BRAF Fusion Confers Resistance to c-Met Inhibitor Figure 2. METi inhibits c-Met phosphorylation in both GTL16 and resistant GTL16R1 and GTL16R3 clones. Heatmap of Reverse Phase Protein Array (RPPA) showing GTL16 and resistant clones GTL16R1 and GTL16R3 relative level of total and phosphorylated proteins. Red indicates higher intensity vs green which indicates lower intensity. Cells were treated with DMSO vehicle or METi (2.5 mM) for 1 hr. doi:10.1371/journal.pone.0039653.g002 catalytic region [24,25,26,27,42]. We probed GTL16, GTL16R1, GTL16R3, and the squamous-like (GTL16S5) resistant clone lysates by Western immunoblot with N-terminal-, C-terminal-, and phospho-specific BRAF antibodies. All three BRAF antibodies identified wild-type BRAF in all samples (Figure 4A, arrows). By contrast, the C-terminal and phospho-specific BRAF antibodies identified a higher molecular weight species in clones GTL16R1 and GTL16R3 that was absent in GTL16 and GTL16S5 (Figure 4A, arrowheads). The N-terminal-specific BRAF antibody was not able to recognize the higher molecular weight species, suggesting that both the GTL16R1 and GTL16R3 clones express a species of BRAF lacking the N-terminal domain. Interestingly, the higher molecular weight BRAF variant appears to be highly phosphorylated at S445 compared to wildtype BRAF. Based on these observations, we suspected a putative BRAF fusion protein and sought to identify the N-terminal fusion partner. Using 59 RACE of GTL16R1 and GTL16R3 transcripts with a 39 primer specific for BRAF exons 11 and 12 produced a 2.1 kb product. Sequencing of the amplicon revealed SND1 (staphylococcal nuclease and tudor domain containing 1) to be the fusion partner (Figure 4B). SND1 is normally located on chromosome 7q31 approximately 12 Mb upstream of BRAF (7q34). Here, SND1 happens to be bisected by the small 7q31 amplification observed in the GTL16R1 and GTL16R3 clones. The translocation yields a SND1-BRAF fusion transcript with exon 16 of SND1 fused in-frame to exon 9 of BRAF, resulting in a fusion transcript coding for SND1 amino acids 1-593 coupled to BRAF amino acids 381–766 (Figure 4B and C). Global copy number analysis confirms that the exons adjacent to the amplification breakpoints of SND1 and BRAF are consistent with this junction (Figure S4). Confirmation of SND1-BRAF Fusion Sequence Using Illumina sequencing with GTL16, GTL16R1, and GTL16R3 transcripts, we confirmed that the fusion junction was identical to the sequence obtained by 59 RACE, and no other nucleotide changes were observed for SND1 or BRAF. De novo assembly of read pairs mapping to either SND1 or BRAF produced a fusion transcript that consisted of nucleotides 1-2006 of RefSeq transcript NM_014390.2 (SND1) and 1202–2947 from RefSeq transcript NM_004333.4 (BRAF). Wild type BRAF transcripts were observed in the GTL16R1 and GTL16R3 clones as evidenced by paired reads spanning between exons 8 and 9 (data not shown). GTL16 did not contain any paired reads where one end mapped to SND1 and the other mapped to BRAF. In contrast, GTL16R1 contained 628 chimeric pairs and GTL16R3 contained 702 chimeric pairs (Figure S4). Consistent with a fusion transcript of SND1 and BRAF, we observed uneven coverage distribution of the N-terminal region of SND1 and the C-terminal region of BRAF. We computed normalized reads per base per million reads (RBM) to compare transcript coverage between samples. Relative to GTL16, the RBM of bases 1-2006 of SND1 in GTL16R1 and GTL16R3 is PLoS ONE | www.plosone.org 5 June 2012 | Volume 7 | Issue 6 | e39653