MET-Independent Lung Cancer Cells Evading EGFR Kinase … · Therapeutics, Targets, and Chemical Biology MET-Independent Lung Cancer Cells Evading EGFR Kinase Inhibitors Are Therapeutically

Therapeutics, Targets, and Chemical Biology

MET-Independent Lung Cancer Cells Evading EGFR KinaseInhibitors Are Therapeutically Susceptible to BH3 MimeticAgents

Weiwen Fan1, Zhe Tang2, Lihong Yin1, Bei Morrison1, Said Hafez-Khayyata3, Pingfu Fu4,8,Honglian Huang6, Rakesh Bagai2, Shan Jiang2, Adam Kresak8, Scott Howell9, Amit Vasanji7,Chris A. Flask8,10, Balazs Halmos11, Henry Koon2,8, and Patrick C. Ma1,5,8

AbstractTargeted therapies for cancer are inherently limited by the inevitable recurrence of resistant disease after

initial responses. To define early molecular changes within residual tumor cells that persist after treatment, weanalyzed drug-sensitive lung adenocarcinoma cell lines exposed to reversible or irreversible epidermal growthfactor receptor (EGFR) inhibitors, alone or in combination with MET-kinase inhibitors, to characterize theadaptive response that engenders drug resistance. Tumor cells displaying early resistance exhibited dependenceon MET-independent activation of BCL-2/BCL-XL survival signaling. Further, such cells displayed a quiescence-like state associated with greatly retarded cell proliferation and cytoskeletal functions that were readily reversedafter withdrawal of targeted inhibitors. Findings were validated in a xenograft model, showing BCL-2 inductionand p-STAT3[Y705] activation within the residual tumor cells surviving the initial antitumor response totargeted therapies. Disrupting the mitochondrial BCL-2/BCL-XL antiapoptotic machinery in early survivor cellsusing BCL-2 Homology Domain 3 (BH3) mimetic agents such as ABT-737, or by dual RNAi-mediated knockdownof BCL-2/BCL-XL, was sufficient to eradicate the early-resistant lung-tumor-cells evading targeted inhibitors.Similarly, in a xenograft model the preemptive cotreatment of lung tumor cells with an EGFR inhibitor and aBH3 mimetic eradicated early TKI-resistant evaders and ultimately achieved a more durable response withprolonged remission. Our findings prompt prospective clinical investigations using BH3-mimetics combinedwith targeted receptor kinase inhibitors to optimize and improve clinical outcomes in lung-cancer treatment.Cancer Res; 71(13); 4494–505. �2011 AACR.

Introduction

Lung cancer is the second most common cancer andcontinues to have the highest cancer-mortality rates. Receptortyrosine kinase (RTK) is a main class of druggable molecular

targets, such as epidermal growth factor receptor (EGFR; 1, 2),MET (3, 4), which can be therapeutically inhibited in humancancer therapy. EGFR tyrosine kinase inhibitors (TKIs), gefi-tinib and erlotinib, are approved targeted agents againstnonsmall cell lung cancer (NSCLC), with enhanced efficacytoward tumors that express somatic sensitizing kinasedomain mutations (e.g., L858R, exon 19 deletions; 5–7). Oneof the most formidable challenges of targeted therapy is theinvariable tumor secondary resistance after initial response.MET genomic amplification has been implicated in about 20%of acquired EGFR TKI resistance (8, 9) whereas the EGFRgatekeeper T790M kinase mutation (10–12) accounts forapproximately half of the resistant cases. Further targetingstrategies to overcome EGFR TKI resistance include the use ofirreversible TKIs (10, 13, 14), pan-EGFR/ERBB kinase inhibi-tors (15), and MET inhibitors (8, 16). The MET receptor hasbeen shown to be an important molecule in a variety ofmalignancies (3, 17) and has recently been validated as anattractive therapeutic target in cancer therapy, including lungcancer (4, 18–23). Reversible small molecule inhibitors totarget against MET have been developed for novel anticancertherapeutic intervention (20, 21, 24–26). Studies from ourgroup and others have recently showed the cross-talk signal-ing network between EGFR andMET, and also the role of MET

Authors' Affiliations: 1Department of Translational Hematology andOncology Research, Taussig Cancer Institute, Cleveland Clinic; 2Divisionof Hematology/Oncology, Department of Medicine, 3Department ofPathology, and 4Department of Biostatistics, 5Cleveland Clinic LernerCollege of Medicine, Case Western Reserve University; 6Department ofImmunology, 7Animal Imaging Core, Lerner Research Institute, ClevelandClinic; 8Case Comprehensive Cancer Center; 9Center of Visual Research,10Case Center of Imaging Research, University Hospitals Case MedicalCenter, Cleveland, Ohio; 11Division of Hematology/Oncology, Departmentof Medicine, Columbia University, New York, New York

Note: W. Fan and Z. Tang have contributed equally to the work.

Current address for Z. Tang: First Affiliated Hospital of Zhengzhou Uni-versity, Zhengzhou, Henan, China 450001.

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

Corresponding Author: Patrick C. Ma, Taussig Cancer Institute, Cleve-land Clinic, 9500 Euclid Avenue, R40, Cleveland, OH 44195. Phone: 216-445-5545; Fax: 216-636-2498; E-mail: [email protected]

doi: 10.1158/0008-5472.CAN-10-2668

�2011 American Association for Cancer Research.

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inhibition in combination with EGFR inhibitor in lung cancerin overcoming MET amplified resistance (8) or T790M–EGFRmediated resistance (16) to EGFR–TKI.Further knowledge into additional mechanisms of tumor-

cell resistance to targeted inhibitors should prove to be of greatsignificance in the quest for novel effective treatment strategiesto impact the long-term prognosis of lung cancer. Majority ofthe reported studies investigating mechanisms of tumor resis-tance centered on late time window after chronic exposure toTKIs at escalating dosing concentrations when secondaryresistant clones ultimately arose and propagated from theparental drug-sensitive cell populations. Nonetheless, a deepunderstanding of the entire spectrum of tumor cells mechan-istic strategies to escape or evade targeted therapeutics inresistance, especially during the early inhibitory phase, remainsto be better defined at present (27, 28). Here, we investigatedthe "early" molecular events in lung tumor cells under targetedEGFR alone or combined with MET-kinase inhibitors treat-ment. Our results identified that a resurgence of prosurvival-antiapoptotic signalingwas evident in the surviving tumorwithearly evasion against the targeted kinase inhibitors, thatinvolved a TKI-induced dependence of activated STAT3, andits transcriptional target BCL-2/BCL-XL, with therapeutictranslational values. Our results show that proapoptoticBCL-2 Homology Domain 3 (BH3)-mimetic, such as ABT-737, can be effective in eradicating these "early" TKI-resistantlung tumor evader cells, thereby potentially enhancing thelong-term efficacy of targeted EGFR lung cancer therapy.

Materials and Methods

Cell culture and immunoblottingLung cancer cell lines were obtained directly from American

Type Culture Collection (ATCC) and grown under standardcell culture conditions. Cell lines characterization and authenti-cation were carried out by the ATCCMolecular AuthenticationCenter, using cytochrome c oxidase subunit I (COI) for inter-species identification and short tandem repeat (STR) anlaysis(DNA fingerprinting) for intraspecies identification. Sodiumdodecylsulfatepolyacrylamidegelelectrophoresis (SDS—PAGE)and Western blotting were carried out as previously described(16, 29). The primary antibodies used are as follows: phospho-MET[Y1234/1235]: Cell Signaling Technology (CST), MET(C-12, Santa Cruz Biotechnology), phospho-EGFR[Y1068]; CST,EGFR (Santa Cruz), phosphotyrosine (p-Tyr), phospho-AKT[S473], AKT, phospho-MAPK(ERK1/2)[T202/Y204]—all fromCST,MAPK(ERK1/2):Biosource,phospho-STAT3[Y705];CST, STAT3, BCL-2, BCL-XL (all fromZymed), cleaved-Caspase-3[Asp175], cleaved-PARP[Asp214], phospho-STAT5[Y694]—allfrom CST, survivin (Zymed) and actin (Santa Cruz).

Chemicals and inhibitorsEGFR inhibitor (reversible) erlotinib was prepared as pre-

viously described (29, 30). MET inhibitors SU11274,PHA665752 and EGFR inhibitor (irreversible) CL-387,785 wereobtained from EMD–Calbiochem. BCL-2 family inhibitorsABT-737, obatoclax mesylate (GX15–070), and HA14–1 wereobtained from Selleck.

Cellular cytotoxicity, viability, and survival assaysCellular cytotoxicity and viability assays were carried out

using CellTiter 96 AQueous. One Solution Cell Proliferation(MTS) assay (Promega), according to the manufacturer'sinstruction at 72 hours after treatment with indicated inhi-bitors in 10% fetal bovine serum (FBS) media. For the studiesof cells under 9 days of pretreatment under targeted (EGFR/MET) inhibitors, the indicated inhibitors in the culture mediawere replenished at least every 2–3 days (which was verified tobe indistinguishable from daily TKI replacement) prior to cellharvesting at the end of the inhibitory culture for subsequentcellular assays.

For cell survival assay using crystal violet staining method,H1975 cells or HCC827 cells were treated as described inSupplementary Materials and Methods with indicated TKIsfor 6 days, followed by indicated BH3-mimetic inhibition withor without concurrent TKI for 3 extra days.

Time-lapsed video microscopy: Image analysis ofcytoskeletal functions

HCC827 cells were plated on cell-culture dishes in a tem-perature-controlled chamber at 37�C in an atmosphere of 5%CO2 for time-lapsed video microscopy (TLVM) analysis ofcytoskeletal functions and determination of cellular mitoticactivities as previously described (16) and also in Supplemen-tary Materials.

In vivo xenograft model and bioluminescence imagingof human lung cancer

Lung cancer xenograft. Firefly-luciferase(luc)-expressingHCC827 and H1975 lung cancer cells, and their correspondingmurine xenograft models were established as previouslydescribed (see also Supplementary Materials and Methods)according to institution-approved protocols and guidelines(16). Immunohistochemical (IHC) analysis of the tumor xeno-graft was carried out in the Tissue Procurement and HistologyCore Facility, Case Comprehensive Cancer Center, using anti-human BCL-2 (Abcam), antihuman p-STAT3[Y705] (rabbitmonoclonal antibody, D3A7, CST) primary antibodies. Fordetails see also Supplementary Materials and Methods.

Tumor microarrayHuman lung cancer tumor microarray was purchased from

Zymed-Invitrogen (MaxArray Human Lung Cancer TissueMicroarray Slides, Cat. No. 75–4083). IHC staining usingantihuman BCL-2 antibody was carried out as describedabove, and graded using 4-tier scoring system (0, 1þ, 2þ,and 3þ) by a dedicated thoracic pathologist (S. H.-K.). For thelung cancer tumor microarray (TMA) analysis, the TMAused in the analysis consisted of the followings: SquamousCell (n ¼ 25), Adenocarcinoma (n ¼ 21), Large Cell (n ¼ 3),SCLC (n ¼ 5), Carcinoid (n ¼ 2), Mesothelioma (n ¼ 2).

BCL-2/BCL-XL DNA transfection and RNA interferencestudies

Human BCL-2 plasmid vector was a generous gift from Dr.Clark Distelhorst (Case Western Reserve University). Trans-fection of the BCL-2 expression vector into HCC827 cells was

BH3-Mimetic Susceptible Early TKI-Resistant Tumor Cells

www.aacrjournals.org Cancer Res; 71(13) July 1, 2011 4495




carried out using Fugene 6 according to the manufacturer'sinstructions (Roche). RNA interference (RNAi) knockdownstudies were conducted using the Thermo Scientific/Dharma-con RNAi Technologies, including siGENOME siRNA-NT (non-targeting; Cat.#D-001210–02), siRNAs against human BCL-2(Cat.#L-003307–00), and BCL-XL (Cat.#L-003458–00). ForHCC827 cells (Fig. 6A–B), cells were plated at full confluenceon 48-well plates, then cultured for 9 days in serum-containingmedia (a) without inhibitor, or with treatment of (b) Erlotinibalone for 9 days, or (c–f) Erlotinib together with the followingsin combination: (c) ABT-737 (2 mmol/L) concurrently at Day 0,(d) siRNA-nontargeting (siNT), (e) siRNA-BCL-2, and (f) dualsiRNA-BCL-2/BCL-XL RNAi knockdown. Cells were then fixedin methanol and stained with 0.1% crystal violet as above atthe end of day 9 to visualize the early TKI-resistant tumorsurvivor cells emerged under various conditions. Experimentswere carried out in triplicate.

Statistical analysisIn the BCL-2 transfection study and erlotinib cellular cyto-

toxicity assay in the HCC827 cells (Fig. 5D), the results undereach transfection condition were first summarized by the areaunder the curve (AUC). The differences of AUC betweentransfection conditions were then examined by Z-test. Statis-tical data analysis of the in vivo study using HCC827-lucxenograft murine model (Fig. 6E) was carried out using themixed model to examine the difference of read-out[bioluminescence imaging (BLI) region of interest (ROI)]among the four study groups (I–IV), by the in vivo xenograftgrowth rate–changing rate over time [i.e., the change of read-out (BLI ROI) divided by time (day)]. To ensure the normalityassumption for the mixed model used is satisfied, the read-outs were transformed by natural log function, i.e., loge(read-out), prior to fitting the data using Mixed Model. Tumorrecurrence was defined as 20% increase of tumor BLI-fluxfrom the nadir and the difference of recurrence rates betweenGroup II (ABT-737 alone), Group III (Erlotinib alone), andGroup IV (ABT-737 plus Erlotinib) was examined by Fisher'sexact test. All tests were two-sided and P-values � 0.05 wereconsidered statistically significant.

Results

Tumor resistance emerged "early" from EGFR-reversible-TKI-sensitive lung adenocarcinoma evadingerlotinib:MET-independent BCL-2/BCL-XL signaling

The lung adenocarcinoma cell lines HCC827 and PC-9 areboth highly sensitive to reversible-EGFR inhibitors (erlotinib/gefitinib), owing to the oncogenic sensitizing EGFR exon 19deletion (E746_A750 del). Here, we focused to study the "early"molecular alterations in tumor cells under TKI treatment, inan attempt to uncover potential therapeutic "Achilles’ heel"for the tumor cells that may survive the TKI within the earlytime-window.

We first adopted the HCC827 cell line in the in vitro "early"TKI-resistance studies, with the cells cultured under ongoingerlotinib (1 mmol/L) inhibitory treatment up to 9 days.We chose the concentration of erlotinib to be used at

approximately IC70–75 in the 72-hrs cell viability assay. Byday 9 of inhibition, there were cell subpopulations ("early"survivors, HCC827_ERL-D9.R) that evaded and survived theTKI treatment. These "early" survivor cells exhibited a dra-matic shift of TKI-sensitivity phenotype toward higherresistance (�100-fold), compared with the TKI-naïve par-ental cells (Fig. 1A). After an initial inhibited state, there wasalso reactivated BCL–2/BCL–XL, within the background of atyrosine-phosphoproteomic reactivated cellular state of aunique profile different from the parental cells (Fig. 1B–C).Importantly, the tumor cells that survived up to days 6–9 ofthe EGFR–TKI treatment evidently signaled independentlyof EGFR and MET (Fig. 1C). Following an initial inhibitoryperiod, we observed a rather early p-STAT3[Y705] reactiva-tion despite ongoing erlotinib treatment. These restoredprosurvival-antiapoptotic markers correlated well withtime-dependent downregulated cleaved-caspase 3 andcleaved-PARP, indicative of a suppressed caspase-dependentintrinsic apoptosis mechanism among these TKI-evadercells. These results were further verified in PC-9 cell line,with the erlotinib-surviving PC-9 cells (PC-9_ERL-D9.R) atDay-9 inhibition exhibiting MET-independent upregulatedp-STAT3[Y705]/BCL-2/BCL-XL signaling, and "early" TKI-resistance (Supplementary Fig. S1).

EGFR-irreversible-TKI-sensitive lung adenocarcinomaH1975 cells also showed "early" tumor resistant evasionfrom CL-387,785

We also tested the H1975 cell line against the irreversible-EGFR-TKI CL-387,785 as an alternate model. Similarly, the"early" TKI-surviving H1975 cells (H1975_CL-D9.R) that wereharvested after 9 days of CL-387,785 (1mmol/L) exposure werefound remarkably more resistant (Fig. 2A), accompanied witha prosurvival-antiapoptotic signaling upregulation as early asbeyond day 3 of inhibition (Fig. 2B). Consistent with thefindings in both HCC827 and PC-9 cells, the H1975_CL-D9.R cells also signaled independent of MET. Intriguingly, theH1975_CL-D9.R cells were found to exhibit reactivated p-EGFR[Y1068] beyond day 3 and onward despite ongoingCL-387,785, in the presence of complete p-MET inhibition.Thus, the downstream prosurvival-antiapoptotic signaling inthe survivor cells may still be at least partially driven by EGFRsignaling. We verified that the reactivated EGFR phosphoryla-tion, and its downstream signals emerged in the H1975_CL-D9.R cells, could be inhibited by a higher concentration (5mmol/L) of the same inhibitor (Fig. 2C). Nonetheless, p-STAT3and BCL-2 were not effectively inhibited by the retreatment,highlighting their potential importance in promoting thecellular resistant survival, independent of both EGFR andMET signaling.

Early EGFR–TKI resistance exhibited "adaptive"phenotypes that were highly "reversible"

Using TLVM analysis, we found that the HCC827_ERL-D9.R exhibited a cellular "quiescence-like" state, with dramati-cally inhibited proliferative and cytoskeletal functions whilein evasion against erlotinib. Promptly after erlotinib-with-drawal, we found that the early resistant cells could readily

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Cancer Res; 71(13) July 1, 2011 Cancer Research4496




be reverted to a highly activated state of cellular motility(Fig. 3A) and mitotic proliferation (Fig. 3B; see also Supple-mentary Movies). We further tested to see if the Day-9 "early"resistant cells could maintain their resistant phenotype aftera brief period of TKI withdrawal. Interestingly, after only7 days of withdrawal of the corresponding TKIs, bothHCC827_ERL-D9.R and H1975_CL-D9.R cells quicklyreverted back to a highly TKI-sensitive phenotype, indis-tinguishable from parental-cell populations, respectively(Fig. 3C and E). Importantly, upon a washout period ofTKI-withdrawal, these early resistant escape survivor tumorcells reexhibited TKI-induced p-EGFR inhibition as in theTKI-naïve parental cells (Fig. 3D).

In vivo activated STAT3/BCL-2 prosurvival-antiapoptotic signal axis in "early" TKI-resistant lungtumor survivor cells

We extended our studies using in vivo xenograft model toexamine tumor cells that survived initial treatment withtargeted RTK-inhibitors. HCC827 xenograft was inhibited witherlotinib for 3 days, during which remarkable tumor responsewas evident as expected. Consistent with our in vitro data, theSTAT3 downstream transcriptional target BCL-2 expressionwas found induced in the TKI-evading survivor cells (Fig. 4A).Interestingly, these early TKI-resistant cells were localizedalong the peripheral rind of the tumor xenograft (Fig. 4A–B). P-STAT3[Y705] was predominantly membranous and less

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Figure 1. Targeted inhibition of EGFR-TKI-sensitive HCC827 lung adenocarcinoma cells resulted in emergence of "early" tumor TKI-resistant evadercells with MET-independent prosurvival-antiapoptotic signaling. A, significant shift of cell viability toward insensitivity in HCC827 cells that survived 9 days oferlotinib (1mmol/L) treatment. HCC827_ERL-D9.R cells displayed �100-fold increase in IC50 corresponding with a dramatically higher resistant phenotypeagainst the EGFR-TKI. B, activated tyrosine-phosphoproteome of early resistant HCC827 cells (HCC827_ERL-D9.R) evading erlotinib up to 9 days.C, activation of p-STAT3[Y705]/BCL-2/BCL-XL signaling in HCC827_ERL-D9.R cells.






so cytoplasmic in the untreated HCC827 cells. Conversely, theactivated p-STAT3 signal was predominantly nuclear in theresidual HCC827 tumor early survivor cells circumscribingalong the xenograft periphery (Fig. 4B).

Past studies suggested that non-T790M-EGFR mediatedacquired gefitinib/erlotinib resistance in sensitive-lung can-cer cells may include genomic MET amplification (8, 9) orhepatocyte growth factor (HGF) overexpression (31). Cur-rently, there are various clinical trials investigating thestrategy of combining EGFR- and MET-inhibitors to enhancetherapeutic efficacy and overcome acquired EGFR-TKI resis-tance in NSCLC. We have previously characterized the METinhibitors SU11274 (20) and PHA665752 (21) in lung cancernovel therapy, which were utilized in the present study.Similar to the EGFR-TKI monotherapy model above, therewas an upregulated BCL-2/BCL-XL prosurvival-antiapopto-tic signaling in H1975 TKI-evader cells after 9 days ofdual CL-387,785/PHA665752 inhibition, alongwith restoredp-STAT3 activation (Fig. 4C), and associating with a moreTKI-resistant phenotype in the evader cells (P ¼ 0.0118;Supplementary Fig. S2).

We recently reported the efficacy of combined SU11274-erlotinib (MET-EGFR/ERBB TKI) in vivo H1975 xenograftmodel in overcoming T790M-EGFR drug-resistance, withresultant near-complete BLI-radiographic complete response(16). Here, we further evaluated the microscopic residualTKI-evading H1975-luc tumor cells and found they residedprimarily along the tumor periphery juxtaposing the murinehost-microenvironment. These TKI-evading survivor tumorcells also exhibited upregulated BCL-2 expression (Fig. 4D).

BCL-2 signal pathway expression and its potentialtherapeutic utility in NSCLC inhibition by BH3-mimetic

BCL-2 was found to be expressed at varying levels in NSCLCcell lines and lung tumor tissues, albeit at a significantlylower level range than in SCLC (Fig. 5A–B). Interestingly,unlike BCL-2, the expression levels of BCL-XL appeared tobe more comparable among NSCLC and SCLC cell lines(Fig. 5A). TMA analysis showed that BCL-2 expression inNSCLC was primarily nuclear, whereas that in SCLC wasstrongly positive both nuclear and cytoplasmic. Stronger

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Figure 2. Targeted inhibition of irreversible EGFR-TKI-sensitive H1975lung adenocarcinoma cells also results in "early’ emergence of tumorresistant escape survivor cells with activated prosurvival-antiapoptoticsignaling. A, significant resistant shift of cell viability in H1975 cellsthat survived 9 days of CL-387,785 treatment. B, activation ofp-STAT3[Y705]/BCL-2/BCL-XL signaling in H1975 cells that survived9 days of CL-387,785 treatment. C, reactivated p-EGFR anddownstream signaling in day 9-resistant-survivors H1975 cells againstCL-387,785 (1 mmol/L) could be inhibited by higher dose ofCL-387,785 (5 mmol/L) in retreatment, but not p-STAT3 orBCL-2.

Fan et al.





BCL-2 nuclear expression was observed in squamous cellcomparing with adenocarcinoma subtype. Our results aboveprovide a rationale to target BCL-2 family signaling throughproapoptotic BH3-mimetic, such as ABT-737 (32–34), in orderto optimize targeted therapies. ABT-737 has been well-char-acterized recently and shown to antagonize BCL-2/BCL-XL,thereby inducing a proapoptotic effect through the mitochon-drial intrinsic apoptosis pathway. NSCLC cell lines wererelatively insensitive to ABT-737 (IC50 > 5 mmol/L), whereasthe SCLC H345 cell line tested was as expected highly sensitive(IC50 < 0.2 mmol/L; Fig. 5C). HCC827 cells with forced over-expression of transfected BCL-2 was sufficient to induce a

significantly higher erlotinib-resistance, with �100-foldincrease in IC50 (Fig. 5D).

ABT-737 inhibition in concert with targeted kinaseinhibitors, both in vitro and in vivo, eradicated "early"TKI-resistant tumor evaders and further inhibitedsubsequent tumor recurrence

We hypothesized that preemptive inhibition and eradica-tion of "early" resistant tumor cells in RTK targeted therapymay impact on the long-term outcome of targeted therapy. Weadopted RNAi knockdown of BCL-2/BCL-XL using siRNAmethods here to test in parallel with ABT-737 (Fig. 6A–B).

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Figure 3. "Adaptive" emergence of "early" TKI-resistant tumor survivor cells in HCC827 under erlotinib, and H1975 under CL-387,785 inhibitorypressure. HCC827 cells (A–C): A, TLVM: HCC827_ERL-D9.R cells exhibited a "quiescence-like" state with highly inhibited cell proliferation as wellas cellular cytoskeletal functions that were both readily reversible upon TKI-washout for 11 days. B, cell mitotic index (M.I.) of HCC827 cells under varioustreatment conditions. Error bar, S.E.M. (n¼ 4). *, P < 0.0001; **, P¼ 0.002; †, P¼ 0.03; z, P¼ 0.008. C, HCC827_ERL-D9.R cells were resensitized to erlotinibinhibition after 7 days of TKI-washout culture conditions, indistinguishable from the parental HCC827 cells sensitivity (P > 0.05, N.S.). H1975 cells (D–E):D, H1975_CL-D9.R cells were resensitized to TKI in cellular signaling inhibition promptly upon withdrawal of CL-387,785. E, H1975_CL-D9.R cells wereresensitized to CL-387,785 inhibition upon 7 days of TKI-washout, indistinguishable from the parental H1975 cells sensitivity (P > 0.05, N.S.).






Dramatic reduction in the early TKI-resistant tumor survivorcells was achievable by dual BCL-2/BCL-XL RNAi knockdownin conjunction with erlotinib, but not by mere knockdown ofBCL-2 alone. ABT-737, when used concurrently with erlotinibto inhibit HCC827 cells, also dramatically reduced emergenceof early TKI-resistant tumor survivor cells against erlotinib(Fig. 6B).

We further carried out in vitro ABT-737 inhibition studieson the NSCLC H1975 TKI-evading tumor cells that wereprimed to upregulate BCL-2/BCL-XL prosurvival signalingby (i) EGFR/ERBB inhibitor (CL-387,785; Fig. 6C), and (ii) dualEGFR–MET inhibitors (erlotinib plus SU11274; Fig. 6D). ABT-737, at a concentration relatively insensitive against H1975parental cells, completely eradicated the early CL-387,785-resistant H1975 evader cells (Fig. 6C, top), either alone or incombination with CL-387,785. Importantly, we showed thatthe "early" TKI-resistant tumor cells were primed to be moresusceptible to ABT-737 inhibition, exhibiting a muchenhanced proapoptotic marker cleaved-PARP induction bythe BH3-mimetic (Fig. 6C, bottom). Moreover, the dual-TKI-resistant H1975_ERL/SU-D9.R tumor cells could also be tar-

geted by ABT-737 to further induce apoptosis (Fig. 6D). OtherBH3-mimetic BCL-2 family inhibitors tested in our study,obatoclax (35) and HA14–1 (36), also showed efficacy(Fig. 6D). Similar to H1975 cells, early TKI-evading resistantHCC827 cells also displayed therapeutic susceptibility to BH3-mimetic in vitro (Fig. 6E).

Finally, we tested if the addition of the BH3-mimetic ABT-737 in vivo would prolong the duration of response inerlotinib-treated drug-sensitive HCC827-luc xenograft(Fig. 6E). The in vivo ABT-737 treatment dosage in our studywas chosen based on reported literature on the therapeuticrange in the tested sensitive cancer models (37). The tumorgrowth rate in recurrence of the ABT-737þErlotinib-treatedgroup (IV) was significantly lower than that of the Erlotinib-alone group–III [P ¼ 0.0009; Fig. 6E, Table 1; also Supple-mentary Fig. S3]. ABT-737-alone (Group II) did not accountfor the tumor regression and abrogation of tumor recurrenceas seen in the ABT-737þErlotinib group (Group IV; P ¼0.0004). Finally, the HCC827 tumor recurrence rates atday 18 and day 32 for Group III (Erlotinib-alone) animalswere 50% (P ¼ 0.014) and 62.5% (P ¼ 0.004), respectively,

A

HC

C82

7H

CC

827

B

C

D

BCL-2Control

H1975

BCL-2

BCL-2

Erlotinib

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MET/EGFR-TKIs:

PHA665752 (1 µmol/L)CL-387,785 (1 µmol/L)

P-EGFR

EGFR

P-MET

MET

P-STAT3

STAT3

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Actin

W.B.

ControlControl

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Erlotinib

BCL-2P-STAT3

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75 BCL-2

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10x

10x

Hr

0 1 4 24 72 9 D

ays

5x

5x

5x

20x

10x

10x

P-STAT3

P-STAT3

10x

10x20x 5x

Figure 4. In vivo xenograft lung tumor survivor cells evading against targeted inhibitors, with activated p-STAT3/BCL-2 antiapoptotic signaling. In vivoHCC827 xenograft model treated with erlotinib (n ¼ 6) revealed "early" TKI-resistant tumor survivor cells (A) with induced BCL-2 expression especiallyin the nuclear subcellular localization (red arrows), and (B) with nuclear translocation (red arrows) of p-STAT3[Y705] from the untreated cytoplasmic localization(yellow arrows). C, combined MET–EGFR TKIs treatment of H1975 cells in vitro also led to an induction of MET-independent activated p-STAT3[Y705],and BCL-2/BCL-XL signaling in TKI-evading survivor cells against 9 days of combined-TKIs (PHA665752 [1 mmol/L] and CL-387,785 [1 mmol/L]). D, inducedBCL-2 expression in H1975-xenograft residual dual SU11274/erlotinib-TKI-evader survivor cells.

Fan et al.





both significantly higher than Group IV (ABT-737þErlotinib;0%; Fig. 6E; Table 1).

Discussion

In recent years, molecularly-targeted cancer therapy hasrenewed our hope for cancer cure. Nonetheless, the challengesof clinical tumor resistance, both intrinsic and acquired,remain formidable and substantially limit long-term efficacy.Classic secondary mutational resistance [e.g., T790M-EGFRagainst gefitinib/erlotinib in lung cancer (11)], and receptorkinase class-switching [e.g., from EGFR-addiction to MET/HGF (8, 9, 16), IGF1-R (27, 38), or AXL (39) signaling] have beenidentified in earlier studies that emphasized on "acquired"drug-resistance at "late-stages" of chronic drug treatment. Ourstudy here focuses on the molecular changes of drug-sensitivetumor cell population within the "early" time-window oftargeted TKI treatment. We identified and further character-ized the "early" adaptive functional TKI-resistant lung adeno-carcinoma cells that survived and evaded EGFR/MET-TKI,as early as within 9 days of therapy. During our manu-script preparation, Settleman and colleagues reported theidentification of "drug-tolerant state" in cancer cell subpopu-lations that was maintained through engagement of IGF-1Rsignaling and an epigenetic alteration of chromatin state that

requires the histone demethylase RBP2/KDM5A/Jarid1A (27).Our report here lends further support to the emerging evi-dence of the existence of tumor cell subpopulations withadaptive resistant-escape under therapeutic inhibitory stress.These early adaptive resistant survivors likely serve as thefounder population as minimal residual disease in solidcancers under therapeutic pressure, which ultimately leadsto frankly recurrent resistant disease on therapy in the future.

Despite the new insights into nonmutational early resis-tance (28), detailed underlying regulatory mechanism(s) thatdirectly mediate the emergence of such early resurgent resis-tant cells against the inhibitors remain to be fully defined. Ourstudy here provided the first evidence that the "early" emer-gence of resistant tumor survivors evading EGFR/ERBB-METTKIs is independent of MET receptor signaling activation,contrasting previous reports ofMET genomic amplification asacquired resistance mechanism in HCC827 cells that escape"chronic" dose-escalating gefitinib inhibition at "late stages"after many months of treatment (8, 9). We present findingshere that the BCL-2-family signaling in the mitochondrial(intrinsic) programmed-cell death pathway may indeed repre-sent the central mechanism as tumor cells’ newly-dependentaddiction, in promoting "early tumor evasion" to survivetargeted therapeutics. Here, we also provide additional in vivotherapeutic study evidence to validate the efficacy of targeting

ANSCLC

Exposure:

(–)

“Light”

“Heavy”

BCL-2

BCL-2

TMA: IHC

TMA analysis

W.B.

Small cell Adenocarcinoma Squamous cell

Human lung cancer

Lung cancer TMA: BCL-2

BCL-2 Expression

HCC827-BCL-2

ABT-737 (µmol/L)

00.

078

0.15

60.

313

0.62

51.

25 2.5 5

HCC827

(†, ‡)

(*)

BCL-2

ParentalMock

SCLC

NSCLC

0 1+ 2+ 3+

3+

2+

1+

0

BCL-XL

HC

C82

7

PC

-9

A54

9

H59

5

H14

37

H19

75

H19

93

H69

H28

H20

52

MS

TO-2

11H

H34

5

H69

H28

H20

52

MS

TO-2

11H

H34

5

H18

38

H14

37

H19

75

H19

93

H18

38

H44

1

HC

C82

7

PC

-9

A54

9

H44

1

SCLC MesotheliomaB C

D1009080

7060

5040

3020

100

Per

cent

TM

A tu

mor

sam

ple

(%)

Per

cent

cel

l via

bilit

y (M

TS

)

Squa

mou

s cell

Larg

e ce

ll

Aden

ocar

cinom

a

100

50

00 0.001 0.01

Erlotinib (µmol/L)0.1 1 10

Moc

kPa

rent

al

BCL-

2 Tra

nsfec

ted

BCL-2

Actin

0

25

50

75

100

H441

H1975

H345

HCC827

Cel

l via

bilit

y (%

Con

trol

)

Figure 5. BCL-2/BCL-XL signaling expression and inhibition in lung cancer. A, BCL-2/BCL-XL expression in lung cancer. Top, BCL-2 and BCL-XL expressionin thoracic malignancy cell lines. Bottom, BCL-2 TMA-IHC staining in SCLC, adenocarcinoma, and squamous cell carcinoma of the lung. B, pattern of lungcancer TMA BCL-2 expression. C, cell viability assay by ABT-737 in HCC827, H441 and H1975 (NSCLC), and H345 (SCLC) cells. D, forced BCL-2overexpression in HCC827 cells desensitized the cells to erlotinib. BCL-2-transfected versus mock-transfected cells: †, P < 0.0001, or versus parental cells:z, P < 0.0001. Mock-transfected versus parental cells: *, P ¼ 0.99.






HCC827A

B

E

C DR

NA

i

Moc

ksi

-NT

si-B

CL-

2si

-BC

L-2/

BCL-

XL

Moc

ksi

-NT

si-B

CL-

2si

-BC

L-2/

BCL-

XL

BCL-2

BCL-XL

Actin

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6 Days

Treatment

H1975 cells

H1975

HCC827-luc in vivo bioluminescence imaging

W.B.

– – + +

– – + +– + – +

W.B.

DAY:Groups

I

I II

II

III

III A

B

A

B

IV

IV

I

II

III

A

B

A

B

Time

IV

0 4 18 32

– + +

– + ––

+

––– +

+

++

+

+

– – – +– +–

H1975

H1975 cells

U

U

U ER

L/S

U

Oba

tocl

ax

HA

14-1

HA

14-1

+ E

RL/

SU

Oba

tocl

ax +

ER

L/S

U

AB

T-73

7 +

ER

L/S

U

AB

T-73

7

UCL-387,785 x 6D

CL-387,785 (1 µmol/L)x 9 Days

SU11274 + Erlotinibx 9 Days

SU11274 + Erlotinib

Cleaved-PARPCleaved-PARP

ActinActin

CL-387,785 (1 µmol/L)

ABT-737 (2 µmol/L) ABT-737 (2 µmol/L)Oblatoclax (2 µmol/L)

Treatment at Days 9 (x8h): Treatment at Days 9 (x8h):

Erlotinib + SU11274 x 6D

CL-3

87,7

85

ABT-7

37 +

CL-

387,

785

ABT-7

37

Erlot

inib

Oblat

oclax

HA14

-1

HA14

-1 +

Erlo

tinib

Oblat

oclax

+ E

rlotin

ib

ABT-7

37 +

Erlo

tinib

ABT-7

37

Day 7–9:

Day 1–6: Day 1–6:

Treatment

Day 7–9:

Post-Transfection RNAi

(a) (b) (c)

(d) (e) (f)

2 Days

U ERL ERL+ABT-737

ERL+si-BCL-2/BCL-XL

HCC827 Cells

Treatment

(–)

(–) Erlotinib (1 µmol/L)

Day 7−9:

Day 1−6:

ERL+si-BCL-2ERL+si-NT

In vivo treatment groups:

BCL-XL

Actin

ImageMin=-2.8587e+06Max=8.2892e+06

p/sec/cm2/sr

Color barMin=-2000

Max=2.9905e+06

0

0.5

1.0

1.5

x106

2.0

2.5

bkg subflat-fieldedcosmic

Erlotinib

ABT-737

HCC827-luc xenograft

4000000

3000000

BLI

Flu

x (R

OI)

1600000

1200000

800000

400000

00 1 2 3 4

Weeks

EGFR-TKI ± BH3-Mimetics

5 6 7 8

* P = 0.0009† P < 0.0001†† P = 0.0004

* P = 0.0009† P < 0.0001

Figure 6. BH3-mimetic therapeutic inhibition of the BCL-2/BCL-XL programmed cell death pathway "Achilles’ heel" to eradicate "early" TKI-resistantlung tumor survivor cells. A, siRNA-mediated knockdown of BCL-2 and BCL-XL in HCC827 cells. WCLs at day 2 and day 6 post-siRNA transfection were thenextracted for Western blotting to verify efficient gene knockdown of the target protein(s) expression. B, BCL-2/BCL-XL RNAi knockdown or BH3-mimeticABT-737 (2 mmol/L) in conjunction with erlotinib (1 mmol/L) remarkably suppressed the emergence of "early" EGFR–TKI resistant tumor-evader cells inHCC827. Representative photomicrographs from the triplicate experiments are shown here. Mag: 50�. C, proapoptotic BH3-mimetic ABT-737 eradicated theH1975 early tumor prosurvival resistance against CL-387,785. H1975 cells that were pretreated with 6 days of CL-387,785 (1 mmol/L) were replatedat full confluence, followed by further treatments as indicated for 3 additional days in triplicate, either with CL- 387,785 (1 mmol/L), ABT-787 (2 mmol/L),or ABT-737þCL-387,785, followed by crystal violet cell staining (top). U, untreated control. Bottom, induction of proapoptotic marker cleaved-PARPby BH3-mimetic in the CL-387,785-resistant early tumor survivor H1975 cells. D, ABT-737, Obatoclax, and HA14-1 eradicated the H1975 early tumorprosurvival resistance against dual-TKIs inhibition by erlotinib/SU11274 (ERL/SU). The experiment was carried out with H1975 cells similar to (C)above, except that cells were pretreated with dual EGFR–MET inhibitors here, i.e., erlotinib (1 mmol/L)/SU11274 (1 mmol/L). BH3-mimetic used in treatmentdays 7–9 were all 2 mmol/L in concentration. Top, crystal violet cell survival staining assay. Bottom, BH3-mimetic treatment of the dual ERL/SU-resistanttumor cells induced a proapoptotic response. E, in vivo EGFR inhibition with erlotinib in conjunction with BH3-mimetic ABT-737 led to significantlymore durable tumor response and prolonged remission in HCC827-luc lung adenocarcinoma xenograft. Left, schematic outline of treatment conditions of thein vivo HCC827-luc xenografts. Middle top, in vitro HCC827_ERL-D9.R early resistant TKI-evader cells emerged after 6 days of erlotinib (1 mmol/L) treatment,and were eradicated by cotargeting BH3-mimetic inhibition using ABT-737 (2 mmol/L), Obatoclax (2 mmol/L), or HA14-1 (2 mmol/L)þ/-ongoing erlotinibfrom days 7 to 9. Middle bottom, in vivo HCC827- luc tumor xenograft growth, under treatment conditions as in Groups I–IV, was monitored by BLI asdescribed in the section Materials and Methods. Error bar, � SEM. Erlotinib-alone (III) versus ABT-737þErlotinib (IV): *, P ¼ 0.0009. Erlotinib-alone (III) versusDiluent Control (I), P < 0.0001 (†); ABT-737þErlotinib (IV) versus Diluent Control (I), P < 0.0001 (†). Group II (ABT-737-alone) versus Group IV(ABT-737þErlotinib), P ¼ 0.0004 (††). Right, BH3-mimetic ABT-737 in vivo treatment in conjunction with EGFR-TKI (Group IV) significantly abolishedlung tumor recurrence.

Fan et al.





the BCL-2 family antiapoptotic machinery in the residualtumor survivors under TKI(s) therapeutic inhibition(Fig. 6E). Collectively, our results further raise the promiseof the feasibility in "drugging" the drug-resistant residualtumor cells (40), particularly within an early therapeuticwindow-of-opportunity. Hence, targeting the mitochondrialantiapoptotic machinery as the secondary "Achilles’ heel"newly-emerged in the early-resistant tumor cells appears tobe an attractive therapeutic strategy. On the other hand,concurrent EGFR-TKI and ABT-737 treatment also signifi-cantly suppressed the emergence of early TKI-resistantHCC827 cells, evident as early as within 6 to 9 days (Fig. 6)with the efficacy lasting up to 4 to 6 weeks (data not shown).We believe that the novel therapeutic strategy in targeting theadaptive drug-evader tumor survivor cells emergent withinthe "early treatment time-window" is attractive, as theseevader cells are most likely more "homogeneous" molecularlythan those found eventually as overtly resistant disease after"chronic" TKI inhibition for months. "Late" TKI-resistanttumor cells likely already had undergone divergent resistantmolecular evolution in progression, hence more heteroge-neous, during the long time lapsed under chronic TKI stress.Our data suggest that the early tumor survival against TKI is

an "adaptive"mechanism, rather than a selection of preexistingresistant cell clones. It remains unclear at present as to whatdefinitively regulates and determines the cell fates early undertargeted inhibition, and which cells among the parental drug-sensitive cell populationwould emerge as resistant survivors inthe beginning of the tumor evolution under therapeutic stres-sors. Nonetheless, the contribution of intrinsic molecularheterogeneity and nongenetic variation within individual cellsamong the parental cell population may still play at least apartial role in the ultimate cell-fate determination. Interest-ingly, BCL-2 has recently been implicated as inhibitor of DNArepairmechanism (41–44), whichmay potentially enhance andfacilitate themolecular evolution of tumor progression beyondthe "early" nonmutational resistance.Persistent STAT3 activation has been detected in a variety

of hematopoietic malignancies and solid tumors (45–47). Weobserved phosphorylated-STAT3(p-STAT3) in the residualtumor survivor cells both in vitro and in vivo under targetedkinase inhibitors. Our results suggest that "early" reactivationof STAT3 at tyrosine-705 (important in STAT3 dimerizationand subsequent nuclear translocation) may be an important

central transcriptional programming event prior to the ulti-mate resurgence of resistant tumor survivors. Recent attemptsto develop therapeutic inhibitors to target STAT3 have provento be rather difficult. Nonetheless, a number of BH3-mimeticthat target the key STAT3 downstream transcriptional targets,such as BCL-2/BCL-XL, have shown promise in preclinical andclinical studies, including ABT-737, and the newer pan-BCL-2family inhibitors ABT-263, and obatoclax (37, 48, 49). Thelatter pan-BCL-2 family inhibitors may potentially be moreadvantageous over ABT-737 in their effective inhibition ofMCL-1, shown to induce ABT-737 resistance (50).

To our knowledge, our study represents the first in vivoevidence that therapeutic targeting early resurgent resistanttumor survivor cells evading cancer targeted inhibitors isfeasible through inhibiting the mitochondrial antiapoptoticBCL-2/BCL-XL signaling in NSCLC, impacting on the thera-peutic outcome. Our results here provide support to furtherdevelop BH3-mimetic beyond basally BCL-2 overexpressingtumors such as SCLC and lymphomas, and extend to NSCLCas a therapeutic strategy to unleash the full potential and tooptimize long-term clinical outcome of oncogenic kinaseinhibitors. We propose that the combinational approach usingBH3-mimetic and RTK inhibitors should be investigatedfurther in the context of NSCLC human clinical trial studies.

Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed.

Acknowledgments

We thank Drs. Zhenghe Wang, Stan Gerson, and George Stark for helpfuldiscussion and suggestions. We also thank Mr. Joseph Molter for technicalassistance.

Grant Support

This work is supported by National Institutes of Health/National CancerInstitute–K08 Award (5K08-CA102545–05, 3K08-CA102545–05S1; P.C. Ma), Cle-veland Clinic Taussig Cancer Institute, Sol Siegal Lung Cancer Research GrantProgram, Case Comprehensive Cancer Center (Gene Expression and Genotyp-ing Core, Confocal Microscopy Core, Xenograft and Athymic Animal Core,Tissue Procurement and Histology Core, and Animal Imaging Core Facilities;P30-CA43703–12), and Northern-East Ohio Small Animal Imaging ResourcesProgram (R24-CA110943).

Received July 21, 2010; revised May 3, 2011; accepted May 3, 2011;published OnlineFirst May 9, 2011.

Table 1. Inhibition of HCC827-luc tumor in vivo xenograft recurrence rates by BH3-mimetic ABT-737treatment in conjunction with EGFR-inhibitor (Fig. 6E)

Treatment Groups Xenograft Recurrence Rates(%)

Day 18 Day 32

III A 50% (2/4) 50%a (4/8) 75% (3/4) 62.5%b (5/8)B 50% (2/4) 50% (2/4)

IV A 0% (0/6) 0% (0/12) 0% (0/6) 0% (0/12)B 0% (0/6) 0% (0/6)

aP ¼ 0.014.bP ¼ 0.004.






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2011;71:4494-4505. Published OnlineFirst May 9, 2011.Cancer Res Weiwen Fan, Zhe Tang, Lihong Yin, et al. AgentsInhibitors Are Therapeutically Susceptible to BH3 Mimetic MET-Independent Lung Cancer Cells Evading EGFR Kinase

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