BRAF gene amplification was evident in 98 and 86% of COLO201AR and COLO206FAR cells, respectively, demonstrating that this molecular event is existing in virtually all resistant cells. In contrast, there was no expand in CRAF gene copy number in AR cells, suggesting the modest increases in CRAF abundance observed in AR cells reflected a distinct mechanism . To verify and more quantify the degree of BRAF amplification, we performed quantitative polymerase chain response from genomic DNA. This revealed that BRAF copy quantity was 5 to seven instances higher in AR cells relative to parental cells . BRAF copy amount was determined to get ~20 to 25 in COLO201AR cells and ~10 to 15 in COLO206F cells. Quantitative PCR did not show a rise in CRAF DNA in AR cells, in agreement together with the FISH information. Sequencing of parental and AR cells did not reveal any MEK1 mutations or new BRAF exon 15 mutations, but sequencing chromatograms showed the peak height ratio from the mutant allele towards the wildtype allele was tremendously improved while in the AR cells, suggesting selective amplification within the mutant BRAF allele .
Notably, in both parental cell lines, occasional cells showed amplification of BRAF, suggesting full report that AR cells may well come up by growth of clones with preexisting amplification of BRAF . These cells represented 4% of COLO201 cells and three.5% of COLO206F cells. We also evaluated 11 human colorectal cancer specimens identified to harbor BRAF V600E mutations by FISH to find out whether or not equivalent populations of cells with preexisting BRAF amplification may possibly exist in human tumors. In 1 tumor, we noticed that 28% of cells had substantial BRAF gene amplification . In this tumor, 10% of tumor cells had a BRAF copy number of 10 or better, that’s very similar for the BRAF copy amount observed in AR cells, implying that these clones would possible be resistant to MEK or BRAF inhibitor therapy.
This finding confirms that BRAF amplification takes place in human tumors harboring V600E mutations and supports the notion that BRAF amplification may possibly exist ahead of remedy in patients and has the probable to get a mechanism for either de novo resistance NVP-BGJ398 or acquired resistance to MEK or BRAF inhibitors. To find out regardless if elevated BRAF abundance is adequate to induce resistance to MEK inhibitors, we overexpressed either wildtype or mutant V600E BRAF in parental COLO201 cells. Very similar on the AR cells, the COLO201 cells overexpressing V600E BRAF had increased quantities of phosphoMEK and demonstrated resistance for the effects of AZD6244 on viable cell titer . Overexpression of V600E BRAF within a BRAFmutated melanoma cell line, WM164, also led to increased phosphoMEK and resistance to MEK inhibitors .
This suggests that BRAF amplification could probably cause MEK inhibitor resistance in other BRAFmutant cell lines along with other tumor styles.