RAF inhibitors transactivate RAF dimers and ERK signalling in cells with wild-type BRAF

Abstract

Tumours with mutant BRAF are dependent on the RAF-MEK-ERK signalling pathway for their growth. We found that ATP-competitive RAF inhibitors inhibit ERK signalling in cells with mutant BRAF, but unexpectedly enhance signalling in cells with wild-type BRAF. Here we demonstrate the mechanistic basis for these findings. We used chemical genetic methods to show that drug-mediated transactivation of RAF dimers is responsible for paradoxical activation of the enzyme by inhibitors. Induction of ERK signalling requires direct binding of the drug to the ATP-binding site of one kinase of the dimer and is dependent on RAS activity. Drug binding to one member of RAF homodimers (CRAF-CRAF) or heterodimers (CRAF-BRAF) inhibits one protomer, but results in transactivation of the drug-free protomer. In BRAF(V600E) tumours, RAS is not activated, thus transactivation is minimal and ERK signalling is inhibited in cells exposed to RAF inhibitors. These results indicate that RAF inhibitors will be effective in tumours in which BRAF is mutated. Furthermore, because RAF inhibitors do not inhibit ERK signalling in other cells, the model predicts that they would have a higher therapeutic index and greater antitumour activity than mitogen-activated protein kinase (MEK) inhibitors, but could also cause toxicity due to MEK/ERK activation. These predictions have been borne out in a recent clinical trial of the RAF inhibitor PLX4032 (refs 4, 5). The model indicates that promotion of RAF dimerization by elevation of wild-type RAF expression or RAS activity could lead to drug resistance in mutant BRAF tumours. In agreement with this prediction, RAF inhibitors do not inhibit ERK signalling in cells that coexpress BRAF(V600E) and mutant RAS.

Figure 1. RAF inhibitors rapidly activate MEK/ERK in cells with wild-type BRAF

a, Calu-6 cells (BRAF^wild-type/K-RAS^Q61K) were treated with increasing doses of the indicated RAF inhibitors and the effects on ERK signaling were determined by immunoblotting for pMEK and pERK. b, Cells with wild-type BRAF (Calu-6) or mutant BRAF (Malme-3M) were treated with vehicle or PLX4720 (1μM/1 hour). Phosphorylation and expression of the indicated proteins were assayed by immunoblotting. c, Calu-6 cells treated with 1μM PLX4720 for the indicated time points. d, Calu-6 cells were treated with 1μM PLX4720 for 60 minutes, then medium was replaced with medium containing 1μM PLX4720 (lanes 3-5) or vehicle (lanes 8-10) for the indicated time points.

Figure 2. MEK/ERK activation requires binding of drug to the catalytic domain of RAF

a, 293H cells transfected with EGFP (control), HA-tagged RAS^G12V, the catalytic domain of CRAF (V5-tagged catC) and catC carrying a mutation at the gatekeeper residue (V5-tagged catC^T421M), treated with vehicle or PLX4720 (1μM/1 hour). Lysates were subjected to immunoblot analysis for pMEK and pERK. b, Wild-type (+/+), BRAF knock-out (BRAF −/−) or CRAF knock-out (CRAF −/−) mouse embryonic fibroblasts (MEFs) were treated with the indicated concentrations of PLX4720 for 1 hour. c, Sorafenib inhibits the gatekeeper mutant catC^T421M protein in vitro (Supplementary Fig. 8c) and activates MEK/ERK in cells expressing it. 293H cells overexpressing catC^T421M were treated with the indicated concentrations of sorafenib for 1 hour. Lysates were subjected to analysis for pMEK and pERK.

Figure 3. RAF inhibitor induces the active, phosphorylated state of wild-type and kinase-dead RAF

a, 293H cells over-expressing catC were treated with the indicated amounts of PLX4720 for 1 hour. Cells were lysed, catC was immunoprecipitated, washed extensively and subjected to kinase assay. Kinase activity was determined by immunoblotting for pMEK. b, Calu-6 cells were treated with PLX4720 (1μM/1 hour). Endogenous BRAF and CRAF were immunoprecipitated, washed and assayed for kinase activity. c, Treatment with RAF inhibitor results in elevated phosphorylation at activating phosphorylation sites on RAF. V5-tagged wild-type CRAF or kinase-dead CRAF^D486N were overexpressed in 293H cells. After 24 hours cells were treated with vehicle or PLX4720 (5μM/1hour) and lysates were immunoblotted for p338CRAF and p621CRAF. The gatekeeper mutant CRAF^T421M was used as negative control. d. Samples as in Fig. 1a, immunoblotted for pS338CRAF. Note that phosphorylation at S338 steadily increased, even when concentrations were reached that inhibited MEK/ERK.

Figure 4. MEK/ERK induction occurs via transactivation of RAF dimers

a, Similarly to RAF inhibitors, JAB34 inhibits MEK/ERK at higher concentrations. 293H cells expressing V5-tagged catC or catC^S428C were treated with either vehicle or 10μM JAB34 for 1 hour. b, Coexpression of drug-sensitive V5-tagged catC with drug-resistant FLAG-catC reveals that activation in the homodimer occurs in trans. 293H cells expressing the indicated mutants V5-tagged catC and FLAG-tagged catC were treated with a dose of JAB34 (10μM/1 hour) that inhibits catC^S428C when expressed alone. c, Activation in the context of the heterodimer BRAF/CRAF occurs in trans. 293H cells co-expressing FLAG-tagged wild-type BRAF and V5- tagged kinase-dead catC (catC^D486N) (lanes 3,4) or JAB34-sensitive/kinase-dead catC (catC^S428C/D486N) (lanes 5,6) treated with vehicle or 10μM JAB34 for 1 hour. d, HT-29 cells (colorectal – BRAF^V600E) were transfected with EGFP or HA-tagged N-RAS^G12V and treated with PLX4720 (1μM/1hour). Lysates were blotted for pMEK and pERK.