activated BRAF can bypass upstream c-Met inhibition by hyperactivating the MAPK pathway in a c-Met independent manner. As summarized in figure 7, several signaling cascades are activated by the c-Met receptor including PI3K, SRC, and Ras signaling. By inhibiting tyrosine phosphorylation of critical c-Met residues used to recruit adapter proteins GRB2 and GAB1, METi inhibits the recruitment and activation of Ras and prevents activation of the MAPK pathway by the c-Met receptor. The SND1-BRAF fusion, however, circumvents METi by signaling downstream of the c-Met receptor to MEK. The resistance can be overcome by treating with MEKi, or a combination of METi and RAFi to target the SND1-BRAF fusion protein. Like previously described BRAF fusion proteins, the specific role of the N-terminal partner seems limited. Despite containing an intriguing array of tandem Staphylococcal nuclease homologue domains which function to capture RNA targets, the SND1 portion of the fusion protein likely does not directly contribute to c-Met resistance because use of RAFi was able to restore sensitivity to METi. Rather, we think transcription of the gene fusion is driven by the SND1 promoter and SND1 replaces the N-terminal regulatory region of BRAF which normally regulates BRAF catalytic activity [49]. Importantly, the simultaneous combination treatment with both METi and RAFi is required to reduce the phosphorylation of ERK in GTL16R1 and GTL16R3 more than 50% compared to basal pERK levels in GTL16 (Figure 6B). The inability of RAFi to completely inhibit ERK phosphorylation and reduce cell prolif- eration as a single agent suggests that c-Met signaling activates MEK independent of RAF catalytic activity. In this model, both the c-Met and BRAF pathways would need to be blocked to reduce ERK phosphorylation, enough to inhibit cell proliferation. Additionally, as revealed by RPPA in figure 2, METi treatment decreases SRC activity, and may suggest that SRC dependent BRAF Fusion Confers Resistance to c-Met Inhibitor signaling is another pathway active in GTL16 cells. Bertolli et al. demonstrates that adding SRC inhibitors enhances inhibition of cell viability by c-Met inhibition [50]. The role of PI3K activation in our particular resistant model seems minor. Lower basal pAKT S473 levels in GTL16R1 and GTL16R3 suggest less dependence upon PI3K signaling. Addi- tionally, while METi treatment suppresses pAKT S473, MEKi treatment dramatically increases pAKT S473, and yet still inhibits cell viability, suggesting that suppressing AKT phosphorylation is not a requirement for blocking cell proliferation (Figure 5A and C). Indeed, these results are consistent with experiments by Hoeflich et.al. where increases in AKT p473 were seen in response to MEKi in several cell lines. While increases in AKT/PI3K activity are associated with anti-apoptosis and enhanced cell survival, Hoeflich et.al. postulated that AKT/PI3K pathway activation in response to MAPK pathway inhibition is delayed and may be too late to maintain cell viability [51]. Hence, in our model, signaling through MEK seems sufficient for rescue from c- Met inhibition. It is unclear if prolonged exposure of GTL16 cells to METi resulted in de novo SND1-BRAF fusion translocations, or if resistant clones arose from an enrichment of pre-existing cells with these translocations. The GTL16R1 and GTL16R3 are not identical clones despite possessing the same SND1-BRAF fusion protein product. RNA-Seq and exome sequencing data show clone- specific SNPs. Furthermore, the CNV segmentation pattern appears to be distinct in the 7q32–7q34 region. However, because the breakpoints are so similar, it is possible that GTL16R1 and GTL16R3 are subvariants of a common precursor cell that was present in the GTL16 tumor which has since diverged over time creating a subpopulation of SND1-BRAF cells in culture. Additionally, the SND1-BRAF fusion is highly amplified in GTL16R1 and GTL16R3, but is not detected via Next Figure 7. A model highlighting the resistance mechanism in GTL16 GTL16R1 and GTL16R3. Highly expressed SND1-BRAF fusion protein signals through the MEK/ERK pathway and short-circuits c-Met inhibition. doi:10.1371/journal.pone.0039653.g007 PLoS ONE | www.plosone.org 10 June 2012 | Volume 7 | Issue 6 | e39653