Vemurafenib Characterization
November 28, 2011
Having
validated BRAF as a possible therapeutic target, we proceeded to the
biochemical characterization of a new BRAF inhibitor, Vemurafenib, in order to confirm the shRNA results with a pharmacologic approach that is more applicable from a clinical standpoint. Vemurafenib
is a highly selective inhibitor of BRAF kinase activity, with an IC50
of 44 nmol/L against V600E-mutant BRAF. From a panel of 65 non-RAF
kinases covering much of the kinome, only one kinase—BRK (also known as
PTK6)—showed inhibition in the nanomolar range (IC50 = 240 nmol/L). Most
of the kinases tested showed >100-fold higher IC50 (data not shown).
Vemurafenib is currently undergoing clinical evaluation. We tested this compound on the melanoma cell line A375 and on
three thyroid carcinoma cell lines (ARO, NPA, and TPC-1). Dose-response
curves obtained using a proliferation read-out indicated an efficient
antiproliferative activity of Vemurafenib in all
BRAF-dependent cellular systems at nanomolar doses.
The compound was most potent in A375 cells (IC50 = 47 nmol/L). Thyroid
cancer ARO and NPA cells were less sensitive and showed similar
inhibition (IC50 = 205 nmol/L and IC50 = 126 nmol/L, respectively). TPC1
showed an approximately 50-fold higher IC50 value (IC50 = 10.77
μmol/L), likely due to the presence of alternative signaling pathways
activated by RET/PTC1. Western blot analysis confirmed the block of
BRAF-mediated MEK1/2 phosphorylation in a dose-dependent manner in A375,
NPA, and ARO cells. In line with the proliferation data,
MEK1/2 phosphorylation was not affected by Vemurafenib in TPC1 cells.
Vemurafenib promoted apoptotic death in A375 cells in a dose-dependent manner, as assessed by Annexin V–staining, by the appearance of a subdiploid peak in cell cycle analysis and by activation of caspase-3. These results confirm that BRAF provides a survival signal in this melanoma cell line. By contrast, in thyroid cancer cell lines NPA and ARO, very little evidence of apoptosis was observed in several experiments. An arrest in G1 and a decrease of S and G2-M phases were observed in these cells, in accordance with results obtained by shRNA (data not shown). In TPC1 cells, neither apoptosis nor cell cycle alterations were observed at up to 10 μmol/L of Vemurafenib. These results on thyroid cell lines are consistent with recently published BRAF inhibitor data. In ARO cells, long-term treatment (6 days) with Vemurafenib induced the reexpression of the NIS pump, again confirming shRNA data.
From these results, we can conclude that the pharmacologic inhibition of BRAF enzymatic activity has the same consequences as down-regulation of its expression, in terms of cell growth and alterations of the cell cycle. These data also indicate that Vemurafenib is specifically active on BRAF-mutated cell lines.
Vemurafenib promoted apoptotic death in A375 cells in a dose-dependent manner, as assessed by Annexin V–staining, by the appearance of a subdiploid peak in cell cycle analysis and by activation of caspase-3. These results confirm that BRAF provides a survival signal in this melanoma cell line. By contrast, in thyroid cancer cell lines NPA and ARO, very little evidence of apoptosis was observed in several experiments. An arrest in G1 and a decrease of S and G2-M phases were observed in these cells, in accordance with results obtained by shRNA (data not shown). In TPC1 cells, neither apoptosis nor cell cycle alterations were observed at up to 10 μmol/L of Vemurafenib. These results on thyroid cell lines are consistent with recently published BRAF inhibitor data. In ARO cells, long-term treatment (6 days) with Vemurafenib induced the reexpression of the NIS pump, again confirming shRNA data.
From these results, we can conclude that the pharmacologic inhibition of BRAF enzymatic activity has the same consequences as down-regulation of its expression, in terms of cell growth and alterations of the cell cycle. These data also indicate that Vemurafenib is specifically active on BRAF-mutated cell lines.
Posted by kinsxu.