Discovery and Therapeutic Exploitation of …...Baomin Wang1, Ciana Deveau1, Karim Saoud1, Isela Gallagher3, Julia Wulfkuhle3, David Schiff 4 , See Phan 5 , Emanuel Petricoin 3 , and

Translational Cancer Mechanisms and Therapy

Discovery and Therapeutic Exploitation ofMechanisms of Resistance to MET Inhibitors inGlioblastomaNichola Cruickshanks1, Ying Zhang1, Sarah Hine1, Myron Gibert1, Fang Yuan1,Madison Oxford1, Cassandra Grello1, Mary Pahuski1, Collin Dube1, Fadila Guessous1,2,Baomin Wang1, Ciana Deveau1, Karim Saoud1, Isela Gallagher3, Julia Wulfkuhle3,David Schiff4, See Phan5, Emanuel Petricoin3, and Roger Abounader1,4,6

Abstract

Purpose: Glioblastoma (GBM) is the most common andmost lethal primary malignant brain tumor. The receptortyrosine kinaseMET is frequently upregulated or overactivatedin GBM. Although clinically applicable MET inhibitors havebeen developed, resistance to single modality anti-MET drugsfrequently occurs, rendering these agents ineffective.We aimedto determine the mechanisms of MET inhibitor resistance inGBM and use the acquired information to develop noveltherapeutic approaches to overcome resistance.

Experimental Design: We investigated two clinicallyapplicable MET inhibitors: crizotinib, an ATP-competitivesmall molecule inhibitor of MET, and onartuzumab, amonovalent monoclonal antibody that binds to the extra-cellular domain of the MET receptor. We developed newMET inhibitor–resistant cells lines and animal models andused reverse phase protein arrays (RPPA) and functional

assays to uncover the compensatory pathways in METinhibitor–resistant GBM.

Results: We identified critical proteins that were altered inMET inhibitor–resistant GBM includingmTOR, FGFR1, EGFR,STAT3, and COX-2. Simultaneous inhibition of MET and oneof these upregulated proteins led to increased cell death andinhibition of cell proliferation in resistant cells compared witheither agent alone. In addition, in vivo treatment of micebearing MET-resistant orthotopic xenografts with COX-2 orFGFR pharmacological inhibitors in combination with METinhibitor restored sensitivity to MET inhibition and signifi-cantly inhibited tumor growth.

Conclusions: These data uncover the molecular basis ofadaptive resistance to MET inhibitors and identify new FDA-approved multidrug therapeutic combinations that can over-come resistance.

IntroductionGlioblastoma (GBM) is the most common and most lethal

primary malignant brain tumor (1). Current standard of careincludes surgical resection, radiotherapy, and chemotherapy.Prognosis remains poor with a median survival of 15 months(2). New therapies targeting common aberrations in signal trans-duction pathways in GBM are currently being investigated (3).Dysregulation of receptor tyrosine kinases (RTK) have been found

in approximately 90% of GBM (4, 5). Consequently, tyrosinekinase inhibitors have been developed for anticancer therapy.

MET is an RTK that is essential for embryonic development andtissue repair (2, 6). Hepatocyte growth factor (HGF), the onlyknown ligand for MET, activates MET and downstream signalingpathways including RAS/MAPK, PI3K/AKT and STAT (7–9). METis commonly dysregulated in GBM via various mechanismsincluding somatic mutations, rearrangement, amplification andoverexpression of MET and HGF that leads to autocrine loopformation (10–12). Furthermore, MET expression inversely cor-relates with patient survival (12, 13) and is upregulated in GBM(5, 14, 15).

Several MET inhibitors are under investigation with mixedresults in clinical trials (14). Crizotinib is an FDA-approvedATP-competitive small-molecule inhibitor of MET. Approved forthe treatment of advanced andmetastatic ALK-positive non–smallcell lung cancer (NSCLC), crizotinib is in phase II clinical trials forCNS and solid brain tumors (5, 16). InNSCLCpatients, crizotinibdisplays initial potent anticancer activity. However, acquiredresistance frequently ensues, rendering this drug ineffective as amonotherapy (17). Onartuzumab is a monovalent monoclonalantibody that competes with HGF for binding to MET (18).Previous clinical trials involving onartuzumab as a monotherapyin NSCLC patients have been disappointing, highlighting theimportance of understanding resistance mechanisms to the drug.

1Department of Microbiology, Immunology, and Cancer Biology, University ofVirginia, Charlottesville, Virginia. 2University Mohammed 6 for Health Sciences,Casablanca, Morocco. 3George Mason University Center for Applied Proteomicsand Molecular Medicine, Manassas, Virginia. 4Department of Neurology, Uni-versity of Virginia, Charlottesville, Virginia. 5Genentech Inc. South San Francisco,California. 6The Cancer Center, University of Virginia, Charlottesville, Virginia.

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

N. Cruickshanks and Y. Zhang contributed equally to the article.

Corresponding Author: Roger Abounader, University of Virginia, Old MedicalSchool, Room 4813, PO Box 800168, Charlottesville VA 22908. Phone: 434-982-6634; E-mail: [email protected]

doi: 10.1158/1078-0432.CCR-18-0926

�2018 American Association for Cancer Research.

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Although MET inhibitors have displayed initial efficacy,acquired resistance to single agent modalities invariably occurs(19). The development of acquired resistance to monotherapyencompasses multiple mechanisms such as acquisition of sec-ondary mutations in therapeutic targets, activation of bypasssignaling pathways, or immune evasion (20). Deciphering theexact mechanism of acquired resistance would be advantageousto halt disease progression in GBM patients by using combi-natorial therapies (20–22). This study had two aims: (i) toelucidate the bypass signaling pathways that are activated whenGBM cells acquire MET inhibitor resistance; (ii) to developcombinatorial drug therapies against MET inhibitor–resistantGBM. Our data uncovered a number of signaling moleculesthat are altered in MET inhibitor–resistant cell lines comparedwith sensitive cell lines. Upregulation of RTKs such as fibroblastgrowth factor receptor (FGFR) and EGFR, signaling moleculessuch as mTOR and signal transducer and activator of transcrip-tion 3 (STAT3), as well as elevation of cyclooxygenase 2(COX-2) emphasized the extent of cross-talk between multiplesignaling pathways in MET inhibitor–resistant GBM. Multi-targeted combinational therapies against these molecules over-came single-agent MET inhibitor resistance, paving the way fornew therapeutic approaches that could be tested in clinicaltrials.

Materials and MethodsCells and tumor specimens

Human GBM cell lines (U87 and U373) and stem cell line(GSC827) were used. U87 and U373 were from the ATCC andwere authenticated through short tandem repeat (STR) pro-filing. GSC827 was isolated from GBM specimens and char-acterized for tumorigenesis, pluripotency, self-renewal, stemcell markers, and neurosphere formation (23). The specimenswere obtained with written informed consent by the patientsand the studies conducted in accordance with recognizedethical guidelines and approved by the institutional reviewboard of the Cleveland Clinic. All cell lines were tested formycoplasma.

Developing MET inhibitor–resistant cell linesU87, U373, and GSC827 cells were exposed to increasing

concentrations of either crizotinib (from 1 to 100 nmol/L) orOnartuzumab (form 1 to 300 nmol/L) over a period of 6months.Cell lines were then tested for MET inhibitor resistance throughtrypanblue death assay as previously described (24). For this, cellswere treated with MET inhibitor [crizotinib 100 nmol/L or Onar-tuzumab 300 nmol/L] for 48 hours, collected and stained withtrypan blue reagent. MET inhibitor–resistant cells were growncontinually in the presence of either 100 nmol/L crizotinib or300 nmol/L Onartuzumab.

Cell death and cell proliferation assaysCell death was assessed by trypan blue assay as previously

described (24). Cell proliferation was assessed by cell countingas previously described (25). All experiments were performedin triplicate. Apoptotic cell death was assessed by pre-treatingcells with ZVAD (20 nmol/L) followed by combinational drugtreatment for 48 hours. Cell death was determined as describedabove.

Reverse phase protein arraysProteomic screening was performed by reverse phase protein

array (RPPA) as previously described (26–28). The cells weretreated as described, in triplicates. Protein signaling analyteswere chosen based on their previously described involvementin key aspects of tumor biology. Detection was performedusing a fluorescence-based tyramide signal amplification strat-egy using Streptavidin-conjugated IRDye680 (LI-COR Bios-ciences, Lincoln NE) detection reagent. All antibodies werevalidated for single band specificity and for ligand-induction(for phospho-specific antibodies) by immunoblotting beforeuse on the arrays as previously described (26–28). Each arraywas scanned using a TECAN LS (Vidar Systems Corporation).Spot intensity was analyzed, data were normalized to totalprotein and a standardized, single data value was generated foreach sample on the array by MicroVigene software V2.999(VigeneTech).

ImmunoblottingImmunoblotting was performed as previously described (29).

Antibodies used were p.FGFR1, FGFR1, p.ERK, ERK, p.AKT, AKT,p.MET, p.mTOR, mTOR, COX-2 and p.STAT3 (Cell SignalingTechnology), MET and GAPDH (Santa Cruz Biotechnology).

Mechanistic studies of MET-inhibitor resistanceFunctional rescue experiments were performed to determine

whether molecules identified through RPPA screenings mediateresistance to MET inhibitors. Cells were pretreated with celecoxib(inhibits COX-2), debio-1347 (inhibits FGFR1), erlotinib (inhi-bits EGFR), rapamycin (inhibits mTOR) or STAT3 inhibitor asdescribed for 2 hours then treated with either crizotinib orOnartuzumab for 48 hours. Cell death and proliferation wasassessed as described above.

Cell transfectionsU87 cells were transfected with either scrambled siRNA

(control), si-FGFR1, si-COX2, si-RON, si-RET, si-Vimentin orsi-ERBB3 (Thermo Fisher Scientific) then treated with crizotinib(100 nmol/L) for 48 hours. Knockdownwas confirmed by immu-noblotting. Cells death was determined as described above.

Translational Relevance

The receptor tyrosine kinase MET is frequently upregulatedor overactivated in many cancers, including GBM. Conse-quently, several clinically applicableMET inhibitors have beendeveloped. Although MET inhibitors initially display antican-cer activity, resistance to the drugs frequently occurs, renderingthese agents ineffective. Elucidating the mechanisms ofacquired resistance to MET inhibitors is a challenge that mustbe overcome to halt disease progression. We used proteomicscreenings to identify pathways altered in response to acquiredMET inhibitor resistance inGBM.Weuncovered several criticalsignaling molecules that mediated resistance to two clinicallyapplicable anti-MET drugs that were previously tested inclinical trials. Inhibition of these molecules with clinicallyapplicable drugs reversed resistance to MET inhibitors. Ourdata uncover the mechanisms of adaptive resistance to METinhibitors and describe new combination therapies that over-come resistance and that could be tested in clinical trials.

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In vivo drug combination studiesCombination therapies to overcome resistance were assessed

using an orthotopic xenograft mouse model. U87 cells (3 � 105)were stereotactically implanted into the right corpus striatum ofimmunodeficient mice (n ¼ 10 per treatment group). Six daysafter implantation, the animals were treated with either controlvehicle (DMSO), agent either alone [debio-1347 (25 mg/kg) orcelecoxib (10 mg/kg), crizotinib (25 mg/kg)] or in combination[debio-1347þcrizotinib or celecoxibþcrizotinib] by oral gavagedaily from day 6 post-tumor implantation for 7 days. Tumorvolumes were visualized and quantified by MRI. These studieswere approved by the University of Virginia Animal Care and UseCommittee.

Statistical analysesThe continuous variable RPPA data generatedwere subjected to

both unsupervised and supervised statistical analyses, as previ-ously described (24). Statistical analyses were performed on finalmicroarray intensity values obtained using R version 2.9.2software (The R Foundation for Statistical Computing). If thedistribution of variables for the analyzed groups were normal, a

two-sample t test was performed. If the variances of two groupswere equal, two-sample t test with a pooled variance procedurewas used to compare the means of intensity between two groups.Otherwise, two-sample t test without a pooled variance procedurewas adopted. For non-normally distributed variables, theWilcoxon rank sum test was used. Significance levels were set atP < 0.05. To evaluate the statistical significance of the differencebetween wildtype and resistant GBM cell proliferation and death,treated and control, we used a two sample t test and significancelevels were set at P < 0.05. To evaluate the statistical significance ofthe difference between each treated and control animal groupsin vivo, we used both two-sample t test and non-parametricWilcoxen rank-sum test.

ResultsGeneration of MET inhibitor–resistant GBM cell lines

Crizotinib-resistant and onartuzumab-resistant GBM cell linesfrom U87, U373, and GSC827 were generated through doseescalation of each drug over 6 months until the cells were nolonger sensitive to crizotinib at a concentration of 100 nmol/L or

Figure 1.

Generation and testing of MET-resistant GBM cells and GSCs. A, U87; B, U373; and C, GSC827 cells were exposed to increasing concentrations of either crizotinib oronartuzumab (Onart) until resistancewas confirmed at 100 nmol/L for crizotinib-resistant (CR) and 300nmol/L forOnart-resistant (OR), respectively, by trypan blueassay. The data confirm the generation of MET inhibitor–resistant cells. D, U87 WT and CR cells were treated with crizotinib (100 nmol/L) for 48 hours. Cellproliferation was assessed by cell counting over a period of 5 days and growth curves were established. � , P < 0.05.

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onartuzumab at a concentration of 300 nmol/L (Fig. 1A–D).When compared with the wild-type cells, the resistant cell linesexhibited no change in cell survival and proliferation whentreated with either crizotinib or onartuzumab for 48 hours.Treatment of wildtype GBM cells with crizotinib or onartuzumabresulted in an antiproliferative effect and induction of apoptosis.Conversely, in crizotinib-resistant and onartuzumab-resistantGBM cells, cell survival and proliferation remained unaffected.

Discovery of bypass signaling pathways in resistant cellsTo elucidate the mechanism(s) of resistance of GBM cells to

crizotinib and onartuzumab, we used proteomic screening withRPPA to compare protein expression and activation changesbetween wildtype and resistant GBM cells when treated with theMET inhibitors for 48 hours. The experiment was performed intriplicate. The data revealed several upregulated signaling path-ways in the resistant cells. The most changed molecules were: p.

Figure 2.

MET inhibitor resistance is mediatedby activation and expression changesin vital oncogenic molecules/pathways. A, U87 wild type (WT),crizotinib-resistant (CR) andonartuzumab-resitant (OR) cells weretreated with either control, crizotinib(100 nmol/L) or onartuzumab (Onart;300 nmol/L) for 48 hours and the celllysate was subjected to RPPA. Themost significantly changed moleculesare shown in this figure. Among other,MET inhibitor resistance resulted in theupregulation of receptor tyrosinekinase FGFR1 and important survivalsignalingmolecules mTOR and COX-2.B, U373 WT and CR cells were treatedwith either control or crizotinib (100nmol/L) for 48 hours and the celllysate was subjected to RPPA.Notably, resistance resulted in theupregulation of EGFR and STAT3. C,RPPA data were verified byimmunoblotting. These data show thatMET inhibitor resistance is mediatedvia multiple signaling molecules.

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EGFR, cABL, ALDH, Cyclin A and PDL1 increased 26.7, 4.3, 3.8,3.3, and 3 fold respectively in crizotinib-resistant U373 cells; p.EGFR, p.ATPCL, and PDL1 increased 7.7, 2.6, 2.3-fold, respec-tively, in onartuzumab-resistant U373 cells; p.NFkB p65 andERBB3 increased 2.7, 2-fold, respectively, in crizotinib-resistantU87 cells and COX-2, p.SRC, and p.ERK increased 2.7, 2.4, and2.3-fold, respectively, in onartuzumab-resistant U87 cells com-pared with the wildtype after MET inhibitor treatment (Fig. 2).Additional significantly changed molecules are shown in Supple-mentary Figs. S2 and S3. In particular, MET inhibitor resistanceresulted in the activation of the EGFRpathway, the FGFRpathway,the mTOR pathway, STAT3, and COX-2. Increased activation ofthese molecules in resistant cells was further induced by addi-tional treatment with MET inhibitors, forcing the resistant cells toupregulate existing bypass signaling pathways. These expressionchanges were validated by immunoblotting (Fig. 2C). Severalother changed molecules were also assessed but could either notbe confirmed via immunoblotting, had no effect on the reversal ofMET resistance, or had no available specific pharmacologicalinhibitors. These molecules were therefore not further investigat-ed (Supplementary Table S1). Overall, the data demonstrate thatMET inhibitor resistance is mediated through multiple key reg-ulatory pathways that compensate for inhibition of the METpathway (Fig. 2A and B, Supplementary Figs. S2 and S3). Cel-ecoxib and debio-1347 were selected for evaluation in vivo,attributed to this, inhibition of COX-2 by celecoxib and FGFRby debio-1347, along with the effect on downstream pathways,was confirmed though immunoblotting (Supplementary Fig. S1).

Inhibition of bypass signaling pathways overcomes acquiredresistance to MET inhibitor

To determine whether inhibition of select bypass-signalingpathways can re-sensitize the resistant cells to MET inhibitors,we treated the cells with MET inhibitors in combination with aninhibitor of one bypass signaling pathway and assessed the cellproliferation and death. A total of nine bypass signaling targetsand their inhibitors were selected on the basis of the availability ofdrugs that inhibit them. These targets included FGFR1, EGFR,mTOR, STAT3,COX-2, RON,RET, Vimentin andERBB3.Wildtypeand resistant U87, U373, and GSC827 cells were pretreated for2 hours with (i). COX-2 inhibitor, celecoxib (100 nmol/L, 25nmol/L or 5 nmol/L, respectively), (ii). FGFR1 inhibitor, debio-1347 (10 mmol/L for U87 or 5 mmol/L for U373 and GSC827),(iii). mTOR inhibitor, rapamycin (100 nmol/L for U87 or25 nmol/L for U373 andGSC827), (iv). STAT3 inhibitor (STAT3i;50 mmol/L for U87 or 25 mmol/L for U373 and GSC827) or (v).EGFR inhibitor, erlotinib (100 nmol/L for U87 or 25 nmol/L forU373 and GSC827) then subjected to either crizotinib (100nmol/L) or onartuzumab (300 nmol/L) for 48 hours. When usedalone, all five inhibitors decreased cell proliferation and increasedcell death in wildtype cells but not in the resistant cells. However,when the resistant cells were treated with aMET inhibitor and oneof thefive inhibitors, weobserved increased cell death (Fig. 3A andB, 4A and B and 5A and B, Supplementary Figs. S4A–S4C, S5A–S5B, and S6A–S6E) and decreased proliferation (Fig. 3C–F, 4C–Fand 5C andD, Supplementary Figs. S4D–S4I, S5C–S5F, and S6F–S6O), indicating restored sensitivity to MET inhibitors. Inhibi-tions of RON, RET, Vimentin and ERBB3 either did not success-fully restore sensitivity to MET inhibitors or clinically applicableinhibitors were not available for use (Supplementary Fig. S1H–

S1K). In addition, to determine the predominant mode of cell

death mediated by the combination of celecoxib and crizotinib,we pretreated U87 WT and CR cells with ZVAD for 30 minutesbefore treating with celecoxib followed by crizotinib for 48 hoursthen assessed the effect on cell death.We show that ZVAD reducedcell death caused by the combinational treatment, indicating thatapoptosis as the preliminary mode of cell death (SupplementaryFig. S4J and S4K). The above data demonstrate that when com-bined with either crizotinib or onartuzumab, celecoxib, debio-1347, rapamycin, STAT3i and erlotinib are all partially butsignificantly effective at overcoming MET inhibitor resistance inGBM cells. P values are stated in the figure legends.

COX2 and FGFR1 siRNA-mediated knockdown induces celldeath in crizotinib-resistant cells

To confirm that the cell death induced by celecoxib and debio-1347 was attributed to inhibition of COX2 and FGFR1 respec-tively, we silenced both COX2 and FGFR1 with siRNA andanalyzed the effect on cell death. Cells were transfected witheither si-control, si-COX2 or si-FGFR1 then treatedwith crizotinibfor 48 hours. siRNA-mediated knockdown of COX2 and FGFR1was verified by immunobloting (Supplementary Fig. S1G). Toassess whether COX2 or FGFR1 silencing sensitized-resistant cellsto MET inhibitors as celecoxib (inhibits COX2) and debio-1347(inhibits FGFR1), cellswere transfected as above anda trypanblueassaywas performed. Thedata showed that silencing ofCOX2andFGFR1 restored MET inhibitor sensitivity in crizotinib-resistantcells (Supplementary Fig. S1E and SF). The above data show thatsilencing of either COX2 or FGFR1 expression leads to compara-ble anticancer effects on crizotinib-resistant U87 cells as celecoxibor debio-1347 treatment.

Combinational treatment is also effective against GSCsGSCs play an important role in mediating resistance to cyto-

toxic therapies (5). MET inhibition reduces GSC populationsensitizing the tumor to therapies (5). With this in mind, wetested these combinational therapies on resistant and wildtypeGSC827 and assessed the effect on cell death and proliferation.Although the concentration of all inhibitors (except MET inhibi-tors) had to be decreased due to toxicity, we observed a similarpattern as seen with U87 and U373 cells. All single-agent treat-ments, although effective in wildtype GSC827 cells, were inade-quate in MET inhibitor–resistant GSC827 cells whilst simulta-neous inhibition of a bypass signaling pathway (EGFR, FGFR,mTOR, STAT3 or COX-2) and MET inhibitor demonstrated sig-nificant induction of cell death and a dramatic suppression of cellproliferation. Importantly, combinational treatment with cele-coxib and crizotinib decreased tumor cell proliferation andincreased cell death significantly in crizotinib-resistant GSC827cells. These data suggest that similar pathways are implicated inMET inhibitor resistance in GSCs and GBM cells, indicating thatthese therapies may prove to be an extremely effective therapy forresistant GBM (Supplementary Fig. S6).

COX2 or FGFR1 inhibitions reverse resistance and cooperatewith MET inhibitor to inhibit GBM xenograft growth in vivo

To examine whether these bypass signaling pathways canovercome MET inhibitor resistance in vivo, we assessed the effectof crizotinib alone and in combination with celecoxib and debio-1347 on tumor growth in immunodeficient mice bearing GBMxenografts. Celecoxib is an FDA-approved drug that demonstratespotent anti-cancer properties through themodulation of both the






pro-survival BCL-2 (30) family and COX-2, and is currently inclinical trials for the treatment of numerous neoplasms. Debio-1347 is in clinical trials for the treatment of advanced solid tumorsand metastatic breast cancer with FGFR alterations. We injectedeither wildtype or resistant U87 cells into the striata of immuno-deficient mice, six days after implantation the animals weretreated with either control (DMSO), either agent alone[Debio-1347 (25 mg/kg), celecoxib (10 mg/kg) or crizotinib(25 mg/kg)] or in combination [Debio-1347þcrizotinib or cel-ecoxibþcrizotinib] by oral gavage daily for 7 days. Tumors werevisualized by MRI and volumes were quantified. The data showthat both therapeutic combinations inhibited tumor growth incrizotinib-resistant mice significantly more than either agentalone (Fig. 6A and B). The resistant cells displayed significant

tumor volume reduction when subjected to combinational treat-ment indicating restored sensitivity to MET inhibitors.

DiscussionAlthough the MET pathway is often dysregulated in GBM, MET

inhibitors have not been particularly effective in treating patientswith cancer due to acquired resistance. Onemechanism of acquir-ing resistance against MET inhibitors is via activation of bypasspathways that compensate for the loss of survival signaling whenMET is inhibited. Elucidating these bypass pathways offers thepotential to develop combinatorial drug therapy to re-sensitizeGBMcells toMET inhibitors. In this study, we developedGBMcelland animalmodels of resistance toMET inhibitors. Resistant cells

Figure 3.

Combination therapy with FGFR and COX-2 inhibitors restores sensitivity to MET inhibitors in resistant GBM cells. U87 wild type (WT), crizotinib-resistant (CR), andonartuzumab-resistant (OR) cells were pretreated with (A) COX-2 inhibitor celecoxib (100 nmol/L) or (B) FGFR1 inhibitor Debio-1347 (10 mmol/L) for2 hours then subsequently treated with either crizotinib (100 nmol/L) or onartuzumab (Onart; 300 nmol/L) for 48 hours. Cell death was assessed via trypan blueassay. U87 WT, CR and OR cells were pretreated with (C) and (D) celecoxib or (E) and (F) Debio-1347 for 2 hours then subsequently treated with eithercrizotinib (100 nmol/L) or Onart (300 nmol/L) for 48 hours. Cell proliferation was assessed by cell counting over a period of 5 days and growth curves wereestablished. � , P < 0.05.

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revealed increased levels of active p.MET that could not besuppressed by the MET inhibitors, thus proving resistance andsuggesting that MET receptor activation could contribute to thisresistance. This finding differs from published mechanisms ofresistance to EGFR inhibitors which involve loss of oncogenicmutant EGFRvIII (31). Using this model, we investigated theproteomic changes that occur when GBM cells become resistantto two clinically applicable MET inhibitors (crizotinib and onar-tuzumab), uncovered several important bypass pathways thatinclude mTOR, FGFR1, EGFR, STAT3, and COX-2 and showedthat targeting these pathways in combination with MET inhibi-tors, reverses resistance to the MET inhibitors.

The mTOR pathway is highly activated in GBM (32) and,although mTOR has been implicated in acquired resistance in

small cell lung cancer (33), less is known about its role in GBMtherapy resistance. MET inhibition leads to downregulation ofPI3K signaling, which, in turn, leads to decreased activation ofmTOR resulting in the induction of apoptosis and decreased cellproliferation (34). However, aberrant activation of PI3K/AKTsignaling as a bypass mechanism results in increased mTORactivation that promotes cancer progression, metastasis, andinvasion (35). We demonstrate that mTOR phosphorylation issignificantly increased in MET inhibitor–resistant GBM cells,suggesting a role for the mTOR pathway in MET inhibitor resis-tance. Rapamycin, an FDA-approved inhibitor of mTOR, alonedid not significantly enhance cell death but did have antiproli-ferative effects in MET inhibitor–resistant GBM cells. Combina-tional treatment of resistant GBM cells with rapamycin and either

Figure 4.

Combination therapy with EGFR and mTOR inhibitors restores sensitivity to MET inhibitors in resistant GBM cells. U373 wild type (WT), crizotinib-resistant (CR),and onartuzumab-resistant (OR) cells were pretreated with (A) EGFR inhibitor erlotinib (25 nmol/L) or (B) mTOR inhibitor rapamycin (25 mmol/L) for2 hours then subsequently treated with either crizotinib (100 nmol/L) or onartuzumab (Onart) (300 nmol/L) for 48 hours. Cell death was assessed via trypan blueassay.U373 cellswerepretreatedwith (C) and (D) erlotinib or (E) and (F) rapamycin for 2 hours then subsequently treatedwith either crizotinib (100nmol/L) orOnart(300 nmol/L) for 48 hours. Cell proliferation was assessed by cell counting over a period of 5 days and growth curves were established. � , P < 0.05.






MET inhibitor induced apoptosis and further suppressed cellproliferation indicating restored MET inhibitor sensitivity.

Aberrant activation of RTKs such as FGFR1 and EGFR is arecognizedmechanismbywhichmalignant cells acquire resistanceto other RTK monotherapies (36). FGFR1 and EGFR compensatefor the loss of MET-mediated survival signaling through reactiva-tion of downstream PI3K and STAT signaling (37). We demon-strate that FGFR1 is implicated inMET inhibitor resistance inGBM.FGFR1 is upregulated in MET inhibitor–resistant GBM cells andshows a trend towards correlationwithGBMpatient survival basedon TCGA data analysis. Therefore, we assessed the effect of debio-1347, an FGFR1 inhibitor, in combination with crizotinib oronartuzumab on MET inhibitor–resistant GBM cells and weshowed that FGFR1 inhibition can circumvent MET inhibitorresistance and simultaneous inhibition of both FGFR1 and METis advantageous for reversal of MET inhibitor resistance (38).

EGFR and MET are frequently co-expressed in cancer and HGFcan transactivate EGFR, which in turn, activates MET resulting insynergistic tumor growth (36, 37). Furthermore, MET has beenreported to play a role in acquired resistance to EGFR-targetedtherapies in many cancers (17, 39). Attributed to this and the factthat cross-talk exists between MET and EGFR, MET inhibitor–resistant GBM cells were subjected to combined treatment with

the EGFR inhibitor, erlotinib, and either crizotinib or onartuzu-mab. Erlotinib alone did not significantly induce apoptotic celldeath in MET inhibitor–resistant GBM cells; however, concom-itant treatment with erlotinib and either crizotinib or onartuzu-mab restored MET inhibitor sensitivity leading to enhanced celldeath and decreased cell proliferation.

Many growth factor receptors, including MET activate STAT3(40, 32). STAT3 is elevated in GBM, driving tumor growth,angiogenesis and invasion (41) through the regulation of down-stream targets including c-myc, Bcl-2, and Bcl-XL and is emergingas a drug resistance mechanism in GBM (42). On the basis of ourscreening data, we assessed the effect of STAT3 inhibition onMETinhibitor resistance and found that combinational treatment ofMET inhibitor–resistant GBM cells with a STAT3 inhibitor andMET inhibitor induced cell death and significantly inhibited cellproliferation indicating restored MET inhibitor sensitivity.

We also found that MET inhibitor resistance resulted in upre-gulation of COX-2, an inducible cyclooxygenase that is over-expressed GBM (43). Elevation of COX-2 stimulates increasedangiogenesis and invasion of tumor cells and correlates with poorprognosis (44) although little is known about COX-2 and METinhibitor resistance in GBM. Celecoxib, a COX-2 inhibitor, wasused in conjunction with either crizotinib or onartuzumab to

Figure 5.

Combination therapy with STAT3 inhibitor restores sensitivity to MET inhibitors in resistant GBM cells. U373 wild type (WT), crizotinib-resistant (CR) andonartuzumab-resistant (OR) cellswere pretreatedwith a STAT3 inhibitor (25 mmol/L) for 2 hours then subsequently treatedwith either (A) crizotinib (100 nmol/L) or(B) onartuzumab (Onart; 300 nmol/L) for 48 hours. Cell death was assessed via trypan blue. U373 cells were pretreated with STAT3 inhibitor (25 mmol/L)for 2 hours then subsequently treatedwith either (C) crizotinib (100 nmol/L) or (D) Onart (300 nmol/L) for 48 hours. Cell proliferation was assessed by cell countingover a period of 5 days and growth curves were established. � , P < 0.05.

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assess the effect of COX-2 inhibition on MET inhibitor–resistantGBM cells. MET inhibitor–resistant GBM cells demonstratedsignificant toxicity to combination therapy. Although COX-2inhibition is thought to be the primary mode of action, celecoxibhas also been reported to act in a COX-2–independent manner

(45). Further evaluation may be needed to completely elucidatethe mechanism by which celecoxib reverts MET inhibitorresistance.

GSCs, a small subpopulation responsible for self-renewal, havebeen implicated in GBM relapse (46, 47). Inhibition of MET,

Figure 6.

Combinational treatment inhibits METinhibitor–resistant xenograft growth.U87 wild-type (WT) and crizotinib-resistant (CR) cells werestereotactically implanted in thestriata of immunodeficient mice (n ¼10). A, Vehicle control, celecoxib,crizotinib or the combination. B,Vehicle control, Debio-1347, crizotinibor the combination was administereddaily by oral gavage starting 6 daysafter tumor implantation. The animalswere subjected to MRI scan at 3 weeksafter tumor implantation and tumorvolumes were quantified. The datashow that both celecoxib and debio-1347 significantly inhibit tumor growthand resensitize tumors to crizotinibtreatment. � , P < 0.05 relative tocontrol and single drug treatment.






which is expressed in GSCs, halts GBM progression and decreasesthe expression of stem markers such as CD133 and Sox2. GSCsdisplay enhanced sensitivity to MET inhibitors, indicating a vitalrole for MET in GSC maintenance (48). Attributed to this, thedevelopment of anti-cancer therapies that target GSCs appearsimperative for optimal GBM treatment (49). We show thattreatment with MET inhibitor and simultaneous inhibition ofone of the bypass proteins reverses MET resistance in GSCs.Interestingly, RPPA exposed considerable overlap in the bypasssignaling involved in resistance to both MET inhibitors. Thesecommonly upregulated pathways are vital for cell survival andinclude important molecules such as EGFR, BCL 2, COX-2, andFGFR1. The ability to target a commonly altered pathway, thatwould be effective at reversing the effects of drug resistance tomultiple inhibitors, is the ultimate goal for personalized therapy(50).

Our data identify the mechanisms of resistance to MET inhi-bitors in GBM and suggest new combination therapies thatovercome resistance. Device-based and PDXmodel-based screensare currently being assessed for optimal, patient-specific onco-genic driver identification. Informed by our findings, individualpatients that display drug resistance could be assessed for theirunique oncogenic driver signature and receive the most effectivecombination therapy.

Disclosure of Potential Conflicts of InterestJ. Wulfkuhle holds ownership interest (including patents) in Theranostics

Health, LLC, and is a consultant/advisory board member for Baylor College ofMedicine. S. Phan holds ownership interest (including patents) in Genentech/Roche. E. Petricoin is an employee of and holds ownership interest (includingpatents) in Perthera, Inc., and is a consultant/advisory board member for

Perthera, Inc. and ADVX Investors Group, LLC. No potential conflicts of interestwere disclosed by the other authors.

Authors' ContributionsConception and design: N. Cruickshanks, Y. Zhang, R. AbounaderDevelopment of methodology:N. Cruickshanks, Y. Zhang, S. Hine, F. GuessousAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): N. Cruickshanks, Y. Zhang, S. Hine, F. Yuan,C. Grello, M. Pahuski, C. Dube, B. Wang, C. Deveau, K. Saoud, J. Wulfkuhle,D. Schiff, S. Phan, E. PetricoinAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): N. Cruickshanks, S. Hine, M. Gibert, F. Yuan,M. Oxford, C. Dube, F. Guessous, C. Deveau, I. Gallagher, J. Wulfkuhle,E. PetricoinWriting, review, and/or revision of the manuscript: N. Cruickshanks,M. Gibert, F. Yuan, M. Oxford, C. Dube, J. Wulfkuhle, D. Schiff, S. Phan,E. Petricoin, R. AbounaderAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): Y. Zhang, S. Hine, M. Gibert, B. WangStudy supervision: R. AbounaderOther (access to onartuzumab and scientific support related toonartuzumab): S. Phan

AcknowledgmentsThisworkwas supported byNIHgrants RO1NS045209 andUO1CA220841

(to R. Abounader).

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

ReceivedMarch 23, 2018; revised June 13, 2018; accepted September 5, 2018;published first September 10, 2018.

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2019;25:663-673. Published OnlineFirst September 10, 2018.Clin Cancer Res Nichola Cruickshanks, Ying Zhang, Sarah Hine, et al. Resistance to MET Inhibitors in GlioblastomaDiscovery and Therapeutic Exploitation of Mechanisms of

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