Signaling, #4051); AKT (Cell Signaling, #4685); actin (Cell Signaling, #4968). Microarrays RNA and DNA were isolated using RNeasy and DNeasy kits (Qiagen, Germantown, MD) for microarray analysis. Whole genome expression profiling and copy number analysis were performed using Affymetrix HGU133 Plus 2.0 and SNP 6.0 arrays (Affymetrix, Santa Clara, CA), respectively, per manufacturer’s protocol. HGU133 Plus 2.0 data were GC Robust Multiarray Average normalized using R and the gcrma package from bioconductor.org [32]. Affymetrix SNP 6.0 data were processed using the aroma.affy- metrix R package according to the methods of H. Bengtsson et al., using the GTL16 as the reference baseline [33]. All array data is MIAME compliant and will be publically available from the Gene Expression Omnibus (GEO) under accession GSE27692. 59 Rapid Amplification of cDNA Ends (RACE) RNA ligase-mediated, rapid amplification of cDNA ends was performed on total RNA from GTL16, GTL16R1, and GTL16R3 clones using a GeneracerTM kit (Invitrogen, Carlsbad, CA). PCR primers specific for 59 generacer oligo sequence [59- cgactggagcacgaggacactga -39] and 39 primer targeting BRAF exons 11 and 12 junction [59-catcaccatgccactttcccttgt-39] were used to amplify a first-strand cDNA generated using random hexamers. PCR was performed using 1X Titanium Taq PCR buffer, 0.2 mM dNTP, 0.4 mM forward generacer and reverse BRAF primers, and 1X Titanium Taq DNA polymerase (Clontech Laboratories, Mountain View, CA). The sequence of cycling was 1 cycle at 95uC for 60 sec; 5 cycles at 95uC for 15 sec, 72uC for 90 sec; 5 cycles at 95uC for 15 sec, 70uC for 90 sec; 25 cycles at 95uC for 15 sec, 68uC for 90 sec, and an additional extension at 68uC for 7 min using Peltier Thermal Cycler 200 (MJ Research, Waltham, MA). The PCR product was run on a 1.2% agarose gel and a 2.1 Kb amplicon was gel-purified, cloned into pCRH4-TOPOHR cloning vector (Invitrogen, Carlsbad, CA), and sequenced using M13 forward and reverse primers on a 3730XL capillary sequencer (Applied Biosystems, Foster City, CA). Sequence data was analyzed using Sequencher (Gene Codes Corporation, Ann Arbor, MI). Kinase Selectivity Screens The METi and RAFi were screened against kinase panels (SelectScreen, Invitrogen; University of Dundee, Division of Signal Transduction Therapy, UK; Upstate Biotechnology/Millipore, Billerica, MA). In these selectivity assays the concentration of ATP was set to the Km value of ATP for each kinase tested to allow for a relative potency comparison between the various kinases. The compounds were tested at 1 mM final concentration. Some of the kinases that showed modest potency in the percent inhibition biochemical assays were further evaluated in a dose response cell based assay. For example METi showed biochemical inhibition of IGF1R but did not inhibit IGF1R kinase activity in the follow-up cell based assay. Next Generation Sequencing RNA-Seq libraries were generated and quality assessed as previously described [34,35] and sequenced on Illumina GA2 instruments. Paired end reads (75 bases each) were aligned to RefSeq transcripts using the BWA alignment tool [36] on default settings and processed to BAM files with samtools [37]. BAM files BRAF Fusion Confers Resistance to c-Met Inhibitor were deposited into the European Nucleotide Archive under accession ERP001432. Aberrantly mapped pairs were identified using the BAM_preprocessingPairsMisMatch.pl script from SVDetect (r0.6d) [38]. Frequency of mismatched transcripts was counted using a custom script. All read pairs for the potential fusion transcript were extracted with samtools from the original BAM file and de novo assembled into contigs using velvet (v1.0.13) [39] and further constructed into transcripts using oases (v0.1.16) (http://www.ebi.ac.uk/˜ zerbino/oases/). Putative fusion tran- scripts were then aligned by BLAST [40] to the original RefSeq transcripts (SND1 NM_014390.2 and BRAF NM_004333.4) to identify the composition of the transcript and the position of the fusion junction. The sequence for the most complete transcript was deposited in GenBank under accession JX013981. Coverage data was generated using the pileup function from samtools. Fusion Search in Public CNV Datasets Computational search was performed using publically available CNV (Copy Number Variation) arrays from: TCGA (http://tcga-data.nci.nih.gov/tcga/tcgaHome2.jsp). Sanger (http://www.sanger.ac.uk/genetics/CGP/Archive/). Broad (http://www.broadinstitute.org/tumorscape/pages/ portalHome.jsf). GEO (http://www.ncbi.nlm.nih.gov/geo/) (GSE13429, GSE14437, GSE14960, GSE14994, GSE15096, GSE15264, GSE15526, GSE15688, GSE16619, GSE17247, GSE17958, GSE18333, GSE18828, GSE19416, GSE19539, GSE20709, GSE21780, GSE21990, GSE22208, GSE22306, GSE22615, GSE23300, GSE23452, GSE25016, GSE25839, GSE27560, GSE7822, GSE9829). GSK (https://cabig.nci.nih.gov/caArray_GSKdata/). Results Generation and Characterization of METi Resistant GTL16 Clones GTL16 cells were cultured in the presence of increasing concentrations of METi up to 2.5 mM over a period of four months. Following treatment, GTL16 cells that grew at a rate similar to untreated GTL16 cells were defined to be resistant. The resulting cells from distinct wells appeared as a homogenous population that appeared either rounded (R) or squamous-like (S) (Figure 1A). Resistant clones, GTL16R1 and GTL16R3 did not respond to growth inhibition by METi at concentrations up to 10 mM, while GTL16 were sensitive with an IC50 of 10 nM (Figure 1B). To determine if resistance was a compound specific or a target specific phenomena, we also treated the GTL16R1 and GTL16R3 clones with the c-Met/ALK inhibitor, crizotinib. We observed an IC50 value .1 mM for the resistant clones, while GTL16 had an IC50 of 3 nM (Figure 1C). Data for the rounded clones (GTL16R1 and GTL16R3) are presented below. The squamous clones are pending further characterization but are resistant to c-Met inhibition by a different but unknown mechanism that does not involve BRAF or the c-Met receptor directly. Molecular Profiling to Understand Resistance Mechanisms To examine the effect of METi on the activation state of signal transduction pathways, protein lysates from GTL16 and the resistant clones were assayed with Reverse Phase Protein Arrays (RPPA) for select total and phosphorylated proteins. Treatment of GTL16, GTL16R1, and GTL16R3 with 2.5 mM METi for 1 hr inhibited phosphorylation of c-Met residue Y1235 (Figure 2). PLoS ONE | www.plosone.org 3 June 2012 | Volume 7 | Issue 6 | e39653