Glesatinib Exhibits Antitumor Activity in Lung Cancer ... · Biology of Human Tumors Glesatinib Exhibits Antitumor Activity in Lung Cancer Models and Patients Harboring MET Exon 14

Biology of Human Tumors

Glesatinib Exhibits Antitumor Activity in LungCancer Models and Patients Harboring MET Exon14 Mutations and Overcomes Mutation-mediatedResistance to Type I MET Inhibitors in NonclinicalModelsLars D. Engstrom1, Ruth Aranda1, Matthew Lee1, Elizabeth A. Tovar2, Curt J. Essenburg2,Zachary Madaj2, Harrah Chiang1, David Briere1, Jill Hallin1, Pedro P. Lopez-Casas3,Natalia Ba~nos3, Camino Menendez3, Manuel Hidalgo3, Vanessa Tassell1, Richard Chao1,Darya I. Chudova4, Richard B. Lanman4, Peter Olson1, Lyudmilla Bazhenova5,Sandip Pravin Patel5, Carrie Graveel2, Mizuki Nishino6, Geoffrey I. Shapiro7, Nir Peled8,Mark M. Awad7, Pasi A. J€anne7, and James G. Christensen1

Abstract

Purpose: MET exon 14 deletion (METex14 del) mutationsrepresent a novel class of non–small cell lung cancer (NSCLC)driver mutations. We evaluated glesatinib, a spectrum-selectiveMET inhibitor exhibiting a type II bindingmode, inMETex14del–positive nonclinical models and NSCLC patients and assessed itsability to overcome resistance to type I MET inhibitors.

Experimental Design: As most MET inhibitors in clinicaldevelopment bind the active site with a type I binding mode, weinvestigated mechanisms of acquired resistance to each METinhibitor class utilizing in vitro and in vivomodels and in glesatinibclinical trials.

Results: Glesatinib inhibited MET signaling, demonstratedmarked regression of METex14 del-driven patient-derivedxenografts, and demonstrated a durable RECIST partialresponse in a METex14 del mutation-positive patient enrolledon a glesatinib clinical trial. Prolonged treatment of nonclinical

models with selected MET inhibitors resulted in differences inresistance kinetics and mutations within the MET activationloop (i.e., D1228N, Y1230C/H) that conferred resistance totype I MET inhibitors, but remained sensitive to glesatinib.In vivo models exhibiting METex14 del/A-loop double muta-tions and resistance to type I inhibitors exhibited a markedresponse to glesatinib. Finally, a METex14 del mutation-positive NSCLC patient who responded to crizotinib but laterrelapsed, demonstrated a mixed response to glesatinib includ-ing reduction in size of a MET Y1230H mutation-positive livermetastasis and concurrent loss of detection of this mutation inplasma DNA.

Conclusions: Together, these data demonstrate that glesa-tinib exhibits a distinct mechanism of target inhibition andcan overcome resistance to type I MET inhibitors. Clin CancerRes; 23(21); 6661–72. �2017 AACR.

IntroductionMET is a receptor tyrosine kinase (RTK) that controls cellular

survival, growth, and motility and functions as an oncogenic

driver in multiple cancer types when amplified or activatedthroughmutation (1–3). A diverse set of somatic pointmutationsand insertions or deletions (indels) flanking the 50 and 30 splicesites of exon 14 of MET have been identified and result inabnormal exon splicing and skipping of exon 14. The exon 14region of MET encodes the juxtamembrane region containing aY1003 ubiquitin ligase CBL binding site and the skipping of thisregion alters receptor processing resulting in prolonged stabili-zation and activation ofMET (4–7).MostMETex14 delmutationshave been identified in lung adenocarcinoma, adenosquamous,or pulmonary sarcomatoid carcinoma subtypes, collectively com-prise approximately 3% of non–small cell lung cancer (NSCLC),and are statistically exclusive with other known oncogenic drivers(4, 8, 9). Genetic, pharmacologic, and clinical studies have furtherestablished that this collection of mutations exhibit the charac-teristics of driver oncogenes and that MET inhibitors are clinicallyactive in NSCLC patients with these mutations (8–13).

Acquired resistance to targeted therapies including EGFR, ALK,and ROS1 RTK inhibitors has been a consistent historicalchallenge in NSCLC. However, the understanding of resistance

1Mirati Therapeutics, Inc., San Diego, California. 2Van Andel Research Institute,Grand Rapids, Michigan. 3Centro Nacional de Investigaciones Oncol�ogicas(Spanish National Cancer Research Centre), Madrid, Spain. 4Guardant Health,Inc., Redwood City, California. 5Moores Cancer Center, University of California,San Diego, San Diego, California. 6Department of Radiology, Brigham andWomen's Hospital and Dana-Farber Cancer Institute, Boston, Massachusetts.7Lowe Center for Thoracic Oncology, Dana-Farber Cancer Institute, HarvardMedical School, Boston, Massachusetts. 8Sheba Medical Center, Tel-Aviv Uni-versity, Ramat-Gan, Israel.

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

Corresponding Author: James G. Christensen, Mirati Therapeutics, Inc., SanDiego, CA, 92121. Phone: 858-332-3426; Fax: 858-597-1009; E-mail:[email protected]

doi: 10.1158/1078-0432.CCR-17-1192

�2017 American Association for Cancer Research.

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mechanisms, including active site mutations and activation ofbypass pathways, has resulted in next-generation inhibitors anddevelopment of combinatorial treatment strategies to simulta-neously target primary and bypass pathways (14–17). Multiplerecent reports of acquired active site resistance mutations inMETfollowing durable clinical responses to crizotinib therapy indicatethat targeting MET is not an exception (18–20).

Small-molecule inhibitors of RTKs can generally be dividedinto three types (I, II, and III) based on their binding interactionsandmode of inhibition of catalytic activity (2, 21, 22). Most METinhibitors (e.g., crizotinib, savolitinib, capmatinib) exhibit type Ibinding to the catalytically inactive state at the ATP-binding siteand stabilize the kinase's activation loop (A-loop) to blockcatalytic activation. In contrast, glesatinib is a unique type II METinhibitor, and predicted to bind either to the DFG-out and auto-inhibited A-loop conformation or to the active form of MET(based on PDB IDs 3U6H and 3U6I) by projecting into a hydro-phobic Ile1145 pocket which resides between the aC helix andcatalytic Lys1110. Both glesatinib bindingmodes are distinct fromtype I inhibitors and independent of interactions with the A-loop(22, 23). MET mutations involving the A-loop residues D1228and Y1230 that confer resistance to type I MET inhibitors havenow been described in model systems and multiple patients(18–20). Because of the differential engagement of the A-loop,type I and type II MET inhibitors are predicted to be differentiallysusceptible to certain active site mutations implicated in drugresistance (19, 24–26). Thus, type II inhibitors are predicted to beeffective against certainMETmutations implicated in resistance totype I inhibitors enabling a sequential treatment strategy in thesetting of target-mediated drug resistance.

We evaluated glesatinib (MGCD265) as a unique MET inhib-itor that demonstrated activity in MET-driven models in vitro andmarked tumor regression of multipleMETex14 del mutant in vivomodels. Interestingly, experimentally-derivedmodels of acquiredresistance to selected type I MET inhibitors were cross-resistant toother type I inhibitors, but remained sensitive to glesatinib. This

observation is consistent with the differential binding modes ofthese inhibitor classes. We also demonstrate that glesatinib hasmarked clinical activity in a patient with METex14 del–mutantNSCLC who experienced a durable partial response to glesatinibafter previously receiving multiple other lines of therapy. Finally,we provide evidence that glesatinib may have activity in patientswith crizotinib-resistantMETex14mutantNSCLCas illustratedbya radiographic response of a lesion harboring a MET A-loopresistancemutationwith concurrent loss of detection of this allelein plasma cell–free DNA (cfDNA). These data suggest that METinhibitors with different bindingmodesmay have distinct clinicalactivity analogous to management of EGFR and ALK alteration–positive cancer with different classes of inhibitors.

Materials and MethodsCell culture and reagents

NIH/3T3, NCI-H441, NCI-H596, and Hs746T cell lines wereobtained from ATCC and SNU-638 was obtained from KoreanCell Line Bank (KCLB). All cell lines were cultured in media andconditions recommended by themanufacturer and authenticatedby STR analysis within 3 months prior to use (IDEXX BioRe-search). Recombinant humanHepatocyte Growth Factor (hHGF)was purchased from R&D Systems (#294-HGN-025). AMG-208(#S1316), crizotinib (#S1068), and capmatinib (#S2788) weresourced from Selleckchem. Savolitinib (#A-1316) was sourcedfrom Active Biochem.

Molecular modeling methodsTwo different binding modes of glesatinib were generated

through initial alignment onto the cognate ligands from theDFG-in PDB ID: 3U6H and DFG-out PDB ID: 3VW8 crystalcomplex structures obtained from The Protein Data Bank(www.rcsb.org; ref. 27).

Engineered cell line generationMET variants (MET, MET Y1230C,METex14 del,METex14 del

D1228N,METex14 del Y1230C, andMETex14 del Y1230H) weresynthesized on the basis of the human MET reference sequence(NM_000245.3), Sanger sequence verified, and cloned into thepMSCVpuro retroviral construct (Clontech). Parental NIH/3T3cells were transduced with retroviral supernatants from thepMSCVpuro MET variants and selected with 1 mg/mL of puro-mycin for at least 14 days. Stably selected pools were maintainedin 0.5 mg/mL puromycin. All cell lines were pools and wereverified by Sanger sequence analysis.

ImmunoblottingProtein lysates were harvested in Cell Signaling Technology

lysis buffer containing protease and phosphatase inhibitors.Lysates were quantified by a BCA protein assay (Pierce) andnormalized prior to gel loading, electrophoresis, and transfer topolyvinylidene difluoride (PVDF) membranes for immunoblot-ting. Blocking buffer, antibody dilutions, and incubation condi-tions were obtained from manufacturers. All primary antibodieswere purchased from Cell Signaling Technology: pMET (#3126),MET (#8198), pAKT (#4060), AKT (#9272), pMEK (#9154),MEK(#9126), pERK (#4370), ERK (#9102), a-Tubulin (#3873), andGAPDH (#5174). Near-infrared secondary antibodies (LI-CORBiosciences) were diluted 1:10,000. Immunoblots were scannedon a LI-COR Odyssey CLx Imager.

Translational Relevance

METex14 del mutations represent a novel class of drivermutations. Multiple MET inhibitors have demonstrated evi-dence of clinical activity in this molecularly defined subset ofNSCLC. However, nonclinical and clinical evidence of resis-tance due to MET A-loop mutations is emerging. Glesatinibdemonstrated marked regression of METex14 del–driventumor models in vivo and clinical activity in METex14 delmutation–positive patients. The current studies indicate thatglesatinib has a type II binding mode distinct from other METinhibitors and that MET A-loop mutations, which conferredresistance to type I MET inhibitors, are sensitive to glesatinib.Glesatinib demonstrated activity inMETex14 del/A-loop dou-ble mutation positive tumor models and in a METex14 delmutation-positive NSCLC patient exhibiting an A-loop-muta-tion positive metastasis after relapse on crizotinib. Together,these data demonstrate glesatinib exhibits a distinct mecha-nism of target inhibition and can overcome resistanceenabling sequential treatment strategies to combat resistancein MET-positive patients.

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MET signalingNIH/3T3 cells expressing full-length human MET or

METex14 del were serum starved for 1 hour and stimulatedwith 400 ng/mL of hHGF for 15 minutes. Cells were rinsed withPBS and replaced with serum-free DMEM for the remainder ofthe time course. Protein lysates were collected at the indicatedtime points, normalized for total protein by BCA and analyzedby immunoblot.

In vivo modelsAll procedures carried out in this study were conducted in

compliance with all applicable laws, regulations, and guide-lines of the NIH regarding animal health and welfare and localInstitutional Animal Care and Use Committee oversight. Foreach study, glesatinib was formulated in 5% DMSO/45%PEG400/50% saline and mice were dosed at 60 mg/kg, oncedaily via oral gavage at a volume of 5 mL/kg and tumors weremeasured twice weekly with Vernier calipers. Hs746t cellssuspended in Matrigel (Corning, reference no. 356237) andDPBS (Gibco, reference no. 14190-135) were injected subcu-taneously into the flank of nu/nu mice (Charles River Labora-tories Inc.). The PDX models LU2503 and LU5381 (CrownBiosciences) harbor c.3082þ2T>C or c.3028G>C mutations,respectively, each resulting in MET exon 14 skipping as con-firmed by RT-PCR (7) were implanted and established atapproximately 500 or 1,000 mm3 and glesatinib (60 mg/kg,once daily) was orally administered as denoted.

For analysis of tumor inhibition in hHGFtg-SCID mice, cellswere harvested and resuspended in serum-free DMEM at 1 �106 cells/mL. Each animal received 100 mL of cell suspensioninjected subcutaneously. Tumor-bearing mice were random-ized into four treatment groups including vehicle, crizotinib(50 mg/kg, once daily), capmatinib (30 mg/kg, twice daily),and glesatinib (60 mg/kg, once daily). Treatment started whenthe average tumor size reached 150 mm3 and drugs wereadministered for 21 days or until mice required euthanasiadue to tumor burden.

Liquid biopsy NGSCell-free DNA (cfDNA), sequencing and analysis was per-

formed at Guardant Health using the Guardant360 version2.10 assay (28). Barcoded sequencing libraries were generatedfrom 5–30 ng of cfDNA and target exons were captured usingbiotinylated custom bait oligonucleotides for 73 genes (Agilent).Samples were paired-end sequenced with average depth ofapproximately 15,000� on an Illumina Hi-Seq 2500 followedby algorithmic reconstruction of unique input molecules andreporting of defined point mutations, amplifications, indels, orrearrangements.

Glesatinib clinical trialsGlesatinib clinical trials (NCT00697632) and patient consents

were conducted in accordance with recognized US ethical guide-lines and as per local institutional review board guidelines.

ResultsMETex14 del mutant variants demonstrate durableHGF-dependent activation and oncogenic transformation ofimmortalized cells

To develop model systems to characterize METex14 del var-iants, we engineered NIH/3T3 cells expressing similar levels offull-length human MET wild-type (WT) or METex14 del variantsand monitored the kinetics of activation of MET and its down-stream signaling pathways. METex14 deletion–expressing cellsexhibited a longer duration of human hepatocyte growth factor(hHGF)-stimulated phosphorylation of MET, ERK, and Akt com-pared with WT MET-expressing cells (Fig. 1A). Moreover, totalMET levels remained constant in hHGF-stimulatedMETex14 del–expressing cells, whereas total MET expression steadily decreasedover 4 hours in WT MET–expressing cells. This observation isreflective of decreased MET protein lysosomal recycling anddegradation in METex14 del, but not WT cells (29). Growth ofcells in anchorage-independent conditions also indicated thatMETex14 deletion–expressing cells produced more and largercolonies compared with WT MET–expressing cells (Fig. 1B). The

Figure 1.

METex14 del leads to prolonged downstream signaling and anchorage-independent growth in NIH/3T3s. A, NIH/3T3 MET or METex14 del–expressing cellswere serum starved and treated with 400 ng/mL HGF for 15 minutes followed by washout for various time points. Phospho-MET (pMET) as well as the PI3Kand MAPK pathways were assessed by Western blot for canonical phospho and total proteins. GAPDH and a-tubulin were included as loading controls.B, NIH/3T3s expressing MET or METex14 del were plated in soft agar and treated with or without 20 ng/mL HGF and colonies were imaged after 41 days.

Glesatinib Circumvents Resistance to Type I MET Inhibitors

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increased cell growth characteristics were only observed inthe presence of hHGF highlighting the requirement for ligand-mediated receptor activation to manifest a transformed pheno-type in METex14 del mutant–expressing cells. These data furtherestablish the oncogenic driver functionofMETex14delmutationsbased on increased duration of HGF/MET–dependent signalingand cell transformation.

Activity of glesatinib in tumor cell lines in vitro and markedregression of cell- and patient-derived tumormodels harboringMETex14 del mutations in vivo

Glesatinib is a small-molecule, ATP-competitiveMET inhibitorwith an IC50 value of 19 nmol/L for inhibition of the catalyticactivity of WT MET in an enzyme-based kinase assay (Supple-mentary Fig. S1A and S1B). Additional profiling of over 200kinases in biochemical and cellular assays indicates that glesatinibonly inhibited MET, Axl, MERTK, and the PDGFR family atconcentrations below 75 nmol/L, a concentration predicted toreflect the highest clinically achievable glesatinib dose andplasma exposure based on clinical pharmacodynamic studies

[Supplementary Fig. S1C and Supplementary Table S1; basedon the observation of complete modulation of plasma shedMET ectodomain and evidence of objective response inpatients exhibiting MET genetic alterations. VEGFR2 is themost potently inhibited RTK target after MET with an IC50 of172 nmol/L. Inhibition of VEGFR2 was not observed inpatients based on the lack of modulation of plasma sVEGFR2and VEGFA and no reported cases of hypertension, protein-uria, and palmar–plantar erythrodysesthesia (J Clin Oncol 33,2015 (suppl; abstr 2589)].

We also assessed the ability of glesatinib to inhibit METphosphorylation and growth in cell lines exhibiting METgenetic alterations. NCI-H441 cells have a low-level gain ofthe MET gene, NCI-H596 harbor a METex14 del mutation andloss of the WT allele, and Hs746T cells exhibit both a METex14del mutation and amplification of the mutated allele. Treat-ment with glesatinib resulted in concentration-dependent inhi-bition of MET phosphorylation and cell viability in each cellline evaluated (Fig. 2A and B). Glesatinib inhibited hHGF-stimulated growth of H441 and H596 cells and completely

Figure 2.

Glesatinib inhibits MET-driven growth in vitro and in vivo. A, Low-level MET-amplified NCI-H441, METex14 del–mutant NCI-H596, and METex14 del–mutantand amplified Hs746T cells were treated with glesatinib with and without 50 ng/mL hHGF. Cells were treated for 3 days, and viability was assessedusing the CellTiter Glo assay. B, Cell lines were serum starved for 2 hours followed by treatment with glesatinib at 0.1 and 1 mmol/L for anadditional 2 hours (in serum-free media). During the last 20 minutes of glesatinib or vehicle treatment, cells were treated with hHGF (100 ng/mL). Proteinlysates were harvested, normalized, and analyzed by Western blot. C–E, Glesatinib exhibits marked responses in METex14 del–mutant xenograft models.The cell line xenograft model Hs746T (C) and the PDX models LU2503 (D) and LU5381 (E) were implanted, randomized, and dosed orally with glesatinibat 60 mg/kg daily starting at average tumor volumes of 400–500 mm3 or 1,000 mm3 for 2 to 4.5 weeks as indicated. Mean tumor volumes are plotted witha sample size per treatment group of 9–12 for Hs746T, 3–10 for LU5203, or 5–10 for LU5381. Error bars represent SEM.

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inhibited growth of Hs746T cells independent of hHGF treat-ment indicating this cell line is completely dependent onconstitutive MET signaling.

Glesatinib was also evaluated in METex14 del mutation–positive tumor models. Glesatinib demonstrated markedregression of both medium (400 mm3) and large established(1,000 mm3) Hs746T xenografts with complete regression (i.e.,no palpable tumor) observed for medium tumors after 11 daysand large tumors after 32 days of oral administration at 60 mg/kgonce daily (Fig. 2C). Glesatinib was also evaluated in theLU2503 and LU5381 NSCLC PDX models, which exhibitedMETex14 del mutations and skipping of exon 14 with varyingdegrees of MET copy gain and demonstrated marked tumorregression in each model. In the LU2503 model, 92% regres-sion of medium sized tumors was observed after 17 days oftreatment and 74% regression of large established tumors wasobserved after 27 days of treatment (Fig. 2D). In the LU5381model, 34% regression of medium established tumors wasobserved after 19 days of treatment and 72% regression oflarge established tumors was observed after 28 days of treat-ment (Fig. 2E). Glesatinib was also tested across a broaderpanel of tumor xenograft models and regression was alsoobserved in the MET amplification-positive MKN-45 model,whereas only partial tumor growth inhibition (40%–80%) wasobserved in eight other models only expressing WT MET(Supplementary Fig. S2). The 60 mg/kg once daily glesatinibdose was generally well tolerated with minimal evidence ofweight loss in all studies and plasma concentrations achievedat this dose level in mice is consistent with clinicallyachievable glesatinib plasma concentrations (data not shown).The marked tumor regression observed with glesatinibfurther establishes METex14 del mutations as a bona fideoncogenic driver in NSCLC and supports the evaluation ofglesatinib as a therapeutic option for patients harboring thismutational class.

Glesatinib case presentation for a METex14 del mutation–positive lung adenocarcinoma patient

A 70-year old never-smoker diagnosed with stage IV lungadenocarcinoma and refractory to multiple lines of therapyincluding carboplatin/paclitaxel/bevacizumab, single-agent nivo-lumab, and carboplatin/pemetrexed was found to have no detect-able actionable mutations following next-generation sequencingof tumor tissue. Subsequently, plasma cell-free DNA (cfDNA)next-generation sequencing demonstrated a small 15-nucleotidedeletion inMET intron 13 adjacent to the exon 14 coding regionsplice acceptor site (c.2942-21_2942-7delTTTCTTTCTCTCTGT at0.1% variant allele fraction) predicted to result in MET exon 14skipping. The only other genomic alteration detected in the73-gene panel was a TP53 mutation (S241F). The patient wasadministered glesatinib starting at the recommended phase IIdose of 1,050 mg twice daily soft gel capsule and had markedclinical improvementwithin 2weeks,with resolution of his coughand increased activity, andmarked radiographic improvement by6 weeks. A maximal RECIST partial response of 66% reduction oftarget lesions was observed at 4months and the patient remainedin partial response for 7 months until he developed progressivepericardial effusion (Fig. 3). Targeted sequencing of MET and 14additional genes implicated in NSCLC pathogenesis inmalignantcells from the effusion revealed no evidence of acquired muta-tions (data not shown).

The role of secondaryMET activation loop (A-Loop) mutationsin acquired resistance to type IMET inhibitors and sensitivity toglesatinib

We also sought to characterize mechanisms of acquired resis-tance to clinically activeMET inhibitors that utilize different activesite bindingmodes.We selected the only available cell line, whichis both MET-addicted and nonamplified, SNU-638, was selectedfor treatment with the type I MET inhibitors crizotinib or capma-tinib or the type II inhibitor glesatinib at increasing concentra-tions over several months. After drug concentrations wereincreased to 1 mmol/L or greater, each pool was cultured for atleast a week without drug to assess stable molecular mechanismsof resistance and evaluate cross-resistance tootherMET inhibitors.For each cell pool, a region of MET encompassing the kinasedomain was amplified by RT-PCR, Sanger sequenced and ana-lyzed for secondary peak calls >25%. In the capmatinib resistant(Cap-res)model, an A toGmutation (c.3689A>G) correspondingto a Y1230C mutation was identified. In the crizotinib-resistantmodel, a T to C mutation (c.3688T>C) corresponding to aY1230H mutation as well as a T to C mutation (c.3598T>C)corresponding to a F1200L mutation were identified on a singleallele in cis with comparable expression of the WT and doublemutant variants (Fig. 4A and B; Supplementary Fig. S3). Capma-tinib- and crizotinib-resistant cell lines exhibited a dramaticincrease in the concentrations required to inhibit cell growth(denoted as IC50 values) and continued to proliferate at2.5 mmol/L capmatinib and 1 mmol/L crizotinib, respectively (Fig.4C–F). In addition, crizotinib was significantly less potent in thecapmatinib-resistant line (IC50 increased �50-fold) and capma-tinibwas inactive in the crizotinib-resistant (IC50 > 3 mmol/L) lineindicating a propensity for cross-resistance for these type I METinhibitors. In contrast, glesatinib exhibited a similar IC50 value forinhibition of cell growth in the capmatinib-resistant model and amodest shift in potency (�4-fold) in the crizotinib-resistantmodel each compared with the parental cell line. The 4-fold shiftin potency in cell viability assays observed for glesatinib in thecrizotinib-resistant but not capmatinib-resistant line also suggeststhat the presence of the F1200L variant in cis with Y1230H mayimpact the activity of both crizotinib and glesatinib. Evaluation ofthe ability of inhibitors to block MET signaling utilizing immu-noblotting indicated all three agents effectively inhibitedMETandAkt phosphorylation at concentrations consistent with their IC50

values in cell viability assays in the parental line (Fig. 4G–I).However, in the crizotinib-resistant line, both capmatinib andcrizotinib exhibited clear shifts in potency and did not demon-strate complete inhibition of MET signaling even at 1 mmol/L. Incontrast, glesatinib demonstrated a similar concentration–response relationship for inhibition of MET signaling in boththe parental and resistant lines with apparent maximal targetinhibition observed at 100 nmol/L in all lines. These data dem-onstrate a MET target–dependent mechanism of resistance inthese acquired resistance models and differential susceptibilityto the distinct MET inhibitor classes.

While we effectively generated resistant cell lines with definedMET mutations after approximately two or three months ofexposure to capmatinib or crizotinib, we were unable to generateclear glesatinib-resistant cells even after four months of exposure.Moreover, we attempted to generate in vivo models of resistanceusing another METex14 del mutant and amplified PDX model,PULM-039. This model was initially sensitive to capmatinib,crizotinib, and glesatinib as all three drugs elicited strong tumor






regression after twoweeks of treatment (Supplementary Fig. S4A).However, in subsequent sequential retreatment studies, capma-tinib and crizotinib drug-resistant tumors emerged between 40 to120 days after initiation of treatment, whereas glesatinib treat-ment did not result in overtly drug-resistant tumors even after180 days on study indicating that the onset of resistance toglesatinib was delayed in comparison (SupplementaryFig. S4B–S4D). Resistant tumors were analyzed by RNAseq;however, no alterations in MET or other oncogenic drivers wereidentified.

Molecular basis of MET mutation–mediated resistance anddifferential activity of glesatinib

MET inhibitors exhibit some atypical binding features com-pared with inhibitors of other RTKs and can be subclassified intotypes Ia, Ib, and II based on their binding mode (2, 21, 22). Thecrystal complex of crizotinib is shown inFig. 5A, demonstrating itstype Ia binding to the MET-active site with an A-loop conforma-tion that positions Y1230 to pi-stack over the tetra-substitutedphenyl ring in a DFG-in catalytically inactive state and projectingthe piperidine group over G1163 into bulk solvent (Supplemen-tary Fig. S5). Capmatinib is also predicted to bind to the auto-inhibited inactive conformation ofMET as a type Ib inhibitor withinteractions that involve pi-stacking with Y1230, but indepen-dently of G1163. In contrast, glesatinib exhibits a structure liketype II inhibitors in the literature (30–32) and extends linearly

toward theaChelix andhydrophobic backDFG-out pocketwith apredicted primary binding mode as a type II inhibitor in catalyt-ically inactive state. Glesatinib also appears capable of adopting asecondary binding mode to a DFG-in catalytically active state,extending back into a hydrophobic I1145 pocket between the aChelix and catalytic K1110. Models for both glesatinib bindingmodes are shown in Fig. 5B and C, each involving A-loop con-formations that are expected to be insensitive to resistance muta-tions observed to date on the A-loop.

Glesatinib and selected type I MET inhibitors were then testedacross a panel of MET WT and clinically relevant mutant enzymeassays including various A-loop mutant variants. Only glesatinibretained activity against all A-loop mutations, whereas the type IMET inhibitors each exhibited a significant loss of potency againstmutant variants involvingD1228 andY1230A-loop residues (Fig.5D). In contrast, all evaluated MET inhibitors except savolitinibexhibited a >10-fold decrease in potency against the F1200Imutation. Although F1200 resides on the N-terminus of the ß6strand distal from the adenine-binding site, mutations of thisresidue to the aliphatic isoleucine (or leucine)maybe predicted toimpart resistance to type I MET inhibitors through altering thesubstrate-binding cleft resulting in enhanced substrate affinity.For type II MET inhibitors, F1200 comprises part of the DFG-outpocket, wherebymutation to a leucine or isoleucine is expected todistort the shape of the hydrophobic back pocket which is pre-dicted to be important for glesatinib binding. The predicted

Figure 3.

Radiographic response to glesatinib ina patient with METex14 del mutation–positive lung adenocarcinoma. Thetop panels show an axial view, andbottom panels show a coronal view ofCT chest images prior to glesatinibtreatment (left) and after 4 months ofglesatinib treatment (right).

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impact of F1200 mutations on both type I and II inhibitorspotentially accounts for the decrease in glesatinib and crizotinibpotency in the F1200I biochemical assay as well as a 4-folddecrease in glesatinib potency in the crizotinib-resistant clones(Fig. 4). An additional MET-mutant variant that is proximal inspace to the F1200 residue; L1195V, was also associated with a5–10-fold decrease in potency for both glesatinib and crizotinibandmore than a 100-fold decrease in potency for capmatinib andother type Ib MET inhibitors. Position 1195 of MET sits one turnfrom the C-terminus of the aE helix. Because leucines are pro-helical and valines anti-helical, the MET L1195V variant mayresult in unravelling of the C-terminus of the aE helix and resultin global alteration of MET topology. Thus, the auto-inhibitedA-loop conformation required for type Ib MET inhibitors may bedestabilized and the shape of extended hydrophobic pocket maybe unfavorably altered for type II MET inhibitors. Finally, crizo-tinib, but not other MET inhibitors, exhibited a 4-fold decrease inpotency against the G1163RMET variant which is consistent withonly crizotinib exhibiting a key binding interaction with thea-carbon of the G1163 linker strand residue. No additionalMETmutations included in the enzyme assay panel including P991S,P992I, V1092I, T1173I, Y1235D, andM1250T resulted in a loss ofactivity for glesatinib or otherMET inhibitors comparedwithMETWT (partial summary in Supplementary Table S2).

Circumvention of MET A-loop mutation–mediated resistanceby glesatinib

To further evaluate the differential activity of each MET inhib-itor class against MET A-loop mutant variants, NIH/3T3 cellsexpressing either MET WT or METex14 del, A-loop individual

mutations, or double A-loop mutations in cis with METex14 del,were grown as 3-dimensional spheroids in ultra-low attachmentplates and treated with glesatinib, crizotinib, or capmatinib. EachMET inhibitor demonstrated potent inhibition of hHGF-stimu-lated growth of MET WT or METex14 del mutant–expressingNIH/3T3 cells; however, only glesatinib retained comparable activ-ity against individual A-loop mutations or double A-loop muta-tions in cis with METex14 del mutations (Fig. 5E; SupplementaryFig. S6). Both crizotinib and capmatinib exhibited a significant lossof potency in all models expressing an A-loop mutation relative tomodels expressing MET WT or METex14 del mutations.

We next evaluated crizotinib, capmatinib, and glesatinib inNIH/3T3 cell line–derived tumor xenograft models engineered toexpress MET Y1230C, the METex14 del Y1230C double mutant,or the METex14 del D1228N double mutant implanted into thehind flank of immunocompromised SCID mice expressing thehumanHGF transgene. Because theseMET variants are dependentonHGFbinding for complete activation and asmouseHGF bindshuman MET with low affinity (33), the hHGFtg-SCID model wasutilized to evaluate the activity of MET inhibitors in modelsexhibiting MET mutation variants (34, 35). Treatment ofestablished tumors with crizotinib (50 mg/kg) or capmatinib(30 mg/kg, twice daily) at previously reported dose regimens didnot appreciably inhibit tumor growth (Fig. 5F–H). In contrast,daily administration of glesatinib (60 mg/kg) resulted in tumorregression of the MET Y1230C (maximum regression of 88% onday 13),METex14 del Y1230C (maximum regression of 52% onday 13), andMETex14delD1228N (maximum regression of 21%on day 3) xenografts. In general, the antitumor activity of glesa-tinib was comparable with the activity seen in METex14 del

Figure 4.

Models of acquired resistance to type I MET inhibitors develop Y1230 and F1200 mutations that are still sensitive to glesatinib. SNU-638 cells were culturedfor several months with capmatinib, crizotinib, or glesatinib and pooled resistant cultures were Sanger sequenced. Chromatogram revealing the MET A-loopY1230C mutation in the SNU-638 capmatinib-resistant cell line (A) and a Y1230H and F1200L mutation in the SNU-638 crizotinib-resistant model (B).The crizotinib-resistant mutations were determined to be in cis (Supplementary Fig. S3). Drug response was assessed in parental (C) and resistant (D and E) cellsusing a Cell Titer-Glo assay. Graphs from a representative experiment are shown, while average data from all experiments are summarized in F. Westernblots of MET and Akt for parental, crizotinib-resistant, or capmatinib-resistant cells treated for 4 hours with a range of doses of capmatinib, crizotinib,or glesatinib (G–I).






mutant and amplified cell line and PDX models; however, thegrowth of some METex14 del D1228N tumors began to recoverafter the initial regression suggesting that this double mutation isnot as sensitive to glesatinib as theMETex14 del Y1230C doublemutation.

Case presentation for glesatinib treatment of a METex14 delmutation–positive lung adenocarcinoma patient progressingon crizotinib with A-loop–resistant mutations

A 64-year-old never smoker diagnosed with stage IV lungadenocarcinoma and extensive metastatic disease harboring aMETex14delmutation (c.3028delG)with concurrentMET ampli-fication was started on crizotinib and experienced a partialresponse for 8 months. At the time of disease progression andacquired resistance to crizotinib, next-generation sequencing of aliver lesion biopsy (asterisk in Fig. 6A) detected a MET Y1230H

mutation (in addition to the originalMETex14 delmutation) thatwas not detected in the pre-crizotinib treatment sample. At thistime, plasma was submitted for cfDNA next-generation sequenc-ing that detected the METex14 del mutation at 53.4% variantallele fraction (VAF), concomitantMET gene amplification, threeMET A-loop mutations D1228N (9.1% VAF), Y1230H (4.3%VAF), Y1230S (1.2% VAF), and the G1163R (0.3% VAF) solventfrontmutation in addition to the originalMETex14 delmutation.The lower % cfDNA allele frequencies of these mutations relativeto the originalMETex14 del mutation implicate them as acquiredresistance mutations. In addition, read mapping indicated thatthe three A-loop mutations were all in trans with each othersuggesting these mutations were present in individual subclonaltumors or tumor cells.

The patient was then started on a phase I study of glesatinib(1,050 mg orally twice daily, soft gel formulation) followed by a

Figure 5.

Structure-based modeling of the distinct binding modes of glesatinib and crizotinib in the MET active site and molecular basis of differential activityagainst MET mutations implicated in resistance to type I MET inhibitors. A, The crystal complex of crizotinib, PDB ID: 2WGJ, indicates a U-shapedbinding mode, demonstrating its type Ia binding in a DFG-in catalytically inactive state with an A-loop conformation that positions Y1230 to pi-stack overthe tetra-substituted phenyl ring of crizotinib. B and C, Models of glesatinib as a non-U-shaped DFG-in type I inhibitor in an extended binding modeprojecting toward the aC helix and hydrophobic I1145 back pocket, using PDB ID: 3U6H with MET in a catalytically active state (B) and as a DFG-out type IIinhibitor, using PDB ID: 3VW8, with MET in a catalytically inactive state (C). The activation loop is shown in red, fully resolved in A, largely unresolved in B,and partially unresolved in C, where Y1230 is >10 Å from the pyridine ring of glesatinib. D, IC50 values of glesatinib and four other MET inhibitors in WTor mutant MET enzymatic assays. E, IC50s of glesatinib in a spheroid growth assay of MET WT or mutant-expressing NIH/3T3s treated with glesatinib,crizotinib, or capmatinib. Representative images of NIH/3T3 variant cells in the spheroid growth assay are included in Supplementary Fig. S6. NIH/3T3 cellsexpressing MET Y1230C (F), METex14 del Y1230C (G), or METex14 del D1228N (H) were implanted into hHGFtg-SCID transgenic mice, randomized,and dosed with crizotinib, capmatinib, or glesatinib. Animals were dosed daily with crizotinib (50 mg/kg) or glesatinib (60 mg/kg) or twice daily withcapmatinib (30 mg/kg), and tumor size was monitored (n ¼ 8–13 per treatment group). Error bars represent SEM.

Engstrom et al.





14-day dose interruption, and then resumed on 500 mg twicedaily spray-dried dispersion formulation). After 6weeks, CT scansof the abdomen demonstrated a decrease in size of a few liverlesions (with a reduction of the sum of the longest diameters by31% of the lesions indicated by � and #); however, most of theother lesions in the liver increased in size (with a 35% increase ofthe sum of the longest diameters of the representative lesions,arrows), overall indicative of tumor progression (Fig. 6A–D).Notably, the lesion that was biopsied and positive for the METY1230H mutation at the time of crizotinib resistance haddecreased in size (asterisk).

Repeat plasma testing after 6 weeks on glesatinib showed thatthe Y1230H and Y1230S mutation were no longer detectable(Fig. 6E). The D1228N and G1163R mutations were still presentand a new L1195Vmutation appeared (Fig. 6E). Also, the plasma-detected MET gene amplification had increased from þþ (>50thpercentile) to þþþ (>90th percentile). The patient discontinuedtreatment with glesatinib and died the following month.

DiscussionOncogenic drivers in NSCLC define targetable disease subtypes

for both approved and clinically active experimental drugs there-by altering the treatment landscape of NSCLC. The recent discov-ery and characterization ofMETex14 del mutations in addition tohigh-level MET gene amplification as defined oncogenic driverevents in NSCLC has renewed interest in MET targeted therapiesfor these genomically defined patient subsets. The clinical rele-vance ofMETex14 delmutations and amplification inNSCLC hasnow been established in multiple case report studies of durableresponses to small-molecule MET inhibitors (8, 11, 36, 37). Thepatient case presentation of a partial response included in thecurrent studies, in addition to previously presented data fromglesatinib clinical trials, provides additional support for the

clinical validity of targeting genetic alterations ofMET in NSCLC.In recognition of recent clinical findings, international guidelinesrecommending routine molecular profiling of NSCLC to identifyoncogenic drivers with promising therapies were updated toinclude METex14 del mutations along with EGFR, ALK, ROS1,BRAF, RET, and ERBB2 alterations.

Despite the impact of targeted therapies inmolecularly definedpatient subsets, most patients will develop resistance to therapyand will require alternative treatment strategies. Although mul-tiple resistance mechanisms have been identified, mutationsinvolving the drug target have emerged as a nearly universaltheme with key examples including kinase domain gatekeepermutations such as EGFR T790M or ABL T315I, solvent frontmutations such asALKG1202RorROS1G2032R, and irreversibleinhibitor binding site mutations such as EGFR C797S and BTKC481S (38).Ourobservations ofmutations atD1228, Y1230, andF1200 either linked to intrinsic resistance or following long-termdrug exposure to type I MET inhibitors are consistent withprevious studies. Multiple clinical case reports implicatingD1228 and Y1230 mutations in resistance to crizotinib haveemerged recently indicating that these mutations have clinicalrelevance (18–20).

A unique finding forMETmutation-mediated resistance is thatthe D1228 and Y1230 resistance mutations are located in theA-loop in contrast to the gatekeeper, solvent front, and bindingsite mutations implicated in resistance to other tyrosine kinaseinhibitors (38). The molecular basis of resistance of these uniqueA-loop mutations is evident from crystal structures of type Iinhibitors such as crizotinib and capmatinib but not type IIinhibitors including glesatinib, which bind independently ofA-loop interactions. Moreover, molecular modeling of glesatinibbound toMETand the ability to adopt twodistinct bindingmodesprovides a molecular explanation as to why glesatinib retainsactivity against mutations within the A-loop and why the onset of

Figure 6.

Glesatinib reduced the diameter of a subset of metastatic liver lesions and the percentage of cfDNA of a METex14 del and A-loop–mutant liver metastases.A–D, CT scans after crizotinib treatment prior to glesatinib treatment (A and B, top) and after 6 weeks of glesatinib treatment (C and D, bottom). "�" indicatesa liver metastasis that was biopsied after crizotinib therapy, sequenced, and found to harbor a Y1230 mutation; "#" indicates a second liver metastasis thatdecreased during glesatinib treatment; arrows indicate other lesions in the liver increased in size during glesatinib treatment. E, Graphic and tabular depictionof detectable MET variant allele as percent of total cfDNA at selected times prior to glesatinib administration and after glesatinib disease progression.






resistance to glesatinib was delayed compared with type I METinhibitors. Interestingly, the MET L1195V and F1200L/I muta-tions that were associated with resistance to both inhibitor classesin MET-mutant variant enzyme screens and/or resistance studiesoccur distal from the ligand binding site of type I inhibitors andmay result in enhanced substrate affinity or destabilization of theautoinhibited A-loop conformation. Consistent with theobserved resistance, biochemical screens in the current studiesindicated a 10-fold loss of activity for crizotinib for MET F1200Icompared with WT and this was the same amino acid that wasfound mutated in a crizotinib drug resistance screen (39).Although the MET F1200I mutation did not emerge as anacquired resistance mechanism during glesatinib drug resistancescreens, there is direct involvement of this residue in the type IIDFG-out binding mode, which would provide a clear molecularbasis for attenuated activity for glesatinib and other type IIinhibitors and is consistent with the 4-fold decrease in potencyin crizotinib-resistant cells that harbored the Y1230H/F1200Ldouble mutation. Interestingly, the type Ib inhibitor, savolitinib,demonstrated potent inhibitory activity against the F1200I var-iant suggesting it has a binding mode distinct from other type IbMET inhibitors and supports the utility of this agent as anotherpotential option to combat mutation mediated resistance viasequential treatment strategies. The relatively modest loss ofglesatinib activity against the L1195V and F1200L mutationsrelative to most other type I inhibitors suggests that glesatinibmay not rely solely on a single binding mode and may be able toadopt a conformation that attenuates loss of activity. Glesatinibwas also evaluated against a panel of twelve other known METmutations and activity was comparable with WT MET; thus, noadditional anticipated glesatinib resistance mutations have beenidentified (partial data summary in Supplementary Table S2).

The clinical case presentation of a Y1230H mutation–positivemetastatic lesion responding to glesatinib in the current studiesindicates that observations from nonclinical resistance studieshave clinical relevance. Although the overall therapeutic outcomefor this patient was cancer progression, it was notable that thispatient had extensivemetastatic disease burden and that multiplegenetic alterations were present in trans configuration at the timeof progression on both crizotinib and glesatinib. These observa-tions suggest that distinct resistance mutations present in sub-clonal metastatic lesions contributed to disease progression whilethe Y1230H-positive metastasis responded. The loss of detectionfor Y1230H and Y1230S allelic variants from the cfDNA analysisat the time of glesatinib progression likely reflect the impact oftreatment on the subclonal lesions bearing these mutations. Incontrast, the increased allelic cfDNAburden forMETD1228NandG1163R, increased MET amplification, and emergence of METL1195V suggest that subclonal lesions harboring these geneticvariants were nonresponsive to glesatinib treatment. The obser-vation ofMET G1163R prior to glesatinib treatment is consistentwith the decreased activity of crizotinib against this MET muta-tion, which is analogous to the ALK G1202R and ROS1 G2302Rmutations implicated in crizotinib resistance in ALK and ROS1rearrangement positive cancers, respectively (40). The emergenceof the L1195V mutation during glesatinib treatment is consistentwith the 5–7-fold loss of potency for both glesatinib and crizo-tinib and suggestive of the continued progression of the lesion(s)harboring this variant despite continued treatment. It is less clearwhy the MET D1228N variant continued to increase duringglesatinib treatment as this variant is predicted to be responsive

to glesatinib. However, this mutation was relatively less sensitiveto glesatinib comparedwith Y1230C/H and it is also possible thatsubclones bearing this and other nonresponding variants exhib-ited cooccurring non-MET genetic alterations that contributed toprogression and/or that local drug levels present at these sub-clonal lesions were insufficient to facilitate a response.

Finally, both clinical case presentations in the current studieswere identified through commercially available NGS-basedcfDNAprofiling. The clinical treatment outcomes in these patientssupports the potential utility of identifying patients harboringoncogenic driver alterations in settings for which tumor tissue isnot available for analysis or negative on tissue-based genotyping.This is further illustrated by the clinical case presentation of apatient responding to glesatinib with a low variant allele fraction(0.1%) which suggests that even patients with low detectablemutation burden may be suitable candidates for targeted thera-pies. In addition, the longitudinal cfDNAanalysis in the resistancecase study demonstrates the utility of cfDNA analysis in under-standing mechanisms of resistance over the course of treatmentand in identifying other potential therapeutic strategies to combatresistance. Furthermore, the presence of multiple transmutationsin the second clinical case presentation highlights the ability ofcfDNA to act as a global measure of tumor heterogeneity consis-tent with mutational heterogeneity reports in multiple metastaticlesions at rapid autopsy.

Collectively, the current studies further the understanding andsupport the clinical relevance of METex14 del mutations as anoncogenic driver class in NSCLC. In addition, these studiesindicate that although targeting MET driver mutations with selec-tive inhibitors has the potential to be an effective therapeuticstrategy, this strategy will also be subject to drug resistance likeother targeted therapies. MET active site mutations, particularlyA-loopmutations, are predicted to be an importantmechanismofresistance based on these and other emerging data as well asstructure-based modeling of the MET active site. Glesatinib andother type II inhibitors appear to be positioned to address resis-tance mediated by these mutations. Although other type II inhi-bitors that target MET such as cabozantinib and merestinib havebeen reported, glesatinib is the only type II MET inhibitor thatspares VEGF receptors at clinically achievable drug concentrationsindicating it may be uniquely positioned to address resistancewhile avoiding well-known dose-limiting toxicities associatedwith VEGFR inhibition. Another important observation is thattype I and type II inhibitors may have utility in sequentialtherapeutic strategies if orthogonal resistance mechanismsemerge for each inhibitor mode. Together, these studies demon-strate that glesatinib exhibits a differentiatedmechanism of targetinhibition, is active against METex14 del–mutant lung cancer,including tumors exhibiting certain acquired resistance to otherMET inhibitor classes, and glesatinib may represent a therapeuticoption for these cancers.

Disclosure of Potential Conflicts of InterestC. Essenburg, Z.B. Madaj, and C.R. Graveel report receiving commercial

research grants fromMirati Therapeutics. R.B. Lanman holds ownership interest(including patents) in Guardant Health, Inc. M. Nishino reports receivingcommercial research grants fromMerck and Toshiba Medical Systems, speakersbureau honoraria from Bayer, and is a consultant/advisory board member forBristol-Myers Squibb, Toshiba Medical Systems, and WorldCare Clinical. G.I.Shapiro is a consultant/advisory boardmember for Lilly and Pfizer. M.M. Awadreports receiving speakers bureau honoraria from Pfizer. P.A. Janne reportsreceiving commercial research grants from Astellas, AstraZeneca, Daiichi

Engstrom et al.





Sankyo, Eli Lilly, and PUMA, holds ownership interest (including patents) inGatekeeper Pharmaceuticals, and is a consultant/advisory board member forAriad, AstraZeneca, Boehringer Ingelheim, Chugai Pharmaceuticals, Eli Lilly,Ignyta, LOXO Oncology, Pfizer, and Roche/Genentech. J.G. Christensen holdsownership interest (including patents) in Mirati Therapeutics. No potentialconflicts of interest were disclosed by the other authors.

Authors' ContributionsConception and design: L. Engstrom, M. Hidalgo, P. Olson, L. Bazhenova,M. Nishino, N. Peled, M.M. Awad, J.G. ChristensenDevelopment of methodology: L. Engstrom, E.A. Tovar, N. Banos,C. Menendez, M. Hidalgo, R.B. Lanman, N. PeledAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): L. Engstrom, R. Aranda, E.A. Tovar, C. Essenburg,H. Chiang, D. Briere, J. Hallin, P.P. Lopez-Casas, N. Banos, C. Menendez,M. Hidalgo, V. Tassell, D.I. Chudova, R.B. Lanman, L. Bazhenova, S.P. Patel,C.R. Graveel, M. Nishino, G.I. Shapiro, M.M. AwadAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): L. Engstrom, R. Aranda, M. Lee, E.A. Tovar,Z.B. Madaj, H. Chiang, M. Hidalgo, R. Chao, R.B. Lanman, P. Olson,L. Bazhenova, S.P. Patel, G.I. Shapiro, N. Peled, M.M. Awad, P.A. Janne,J.G. Christensen

Writing, review, and/or revision of the manuscript: L. Engstrom, M. Lee,Z.B. Madaj, P.P. Lopez-Casas, M. Hidalgo, V. Tassell, R. Chao, R.B. Lanman,P. Olson, L. Bazhenova, S.P. Patel, M. Nishino, G.I. Shapiro, N. Peled,M.M. Awad, P.A. Janne, J.G. ChristensenAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): L. Engstrom, P.P. Lopez-Casas, N. Banos,R.B. Lanman, L. Bazhenova, S.P. Patel, J.G. ChristensenStudy supervision: L. Engstrom, M. Hidalgo, P. Olson, L. Bazhenova,J.G. Christensen

Grant SupportE.A. Tovar, C.J. Essenburg, and C. Graveel (Van Andel Research Institute) and

P.P. Lopez-Casas, N. Ba~nos, C. Menendez, andM. Hidalgo (Centro Nacional deOncol�ogicas) received research funding from Mirati Therapeutics.

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 April 24, 2017; revised June 13, 2017; accepted July 24, 2017;published OnlineFirst August 1, 2017.

References1. Furlan A, Kherrouche Z,Montagne R, CopinMC, Tulasne D. Thirty years of

research on met receptor to move a biomarker from bench to bedside.Cancer Res 2014;74:6737–44.

2. Gherardi E, BirchmeierW, Birchmeier C, VandeWoudeG. TargetingMET incancer: rationale and progress. Nat Rev Cancer 2012;12:89–103.

3. Trusolino L, Bertotti A, Comoglio PM. MET signalling: principles andfunctions in development, organ regeneration and cancer. Nat Rev MolCell Biol 2010;11:834–48.

4. Frampton GM, Ali SM, Rosenzweig M, Chmielecki J, Lu X, Bauer TM, et al.Activation of MET via diverse exon 14 splicing alterations occurs inmultiple tumor types and confers clinical sensitivity to MET inhibitors.Cancer Discov 2015;5:850–9.

5. Kong-Beltran M, Seshagiri S, Zha J, Zhu W, Bhawe K, Mendoza N, et al.Somatic mutations lead to an oncogenic deletion of met in lung cancer.Cancer Res 2006;66:283–9.

6. Liu X, Jia Y, Stoopler MB, Shen Y, Cheng H, Chen J, et al. Next-generationsequencing of pulmonary sarcomatoid carcinoma reveals high frequencyof actionable MET gene mutations. J Clin Oncol 2016;34:794–802.

7. Onozato R, Kosaka T, Kuwano H, Sekido Y, Yatabe Y, Mitsudomi T.Activation of MET by gene amplification or by splice mutations deletingthe juxtamembrane domain in primary resected lung cancers. J ThoracOncol 2009;4:5–11.

8. AwadMM,Oxnard GR, JackmanDM, Savukoski DO,Hall D, Shivdasani P,et al. MET exon 14 mutations in non-small-cell lung cancer are associatedwith advanced age and stage-dependentMET genomic amplification and c-Met overexpression. J Clin Oncol 2016;34:721–30.

9. Tong JH, Yeung SF, Chan AW, Chung LY, Chau SL, Lung RW, et al. METamplification and exon 14 splice site mutation define unique molecularsubgroups of non-small cell lung carcinoma with poor prognosis. ClinCancer Res 2016;22:3048–56.

10. Jorge SE, Schulman S, Freed JA, VanderLaan PA, Rangachari D, KobayashiSS, et al. Responses to the multitargeted MET/ALK/ROS1 inhibitor crizo-tinib and co-occurring mutations in lung adenocarcinomas with METamplification or MET exon 14 skipping mutation. Lung Cancer 2015;90:369–74.

11. Paik PK,DrilonA, FanPD, YuH, RekhtmanN,GinsbergMS, et al. Responseto MET inhibitors in patients with stage IV lung adenocarcinomas harbor-ing MET mutations causing exon 14 skipping. Cancer Discov 2015;5:842–9.

12. Schrock AB, Frampton GM, Suh J, Chalmers ZR, Rosenzweig M, Erlich RL,et al. Characterization of 298 patients with lung cancer harboring METexon 14 skipping alterations. J Thorac Oncol 2016;11:1493–502.

13. Zheng D, Wang R, Ye T, Yu S, Hu H, Shen X, et al. MET exon 14 skippingdefines a unique molecular class of non-small cell lung cancer. Oncotarget2016;7:41691–702.

14. Awad MM, Shaw AT. ALK inhibitors in non-small cell lung cancer: crizo-tinib and beyond. Clin Adv Hematol Oncol 2014;12:429–39.

15. KwakEL, SordellaR, BellDW,Godin-HeymannN,OkimotoRA, BranniganBW, et al. Irreversible inhibitors of the EGF receptor may circumventacquired resistance to gefitinib. Proc Natl Acad Sci U S A 2005;102:7665–70.

16. Pao W, Miller VA, Politi KA, Riely GJ, Somwar R, Zakowski MF, et al.Acquired resistance of lung adenocarcinomas to gefitinib or erlotinib isassociated with a second mutation in the EGFR kinase domain. PLoS Med2005;2:e73.

17. Zhang Z, Lee JC, Lin L,Olivas V, AuV, LaFramboise T, et al. Activation of theAXL kinase causes resistance to EGFR-targeted therapy in lung cancer. NatGenet 2012;44:852–60.

18. Dong HJ, Li P, Wu CL, Zhou XY, Lu HJ, Zhou T. Response and acquiredresistance to crizotinib in Chinese patients with lung adenocarcinomasharboring MET Exon 14 splicing alternations. Lung Cancer 2016;102:118–21.

19. Heist RS, Sequist LV, Borger D, Gainor JF, ArellanoRS, Le LP, et al. Acquiredresistance to crizotinib in NSCLC with MET exon 14 skipping. J ThoracOncol 2016;11:1242–5.

20. Ou SI, Young L, Schrock AB, Johnson A, Klempner SJ, Zhu VW, et al.Emergence of preexisting MET Y1230C mutation as a resistance mecha-nism to crizotinib in NSCLC with MET exon 14 skipping. J Thorac Oncol2017;12:137–40.

21. Backes A, Zech B, Felber B, Klebl B, Muller G. Small-molecule inhibitorsbinding to protein kinase. Part II: the novel pharmacophore approachof type II and type III inhibition. Expert Opin Drug Discov 2008;3:1427–49.

22. Cui JJ. Targeting receptor tyrosine kinase MET in cancer: small moleculeinhibitors and clinical progress. J Med Chem 2014;57:4427–53.

23. Zhang J, Yang PL, Gray NS. Targeting cancer with small molecule kinaseinhibitors. Nat Rev Cancer 2009;9:28–39.

24. Henry RE, Barry ER, Castriotta L, Ladd B, Markovets A, Beran G, et al.Acquired savolitinib resistance in non-small cell lung cancer arises viamultiplemechanisms that converge onMET-independentmTORandMYCactivation. Oncotarget 2016;7:57651–70.

25. Qi J, McTigue MA, Rogers A, Lifshits E, Christensen JG, Janne PA, et al.Multiple mutations and bypass mechanisms can contribute to develop-ment of acquired resistance to MET inhibitors. Cancer Res 2011;71:1081–91.

26. Weber H, Muller D, Muller M, Ortiz A, Birkle M, Umber S, et al. Cell linesexpressing recombinant transmembrane domain-activated receptorkinases as tools for drug discovery. J Biomol Screen 2014;19:1350–61.

27. Berman HM, Westbrook J, Feng Z, Gilliland G, Bhat TN, Weissig H, et al.The protein data bank. Nucleic Acids Res 2000;28:235–42.






28. Lanman RB, Mortimer SA, Zill OA, Sebisanovic D, Lopez R, Blau S, et al.Analytical and clinical validation of a digital sequencing panel for quan-titative, highly accurate evaluationof cell-free circulating tumorDNA. PLoSOne 2015;10:e0140712.

29. Abella JV, Peschard P, Naujokas MA, Lin T, Saucier C, Urbe S, et al. Met/Hepatocyte growth factor receptor ubiquitination suppresses transforma-tion and is required for Hrs phosphorylation. Mol Cell Biol 2005;25:9632–45.

30. Liu L, SiegmundA, XiN, Kaplan-Lefko P, RexK, ChenA, et al.Discovery of apotent, selective, and orally bioavailable c-Met inhibitor: 1-(2-hydroxy-2-methylpropyl)-N-(5-(7-methoxyquinolin-4-yloxy)pyridin-2-yl)-5-methyl-3-oxo-2-phenyl-2,3-dihydro-1H-pyrazole-4-carboxamide (AMG 458).J Med Chem 2008;51:3688–91.

31. Munshi N, Jeay S, Li Y, Chen CR, France DS, Ashwell MA, et al. ARQ 197, anovel and selective inhibitor of the human c-Met receptor tyrosine kinasewith antitumor activity. Mol Cancer Ther 2010;9:1544–53.

32. Pennacchietti S, Cazzanti M, Bertotti A, Rideout WM III, Han M, Gyuris J,et al. Microenvironment-derived HGF overcomes genetically determinedsensitivity to anti-MET drugs. Cancer Res 2014;74:6598–609.

33. Rong S, Bodescot M, Blair D, Dunn J, Nakamura T, Mizuno K, et al.Tumorigenicity of the met proto-oncogene and the gene for hepatocytegrowth factor. Mol Cell Biol 1992;12:5152–8.

34. Zhang YW, Su Y, Lanning N, Gustafson M, Shinomiya N, Zhao P, et al.Enhanced growth of human met-expressing xenografts in a new strain of

immunocompromised mice transgenic for human hepatocyte growthfactor/scatter factor. Oncogene 2005;24:101–6.

35. Zhang YW, Staal B, Essenburg C, Su Y, Kang L, West R, et al. MET kinaseinhibitor SGX523 synergizes with epidermal growth factor receptor inhib-itor erlotinib in a hepatocyte growth factor-dependent fashion to suppresscarcinoma growth. Cancer Res 2010;70:6880–90.

36. Mendenhall MA, Goldman JW.MET-mutatedNSCLCwithmajor responseto crizotinib. J Thorac Oncol 2015;10:e33–4.

37. Ou SH, Kwak EL, Siwak-Tapp C, Dy J, Bergethon K, Clark JW, et al. Activityof crizotinib (PF02341066), a dual mesenchymal-epithelial transition(MET) and anaplastic lymphoma kinase (ALK) inhibitor, in a non-smallcell lung cancer patient with de novo MET amplification. J Thorac Oncol2011;6:942–6.

38. Mancini M, Yarden Y. Mutational and network level mechanisms under-lying resistance to anti-cancer kinase inhibitors. Semin Cell Dev Biol2016;50:164–76.

39. Tiedt R, Degenkolbe E, Furet P, Appleton BA,Wagner S, Schoepfer J, et al. Adrug resistance screen using a selective MET inhibitor reveals a spectrum ofmutations that partially overlap with activating mutations found in cancerpatients. Cancer Res 2011;71:5255–64.

40. Davare MA, Vellore NA, Wagner JP, Eide CA, Goodman JR, Drilon A,et al. Structural insight into selectivity and resistance profiles ofROS1 tyrosine kinase inhibitors. Proc Natl Acad Sci U S A 2015;112:E5381–90.


Engstrom et al.




2017;23:6661-6672. Published OnlineFirst August 1, 2017.Clin Cancer Res Lars D. Engstrom, Ruth Aranda, Matthew Lee, et al. Nonclinical ModelsMutation-mediated Resistance to Type I MET Inhibitors in

Exon 14 Mutations and OvercomesMETPatients Harboring Glesatinib Exhibits Antitumor Activity in Lung Cancer Models and

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Glesatinib Exhibits Antitumor Activity in Lung Cancer ... · Biology of Human Tumors Glesatinib Exhibits Antitumor Activity in Lung Cancer Models and Patients Harboring MET Exon 14