Tumors with mutant BRAF are dependent on the RAF/MEK/ERK signaling pathway for their
growth1-3. We found that ATP-competitive RAF inhibitors inhibit ERK signaling in cells
with mutant BRAF, but unexpectedly enhance signaling in cells with wild-type BRAF.
Here we demonstrate the mechanistic basis for these findings. We employed 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 signaling
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 homo-(CRAF/CRAF)
or heterodimers (CRAF/BRAF) inhibits one protomer, but results in transactivation
of the drug-free protomer. In BRAFV600E tumors, RAS is not activated, thus transactivation
is minimal and ERK signaling is inhibited in cells exposed to RAF inhibitors. These
results imply that RAF inhibitors will be effective in tumors in which BRAF is mutated.
Furthermore, since they do not inhibit ERK signaling in other cells, the model predicts
that they would have a higher therapeutic index and greater antitumor activity than
MEK inhibitors, but could also cause toxicity due to MEK/ERK activation. These predictions
have been borne out strikingly in a recent clinical trial of the RAF inhibitor PLX40324-5.
Finally, the model suggests that promotion of RAF dimerization by elevation of wild-type
RAF expression or RAS activity could lead to drug resistance in mutant BRAF tumors.
In agreement with this prediction, RAF inhibitors do not inhibit ERK signaling in
cells that coexpress BRAFV600E and mutant RAS.
Six distinct ATP-competitive RAF inhibitors induced ERK activation in cells with wild-type
BRAF, but inhibited signaling in mutant BRAFV600E cells (Fig. 1a, b, Supplementary
Fig. 2a, b, Data Not Shown (DNS), structures of compounds shown in Supplementary Fig.
3, except that of PLX4032, which is unavailable). PLX47206, and its analog in clinical
trial PLX4032, were studied in more detail. PLX4032 inhibited ARAF, BRAF and CRAF
immunoprecipitated from 293H cells (Supplementary Fig. 4) and purified catalytic domains
of BRAFV600E, wild-type BRAF and CRAF (IC50s: 35, 110 and 48nM) (Supplementary Table
1). PLX4032 was assayed against 62 additional kinases that span the kinome, and had
IC50s of 1μM-10μM against eight of these and greater than 10μM against the rest (G.B.,
unpublished data). Induction of ERK signaling by PLX4720 was rapid (Fig. 1c), reversible
(Fig. 1d), and associated with increased phosphorylation of the ERK substrate RSK
(Fig 1b). MEK and ERK phosphorylation were induced at intermediate concentrations
of RAF inhibitor, and inhibited at much higher doses (Fig. 1a).
Physiologic induction of ERK signaling depends on upstream activation of RAS by receptor-induced
signaling7-8. PLX4032 induced ERK signaling in SKBR3 breast cancer cells, in which
RAS activation is HER2-dependent9. The HER2 inhibitor Lapatinib abolished basal and
PLX4032-induced ERK signaling in these cells (Supplementary Fig. 5a). In 293H cells,
induction of MEK and ERK phosphorylation by either PLX4032 or PLX4720 was barely detectable
(PLX will refer to data obtained with both compounds). HA-tagged wild-type RAS overexpression
resulted in enhanced MEK/ERK activation by RAF inhibitor, which was more pronounced
when mutant RAS was overexpressed (Fig. 2a and Supplementary Fig. 5b). The results
suggest that RAS activity is required for MEK/ERK activation by RAF inhibitors. In
contrast, in 293H cells expressing FLAG-tagged BRAFV600E, ERK signaling was inhibited
by PLX4032 (Supplementary Fig. 5c). These results suggest that RAF inhibitors will
inhibit the growth of tumors with mutant BRAF, but not those with wild-type BRAF,
including those with RAS mutation. This is indeed the case: MEK-dependent tumors with
RAS mutation are unaffected by PLX4032 (unpublished data).
BRAF and CRAF kinases form homo- and heterodimers upon RAS activation10-12. PLX induced
pronounced phosphorylation of MEK and ERK in wild-type MEFs and BRAF (−/−) MEFs. The
response was diminished markedly in CRAF (−/−) MEFs (Fig. 2b, Supplementary Fig. 6a).
Coexpression of CRAF and active RAS in CRAF (−/−) MEFs reconstituted the wild-type
phenotype (Supplementary Fig. 6b, c). We conclude that BRAF is dispensable for MEK/ERK
activation by PLX, and that CRAF expression is required for significant induction.
We therefore investigated the mechanism of CRAF-dependent induction of ERK signaling
in response to the drug.
Autoinhibition of RAF by its N-terminal domain13 is relieved upon binding to activated
RAS7. We asked whether overexpression of an N-truncated form of CRAF would bypass
the requirement for RAS activity. In 293H cells expressing the catalytic domain of
CRAF (catC), PLX caused dramatic induction of MEK and ERK phosphorylation (Fig. 2a,
Supplementary Fig. 7a). We focused mechanistic investigations on catC, in which PLX-induced
MEK/ERK activation is RAS-independent. To test whether direct binding of PLX to CRAF
is required for induction of signaling, we generated a catC carrying a mutation at
the gatekeeper position (T421) in the kinase domain (mutations used and their properties
are in Supplementary Fig. 1a). Structural studies6 predict that the T421M mutation
should prevent drug binding and catCT421M was indeed resistant to inhibition by PLX
in vitro (Supplementary Fig. 8a, b). ERK signaling was not induced by PLX in cells
expressing catCT421M (Fig. 2a, Supplementary Fig. 7b). Thus, activation of MEK/ERK
by PLX depends on its direct binding to the RAF kinase active site. Sorafenib inhibited
catCT421M in vitro (Supplementary Fig. 8c) and induced ERK signaling in cells expressing
catCT421M (Fig. 2c), demonstrating that this mutant is capable of inhibitor-induced
MEK/ERK activation. Thus, direct binding of an ATP-competitive inhibitor to CRAF is
required for induction of ERK signaling.
Recent work shows that binding of ATP-competitive inhibitors to AKT and PKC inhibits
their activity, but induces the active, phosphorylated state of these kinases14-15.
Washed catC immunoprecipitated from PLX-treated cells was more active than that isolated
from untreated cells (Fig. 3a, Supplementary Fig. 9a). The same was true for endogenous
BRAF and CRAF immunoprecipitated from treated Calu-6 cells (Fig. 3b, Supplementary
Fig. 9b). Phosphorylation of CRAF at S338 and S621 has been correlated with its activation7.
PLX caused increased phosphorylation of both sites on wild-type and kinase-dead CRAF
in 293H cells. In contrast, it did not affect the phosphorylation of the PLX-resistant
CRAFT421M mutant. (Fig. 3c, Supplementary Fig. 9c). All RAF inhibitors tested induced
phosphorylation at p338 of endogenous CRAF (Fig. 3d). The data suggest that binding
of PLX to CRAF induces activation of the enzyme and, subsequently, ERK signaling.
The result seems paradoxical: binding of ATP-competitive inhibitors to the catalytic
domain of CRAF activate its function.
RAF isoforms form dimers in cells10-12,16. Since binding of both the drug and ATP
to the catalytic domain would be required for activation and cannot occur simultaneously
on the same molecule, we hypothesized that RAF inhibitors activate CRAF dimers in
trans. (Supplementary Fig. 1b). To test this model, we generated mutant catCS428C
that binds to quinazoylacrylamide-based inhibitors17, whereas catC does not. Two inhibitors
JAB1317 and JAB34 (PD-168393)18 both inhibited catCS428C, but up to 30μM had no effect
in vitro on catC (Supplementary Fig. 10a, b). JAB13 and JAB34 selectively affected
ERK signaling in cells expressing catCS428C, and were inactive in those expressing
catC (Supplementary Fig. 11). Like the other inhibitors (Fig. 1a), lower doses (40nM
- 1μM) induced ERK signaling (Supplementary Fig. 11), whereas higher doses (10μM)
inhibited (Fig. 4a). The specificity of this system allows us to test the dimer transactivation
model. We coexpressed a V5-tagged, JAB-sensitive, kinase-dead catC, V5-catCS428C/D486N
and FLAG-catC in 293H cells. V5-catCS428C/D486N is deficient in catalytic activity;
it can bind to the inhibitor (JAB34) but cannot phosphorylate MEK, while FLAG-catC
is catalytically active, but cannot bind JAB34. Treatment of cells expressing both
constructs with a concentration of JAB34 that inhibited ERK signaling in cells expressing
catCS428C alone (10μM JAB34, Fig. 4a) resulted in marked induction of ERK signaling
(Fig. 4b, lanes 5-6). Thus, binding of JAB34 to kinase-dead, V5-catCS428C/D486N transactivated
the catalytically competent FLAG-catC. When the catalytically active drug-binding
mutant V5-catCS428C is coexpressed with catalytically inactive catC (FLAG-catCD486N),
10μM JAB34 inhibited, rather than activated ERK signaling. (Fig. 4b, lanes 9-10).
When both constructs were insensitive to JAB, JAB34 had no effect on ERK signaling
(Fig. 4b, lanes 1-2). When both constructs were catalytically active, we observed
moderate MEK/ERK activation, likely resulting from inhibition of V5-catCS428C and
transactivation of FLAG-catC (Fig. 4b, lanes 3-4).
Transactivation from CRAF to BRAF can occur as well. JAB34 activated ERK signaling
in cells coexpressing FLAG-BRAF with V5-catCS428C/D486N (Fig. 4c). Finally, JAB34
induced ERK activation in cells coexpressing full-length V5-CRAFS428C/D486N and wild-type
FLAG-CRAF, confirming that our model is valid in the context of full-length CRAF (Supplementary
Fig. 12).
Thus, activation of RAF by ATP-competitive inhibitors can be explained by transactivation:
binding of drug to one RAF in the dimer activates the other. This is consistent with
the enhancement of induction by active RAS, which promotes homo- and hetero-dimerization
of BRAF and CRAF10,12. Our model suggests that transactivation will be dependent on
formation of RAF dimers. A side-to-side dimer of the kinase domain is observed in
crystal structures of BRAF11 and the residues at the dimer interface are conserved
in all RAF isoforms. Based on the BRAF crystal structures, we identified a conserved
Arg (R509) at the center of the dimer interface. Structural analysis predicts that
mutation of R509 will diminish contacts between the two interacting proteins and reduce
dimer formation, as also recently reported19. In that study, mutation of BRAF at R509
to Histidine resulted in dramatic loss of activity. The corresponding mutation in
catC (R401H) results in severe loss of both expression and activity (DNS). We therefore
mutated R401 to Alanine in V5-catCS428C and FLAG-catC. This mutation diminished dimerization
(Supplementary Fig. 13), but retained expression and activity. In cells coexpressing
these mutants, JAB34 failed to induce ERK signaling (Fig. 4b, lanes 7-8). Thus, a
mutation that affects dimerization prevents transactivation.
The transactivation model explains the observation that inhibitors of RAF activate
ERK signaling at low concentrations, but inhibit at higher concentrations in BRAFwild-type
cells. Binding of an ATP-competitive inhibitor to one protomer within a RAF dimer
results in both abolition of the catalytic activity of the inhibitor-bound RAF and
transactivation of the other. Transactivation of RAF homo- and heterodimers is therefore
responsible for induction of MEK/ERK phosphorylation by RAF inhibitors in cells with
wild-type BRAF. Our model explains the paradoxical phenomenon of ERK activation by
RAF inhibitors, previously reported by others20-22. Other kinases that exist in dimeric
or multimeric complexes may behave in a similar manner. Recently, another model to
explain these phenomena has been proposed23. They report that only selective BRAF
inhibitors activate CRAF and ERK signaling, whereas pan-RAF inhibitors do not. Our
data that all RAF inhibitors activate ERK signaling at low concentrations, that the
phenomenon occurs in BRAF-null cells and that binding to CRAF activates CRAF and BRAF-dependent
ERK signaling render that model unlikely.
Nevertheless, the clinical utility of these inhibitors depends on their inhibition
of ERK signaling in tumor cells with BRAFV600E. Since transactivation of wild-type
RAF requires dimerization and depends on RAS activity, we hypothesized that the levels
of RAS activity in BRAFV600E mutant tumors may not be sufficient to support transactivation.
If so, activation of RAS in BRAFV600E cells should prevent inhibition of ERK signaling
by RAF inhibitors. In 293H cells overexpressing BRAFV600E and in HT29 tumor cells
with endogenous BRAFV600E, ERK signaling was inhibited by either PLX or a MEK inhibitor.
In contrast, when mutant RAS was coexpressed with BRAFV600E in either cell, ERK signaling
became resistant to PLX, but remained sensitive to the MEK inhibitor (Fig. 4d, Supplementary
Fig. 14a, b).
The data are consistent with the idea that RAF inhibitors suppress ERK signaling in
BRAFV600E tumors because the level of RAS activation in these cells is insufficient
to support transactivation of wild-type RAF and inhibition of BRAFV600E activity becomes
the dominant effect of the drug. The findings suggest that increases in RAS activation
or RAF dimerization may be sufficient to cause drug resistance.
The clinical implications of these findings are profound. BRAFV600E tumors and some
with RAS mutation are dependent on ERK signaling. However, in clinic, a MEK inhibitor
had only a 12% response rate in melanomas with BRAF mutation24. MEK inhibitors block
ERK signaling in all tumor and normal cells and the dose of the drug that can be administered
is limited by toxicity. RAF inhibitors and MEK inhibitors might have been expected
to have similar biologic effects. Our findings show otherwise. RAF inhibitors will
be useful for the treatment of tumors driven by BRAFV600E, but could have deleterious
effects in some contexts due to ERK activation. However, the absence of ERK inhibition
in normal cells may allow administration of high doses of RAF inhibitors and thus
more complete inhibition of ERK signaling in BRAFV600E tumors, than is possible with
MEK inhibitors.
The recent phase I clinical trial of PLX4032 in metastatic melanoma strikingly confirmed
these predictions4-5. High serum levels of drug were achieved with modest toxicity
and resulted in profound inhibition of ERK signaling in tumors. Tumor regression was
observed in more than 90% of patients with BRAFV600E mutation, with 64% achieving
a partial response by RECIST criteria. We believe that the remarkable activity of
this drug, compared to that of MEK inhibitors, is due to its ability to inhibit ERK
signaling in tumors more completely because of the absence of ERK inhibition in normal
tissue.
Resistance to PLX4032 does develop, with a median time to disease progression of 8-9
months5. Potential mechanisms include gatekeeper mutations in BRAF and activating
mutations in parallel signaling pathways. Our results suggest the possibility of novel
mechanisms as well. Lesions that activate RAS or, as recently reported, overexpression
of wild type RAF isoforms25 could result in inability of RAF inhibitors to suppress
ERK signaling in the tumor and thus lead to resistance.
Methods Summary
Compounds and cell culture
PLX4032 and PLX4720 were obtained from Plexxikon, Inc. PD0325901 was synthesized in
the MSKCC Organic Synthesis Core Facility by Dr. Ouathek Ouerfelli. Sorafenib was
synthesized using published procedures26. JAB13, JAB34 were synthesized as previously
described17. All other drugs were obtained from Calbiochem. Drugs were dissolved in
DMSO and stored at −20°C. Cells were maintained in either DMEM or RPMI, supplemented
with 2mM glutamine, antibiotics, and 10% fetal bovine serum. Wild-type, BRAF (−/−)
and CRAF (−/−) MEFs were kindly provided by Dr. Manuela Baccarini, University of Vienna,
Austria. 293H cells were from Invitrogen. All other cell lines were from ATCC.
Antibodies
Western blot analysis was performed as described1. The following antibodies were used:
p217/p221MEK (pMEK), p202/p204ERK (pERK), p338CRAF, p380RSK, p573RSK, MEK, ERK, myc-tag
(Cell Signaling), p621CRAF, V5-tag (Invitrogen), ARAF, BRAF (Santa Cruz Biotechnology),
FLAG-tag (Sigma), CRAF (BD Transduction Laboratories), HA-tag (Covance). For immunoprecipitations
of tagged proteins, the following reagents were used: Anti-V5 agarose affinity gel,
Anti-FLAG M2 affinity gel, anti-c-myc agarose affinity gel (all from Sigma).
Plasmids
Plasmids encoding HA-tagged wild-type and mutant N-RAS were obtained from Biomyx.
Plasmids for wild-type BRAF and BRAFV600E were kindly provided by Dr. Walter Kolch,
(University of Glasgow, UK), and were used as template to create FLAG-tagged constructs.
All other plasmids were created using standard cloning methods, with pcDNA3.1 (Invitrogen)
as a vector. Mutations were introduced using site-directed Mutagenesis Kit (Stratagene).
The catalytic domain of CRAF (catC) was created by truncating the first 305 amino-acids
of CRAF.
Kinase assays
RAF kinase assays were conducted in the presence of 100μM ATP, at 30°C for 20 minutes.
Recombinant inactive K97R MEK (MIllipore) was used as a substrate and kinase activity
was estimated by immunoblotting for pMEK.
Transfections
Cells were seeded at 35mm or 100mm plates and transfected the following day using
Lipofectamine 2000 (Invitrogen).
Methods
Compounds and cell culture
PLX4032 and PLX4720 were obtained from Plexxikon, Inc. PD0325901 was synthesized in
the MSKCC Organic Synthesis Core Facility by Dr. Ouathek Ouerfelli. Sorafenib was
synthesized using published procedures26. JAB13, JAB34 were synthesized as previously
described17. All other drugs were obtained from Calbiochem. Drugs were dissolved in
DMSO to yield 10 mM stock and stored at −20°C. Cells were maintained in DMEM (MEFs,
293H, NIH3T3 and Hela) or RPMI (all other cell lines), supplemented with 2mM glutamine,
antibiotics, and 10% fetal bovine serum. Wild-type, BRAF (−/−) and CRAF (−/−) MEFs
were kindly provided by Dr. Manuela Baccarini, University of Vienna, Austria. 293H
cells were from Invitrogen. All other cell lines were from ATCC.
Antibodies
Western blot analysis was performed as described1. The following antibodies were used:
p217/p221MEK (pMEK), p202/p204ERK (pERK), p338CRAF, p380RSK, p573RSK, MEK, ERK, myc-tag
(Cell Signaling), p621CRAF, V5-tag (Invitrogen), ARAF, BRAF (Santa Cruz Biotechnology),
FLAG-tag (Sigma), CRAF (BD Transduction Laboratories), HA-tag (Covance). For immunoprecipitations
of tagged proteins, the following reagents were used: Anti-V5 agarose affinity gel,
Anti-FLAG M2 affinity gel, anti-c-myc agarose affinity gel (all from Sigma).
Plasmids
Plasmids encoding HA-tagged wild-type and mutant N-RAS were obtained from Biomyx.
Plasmids expressing myc-tagged wild-type BRAF and BRAFV600E were kindly provided by
Dr. Walter Kolch, (University of Glasgow, UK), and were used as template to create
FLAG-tagged constructs. All other plasmids were created using standard cloning methods,
with pcDNA3.1 (Invitrogen) as a vector. Mutations were introduced using site-directed
Mutagenesis Kit (Stratagene). The catalytic domain of CRAF (catC) was created by truncating
the first 305 amino-acids of CRAF.
Immunoprecipitations and Kinase assays
Cells were lysed in lysis buffer (50 mM Tris (pH 7.5), 1% NP40, 150 mM NaCl, 10% glycerol,
1mM EDTA, supplemented with protease and phosphatase inhibitor cocktail tablets (Roche).
Immunoprecipitations were carried out at 4°C for 4 hours, followed by 3 washes with
lysis buffer and, in cases of subsequent kinase assay, one extra wash with kinase
buffer (25mM Tris, pH 7.5, 10mM MgCl2). RAF kinase assays were conducted in the presence
of 100μM ATP, at 30°C for 20 minutes. Recombinant inactive K97R MEK (MIllipore) was
used as a substrate and the reaction was terminated with the addition of sample buffer
and boiling. Kinase activity was estimated by immunoblotting for pMEK.
Transfections
Cells were seeded at 35mm or 100mm plates and transfected the following day using
Lipofectamine 2000 (Invitrogen). 24 hours later, cells were collected for subsequent
analysis.
Supplementary Material
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