ANovelALKSecondary Mutation andEGFRSignalingCause ...Therapeutics, Targets, and Chemical Biology ANovelALKSecondary Mutation andEGFRSignalingCause Resistance to ALK Kinase Inhibitors

Therapeutics, Targets, and Chemical Biology

A Novel ALK Secondary Mutation and EGFR Signaling CauseResistance to ALK Kinase Inhibitors

Takaaki Sasaki1,4, Jussi Koivunen1,4, Atsuko Ogino1,4, Masahiko Yanagita1,4, Sarah Nikiforow1,4,6,Wei Zheng2,3, Christopher Lathan1,4,6, J. Paul Marcoux1,4,6, Jinyan Du7, Katsuhiro Okuda1,4, Marzia Capelletti1,4,Takeshi Shimamura1,4,6, Dalia Ercan1,4, Magda Stumpfova1,4, Yun Xiao5, Stanislawa Weremowicz5, Mohit Butaney1,4,Stephanie Heon1,4,6, Keith Wilner8, James G. Christensen8, Michel J. Eck2,3, Kwok-Kin Wong1,4,6, Neal Lindeman5,Nathanael S. Gray2,3, Scott J. Rodig5, and Pasi A. J€anne1,4,6

AbstractAnaplastic lymphoma kinase (ALK) tyrosine kinase inhibitors (TKI), including crizotinib, are effective

treatments in preclinical models and in cancer patients with ALK-translocated cancers. However, their efficacywill ultimately be limited by the development of acquired drug resistance. Here we report two mechanisms ofALK TKI resistance identified from a crizotinib-treated non–small cell lung cancer (NSCLC) patient and in a cellline generated from the resistant tumor (DFCI076) as well as from studying a resistant version of the ALK TKI(TAE684)–sensitive H3122 cell line. The crizotinib-resistant DFCI076 cell line harbored a unique L1152R ALKsecondary mutation and was also resistant to the structurally unrelated ALK TKI TAE684. Although the DFCI076cell line was still partially dependent on ALK for survival, it also contained concurrent coactivation of epidermalgrowth factor receptor (EGFR) signaling. In contrast, the TAE684-resistant (TR3) H3122 cell line did not containan ALK secondary mutation but instead harbored coactivation of EGFR signaling. Dual inhibition of both ALKand EGFR was the most effective therapeutic strategy for the DFCI076 and H3122 TR3 cell lines. We furtheridentified a subset (3/50; 6%) of treatment naive NSCLC patients with ALK rearrangements that also hadconcurrent EGFR activating mutations. Our studies identify resistance mechanisms to ALK TKIs mediated byboth ALK and by a bypass signaling pathway mediated by EGFR. These mechanisms can occur independently, orin the same cancer, suggesting that the combination of both ALK and EGFR inhibitors may represent an effectivetherapy for these subsets of NSCLC patients. Cancer Res; 71(18); 6051–60. �2011 AACR.

Introduction

The emerging impact of targeted therapies as cancer treat-ments has led to a therapeutic paradigm shift in the field ofoncology. Several kinase inhibitors have been identified aseffective clinical therapies for a broad range of cancers and,specifically, in those in which the target of the kinase inhibitorhas undergone a gain of function genomic alteration (1, 2).However, the clinical success of treatment with kinase inhi-

bitors is uniformly limited by the development of acquireddrug resistance. Two common mechanisms of acquired drugresistance have been identified. These include secondarymutations in the target of the kinase itself, which abrogatethe inhibitory activity of the drug, and activation of alternativesignaling pathways that bypass the continued requirement forinhibition of the original target (3–5). The understanding ofthe mechanistic bases for drug resistance will continue toinform the development of strategies to overcome or preventclinical drug resistance, thereby providing a greater therapeu-tic benefit for cancer patients (6, 7).

Chromosomal rearrangements in the anaplastic lymphomakinase (ALK) gene have been detected in anaplastic large celllymphoma (ALCL), inflammatory myofibroblastic tumor(IMT), and in non–small cell lung cancer (NSCLC; refs. 8–10). In NSCLC, ALK rearrangements have been detected in 3%to 13% of patients, are more common in never-smokers and inthose with adenocarcinoma (11). In addition, they are oftenmutually exclusive with other oncogenic alterations detectedin NSCLC, including epidermal growth factor receptor (EGFR)mutations. ALK kinase inhibitors are effective therapies inboth preclinical in vitro and in vivo models and in NSCLCpatients harboring ALK rearrangements (2, 12, 13). In thephase I clinical trial of crizotinib, a radiographic tumor

Authors' Affiliations: Departments of 1Medical Oncology, 2Cancer Biol-ogy, 3Biological Chemistry and Molecular Pharmacology, 4Lowe Centerfor Thoracic Oncology, Dana-Farber Cancer Institute; Departments of5Pathology and 6Medicine, Brigham and Women's Hospital, Boston;7The Broad Institute, Cambridge, Massachusetts; and 8Pfizer GlobalResearch and Development, Department of Research Pharmacology,La Jolla, California

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

Corresponding Author: Pasi A. J€anne, Lowe Center for ThoracicOncology, Dana-Farber Cancer Institute, D820, 450 Brookline Avenue,Boston, MA 02215. Phone: 617-632-6076; Fax: 617-582-7683; E-mail:[email protected]

doi: 10.1158/0008-5472.CAN-11-1340

�2011 American Association for Cancer Research.

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response rate of 55% was observed in ALK rearranged NSCLCpatients (2). This agent is currently in phase III clinicaldevelopment in this genomically defined patient population.

Recent studies have also identified and studied crizotinibresistance mechanisms. To date 3 secondary mutations, allidentified from crizotinib-treated NSCLC or IMT patients,have been reported (14, 15). These mutations either involvethe "gatekeeper" residue (L1196) or sites away from crizotinibbinding (F1174L and C1156Y; refs. 14, 15). The mechanisticbasis for how the different mutations lead to crizotinibresistance is not fully understood. The L1196 mutation maycreate a steric hindrance for crizotinib binding, whereas theF1174L mutation likely promotes the active conformation ofALK, thus disfavoring crizotinib binding which preferentiallybinds the inactive conformation of ALK (14). Continuedstudies of these and other resistance mechanisms will becritical to the design of subsequent treatments for NSCLCpatients with ALK rearrangements.

In this study, using cell line models of ALK inhibitorresistance, either derived from a crizotinib-resistant patientor generated in vitro, we uncover additional mechanisms ofALK kinase inhibitor resistance. Our findings underscore thecomplexity of drug resistance mechanisms and the therapeu-tic challenges of treating multiple concurrent resistancemechanisms.

Materials and Methods

PatientsPatients were either identified from the Thoracic Oncology

Program at DFCI (n ¼ 3) or were treated in a clinical trial(NCT00932893) with crizotinib that was sponsored by Pfizer,Inc (n ¼ 1). Tumor biopsies were obtained under an Institu-tional Review Board approved protocol. All patients providedwritten informed consent.

ALK and EGFR genomic analysesThe ALK kinase domain was sequenced from all of the

available specimens. The PCR primers and conditions areavailable upon request. ALK FISH was carried out using thebreak apart probe (Vysis LSI ALK Dual Color; Abbott Molec-ular) as previously described (14, 16). EGFR mutation detec-tion was done in a CLIA certified laboratory using previouslydescribed methods (17).

Cell lines and expression constructsThe NSCLC cell lines H3122 (EML4-ALK variant 1 E13;A20)

and DFCI-032 (EML4-ALK variant 1 E13:A20), A549, HCC827(EGFR del E746_A750) have been previously published (13).The H3122 cells were obtained from the NIH and confirmed byfingerprinting using the Power Plex 1.2 system (Promega) inOctober 2010. The DFCI076 (EML4-ALK variant 3 (E6;A20) cellwas established at Dana-Farber Cancer Institute from pleuraleffusion from a patient who had developed acquired resis-tance to crizotinib. The DFCI076 cells were cultured in RPMI1640 (GIBCO) supplemented with 10% FBS, 100 units/mLpenicillin, 100 mg/mL streptomycin, and 1 mmol/L sodiumpyruvate (RPMI 10% medium).

The EML4-ALK (Variant 1) cDNA from the H3122 cell lineand the EGFR-del (EGFR delE746_A750) cDNA were clonedinto pDNR-Dual (BD Biosciences) as described previously (14).To generate EML4-ALK mutants, L1152R, L1196M, C1156Y, orF1174L mutations were introduced using site-directed muta-genesis (Agilent) with mutant specific primers according tothe manufacturer's instructions and as previously described(14). All constructs were confirmed by DNA sequencing.Retroviral infection and culture of Ba/F3 cell were done usingpreviously described methods (18). Polyclonal cell lines wereestablished by puromycin selection and subsequently culturedin the absence of interleukin-3 (IL-3). Uninfected Ba/F3 cellsor cell lines expressing green fluorescent protein (GFP) wereused as controls

Cell proliferation and growth assaysCrizotinib and the pan-ERBB inhibitor PF299804 were

provided by Pfizer. TAE684 and BMS-536,924 were synthesizedas previously described (19, 20). Recombinant human EGF(PHG0314) was purchased from Invitrogen. Growth andinhibition of growth was assessed by MTS assay accordingto previously established methods (18). All experimentalpoints were set up in 6 to 12 wells and all experiments wererepeated at least 3 times. For clonogenic assays, cells wereplated in triplicate on the 6-well plates and subject to drugexposure for 14 days, the colonies were fixed and stained with0.5% crystal violet in 25% methanol, and the numbers ofcolonies were counted.

ALK and EGFR shRNA constructs and lentiviralinfection

ALK and EGFR short hairpin RNA (shRNA) constructscloned into the pLKO.1 puro vector as previously described(21). A vector containing a nontargeting (NT) shRNA and GFPshRNAwas used as a control. Lentivirus production, titrations,and infections were done as in ref. 21. The specific shRNAsequences are available upon request.

Generation of in vitro drug-resistant H3122 cellsTo generate drug-resistant cells, the H3122 cells were ex-

posed to increasing concentrations of TAE684, similar to ourpreviously described methods (3, 21). TAE684 concentrationswere increased stepwise from 1 to 100 nmol/L when the cellsresumedgrowthkinetics, similar to untreatedparental cells. Toconfirm the emergence of a resistant clone, MTS assays weredone following growth at each concentration. The in vitrodrug-resistant cells were subcloned by single-cell isolation.

Antibodies and Western blottingCell lysis, Western blotting, and immunoblotting was done

as previously described (18). Anti-ALK (DF53), anti–phospho-EGFR, anti–total-EGFR, anti-EGFR L858R, anti-EGFRdelE746_A750, anti-PARP, anti–a�tubulin, anti–phospho-Akt (Ser-473), and anti–total-Akt, were obtained from CellSignaling Technology. Total ERK1/2 and phospho-ERK1/2(pT185/pY187) antibodies were from Invitrogen. Immunopre-cipitations were done using anti–Flag-M2 agarose (Sigma-Aldrich Co.). The receptor tyrosine kinase (RTK) array was

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Cancer Res; 71(18) September 15, 2011 Cancer Research6052




purchased from R&D Systems and used according to themanufacturer's recommended conditions. ALK immunohis-tochemistry (IHC) was done using the mouse monoclonalanti-human ALK (clone: 5A4; Abcam) as previously described(16). EGFR IHC was done as previously described (22).

Phosphotyrosine profilingPhosphotyrosine profiling was done using a bead based

assay using previously described methods (23). Parental anddrug-resistant cells were treated with either vehicle or drug for6 hours followed by cell lysis prior to analysis.

Detection of EGFR ligandsEGF, amphiregulin, TGF-a, HB-EGF, and epiregulin were

measured in cell culture medium using an ELISA, were doneaccording to the manufacturer's recommended procedures(Quantikine; R&D Systems) and as described previously (24).All samples were run in triplicate. Color intensity wasmeasured at 450 nm using a spectrophotometric plate reader.Growth factor concentrations were determined by compari-son with standard curves.

Cell-cycle analysis and apoptosis assaysA total of 1� 106 cells treated with each of the compound at

indicated concentration or dimethyl sulfoxide were washed inPBS, fixed in 70% ethanol, incubated with RNAse in PBS, andresuspended in 500 mL propidium iodide. The percentage ofcells in G1, S-phase, and G2-M phases were analyzed by flowcytometry. Apoptosis was analyzed by terminal deoxynucleo-tidyl transferase–mediated dUTP nick end labeling (TUNEL)assay using the APO-BRDU Kit (BD Biosciences) according tothe manufacturer's instructions.

Results

The novel somatic ALK L1152R mutation results in drugresistance to ALK inhibitors

To identify additional mechanisms of resistance to crizo-tinib, we first studied a NSCLC patient with an ALK rear-rangement [EML4-ALK variant 3 (E6;A20)] who had developedclinical acquired resistance to crizotinib, following a briefradiographic response after 3 months of treatment. Sequenc-ing of the ALK gene from the clinically progressing tumor

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Figure 1. The ALKmutation L1152R causes the ALK tyrosine kinase inhibitor resistance. A, sequence tracing from posttreatment tumor specimens. There is aT to G mutation in codon 3455 in exon 23 resulting in the L1152R mutation. B, Ba/F3 cells were treated with crizotinib at the indicated concentrations, andviable cells were measured after 72 hours of treatment and plotted relative to untreated controls. There is a significant effect of the L1152R mutation at 300nmol/L (P < 0.001). C, Ba/F3 cells with indicated genotypes were treated with increasing concentrations of crizotinib for 6 hours. Cell extracts wereimmunoprecipitated with an anti-FLAG antibody followed immunoblotting to detect the indicated proteins. D, ribbon diagram depicting the crystal structure ofALK kinase in complex with crizotinib. The side chains of residues that are sites of resistance mutations, including the L1152R mutation described here,are shown in green. Note that L1152 and C1156 are not in contact with the ATP-binding cleft but are adjacent to each other and to the C-helix (pink).Both the L1152R and C1156Y mutations could introduce hydrogen bond interactions with E1161 on the C-helix. All of the resistance mutations identifiedto date cluster around the conformationally sensitive C-helix and activation loop, suggesting that they may affect kinase activity and inhibitor bindingthrough alterations in the structure or stability of these elements. The activation loop (A-loop, colored orange) is partially disordered in this structure asdenoted by the dashed line. Figure is drawn from PDB ID 2XP2.

Resistance Mechanisms in ALK Kinase Inhibitors

www.aacrjournals.org Cancer Res; 71(18) September 15, 2011 6053




(pleural effusion) revealed the presence of a novel mutation(Fig. 1A). This mutation resulted in a change from a leucine toan arginine at position 1152 (L1152R). The recurrent tumorwas wild-type for EGFR and KRAS (data not shown). Thismutation was not detected in the tumor of the patientobtained prior to crizotinib treatment (data not shown).

We evaluated the biological impact of the L1152R mutationby introducing it into EML4-ALK and generating Ba/F3 cells.BothEML4-ALK andEML4-ALKL1152R led to IL-3 independentgrowth of Ba/F3 cells (Supplementary Fig. S1A). The EML4-ALK L1152R cells were significantly more resistant than theparental cells to both crizotinib (Fig. 1B) and ALK inhibitorTAE684 (Supplementary Fig. S1B). The L1152R mutation di-minished crizotinib-mediated inhibition of downstream AKTand ERK 1/2 phosphorylation (Supplementary Fig. S1C). Con-sistentwith these findings on growth, greater concentrations ofcrizotinib were required to inhibit ALK phosphorylation in theEML4-ALK L1152R cells compared with those with EML4-ALKalone (Fig. 1C). Furthermore, to compare the impact of resis-tance in endogenous EML4-ALK NSCLC cells, the L1152R andpreviously identified resistance mutations (C1156Y, L1196M,and F1174L) were stably expressed in H3122 cells and the cellswere examined for crizotinib resistance (14, 15). All of theresistance mutations, C1156Y, L1196M, L1152R, and F1174L,resulted in the significant elevation of IC50 compared with thecontrol cells (GFP or EML4-ALK wild-type), but there were nosignificant difference among the C1156Y, L1196M, and L1152Rmutations (Supplementary Fig. S1D and Table 1). Analogous tothe known resistance mutation C1152Y, examination of thepublished crystal structure of ALK in an inactive conformationreveals that the L1152R mutation is not in direct contact withthe ATP-binding pocket, where both crizotinib and TAE684 areexpected to bind (Fig. 1D, ref. 25). The currently availablestructures do not reveal a clear mechanistic basis as to howL1152R may mediate ALK inhibitor resistance.

A NSCLC cell line harboring the L1152R mutation isALK and EGFR dependent

We successfully established a cell line, DFCI076, from thepleural effusion of the patient harboring the ALK L1152R

mutation. Similar to the Ba/F3 cells harboring the L1152Rmutation, the DFCI076 cells were resistant to both crizotiniband TAE684 (Fig. 2A and Supplementary Fig. S2A). However,these cells were still dependent on ALK for their growth asdownregulation of ALK using an ALK-specific shRNA resultedin significant growth inhibition compared with either a NT- oran EGFR-specific shRNA (Fig. 2B, Supplementary Fig. S2B andC). Similarly, the ALK shRNA, but not the EGFR shRNA, waseffective in the crizotinib and TAE684-sensitive H3122 cell line(Fig. 2B). However, the degree of growth inhibition by the ALKshRNA was not as dramatic in the DFCI076 cells comparedwith the H3122 cells. This prompted us to evaluate whetherthe DFCI076 cells might contain other concurrent resistancemechanisms. We assessed the activation status of multipleRTKs using the human phospho-RTK arrays as in our priorstudy (3). Using this approach, we observed strong EGFR andMET phosphorylation in the DFCI076 cells (Fig. 2C). TheDFCI076 cells did not contain an EGFR mutation or an EGFRamplification (data not shown) but secreted the EGFR ligandamphiregulin (Supplementary Fig. S2D). Although crizotinib isa potent MET inhibitor and successfully inhibited phospho-MET, it does not inhibit EGFR activity and, even at highconcentrations, did not lead to downregulation of pAKT andpERK1/2 to the extent observed in H3122 cells (Fig. 2D).Combined inhibition of ALK (using a shRNA) and EGFR, usingthe pan-ERBB inhibitor PF299804, was significantly moreeffective (P < 0.001) than either strategy alone in the DFCI076cells (Fig. 2B). In addition, the growth curve of DFCI076 cellstreated with both PF299804 and crizotinib was similar to theH3122 cells engineered to express the L1152R mutation andsubjected to crizotinib treatment (Fig. 2A). Collectively, thesefindings suggest that although the DFCI076 cells remainlargely ALK dependent for their growth, concurrent EGFRinhibition may provide additive growth inhibition. Thesefindings are similar to our prior studies of the DFCI032 cellline generated from a NSCLC patient with EML4-ALK who wasnever treated with an ALK inhibitor (13). We further con-firmed that the DFCI032 cells were sensitive to the combina-tion of the ALK shRNA and PF299804 (Supplementary Fig. S2Eand F).

Table 1. Summary of in vitro growth assays

Cell line Vector Crizotinib IC50 (nmol/L) TAE684 IC50 (nmol/L)

Ba/F3 EML4-ALK 68 � 4 3 � 1Ba/F3 EML4-ALK L1152R 853 � 27 66 � 6H3122 GFP 88H3122 EML4-ALK 70H3122 EML4-ALK L1152R 260H3122 EML4-ALK C1156Y 240H3122 EML4-ALK L1196M 240H3122 EML4-ALK F1174L 620DFCI076 1,008 � 37 296 � 29DFCI076 þPF299804 (1 mmol/L) 314 � 37H3122 100 7 � 2H3122 TR3 2,564 � 107 291 � 18

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ALK inhibitor–resistant H3122 cells contain activationof EGFR signalingTo identify additional mechanisms of resistance to ALK

kinase inhibitors, we generated a TAE684-resistant versionof the EML4-ALK H3122 NSCLC cell line. We have used asimilar approach to identify known and previously unknownEGFR kinase inhibitor resistance mechanisms (3, 21). After6 months of gradually increasing TAE684, we were able toisolate cells that proliferated in 100 nmol/L of TAE684. Inour prior studies, we showed that 100 nmol/L of TAE684inhibited ALK signaling and significantly decreased cellviability in H3122 cells, but this concentration was notgenerally toxic in non-ALK rearranged NSCLC cell lines(13). We subcloned the TAE-resistant (TR) cells from singlecells (H3122 TR3) and these cells were resistant to bothTAE684 and crizotinib (Fig. 3A, Supplementary Fig. S3A).DNA fingerprinting confirmed that the H3122 TR3 cells werederived from the H3122 parental cells (data not shown). Wesequenced the entire ALK kinase domain from the H3122TR3 cells and did not detect any secondary ALK mutations(data not shown). To determine whether the H3122 TR3 cellswere still ALK dependent for their growth, we downregu-lated ALK using an ALK-specific shRNA (Fig. 3B). However,unlike the parental H3122 cells, the H3122 TR3 cells wereonly minimally growth inhibited by ALK downregulation(Fig. 3B). We further evaluated the ALK locus using FISH.Although all of the H3122 cells contained the EML4-ALKinversion, this was only detected in a small fraction (5%) ofthe H3122 TR3 (Supplementary Fig. S3B and C). The cellsthat retained the inversion also harbored a concurrentamplification in the ALK locus (Supplementary Fig. S3C).

Together, these findings suggest that the H3122 TR3 cellshave evolved to lose their ALK dependence for growth.

To further characterize the H3122 TR3 cells, we carried outphospho-RTK arrays in both the parental and drug-resistantcells with and without TAE684 treatment (Fig. 3C). Comparedwith the parental cells, the H3122 TR cells contained greaterEGFR, IGF1R, and MET phosphorylation and these proteinsremained persistently phosphorylated despite TAE684 treat-ment (Fig. 3C). We also used a previously described quanti-tative bead based phospho-tyrosine assay to specifically studythese 3 proteins in further detail (23). Consistent with thegenomic findings, ALK phosphorylation was greater in theH3122 compared with the H3122 TR3 cells (Fig. 3D). TAE684still effectively inhibited ALK phosphorylation in both celllines. In contrast and consistent with the RTK array, EGFRphosphorylation was markedly elevated in the H3122 TR3 cells(Fig. 3D). This was inhibited by the EGFR kinase inhibitorgefitinib but not TAE684 (Fig. 3D). We also observed phos-phorylated ERBB2 and IGF1R in H3122 TR3 clone using thisassay (Supplementary Fig. S3D). Of note, the ectopic expres-sion of ALK secondary mutations (Supplementary Fig. S1D)did not lead to an increase in EGFR expression in the H3122cells (data not shown).

Next, we examined whether activated EGFR had a func-tional role in the H3122 TR3 cells. We first downregulatedEGFR using 2 different (A3 and D) EGFR shRNAs. Comparedwith a control shRNA, EGFR knockdown led to significant(P < 0.05) decrease in cell proliferation by day 6 in theH3122 TR3 but not the parental cell line (Fig. 3E). Thisobservation was mirrored in a colony formation assay inwhich treatment with PF299804 resulted in a significant

Figure 2. DFCI076 cells arecodependent on ALK and EGFR.A, the H3122 or DFCI 076 cellswere treated with crizotinib, aloneor concurrently with 1 mmol/LPF299804, at the indicatedconcentrations, and viable cellswere measured after 72 hours oftreatment and plotted relative tountreated controls. B,downregulation of ALK or EGFRusing shRNAs in H3122 andDFCI076 cells. Further growthinhibition is observed with theaddition of 1 mmol/L PF2990804.Cell viability is measured relativeto the NT control shRNA. C, aphospho-RTK array reveals thatthe DFCI076 cells contain strongphosphorylation of both EGFRand MET. Cells were grown inmedium and the cell lysates werehybridized to a phospho-RTKarray. Hybridization signals at thecorners serve as controls.D, H3122 and DFCI076 cells aretreated with indicatedconcentrations of crizotinib for 6hours. Immunoblotting was usedto detect the indicated proteins.

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decrease (P < 0.001) in H3122 TR3 but not H3122 colonies(Fig. 3F) compared with untreated cells. The combination ofthe pan-ERBB inhibitor PF299804 and crizotinib was mosteffective in the H3122 TR3 cells leading to complete inhibi-tion of colony formation. However, the effects of PF299804and crizotinib were mostly cytostatic as judged by onlyminimal changes in cleaved PARP and by using a TUNELassay (Fig. 3G and Supplementary Fig. S3E). There was noeffect on growth using the combination of an IGF1R kinaseinhibitor BMS-536,924 and crizotinib in the H3122 TR3 cells

despite harboring evidence of IGF1R activation (Supplemen-tary Fig. S3F).

We further determined how EGFR was activated in theH3122 TR3 cells. We did not identify evidence of an EGFRmutation or amplification as detected by FISH (data notshown). However, the supernatant of the H3122 TR3 cellscontained significantly (P < 0.05) greater amounts of knownEGFR ligands including amphiregulin andEGF (SupplementaryFig. S3G), suggesting that themechanism of EGFR activation inthese cells is through a ligand-mediated autocrine activation.

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Figure 3. H3122 TR3 cells are ALK inhibitor resistant and contain coactivation of EGFR signaling. A, H3122 and TR3 cells were treated with indicatedconcentrations of TAE684. Viable cells were measured after 72 hours of treatment and plotted relative to untreated controls. B, ALK shRNA has minimal effecton H3122 TR3 cell viability. Cell viability is measured relative to NT control. H3122 and A549 cells serve as positive and negative controls, respectively.Successful ALK knockdown in H3122 TR3 cells is confirmed byWestern blotting. C, summary of RTK array from H3122 and H3122 TR3 cells with and withoutTAE684 (100 nmol/L; 6 hours) treatment. The protein lysates were exposed to the RTK array (R&D). Each spot of membrane were calculated as signal intensityand shown in the bar graph. D, ALK and EGFR phosphorylation in H3122 and H3122 TR3 cells with and without TAE684 and gefitinib treatment.Phosphorylation wasmeasured using a bead based assay (methods). E, proliferation of H3122 TR3 but not H3122 cells is effected following EGFR knockdownusing 2 different shRNAs. F, results of colony formation assay after 14 days treatment with indicated compounds with both H3122 and H3122 TR3. Thecombination of crizotinib (1 mmol/L) and PF299804 (1 mmol/L) effectively inhibited colony formation in H3122 TR3 cells. G, H3122 and H3122 TR3 cells weretreated with indicated compounds for 6 hours, and immunoblotting was used to detect the indicated proteins. For the PARP blot, cells were treated for 24hours.

Sasaki et al.





Activation of EGFR signaling induces resistance tocrizotinibOur studies from the ALK inhibitor–resistant DFCI032,

DFCI 076, and H3122 TR3 cells suggest a role for EGFRsignaling in mediating crizotinib resistance. To formally eval-uate this hypothesis, we activated EGFR signaling in H3122cells using EGFR ligands and by oncogenic forms of EGFR anddetermined the effects on crizotinib sensitivity. We observedthat exogenous EGF was indeed sufficient to promote resis-tance to ALK inhibition, and resistance could be reversedusing a combination of ALK and EGFR inhibitors (Fig. 4A). Inthe presence of EGF, crizotinib was still able to inhibit ALKphosphorylation but not AKT, S6, and ERK 1/2 phosphoryla-tion (Fig. 4B). Similarly, introduction of EGFR E746_A750 intoH3122 cells promoted crizotinib resistance (Fig. 4C) which is

reversed by the concurrent administration of the EGFR in-hibitor gefitinib or PF299804 (Fig. 4D and SupplementaryFig. S4A). Analogously EML4-ALK promotes gefitinib resis-tance in the HCC827 EGFR mutant NSCLC cell line (Supple-mentary Fig. S4B), which was reversed by concurrenttreatment with TAE684 (Supplementary Fig. S4C).

A subset of EML4-ALK NSCLC patients harborconcurrent EGFR mutations

Our in vitro studies suggest that EGFR signaling can con-tribute to ALK kinase inhibitor resistance in EML4-ALKNSCLC. In addition, we show that a cancer cell line thatharbors a concurrent ALK rearrangement and an EGFRmutation would be expected to be resistant to both single-agent ALK and EGFR inhibitors. Acquired drug resistance

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Figure 4. Concurrent ALK and EGFR signaling leads to drug resistance. A, H3122 cells were treated with crizotinib at the indicated concentrations in thepresence or absence of EGF (10 ng/mL) and PF299804 (1 mmol/L). Viable cells were measured after 72 hours of treatment and plotted relative tountreated controls. B, H3122 cells were treated with crizotinib for 6 hours at indicated concentrations in the absence or presence of EGF (10 ng/mL).Immunoblotting was used to detect the indicated proteins. C, H3122 cells stably expressing GFP or EGFR E746_A750 were treated with crizotinib in theabsence or presence of gefitinib (1 mmol/L). Viable cells were measured after 72 hours of treatment and plotted relative to untreated controls. D, resultsof colony formation assay after 14 days treatment with indicated compounds using H3122 and H3122 EGFR E746_A750. The combination of crizotiniband PF299804 effectively inhibited colony formation. E, EGFR sequence tracing (left) and ALK FISH analysis (right) from patient 1. Asterisk, T to G mutationresulting in L858R; arrows, split red and green FISH signals. F, immunohistochemistry for EGFR L858R (left) and ALK (right) from patient 1.






mechanisms can sometimes also occur de novo and both EGFRT790M andMET amplification have been described in cancersfrom EGFR tyrosine kinase inhibitor (TKI) naive patients (26).In our prior study, we identified one treatment naive NSCLCpatient that harbored a concurrent EGFRmutation and EML4-ALK (13). However, this tumor was obtained from a patientthat had undergone surgery and thus never received systemictherapy. Subsequently, in 50 crizotinib treatment naive NSCLCpatients harboring ALK rearrangements at DFCI, all detectedin a clinical laboratory, we have now identified 3 (6%) patientsthat also harbor concurrent EGFRmutations (Table 2). Two ofthe three patients have undergone therapy with erlotinib andboth have achieved partial responses (Table 2). Evaluation ofthe pretreatment tumor from patient 1 using the EGFRL858R–specific antibody shows EGFR staining (Fig 4E), where-as no staining was observed with the ALK-specific antibody(Fig 4E) despite the presence of the ALK genomic rearrange-ment (Table 2).

Discussion

ALK TKIs are emerging as effective clinical therapies forcancers containing genetic rearrangements in ALK includingNSCLC, IMT, and ALCL (8, 10, 27). However, the clinicalsuccess of this therapeutic approach is uniformly limited bythe development of drug resistance. The mechanistic under-standing of drug resistance may help to develop effectivesubsequent clinical treatments and/or rational combinationtherapeutic strategies.

In this study, by studying patient-derived tumors and celllines, we uncover novel ALK TKI resistance mechanisms.These include both a secondary mutation (L1152R) in ALKand activation of EGFR signaling. Importantly, these can occurtogether in the same tumor (i.e., the DFCI 076 cell line)highlighting both the complexity of drug resistance mechan-isms and the therapeutic challenges in developing strategiesto overcome clinical drug resistance.

Secondary mutations in kinases are a common mechanismof drug resistance to kinase inhibitors (4, 5, 28, 29). A fewdistinct categories of mutations have so far been identified.These include secondary mutations that alter drug contactresidues, thus creating a steric hindrance for drug binding(30). Alternatively, secondary mutations can promote confor-mational changes in the kinase and thus disfavoring thebinding of a kinase inhibitor (30). The L1152R mutation isnot located in the kinase domain. The currently available

crystal structures of ALK do not provide a clear explanation ofthe mechanistic basis of drug resistance imparted by thismutation. Furthermore, despite different EML4-ALK variantsthat have been described to date, there is no evidence tosuggest that the mechanisms of acquired resistance will varyon the basis of specific EML4-ALK variant (11). Notably theL1152Rmutation, unlike the F1174L mutation, is also resistantto TAE684 (14). Thus structurally distinct ALK inhibitors areneeded to overcome this mutation and several are underpreclinical development. Additional studies, including solvingthe crystal structure for the ALK L1152R, will be necessary toobtain further insight into how this mutation causes drugresistance.

Prior studies have generated crizotinib-resistant H3122cells and detected both evidence of an ALK amplificationand the presence of the L1196M gatekeeper mutation (31).We also identify ALK amplification in a subset of the H3122TR3 cells (Supplementary Fig. S3B) but not the L1196Mmutation. Because TAE684, unlike crizotinib, can effectivelyinhibit the growth of H3122 EML4-ALK L1196M cells, ourfindings are consistent with the prior studies (31). In fact,they suggest that a more potent ALK inhibitor may be able toprevent the emergence of this specific drug resistancemechanism. Whether this will ultimately translate into aclinical benefit (such as a prolongation if progression-freesurvival) for NSCLC patients can only be determined fromclinical trials.

Our studies identify activation of EGFR signaling as abypass signaling mechanisms that contributes to ALK inhib-itor resistance. Concurrent inhibition of both EGFR and ALKis therapeutically effective in all of the resistant models.Intriguingly, different models have differing degrees of appar-ent EGFR dependence. The DFCI076 cells are mostly ALKdependent (Fig. 2B), whereas the H3122 TR3 cells are moreEGFR dependent (Fig. 3D) for their growth. The DFCI032 cellsare equally codependent with very little effect on growth byonly EGFR or ALK inhibition (Supplementary Fig. S2F and ref.13). These different examples may be representative of adynamic process with variable degrees of adaptation to EGFRsignaling in the presence of ALK inhibition. However, wecannot completely exclude the possibility that activation ofEGFR signaling in these cell lines did not arise in the process ofgenerating the cell lines. Additional evaluation of tumorspecimens for changes in EGFR phosphorylation obtainedfrom patients that have developed crizotinib resistance willbe necessary. Further investigation is also needed to study

Table 2.Characteristics and outcomes of NSCLC patients harboring concurrent EGFRmutations andALKrearrangements

Pt. EGFR mutation ALK FISH EGFR IHC ALK IHC Erlotinib Therapy Outcome

1 L858R þ L858R Ab positive Negative Yes PR; 9 mo2 Del E746_A750 þ Exon 19 del Ab positive Negative Yes PR; 5 þ mo3 A767_V769dupASV þ N.D. Positive No N/A

Abbreviations: IHC, immunohistochemistry; PR, partial response; N.D., not done; N/A, not applicable.

Sasaki et al.





changes in EGFR signaling over time to further understandhow this adaptive process evolves. Furthermore, whether theprocess will revert in the absence of ALK inhibition needs to bedetermined. Such observation may be clinically significant asit would suggest that drug-resistant cancers could regain theirsensitivity following a therapeutic holiday from ALK inhibi-tors. To date we have grown the H3122 TR3 cells 60 passagesin the absence of TAE684 and have not observed a reversion toTAE684 sensitivity (data not shown).It is noteworthy that both in vitro and in NSCLC patients,

activated EGFR signaling occurs concurrently with EML4-ALK.Such cancers could still retain sensitivity to single-agent EGFRor ALK inhibitors if the tumor was heterogenous and con-tained 2 independent populations: one with EML4-ALK andone with an EGFR mutation. Alternatively, a tumor couldcontain both genetic alterations but only expressed one of themutant proteins. In both instances, such patients may achievea transient partial response following therapy with eithersingle agent. Our limited studies of crizotinib naive NSCLCpatients, with both genetically confirmed EML4-ALK andEGFR mutations, suggest that ALK is not expressed as bothpatients treated with erlotinib achieved a clinical response. Incontrast, the in vitro studies would predict that coexpressionof EML4-ALK and mutant EGFR in the same cells would leadto resistance to both single-agent ALK and EGFR inhibitors.Why some cancers harbor an ALK rearrangement which doesnot lead to ALK expression remains to be determined. It willalso be of interest to determine whether the mechanismof erlotinib resistance in our patients, with both an EGFR

mutation and ALK rearrangement, will involve reactivation ofALK expression.

Currently there is an ongoing phase I clinical trial ofcrizotinib and PF299804 (NCT01121575) originally designedto evaluate the therapeutic benefit of inhibiting MET (crizo-tinib is a potent MET inhibitor) and EGFR T790M in erlotinib-resistant EGFR mutant NSCLC patients. However, our studiessuggest that combination of crizotinib and PF299804 mayrepresent a rational therapeutic strategy for at least a subset ofEML4-ALK NSCLC patients that develop acquired crizotinibresistance.

Disclosure of Potential Conflicts of Interest

M.J. Eck: commercial research support, Novartis. P.A. Jänne: consultant/advisory board and clinical trial support, Pfizer. The other authors disclosed nopotential conflicts of interest.

Acknowledgments

The authors thank Pfizer, Inc. for the clinical trial support.

Grant Support

This study was supported by NIH R01CA136851 (N.S. Gray and P.A. J€anne),R01CA135257 (P.A. J€anne), The Cammarata Family Foundation Research Fund(P.A. J€anne), and by the NCI Lung SPORE P50CA090578 (P.A. J€anne).

The costs of publication of this article were defrayed in part by the paymentof page charges. This article must therefore be hereby marked advertisement inaccordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received April 18, 2011; revised June 22, 2011; accepted July 6, 2011;published OnlineFirst July 26, 2011.

References1. Mok TS, Wu YL, Thongprasert S, Yang CH, Chu DT, Saijo N, et al.

Gefitinib or carboplatin-paclitaxel in pulmonary adenocarcinoma. NEngl J Med 2009;361:947–57.

2. Kwak EL, Bang YJ, Camidge DR, ShawAT, Solomon B,Maki RG, et al.Anaplastic lymphoma kinase inhibition in non-small-cell lung cancer.N Engl J Med 2010;363:1693–703.

3. Engelman JA, Zejnullahu K, Mitsudomi T, Song Y, Hyland C, Park JO,et al. MET amplification leads to gefitinib resistance in lung cancer byactivating ERBB3 signaling. Science 2007;316:1039–43.

4. Kobayashi S, Boggon TJ, Dayaram T, Janne PA, Kocher O, MeyersonM, et al. EGFR mutation and resistance of non-small-cell lung cancerto gefitinib. N Engl J Med 2005;352:786–92.

5. Gorre ME, Mohammed M, Ellwood K, Hsu N, Paquette R, Rao PN,et al. Clinical resistance to STI-571 cancer therapy caused by BCR-ABL gene mutation or amplification. Science 2001;293:876–80.

6. Kantarjian H, Shah NP, Hochhaus A, Cortes J, Shah S, Ayala M, et al.Dasatinib versus imatinib in newly diagnosed chronic-phase chronicmyeloid leukemia. N Engl J Med 2010;362:2260–70.

7. Saglio G, Kim DW, Issaragrisil S, le Coutre P, Etienne G, Lobo C, et al.Nilotinib versus imatinib for newly diagnosed chronic myeloid leuke-mia. N Engl J Med 2010;362:2251–9.

8. Soda M, Choi YL, Enomoto M, Takada S, Yamashita Y, Ishikawa S,et al. Identification of the transforming EML4-ALK fusion gene in non-small-cell lung cancer. Nature 2007;448:561–67.

9. Morris SW, Kirstein MN, Valentine MB, Dittmer KG, Shapiro DN,Saltman DL, et al. Fusion of a kinase gene, ALK, to a nucleolar proteingene, NPM, in non-Hodgkin's lymphoma. Science 1994;263:1281–4.

10. Lawrence B, Perez-Atayde A, Hibbard MK, Rubin BP, Dal Cin P,Pinkus JL, et al. TPM3-ALK and TPM4-ALK oncogenes in inflamma-tory myofibroblastic tumors. Am J Pathol 2000;157:377–84.

11. Sasaki T, Rodig SJ, Chirieac LR, Janne PA. The biology and treatmentof EML4-ALK non-small cell lung cancer. Eur J Cancer 2010;46:1773–80.

12. Soda M, Takada S, Takeuchi K, Choi YL, Enomoto M, Ueno T, et al. Amouse model for EML4-ALK-positive lung cancer. Proc Natl Acad SciU S A 2008;105:19893–7.

13. Koivunen JP, Mermel C, Zejnullahu K, Murphy C, Lifshits E, HolmesAJ, et al. EML4-ALK fusion gene and efficacy of an ALK kinaseinhibitor in lung cancer. Clin Cancer Res 2008;14:4275–83.

14. Sasaki T, Okuda K, Zheng W, Butrynski J, Capelletti M, Wang L, et al.The neuroblastoma-associated F1174L ALK mutation causes resis-tance to an ALK kinase inhibitor in ALK-translocated cancers. CancerRes 2010;70:10038–43.

15. Choi YL, Soda M, Yamashita Y, Ueno T, Takashima J, Nakajima T,et al. EML4-ALK mutations in lung cancer that confer resistance toALK inhibitors. N Engl J Med 2010;363:1734–9.

16. Mino-KenudsonM, Chirieac LR, LawK, Hornick JL, LindemanN,MarkEJ, et al. A novel, highly sensitive antibody allows for the routinedetection of ALK-rearranged lung adenocarcinomas by standardimmunohistochemistry. Clin Cancer Res 2010;16:1561–71.

17. Paez JG, Janne PA, Lee JC, Tracy S, Greulich H, Gabriel S, et al. EGFRmutations in lung cancer: correlation with clinical response to gefitinibtherapy. Science 2004;304:1497–500.

18. Zhou W, Ercan D, Chen L, Yun CH, Li D, Capelletti M, et al. Novelmutant-selective EGFR kinase inhibitors against EGFR T790M. Nature2009;462:1070–4.

19. McDermott U, Iafrate AJ, Gray NS, Shioda T, Classon M, MaheswaranS, et al. Genomic alterations of anaplastic lymphoma kinase maysensitize tumors to anaplastic lymphoma kinase inhibitors. CancerRes 2008;68:3389–95.






20. Galkin AV, Melnick JS, Kim S, Hood TL, Li N, Li L, et al. Identification ofNVP-TAE684, a potent, selective, and efficacious inhibitor of NPM-ALK. Proc Natl Acad Sci U S A 2007;104:270–5.

21. Ercan D, Zejnullahu K, Yonesaka K, Xiao Y, Capelletti M, Rogers A,et al. Amplification of EGFR T790M causes resistance to an irrevers-ible EGFR inhibitor. Oncogene 2010;29:2346–56.

22. Yu J, Kane S, Wu J, Benedettini E, Li D, Reeves C, et al. Mutation-specific antibodies for the detection of EGFR mutations in non-small-cell lung cancer. Clin Cancer Res 2009;15:3023–8.

23. Du J, Bernasconi P, Clauser KR, Mani DR, Finn SP, Beroukhim R, et al.Bead-based profiling of tyrosine kinase phosphorylation identifiesSRC as a potential target for glioblastoma therapy. Nat Biotechnol2009;27:77–83.

24. Yonesaka K, Zejnullahu K, Lindeman N, Homes AJ, Jackman DM,Zhao F, et al. Autocrine production of amphiregulin predicts sensitivityto both gefitinib and cetuximab in EGFR wild-type cancers. ClinCancer Res 2008;14:6963–73.

25. BossiRT,SaccardoMB,ArdiniE,MenichincheriM,RusconiL,MagnaghiP, et al. Crystal structures of anaplastic lymphoma kinase in complexwith ATP competitive inhibitors. Biochemistry 2010;49:6813–25.

26. Sequist LV, Martins RG, Spigel D, Grunberg SM, Spira A, Janne PA,et al. First-line gefitinib in patients with advanced non-small-cell lungcancer harboring somatic EGFR mutations. J Clin Oncol 2008;26:2442–9.

27. Gambacorti-Passerini C, Messa C, Pogliani EM. Crizotinib in ana-plastic large-cell lymphoma. N Engl J Med 2011;364:775–6.

28. Pao W, Miller VA, Politi KA, Riely GJ, Somwar R, Zakowski MF, et al.Acquired resistance of lung adenocarcinomas to gefitinib or erlotinibis associated with a second mutation in the EGFR kinase domain.PLoS Med 2005;2:1–11.

29. Heinrich MC, Corless CL, Blanke CD, Demetri GD, Joensuu H,Roberts PJ, et al. Molecular correlates of imatinib resistancein gastrointestinal stromal tumors. J Clin Oncol 2006;24:4764–74.

30. Sherbenou DW, Druker BJ. Applying the discovery of the Philadelphiachromosome. J Clin Invest 2007;117:2067–74.

31. Katayama R, Khan TM, Benes C, Lifshits E, Ebi H, Rivera VM, et al.Therapeutic strategies to overcome crizotinib resistance in non-smallcell lung cancers harboring the fusion oncogene EML4-ALK. Proc NatlAcad Sci U S A 2011;108:7535–40.

Sasaki et al.





2011;71:6051-6060. Published OnlineFirst July 26, 2011.Cancer Res Takaaki Sasaki, Jussi Koivunen, Atsuko Ogino, et al. Resistance to ALK Kinase InhibitorsA Novel ALK Secondary Mutation and EGFR Signaling Cause

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