Abstract
Mitogen-Activated Protein Kinase (MAPK) pathway activation has been implicated in many types of human cancer. BRAF mutations that constitutively activate MAPK signalling and bypass the need for upstream stimuli occur with high prevalence in melanoma, colorectal carcinoma, ovarian cancer, papillary thyroid carcinoma, and cholangiocarcinoma. In this report we characterize the novel, potent, and selective BRAF inhibitor, dabrafenib (GSK2118436). Cellular inhibition of BRAFV600E kinase activity by dabrafenib resulted in decreased MEK and ERK phosphorylation and inhibition of cell proliferation through an initial G1 cell cycle arrest, followed by cell death. In a BRAFV600E-containing xenograft model of human melanoma, orally administered dabrafenib inhibited ERK activation, downregulated Ki67, and upregulated p27, leading to tumor growth inhibition. However, as reported for other BRAF inhibitors, dabrafenib also induced MAPK pathway activation in wild-type BRAF cells through CRAF (RAF1) signalling, potentially explaining the squamous cell carcinomas and keratoacanthomas arising in patients treated with BRAF inhibitors. In addressing this issue, we showed that concomitant administration of BRAF and MEK inhibitors abrogated paradoxical BRAF inhibitor-induced MAPK signalling in cells, reduced the occurrence of skin lesions in rats, and enhanced the inhibition of human tumor xenograft growth in mouse models. Taken together, our findings offer preclinical proof of concept for dabrafenib as a specific and highly efficacious BRAF inhibitor and provide evidence for its potential clinical benefits when used in combination with a MEK inhibitor.
Highlights
The Mitogen-Activated Protein Kinase (MAPK) signal transduction pathway plays a central role in cellular growth, differentiation, and stress response [1,2,3,4,5]
While dabrafenib inhibits a truncated CRAF kinase in vitro, this does not translate into cell culture, as cell lines with CRAF-dependent MEK activation (MEK inhibitor-sensitive) are insensitive to dabrafenib
We demonstrated high selectivity of dabrafenib for 80% of the BRAFV600E cell lines tested and hypothesize that the relative lack of activity against 3 of the BRAFV600E cell lines may be due to the presence of additional mutations (PTEN in GCT cells, PI3K in RKO cells, and p53 in A673 cells) that were confirmed by in-house sequencing
Summary
The MAPK signal transduction pathway plays a central role in cellular growth, differentiation, and stress response [1,2,3,4,5] This pathway is normally activated by the binding of extracellular growth factors to membrane-bound receptors, which recruit intracellular proteins to the cell membrane, leading to the activation of the small guanosine triphosphate-binding protein, RAS. RAS adopts an activated conformation that stimulates downstream signalling, resulting in the phosphorylation and activation of ERK, which regulates a wide range of cellular processes. This pathway can be constitutively activated by mutation of specific proteins, including BRAF. The high frequency of activating mutations in tumors and ensuing MAPK pathway addiction make BRAF an attractive therapeutic target, where inhibition of the kinase activity of BRAFV600E and other activated BRAF mutants could provide an effective therapy
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