Recurrent BRAF kinase fusions in melanocytic tumors offer an opportunity for targeted therapy

Abstract

BRAF is the most prevalent oncogene and an important therapeutic target in melanoma. In some cancers, BRAF is activated by rearrangements that fuse its kinase domain to 5' partner genes. We examined 848 comparative genomic hybridization profiles of melanocytic tumors and found copy number transitions within BRAF in 10 tumors, of which six could be further characterized by sequencing. In all, the BRAF kinase domain was fused in-frame to six N-terminal partners. No other mutations were identified in melanoma oncogenes. One of the seven melanoma cell lines without known oncogenic mutations harbored a similar BRAF fusion, which constitutively activated the MAP kinase pathway. Sorafenib, but not vemurafenib, could block MAP kinase pathway activation and proliferation of the cell line at clinically relevant concentrations, whereas BRAF(V) (600E) mutant melanoma cell lines were significantly more sensitive to vemurafenib. The patient from whom the cell line was derived showed a durable clinical response to sorafenib.

Keywords: BRAF; kinase; melanoma; oncogenes; translocation.

Figure 1. Representative case with MAD1L1-BRAF fusion

Array CGH demonstrates copy number changes limited to chromosome 7 (top row and second row). There is a copy number transition within both MAD1L1 and BRAF (third row). The y-axes represent the average log₂ ratio of tumor to normal fluorescence intensity and the genomic coordinates are on the x-axis. The copy number data supports a fusion between the 5’ end of MAD1L1 and the 3’ end of BRAF as they have the same copy number level. The predicted gene model fuses intron 16 of MAD1L1 and intron 7 of BRAF (fourth row) as supported by the breakpoint sequence.

Figure 2. Predicted BRAF fusion proteins

Conserved region 1 (CR1), conserved region 2 (CR2) and the kinase domain of BRAF are highlighted in dark blue. For each fusion protein, the contribution from 5’ partner gene is depicted in red and the contribution from the BRAF gene is depicted in blue. In all cases, the kinase domain of BRAF is intact and the auto-inhibitory domain CR1 is lost. The PLIN3-BRAF fusion leads to a truncated version of BRAF.

Figure 3. Activation of the MAP kinase pathway by AGK-BRAF and increased sorafenib sensitivity of AGK-BRAF fusion containing cell line compared to BRAF^V600E mutant melanoma cell lines

a) Comparable activation of the MAP kinase pathway in the C0902 cell line, which harbors an AGK-BRAF kinase fusion to two BRAF^V600E mutant melanoma cell lines. The AGK-BRAF fusion is detected exclusively in the C0902 cell line at its predicted molecular weight of 53 kDa (asterisk). The bands corresponding to the various known isoforms of BRAF are indicated by arrowheads. b) Transient transfection of the AGK-BRAF fusion in 293FT cells activates the MAP kinase pathway. c) Dose response curves for sorafenib and vemurafenib for the C0902 and two BRAF^V600E mutant melanoma cell lines (5 days of treatment). d) Western Blot analysis of MAP-kinase pathway status. Cell lysates were prepared after serum starvation and HSP60 was used as the loading control.