Dabrafenib; preclinical characterization, increased efficacy when combined with trametinib, while BRAF/MEK tool combination reduced skin lesions

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 BRAF(V600E) 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 BRAF(V600E)-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.

Figure 1. Dabrafenib structure and activity against RAF kinases.

Dabrafenib chemical structure (A). Dabrafenib potency (IC₅₀) was measured against the kinase activity of various BRAF orthologs, human BRAF mutants, and human CRAF (B).

Figure 2. Dabrafenib inhibits MAPK signalling in BRAF^V600E cells and is abrogated by ARAF or CRAF depletion.

The inhibition of MAPK signalling by dabrafenib in a BRAF^V600E cell line was examined in comparison with knockdown of RAF paralogs using siRNA. A375P cells were transfected with the indicated siRNA for 72 h and treated with 8 nM dabrafenib (+) or DMSO control (−) for 1 h. Lysates were immunoblotted for the proteins indicated.

Figure 3. Dabrafenib shows functional selectivity for BRAF^V600D/E/K tumor cell growth inhibition.

The functional selectivity of dabrafenib was evaluated by measuring the growth of a range of human tumor cell lines of different genetic mutation status. Cells were treated with a concentration range of dabrafenib in a 3-day proliferation assay and growth IC₅₀ (gIC₅₀) values were measured.

Figure 4. Modulation of pharmacodynamic markers by dabrafenib in BRAF^V600E tumors.

Mice bearing A375P tumor xenografts were treated orally with 30 mg/kg dabrafenib, once daily for 14 days. Blood and tumors from vehicle- and dabrafenib-treated animals were analyzed for compound concentration and pERK inhibition, respectively (A). Phospho-ERK (pERK) is normalized to total ERK (tERK). Tumors harvested 6h post-last 6^th dose were stained for Ki67, p27, and ppERK by immunohistochemistry and compared with pre-treatment controls (B). Data are representative of n = 3 studies and percent changes were calculated from the ratio of positively stained cells following drug treatment to those following vehicle control treatment, each as a percentage of the total cell population.

Figure 5. Inhibition of BRAF^V600E tumor xenograft growth by dabrafenib.

Growth of Colo 205 tumor xenografts was measured in mice during and for a period following oral q.d. × 14 treatment with 0, 3, 10, 30, and 100 mg/kg dabrafenib. Mean tumor volumes are plotted with their standard error mean and 4 partial regressions out of 8 mice were observed at the 100 mg/kg dose after the 14-day treatment period. The 14-day period of dosing is indicated by the shaded gray bar.

Figure 6. Dabrafenib-induced MAPK activation in wild-type BRAF/mutant RAS cells is CRAF-dependent and abrogated by MEK inhibition.

In order to assess the individual contributions of ARAF, BRAF, and CRAF to paradoxical MAPK activation caused by dabrafenib in HCT-116 (wild-type BRAF, mutant KRAS) cells, phospho and total MEK (pMEK, tMEK) and ERK (pERK, tERK) were evaluated by immunoblot after a 72-hour incubation with siRNA towards ARAF, BRAF, or CRAF, or medium (none), and treatment with 0, 100, or 300 nM dabrafenib for 1 h (A). Sensitivity of this paradoxical activation to MEK inhibition was evaluated in HCT-116 cells, following treatment with 0, 100, or 300 nM dabrafenib for 1 h in the presence of 0, 0.5, 5.0, or 50 nM MEK inhibitor (trametinib). Lysates were immunoblotted for total ERK (tERK) and dual-phosphorylated ERK (ppERK) (B).

Figure 7. BRAF and MEK inhibitor combination decreases rat skin lesion formation and increases tumor growth inhibition.

Macroscopic and light microscopic photographs of skin (ventral forepaw) from rats given vehicle (a/d), 150 mg/kg/d BRAF tool inhibitor (b/e), or 150 mg/kg/d BRAF tool inhibitor with 0.75 mg/kg/d MEK tool inhibitor (c/f) by oral gavage for 12 consecutive days (A). Stratum spinosum/stratum granulosum (epithelial layer, SS/SG) and stratum corneum (keratin layer, SC) are indicated. Mice bearing A375P tumors were treated orally, once daily, with the indicated doses of dabrafenib, trametinib, or a combination of both agents (B). Treatment continued until the mean tumor volume of each group reached 1 200 mm³ or one death occurred. Mean tumor volumes are plotted with their standard error mean and complete (CR) or partial (PR) regressions are indicated for each group of n = 8 mice after 14 days of treatment. The dotted line for the combination group indicates where one animal was euthanized due to tumor necrosis.