BRAF Fusion Confers Resistance to c-Met Inhibitor Figure 4. Chromosomal rearrangement at 7q32 and 7q34 results in a highly expressed SND1-BRAF fusion protein. (A) Western Immunoblot of total protein lysates identify a higher molecular weight band (arrowhead) recognized only by the anti-C-terminal BRAF antibody, and present exclusively in GTL16R1 and GTL16R3, consistent with a fusion event within BRAF. Additionally, the putative fusion BRAF is highly expressed and hyperphosphorylated compared to wild-type BRAF (arrow in BRAF panels). (B) 59 RACE identified the nucleotide sequence of fusion junction spanning exon 16 of SND1 and exon 9 of BRAF. (C) Schematic representation of CNV amplification of the N-terminal portion of SND1 (mapping from exon 1 to 16) and the C-terminal protein of BRAF (mapping from exon 9 to exon 18). doi:10.1371/journal.pone.0039653.g004 GTL16 and the Resistant Clones are Dependent upon MAPK Signaling To further confirm that the BRAF fusion protein is indeed signaling through the MAPK pathway, we treated GTL16R1 and GTL16R3 with MEKi as a single agent or in combination with METi. Surprisingly, unlike RAFi, single agent MEKi was very potent and was able to inhibit tumor cell growth with an IC50 value of 1.5 and 4.5 nM for GTL16R1 and GTL16R3, respectively (Fig 5A). GTL16 cells were also inhibited by single agent MEKi with an IC50 value of 8.2 nM (Figure 5C, x-axis). To determine if MEKi can synergize with METi, we tested with a sub-optimal dose of MEKi (ranging from 10 nM to 156 pM). Combination with 2.5 nM of MEKi yielded the best synergy as demonstrated by the DBLISS plot (Figure 5C). A Combination of METi and RAFi Increased Inhibition of ERK Phosphorylation in the Resistant Clones To elucidate the mechanism of synergy, we examined modulation of signal transduction pathways by Western immuno- staining total cell lysates from GTL16, GTL16R1 and GTL16R3 PLoS ONE | www.plosone.org 7 June 2012 | Volume 7 | Issue 6 | e39653