Activating RAS and BRAF mutations typically demonstrate mutual exclusivity in tumours1–3
. This suggests an epistatic relationship whereby either mutation is sufficient to deregulate a common effector pathway such as the MEK–ERK kinase cascade. If so, tumours arising as a result of mutation to either RAS or BRAF should harbour similar downstream dependencies that might represent useful therapeutic targets. To test this hypothesis, we examined the consequences of MEK–ERK pathway inhibition in a collection of cancer cell lines that exhibited differing mechanisms of MAP kinase pathway deregulation. Cell lines containing the NRAS(Q61R) or BRAF(V600E) mutations (present in ~15% and ~50% of melanomas, respectively) were analysed alongside a panel of cancer cell lines that lacked both mutations (hereafter referred to as RAS/BRAF-WT). Several of these RAS/BRAF-WT cell lines exhibit levels of ERK phosphorylation comparable to those observed in the setting of RAS or RAF mutation.
MEK1/2 are dual-specificity kinases that phosphorylate and activate ERK, the classical MAP kinase4
. To inhibit MEK–ERK, we used the potent and selective MEK inhibitor CI-1040 (ref. 5
). CI-1040 is a non-competitive inhibitor of MEK1/2 with a Ki
of 300nM in vitro5,6
. The only other known CI-1040 target is the MEK5 kinase; however, its inhibition occurs at a 100-fold greater concentration than that required for inhibition of MEK1/2 (ref. 7
). Because ERK is the only known MEK substrate, we reasoned that selective MEK inhibition might clarify the role of the MAP kinase pathway in differing genetic contexts.
CI-1040 inhibited MEK (as measured by phosphorylated ERK (p-ERK) levels) with a half-maximal inhibitory concentration (IC50) of 100–500nM in all cell lines tested ( and data not shown). In contrast, the IC50 for growth inhibition by CI-1040 differed markedly in a manner that correlated with the mechanism of ERK activation (). Whereas RAS/BRAF-WT cells exhibited resistance to CI-1040 even at concentrations in vast excess of those required for ERK inhibition, cells harbouring a BRAF mutation were exquisitely sensitive, with IC50 values of 0.024–0.111 µM (). Surprisingly, RAS mutant cells did not demonstrate the same sensitivity despite effective inhibition of p-ERK ( and data not shown). These data raised the possibility that RAS and BRAF mutant cancer cells might be differentially dependent on signalling mechanisms that involve MEK, despite their known epistatic relationship in human cancers.
The BRAF(V600E) mutation confers sensitivity to the MEK inhibitor CI-1040
To explore this hypothesis in an unbiased manner, we interrogated the large-scale chemical sensitivity data available for the NCI60 cancer cell lines8
using supervised learning methods previously applied to the analysis of gene-expression data. NCI60 cell lines were partitioned into two classes according to the presence or absence of the BRAF(V600E) mutation. We then performed a supervised analysis9
where the mean −log10
(GI50) values for each compound in the BRAF(V600E) and non-mutant classes were compared using a variance fixed t
-test metric and ranked according to T-score (the GI50 is the concentration that inhibits cell growth by 50%). Thirty-six compounds exhibited significantly increased potency against the BRAF(V600E) class distinction ( and Supplementary Table S1
; false discovery rate (FDR) = 0.25, nominal P
value <3 × 10−4
). The top-scoring compound against the BRAF(V600E) class was hypothemycin (a resorcylic acid lactone, the homologues of which possess potent and selective MEK inhibitory activity), which was found to inhibit p-ERK at a potency comparable to CI-1040 (Supplementary Fig. S1
. Additional top-scoring compounds included protein LF (anthrax lethal factor), a zinc metalloproteinase known to inactivate MEK through enzymatic cleavage12
, and PD98059 (ref. 13
), a well-characterized MEK inhibitor. Thus, at least three of the most potent compounds against the BRAF(V600E) class distinction appeared to exert their effects through MEK inhibition. These results were consistent with the CI-1040 analysis and suggest that BRAF mutation might confer a preferential sensitivity to MEK inhibition in human cancer cells.
Chemical sensitivity associated with mutant BRAF and RAS class distinctions
NCI60 cell lines that harbour RAS mutations are non-overlapping with respect to the BRAF(V600E) mutation, supporting the notion of a redundant pathway function8
. To explore this further, supervised analysis of the NCI60 data was repeated, using the class distinction RAS mutant versus wild-type RAS. Surprisingly, and in contrast to the results observed for the BRAF(V600E) class distinction, no compound surpassed the Bonferroni significance threshold in the RAS mutant class (). Conceivably, RAS and BRAF mutations might elicit similar dependencies despite these results, and our failure to identify compounds in the RAS analysis might reflect confounding genetic heterogeneity. Thus, we performed an additional supervised analysis that directly compared only the BRAF and RAS mutant lines. If these classes do indeed manifest common genetic dependencies, compounds that target the relevant mechanisms (for example, the MEK–ERK pathway) should fail to score by this class distinction. However, hypothemycin again distinguished BRAF and RAS mutant cells (); protein LF also retained a high rank.
Because most BRAF(V600E) cell lines analysed were melanoma-derived, the enhanced sensitivity to MEK inhibition may have reflected a melanocytic lineage effect independent of the BRAF(V600E) mutation; however, several lines of evidence rendered this possibility unlikely. First, in the NCI60 analyses all BRAF(V600E) cell lines exhibited markedly reduced hypothemycin GI50 values relative to the mean across the sample set, regardless of tissue type (). Colo205, an NCI60 colon cancer line with the BRAF(V600E) mutation, was also found to exhibit sensitivity to CI-1040 at an equivalent level to the melanoma cells (). In addition, the two melanoma lines lacking a BRAF mutation were clearly indifferent to the effects of hypothemycin. Finally, only one of the breast/prostate cell lines demonstrated similar sensitivity to the drug: the breast cancer line DU-4475 (IC50 24 nM). Notably, sequencing of BRAF in this cell line showed that it also contained a V600E mutation. Thus, the sensitivity of cancer cell lines to MEK inhibition correlated most closely with BRAF mutation status.
In many cell types, RAS–RAF–MEK–ERK signalling is required for both D-cyclin expression and assembly of the cyclin D–cdk4 complex14
. The marked sensitivity of BRAF mutant cells to MEK inhibitors allowed us to examine the functional consequences of MAP kinase blockade in this context. Treatment of BRAF mutant cell lines with CI-1040 caused a marked decline in D-cyclin protein levels ( and ). In the SKMEL28 cell line, this decline was followed by loss of RB phosphorylation and a profound G1 cell cycle arrest (). G1 arrest was accompanied by apoptosis in several BRAF mutant cell lines (), suggesting that MEK inhibition in a BRAF-mutant context exerts both cytocidal and cytostatic effects. In contrast, CI-1040 concentrations that completely inhibited p-ERK had no effect on cyclin D1 protein expression in the vast majority of RAS/BRAF-WT cells, as shown in for the MCF7, BT-474 and A431 cell lines. BT-474 and A431 exhibited robust MAPK activity driven by HER2/neu and EGFR, respectively, suggesting that cyclin D1 expression and G1 progression are driven by MEK/ERK-independent mechanisms in certain RAS/BRAF-WT cells.
MEK inhibition causes loss of D-cyclin expression, RB hypophosphorylation and G1 arrest in BRAF mutant cancer cells
To determine whether the differential sensitivity to MEK inhibition observed for BRAF mutant cancer cells was re-capitulated in vivo
, mice harbouring xenograft tumours were treated with the MEK inhibitor PD0325901. PD0325901 is a derivative of CI-1040 that has improved oral bioavailability and induces a longer duration of target suppression15
. The effects of PD0325901 on tumour cells in vitro
are qualitatively identical to those of CI-1040, including the marked selectivity for BRAF mutant cell lines, but occur at 100-fold lower concentrations (Supplementary Fig. S2
Daily treatment with PD0325901 at doses of 5 and 25 mg kg−1
completely suppressed the growth of SKMEL28 and Colo205 BRAF(V600E) mutant xenografts ( and Supplementary Fig. S3
< 0.01 for both 5 and 25 mg kg−1
versus control, P
= 0.16 for 5 versus 25 mg kg−1
). Growth suppression was associated with loss of D-cyclin expression, induction of p27 and hypophosphorylation of RB ( and Supplementary Fig. S4
). In contrast, PD0325901 treatment of SKMEL103 (NRAS(Q61R)), SKMEL30 (NRAS(Q61R)) and SKMEL31 (RAS/BRAF-WT) xenografts at a dose of 5 mg kg−1
only delayed tumour growth, with complete growth suppression requiring 25 mg kg−1
( and Supplementary Fig. S3b
< 0.01 for 5 versus 25 mg kg−1
, and 5 and 25 mg kg−1
versus control). BT-474 xenografts (BRAF/RAS-WT) were completely insensitive to PD0325901 (). PD0325901 treatment at the doses studied was non-toxic and resulted in profound p-ERK inhibition in all xenograft models studied; however, RB phosphorylation, cyclin D expression and proliferation as measured by Ki67 were unaffected by MEK inhibition in PD0325901-resistant BT-474 xenografts (), and there was no correlation between basal p-ERK levels and PD0325901 sensitivity (Supplementary Fig. S4
). Thus, the MEK dependency characteristic of BRAF mutant tumour cells in vitro
was also apparent in vivo
PD0325901 completely suppresses the growth of BRAF(V600E) mutant xenografts
Excess MAP kinase pathway activation occurs commonly in human tumours. In melanoma and other solid tumours, mutation of BRAF and RAS occurs frequently and tends to exhibit mutual exclusivity, suggesting that each mutation confers a similar selective advantage1
. However, our findings suggest that tumour cells carrying BRAF mutations are much more reliant on MEK–ERK signalling than are RAS mutant cells, or cells that activate MAP kinase by other means. Thus, BRAF mutant cancer cells may harbour a critical dependency on MEK–ERK that renders them highly sensitive to pharmacological MEK inhibition.
BRAF mutations occur at a high frequency in melanomas, but are also observed in colon, lung and several other tumour types1,2
. Expression of BRAF(V600E) in non-transformed melanocytes leads to constitutive ERK activation and tumorigenicity in mice, and depletion of BRAF but not A-RAF or C-RAF in BRAF(V600E) mutant melanoma cells reduces ERK activity16,17
. Our data suggest that D-cyclin expression is also deregulated and ERK-dependent in BRAF-mutant tumours. Cyclin D downregulation may therefore mediate at least some of the anti-proliferative effects observed after MEK inhibition. On the other hand, MEK inhibition had little effect on D-cyclin expression in most BRAF/RAS-WT tumour cells. In these cells, mutations in the PTEN or phosphatidylinositol-3-OH kinase (PI(3)K) genes, or activation of other pathways, may drive D-cyclin expression in an ERK-independent fashion18,19
. Our results are also consistent with a model in which ERK regulates G1 progression only in certain lineages (for example, melanocytes); presumably, such lineage differences in cell growth control contribute to the imbalanced frequency of RAS and BRAF mutations observed across tumour types.
RAS-dependent transformation has been found previously to require activation of cyclin D1 (refs 20–23
). As both oncogenic RAF and activated ERK also induce cyclin D1 expression24,25
, it has been presumed that in human tumours with RAS mutation, cyclin D1 expression was controlled by RAS-mediated MEK–ERK activation. However, our results suggest that in certain genetic contexts, including some tumours with RAS mutation, ERK signalling may be dispensable for cyclin D1 expression and cell proliferation. RAS family members have multiple other targets, such as PI(3)K and RalGDS; these may exert more prominent oncogenic effects in certain tumour subtypes, thereby reducing the requirement for MEK–ERK activation26,27
. Our findings therefore raise the possibility that single-agent therapeutic strategies may prove insufficient in RAS mutant tumours. Instead, direct RAS inhibitors or combinatorial strategies may be required.
Thus far, the use of BRAF inhibitors in clinical trials has met with mixed results. On the other hand, the favourable therapeutic index and selectivity of MEK inhibitors may provide an appealing therapeutic strategy for BRAF mutant cancers. We therefore propose clinical trials of MEK inhibitors in which patients are stratified based on BRAF mutational status.