Synthetic Lethal Screening Reveals FGFR As One of the Combinatorial Targets to Overcome Resistance to Met-Targeted Therapy

ORIGINAL ARTICLE Synthetic lethal screening reveals FGFR as one of the combinatorial targets to overcome resistance to Met-targeted therapy

B Kim1,4, S Wang2,4, JM Lee1, Y Jeong1, T Ahn1, D-S Son1, HW Park1, H-s Yoo1, Y-J Song1, E Lee1,YMOh1, SB Lee1, J Choi1, JC Murray2, Y Zhou3, PH Song1, K-A Kim1 and LM Weiner2

Met is a receptor tyrosine kinase that promotes cancer progression. In addition, Met has been implicated in resistance of tumors to various targeted therapies such as epidermal growth factor receptor inhibitors in lung cancers, and has been prioritized as a key molecular target for cancer therapy. However, the underlying mechanism of resistance to Met-targeting drugs is poorly understood. Here, we describe screening of 1310 genes to search for key regulators related to drug resistance to an anti-Met therapeutic antibody (SAIT301) by using a small interfering RNA-based synthetic lethal screening method. We found that knockdown of 69 genes in Met-amplified MKN45 cells sensitized the antitumor activity of SAIT301. Pathway analysis of these 69 genes implicated fibroblast growth factor receptor (FGFR) as a key regulator for antiproliferative effects of Met-targeting drugs. Inhibition of FGFR3 increased target cell apoptosis through the suppression of Bcl-xL expression, followed by reduced cancer cell growth in the presence of Met-targeting drugs. Treatment of cells with the FGFR inhibitors substantially restored the efficacy of SAIT301 in SAIT301-resistant cells and enhanced the efficacy in SAIT301-sensitive cells. In addition to FGFR3, integrin β3 is another potential target for combination treatment with SAIT301. Suppression of integrin β3 decreased AKT phosphorylation in SAIT301-resistant cells and restored SAIT301 responsiveness in HCC1954 cells, which are resistant to SAIT301. Gene expression analysis using CCLE database shows that cancer cells with high levels of FGFR and integrin β3 are resistant to crizotinib treatment, suggesting that FGFR and integrin β3 could be used as predictive markers for Met-targeted therapy and provide a potential therapeutic option to overcome acquired and innate resistance for the Met-targeting drugs.

Oncogene (2015) 34, 1083–1093; doi:10.1038/onc.2014.51; published online 24 March 2014

INTRODUCTION drug being tested in phase 3 clinical trials targets Met and other Met, a typical receptor tyrosine kinase (RTK) present on cell targets simultaneously or in combination with another drug.10 surfaces, is overexpressed in various tumors and may contribute Cabozantinib was approved by the United States Food and to the poor prognosis of several malignancies.1,2 Met functions Drug Administration to treat medullary thyroid cancer; this drug to mediate a wide spectrum of signals driven by binding with targets Met and vascular endothelial growth factor receptor 2 its ligand hepatocyte growth factor/scatter factor (HGF/SF) simultaneously.11 Onartuzumab, an anti-Met monoclonal human and promotes cancer progression, metastasis, cancer cell antibody, and tivantinib, a small-molecule Met inhibitor, are in migration and angiogenesis.3 HGF/SF binding to Met induces phase 3 clinical trials in patients with non-small-cell lung cancer – Met dimerization followed by the activation of intracellular (NSCLC) in combination with erlotinib.12 14 signal transduction such as mitogen-activated protein kinase Met has been validated as an oncogenic kinase in preclinical – and AKT phosphorylation.4,5 Among signal cascades induced by models through the use of selective kinase inhibitors.15 17 Met activation, AKT phosphorylation is related to cell survival;6 Although these inhibitors may induce early responses, the treatment of cancer cells with an anti-Met antibody results in a emergence of drug resistance is common and limits their marked antiproliferative effect, as well as a concomitant decrease effectiveness.18 The Met pathway was associated with acquired of AKT phosphorylation and an increase of apoptosis.7 resistance to epidermal growth factor receptor (EGFR) inhibitors in Because of the relevance of Met to cancer biology, it has been EGFR mutant NSCLCs.19 In turn, the activation of the HER family a popular target for cancer drug development.8,9 Several was shown to be responsible for the resistance of PHA665752, Met-targeted drugs such as cabozantinib, onartuzumab and a Met-speciﬁc inhibitor, in Met-addicted gastric cancer cells.20,21 tivantinib are in phase 3 registration trials. Every Met-targeted It was also reported that resistance to Met-targeting inhibitors

1BioTherapeutics Lab, Samsung Advanced Institute of Technology (SAIT), Giheung-gu, Yongin-si, Gyeonggi-do, South Korea; 2Lombardi Comprehensive Cancer Center, Department of Oncology, Georgetown University Medical Center, Washington, DC, USA and 3Biostatistics and Bioinformatics Facility, Fox Chase Cancer Center, Philadelphia, PA, USA. Correspondence: Dr K-A Kim, BioTherapeutics Lab, Samsung Advanced Institute of Technology (SAIT), 95 Samsung2-ro, Giheung-gu, Yongin-si, Gyeonggi-do 446-712, South Korea. E-mail: [email protected] or Dr LM Weiner, Lombardi Comprehensive Cancer Center, Department of Oncology, Georgetown University Medical Center, 3970 Reservoir Road NW, Research Building, Washington, DC 20057, USA. E-mail: [email protected] 4These authors contributed equally to this work. Received 16 September 2013; revised 30 December 2013; accepted 14 January 2014; published online 24 March 2014 FGFR in Met-targeting therapy B Kim et al 1084 can occur through MET point mutations, especially at Y1230,22 The MKN45 gastric cancer cell line is dependent on Met MET gene amplification followed by KRAS overexpression in signaling for proliferation and survival. This cell line was screened Met-addicted gastric and lung cancer cells,23 and overexpression with the siRNA library targeting 1310 genes in combination with 24 of constitutively active SND1-BRAF fusion protein. In NSCLC, the vehicle or SAIT301 at IC20 (inhibitory concentration 20). Primary mechanism of acquired resistance to EGFR/Met tyrosine kinase hits were identified as genes that, when knocked down, cause inhibitor was attributed to the activation of mammalian target of reduction of cell viability by >15% of normalized cell viability in rapamycin and the Wnt signaling pathway.25 the presence of SAIT301 compared with vehicle control (false However, the underlying mechanism of acquired or inherent discovery rate (FDR) o20% and sensitization index (SI) o0.85) in resistance to Met-targeted antibodies has not been fully three independent experiments. elucidated.26–28 Although the relationship between Met and other To further validate the positive hits, MKN45 cells were screened RTKs in the survival of Met drug-resistant cancer cells remains using the deconvoluted siRNAs that target candidate genes uncertain, it has been shown that Met inhibitor-driven resistance identified in the initial screening studies. Statistical analysis could be rescued by inactivation of fibroblast growth factor confirmed that 69 validated genes significantly increased the receptor (FGFR) by small molecules.29,30 Recently, many sensitivity of MKN45 cells to SAIT301 treatment (Table 1). Therefore, approaches have focused on discovering biomarkers for patient the 69 genes were considered to be potential mediators of selection and exploring novel combination therapies.31 To identify resistance to SAIT301. Figure 1a demonstrates that the distribution systematically targets whose inhibition would increase the of viability and SI of 69 hits was independent of the viability response of cancer cells to Met inhibitors, we performed reduction induced by siRNA knockdown in the absence of antibody medium-throughput small interfering RNA (siRNA) library syn- treatment. Most of the sensitizing hits were connected in a thetic lethal screening targeting genes associated with systems physically interacting network (Figure 1b), and the distribution of biology-derived EGFR and Met signaling pathways.32 Here, we these hits was random in the context of the full library. show that FGFR could have a role as an alternative driver kinase We next assessed the efficacy of these 69 putative sensitizers to for Met because dependence on either FGFR or Met can be SAIT301 treatment in seven other cell lines: NCI-N87 (gastric compensated by activation of the other kinase. Therefore, cancer), HCC827 (lung cancer), BxPC-3 (pancreatic cancer), H1993 simultaneous inhibition of FGFR and Met or intervention at a (lung cancer), A549 (lung cancer), RKO (colon cancer) and common downstream effector such as AKT is required for effective HCC1954 (breast cancer). As shown in Figure 1c, 24 genes Met-targeted anticancer therapeutics. sensitized at least three of these cell lines to the effects of a Previous studies have shown that integrin β1mediatesEGFR Met-targeting antibody. drug resistance and its association with the Met signaling pathway 33 β in NSCLCs. Integrin subunits are adhesion molecules involved in Pathway analysis of the targets from the screen implicates the cell survival and cancer resistance to chemotherapy in breast FGFR and integrin pathways as key regulators of the efficacy of a 34,35 fi cancers. Here, we identify signi cant cross-talk between Met-targeting antibody integrin β3 and Met in HCC1954 breast cancer cells and investigate To delineate the functional associations and identify potential the mechanism of Met drug resistance related to integrin signaling. interacting partners of the 69 validated hits, we evaluated the hit We also demonstrate that perturbation of integrin β3 and FGFR network using the Ingenuity Pathway Analysis (IPA) software signaling significantly inhibits proliferation of SAIT301-resistant (http://www.ingenuity.com/). Gene set enrichment analysis MKN45 cells. These data provide a strong rationale for the use of showed that seven canonical signaling pathways were used by integrin β3 and FGFR inhibitors in Met-amplified tumors that have the identified 69 hits (Po0.015). The genes involved in each become resistant to selective Met inhibition, or to combined individual pathway are summarized in Figure 2a. As these therapy to prevent these resistance mechanisms. Our findings pathways were all statistically significantly enriched by the demonstrate a specific cross-talk of integrin, FGFR and Met analysis, we examined the frequency of the genes shared by pathways and suggest the partial overlap of downstream signaling different pathways. Genes such as RAF1, AKT2, SOS1 and KRAS and common cellular effects of each pathway. (shaded in blue) are heavily shared by six out of seven pathways, indicating that the RAS/RAF/MEK and PI3K/AKT signaling path- RESULTS ways are common downstream pathways. ITGB3, FGFR3 and SGK1 Synthetic lethal screening to identify sensitizers of cellular (red, orange shades) are not shared by more than two signaling response to a Met inhibitor pathways; therefore, representing the involvement of individual signaling pathways. Subsequent network analysis corroborated To identify molecular determinants that modulate cellular responses several interacting partners of Met signaling that potentially to Met-targeted therapies, we developed an siRNA library and mediate resistance to Met inhibitors. Among them, highly performed synthetic lethal screening using a Met-specificmono- ranked partners are cell surface receptors triggering intracellular clonal antibody, SAIT301.7,36 Previously, we reported that SAIT301 signaling system, such as FGFR3, involved in the FGFR signaling promotes Met degradation via an LRIG1-mediated pathway. SAIT301 pathway, and ITGB3, triggering the integrin signaling pathway. treatment promoted the binding of Met with LRIG1, bypassing the Depletion of these two genes significantly reduces cancer cell Cbl-mediated Met degradation pathway that requires Met activation. viability when used in combination with SAIT301 treatment. IPA This unique mechanism permits SAIT301 to induce Met degradation analysis of the distribution of connections indicates potential without triggering Met signaling activation, and consequently interactions among the FGFR, integrin and Met signaling pathways activate cellular apoptosis.7 The siRNA library used in our studies (Figure 2b), which may serve as compensatory pathways for escape comprised of siRNAs targeting 1310 genes. We used Met as a seed from Met blockade. node to collect data from public archives reporting curated pathway On the basis of these findings, FGFR3 and ITGB3 were selected information, protein–protein interactions, association in protein for further studies that explore the potential for SAIT301-based complexes and putative genes responsive to Met antibodies combination therapy. (Supplementary Figure S1). The data mining provided 828 genes in the Met-centered network. As there is good evidence of cross-talk among the Met and EGFR pathways, we included the 638 genes FGFR3 suppression increases the antiproliferative effects of from an EGFR-centered network described by one of us (LMW).32 A SAIT301 by induction of apoptosis in MKN45 cells total of 1310 genes comprised the final network, which included 156 FGFR signaling is known to be responsible for acquired resistance genes shared by the two networks (Supplementary Figure S1). to RTK inhibitors, such as crizotinib in acute myeloid leukemia29

Table 1. Sixty-nine validated MET antibody-sensitizing genes

Gene symbol Entrez ID Location Type(s) Drug(s)

AKT2 208 Cytoplasm Kinase Enzastaurin AREG/AREGB 374 Extracellular space Growth factor ATP1A2 477 Plasma membrane Transporter Digoxin, omeprazole, ethacrynic acid, perphenazine BCL2L1 598 Cytoplasm Other BCR 613 Cytoplasm Kinase Imatinib BMPR1A 657 Plasma membrane Kinase CALR 811 Cytoplasm Transcription regulator CASP1 834 Cytoplasm Peptidase CASP2 835 Cytoplasm Peptidase CCND2 894 Nucleus Other CD151 977 Plasma membrane Other CD1D 912 Plasma membrane Other CD247 919 Plasma membrane Transmembrane receptor Visilizumab, blinatumomab CD3E 916 Plasma membrane Transmembrane receptor Visilizumab, blinatumomab, muromonab-CD3 CDKN1B 1027 Nucleus Kinase CDKN2C 1031 Nucleus Transcription regulator CHRNA7 1139 Plasma membrane Transmembrane receptor Varenicline, ABT-089, isoflurane,mecamylamine, CRK 1398 Cytoplasm Other CTSD 1509 Cytoplasm peptidase CTTN 2017 Plasma membrane Other DIO1 1733 Cytoplasm Enzyme Propylthiouracil DOK2 9046 Plasma membrane Other DUSP2 1844 Nucleus Phosphatase E2F1 1869 Nucleus Transcription regulator EGR1 1958 Nucleus Transcription regulator EPB41L2 2037 Plasma membrane Other EPHB1 2047 Plasma membrane Kinase EPS15L1 58513 Plasma membrane Other FGFR3 2261 Plasma membrane Kinase Pazopanib FOS 2353 Nucleus Transcription regulator GAB1 2549 Cytoplasm Other GBP1 2633 Cytoplasm Enzyme GRB7 2886 Plasma membrane Other HIC1 3090 Nucleus Transcription regulator HOPX 84525 Nucleus Transcription regulator HSF4 3299 Nucleus Transcription regulator HSP90B1 7184 Cytoplasm Other 17-Dimethylaminoethylamino -17-demethoxygeldanamycin, IPI-504, cisplatin IL24 11009 Extracellular space Cytokine INSRR 3645 Plasma membrane Kinase ITGB3 3690 Plasma membrane Transmembrane receptor Abciximab, TP 9201, EMD121974, tirofiban KDM1A 23028 Nucleus Enzyme KRAS 3845 Cytoplasm Enzyme MAP3K11 4296 Cytoplasm Kinase MCM2 4171 Nucleus Enzyme MCM7 4176 Nucleus Enzyme MYB 4602 Nucleus Transcription regulator NFKB2 4791 Nucleus Transcription regulator PARP1 142 Nucleus Enzyme ABT-888, olaparib, INO-1001 PDX1 3651 Nucleus Transcription regulator PLAU 5328 Extracellular space Peptidase PRKAB1 5564 Nucleus Kinase PTPN11 5781 Cytoplasm Phosphatase RAB5A 5868 Cytoplasm Enzyme RAC1 5879 Plasma membrane Enzyme RAC2 5880 Cytoplasm Enzyme RAF1 5894 Cytoplasm Kinase CHIR-265, vemurafenib, regorafenib, sorafenib RGS16 6004 Cytoplasm other RPS6KA1 6195 Cytoplasm kinase RPS6KA2 6196 Nucleus kinase SERPINA3 12 Extracellular space Other SGK1 6446 Cytoplasm Kinase SIN3A 25942 Nucleus Transcription regulator SOS1 6654 Cytoplasm Other SPEN 23013 Nucleus Transcription regulator SRF 6722 Nucleus Transcription regulator STK3 6788 Cytoplasm Kinase TNIP2 79155 Cytoplasm Other TYR 7299 Cytoplasm Enzyme Hydroquinone, azelaic acid WDR1 9948 Extracellular Space Other Hits are listed alphabetically by the official Entrez gene symbol.

Figure 1. Design and screening of a targeted library. (a) Distribution of normalized viability and SI of 69 validated hits. The siRNAs for the 69 validated target genes are listed in the order of intrinsic impact on viability of MKN45 cells treated with the medium (blue circles). Blue circles, normalized viability with the siRNA knockdown. Red circles, SI for validated hits with SAIT301. (b) Network of 69 validated hits (red squares) sensitizing to Met-targeting SAIT301 in the context of the full library (blue squares). Lines (edges) represent connections based on protein–protein interactions. Hits and genes from the starting set that were not connected in the network are shown below the network. (c) Heatmap of the SI of the 69 validated genes in MKN45, H1993, A549, BxPC-3, HCC827, NCI-N87, HCC1954 and RKO cells. SI on log scale is shown on the heatmap. Gene with SI o1 are depicted in green, and those with SI >1 are shown in red.

and vemurafenib in melanoma.37 In the synthetic lethal screening mechanism for the regulation of Bcl-xL expression, we tested the studies described here, the growth inhibitory effect of SAIT301 was mitogen-activated protein kinase inhibitors (SB203580 and U0126) increased with concomitant knockdown of the FGFR3 gene in and an AKT inhibitor (MK-2206). Interestingly, a p38 inhibitor, MKN45 cells compared with control siRNA-transfected MKN45 cells SB203580, in the presence of a Met-targeting antibody, suppressed (Figure 3a). Knockdown of FGFR3 gene also increased the growth Bcl-xL expression, followed by induction of apoptosis (Figure 3f). inhibitory effect of other Met inhibitors such as PHA665752 and Consistent with this finding, BCL2L1 knockdown also increased the XL-184 (Figures 3b and c); PHA665752 is a small-molecule inhibitor growth inhibitory effect of SAIT301 (Figure 3g). In addition, FGFR that specifically targets Met,17 and XL-184 is a small-molecule signaling activation by basic fibroblast growth factor treat- inhibitor that targets the Met, vascular endothelial growth factor ment restored the growth inhibition induced by SAIT301 receptor 2 and Ret kinases.38 We then investigated the mechan- (Supplementary Figure S3), further confirming the role of FGFR isms through which FGFR3 knockdown promotes the antitumor activity in cellular sensitivity to a Met-targeting antibody. These activity of SAIT301 in MKN45 cells. FGFR3 knockdown increased the data strongly support that FGFR signaling has an antiapoptotic role apoptosis of MKN45 cells compared with treatment with SAIT301 in MKN45 cells by sustaining Bcl-xL expression, and FGFR alone (Figure 3d). Apoptosis induction by FGFR3 suppression was suppression makes these cells susceptible to apoptosis induced further confirmed by cleavage of poly ADP ribose polymerase by Met-targeting drugs. (PARP) and caspase-3 (Supplementary Figure S2). It is reported that FGFR3 inhibition leads to apoptosis due to decreased expression of Bcl-2.39 We therefore examined the expression of Bcl-xL, which is Combinations of selective FGFR inhibitors and Met inhibitors transcribed by BCL2L1, one of the 69 sensitizers identified by synergistically inhibit cancer cell growth accompanied by our synthetic lethal screening. FGFR3 knockdown substantially induction of apoptosis enhanced reduction of Bcl-xL protein and SAIT301-induced PARP Because genetic knockdown of FGFR3 sensitizes MKN45 cells to cleavage (Figure 3e). To determine the underlying signaling SAIT301 treatment, we assessed whether pharmacological

Figure 2. Canonical pathway enrichment and network analysis of 69 validated hits reveal links between Met and FGFR, integrin signaling pathway. (a) In silico analysis was performed using the IPA software and categorized the hits according to their proposed enrichment for canonical pathways. The top seven signaling pathways are plotted based on the number of genes identified within each pathway (Po0.015). Hits were included in one signaling pathway (red shades); two signaling pathways (orange shades); three signaling pathways (yellow shades); four signaling pathways (green shades); and six signaling pathways (blue shades). (b) Interactions among hits and potential interaction partners were accessed using IPA. Nodes represent genes, with their shape representing the functional class of the gene product, and the edges indicate the biological relationship between the nodes. Hits are represented in pink outlined symbols, and interaction partners not in the hit list are in black. inhibitors of the FGFR family have similar effects. The small- PHA665752 and XL-184, in the presence of PD173074 compared molecule tyrosine kinase inhibitor, PD173074, is a specific and with the treatment with either drug alone (Supplementary Figures potent inhibitor of the FGFR family.40 PD173074 potently inhibited S5a and b). We tested another selective FGFR inhibitor, tyrosine phosphorylation of FGFR in MKN45 cells and siRNAs NVP-BGJ398 (hereinafter referred to as BGJ398), that is currently directed against other FGFR family members also increased Met- in phase I clinical trials.41 BGJ398 combined with SAIT301 also has targeted antibody-mediated efficacy (Supplementary Figure S4). a synergistic effect on growth inhibition of both EBC1 and MKN45 MKN45 cell viability was reduced by Met inhibition using SAIT301 cells (CI o1; isobologram analysis; Supplementary Figure S6). alone, and this inhibition was significantly enhanced by the We then determined whether the effects of combining FGFR addition of PD173074 (combination index (CI) o1; isobologram inhibitors with SAIT301 were because of induction of apoptosis. analysis; Figure 4a). PD173074 treatment also promoted the cell SAIT301-mediated cell apoptosis in EBC1 cells was markedly growth inhibitory effect of crizotinib, a small-molecule targeting enhanced with the addition of PD173074 or BGJ398 (Figure 4f). Met, as well as ALK,16 PHA665752 and XL-184 (Figures 4b–d). To Furthermore, as measured by immunoblot analysis, PD173074 further confirm this effect, we tested EBC1, a lung cancer cell line significantly induced PARP cleavage when combined with SAIT301 that is known to be addicted to Met signaling for its survival.7 Cell (Figure 4g). PD173074 alone had no effect on the protein levels of growth inhibitory effect of SAIT301 was increased by the addition Bcl-xL, but exposure to the combination of PD173074 and SAIT301 of PD173074 in EBC1 cells (CI o1; isobologram analysis; induced a nearly complete ablation of Bcl-xL. As BCL2L1 knock- Figure 4e). Like MKN45 cells, EBC1 cells showed marked reduction down improved SAIT301 efficacy in MKN45 cells, combined of cell viability when treated with Met-targeting inhibitors, treatment with SAIT301 and ABT-263, a potent inhibitor of

Figure 3. FGFR3 suppression enhances the efﬁcacy of an anti-Met antibody through decreased expression of Bcl-xL. (a) The viability of MKN45 cells was measured by CTG assay after reverse transfection of control or FGFR3 targeting siRNAs for 24 h and treating with the medium or 0.016 μg/ml of SAIT301 for 72 h. The relative cell viability (%) represents percent growth compared with the control group (no antibody treatment). (b) The viability of MKN45 cells was measured by CTG assay after reverse transfection of control or FGFR3 targeting siRNAs for 24 h and treating with medium or 10 nM of PHA665752 for 72 h. The relative cell viability (%) represents percent growth compared with the control group (no inhibitor treatment). (c) The viability of MKN45 cells was measured by CTG assay after reverse transfection of control or FGFR3 targeting siRNAs for 24 h and treating with the medium or 62.5 nM of XL-184 for 72 h. The relative cell viability (%) represents percent growth compared with the control group (no inhibitor treatment). (d) The apoptotic rate of MKN45 cells was determined by caspase activation using Caspase 3/7 Glo assay (Promega, Madison, WI, USA), after reverse transfection of control or FGFR3 targeting siRNAs for 24 h and treating with the medium or 0.016 μg/ml of SAIT301 for 72 h. Cell numbers were normalized in parallel wells using CTG assay. The relative caspase-3/7 activity (%) is represented as a percentage comparison with the control group (no antibody treatment). (e) Cleaved PARP and Bcl-xL were detected by immunoblotting assay in MKN45 cells. Twenty-four hours after reverse transfection of control, FGFR3 or BCL2L1 targeting siRNAs, MKN45 cells were treated with 2 μg/ml of SAIT301 for 72 h. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was a loading control. (f) Cleaved PARP and Bcl-xL were detected by immunoblotting assay with MKN45 cells treated with 2 μg/ml of SAIT301 alone or in combination with p38 inhibitor (SB203580, 10 μM) for 48 h. (g) The viability of MKN45 cells was measured by CTG assay after reverse transfection of control or BCL2L1 targeting siRNAs for 24 h and treating with the medium or 0.016 μg/ml of SAIT301 for 72 h. The relative cell viability (%) represents percent growth compared with the control group (no antibody treatment).

Bcl-xL,42 synergistically inhibited cell growth of EBC1 cells that originally were sensitive to this antibody.7 The viability of (Figure 4h). Crizotinib also synergized with BGJ398 in EBC1 cells SAIT301-resistant clone nos. 1 and 24 was not affected by SAIT301 (Figures 4i and j). BGJ398 treatment increased the apoptotic rate treatment at up to 2 μg/ml concentrations that induced ~ 50% induced by Met inhibitors, PHA665752 or XL-184 (Figures 4k growth inhibition in parental MKN45 cells (Figure 5a). To and l). These data suggest that FGFR activation maintains characterize underlying molecular mechanisms of resistance in antiapoptotic regulation in Met-addicted cancer cells. We further these SAIT301-resistant clones, we investigated the activation confirmed the combination effect using a nude mouse hetero- status of several signaling mediators. As shown in Figure 5b, the tropic MKN45 xenograft model. SAIT301 demonstrated an phosphorylation of FGFR3 and AKT is higher in SAIT301-resistant inhibition of tumor growth, resulting in the reduction of tumor clones compared with the parental MKN45 cells. To test if volume by 27.8%. Combination treatment of SAIT301 and BGJ398 insensitivity of resistant cells to SAIT301 is due to the activation resulted in the reduction of tumor volume by 41.2% of AKT, we performed cell viability assays with SAIT301 in the (Supplementary Figure S7). These findings further support presence of an AKT inhibitor, MK-2206.44,45 AKT inhibition using the concept that FGFR inhibitors and Met-targeted drugs MK-2206 resensitized MKN45-SAIT301-resistant cells to SAIT301 cooperatively inhibit cancer cell growth. in a dose-dependent manner (Figure 5c and Supplementary Figure S8a). Because AKT is commonly involved in the signaling of several MKN45-SAIT301-resistant cells display activation of FGFR3, which RTKs, FGFR could be an upstream RTK of activated AKT in SAIT301- confers resistance to Met inhibition through reactivation of AKT resistant clones. As shown in Figure 5d, SAIT301-mediated signaling inhibition of AKT phosphorylation was significantly enhanced by As acquired resistance is a general phenomenon in targeted cotreatment with PD173074. To investigate the role of FGFR in therapy, identifying the relevant resistance mechanisms can acquired resistance for SAIT301, MKN45-SAIT301-resistant clone improve the clinical benefit of targeted cancer drugs.43 We no. 24 cells were treated with SAIT301 and BGJ398. Concomitant created SAIT301-resistant clones of MKN45 cells and EBC1 cells inhibition of Met and FGFR signaling using SAIT301 and BGJ398 FGFR in Met-targeting therapy B Kim et al 1089

Figure 4. FGFR inhibitor and anti-Met antibody synergize to activate apoptosis. (a) The viability of MKN45 cells was measured by CTG assay after treating with SAIT301 (0.04 μg/ml) alone or in combination with the FGFR inhibitor (PD173074, 10 μM) for 72 h. The relative cell viability (%) represents the percent growth as compared with the control group (no antibody and inhibitor treatment). The CI o1.0 indicates synergistic effects. (b) The viability of MKN45 cells was measured by CTG assay after treating with 16 nM of PHA665752 alone or in combination with the FGFR inhibitor (PD173074, 10 μM) for 72 h. The relative cell viability (%) represents the percent growth as compared with the control group (no inhibitor treatment). (c) The viability of MKN45 cells was measured by CTG assay after treating with 4 nM of PHA665752 alone or in combination with the FGFR inhibitor (PD173074, 10 μM) for 72 h. The relative cell viability (%) represents the percent growth as compared with the control group (no inhibitor treatment). (d) The viability of MKN45 cells was measured by CTG assay after treating with 4 nM of XL-184 alone or in combination with the FGFR inhibitor (PD173074, 10 μM) for 72 h. The relative cell viability (%) represents the percent growth as compared with the control group (no inhibitor treatment). (e) The viability of EBC1 cells was measured by CTG assay after treating with 0.04 μg/ml of SAIT301 alone or in combination with the FGFR inhibitor (PD173074, 5 or 10 μM) for 72 h. The relative cell viability (%) represents the percent growth as compared with the control group (no antibody and inhibitor treatment). (f) The apoptotic rate of EBC1 cells was determined by caspase activation using Caspase 3/7 Glo assay after treating with 0.08 μg/ml of SAIT301 alone or in combination with the FGFR inhibitors (PD173074, 10 μM; BGJ398, 5 μM) for 72 h. Cell numbers were normalized in parallel wells using CTG assay. The relative caspase-3/7 activity (%) is represented as a percentage comparison with the control group (no antibody treatment). (g) Cleaved PARP and Bcl-xL were detected by immunoblotting assay with EBC1 cells treated with 0.4 μg/ml of SAIT301 alone or in combination with PD173074 (10 μM) for 72 h. (h) The viability of EBC1 cells was measured by CTG assay after treating with 0.08 μg/ml of SAIT301 alone or in combination with the Bcl-xL inhibitor (ABT-263, 0.5 μM) for 72 h. The relative cell viability (%) represents the percent growth as compared with the control group (no antibody and inhibitor treatment). (i) The viability of EBC1 cells was measured by CTG assay after treating with 10 nM of crizotinib alone or in combination with the FGFR inhibitor (BGJ398, 5 μM) for 72 h. The relative cell viability (%) represents the percent growth as compared with the control group (no inhibitor treatment). (j) The apoptotic rate of EBC1 cells was determined by caspase activation using Caspase 3/7 Glo assay after treating with 20 nM of crizotinib alone or in combination with the FGFR inhibitor (BGJ398, 5 μM) for 72 h. Cell numbers were normalized in parallel wells using CTG assay. The relative caspase-3/7 activity (%) is represented as a percentage comparison with the control group (no antibody treatment). (k) The apoptotic rate of EBC1 cells was determined by caspase activation using Caspase 3/7 Glo assay after treating with 20 nM of PHA665752 alone or in combination with the FGFR inhibitor (BGJ398, 5 μM) for 72 h. Cell numbers were normalized in parallel wells using CTG assay. The relative caspase-3/7 activity (%) is represented as a percentage comparison with the control group (no antibody treatment). (l) The apoptotic rate of EBC1 cells was determined by caspase activation using Caspase 3/7 Glo assay after treating with 1 μM of XL-184 alone or in combination with the FGFR inhibitor (BGJ398, 5 μM) for 72 h. Cell numbers were normalized in parallel wells using CTG assay. The relative caspase-3/7 activity (%) is represented as a percentage comparison with the control group (no antibody treatment).

Figure 5. FGFR activation is required for acquired resistance to anti-Met antibody. (a) The viability of MKN45-resistant cells and MKN45- SAIT301-resistant clone nos. 1 and 24 was measured by CTG assay after treating with various concentrations (0.016, 0.08, 0.4 and 2 μg/ml) of SAIT301 for 72 h. The relative cell viability (%) represents the percent growth as compared with the control group (no antibody treatment). (b) Phosphorylation of FGFR3 and AKT was detected by immunoblot assay in MKN45-resistant cells and MKN45-SAIT301-resistant clone nos. 1 and 24. GAPDH was a loading control. (c) The viability of MKN45-SAIT301-resistant clone no. 1 cells was measured by CTG assay after treating with various concentrations (0.016, 0.008, 0.4 and 2 μg/ml) of SAIT301 alone or in combination with the AKT inhibitor (MK-2206, 5 μM) for 72 h. The relative cell viability (%) represents percent growth compared with the control group (no antibody and inhibitor treatment). (d) AKT phosphorylation was detected by immunoblot assay in MKN45-SAIT301-resistant clone no. 24 cells. Twenty-four hours after pre-treatment of FGFR inhibitor (PD173074, 10 μM), MKN45-resistant clone no. 24 cells were treated with medium or 2 μg/ml of SAIT301 for 1 h. (e) The viability of MKN45-SAIT301-resistant clone no. 24 cells was measured by CTG assay after treating with various concentrations (0.016, 0.008, 0.4 and 2 μg/ml) of SAIT301 alone or in combination with the FGFR inhibitor (BGJ398, 5 μM). The relative cell viability (%) represents percent growth compared with the control group (no antibody and inhibitor treatment). (f) The viability of EBC1-SAIT301-resistant clone no. 20 cells was measured by CTG assay after treating with various concentrations (8, 12, 16 and 20 nM) of crizotinib alone or in combination with the FGFR inhibitor (BGJ398, 3 μM) for 72 h. The relative cell viability (%) represents percent growth compared with the control group (no antibody and inhibitor treatment).

led to a dose-dependent inhibition of viability of MKN45-SAIT301- shown in Figure 6a, ITGB3 suppression by siRNA enhanced the resistant cells (Figure 5e). Because parental MKN45 cells displayed effects of SAIT301 on cell viability. A potent integrin β3 inhibitor, decreased levels of Bcl-xL following FGFR inhibition (Figure 3e), cilengitide,19,48 also enhanced the effects of SAIT301 in MKN45- we tested whether Met inhibition in combination with ABT-263 SAIT301-resistant cells (Figure 6b). Combination treatment of could inhibit the viability of MKN45-SAIT301-resistant cells. cilengitide further enhanced SAIT301-mediated inhibition of AKT ABT-263 as well as BGJ398 significantly reduced the cell viability phosphorylation compared with treatment with SAIT301 alone of MKN45-SAIT301-resistant no. 1 cells treated with SAIT301 (Figure 6c). (Supplementary Figure S8b). To confirm the role of FGFR function HCC1954 breast cancer cells have high Met protein expression, in acquired resistance to Met-targeting therapy, we tested and were used to test the efficacy of Met-targeting drugs.49 EBC1-SAIT301-resistant clones, and found that AKT phosphoryla- HCC1954 cells did not respond to SAIT301 treatment and were tion was elevated in these cells (Supplementary Figure S9a). shown to have very low protein levels of FGFR3 but relatively high Combined inhibition of Met and FGFR led to a reduction of levels of integrin β3 (data not shown). It is worth noting that an cell viability in a dose-dependent manner (Figure 5f and FGFR inhibitor, PD173074, yields no synergistic inhibition of Supplementary Figure S9b. These data further support FGFR and proliferation with SAIT301 in these cells (data not shown). The its downstream pathway-mediated signaling as a potentially reason might be low levels of FGFR3 phosphorylation compared important mechanism of acquired resistance to Met inhibition. with MKN45 cells in which FGFR inhibitors have synergistic effects with SAIT301 (Supplementary Figure S10). To investigate the role of ITGB3 in innate resistance to SAIT301, the effect of knockdown ITGB3 inhibition improves the antitumor efficacy of SAIT301 in of ITGB3 was tested in HCC1954 cells. The combination of SAIT301 cancer cells with acquired and innate resistance to Met inhibition treatment and ITGB3 knockdown resulted in greater inhibition of ITGB3 is another highly ranked hit from our synthetic lethal cell growth (Figure 6d) and AKT phosphorylation (Figure 6e) as screening study, and ITGB3 is known to influence the develop- compared with SAIT301 treatment alone. ment and progression of breast cancers.46,47 To investigate the In addition, to elucidate the potential implication of FGFR and relationship of ITGB3 expression to Met resistance, knockdown ITGB3 in cancer cell responses to Met-targeting therapy in general, assays were conducted with MKN45-SAIT301-resistant cells. As we first analyzed IC50 (half-maximal inhibitory concentration)

Figure 6. ITGB3 suppression increases antiproliferative effects of anti-Met antibody. (a) The viability of MKN45-SAIT301-resistant clone no. 1 cells was measured by CTG assay after reverse transfection of control or ITGB3 targeting siRNAs for 24 h and treating with various concentrations (0.016, 0.008, 0.4 and 2 μg/ml) of SAIT301 for 72 h. (b) The viability of MKN45-SAIT301-resistant clone no. 1 cells was measured by CTG assay after treating with various concentrations (0.016, 0.008, 0.4 and 2 μg/ml) of SAIT301 alone or in combination with the integrin β3 inhibitor (cilengitide, 10 μM) for 72 h. The relative cell viability (%) represents percent growth compared with the control group (no antibody and inhibitor treatment). (c) AKT phosphorylation was detected by immunoblot assay in MKN45-SAIT301-resistant clone no. 1 cells. Twenty-four hours after pre-treatment of the integrin β3 inhibitor (cilengitide, 10 μM), MKN45-resistant clone no. 1 cells were treated with medium or 2 μg/ml of SAIT301 for 1 h. (d) The viability of HCC1954 cells was measured by CTG assay after reverse transfection of control or ITGB3 targeting siRNAs for 24 h and treating with various concentrations (0.016, 0.008, 0.4 and 2 μg/ml) of SAIT301 for 72 h. (e) AKT phosphorylation was detected by immunoblot assay in HCC1954 cells. Forty-eight hours after reverse transfection of control or ITGB3 targeting siRNAs, MKN45-resistant clone no. 1 cells were treated with medium or 2 μg/ml of SAIT301 for 30 min. (f) CCLE mRNA expression data with crizotinib-treated 504 cell lines. Group A consists of cell lines with ‘low’ expression of FGFR2, FGFR3 and ITGB3, and Group B has cell lines with ‘high’ expression for at least one of the three genes. values for 504 cell lines following treatment with crizotinib of siRNAs targeting the 1310 Met- and EGFR-centered genes. By (PF-2341066) from the CCLE database (http://www.broadinstitute. focusing on the statistically significant viability changes and by org/ccle/) and categorized each cell line into three tiers (low, filtering off-target effects by requiring two or more siRNAs to medium and high) based on the expression levels of FGFR2, replicate the effects, we were able to identify 69 genes connected FGFR3 and ITGB3, with cutoffs at 33 and 67 percentile values. Cell to several signaling pathways that likely mediate SAIT301 lines that have ‘low’ expression for all three genes were assigned resistance. By examining the frequency of the genes shared by to Group A, whereas Group B consisted of cell lines that have these seven putative signaling pathways, we found many heavily ‘high’ expression for at least one of the three genes. The median shared hits (RAF1, AKT2, SOS1 and KRAS), located at the mediator IC50 values for Groups A and B are 6.81 and 8.00, respectively level of cross-talk involved in resistance, did not represent any (P = 0.045 by Mann–Whitney U-test) (Figure 6f). These data provide specific pathway. In contrast, canonical pathway enrichment and molecular validation of ITGB3 or FGFR family as being synthetically network analysis of the 69 gene validated hit set revealed lethal with a Met inhibitor under defined conditions. enrichment of the integrin and FGFR signaling pathways. ITGB3 and FGFR3 regulate the integrin and FGF signaling pathways, respectively, are well-enriched surface molecules and are DISCUSSION easily accessible to targeted therapies. Furthermore, potential As it is well known that repeated treatment with targeted cancer interaction indicated compensatory pathways among the FGFR, drugs eventually leads to drug resistance,18 investigating the integrin and Met signaling pathways. mechanism of acquired resistance is important in the develop- Recent reports have referred to signaling cross-talk between ment of cancer drugs. siRNA screening has been demonstrated as FGFR and Met. In acute myeloid leukemia, FGFR1 is responsible for an effective method for identifying the potential mechanisms of innate resistance to crizotinib by inducing HGF transcription.29 In acquired or inherent resistance to certain drugs.50,51 We devel- turn, HGF secretion compensates for cancer cell growth inhibition oped an siRNA library centered to Met and EGFR signaling and by BGJ398, a selective FGFR inhibitor.52 The results from the performed synthetic lethal screening to search for key regulators present study support previous findings, as we show that related to drug resistance against an anti-Met therapeutic suppression of FGFR can significantly improve the antiproliferative antibody. The siRNA library used in our studies was comprised effects of Met-targeting drugs in Met-addicted cell lines such as

© 2015 Macmillan Publishers Limited Oncogene (2015) 1083 – 1093 FGFR in Met-targeting therapy B Kim et al 1092 MKN45 (gastric cancer) and EBC1 (lung cancer). In MKN45, FGFR Biocarta, Linnea, Protein Lounge, STKE and literature searches (Figure S1). signaling has an antiapoptotic function by retaining Bcl-xL This resulted in an siRNA library comprising siRNAs targeting 1310 genes. expression through constitutively active p38, and suppresses the transcription of proapoptotic genes such as TRAIL and BIM (data Further testing of identified sensitizing genes in responsive and not shown). Pharmacological inhibitors targeting FGFR synergize non-responsive cell lines with Met-targeting drugs in preventing cancer cell growth in vitro We further tested the 69 validated hits for sensitization to SAIT301 in three and in vivo. These data suggest that a Met-targeting drug in SAIT301-responsive cell lines (BxPC-3, HCC827 and H1993) versus four combination with an FGFR inhibitor warrants further interrogation SAIT301-non-responsive cell lines (A549, NCI-N87, RKO and HCC1954). Cells in combination therapy. were transfected with siRNA pools comprising two most effective siRNAs In the present study, we demonstrated that SAIT301-resistant identified during the validation. SI and statistical significance were clones obtained through prolonged treatment with SAIT301 calculated as in the Supplementary Information. All experiments were exhibit enhanced FGFR3 activation and increased AKT phosphor- performed at least three times independently. ylation. AKT activation appears to be a key mechanism for resistance to the Met-targeting antibody, and MK-2206, an AKT Network analysis with 69 validated hits inhibitor, restores the sensitivity of resistant cells to SAIT301 For canonical pathway enrichment, network analysis and gene interaction (Figure 5c and Supplementary Figure S8a). FGFR signaling networks were generated using IPA (Ingenuity Systems, Redwood City, CA, compensates for the inhibition of Met in two ways: by retaining USA; http: \\www.Ingenuity.com). Sixty-nine validated hits were uploaded antiapoptotic molecules and by activating PI3K/AKT signaling. into the application. MiMI plugin for Cytoscape was used to access the These data provide the rationale for further investigation integrated molecular data and to retrieve interaction map that display combinations of Met and FGFR inhibitors to overcome acquired protein interactions. All 69 genes were hierarchically clustered as distance resistance. measure with Cluster 3.0, and then visualized using Java TreeView (version1.1.6r2, Jtreeview.sourceforge.net). We also examined integrin signaling, which emerged as another highly ranked resistance-promoting signaling pathway in the analysis of our screening studies. Met binds to integrin β3 and Generation of SAIT301-resistant MKN45 and EBC1 cells in vitro promotes the migration and invasion of mammary epithelial To generate SAIT301-resistant clones, MKN45 and EBC1 cells were exposed cells.47 Suppression of ITGB3 improved the efficacy of SAIT301 to increasing concentrations (range 1–10 μg/ml) of SAIT301 over 3 months fi in vitro, through antiproliferative effects but not by facilitating in vitro. To con rm that these clones are durably resistant to SAIT301, cell apoptosis in MKN45 cells (data not shown). Cilengitide, an integrin viability tests were performed after the resistant clones had been cultured in the presence or absence of SAIT301 for 6 weeks. β3 inhibitor, also restored sensitivity to SAIT301 in SAIT301- resistant MKN45 cells. In addition, integrin signaling may be related to innate resistance to SAIT301. SAIT301 alone has no Cell viability and apoptosis assay antiproliferative effect on HCC1954, even though this cell line Cell viability test and apoptosis assay were performed as reported 7,36 possesses a relatively high level of Met expression compared with previously. Synergism for drug combination was quantified by the CI, o other breast cancer cell lines. However, ITGB3 inhibition combined where CI 1 and CI = 0 indicate synergistic and additive effects, with SAIT301 had significant antitumor effects. Interestingly, respectively. CI was calculated with the CalcuSyn software (Biosoft, Cambridge, UK). transcript levels of ITGB3 are high in HCC1954 compared with other Met-addicted cell lines, such as MKN45 and EBC1 (data not shown). These findings imply that ITGB3 can be used as a putative CONFLICT OF INTEREST predictive marker as well as a target for combination therapy fi The following authors are employed by the Samsung Advanced Institute of using SAIT301. Although future studies will be required to con rm Technology: BK, JML, YJ, TA, D-SS, HWP, H-sY, Y-JS, EL, YMO, SBL, JC, PHS and K-AK. the hypothesis that the expression of FGFR and ITGB3 can both predict the efficacy of SAIT301 and comprise a new combination treatment strategy, our synthetic lethal screening approach ACKNOWLEDGEMENTS appears to be a useful way to identify potential candidates for LMW, SW and JCM are supported in part by NCI Grants CA51008 and CA50633. We combination with a tumor-targeting monoclonal antibody that thank Sandra A Jablonski, Wei Xu and Kyutaeg Lee for technical assistance. We are perturbs cancer cell signaling. grateful for the support of the Lombardi Comprehensive Cancer Center’s Genomics In summary, we have identified a number of key regulators of and Epigenomics Shared Resource. resistance to a Met-targeting antibody by synthetic lethal screening. Among them, we have validated that FGFR and integrin are important components for resistance mechanism toward SAIT301, REFERENCES and possibly for Met inhibitors in general. Combining Met 1 Lee HE, Kim MA, Lee HS, Jung EJ, Yang HK, Lee BL et al. MET in gastric carcinomas: inhibitors with FGFR or integrin inhibitors may be useful to comparison between protein expression and gene copy number and impact on mitigate the development of clinical resistance. In addition, both clinical outcome. Br J Cancer 2012; 107:325–333. FGFR and integrin may be useful as predictive markers for clinical 2 Garcia S, Dales JP, Charafe-Jauffret E, Carpentier-Meunier S, Andrac-Meyer L, response to Met inhibitors. Jacquemier J et al. Poor prognosis in breast carcinomas correlates with increased expression of targetable CD146 and c-Met and with proteomic basal-like phenotype. Hum Pathol 2007; 38: 830–841. MATERIALS AND METHODS 3 Birchmeier C, Birchmeier W, Gherardi E, Vande Woude GF. Met metastasis, motility and more. Nat Rev Mol Cell Biol 2003; 4:915–925. siRNA library construction 4 Wickramasinghe D, Kong-Beltran M. Met activation and receptor dimerization in We have previously reported on an EGFR network-targeted library,32 using cancer: a role for the Sema domain. Cell Cycle (Georgetown, TX) 2005; 4: 683–685. EGFR as the seed protein. This network was involved in this study based on 5 Xiao GH, Jeffers M, Bellacosa A, Mitsuuchi Y, Vande Woude GF, Testa JR. Anti- the evidence of cross-talk among the Met and EGFR pathways. A distinct apoptotic signaling by hepatocyte growth factor/Met via the phosphatidylinositol Met-centered network containing 828 genes was developed using the 3-kinase/Akt and mitogen-activated protein kinase pathways. Proc Natl Acad Sci open-source software tool, Cytoscape (www.cytoscape.org), with Met as USA 2001; 98:247–252. the seed protein. Bioinformatic databases were mined for protein–protein 6 Nicholson KM, Anderson NG. The protein kinase B/Akt signalling pathway in interactions, protein complexes, members of canonical pathways linked human malignancy. Cell Signal 2002; 14:381–395. with Met and additional genes include genes encoding RTKs and 7 Lee JM, Kim B, Lee SB, Jeong Y, Oh YM, Song YJ et al. Cbl-independent degra- differentially expressed genes in SAIT301-treated NCI-H441 cells. These dation of Met: ways to avoid agonism of bivalent Met-targeting antibody. sources included BIND, BioGRID, DIP, HPRD, IntAct, MiMI, MINT, STRING, Oncogene 2014; 33:34–43.

Oncogene (2015) 1083 – 1093 © 2015 Macmillan Publishers Limited FGFR in Met-targeting therapy B Kim et al 1093 8 Landi L, Minuti G, D'Incecco A, Cappuzzo F. Targeting c-MET in the battle against survival of head and neck squamous carcinoma cell lines. Mol Pharmacol 2013; advanced nonsmall-cell lung cancer. Curr Opin Oncol 2013; 25: 130–136. 83:882–893. 9 Scagliotti GV, Novello S, von Pawel J. The emerging role of MET/HGF inhibitors in 31 Morse DL, Gillies RJ. Molecular imaging and targeted therapies. Biochem oncology. Cancer Treat Rev 2013; 39:793–801. Pharmacol 2010; 80: 731–738. 10 Cecchi F, Rabe DC, Bottaro DP. Targeting the HGF/Met signaling pathway in 32 Astsaturov I, Ratushny V, Sukhanova A, Einarson MB, Bagnyukova T, Zhou Y, et al. cancer therapy. Exp Opin Ther Targets 2012; 16:553–572. Synthetic lethal screen of an EGFR-centered network to improve targeted 11 Nagilla M, Brown RL, Cohen EE. Cabozantinib for the treatment of advanced therapies. Sci Signal 2010; 3: ra67. medullary thyroid cancer. Adv Ther 2012; 29: 925–934. 33 Ju L, Zhou C. Association of integrin beta1 and c-MET in mediating EGFR TKI 12 Basilico C, Pennacchietti S, Vigna E, Chiriaco C, Arena S, Bardelli A et al. Tivantinib gefitinib resistance in non-small cell lung cancer. Cancer Cell Int 2013; 13:15. (ARQ197) displays cytotoxic activity that is independent of its ability to bind MET. 34 Huang C, Park CC, Hilsenbeck SG, Ward R, Rimawi MF, Wang YC et al. beta1 Clin Cancer Res 2013; 19: 2381–2392. integrin mediates an alternative survival pathway in breast cancer cells resistant 13 Sequist LV, von Pawel J, Garmey EG, Akerley WL, Brugger W, Ferrari D et al. to lapatinib. Breast Cancer Res 2011; 13:R84. Randomized phase II study of erlotinib plus tivantinib versus erlotinib plus 35 Lesniak D, Xu Y, Deschenes J, Lai R, Thoms J, Murray D et al. Beta1-integrin placebo in previously treated non-small-cell lung cancer. J Clin Oncol 2011; 29: circumvents the antiproliferative effects of trastuzumab in human epidermal 3307–3315. growth factor receptor-2-positive breast cancer. Cancer Res 2009; 69: 8620–8628. 14 Surati M, Patel P, Peterson A, Salgia R. Role of MetMAb (OA-5D5) in c-MET active 36 Oh YM, Song YJ, Lee SB, Jeong Y, Kim B, Kim GW et al. A new anti-c-Met antibody lung malignancies. Exp Opin Biol Ther 2011; 11: 1655–1662. selected by a mechanism-based dual-screening method: therapeutic potential 15 Lennerz JK, Kwak EL, Ackerman A, Michael M, Fox SB, Bergethon K et al. MET in cancer. Mol Cells 2012; 34:523–529. amplification identifies a small and aggressive subgroup of esophagogastric 37 Yadav V, Zhang X, Liu J, Estrem S, Li S, Gong XQ et al. Reactivation of mitogen- adenocarcinoma with evidence of responsiveness to crizotinib. J Clin Oncol 2011; activated protein kinase (MAPK) pathway by FGF receptor 3 (FGFR3)/Ras mediates 29: 4803–4810. resistance to vemurafenib in human B-RAF V600E mutant melanoma. J Biol Chem 16 Ou SH, Kwak EL, Siwak-Tapp C, Dy J, Bergethon K, Clark JW et al. Activity of 2012; 287: 28087–28098. crizotinib (PF02341066), a dual mesenchymal-epithelial transition (MET) and 38 Zhang Y, Guessous F, Kofman A, Schiff D, Abounader R. XL-184 a MET, VEGFR-2 anaplastic lymphoma kinase (ALK) inhibitor, in a non-small cell lung cancer and RET kinase inhibitor for the treatment of thyroid cancer, glioblastoma patient with de novo MET amplification. J Thorac Oncol 2011; 6: 942–946. multiforme and NSCLC. IDrugs 2010; 13: 112–121. 17 Smolen GA, Sordella R, Muir B, Mohapatra G, Barmettler A, Archibald H et al. 39 Zhu L, Somlo G, Zhou B, Shao J, Bedell V, Slovak ML, et al. Fibroblast growth Amplification of MET may identify a subset of cancers with extreme sensitivity to factorreceptor 3 inhibition by short hairpin RNAs leads to apoptosis in multiple the selective tyrosine kinase inhibitor PHA-665752. Proc Natl Acad Sci USA 2006; myeloma. Mol Cancer Ther 2005; 4: 787–798. 103: 2316–2321. 40 Mohammadi M, Froum S, Hamby JM, Schroeder MC, Panek RL, Lu GH, et al. Crystal 18 Holohan C, Van Schaeybroeck S, Longley DB, Johnston PG. Cancer drug structure of an angiogenesis inhibitor bound to the FGF receptor tyrosine resistance: an evolving paradigm. Nat Rev Cancer 2013; 13:714–726. kinase domain. EMBO J 1998; 17: 5896–5904. 19 Dechantsreiter MA, Planker E, Matha B, Lohof E, Holzemann G, Jonczyk A et al. 41 Guagnano V, Furet P, Spanka C, Bordas V, Le Douget M, Stamm C et al. N-methylated cyclic RGD peptides as highly active and selective alpha(V)beta(3) Discovery of 3-(2,6-dichloro-3,5-dimethoxy-phenyl)-1-{6-[4-(4-ethyl-piperazin-1- integrin antagonists. J Med Chem 1999; 42: 3033–3040. yl)-phenylamin o]-pyrimidin-4-yl}-1-methyl-urea (NVP-BGJ398), a potent and 20 Bachleitner-Hofmann T, Sun MY, Chen CT, Tang L, Song L, Zeng Z et al. selective inhibitor of the fibroblast growth factor receptorfamily of receptor HER kinase activation confers resistance to MET tyrosine kinase inhibition in tyrosine kinase. J Med Chem 2011; 54: 7066–7083. MET oncogene-addicted gastric cancer cells. Mol Cancer Ther 2008; 7: 42 Vogler M, Furdas SD, Jung M, Kuwana T, Dyer MJ, Cohen GM. Diminished 3499–3508. sensitivity of chronic lymphocytic leukemia cells to ABT-737 and ABT-263 due to 21 Corso S, Ghiso E, Cepero V, Sierra JR, Migliore C, Bertotti A et al. Activation of HER albumin binding in blood. Clin Cancer Res 2010; 16: 4217–4225. family members in gastric carcinoma cells mediates resistance to MET inhibition. 43 Janne PA, Gray N, Settleman J. Factors underlying sensitivity of cancers to Mol Cancer 2010; 9: 121. small-molecule kinase inhibitors. Nat Rev Drug Discov 2009; 8: 709–723. 22 Tiedt R, Degenkolbe E, Furet P, Appleton BA, Wagner S, Schoepfer J et al. A drug 44 Hirai H, Sootome H, Nakatsuru Y, Miyama K, Taguchi S, Tsujioka K et al. MK-2206, resistance screen using a selective MET inhibitor reveals a spectrum of mutations an allosteric Akt inhibitor, enhances antitumor efficacy by standard chemo- that partially overlap with activating mutations found in cancer patients. Cancer therapeutic agents or molecular targeted drugs in vitro and in vivo. Mol Cancer Res 2011; 71: 5255–5264. Ther 2010; 9: 1956–1967. 23 Cepero V, Sierra JR, Corso S, Ghiso E, Casorzo L, Perera T et al. MET and KRAS gene 45 Iida M, Brand TM, Campbell DA, Starr MM, Luthar N, Traynor AM et al. Targeting amplification mediates acquired resistance to MET tyrosine kinase inhibitors. AKT with the allosteric AKT inhibitor MK-2206 in non-small cell lung cancer cells Cancer Res 2010; 70: 7580–7590. with acquired resistance to cetuximab. Cancer Biol Ther 2013; 14:481–491. 24 Lee NV, Lira ME, Pavlicek A, Ye J, Buckman D, Bagrodia S et al. A novel SND1-BRAF 46 Langsenlehner U, Renner W, Yazdani-Biuki B, Eder T, Wascher TC, Paulweber B fusion confers resistance to c-Met inhibitor PF-04217903 in GTL16 cells through et al. Integrin alpha-2 and beta-3 gene polymorphisms and breast cancer risk. [corrected] MAPK activation. PLoS One 2012; 7: e39653. Breast Cancer Res Treat 2006; 97:67–72. 25 Fong JT, Jacobs RJ, Moravec DN, Uppada SB, Botting GM, Nlend M et al. 47 Tuck AB, Elliott BE, Hota C, Tremblay E, Chambers AF. Osteopontin-induced, Alternative signaling pathways as potential therapeutic targets for overcoming integrin-dependent migration of human mammary epithelial cells involves acti- EGFR and c-Met inhibitor resistance in non-small cell lung cancer. PLoS One 2013; vation of the hepatocyte growth factorreceptor (Met). J Cell Biochem 2000; 8: e78398. 78:465–475. 26 Engelman JA, Zejnullahu K, Mitsudomi T, Song Y, Hyland C, Park JO et al. MET 48 Burke PA, DeNardo SJ, Miers LA, Lamborn KR, Matzku S, DeNardo GL. amplification leads to gefitinib resistance in lung cancer by activating ERBB3 Cilengitide targeting of alpha(v)beta(3) integrin receptor synergizes with radio- signaling. Science (New York, NY) 2007; 316: 1039–1043. immunotherapy to increase efficacy and apoptosis in breast cancer xenografts. 27 Turke AB, Zejnullahu K, Wu YL, Song Y, Dias-Santagata D, Lifshits E et al. Cancer Res 2002; 62: 4263–4272. Preexistence and clonal selection of MET amplification in EGFR mutant NSCLC. 49 Zhang YW, Staal B, Essenburg C, Su Y, Kang L, West R et al. MET kinase inhibitor Cancer Cell 2010; 17:77–88. SGX523 synergizes with epidermal growth factor receptorinhibitor erlotinib in a 28 Yano S, Wang W, Li Q, Matsumoto K, Sakurama H, Nakamura T et al. Hepatocyte hepatocyte growth factor-dependent fashion to suppress carcinoma growth. growth factor induces gefitinib resistance of lung adenocarcinoma with Cancer Res 2010; 70: 6880–6890. epidermal growth factor receptor-activating mutations. Cancer Res 2008; 68: 50 Corcoran RB, Cheng KA, Hata AN, Faber AC, Ebi H, Coffee EM et al. Synthetic lethal 9479–9487. interaction of combined BCL-XL and MEK inhibition promotes tumor regressions 29 Kentsis A, Reed C, Rice KL, Sanda T, Rodig SJ, Tholouli E et al. Autocrine activation in KRAS mutant cancer models. Cancer Cell 2013; 23:121–128. of the MET receptor tyrosine kinasein acute myeloid leukemia. Nat Med 2012; 18: 51 Prahallad A, Sun C, Huang S, Di Nicolantonio F, Salazar R, Zecchin D et al. Unre- 1118–1122. sponsiveness of colon cancer to BRAF(V600E) inhibition through feedback acti- 30 Singleton KR, Kim J, Hinz TK, Marek LA, Casas-Selves M, Hatheway C et al. A vation of EGFR. Nature 2012; 483:100–103. receptor tyrosine kinasenetwork composed of fibroblast growth factor receptors, 52 Harbinski F, Craig VJ, Sanghavi S, Jeffery D, Liu L, Sheppard KA et al. Rescue epidermal growth factor receptor, v-erb-b2 erythroblastic leukemia viral screens with secreted proteins reveal compensatory potential of receptor tyrosine oncogene homolog 2, and hepatocyte growth factorreceptor drives growth and kinases in driving cancer growth. Cancer Discov 2012; 2:948–959.

Supplementary Information accompanies this paper on the Oncogene website (http://www.nature.com/onc)