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Models and Technologies

NSCLC Driven by DDR2 Mutation Is Sensitive toDasatinib and JQ1 Combination TherapyChunxiao Xu1,2, Kevin A. Buczkowski1, Yanxi Zhang1,2, Hajime Asahina1,2,Ellen M. Beauchamp1, Hideki Terai1, Yvonne Y. Li1,3, Matthew Meyerson1,3,4,5,Kwok-kin Wong1,2, and Peter S. Hammerman1,3

Abstract

Genetically engineered mouse models of lung cancer havedemonstrated an important role in understanding the functionof novel lung cancer oncogenes and tumor-suppressor genesidentified in genomic studies of human lung cancer. Furthermore,these models are important platforms for preclinical therapeuticstudies. Here, we generated a mouse model of lung adenocarci-noma driven by mutation of the discoidin domain receptor 2(DDR2) gene combined with loss of TP53. DDR2L63V;TP53L/L

micedevelopedpoorly differentiated lung adenocarcinomas in alltransgenic animals analyzedwith a latency of 40 to 50weeks and amedian survival of 67.5 weeks. Mice expressing wild-type DDR2with combined TP53 loss did not form lung cancers. DDR2L63V;

TP53L/L tumors displayed robust expression of DDR2 and immu-nohistochemical markers of lung adenocarcinoma comparablewith previously generated models, though also displayed con-comitant expression of the squamous cell markers p63 and SOX2.Tumor-derived cell lines were not solely DDR2 dependent anddisplayed upregulation of and partial dependence on MYCN.Combined treatment with the multitargeted DDR2 inhibitordasatinib and BET inhibitor JQ1 inhibited tumor growth in vitroand in vivo. Together, these results suggest that DDR2 mutationcan drive lung cancer initiation in vivo and provide a novel mousemodel for lung cancer therapeutics studies.Mol Cancer Ther; 14(10);2382–9. �2015 AACR.

IntroductionLung cancer is the leading cause of cancer-related death in the

United States and worldwide (1). More than 85% of lung cancerpatients are diagnosed with non–small cell lung cancer (NSCLC),including adenocarcinoma (50% of lung cancers), squamous cellcarcinoma (SCC; 30%), and large-cell carcinoma (10%; ref. 2).Technologic advances in recent years, including the application ofnext-generation sequencing (NGS), have allowed researchers toconstruct large databases describing the molecular features ofhuman lung tumors. These efforts have been accompanied bythe generation of a number of newmodels of lung cancer, notablygenetically engineeredmouse models (GEMM), which have facil-itated the study of candidate human lung cancer oncogenes andtumor-suppressor genes in vivo. Thesemodels have beenused for a

number of purposes, including studies of tumor formationusing one or more genetic alterations found in human cancers,studies of the impact of specific genetic changes on the tumorand its microenvironment, and for studies of anticancer ther-apies. EGFR-, Kras-, BRAF-, and ERBB2-mutated and -translo-cated EML4-ALK lung adenocarcinoma models have been gen-erated and used for studies providing important insights intothe mechanisms of response and resistance to clinically rele-vant–targeted lung cancer therapies (3–7). Lung cancer genomestudies continue to nominate an increasing number of candi-date lung cancer oncogenes and tumor suppressors in lungadenocarcinoma, and more recently, in lung SCC. Some can-didate genomic alterations from studies of squamous cell lungcancers include amplification of SOX2, PDGFRA, and FGFR1and mutations of KEAP1, STK11, ERBB4, and DDR2 (Discoidindomain receptor 2; refs. 8, 9).

DDR2 is a type I transmembrane receptor tyrosine kinase(RTK). DDR kinases are widely expressed in human tissues, areactivated by collagens, and have roles in cell adhesion, migration,proliferation, and survival when activated by ligand binding andphosphorylation (10). DDR kinases play a role in cancer progres-sion by regulating the interactions of tumor cells with theirsurrounding collagen matrix. DDR2 mutations have beenreported in multiple tumor types, including lung cancer, breastcancer, brain cancer, gynecologic cancer, and prostate cancer (10).We previously reported the identification of novel somatic muta-tions in the DDR2 gene at a frequency of 3.8% in a sample set of290 squamous cell lung cancer samples (11). Overall, 11 muta-tions were found throughout the entire gene and located invarious DDR2 domains, including L63V, I120M, and D125Y,within the collagen-binding discoidin 1 domain; L239R andG253C within the discoidin 2 domain; G505S in the cytosolic

1Department ofMedical Oncology, Dana-Farber Cancer Institute, Bos-ton, Massachusetts. 2Belfer Institute for Applied Cancer Sciences,Boston, Massachusetts. 3Cancer Program, Broad Institute of HarvardandMIT, Cambridge, Massachusetts. 4Department of Pathology, Brig-ham and Women's Hospital, Boston, Massachusetts. 5Center forCancer Genome Discovery, Dana-Farber Cancer Institute, Boston,Massachusetts.

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

C. Xu and K.A. Buczkowski contributed equally to this article.

Corresponding Authors: Peter Hammerman, Dana-Farber Cancer Institute, 450Brookline Avenue, Boston, MA 02215. Phone: 617-632-6335; Fax; 617-582-7880;E-mail: [email protected]; and Kwok-kin Wong,[email protected]

doi: 10.1158/1535-7163.MCT-15-0077

�2015 American Association for Cancer Research.

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JMdomain; andC580Y, I638F, T765P,G774E, andG774Vwithinthe kinase domain.DDR2 L63V and I638Fmutationswere shownto confer oncogenicity inNIH-3T3 fibroblasts in a colony formingassay in soft agar (11).

DDR2 can interact with multiple proteins resulting in complexsignaling processes. Src has been shown to phosphorylate DDR2resulting in subsequent DDR2 autophosphorylation (12). Thus,DDR2–Src interactions may play a key role in DDR2-intiatedsignaling. The signaling networks downstream of DDR2 includeMAPK (13) andphosphoproteomic studies havenominated SHP-2 as a critical mediator of DDR2 signaling (14). Acquisition of anEMT phenotype in MDCK and human breast epithelial cells hasalso been shown to induce DDR2 expression (15). Activation ofDDR2 regulates the EMT driver SNAIL1 stability by stimulatingERK2 activity, in a Src-dependent manner. DDR2-mediated sta-bilization of SNAIL1 promotes breast cancer cell invasion andmetastasis in vivo (16). However, the transcription factors andmechanisms involved in upregulation ofDDR2 during EMT havenot been elucidated.

Four kinase inhibitors, dasatinib, imatinib, ponatinib, andnilotinib were identified initially as inhibitors of DDR1 andDDR2 by chemical proteomic profiling studies (17, 18), suggest-ing that tumors with activatedDDR2 signalingmay be targeted byagents that are already FDA approved for other indications. Inpreclinical studies, dasatinibwas shown to inhibit proliferation intwo lung cancer cell lineswithDDR2mutations both in vitro and invivo and two case reports of response to dasatinib in lung cancerpatients with DDR2 mutations have been published (11, 19).However, studies have shown that DDR2 inhibition with dasa-tinib leads to both adaptive (20) and acquired resistance withresistance mechanisms, including DDR2 gatekeeper mutation,NF1 loss and activationof parallel RTKpathways, including EGFR,IGF1R, and MET (13).

Although several studies performed in cellular models havesuggested that DDR2 mutations may be clinically relevant, dataare lacking to prove thatDDR2mutations can drive lung cancer invivo. Here, we report that the conditional overexpression of theDDR2 L63V-mutant downstream of amurine CCSP promoter canpromote tumorigenesis in genetically engineered mice with aphenotype of lung adenocarcinoma. In addition, MYCN waselevated in the tumors driven by this DDR2 mutation and celllines derived from themurine tumorswere partially dependent onNYMC. We showed that the bromodomain inhibitor JQ1, aninhibitor of MYC-driven malignancies, plus the nonselectiveDDR2 inhibitor dasatinib as combination therapy could suppressgrowth of these tumors. Together, these data suggest that DDR2mutations can contribute to lung cancer formation in vivo.

Materials and MethodsGeneration of the DDR2wt- and L63V-mutant mouse cohort

The full-lengthDDR2wt cDNAwas obtained fromOrigene andcloned into expression vector pBS31 (21). L63V and I638F muta-tions were generated by site-directed mutagenesis using theQuickchange Site Directed Mutagenesis Kit (Strategene) andfurther verified by DNA sequencing.

For the generation of transgenic mice with lung-specific doxy-cycline-inducible DDR2 expression, we used a previouslydescribed method (22). The CCSP-rtTA and conditional Trp53-deficient allele (p53L/L) mice were kept at DFCI. DDR2wt andDDR2L63V mice were crossed with CCSP-rtTA and p53L/L mice.

Progeny of DDR2wt;p53, DDR2L63V;p53 and DDR2I638F;p53were genotyped as described in the Supplementary Methods. TheDDR2mice were fed a doxycycline diet at 6 weeks of age to induceDDR2 expression and intranasally instilled with Ad-Cre (Univer-sity of Iowa viral vector core) to delete Trp53 as described (23).Mouse health condition was monitored weekly and evaluated byMRI as described previously (6).

All care of experimental animals was in accordance with Har-vard Medical School/Dana-Farber Cancer Institute (DFCI) insti-tutional animal care and use committee (IACUC) guidelines. Allmice were housed in a pathogen-free environment at a DFCIanimal facility and handled in strict accordance with GoodAnimal Practice as defined by the Office of Laboratory AnimalWelfare.

Establishment of GEMM-derived primary cell linesWhenDDR2L63V; p53mice developed lung tumors confirmed

by MRI, the mice were sacrificed and lung tumor nodules wereharvested, finely minced, and cultured in 100-mm dishes withRPMI-1640/10% FBS/1% penicillin–streptomycin (pen–strep)/2 mmol/L L-glutamine. After 3 passages, frozen stocks of theseshort-term cultures were prepared, and further characterized bygenotyping. DDR2 expression was induced by treating cells with2 mg/mL doxycycline every 2 to 3 days and confirmed by Westernblot analysis. Cells (634, 855, and 858) were primary murine celllines with Kras;p53 mutation established in the Wong laboratory(24). The cells were cultured in RPMI-1640/10% FBS/1% pen-strep/2mmol/L L-glutamine. All cells were cultured at 37�C in ahumidified incubator with 5% CO2.

Histology and immunohistochemistryMicewere sacrificedwithCO2; half of the dissected tumorswere

snap-frozen in liquid nitrogen for preparation of protein lysatesand the left lung tissuewasfixed in 10%neutral-buffered formalinfor 24 hours at room temperature, and then transferred to 70%ethanol, embedded in paraffin, and sectioned at 5 mm for IHCstaining. Hematoxylin and eosin (H&E) stains were performed inthe Department of Pathology in Brigham and Women's Hospital(Boston, MA). Immunohistochemistry was performed using pre-viously described methods (23). Anti-DDR2 antibody was fromBethyl Laboratories. All the other antibodies used for dynamicmarkers are listed in Table 1.

Gene expression profiling analysisRNA was extracted from snap-frozen Kras;p53 andDDR2L63V;

p53 tumor tissue using TRizol (Invitrogen) and further purified bythe RNeasy MinElute Cleanup Kit (Qiagen). Arrays were per-formed at Dana-Farber Cancer Institute facility on AffymetrixmouseGene1.0ST arrays.Datawere preprocessed andnormalizedusing the Genepattern Preprocess module with default para-meters followed by Genepattern Comparative Marker Selection

Table 1. Antibodies used for immunohistochemistry

Antibodies Companies Cat. ID

TTF1 DAKO M3575p63 Abcam ab53039SOX2 (C70B1) Cell Signaling Technology 3728SSPC Millipore AB3786F4/80 eBioscience 14-4801-82MPO Novus R-1073CCSP Santa Cruz Biotechnology sc-9772

NSCLC Mouse Model Driven by DDR2 Mutation

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(25). GEO accession number for the microarray data reported inthis article is GSE64907.

Cell growth and proliferation assaysDDR2 doxycycline induced lung cancer cell lines (3941 and

3942) were obtained fromDDR2L63Vmice and were cultured inRPMI-1640 (Invitrogen) and 10% FBS (Gibco). Kras-mutated celllines were previously generated (24) and maintained in the sameconditions.DDR2 expression was induced by treating cells with 2mg/mL doxycycline every 2 to 3 days.

Proliferation of MYCN and control siRNA tranfected cells wasmeasured in triplicate after 24 and 48 hours using a Vi-CELL CellViability Analyzer (Beckman Coulter). Proliferation of cells (634,855, 858, 3941 DDR2, and 3942 DDR2) was measured with theCell-TiterGlo reagent (Promega) per the manufacturer's instruc-tions. Cells were seeded in triplicate at 1,500 cells per well in 96-well clear-bottomed plates. Single drugs and combinations wereadded the following day at concentrations of 10 nmol/L, 50nmol/L, 100 nmol/L, 500 nmol/L, 1 mmol/L, and 10 mmol/L andafter 5 days a standard 96-well plate luminometer was used tomeasure cell proliferation. Comparison of untreated cells withthose treated at a given concentration was used to determine thepercentage of survival. For proliferation analysis, mean valueswere calculated from samples in triplicate and SEs were calculatedby Microsoft Excel. GraphPad Prism software was used to deter-mine IC50 values.

RNAi and cell cultureMYCN expression was knocked down usingMYCN siRNA (m)

obtained from Santa Cruz Biotechnology, Inc. Cell lines 3941DDR2 and 858 were seeded at 2 � 105 cells per well in 6-welltissue culture plates. Control- and N-Myc siRNA–transfected cellswere used for immunoblot analysis and counted at 24 and 48hours requiring a total of 12 seeded wells. Control siRNA wasobtained from the siRNA Reagent System (Santa Cruz Biotech-nology) and cells were transfected according to the manufac-turer's protocol (Santa Cruz Biotechnology). For DDR2 siRNAthe same protocol was followed using siRNAs s9761 and s9762from Santa Cruz Biotechnology. siRNA efficiency was deter-mined by Western blot analysis as below or real-time PCR. Forreal-time PCR total cellular RNA was prepared from the cells byusing an RNeasy Mini Kit (Qiagen) and 1.0 mg of the RNA wasthen reverse transcribed to cDNA using TaqMan Reverse Tran-scription Reagents (Life Technologies). Mouse GAPDH was usedfor normalization of input cDNA. The human NSCLC cell linesNCI-H2286 and HCC-366 were obtained from the ATCC in2009 and not further authenticated.

Western blottingSnap-frozen tissues or cells cultured in 6-well plates were

homogenized with RIPA buffer containing phosphatase andprotease inhibitors (ThermoFisher Scientific), lysateswere clearedby centrifugation, protein concentration was determined usingthe Bradford reagent (Bio-Rad), and 100 mg of lysate was loadedper sample. Immunoblots were then performed using theNupageSystem (Invitrogen) per the manufacturer's instructions. Primaryantibodies used were NMYC (Santa Cruz Biotechnology), DDR2(Bethyl Laboratories), b-actin (Sigma), and Vinculin (Sigma).Secondary antibodies used were horseradish peroxidase mouseand rabbit (Pierce) and ECL prime (Thermo Fisher Scientific) wasused for protein detection.

Xenografts and in vivo treatmentNu/Nu mice were purchased from Charles River Laboratories

International Inc. 3941-DDR2 was detected as pathogen free inCharles River Laboratories International Inc. and cultured inRPMI-1640 with 10% FBS. The cells were washed with serum-free medium and resuspended in serum-free medium mixedwith an equal amount of Matrigel (BD Biosciences). Mice wereinjected with 1 million cells per tumor and three locations permouse. The mice were randomly grouped and treatment wasstarted the second day after inoculation. Each cohort included5mice.Dasatinibwas dissolved inHKI solution and administeredorally at 50mg/kg daily. HKI solutionwas prepared as same as theprevious publication (22). JQ1 was formulated in 10% DMSO,90% 10%-hyroxypropyl beta cyclodextrin and given intraperito-neal route at 50mg/kg daily. Both oral and intraperitoneal vehiclewere administered to the control group in the same way as thetreatment mice. Tumor sizes were monitored twice weekly andvolumes were calculated with the formula: (mm3) ¼ length �width � width � 0.5.

Statistical analysisStatistical analysis was performed with GraphPad Prism

software. Proliferation assays are represented as the mean �SD. In vivo experiments are represented as the mean � SEM.Significance of in vivo treatment was assessed by two-wayANOVA and Bonferonni's multicomparison test. Survival anal-ysis of DDR2wt;p53 and DDR2L63V;p53 mice was assessed bythe log-rank test.

ResultsDDR2L63V expression is oncogenic in the lung

Given that somatic DDR2 mutants have been observed inhuman NSCLC and shown to be potential therapeutic targets incellular studies, we sought to generate a mouse model of lungcancer driven bymutatedDDR2 to examinewhether expression ofDDR2 mutants could initiate lung tumorigenesis in vivo. Toaddress this question, we selected the L63V and I638F mutationsfor characterization, given that they appeared to be the mostoncogenic in NIH-3T3 assays reported previously (11). TheseDDR2 mutations were introduced in the human DDR2 gene bysite-directed mutagenesis and cloned into pBS31 flp-in vectorfollowed by a tetO minimal CMV promoter and an ATG and anFRT site (21).DDR2wt,DDR2L63V, andDDR2I638F transgenic micewere then generated by injection of the construct into FVB/Nfertilized eggs. Progeny were genotyped by PCR. Founders werecrossed with CCSP-rtTA as well as conditional Trp53-deficientallele (p53L/L) mice to accelerate tumor formation as no tumorswere observed in the DDR2 transgenics alone with 2 years ofobservation. The complexmodel tet-op-DDR2wt;CCSP-rtTA;p53L/L

(DDR2wt;p53 hereafter), tet-op-DDR2L63V;CCSP-rtTA;p53L/L

(DDR2L63V;p53 hereafter), and tet-op-DDR2I638F;CCSP-rtTA;p53L/L (DDR2I638F;p53 hereafter) were confirmed by genotypingand expanded for subsequent analyses and experiments (Fig. 1A).DDR2wt;p53, DDR2L63V;p53, and DDR2I638F;p53 mice wereplaced on a continuous doxycycline diet at 6 weeks of age toinduce DDR2 expression, and Adenovirus-Cre intranasallyinstilled to delete Trp53 on the same day. Lungs of induced micewere visualized by serial MRI every 8 weeks and mice were alsosacrificed every 8 weeks for histologic examination.

Expression ofDDR2was targeted to lung type II alveolar cells bycrossing mice carrying club (originally known as Clara) cell

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secretory protein (CCSP)–regulated reverse tetracycline transac-tivator (rtTA) transgene, regulated by tetracycline (tet)-respon-sive elements, which allowed for DDR2 expression in lungpneumocytes after doxycycline administration. The median sur-vival of DDR2L63V;p53 mice was 67.5 weeks (Fig. 1B) and therewas no mortality observed in the DDR2wt;p53 or DDR2I638F;p53 models at 2 years of observation. After 20 to 30 weeks ofdoxycycline treatment, histologic examination of DDR2L63V;p53 mice demonstrated the development of focal or diffusebronchioloalveolar carcinoma (BAC), in some cases precededby precancerous adenomatous lesions in the airway epithelia(Fig. 1C). After 30 to 40 weeks on doxycycline, the DDR2L63V;p53 mice developed small intrabronchial carcinomas in thedistal (Fig. 1C) and proximal (Fig. 1C) bronchioloalveolarlocations. After 50 to 60 weeks, all of the tested DDR2L63V;p53 mice developed lung adenocarcinoma (Fig. 1C). In agree-ment with the pathologic observations, MRI images also showedgradual tumor development of DDR2L63V;p53 mice after 40weeks of doxycycline induction (Supplementary Fig. S1A). TheDDR2wt;p53 and DDR2I638F;p53 models only showed epithe-lial hyperplasia (Supplementary Fig. S1B); none of tested micedeveloped malignant tumors within 2 years. Both immunohis-tochemical (IHC) stains and Western blotting confirmed DDR2expression in tumor nodules (Fig. 1D and Supplementary Fig. S1C and S1D) and lack of p53 (Supplementary Fig. S2A). Themajority of DDR2L63V tumors showed marked SPC staining,implying a type II pneumocyte origin, as expected, given the useof the CCSP promoter. Interestingly, CCSP staining was nearlynegative (Supplementary Fig. S2B). One possibility is that these

tumors are of club cell origin, which is then followed by altereddifferentiation leading to loss of the CCSP expression marker.The same phenomenon was observed previously for the tet-op-EGFR-T790M-L858R;CCSP-rtTA mice (22).

NSCLC driven by DDR2L63V mutant is morphologicallyadenocarcinoma but with p63 and SOX2 expression

Because somaticDDR2mutations aremore frequently found inhuman SCC, we explored whether the DDR2L63V model wegenerated displayed any markers of lung SCC. p63 is a well-knownmarker of squamous differentiation andoverexpression ofthis gene has been consistently identified in lung SCCs by globalgene-expression profiling or by IHC (26, 27). Several othermarkers are widely used in the subclassification of lung carcino-mas, including CK5/6 and TTF1 (26–28). Most human SCC casesdisplay a p63þ, TTF1� immunophenotype, whereas most ade-nocarcinoma cases show the opposite expression pattern: TTF1þ,p63�. SOX2 is a transcription factor reported as an important genein SCC and is also used as histologic marker of human SCC (29,30). As controls for this analysis, we used LSL-KrasG12D;p53L/L

(Kras;p53 hereafter) genetically engineered mice that developtypical adenocarcinoma and mice with biallelic inactivation ofLkb1 and Pten, which leads to SCC (Fig. 2; ref. 23). Interestingly,DDR2L63V tumors displayed typical adenocarcinoma morphol-ogy with strong TTF1 expression but also displayed some p63 andSOX2 staining (Fig. 2). Thismixed phenotype is not unique to thetumors induced by DDR2L63V and has been previously reportedwith inducible deletion of the tumor-suppressor Lkb1 along withKrasG12D mutant (5).

Figure 1.Generation and characterization of thegenetically engineered mice withinducible expression of DDR2L63V andTP53 deletion in the lung. A, schema oftheDDR2 construct, cross strategy, andinduction for the conditional transgenicmice. B, Kaplan–Meier survival analysisof DDR2;p53mice following intra-nasalAd-Cre instillation and doxycyclineadministration. All deathwas caused bytumor burden. Median survival ofDDR2L63V;p53 ¼ 67.5 weeks. C,representative H&E-stained sectionsderived from tumors arising in theDDR2L63V;p53 mouse model atdifferent time points after doxycyclineand Ad-Cre administration. D,immunohistochemical staining ofDDR2on tumor nodules driven byDDR2L63V;p53 and EGFRT 790M-L858R; scalebars, 100 mm for all panels.






MYCN is elevated in DDR2L63V tumorsGiven the long latency of the DDR2L63V model, we generated

two tumor-derived cell lines (3941 and 3942) to study signalingand therapeutics in more detail. Both cell lines demonstratedexpression ofDDR2 thatwas augmented by culture in doxycycline,as would be expected (Fig. 3A). The cell line 855 displayed in thisfigure is one of several previously described cell lines generatedfrom murine Kras;p53 tumors (24). Interestingly, both the 3941and 3942 cell lines proliferatedwell in culture in the presence of orabsence of doxycycline with no discernible difference in pheno-type, suggesting that they were not dependent on ongoing DDR2expression and raising the possibility that additional genomicalterations may be contributing to transformation in the DDR2-mutatedmousemodel. To support this observation, weperformedsiRNA–mediated knockdown of DDR2 in the 3941 and 3942 celllines. Although the degree of knockdown in these lines was onlyapproximately50%asmeasuredby real-timePCR (SupplementaryFig. S3A), there was no effect seen on the proliferation of these celllines or of theKras;p53 cell line858,whichwas selectedon thebasisof transfectibility (Supplementary Fig. S3B).

To address the possibility that other genetic alterations may beplaying an important role in the tumors from the DDR2L63V;p53–mutated mice, we performed microarray analyses to com-pare the gene-expression profiles of these two tumor-derived celllines from the DDR2L63V;p53–mutated mice to lines from threemouse Kras;p53 tumors (24). This was performed after compar-ative immunoblot analysis of these cell lines did not show anydifferential phosphorylation of RAS/MAPK or PI3K effectors ascompared with the Kras-mutated cell lines (data not shown). Ofnote, the cell lines studied were derived from tumors that devel-oped typical adenocarcinoma on pathology, and p53 was condi-tionally deleted at the same ages. Differential gene-expressionanalysis using the Genepattern module identified 541 genesdifferentially upregulated in the DDR2L63V tumors as comparedwith the Kras tumors using a fold-change cutoff of 2.0 and acorrected P value of less than 0.001 (Supplementary Data File). Arepresentative heatmap of the top differentially expressed genes isshown in Fig. 3B. Manual inspection of these top differentiallyexpressed genes focused our attention onMYCN, amember of themyc family of proto-oncogenes. The MYC gene is expressed in awide variety of tissues, whereas MYCN expression is restricted toearly stages of embryonic development, amplification ofMYCN isfrequently found in a number of advanced-stage tumors, includ-

ing lung cancer, contributing to a myriad of phenotypes associ-ated with growth, invasion, and drug resistance (31).

In themicroarray data,MYCN expressionwas 37-fold higher inthe DDR2L63V tumor-derived cell lines, and immunoblottingdemonstrated elevated MYCN protein levels in 3941 and 3942cell lines as compared with Kras lines 634, 855, and 858 (Fig. 3C).Analysis of gene-expression data from The Cancer Genome Atlas(TCGA) demonstrated a trend toward higher expression ofMYCNin DDR2-mutated lung SCCs (mean RSEM 176 vs. 127), but thisdifference was not statistically significant. DDR2 and MYCNexpression levels were weakly correlated across the TCGA cohort(Pearson correlation 0.22). We did not note any upregulation ofMYCN in two previously described NSCLC cell lines with DDR2mutations (NCI-H2286 and HCC-366) as compared with otherNSCLC cell lines described in the Cancer Cell Line Encylopedia.

Given increased MYCN expression in the DDR2-mutated lines(Fig. 3C),we assessedwhetherMYCN losswould have an effect ontheir proliferation. siRNA was used to knockdown MYCN (Fig.3D) and cellular viability was counted after 24 and 48 hours. Nosubstantial difference was observed in the 858 Kras–mutated cellline withMYCN depletion. However, we observed a reduction inproliferation of the 3941 DDR2-mutated cell line with MYCNdepletion (Fig. 3E), indicating partial dependence on MYCN inDDR2L63V mutated cell lines.

NSCLC driven by DDR2L63V is sensitive to dasatinib and JQ1combination therapy

We next probed the 3941 and 3942 cell lines for sensitivity totwo previously reported nonselective DDR2 inhibitors, dasatinibandponatinib, and compared the sensitivitywith three previouslygenerated Kras;p53 cell lines (634, 855, and 858). We did notobserve any differences in the sensitivity of these cell lines to thesetwo chemical inhibitors (dasatinib: 3941 and 3942 DDR2 IC50

1.96 and 0.682 mmol/L, respectively, as compared with Kras 634,855, 858 with IC50 0.484, 1.76, and 0.557 mmol/L, respectively.Ponatinib: 3941 and 3942 DDR2 IC50 0.579mmol/L and0.978mmol/L, respectively, as compared with Kras 634, 855, 858IC50 2.26, 2.31, and 2.57 mmol/L, respectively; SupplementaryFig. S4). These two cell lines were also probed for sensitivity to apreviously reported SRC inhibitor sarcatinib, given studies linkingSRC and DDR2 activity and no differences in sensitivity wereobserved (saracatinib: 3941 and 3942 DDR2 IC50 2.69 and 5.49mmol/L, respectively, as compared with Kras 634, 855, 858 with

Figure 2.NSCLC driven by DDR2L63V displays amixed phenotype. Immunohistochemicalstaining of tumor nodules of DDR2L63V;p53mice, the SCC canonical markers p63and SOX2, and the adenocarcinomacanonical marker TTF1 were used todistinguish the tumor types; scale bars,100 mm for all panels.

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IC50 4.69, 11.67, and 8.86 mmol/L, respectively; SupplementaryFig. S4). To control for any intrinsic drug resistance of the 3941 or3942 cell lines as compared with the Kras-mutated lines, wecompared their sensitivity with etoposide and noted no inherentchemoresistance in the DDR2-mutated lines (3941 and 3942DDR2 IC50 0.840 and 0.439 mmol/L, respectively, as comparedwith Kras 634, 855, 858 with IC50 2.49, 1.53, and 1.00 mmol/L,respectively; Supplementary Fig. S4).

Given evidence that MYCN knockdown in DDR2-mutatedcell lines decreased cellular proliferation, we reasoned thatcombined inhibition of DDR2 and NYMC might further sup-press proliferation of these models. To this end, we assessed theimpact of the compound JQ1, a prototype bromodomain andextraterminal bromodomain inhibitor that has been previouslyshown to suppress MYC activity (32, 33), in the 3941 and858 cell lines. We observed enhanced sensitivity of 3941 toJQ1 alone (3941 DDR2 IC50 of 160 nmol/L as compared withKras 858 IC50 of 761 nmol/L) and to the combination of JQ1and dasatinib as compared with 858 (3941 DDR2 IC50 of1.0 nmol/L as compared with Kras 858 IC50 of 10.8 nmol/L; Fig. 4A). To assess this combination treatment in a morephysiologic setting, we performed a xenograft study where Nu/Nu mice were injected with 3941 DDR2 cells and treated withvehicle, dasatinib, JQ1, or dasatinib plus JQ1 combination.Tumor formation was monitored twice weekly for 3 continuousweeks. Consistent with our previous observation (11), dasati-nib showed antitumor efficacy on the DDR2L63Vmouse modelas compared with vehicle control. JQ1 also showed antitumorefficacy; however, the combination treatment of JQ1 and dasa-tinib displayed the most potent effect with growth inhibitionof all tested tumors (Fig. 4B and Cand Supplementary Fig. S5,P < 0.001; individual tumor responses are shown in Supple-mentary Fig. S5). Although they were not as sensitive as themurine lines to JQ1 alone, JQ1 potentiated the effects ofdasatinib in the DDR2-mutated NSCLC cell lines NCI-H2286and HCC366 with IC50 values of 10 and 14 nmol/L, respec-tively, for dasatinib with 1 mmol/L JQ1, a one-log reductionfrom their typical dasatinib IC50 (Supplementary Fig. S6).

DiscussionHere, we have presented a novel genetically engineered

mouse model of lung cancer driven by L63Vmutation inDDR2.These studies were motivated by prior work suggesting thatDDR2 mutations may be oncogenic and confer sensitivity toFDA-approved tyrosine kinase inhibitors, including dasatinib,imatinib, nilotinib, and ponatinib. Because all of the priorwork was completed in lung cancer cell lines or other cellularmodels, we sought to explore whether mutated DDR2 coulddrive lung cancer formation in an organism and observed aphenotype of lung adenocarcinoma with a relatively longlatency. The adenocarcinoma phenotype was not surprising,given our use of the CCSP promoter to drive expression ofmutated DDR2 in cell types known to be precursors for lungadenocarcinoma as well as prior reports of DDR2 mutations inlung adenocarcinoma (34). The recent publication of severalmodels of mouse lung SCCs also suggests that an inflammatoryinsult and/or a genetic lesion–driving squamous metasplasia isrequired for SCC formation (23, 35), neither of which waspresent in this model.

One of the challenges in advancing DDR2 mutations as apotential therapeutic target in lung cancer has been the lack ofrecurrent pointmutations in the gene aswell as the infrequency ofDDR2mutations overall. Studies in cell lines have suggested thatonly a subset of patient-derived mutations confer gain-of-func-tion phenotypes, and the ability to discern "driver" from "pas-senger" events in DDR2 has not been well established in anexperimental system with conflicting reports in some cases, suchasDDR2 I638F,whichwas not oncogenic in ourmousemodel norin prior proteomic studies, but was transforming in some cellularsystems (11, 14).Our results, therefore, call intoquestionwhetherI638F is really a gain-of-function mutation or is operating via adifferent mechanism as compared with L63V. Interestingly, twocases of response to dasatinib in patients with DDR2 S768Rmutations have been reported, suggesting that at least somepatient-derived mutations are likely to be therapeutic targets,though dasatinib therapy has been associated with toxicity in lungcancer patients (36). In this work, we established a single point

Figure 3.MYCN is elevated in DDR2L63V tumors. A,immunoblot analysis of DDR2 levels inuninduced and doxycycline (DOX)-induced celllines (3941 and 3942) from DDR2 transgenictumors. The 855 linewas previously derived froma murine lung adenocarcinoma from a Kras;p53mouse. B, microarray expression profiling ofmouseDDR2L63V;p53 (3941 and 3942) andKras;p53 cell lines (634, 855, and 858). Heatmapdepicts elevated expression of selected genes inDDR2L63V;p53 lines. C, immunoblot analysisof NMYC in DDR2L63V;p53 cell lines versusKras;p53 cell lines. D, immunoblot analysisof NMYC knockdown (þ, siRNA knockdown;�, nontargeting siRNA). E, proliferation assay ofa Kras;p53 cell line (858) compared with theDDR2L63V;p53 cell line (3941) 24 and 48 hoursafter siRNA mediated NMYC knockdown.Control, a nontargeting siRNA control.






mutation (L63V) ofDDR2 as oncogenic in vivo, and the generationof additional cellular and animal models will be necessary todiscern which DDR2 mutations are truly oncogenic, given that itis likely that manyDDR2mutations will be passenger events giventhehighbackgroundmutation rate of lung cancers. Thisworkdoes,however, serve as a proof-of-concept of the oncogenicity of mutat-ed and not wild-type DDR2 in a mouse model.

Although we observed lung cancer formation at a high pene-trance in theDDR2 L63Vmodel, the latency was long and the factthat we were able to grow tumor-derived cell lines in the absenceof doxycycline induction suggests that, although DDR2 mutantscan play a role in lung cancer initiation, there are likely to be otherimportant cooperating oncogenic alterations that facilitate ongo-ing proliferation of DDR2-mutated tumors. This idea is in agree-mentwith human cancer-derived cell line studies that suggest thata number of oncogenic pathways are activated in DDR2-mutatedcell lines and can substitute for DDR2 in the context of DDR2inhibition by small-molecule inhibitors. Although these studieshave largely focused on compensatory RTK pathways, our obser-vation that MYCN can play a role in DDR2-mutated tumors alsoargues that, although DDR2 mutations can facilitate tumor for-mation, specific and selective inhibition of DDR2 may not besufficient to eradicate DDR2-driven cancers. It is likely that addi-tional genomic alterations beyond overexpression of MYCN arerelevant in DDR2-mutated lung cancers as the TCGA datasetsshow overlap with KRAS mutation and NKX2-1 amplification inthe limited number of samples with DDR2 mutation. Limitedsequence analysis of the DDR2-mutated nodules in this report

also indicated that other mutations are present along with DDR2and include mutations in Kras (codon 61), Fgfr2, Irs2, Csf1r, andDaxx, suggesting that DDR2mutations are not sufficient alone todrive tumorigenesis and require cooperating events.

In conclusion, our work extends prior cellular studies that havedemonstrated that DDR2 mutations are found in NSCLCs, andthat a subset of these mutations are oncogenic both in culturesystems and in the context of a transgenic mouse model. Ourmodel can facilitate additional studies of the role of mutatedDDR2 in lung cancer as well as serve as a platform for therapeuticstudies aimed at targeting DDR2.

Disclosure of Potential Conflicts of InterestM. Meyerson reports receiving a commercial research grant from Bayer,

has ownership interest (including patents) in Foundation Medicine andLabCorp, and is a consultant/advisory board member for FoundationMedicine. P.S. Hammerman is a consultant/advisory board member forMolecular MD. No potential conflicts of interest were disclosed by the otherauthors.

Authors' ContributionsConception and design: C. Xu, M. Meyerson, K.-k. Wong, P.S. HammermanDevelopment of methodology: C. Xu, K.-k. Wong, P.S. HammermanAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): C. Xu, Y. Zhang, H. Asahina, E.M. Beauchamp,K.-k. Wong, P.S. HammermanAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): C. Xu, K.A. Buczkowski, E.M. Beauchamp, Y.Y. Li,K.-k. Wong, P.S. Hammerman

Figure 4.NSCLC driven by DDR2L63V is sensitive to dasatinib and JQ1 combination therapy. A, proliferation of Kras;p53 cell lines 858 and DDR2L63V;p53 cell line 3941 grownfor 5 days in the presence of varying concentrations of dasatinib, JQ1, and a combination of 1 mmol/L JQ1 and varying dasatinib concentrations. B and C,tumor volumemeasurement in a xenograft study of the DDR2L63V;p53 (3941) lung cancer cell line. Nu/Numice were treated with dasatinib, JQ1, and dasatinib plusJQ1 combination for 3 weeks following tumor formation. B, average tumor volumes. C, harvested tumor nodule sizes.

Xu et al.





Writing, review, and/or revision of the manuscript: C. Xu, K.A. Buczkowski,H. Asahina, K.-k. Wong, P.S. HammermanAdministrative, technical, ormaterial support (i.e., reportingororganizingdata,constructing databases): C. Xu, K.A. Buczkowski, H. Terai, P.S. HammermanStudy supervision: P.S. Hammerman

Grant SupportThis work was supported in part by United against Lung Cancer and Susan

Spooner Research Fund (to K.-K. Wong); NIH grants NCI P01 CA154303

(to K.-K. Wong and M. Meyerson); and NCI K08 CA163677 (to P.S.Hammerman).

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

Received January 28, 2015; revised May 29, 2015; accepted June 24, 2015;published OnlineFirst July 23, 2015.

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2015;14:2382-2389. Published OnlineFirst July 23, 2015.Mol Cancer Ther Chunxiao Xu, Kevin A. Buczkowski, Yanxi Zhang, et al. Combination Therapy

Mutation Is Sensitive to Dasatinib and JQ1DDR2NSCLC Driven by

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