BRAF Fusion Confers Resistance to c-Met Inhibitor Figure 3. Copy Number Variation (CNV) indicates that BRAF is amplified and bisected in resistant clones. Relative to GTL16, CNV analysis identifies a region of increased copy number at 7q34, with a breakpoint within the BRAF locus in GTL16R1 and GTL16R3. In conjunction with Affymetrix expression analysis, genes within the amplified regions are coordinately over-expressed (indicated in red) normalized to GTL16. Expression of the BRAF transcript is increased over GTL16. doi:10.1371/journal.pone.0039653.g003 approximately 2-fold higher, while bases 2007–3522 are equiva- lent (Figure S5). Similarly for BRAF, bases 1-1201 have about the same RBM in GTL16, GTL16R1 and GTL16R3, while bases 1202–2947 have about 20-fold more RBM in GTL16R1 and GTL16R3 compared to GTL16. There is a steep change in coverage at the fusion junction point at position 2006 for SND1 and 1202 for BRAF. (Figure S5). Determination of the Functional Relevance of the Fusion Event Utilizing a RAF Inhibitor To test the functional significance of the BRAF fusion, we treated with METi, RAFi, or a combination of both in cell viability assays. Single agent treatment with RAFi did not alter growth of GTL16, GTL16R1 or GTL16R3 (Figure 5A and B, x-axis). However, GTL16R1 and GTL16R3 treated with METi in combination with RAFi demonstrated an effective tumor cell growth inhibition (TGI). To assess whether this combination was additive or synergistic, we calculated the DBLISS score (see methods). As shown in figure 5B, GTL16R1 and GTL16R3 both have a maximal DBLISS score of 1 indicating a synergistic effect. Maximal synergy occurs between .39 nM METi and .625 nM RAFi for GTL16R1 while GTL16R3 achieves max synergy .39 nM METi and .2.5 mM RAFi. Treating GTL16 with this same combination of METi and RAFi did not demonstrate additional growth inhibition compared with METi alone, consis- tent with oncogenic addiction to c-Met signaling. PLoS ONE | www.plosone.org 6 June 2012 | Volume 7 | Issue 6 | e39653