The RAF inhibitor PLX4032 inhibits ERK signaling and tumor cell proliferation in a V600E BRAF-selective manner

Abstract

Tumors with mutant BRAF and some with mutant RAS are dependent upon ERK signaling for proliferation, and their growth is suppressed by MAPK/ERK kinase (MEK) inhibitors. In contrast, tumor cells with human EGF receptor (HER) kinase activation proliferate in a MEK-independent manner. These findings have led to the development of RAF and MEK inhibitors as anticancer agents. Like MEK inhibitors, the RAF inhibitor PLX4032 inhibits the proliferation of BRAF(V600E) tumor cells but not that of HER kinase-dependent tumors. However, tumors with RAS mutation that are sensitive to MEK inhibition are insensitive to PLX4032. MEK inhibitors inhibit ERK phosphorylation in all normal and tumor cells, whereas PLX4032 inhibits ERK signaling only in tumor cells expressing BRAF(V600E). In contrast, the drug activates MEK and ERK phosphorylation in cells with wild-type BRAF. In BRAF(V600E) tumor cells, MEK and RAF inhibitors affect the expression of a common set of genes. PLX4032 inhibits ERK signaling output in mutant BRAF cells, whereas it transiently activates the expression of these genes in tumor cells with wild-type RAF. Thus, PLX4032 inhibits ERK signaling output in a mutant BRAF-selective manner. These data explain why the drug selectively inhibits the growth of mutant BRAF tumors and suggest that it will not cause toxicity resulting from the inhibition of ERK signaling in normal cells. This selectivity may lead to a broader therapeutic index and help explain the greater antitumor activity observed with this drug than with MEK inhibitors.

Conflict of interest statement

Conflict of interest statement: N.R. has major consulting/advisory roles with Roche (>$10,000/y). D.B.S. and P.B.C have minor consulting/advisory roles with Roche (>$10,000/y). J.T. and G.B. are full-time employees of Plexxikon.

Figures

Fig. 1.

Identification of RAF- and MEK-dependent gene expression in BRAFV600E melanoma cells. (A) Heat map representation of 93 probe sets (73 genes), in each of five BRAFV600E cell lines in the presence of DMSO (control), 250 nM PLX4032 (RAFi), or 50 nM PD0325901 (MEKi). Genes were categorized by their change in expression in response to PLX4032 (“RAFi only”), to PD0325901 (“MEKi only”), or to both (“overlap”). (B) The correlation between the average change in expression of each of the 73 ERK-dependent genes across BRAFV600E melanoma cells in response to either RAFi or MEKi. The plot distinguishes genes responding to “RAFi only” (red), to “MEKi only” (blue), or to both (gray).

Fig. 2.

PLX4032 selectively inhibits the growth of BRAFV600E-mutant cell lines. (A) Day five IC50 values for PD0325901 (orange) and PLX4032 (blue) in a panel of 37 cell lines with BRAFV600E mutation, with RAS mutation, and with BRAFWT/RASWT, determined using the Alamar Blue assay (Methods). (B and C) MAPK activity as measured by immunoblot detection of pMEK1/2 (Ser217/221) and pERK1/2 (Thr202/Tyr204) in select BRAFV600E, RAS mutant, and BRAFWT/RASWT cell lines from A, after 2-h exposure to PD0325901 (B) or PLX4032 (C) over a range of concentrations.

Fig. 3.

PLX4032 inhibits ERK phosphorylation only in BRAFV600E-mutant cell lines. Immunoblots of pMEK, pERK, and total ERK for a panel of tumor and primary human keratinocyte cell lines. Cells were treated with 2 μM PLX4032 for 1, 6, and 24 h.

Fig. 4.

PLX4032 inhibited the ERK-dependent transcriptional output of BRAFV600E cell lines. The expression levels of ERK output genes were determined by RT-PCR in the designated cell lines. Cells were treated with 50 nM PD0325901 (A) or 250 nM PLX4032 (B) for 0, 2, 4, and 8 h. Values are expressed as the relative mRNA level in drug-treated samples compared with time 0. (C) Relative mRNA levels of DUSP6, SPRY2, ETV5, and FOSL1 in PLX4032-treated BRAFV600E and BRAFWT cell lines over time. Untreated RNA expression is set at 100% for each individual cell line.

Fig. 5.

Biologic effects of RAF inhibition occur only in BRAFV600E-mutant cell lines. (A) Growth kinetics of BRAFV600E cell lines (Malme3M and SkMel-28) compared with a MEK/ERK-dependent K-RAS–mutant cell line (SW-620) and a BRAFWT/RASWT cell line (SkMel-31) in the presence of PLX4032 (10 nM–2 μM) or vehicle alone (0 nM), as determined by cell count. (B) Percentage of cells in the sub-G1 population, as determined by FACS, following 24-, 48-, and 72-h exposures to 0.5 μM and 2.0 μM PLX4032 or control, for each of the cell lines shown in A. (C) Immunoblot detection of pMEK and pERK in lysate from the same cell lines, following treatment with vehicle (−) or 0.5 μM PLX4032 (+) over a 72-h time course. Cell-cycle regulation and apoptosis induction in the same samples was assessed by immunoblot detection of cyclin D1, p27, RB phosphorylation, and PARP cleavage.