BRAF Fusion Confers Resistance to c-Met Inhibitor Figure 1. The GTL16 gastric carcinoma cell line becomes resistant to c-Met inhibition after 4 months of continuous treatment with METi. (A) METi-resistant cells exhibit distinct squamous-like and rounded morphologies compared to GTL16 cells. 100X magnification. (B) Cell viability response curves to METi. METi inhibits GTL16 (blue) with an IC50 value of 10 nM, while it does not inhibit cell viability in clones GTL16R1 and GTL16R3 (red and green). Inhibition of cell viability is normalized relative to untreated control cells. (C) Cell viability response curves to crizotinib. Crizotinib inhibits GTL16 (blue) with an IC50 value of 3 nM. While IC50 values for GTL16R1 and GTL16R3 are .1 mM. doi:10.1371/journal.pone.0039653.g001 Total c-Met protein levels were slightly elevated in the METi- treated samples compared to vehicle-treated samples, showing that the decrease in phosphorylation was not simply due to a reduction in the amount of total protein (Figure 2). Inhibition of the Y1235 autophosphorylation site, which is critical for c-Met receptor kinase activation [41], indicates that the mechanism of resistance in clones GTL16R1 and GTL16R3 is not due to an inability of METi to bind c-Met kinase and inhibit its catalytic activity. Furthermore, a compensatory increase in c-Met expression in the GTL16R1 and GTL16R3 clones is insufficient to sequester the compound and restore signaling. Interestingly, tyrosine phosphor- ylation of EGFR, ERBB2, ERBB3, ERBB4, and SRC was also inhibited upon METi treatment. One explanation for this decrease is that overexpressed c-Met transactivates these proteins (Figure 2). The relative fold change of phosphorylation by METi treatment is summarized in Figure S3. To further assess the mechanism of resistance to c-Met inhibition, METi-resistant clones were profiled for gene expression with Affymetrix U133 Plus 2.0 chips and for DNA Copy Number Variation (CNV) using Affymetrix SNP 6.0 arrays. GTL16R1 and GTL16R3 clones have a nearly identical CNV profile to GTL16 except for a 4-fold amplification of ,356 KB in 7q31 and ,2 megabases within 7q34. Relative to GTL16, most genes within 7q34 had a corresponding increase in mRNA expression with BRAF expressed more than 32-fold in both GTL16R1 and GTL16R3 (Figure 3). BRAF Fusion Identification Affymetrix SNP 6.0 array analysis indicated a segmentation breakpoint that bisected the genomic BRAF gene structure within intron 8 (Figure 3). BRAF fusions have been reported previously, and these commonly occur at exon 9 with an intact BRAF PLoS ONE | www.plosone.org 4 June 2012 | Volume 7 | Issue 6 | e39653