Published OnlineFirst April 10, 2017; DOI: 10.1158/1535-7163.MCT-17-0104 Cancer Biology and Signal Transduction Molecular Cancer Therapeutics Genomic and Molecular Screenings Identify Different Mechanisms for Acquired Resistance to MET Inhibitors in Lung Cancer Cells Pol Gimenez-Xavier1, Eva Pros1, Ester Bonastre1, Sebastian Moran2, Ana Aza1, Osvaldo Grana~ 3, Gonzalo Gomez-Lopez3, Sophia Derdak4,5, Marc Dabad4,5, Anna Esteve-Codina4,5, Jose R. Hernandez Mora2, Diana Salinas-Chaparro1, Manel Esteller2,6, David Pisano3, and Montse Sanchez-Cespedes1 Abstract The development of resistance to tyrosine kinase inhibitors groups of resistant cells. In one of these, the cells have acquired (TKI) limits the long-term efficacy of cancer treatments involving sensitivity to erlotinib, concomitantly with mutations of the them. We aimed to understand the mechanisms that underlie KIRREL, HDAC11, HIATL1, and MAPK1IP1L genes, among acquired resistance (AR) to MET inhibitors in lung cancer. EBC1 others. In the other group, some cells have acquired inactivation cells, which have MET amplification and are sensitive to TKIs of neurofibromatosis type 2 (NF2) concomitantly with strong against MET, were used to generate multiple clones with AR to a overexpression of NRG1 and a mutational profile that includes MET-TKI. Whole-exome sequencing, RNA sequencing, and global changes in LMLN and TOMM34. Multiple independent and DNA methylation analysis were used to scrutinize the genetic and simultaneous strategies lead to AR to the MET-TKIs in lung cancer molecular characteristics of the resistant cells. AR to the MET-TKI cells. The acquired sensitivity to erlotinib supports the known involved changes common to all resistant cells, that is, phenotypic crosstalk between MET and the HER family of receptors. For the modifications, specific changes in gene expression, and reactiva- first time, we show inactivation of NF2 during acquisition of tion of AKT, ERK, and mTOR. The gene expression, global DNA resistance to MET-TKI that may explain the refractoriness to methylation, and mutational profiles distinguished at least two erlotinib in these cells. Mol Cancer Ther; 16(7); 1–11. Ó2017 AACR. Introduction growth following the administration of specific tyrosine kinase inhibitors (TKI) has been observed in lung cancer cells with Most novel therapeutic approaches in cancer are developed on alterations at various RTKs, including mutations, gene fusions, the basis of our increasing knowledge of underlying oncogenic and strong focal gene amplification (GA) at ALK, EGFR, ERBB2, mutations. In the case of lung cancer, our better understanding of FGFR1, MET, PDGFRA, RET, and ROS genes, among others (1–9). its genetic architecture has enabled novel treatments to be Inasmuch as most of these observations are based on screenings designed, most of which are based on drugs that target growth made in cancer cell lines, these have become essential preclinical factor receptors with tyrosine kinase activity (RTK) that are genet- tools for evaluating how the cancer genotype determines the ically activated in the tumors. A strong effect of decreasing cell response to these specific drugs (1–3). Despite the success of genotype-directed therapies, some 1 tumors with activated target RTK exhibit frequent failures in the Genes and Cancer Group, Cancer Epigenetics and Biology Program (PEBC), fi Bellvitge Biomedical Research Institute-IDIBELL, Hospitalet de Llobregat, Bar- primary response to these speci c TKIs. This is the so-called celona, Spain. 2Cancer Epigenetics Group, Cancer Epigenetics and Biology intrinsic (i.e., primary) resistance, which has been associated with Program (PEBC), Bellvitge Biomedical Research Institute-IDIBELL, Barcelona, the simultaneous activation of parallel signal transduction path- Spain. 3Bioinformatics Unit, Structural Biology and BioComputing Programme, ways that circumvent the targeted pathway blockade (9). Further- 4 Spanish National Cancer Centre (CNIO), Madrid, Spain. CNAG-CRG, Centre for more, in most tumors that are initially sensitive to these therapies, Genomic Regulation (CRG), Barcelona Institute of Science and Technology acquired (i.e., secondary) resistance inevitably develops due to the (BIST), Barcelona, Spain. 5Universitat Pompeu Fabra (UPF), Barcelona, Spain. 6Institucio Catalana de Recerca i Estudis Avancats¸ (ICREA), Barcelona, Spain. acquisition of genetic alterations in the kinase target or in mole- cules acting in related signaling pathways (10–14). The origin of Note: Supplementary data for this article are available at Molecular Cancer the genetic alterations responsible for the acquired resistance (AR) Therapeutics Online (http://mct.aacrjournals.org/). could either be generated de novo or arise as clonal expansions P. Gimenez-Xavier and E. Pros contributed equally to this article. from preexisting minor subclones. In lung cancer, this has been Corresponding Author: Montse Sanchez-Cespedes, Bellvitge Biomedical extensively modeled, especially to study the responses to EGFR Research Institute (IDIBELL), Av. Gran Via de Hospitalet, 199-203, Hospitalet inhibitors, using isogenic pairs of drug-sensitive and drug-resis- de Llobregat, Barcelona 08908, Spain. Phone: 349-3260-7132; Fax: 349-3260- tant human cancer cell lines (10, 12–14). It is well established that 7219; E-mail: [email protected] resistance to TKIs against EGFR can be acquired through the doi: 10.1158/1535-7163.MCT-17-0104 p.T790M mutation at EGFR, which prevents the binding of the Ó2017 American Association for Cancer Research. inhibitor to the receptor, or the amplification or overexpression of www.aacrjournals.org OF1 Downloaded from mct.aacrjournals.org on September 25, 2021. © 2017 American Association for Cancer Research. Published OnlineFirst April 10, 2017; DOI: 10.1158/1535-7163.MCT-17-0104 Gimenez-Xavier et al. RTKs such as MET, EGFR, AXL, and ERBB2 (10–15). Likewise, AR lysis buffer and absorbance was measured at 596 nm. Data were to crizotinib in EML4-ALK-positive lung cancer cell lines has been analyzed by nonlinear regression using GraphPad Prism Soft- associated with point mutations or amplification in ALK or KIT, ware version 5.2 (GraphPad Software, Inc.). Viabilities were among other genes (15, 16). expressed as a percentage of the untreated controls. The IC50 Regarding the MET oncogene, the genetic and molecular was determined from the dose–response curve. Results are mechanisms linking AR to its associated TKI are not fully under- presented as the median of at least three independent experi- stood. Amplification and overexpression of wild-type KRAS or ments performed in triplicate for each combination of cell line activation of BRAF and of the HER pathways are some of the and compound. mechanisms that have been proposed for the AR to MET-TKIs in For wound-healing assays, cells were grown in 6-well plates various types of cancer (17–21). Here, we have used the EBC1 lung with complete medium. After 90% confluence was reached, the cancer cells, which are intrinsically sensitive to the MET-TKIs due medium was replaced with FBS-free media for 24 hours. A wound to genetic activation of MET by GA, as a cancer cell model to was created with a germ-free 100-mL pipette tip in the monolayer. investigate AR. We obtained various clones from the EBC1 paren- The cells were washed with PBS and grown in FBS-free media for tal cells that had become refractory to the MET-TKIs and, taking 72 hours. The wounds were observed under a phase contrast advantage of next-generation sequencing technologies, scruti- microscope (IX81, Olympus). The images were analyzed by nized these various cell subpopulations for genetic and molecular drawing lines at the wound edges. The width of the scratch was alterations responsible for drug resistance. measured using TScratch software (25). For colony formation assays, cells were seeded at a density of 2,000 to 4,000 cells per well, in 6-well plates, in DMEM media Materials and Methods containing 10% (v/v) FBS, with or without the corresponding Generation of AR to PHA665752 and tyrosine kinase inhibitor treatment, and stained 14 to 15 days later with Crystal Violet treatments (Merck Millipore). Each assay was performed in triplicate. Col- The EBC1 lung cancer cell line was obtained from the JCRB onies were quantified using ImageJ software. Cell Bank (National Institutes of Biomedical Innovation, Health and Nutrition, Japan), grown under recommended FISH analysis conditions, and maintained at 37Cinahumidified atmo- To determine gene copy number of the MET gene, we per- sphere of 5% CO2. The cell line was obtained directly from the formed dual-color FISH analysis in interphase nuclei, following repository and passaged for fewer than 4 months after receipt, previously described protocols (26). Briefly, we used a BAC clone before generating the various derived resistant cells. The cells labeled in Spectrum Red for the MET gene (RP11-95I20) and were tested for mycoplasma infection prior to the generation of control BAC, labeled in Spectrum Green and located pericentro- the various resistant cells and periodically thereafter. All the merically (RP11-45N18). cells used in the experiments, tested negative for the infection. Genomic DNA and total RNA were extracted using standard Antibodies and Western blots protocols. The identity of the EBC1 cells, both the parental and The antibodies are indicated in the Supplementary Methods. each of the resistant pools and clones, was verified by deter- For Western blots, cells were lysed into ice-cold RIPA lysis cell mining the presence of the TP53 mutation (5) and amplifica- buffer supplemented with protease and phosphatase inhibitor tion of the MET gene in the cells. cocktails (Complete Mini and Phosphostop), followed by clear- To generate AR to MET inhibitors, the EBC1 cells were cultured ance of lysates by microcentrifugation. Supernatants were for about 16 weeks in a medium with an increasing concentration resolved on an SDS-PAGE gel, transferred onto nitrocellulose of PHA665752 (Ref. 2693, Tocris Bioscience), from a starting membranes (Amersham, GE Healthcare Life Science), and probed concentration of 62.5 nmol/L to a final concentration of 1 mmol/ with the indicated antibodies. Detection was done using a chemi- L.