BRAF mutation predicts sensitivity to MEK inhibition

Abstract

The kinase pathway comprising RAS, RAF, mitogen-activated protein kinase kinase (MEK) and extracellular signal regulated kinase (ERK) is activated in most human tumours, often through gain-of-function mutations of RAS and RAF family members. Using small-molecule inhibitors of MEK and an integrated genetic and pharmacologic analysis, we find that mutation of BRAF is associated with enhanced and selective sensitivity to MEK inhibition when compared to either 'wild-type' cells or cells harbouring a RAS mutation. This MEK dependency was observed in BRAF mutant cells regardless of tissue lineage, and correlated with both downregulation of cyclin D1 protein expression and the induction of G1 arrest. Pharmacological MEK inhibition completely abrogated tumour growth in BRAF mutant xenografts, whereas RAS mutant tumours were only partially inhibited. These data suggest an exquisite dependency on MEK activity in BRAF mutant tumours, and offer a rational therapeutic strategy for this genetically defined tumour subtype.

Figure 1. The BRAF(V600E) mutation confers sensitivity to the MEK inhibitor CI-1040

a, CI-1040 IC₅₀ values as a function of BRAF and NRAS mutational status. b, Immunoblot of p-ERK and total ERK, demonstrating that CI-1040 inhibits MAPK activity with IC₅₀ values ranging from 100 to 500 nM. Cells were treated for 24 h. MEK inhibition caused profound downregulation of cyclin D1 expression in BRAF mutant tumour cells. In contrast, cyclin D1 declined only modestly in SKMEL103 melanoma cells with the NRAS(Q61R) mutation and in SKMEL31 RAS/BRAF-WT cells. Cyclin D1 expression was unaffected by CI-1040 in non-melanoma cells with wild-type RAS and BRAF.

Figure 2. Chemical sensitivity associated with mutant BRAF and RAS class distinctions

a–c, Colourgrams show BRAF mutant (a) or RAS mutant (b) versus the remaining NCI60 cells, or for mutant RAS versus mutant BRAF lines (c). Columns denote NCI60 cell lines; rows denote compounds; colour denotes the number of standard deviations above (red) or below (blue) the mean for all cell lines (top 100 compounds for each class distinction shown; see Methods). NSC numbers, names, variance-fixed T-scores (absolute values; see Methods) and asymptotic P values are shown for top-scoring compounds. Blue font indicates known MEK inhibitors. d, Relative GI50 values for the MEK inhibitor hypothemycin in non-haematological NCI60 cell lines. Black bars indicate BRAF wild-type cells; blue bars indicate BRAF(V600E) cells; asterisks indicate non-melanoma cell lines with the BRAF(V600E) mutation.

Figure 3. MEK inhibition causes loss of D-cyclin expression, RB hypophosphorylation and G1 arrest in BRAF mutant cancer cells

a, Immunoblot showing the kinetics of change in p-ERK, D-cyclin expression and RB in SKMEL28 cells treated with 1 µM CI-1040. b, CI-1040 induced a G1 growth arrest in BRAF mutant tumour cells but not in RAS/BRAF-WT breast cancer cells (BT-474 shown). c, d, CI-1040 induces apoptosis in some but not all cancer cell lines with the BRAF(V600E) mutation as measured by FACS analysis (c) and PARP cleavage (d).

Figure 4. PD0325901 completely suppresses the growth of BRAF(V600E) mutant xenografts

a, PD0325901 suppressed the growth of SKMEL28 (BRAF(V600E)) xenografts at both the 5 and 25 mg kg⁻¹ dose levels. b, c, In contrast, 5 mg kg⁻¹ PD0325901 only delayed the growth of SKMEL103 (RAS(Q61R)) and SKMEL31 (RAS/RAF-WT) xenografts, with complete growth suppression requiring the higher dose. d, BT-474 xenografts (RAS/RAF-WT) were refractory to MEK inhibition (n = 10 mice per group). e, f, PD0325901 reduced p-ERK levels in both SKMEL28 and BT-474 xenograft tumours but caused downregulation of D-cyclins, induction of p27 and hypophosphorylation of RB, and a decline in the proliferative index only in the SKMEL28 xenografts. Tumour lysates were derived from mice euthanized 8 h after the final treatment. All error bars indicate standard error.