BRAF Fusion Confers Resistance to c-Met Inhibitor Figure 5. The c-Met inhibitor synergizes with either RAFi or MEKi. (A) Cell Viability of resistant GTL16R1 and GTL16R3 clones treated with RAFi or MEKi as single agents. Resistance of GTL16R1 and GTL16R3 clones to RAFi single agent treatment suggests that c-Met is involved in signaling independent of Raf. MEKi treatment inhibits cell viability as a single agent with IC50 values of 1.5 nM and 4.5 nM for GTL16R1 and GTL16R3, respectively. (B) METi and RAFi synergistically inhibit tumor growth of GTL16R1 and GTL16R3 resistant lines. Wildtype GTL16 (WT), resistant GTL16R1 and GTL16R3 clones were cultured with a combination of METi and RAFi at the indicated nM concentrations. Matrix grid represents various concentration combinations. Single agent activity can be seen for METi and RAFi on the respective edges of the plot. Tumor cell growth inhibition (TGI) or DBLISS independence is indicated on the Z-axis and shaded according to the value in the legend. TGI ranges from low (no effect on growth) to high (complete suppression of growth). DBLISS ranges from low (complete independence/additivity) to high (synergy). (C) METi and MEKi can synergize in GTL16R1 and GTL16R3. Wildtype GTL16 (WT), resistant GTL16R1 and GTL16R3 clones were cultured with a combination of METi and MEKi at the indicated nM concentrations. Matrix grid represents various concentration combinations. Single agent activity can be seen for METi and MEKi on the respective edges of the plot. Tumor cell growth inhibition (TGI) or DBLISS independence is indicated on the Z-axis and shaded according to the value in the legend. TGI ranges from low (no effect on growth) to high (complete suppression of growth). DBLISS ranges from low (complete independence/additivity) to high (synergy). doi:10.1371/journal.pone.0039653.g005 treated with 200 nM of METi, RAFi, or MEKi as single agents and in combination (Figure 6). Phosphorylation of the c-Met Y1349 docking site was consistently inhibited in GTL16, GTL16R1, and GTL16R3 by METi, but not by RAFi or MEKi alone (Figure 6A). This inhibition was achieved despite moderately increased levels of total c-Met protein upon METi treatment, suggesting that METi blocks c-Met activity in GTL16R1 and GTL16R3 with similar efficacy as GTL16 (Figure 6A). In GTL16R1 and GTL16R3, SND1-BRAF was constitutively phosphorylated on BRAF S445 and levels did not respond significantly relative to total SND1-BRAF protein upon treatment with any inhibitor (Figure 6A). The Serine 445 residue has been reported to be constitutively phosphorylated in BRAF [43], and would not be expected to be affected by inhibitors of BRAF activity. The c-Met tyrosine receptor kinase activates two major signal transduction cascades, the RAS-RAF-ERK MAP Kinase pathway and the PI3K/AKT pathway. We examined AKT activity via phosphorylation of S473 on AKT using Western immunoblot. Compared to GTL16, untreated levels of AKT pS473 were approximately two-fold and six-fold lower in GTL16R1 and GTL16R3, respectively (Figure 6A and C). Treatment with METi reduced AKT S473 phosphorylation to ,10% in all cell lines. RAFi alone reduced AKT pS473 levels in GTL16, while it increased pS473 levels slightly in GTL16R1 and GTL16R3. A combination of METi and RAFi decreased AKT pS473 to levels comparable to METi alone. In contrast, treatment with MEKi induced an approximately three-fold increase of AKT pS473 in all cell types. We also investigated ERK phosphorylation levels in response to compound treatments. The basal phosphorylated ERK levels in GTL16R1 and GTL16R3 were approximately 3 fold higher than GTL16. In response to METi, ERK phosphorylation was completely inhibited in GTL16, however, ERK phosphorylation only decreased slightly in GTL16R1 and GTL16R3 (Figure 6A and B). Treatment with RAFi increased ERK phosphorylation two fold in GTL16 as RAF inhibitors can transactivate homo/heterodi- mers of RAF isoforms to increase MEK signaling [44,45]. Despite the increased level of ERK phosphorylation, we did not observe an increase in cell proliferation. Although treatment of GTL16R1 and GTL16R3 with RAFi decreased ERK phosphorylation by approximately three fold, the level of phosphorylated ERK remained comparable to untreated GTL16 (Figure 6B). This could explain why RAFi alone did not inhibit cell proliferation of the resistant clones (Figure 5B) because the GTL16 cells are being driven by hyperactivation of c-Met signaling which activates MAPK through a RAF independent mechanism. Since METi completely blocks ERK phosphorylation in GTL16 cells, there are likely no activating Ras mutations or other activated downstream mediators of MAPK signaling. Only the treatment of GTL16R1 and GTL16R3 with a combination of METi and RAFi further inhibited ERK phosphorylation to 38% and 10.5% of untreated GTL16 (Figure 6B). Since GTL16R1 and GTL16R3 seemed to shift signaling towards the MAPK pathway and away from the PI3K/AKT pathway, we tested whether the resistance was solely dependent PLoS ONE | www.plosone.org 8 June 2012 | Volume 7 | Issue 6 | e39653