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

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Synthetic Lethal Screening Reveals FGFR As One of the Combinatorial Targets to Overcome Resistance to Met-Targeted Therapy Oncogene (2015) 34, 1083–1093 © 2015 Macmillan Publishers Limited All rights reserved 0950-9232/15 www.nature.com/onc 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
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