprox-imate size of 60 mm3. BI 10N was administered orally viamedicated diet (25) at 15 mg/kg/d. Body mass and body condi-tion score (26) were monitored weekly via caliper measurementsfor the duration of the experiment as a marker for toxicity. Tumorvolume was calculated using the formula: (width2)� (length)/2.

Statistical analysisStatistical analyses of IF data were accomplished using R

software (The R project for Statistical Computing). Mann–Whit-ney andKruskal–Wallis tests were used to compare ITK expressionwith clinical attributes in subsets of nevi, primarymelanoma, andmetastatic melanoma samples. Mann–Whitney tests were used tocompare ITK expression with mutational status in melanomametastases (mutant vs. wild type) and genetic alterations (nega-tive versus positive) in cell lines.

Linear regression analyses were performed for shRNA cellproliferation data using logarithmically transformed baselinenormalized cell counts (y) against days (x). Similar regressionanalyses were performed for mouse tumor growth data using thesquare root-transformed tumor volume (y) against weeks (x).Likelihood ratio tests were conducted to assess whether slopesbetween control and treatment groups were statistically differentfrom each other. Differences in melanoma cell motility amongdifferent treatment groups were assessed using Mann–Whitney Utests. P values were Bonferroni corrected to account for multiplecomparisons of different treatment groups for each cell line.

ITK, a Therapeutic Target in Melanoma

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For RPPA data, a linear regression line was fitted using proteinexpression as the dependent variable and BI 10N concentration asthe independent variable to analyze RPPAdata. t testswere used toassess whether the estimated slopes significantly differed fromzero. The P values of the t tests for slope were adjusted using theFDR control to account for multiple testings. Data were analyzedthrough the use of QIAGEN's Ingenuity Pathway Analysis (IPA).

Microarray analysisAnalysis of microarray data (Gene Expression Omnibus acces-

sion number GSE54623) was performed using BiometricResearch Branch (BRB, National Cancer Institute, Bethesda, MD)array tools software (27). The quantitative trait analysis (QTA)tool used the Spearman correlation coefficient to identify geneswhose expression was correlated with ITK expression in melano-ma cell lines. The correlated (P < 0.005) geneswere investigated todetermine whether they were overrepresented in Biologic Bio-chemical Image Database (28), Kyoto Encyclopedia of Genes andGenomes (29), BioCarta (30), Panther (31), or Reactome (30)pathways.

Study approvalHuman tissue studies were IRB approved under UNCprotocols

07-0450 and 09-0737. Animal experiments were approved by theUNC Institutional Animal Care and Use Committee.

ResultsITK protein expression in melanocytic tissues

Sections of normal skin, benignnevus, primarymelanoma, andmetastatic melanomas were dually probed with antibodiesagainst ITK (red) and S100 (green; a marker for melanocyticlineage cells) and visualized using immunofluorescence (IF;example shown in Fig. 1A). ITK in melanocytic lineage cells ofbenign nevi (n ¼ 30), primary melanomas (n ¼ 20), and met-astatic melanomas (n ¼ 70) was quantified in the S100-positivecells using AQUA (refs. 32, 33; Fig. 1B and Supplementary TableS1). Mean ITK expression was higher in primary melanomas thanbenign nevi, and even higher in metastatic melanomas (P <0.001). The mean ITK protein levels of nevi and melanomas wereeach not significantly associated (all P > 0.05) with demographicor tumor characteristics, and the melanomas' ITK protein levelswere not associated with BRAFV600E status (SupplementaryTable S1).

ITK protein expression in immune cell subsetsBecause ITK has been shown to have a role in Th2-mediated

responses, which have been previously associated with ineffectiveantitumor responses (34), we investigated whether ITK wasexpressed in immune subsets infiltrating the tumors. To accom-plish this, we performed dual color IF analysis of ITK andmarkersof immune cell subsets on three primary melanomas and two

Figure 1.ITK expression in melanocytic tissues. A,representative tissue sections from normal skin(a), benign nevus (b), primary melanoma (c),and metastatic melanoma (d) were stained withH&E (top row) or probed with ITK-Cy5 (red) andS100-Alexa 555 (green) primary and secondaryantibodies and counterstained with DAPI (blue).Orange indicates colocation of ITK with S100, amelanocytic lineage marker. The clinical andhistologic characteristics of the melanocytictissues stained and their ITK levels are inSupplementary Table S1. Staining protocols arein Supplementary Table S2. B, box-and-whiskerplots (median with the 25–75th percentilesand outliers) of ITK expression in S100þ cellsfromnevi (n¼ 30), primarymelanomas (n¼ 20),and metastatic melanomas (n ¼ 70). Of themetastatic melanomas, 91% (64/70) had ITKexpression above that of the range of ITKexpression found in nevi.

Carson et al.




melanomas metastatic to lymph nodes from 5 patients. ITKexpression was observed in a few small mononuclear cells in themelanomamicroenvironment, but did not colocalize with CD3þ,CD19þ, CD68þ, myeloperoxidase (MPO)þ, mast cell tryptase(MCT)þ, CD56þ, or CD57þ markers for T cells, B cells, macro-phages, neutrophilic granulocytes, mast cells, and subsets ofnatural killer cells (Supplementary Fig. S1A). We therefore couldnot confirm ITK expression in the most abundant tumor-infil-trating immune cell subsets.

Melanoma cell line characterization and ITK levelsTo investigate whether ITK expression is associated with a

particular gene expression or mutation signature, we firstprobed a melanocyte-melanoma CLA with the ITK antibody(Supplementary Table S2). These melanoma cell lines hadpreviously undergone next-generation sequencing (35) andgene expression profiling (16). The three different NHM cul-tures in the CLA had a mean ITK signal of 38.5 (range 26.7–52.3) in arbitrary IF units, whereas 38 melanoma cell lines hada mean ITK signal of 278 (range 30.4–4,941). To group themelanoma cell lines in the CLA by ITK levels, we used the ITK IFarbitrary units obtained for each cell line to classify them as ITKlow (ITKLO;

not shown), whereas ITK protein levels in the PMWK (ITKLO) cellline were too low to reliably measure significant changes in ITKprotein levels (Fig. 2A).

The shRNAs were utilized to study the effects of decreasedITK protein expression on melanoma cell proliferation, cell-cycle profile, apoptosis, and motility. Melanoma cell linesVMM 39 and RPMI-8322 (both ITKHI) transduced with shRNAsITK 4, 5, or 7 proliferated at a decreased rate (P < 0.05,Bonferroni corrected) compared with those transduced withSCR (Fig. 2C and Supplementary Table S4). shRNAs ITK 5 and7 had no affect (P > 0.05) on PMWK (ITKLO) proliferation,although ITK 4 slightly decreased the proliferation of PMWKcompared with SCR.

To assess whether ITK suppression induced changes in thecell cycle, incorporation of 5-ethynyl-20-deoxyuridine (EdU)was measured to determine de novo DNA synthesis (S phase). Inaddition, each cell line was stained using FxCycle violet tomeasure DNA content. Inhibition of ITK expression in the VMM39 (ITKHI) and RPMI-8322 (ITKHI) cell lines increased theproportion of cells in G0–G1 (FxCycle

low EdUlow) anddecreased the number of cells in S phase (FxCyclemedium/low-

EdUhigh

). There were no significant cell-cycle changes in PMWK(ITKLO) melanoma cells transduced with any of the shRNAs(data not shown).

Because ITK regulates migration in T cells (36, 37), we inves-tigated the effect of ITK expression onmelanoma cell linemotilityquantified by single-cell tracking analysis (38). Migration rates ofthe ITK 4, 5, or 7- transduced VMM 39 (ITKHI) and RPMI-8322(ITKHI) cell lines were significantly decreased (P < 0.05, Bonfer-roni corrected) compared with the corresponding SCR-trans-duced cell lines (Fig. 2D and Supplementary Table S4). Nosignificant changes in the migration rate of the PMWK (ITKLO)cells were observed following transduction with any of the lenti-viral clones.

BI 10N decreases ITK activity in melanoma cellsTo assess the effect of pharmacologic inhibition of ITK

activity in melanoma cell lines, we used BI 10N, a small-molecule inhibitor of ITK (21, 39). We investigated the potencyand selectivity of BI 10N at a concentration of 200 nmol/Lagainst 56 kinases using a cell-free assay (Supplementary TableS5). IC50s were measured for the kinases most potently inhib-ited by BI 10N in the initial screen, and tight binding studieswere performed as needed. BI 10N is a highly selective inhibitorof ITK, and this affinity is described by an apparent dissociationconstant (KI,apparent) of 8.6 pmol/L. Of the 56 protein kinasestested, BI 10N is at least 1,000-fold more selective for ITK thanfor 51 of the protein kinases and is about 10-fold selective overthree members of the neurotrophic tyrosine receptor kinasefamily (TRKA, TRKB, TRKC) and the SRC family membernonreceptor tyrosine kinase YES. BI 10N is about 1,000-foldmore selective for ITK than for the seven other SRC familytyrosine kinases tested, including SRC and FYN.

To assess whether ITK expressed inmelanoma cells is active, weprobed phosphorylation at Tyr-180 and 511 residues (40) inwhole-cell lysates from high ITK-expressing melanoma cell lineswith the commercially available antibodies 4F10 and 8D11, butcould not identify a correctly migrating band. To quantitate ITKphosphorylation in the melanoma cells, we therefore performedtwo-dimensional electrophoresis on whole-cell lysates obtainedfromRPMI-8322 cells followed byWestern blot analysis using theITK Y401 antibody. Lysates profiled included those from untreat-ed cells, phosphatase-treated cells, and from cells treated withdifferent concentrations of BI 10N for 3 days before harvest (Fig.3A). In untreated RPMI-8322 cells, two differentially chargedimmune reactive bands thatmigratedwith the appropriate weightfor ITK were identified as ITK by Western blot analysis. However,one of the immune reactive bands was absent in extracts treatedwith lambda phosphatase and from extracts from cells treated

0 nmol/L0 nmol/L 0 nmol/L

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A

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Figure 3.Effects of BI 10N on the phosphorylation of ITKand on the proliferation andmotility of melanomacells. A, Western blot analysis on whole-celllysates obtained from RPMI-8322 cells. Cells weretreated with BI 10N for 3 days at the indicatedconcentrations before the analysis. Extracts ofcells that did not receive BI 10N were treated withphosphatase before analysis. Proteins fromlysateswere separated by two-dimensional PAGEbefore Western blot analysis using an antibodyagainst total ITK. The percentages indicate thearea of the smaller band as a percent of the wholesignal. B, effects of increasing BI 10Nconcentrations on the proliferation rates ofPMWK, VMM 39, and RPMI-8322 melanoma cells.Asterisks denote significant decreases inproliferation compared to untreated cells(P < 0.05, Bonferroni corrected; SupplementaryTable S4C). C, effects of increasing BI 10Nconcentrations on the motility of PMWK, VMM 39,and RPMI-8322 melanoma cells treated for 24hours before the assay. Asterisks denotesignificant decreases in motility in BI 10N-treatedcompared with untreated melanoma cells(P < 0.05, Bonferroni corrected; SupplementaryTable S4D).

Carson et al.




with concentrations of BI 10N � 25 nmol/L. In untreated cells,about 14% of ITK is phosphorylated.

To determine whether the ITK's catalytic activity stimulates theproliferation and migration of melanoma cell lines, we inhibitedITK using BI 10N. Proliferation assays were conducted on PMWK(ITKLO), VMM 39 (ITKHI), RPMI-8322 (ITKHI), and other mela-noma cell lines (Fig. 3B, data not shown, and SupplementaryTable S4). In these studies, BI 10N was added at the inception ofthe assay at the indicated concentrations when the cells wereplated. Similar to the equivalent shRNA studies, proliferation ofthe ITKHI cell lines (VMM 39, RPMI-8322, and SKMEL 153) andan ITKINT cell line (A375) decreased by about 50% in the presenceof 50 nmol/L BI 10N, whereas the effects on the proliferation ofPMWK (ITKLO) cells were weaker and required 200 nmol/L BI10N. ITK catalytic activity inhibition by BI 10N reduced ITKHI cellproliferation and migration similar to shRNA suppression of ITKexpression. Isogenic overexpression of ITK was not performed toconfirm these results.

Cell-cycle analysis of VMM 39 (ITKHI) and RPMI-8322 (ITKHI)cell lines using EdU labeling and FxCycle Violet staining indicatedthat, similar to the shRNA studies, treatment of cells with BI 10Nincreased thepercentage of cells in theG0–G1phase anddecreasedthe percentage of cells in the S phase in a dose-dependent manner(Supplementary Fig. S1B). No cell-cycle–specific changes wereobserved between the treated and untreated PMWK (ITKLO) cells.BI 10N did not affect the percentage of cells in pre-G0–G1 inPMWK, RPMI 8322, or VMM39 cells (data not shown). To furtherassess the effects of ITK activity onapoptosis, caspase-3/7 activitiesin PMWK, RPMI, and VMM39were assessed; no consistent effectsby BI 10N on the caspase activity levels were noted (data notshown).

The effects of BI 10N on melanoma cell migration were mea-sured for PMWK (ITKLO), VMM 39 (ITKHI), RPMI-8322 (ITKHI),and additional melanoma cell lines (Fig. 3C, data not shown, andSupplementary Table S4), by single-cell tracking analysis. Similarto the results with the shRNA clones targeting ITK, the migrationby the ITKHI cell lines (VMM39, RPMI-8322, and SKMEL 153)decreased about 50% in the presence of 10 to –25 nmol/L BI 10N,whereas an ITKINT cell line (A375) and the ITKLO cell line PMWKrequired BI 10N concentrations of 100 nmol/L, or greater, tosignificantly decrease migration.

Effects of ITK activity on specific cellular proteinsWesterns were performed on RPMI-8322 cells treated with BI

10N administered at concentrations as high as 50 nmol/L for 3days. Inhibition of ITK resulted in cyclinD2 (CCND2) decrease byabout one-third but no change in LC3 (MAP13LC3A) level(related to autophagy; Supplementary Fig. S2A). AKT and ERKprotein levels and phosphorylation were unchanged by Westernblot analyses (data not shown).

To further investigate the effects of ITK activity on cells, RPPAs(282 proteins or phosphorylated species) were performed ontriplicate extracts of PMWK and RPMI-8322 cells treated with upto 50 nmol/L BI 10N. The RPPA results confirmed the phosphorWestern analyses we performed for AKT and ERK showing nochange in the phoshphorylation state of those proteins (data notshown). IPA using a cutoff FDR value of 0.15 indicated that themost affected pathway was p53 signaling with cell-cycle arrestbeing influenced but not apoptosis or cell survival (Supplemen-tary Table S6 and Supplementary. S2B). RPPA-determined levels

of protein analyzed in the p53 pathway are plotted in Supple-mentary Fig. S2C.

Efficacy of BI 10N in murine melanoma modelsTo investigate whether BI 10N reduces melanoma growth in

vivo, we treated mouse melanoma models with medicated chowcontaining BI 10N (21). Dose-finding studies based on thepublished literature were performed to establish an effectivedose of 15 mg per kg (mpk)/day. No signs of toxicity (e.g.,weight loss, lethargy) were noted in immunodeficient or genet-ically engineered mice at this dose. Human melanoma xeno-graft mice were established from two melanoma cell lines withhigh ITK expression (VMM 39 and RPMI-8322) and from alower ITK-expressing line, A375. Treatment of each xenograftmouse model with BI 10N at 15 mpk significantly slowedtumor growth in the VMM 39 and RPMI-8322 mice, whereasno such effect was seen in the melanoma xenograft miceestablished from A375 cells (Fig. 4A).

Given ITK's possible effect on immune system function, wetested BI 10N in immunocompetent mice with autochthonousmelanoma. For this purpose, we used the Tyr-CRE-ERT2 B-RafCA

PtenL/L genetically engineered murine model (GEMM; ref. 24). Inthis system, endogenous tumors featuring V600E B-Rafmutationand Pten loss are generated by tissue-specific CRE recombinaseactivation inmelanocytes. Melanomas from thismodel expressedhigh levels of ITK in all five excised tumors tested (Fig. 4B). Micewere dosedwith BI 10Nafter the tumors reached a size of about 60mm3. Treatment of mice with BI 10N at 15 mpk resulted in arrestof tumor growth compared with the untreated mice (Fig. 4C).These results suggest that ITK kinase activity is a driver of tumorgrowth in a GEMM of melanoma. GEMM mice survive signifi-cantly longer when treated with BI 10N (Fig. 4D). Similar to thetreated cell lines, cyclin D2 was decreased by at least one-third inBI 10N-treated versus untreated tumors, whereas LC3 showed nochange (data not shown).

DiscussionOur work provides several lines of evidence that ITK, a gene

whose promoter CpG sites are hypomethylated in primary mel-anomas compared with benign nevi, is expressed in a majority ofmetastatic and primary melanoma tumors. Specifically, we showthat ITK protein expression increased with tumor progressionfrom nevus to primary melanoma and evenmore so tometastaticmelanoma. Furthermore, suppression of ITK expression or kinasefunction inmelanoma cells reduced cell attributes associatedwithtumorigenesis, including proliferation andmotility. Finally, inhi-bition of ITK activity using BI 10N reduced tumor growth in bothanimal models and in addition extended the survival of the Tyr-Cre/Ptennull/BrafV600E mice.

To our knowledge, this is the first report that ITK is highlyexpressed in nonhematopoietic tissues and even more so by asolid tumor malignancy, such as melanoma. One group reportedlow-level expression of ITK mRNA in colon cancer tumor tissues,but commented that its expression was probably derived fromcontaminating host cells (41). Previous studies about the putativeoncogenic role of ITK have been limited to its increased oraberrant expression in T-cell malignancies (42–44). It has alsobeen reported that an ITK inhibitor had cytotoxic effects onmalignant T cells (43). We are not aware of previous studiesreporting ITK inhibition of solid tumor malignancies.





Our review of the 226 melanoma samples that have under-gone next-generation sequencing and putative copy-numberanalysis as part of The Cancer Genome Atlas (TCGA) Project(45, 46) using the GISTIC tool revealed no evidence of copynumber alterations or somatic mutations that could account forthe ITK expression in the cells or clinical samples reported here.The lack of ITK gene copy number alterations in the TCGAmelanomas together with our finding that melanomas arehypomethylated in the ITK promoter relative to nevi (10) andexpress ITK protein suggests that epigenetic rather than geneticmechanisms, at least in part, account for the high ITK expressionin melanoma.

Therapeutic targeting of ITK in melanoma should criticallyconsider its bystander effect on the host immune response giventhe overwhelming evidence that a functioning immune system is

fundamental for antitumor responses against this disease. Ofnote, our investigations failed to demonstrate ITK expression inseveral tumor-infiltrating immune cell subsets such as T cells,polymorphonuclear cells, mast cells, natural killer cells, andmacrophages. Importantly, the antitumor effect of BI 10Nremained significant in immunocompetentmicewithmelanoma,suggesting at least the lack of a significant negative effect of ITKinhibition on the host antitumor response.

In this work, we characterized the kinase inhibitor profile of BI10N, a small-molecule selective inhibitor of ITK previously usedin animal studies (21). BI 10N was highly selective for ITK over alarge panel of kinases, but not for three TRK family members andthe SFK member YES. Although TRKA and TRKB are highlyexpressed in melanoma (47), the effects of BI 10N are most likelymediated through the inhibition of ITK as the effects of BI 10N on

Figure 4.Effects of BI 10N on tumor growth in vivo. A, effects of BI 10N (15 mpk) on the growth of human melanoma xenografts (mean and SEM). Asterisks indicatesignificant tumor growth inhibition by BI 10N-treatment comparedwith untreatedmice (P

melanoma cells are similar to those achieved with ITK depletionusing shRNAs.

A limitation to our work is that the mechanism by which ITKincreases cell proliferation and migration remains unknown. BI10N treatment did not change AKT and ERK phosphorylationlevels in RPMI-8322 cells. However, evidence presented indicatesthat themechanism by which ITK activity drives the cell cyclemayinvolve changes in the p53pathway, and in particularmodulationof cyclin levels, but further studies are needed to clarify theseissues.

In summary, ITK appears to be a driver of melanoma asdemonstrated by its expression in most of the metastatic mela-nomas examined and by the significant effects of BI 10N on theproliferation and migration of melanoma cells and its efficacy inmousemelanomamodels. We are currently investigating rationaltreatment combinations between ITK inhibitors and standardtreatments for metastatic melanoma in various preclinical mel-anoma models that may serve as the basis for future clinical trialsin metastatic melanoma. Besides BI 10N, several other ITK inhi-bitors previously have been developed to target Th2 dominantautoimmune, inflammatory, and infectious diseases (48, 49).Notably, ibrutinib (IMBRUVICA, Pharmacyclics, Inc.), an irre-versible inhibitor of BTK and ITK (50), has been granted approvalby the U.S. FDA. We conclude that ITK is a novel target for thetreatment of melanoma that, at least in animal models, is ame-nable to pharmacologic intervention.

Disclosure of Potential Conflicts of InterestS.J. Moschos is a consultant/advisory board member for Merck and Prome-

theus. D.S. Rubenstein is an employee of Stiefel/GlaxoSmithKline. No potentialconflicts of interest were disclosed by the other authors.

Authors' ContributionsConception and design: C.C. Carson, S.J. Moschos, D.S. Rubenstein, J.E. Bear,D.W. Ollila, N.E. Sharpless, N.E. ThomasDevelopment of methodology: C.C. Carson, N. Nikolaishvili-Feinberg,S. Pandey, H. Hao, D.C. Gibbs, D.S. Rubenstein, N.E. Sharpless, N.E. ThomasAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): C.C. Carson, S.J. Moschos, S.N. Edmiston, D.B. Darr,N. Nikolaishvili-Feinberg, P.A. Groben, S. Pandey, K.T. Chan, J.L. Jordan,J.S. Frank, D.A. Hopkinson, D.C. Gibbs, V.D. Alldredge, S.C. Hanna, J.E. Bear,K. Conway, N.E. Thomas

Analysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): C.C. Carson, S.J. Moschos, S.N. Edmiston, N. Niko-laishvili-Feinberg, P.A. Groben, X. Zhou, P.F. Kuan, S. Pandey, K.T. Chan,J.L. Jordan, C.R. Miller, N.E. Sharpless, N.E. ThomasWriting, review, and/or revision of themanuscript:C.C. Carson, S.J. Moschos,S.N. Edmiston, D.B. Darr, S. Pandey, J.S. Frank, D.C. Gibbs, S.C. Hanna,C.R. Miller, D.W. Ollila, K. Conway, N.E. ThomasAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): S.N. Edmiston, N. Nikolaishvili-Feinberg, P.A.Groben, H. Hao, J.S. Frank, D.C. Gibbs, E. Parrish, P. Berkowitz, D.W. Ollila,K. Conway, N.E. ThomasStudy supervision: C.C. Carson, N.E. Thomas

AcknowledgmentsThe authors thankM.Olorvida and B.Midkiff (UNC TPL) for assistance with

staining and tissue imaging analysis of the immunofluorescence experiments;the UNC MP1U personnel for assistance with animal handling, therapeuticstudies, and tumor serial assessment; UNC Dermatopathology (director: Dr.Daniel Zedek) for immunostaining of themetastatic tumor tissues with the VE1BRAFV600E mutation-specific antibody; Dr. Marcus W. Bosenberg M.D., Ph.D.for kindly providing the 4-hydroxytamoxifen Tyr::CreER; BrafCA; Ptenlox/lox

mouse for our in vivo studies; UNC-CH's Lenti-shRNA Core Facility for supply-ing lentiviral small hairpin RNA; and Dr. Alan Houghton and Memorial Sloan-Kettering Cancer Center for providing the SKMEL cell lines used in this research.

Grant SupportThis work was supported by National Cancer Institute (NCI) grants

R33CA10704339 (to N.E. Thomas and K. Conway), R21CA134368 (to N.E.Thomas and K. Conway), R01CA112243 (toN.E. Thomas), and P30CA016086;National Institute of Environmental Health Sciences (NIEHS) grantP30ES010126; University Cancer Research Fund (UCRF) at University of NorthCarolina (UNC) Chapel Hill's Lineberger Comprehensive Cancer Center(LCCC; to N.E. Thomas); Harry J. Lloyd Charitable Trust (to N.E. Thomas andS.J. Moschos); UNC LCCC Developmental Grant; The V Foundation (to N.E.Thomas and D.W. Ollila); Doris Duke Charitable Foundation Clinical ResearchFellowship (to S. Pandey and V.D. Alldredge); National Center for AdvancingTranslational Sciences awardULTR000083 (toN.E. Thomas); and The Irene andRobert Alan Briggaman Distinguished Professorship (to N.E. Thomas). UCRFsupported, in part, work in the Mouse Phase 1 Unit (MP1U) and the UNCTranslational Pathology Laboratory (TPL).

The costs of publication of this articlewere defrayed inpart by the payment ofpage charges. This article must therefore be hereby marked advertisement inaccordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received July 16, 2014; revised January 26, 2015; accepted January 26, 2015;published online May 1, 2015.

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