kinome rnai screens reveal synergistic targeting of mtor ... · the human kinase trc shrna library...

Therapeutics, Targets, and Chemical Biology

Kinome RNAi Screens Reveal SynergisticTargeting of MTOR and FGFR1 Pathways forTreatment of Lung Cancer and HNSCCKatherine R. Singleton1, Trista K. Hinz1, Emily K. Kleczko1, Lindsay A. Marek1, Jeff Kwak1,Taylor Harp1, Jihye Kim2, Aik Choon Tan2, and Lynn E. Heasley1

Abstract

The FGFR1 is a therapeutic target under investigation inmultiple solid tumors and clinical trials of selective tyrosinekinase inhibitors (TKI) are underway. Treatment with a singleTKI represents a logical step toward personalized cancer ther-apy, but intrinsic and acquired resistance mechanisms limittheir long-term benefit. In this study, we deployed RNAi-basedfunctional genomic screens to identify protein kinases control-ling the intrinsic sensitivity of FGFR1-dependent lung cancerand head and neck squamous cell cancer (HNSCC) cells toponatinib, a multikinase FGFR-active inhibitor. We identifiedand validated a synthetic lethal interaction between MTOR andponatinib in non–small cell lung carcinoma cells. In addition,treatment with MTOR-targeting shRNAs and pharmacologicinhibitors revealed that MTOR is an essential protein kinase

in other FGFR1-expressing cancer cells. The combination ofFGFR inhibitors and MTOR or AKT inhibitors resulted insynergistic growth suppression in vitro. Notably, tumor xeno-grafts generated from FGFR1-dependent lung cancer cells exhib-ited only modest sensitivity to monotherapy with the FGFR-specific TKI, AZD4547, but when combined with the MTORinhibitor, AZD2014, significantly attenuated tumor growth andprolonged survival. Our findings support the existence of asignaling network wherein FGFR1-driven ERK and activatedMTOR/AKT represent distinct arms required to induce fulltransformation. Furthermore, they suggest that clinical efficacyof treatments for FGFR1-driven lung cancers and HNSCC maybe achieved by combining MTOR inhibitors and FGFR-specificTKIs. Cancer Res; 75(20); 4398–406. �2015 AACR.

IntroductionOur studies and those of others demonstrate that overex-

pressed, nonmutated FGFR1 participates as an oncogenic drivervia autocrine FGFs in cell lines derived from lung cancers of allhistologies (1–5), head and neck squamous cell carcinomas(HNSCC; refs. 6, 7) and malignant pleural mesothelioma (8).As a result, multiple early-phase clinical trials of FGFR-targetingtyrosine kinase inhibitors (TKI) are now under way, including astudy of the multikinase TKI ponatinib (9) in lung cancer at ourinstitution (NCT01935336).

The clinical efficacy of FGFR TKIs as single anticancer agentsis not fully realized. Yet, the problem of intrinsic and acquiredresistance to TKI monotherapy has emerged as a major limi-tation to long-term control or cure of solid tumors (10–13) andportends similar difficulties with single FGFR TKIs as therapeu-tics. Defining mechanisms of acquired resistance to targeted

therapeutics is an ongoing subject of intense investigation andsets the stage for strategies to deploy inhibitors of the resistancemechanisms following treatment failure of the initial drug.Thus, "serial monotherapy" has emerged as a logical approachin clinical oncology for solid tumors, including lung cancer. Inthis regard, however, it is important to review the lessonslearned from acquired resistance to antimicrobial and antiviralmonotherapy over the past 60 years (reviewed in ref. 12). Thepresent strategy to combat acquired resistance to monotherapyin cancer by deploying sequential therapies to block emergentresistance pathways (i.e., MET inhibitors after resistance toEGFR-specific TKIs) failed as a strategy to cure TB and HIVinfections.

Importantly, therapeutic success in HIV and TB infections wasonly achieved when combinations of inhibitors were deployedthat induced rapid and synergistic suppression of the infectiousagent at the onset of therapy, thereby preventing the emergence ofdrug resistance (12). We hypothesize that the development ofrational, mechanism-based combinations of inhibitors thatsimultaneously inhibit multiple elements within transformingreceptor tyrosine kinase (RTK) coactivation networks (14) activein cancer cells may achieve a similar impact on cancer cure orcontrol. In this study, we deployed functional genomics screenswith a kinome-targeting shRNA library to identify auxiliary path-ways that co-signal with FGFR1 in lung cancer and HNSCC celllines. Our studies establish MTOR as a protein kinase withessential properties in some FGFR1-dependent cancer cell linesas well as auxiliary, synthetic lethal (SL) properties in the contextof FGFR inhibitors in other cell lines. In sum, our findings identify

1Department of Craniofacial Biology, University of ColoradoAnschutzMedical Campus, Aurora, Colorado. 2Department of Medicine, Univer-sity of Colorado Anschutz Medical Campus, Aurora, Colorado.

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

Corresponding Author: Lynn E. Heasley, University of Colorado AnschutzMedical Campus, 12801 East 17th Avenue, Aurora, CO 80045. Phone: 303-724-4578; Fax: 303-724-4580; E-mail: [email protected]

doi: 10.1158/0008-5472.CAN-15-0509

�2015 American Association for Cancer Research.

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MTOR as a protein kinase that contributes to the intrinsic sensi-tivity of cancer cells to FGFR TKIs such that combined treatmentwith MTOR inhibitors and FGFR TKIs elicits synergistic growthinhibition. Thus, direct MTOR kinase inhibitors are attractiveagents to consider combining with FGFR-specific TKIs for treat-ment of FGFR1-dependent lung cancers and HNSCCs.

Materials and MethodsCell culture

All cancer cell lines used in this study were submitted tofingerprint analysis by the University of Colorado CancerCenter DNA Sequencing and Analysis Core to confirm theirauthenticity. Cell lines were routinely cultured in RPMI-1640growth medium (Invitrogen) supplemented with 10% FBS with1% penicillin–streptomycin (Sigma-Aldrich) at 37�C in ahumidified 5% CO2 incubator.

Lentivirus preparationThe Human Kinase TRC shRNA library (obtained from the

Functional Genomics Shared Resource within the University ofColorado Cancer Center) was packaged in 293T cells as follows.293T cells were incubated overnight at 37�C in a 5% CO2

humidified incubator with Turbofect transfection reagent, 5.0 mgpD8.9, 5.0 mg pCMV-VSV-G and 3.0 mg kinome library. The virus-containing media from the 293T cells was then filtered through a0.45-mm filter after adding 1 mg/mL polybrene and either usedimmediately as described or stored at 4�C until ready for use. Inexperiments where MTOR was validated as an essential or SLkinase, two MTOR shRNAs in the pLKO.2 lentiviral vector(TRCN0000332888 and TRCN0000363722) distinct from thoseshRNAs included in the library or an shRNA-targeting GFP as anegative control were packaged with the pCMV-VSV-G and pD8.9component vectors. The virus was titered on NIH3T3 fibroblastsand the effect on cell growth was measured by clonogenic growthassay as described previously.

Functional genomics screensFor details on functional genomics screens, see Supplementary

Information.

In vitro and in vivo growth assaysClonogenic and anchorage-independent growth assays. To measurethe effect of inhibitors or shRNA-mediated knockdown on cellgrowth, cells were seeded at 100 cells per well in 6-well tissueculture plates in full media. After 24 hours, cells were treated asindicated and cultured for 14 days with the addition of freshmedia containing inhibitors every 7 days. Plates were rinsedtwice with PBS, fixed, and stained with a solution of 0.5% (w/v)crystal violet in 6.0% (v/v) gluteraldehyde for 30 minutes atroom temperature. Plates were rinsed in distilled H2O andphotographed. For measurement of anchorage-independentgrowth in soft agar, 20,000 cells were suspended in 1.5 mLmedia and 0.35% noble agar and overlaid on base layerscontaining 1.5 mL media and 0.5% noble agar in 6-well plates.Wells overlaid with 2 mL media containing drugs were fed oncea week and allowed to grow for 14 to 21 days. Viable colonieswere stained for 24 hours with 250 mL 1 mg/mL nitrobluetetrazolium. Digital photographs of both clonogenic and softagar wells were used to quantify total colony area by Meta-morph imaging software (Molecular Devices).

Cell proliferation assay. Cells were plated at 100 cells per well in96-well tissue culture plates and treated with inhibitors atvarious doses. When the DMSO-treated control wells becameconfluent (1,2, weeks), cell numbers were assessed using aCyQUANT Direct Cell Proliferation Assay (Invitrogen) accord-ing to the manufacturer's instructions.

Xenograft tumor studies. H1581 and Colo699 cells were sus-pended in 50% Matrigel/PBS at 10 million cells per mL and 1million cells were injected s.c. in bothflanks of female nu/numiceaccording to protocols approved by the University of ColoradoInstitutional Animal Care and Use Committee (IACUC). When atleast one of the tumors reached a volume of 100 mm3, the micewere randomized into treatment groups (n ¼ 9,10/group) ofdiluent control (1% Polysorbate 80), AZD4547 (12.5 mg/kg),AZD2014 (10 mg/kg) or both AZD4547 and AZD2014. Drugswere delivered daily by oral gavage (�0.25 mL/mouse). Peakplasma levels of AZD4547 following similar dosing are approx-imately 1 to 2 mmol/L (15). Also, a total Cmax of 11 mmol/L (freeCmax of 0.51 mmol/L) is achieved 30minutes after a single 10mg/kg dose of AZD2014 (S. Cosulich, AstraZeneca; unpublisheddata). Tumor volumes were determined by caliper measurementsof the long and short diameter performed twice perweek using themodified ellipsoid formula for volume,V¼1/2(length�width2).Micewere euthanizedwhen tumors reached a volumegreater than2 cm3 or they exhibited signs of morbidity specified in the IACUCprotocol.

Immunoblot analysesPhospho-ERK, total ERK, phospho-AKT S473, total AKT,

p-p70S6K T389, total p70S6K, p-S6 S235/236, total S6,p-Rictor T1135, total Rictor, MTOR, and PARP1 were measuredby immunoblotting using antibodies obtained from Cell Sig-naling Technology, Inc. Aliquots of cell extracts prepared inlysis buffer (0.5% Triton X-100, 50 mmol/L b-glyceropho-sphate (pH 7.2), 0.1 mmol/L Na3VO4, 2 mmol/L MgCl2,1 mmol/L EGTA, 1 mmol/L DTT, 0.3 mol/L NaCl, 2 mg/mLleupeptin, and 4 mg/mL aprotinin) were submitted SDS-PAGE.After electrophoretic transfer to nitrocellulose, filters wereblocked in 3% BSA (Cohn Fraction V; ICN Biomedicals, Inc.)in TBS with 0.1% Tween 20 (TTBS). The filters were thenincubated overnight at 4�C with antibodies, washed threetimes in TTBS, and incubated for 1 hour at room temperaturewith alkaline phosphatase–coupled goat anti-rabbit antibo-dies. The filters were developed using Luminata Classico sub-strate (Millipore Corporation) according to the manufacturer'sinstructions. When blotting phosphorylated proteins, the fil-ters were stripped and probed for the corresponding totalsignaling enzyme level or Na/K-ATPase a-subunit (Santa CruzBiotechnology, Inc.) as a loading control.

Caspase-3 assayH1581 cellswereplated at 200,000 cells perwell in6-well tissue

culture plates in full media. After 24 hours, cells were treated withDMSO, 300 nmol/L AZD4547, 100 nmol/L AZD8055, or com-bination in triplicate and cultured for 3 days. Cells were harvestedand caspase-3 activity was assessed using the Caspase-3 CellularActivity Assay Kit PLUS (Enzo Life Sciences, Inc.) according to themanufacturer's instructions.

FGFR1 and MTOR Coinhibition Synergistically Suppresses Growth

www.aacrjournals.org Cancer Res; 75(20) October 15, 2015 4399




ChemicalsPonatinib was obtained by material transfer agreement from

Ariad Pharmaceuticals, Inc. AZD4547 and AZD2014 wereobtained by material transfer agreement from AstraZeneca.AZD8055, GSK690693 and MK2206 were purchased fromSelleck Chemicals. Stocks of the drugs were prepared in DMSOat concentrations such that the final concentration of DMSOwas 0.1% v/v.

ResultsA functional genomics-based SL screen with a kinome shRNAlibrary identifies MTOR as a collaborator with FGFR1

Our recent studies demonstrate through molecular and phar-macologic approaches an autocrine role for nonmutated FGFR1in multiple cancers, including lung cancer, malignant mesothe-lioma, and HNSCC (1,2,6,8). On the basis of precedent frommonotherapy with TKIs in other oncogene-driven lung cancers,including those bearing mutant EGFR or rearranged ALK(10,11,13), intrinsic and/or acquired resistance mechanisms arepredicted to limit the clinical response of FGFR inhibitors as well.To screen for signal pathways whose activity reduces intrinsicsensitivity of lung cancer cell lines to FGFR inhibitors, we per-formed functional genomics-based SL screens with a kinome-targeting shRNA library (see Materials and Methods) and lungcancer cell lines exhibiting high sensitivity (Colo699, H520, andH1703) and moderate/low sensitivity (H1299 and H157) toponatinib (Table 1; ref. 2). Although ponatinib is a multikinaseinhibitor with IC50 values ranging from 0.2 to 8 nmol/L on ABLand SRC family kinases, FGFR1, 2 and 4, PDGFRs, VEGFRs andRET (16), our recent studies in lung cancer andmesothelioma celllines indicate that sensitivity to the TKI is closely associated withFGFR1 expression and function (2,8). The cell lines were trans-ducedwith lentiviruses encoding the kinome-targeting shRNAs inthe pLKO.1 vector at a multiplicity of infection <1. Followingselection for puromycin resistance, which eliminates nontrans-duced cells and cells expressing an shRNA-targeting an essentialgene, the cells were treated with or without ponatinib for 3 daysfollowed by an additional 3 days of culture inmedium lacking thedrug. The shRNAswere amplified from genomicDNAby PCR andsubmitted to massively parallel deep sequencing, and the readswere analyzed by BiNGS!SL-seq (17) to determine the countfrequency of the individual shRNAs in the control and treatedsamples. Kinase genes that are SL with respect to ponatinibtreatment are defined herein by significantly decreased shRNA

counts in the treated samples with at least two independentshRNAs.

The functional genomics analysis identified MTOR as the top-ranking SL hit in both H157 andH1299 cells, but not in themoreponatinib-sensitive H520 andH1703 cells (Supplementary TableS2) or Colo699 cells (data not shown). To validateMTOR as an SLgene with respect to ponatinib in H157 and H1299 cells, lenti-viral-encoded shRNAs distinct from those used in the kinomeshRNA library were transduced into H157 and H1299 cells. Asshown in Fig. 1A, both MTOR-targeting shRNAs reduced MTORprotein levels relative to a nonsilencing control shRNA-targetingGFP. Although MTOR silencing, alone, exerted little or no effecton clonogenic growth of H157 or H1299 cells, enhanced growthinhibition by ponatinib uponMTORknockdownwas observed inboth cell lines (Fig. 1B-D). Finally, treatment of H157 cells withponatinib and the MTOR inhibitor, AZD8055 (18), yieldedsignificantly greater clonogenic growth inhibition than ponatinibalone (Supplementary Fig. S3). These results validateMTOR as anSL gene with ponatinib.

MTOR is an essential gene inmultiple FGFR1-dependent cancercell lines

MTOR was not identified as an SL protein kinase in H520,H1703, or Colo699 cells (Supplementary Table S2 and data notshown). Because the SL screen format (see Supplementary Fig. S1)eliminates shRNAs targeting essential kinases due to the selectionfor stable puromycin resistant cells, it is possible that MTORshRNAs were eliminated from the transduced cell population inColo699, H520, and H1703 cells. We performed an essentialkinase screen in FGFR1-dependent Colo699 lung adenocarcino-ma cells and FGFR1-dependent 584-A2 HNSCC cells (see Sup-plementary Materials and Methods). The count frequency ofmultiple MTOR shRNAs was determined 2 and 7 days aftertransduction by sequencing of amplified shRNAs and the resultsare presented graphically in Fig. 2A. Five of six independentMTOR-targeting shRNAs were eliminated from the pool of recov-ered shRNAs in Colo699 cells 7 days after transduction. Bycontrast, none of the six shRNAs were eliminated from 584-A2cells after 7 days of incubation. Thus, the results are consistentwith an essential role of MTOR in Colo699 cells, but not 584-A2cells. The essential function ofMTOR in Colo699 cells was furthervalidated with two shRNA distinct from those used in the kinomelibrarywhere strong reduction of clonogenic growth resulted fromtransduction of MTOR shRNAs relative to the GFP-targetingshRNA control (Fig. 2B).

Table 1 Ponatinib, AZD4547, and AZD8055 sensitivity of lung and HNSCC cell lines

Cell line Tissue and histologyPonatinibIC50 (nmol/L)

AZD4547IC50 (nmol/L)



H520 Lung squamous cell carcinoma 53 57 23 21Colo699 Lung adenocarcinoma 14 6 61 4H1703 Lung adenosquamous carcinoma 24 460 60 8CCL30 Nasopharynx, squamous cell carcinoma ND 9 66 NDH1581 Lung large cell carcinoma 1 2 78 40H1299 Lung large cell carcinoma 140 >1,000 178 25H157 Lung squamous cell carcinoma 119 >1,000 632 210584-A2 Larynx, squamous cell carcinoma ND 6 >1,000 ND

NOTE: The tissue source and histology of the cancer cell lines used in the studies is summarized. The sensitivity of the cell lines to ponatinib is from ref. 2. Thesensitivities of the cell lines to the FGFR inhibitor, AZD4547, and theMTOR inhibitor, AZD8055, were determinedwith clonogenic or anchorage-independent growthassays using drug concentrations from 0 to 1 mmol/L. The sensitivity of the cell lines to AZD2014 was measured using cell proliferation assays and the CyQUANTreagent over drug concentrations from 0 to 1 mmol/L. The primary data for AZD8055 sensitivity on six of the cell lines are shown in Fig. 2C and the IC50 values werecalculated with the Prism software program.Abbreviation: ND, not determined.

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Cancer Res; 75(20) October 15, 2015 Cancer Research4400




As a pharmacologic approach to define the relative requirementof MTOR for growth and survival in FGFR1-dependent cancer celllines, clonogenic cell growth was measured in a panel of cell linesin the presence of increasing concentrations of AZD8055.AZD8055, and the congener, AZD2014 are highly selective forMTOR in either the TORC1 or TORC2 complexes, with littleactivity on other members of the phosphatidylinositol kinasesuperfamily (18). In clonogenic/anchorage-independent growthassays, H157 and 584-A2 cells exhibited low sensitivity (IC50 >600 nmol/L) andH1299 cells intermediate sensitivity (IC50�180nmol/L) to AZD8055 (Fig. 2C; Table 1). By contrast, Colo699,H520, and H1703 as well as the FGFR1-dependent lung cancercell line,H1581, and theHNSCC cell line, CCL30,were uniformlysensitive to AZD8055 with IC50 values <80 nmol/L. This rankorder of sensitivity among the cancer cell lines to AZD8055 wasconfirmed with a cell proliferation assay (Supplementary Fig. S4)and was similar to that observed with AZD2014 (Table 1). Thepanel of cell lines was very sensitive to the TORC1-specificinhibitor, rapamycin, although little or no difference in relativesensitivity between the cell lines was observed with IC50 valuesranging from 0.3 to 2 nmol/L (Supplementary Fig. S5). Together,the studies indicate that FGFR1-dependent lung cancer andHNSCC cell lines exhibit variable degrees of MTOR dependencyfor growth and survival and explain the observation that MTORwas not identified as SL with ponatinib in Colo699, H520, andH1703 cells because it exerts an essential function in these celllines relative to H157 and H1299 cells.

Synergistic growth inhibition by FGFR andMTOR inhibitors inColo699 and H1581 cells

Despite the range of MTOR dependencies measured in thepanel of cancer cell lines, we tested whether FGFR and MTOR

inhibitors might still yield additive or synergistic growth inhibi-tion in cell lines even when MTOR exhibits an essential pheno-type. As shown in Supplementary Fig. S6, combination treatmentwith either ponatinib or AZD4547, a specific FGFR1,2,3 inhibitor(15), and AZD8055 resulted in significantly greater clonogenicgrowth inhibition relative to FGFR inhibitor, alone, in Colo699,H1703, and H520 cells. To rigorously test for synergistic growthinhibition resulting from simultaneous blockade of FGFR1 andMTOR, FGFR1-dependent lung cancer cell lines (H1581 andColo699) or an HNSCC cell line (CCL30) exhibiting high sen-sitivity to AZD8055 (Table 1) were treated in a 96-well plateformat (seeMaterials andMethods) withmultiple concentrationsof AZD4547, alone and in combinationwith theMTOR inhibitor,AZD8055. The effects on cell growth measured by the CyQUANTassay are shown in Fig. 3 and Supplementary Fig. S7, and analysisof the resulting data by the method of Chou and Talalay (19)revealed synergistic growth inhibition over multiple concentra-tions of the twodrugs. The greater than additive growth inhibitionby combined AZD4547 and AZD8055 was confirmed with thedistinctMTOR inhibitor, AZD2014 (18), inH1581, Colo699, and584-A2 cells (Fig. 4) and with rapamycin and AZD4547 inColo699 and H1581 cells (Supplementary Fig. S8). Thus, theidentification of MTOR as an SL pathway with FGFR1 is observedpharmacologically in multiple FGFR1-dependent cancer celllines.

To explore the mechanism by which simultaneous inhibitionof FGFR1 and MTOR yield synergistic growth inhibition, theactivity of signaling pathways known to be regulated by theseprotein kinases was monitored by immunoblot analyses.Figure 5A reveals that AZD4547 inhibited ERK phosphorylation,but had little or no effect on phosphorylation of the TORC2 site(S473) of AKT in H1581 and Colo699 cells. In addition, none of

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Figure 1.Validation of MTOR as a synthetic lethal gene with FGFR inhibition by RNAi-mediated knockdown. A, H157 and H1299 cells were transduced with either anegative control shRNA-targeting GFP or one of two independent MTOR targeting shRNAs (TRCN0000363722 or TRCN0000332888), henceforth abbreviatedas MTOR 22 and MTOR 88, respectively. Cells were selected for resistance to puromycin and resulting colonies were harvested and immunoblotted for MTORprotein levels. The protein level of the a-subunit of Na/K-ATPase was measured as a loading control. B, as in A except H157 cells were treated with 300 nmol/Lponatinib or DMSO at the time of puromycin selection. Resulting colonies were stained with crystal violet and photographed. A representative well of threereplicates is shown. Mean total colony area (� SEM, n ¼ 3) for the indicated shRNA transductions and ponatinib treatments were quantified as described inMaterials and Methods, and the data are graphically presented for H157 (C) and H1299 (D) cells, respectively.






the TORC1 targets (p70S6K, S6, and Rictor) showed alteredphosphorylation by FGFR1 inhibition with AZD4547. By con-trast, AZD8055 inhibited phosphorylation of AKT-Ser473,

p70S6K, S6, and Rictor, but not phosphorylation of ERK(Fig. 5A). Finally, combined AZD4547 and AZD8055 treatmentinhibited phosphorylation of ERKs and multiple targets of

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Figure 2.MTOR is an essential kinase in Colo699cells. An essential kinase screen wasperformed with the kinome shRNAlentiviral library as described inMaterials and Methods. In A, the countfrequencies following Illuminasequencing of six MTOR targetingshRNAs is presented for Colo699 and584-A2 cells. The findings show loss offive of the six MTOR shRNAs at 7 daysof culture in Colo699 cells, but not584-A2 cells. B, Colo699 cells weretransduced with either a negativecontrol shRNA targeting GFP or oneof two independent MTOR-targetingshRNAs (MTOR 22 or MTOR 88). Cellswere selected for resistance topuromycin, and resulting coloniesfollowing 2 weeks of culture werestained with crystal violet and totalcolony areawas quantified. � ,P <0.05.C, the indicated cell lines and othersnoted in Table 1 were submitted toclonogenic growth assays (seeMaterials and Methods) with 0to 1 mmol/L AZD8055. Afterapproximately 2 weeks, the colonieswere fixed and stained with crystalviolet and total colony area wasquantified. The IC50 values werecalculated with the Prism softwareprogram and are presented in Table 1.

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Figure 3.Synergistic growth inhibition ofH1581 andColo699 cells byAZD4547and AZD8055. The indicated celllines were seeded at 100 cells perwell in 96-well plates. The next day,the growth medium was replacedwith 100 mL medium containing theindicated combinations of AZD4547and AZD8055 in triplicate andincubation was continued forapproximately 10 to 14 days. Themedium and drugs were replacedevery 7 days. Cell growth wasassessedwith the CyQUANT reagentas described in Materials andMethods and the mean fluorescence(n ¼ 3) for each treatment is plottedon the y-axis. The data weresubmitted to further analysiswith theCalcusyn program for determinationof the degree of synergy achieved bythe combinations relative to themonotherapy treatments. Theresulting combination index (CI)values are tabulated below and thedegree of synergy indicated.

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TORC1 and TORC2, consistent with the functioning of an RTKcoactivation network (14) inwhichMTOR is regulated in parallel,not distal to FGFR1. These results are also observed with rapa-mycin and AZD4547 except that no inhibition of AKT Ser473phosphorylation is observed (Supplementary Fig. S9). Dualblockade of FGFR1 andMTOR, but not inhibition of either targetalone, induced PARP1 cleavage in H1581 cells as measured byimmunoblot analysis, suggesting the induction of apoptosis withthe combination inhibitor treatment (Fig. 5B). As an independentbiochemical measure of apoptosis, the activity of caspase-3 wasassayed in extracts from H1581 cells similarly treated. Figure 5Cshows an approximately 5-fold increase in caspase-3 activity incells treated with combined AZD4547 and AZD8055 relative tocontrol cells or cells treated with the single agents.

It is noteworthy that neither AKT1, 2, nor 3 were identified ashigh-ranking hits in the SL screens (Supplementary Table S2),despite the findings in Fig. 5 showing marked inhibition of AKTphosphorylation by MTOR kinase inhibitors. It is possible thatfunctional redundancy occurs among the distinct AKT gene pro-ducts such that silencing of any one AKT gene fails to exert aphenotype. We tested the ability of two AKT inhibitors, MK2206(20) and GSK690693 (21) to exert synergistic growth inhibitionwith FGFR inhibitors. The sensitivity ofColo699 cells to these AKTinhibitors is shown in Supplementary Fig. S10A and indicatesonly modest sensitivity to the single agents. As shown in Supple-mentary Figs. S10B and S11, strong synergy of MK2206 andponatinib in Colo699 cells and GSK690693 and ponatinib inH1581 cells was observed. Thus, the findings in Figs. 3–5 andSupplementary Figs. S10 to S11 support the parallel activation oftheMTOR–AKT signaling pathway as a modulator of the intrinsicsensitivity of multiple FGFR1-dependent cancer cell lines to FGFRTKIs.

Enhanced tumor growth inhibition by combination AZD4547and AZD2014 in flank xenograft assays

H1581 and Colo699 cells were implanted in the flanks offemale nu/nu mice as described in Materials and Methods. Whenthe tumors reached approximately 100 mm3, the mice wererandomized into treatment groups; diluent control, AZD4547(12.5 mg/kg), AZD2014 (10 mg/kg), or the combination of both

drugs. As shown in Fig. 6B and C, treatment of flank H1581tumors with either AZD4547 or AZD2014 alone yielded littlegrowth inhibition relative to diluent control (Fig. 6A). In fact, themodest effect of AZD4547 monotherapy is surprising, consider-ing the potency with which this cell line is inhibited by AZD4547in vitro (Fig. 3 and Table 1). However, in combination, AZD4547and AZD2014 yielded significant tumor growth inhibition (Fig.6D and E) and significantly prolonged survival (Fig. 6F), consis-tent with the synergistic growth inhibition observed in vitro.Similar relative activities of AZD4547 and AZD2014 as mono-therapies, and in combinationwere observed following treatmentof flank Colo699 xenografts (Supplementary Fig. S12A–S12F),although the survival benefit afforded by the combination ther-apy was not statistically significant (P ¼ 0.06).

DiscussionUsing an unbiased RNAi screen, our study highlights MTOR as

an actionable protein kinase that can be targeted in combinationwith FGFR1 to achieve synergistic growth inhibition in FGFR1-dependent cancer cell lines. Themolecular basis for the synergismappears to involve the collapse of a greater signaling network bythe combination therapy than that achieved by either FGFR1 orMTOR inhibition alone. The findings in Fig. 5 support thedominant regulation of the ERK pathway downstream of FGFR1and activation of TORC1 targets (p70S6K and S6) and TORC2targets (pAKT S473) by MTOR. Moreover, this rationally derivedcombination of MTOR inhibitors with FGFR inhibitors is con-sistent with an extensive literature demonstrating benefit of add-ing MTOR inhibitors to various targeted therapeutics. For exam-ple, a rapamycin analogue increased growth inhibition by pona-tinib in FGFR2-driven endometrial cancer cell lines (22), andcombining a dual PI3K–MTOR inhibitor with TKIs active on BCR-ABL yielded increased growth inhibition of CML cell lines (23).Combination of a BTK inhibitor with the MTOR inhibitor,AZD2014, induced synergistic killing of diffuse large B-cell lym-phoma cells (24), and the benefit of combining IGF inhibitorswith MTOR inhibitors for reducing growth of Ewing sarcoma celllines has been documented (25). The generality of the synergisticgrowth suppression achieved with addition of MTOR inhibitors

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Figure 4.AZD4547 andMTOR inhibitors, AZD2014, alone and combined ongrowth of H1581 andColo699 cells. H1581, Colo699, and 584-A2 cellswere submitted to anchorage-independent growth assays with 100 nmol/L (H1581) or 300 nmol/L (Colo699, 584-A2) AZD4547 with or without 100 nmol/L AZD2014. After 14 to 21 days,viable colonies were stained with NBT and total colony area was measured as described in Materials and Methods. The data were normalized to DMSO controltreatments and are the means and SEM (n ¼ 3), where �� , P < 0.0001; �� , P < 0.001.






indicates the degree to which MTOR participates in cancer sig-naling networks.

The identification of MTOR as an SL protein kinase in thesetting of ponatinib treatment of H157 andH1299 cells as well asthe ability of AZD8055 and AZD2014 to synergize with AZD4547in Colo699 and H1581 cells supports a model where MTORsignals in parallel with, not downstream of FGFR1. In this regard,our results are consistent with the existence of RTK coactivationnetworks as reviewed by Xu and Huang (14) where the ERKpathway distal to FGFR1 and MTOR represents distinct fragilepoints. The ability of theMEK inhibitor, selumetinib, to synergizewith the MTOR inhibitor, AZD8055, supports this hypothesis(26). The identification of receptors or RTKs that reside upstreamofMTOR in FGFR1-dependent cancer cells remains to be defined.Also, MTOR exists within two distinct signaling complexes,TORC1 and TORC2 (27) and the identity of the precise complexmediating the SL, and essential activities measured in our studywere not resolved. The kinome shRNA library lacked shRNAs toRaptor and Rictor that would distinguish TORC1 and TORC2 andspecific silencing of Rictor or Raptor yielded equivocal results(data not shown). A likely scenario is that both complexesparticipate in growth regulation in lung and HNSCC cells andclearly, both TORC1 and TORC2 are inhibited by AZD8055 and

AZD2014 (18). Also, rapamycin strongly inhibited growth (Sup-plementary Figs. S5 and S8), supporting the critical involvementof TORC1. It is noteworthy that AKT1, 2, or 3 were not high-ranking hits in the screens, although redundancy among the threegene products could preclude identification of anywith our RNAi-based approach. Like MTOR inhibitors, two independent AKTinhibitors synergized with ponatinib for growth inhibition ofmultiple cell lines (Supplementary Fig. S10 and S11), providingsupport for AKT as at least one important target of MTOR in thesecancer cells.

In light of the potent in vitro sensitivity of Colo699 andH1581 cells to FGFR TKIs (Figs. 3 and 4), we were surprised bythe modest degree of growth inhibition achieved withAZD4547 alone when these cell lines were propagated as flankxenografts in nu/nu mice (Fig. 6 and Supplementary Fig. S12).Still, this degree of growth inhibition is not inconsistent withthe early results of clinical trials of AZD4547 and BGJ398 insquamous cell lung cancer where only partial responses havebeen observed thus far in less than 20% of patients (28,29). Onthe basis of our present studies showing strong in vivo responseswith flank xenografts that are limited to combination treatmentwith AZD4547 and AZD2014, we wonder whether targetingauxiliary pathways such as MTOR will be required to observe

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Figure 5.Signal pathway inhibition and apoptosis induction by AZD4547 and AZD8055 alone, and in combination. A, H1581 and Colo699 cells were incubated with 0.1%DMSO as a control, AZD4547 (300 nmol/L), AZD8055 (100 nmol/L), or the combination of the two drugs for 2 hours as described in Materials and Methods.Cell extracts were prepared and submitted to immunoblot analyses for the indicated signaling intermediates. Phospho-AKT (S473) is a measure of TORC2 activitywhereas phospho-p70S6K (T389), phospho-S6 (S235/236), and phospho-Rictor (T1135) are measures of activity through the TORC1 pathway. The filters werestripped and reprobed for total ERK, AKT, p70S6K, S6, and Rictor to ensure equal loading of cell protein in each lane. B, H1581 cells were treated for 3 dayswith 0.1% DMSO as a control, AZD4547 (300 nmol/L), AZD8055 (100 nmol/L), or the combination of the two drugs. Cell extracts were prepared andsubmitted to immunoblot analysis of PARP1 and the a-subunit of the NaK-ATPase as a loading control. The cleavage product of PARP1 is indicated. C, cellextracts from H1581 cells treated 3 days with 0.1% DMSO, AZD4547 (300 nmol/L), AZD8055 (100 nmol/L), or the combination of the two drugs weresubmitted to a caspase-3 enzymatic assay as described in Materials and Methods. The data are the initial rates of the assays in triplicate determinations.Caspase-3 activity in combination-treated cells is significantly different (��, P < 0.0005) from activity in either monotherapy-treated extracts.

Singleton et al.





significant clinical responses in FGFR1-dependent lung cancersandHNSCCs. If so, it is important that FGFR1not be immediatelyabandoned as a therapeutic target in these settings based solely onamarginal clinical response to FGFR TKI monotherapy. Althoughthe frequent andprofound tumor shrinkage responsesobserved inlung cancers bearingmutant EGFR or rearranged ALK treated withTKImonotherapies have established a new expectation for clinicalresponsiveness, we hypothesize that cancers driven by nonmu-tated drivers like FGFR1 may inherently depend more on RTKcoactivation networks for full-transforming potential (14).Admittedly, clinical investigation of combination therapies isunwieldy relative to monotherapy and bears increased concernof drug toxicity. Still, the fact that many lung cancers and themajority of HNSCC will present with nonmutated oncogenedrivers necessitates careful consideration of combination thera-pies as a starting point in treatment design.

Disclosure of Potential Conflicts of InterestL.E. Heasley reports receiving a commercial research grant from ARIAD. No

potential conflicts of interest were disclosed by the other authors.

Authors' ContributionsConception and design: K.R. Singleton, A.C. Tan, L.E. HeasleyDevelopment of methodology: K.R. Singleton, A.C. Tan, L.E. HeasleyAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): K.R. Singleton, T.K. Hinz, E.K. Kleczko, L.A. Marek,J. Kwak, A.C. Tan, L.E. HeasleyAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): K.R. Singleton, T.K. Hinz, E.K. Kleczko, L.A. Marek,J. Kwak, J. Kim, A.C. Tan, L.E. HeasleyWriting, review, and/or revision of the manuscript: K.R. Singleton, J. Kwak,J. Kim, A.C. Tan, L.E. HeasleyAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): L.E. HeasleyOther [assisted with execution of experiments and maintenance of laboratorymaterials (making buffers, media, cleaning supplies, etc.) and presentation ofthe data collected (generation of graphs and images] for experiments): T. Harp

AcknowledgmentsThe authors thank the Next-Generation Sequencing Production Core and

staff within the University of Colorado School of Medicine, Department ofBiochemistry and Molecular Genetics for expert assistance with the IlluminaGenome Analyzer II sequencing of the kinome shRNA library. In addition, the

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Figure 6.Combination AZD4547 and AZD2014 provides superior growth inhibition of H1581 xenografts. Flank xenografts derived from H1581 cells were generated asdescribed in Materials and Methods. When the tumors reached 100 mm3, the mice were randomized to treatment by daily oral gavage with diluent (A), 12.5 mg/kgAZD4547 (B), 10 mg/kg AZD2014 (C), or combined AZD4547 and AZD2014 (D). The individual tumor volumes relative to their initial volumes are shownfor the different treatment groups. The average fold change in tumor volume among the groups after 2 weeks of treatment is shown in E and reveals significantlygreater growth inhibition by the combination relative to either monotherapy. F, Kaplan–Meier survival curve analysis of the mice in the four groups demonstratessignificantly longer survival (P ¼ 0.0002) with combination therapy relative to the mice treated with diluent or the monotherapies.






authors acknowledge AstraZeneca for the supply of AZD4547 and AZD2014 aswell as ARIAD Pharmaceuticals for the supply of ponatinib.

Grant SupportThe studies were supported by the NIH (Lung SPORE P50 CA58187, UC

Cancer Center Support grant P30 CA046934) and the VA (Merit Award1BX001994-01).

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 February 19, 2015; revised June 26, 2015; accepted July 20, 2015;published OnlineFirst September 10, 2015.

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Singleton et al.




2015;75:4398-4406. Published OnlineFirst September 10, 2015.Cancer Res Katherine R. Singleton, Trista K. Hinz, Emily K. Kleczko, et al. FGFR1 Pathways for Treatment of Lung Cancer and HNSCCKinome RNAi Screens Reveal Synergistic Targeting of MTOR and

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