In this study, we show that inhibition of BRAF by chemical or genetic means in the presence of oncogenic or growth-factor activated RAS induces BRAF binding to CRAF, leading to CRAF hyperactivation and consequently elevated MEK and ERK signaling. The mechanism we describe is another paradigm of RAF activation downstream of RAS and based on our findings, we propose the following mechanism by which this occurs. We posit that in RAS mutant cells, BRAF maintains itself in an inactive conformation through its own kinase activity, either through auto-phosphorylation, or by phosphorylating a partner protein that then keeps it inactive (A). We are currently using mass-spectrometry and mutagenic approaches to elucidate the underlying mechanism. We propose that when BRAF is inhibited, it escapes this auto-inhibited state and is recruited to the plasma membrane by RAS, where it forms a stable complex with CRAF. Critically, we posit that because it is inhibited, BRAF does not directly phosphorylate MEK, but rather it acts as a scaffold whose function is to enhance CRAF activation, thereby allowing CRAF to hyperactivate the pathway (B). We do not know the stoichiometry of the components in these complexes, but since BRAF and CRAF must both bind to RAS for complex formation, it seems likely that at least two RAS proteins are needed to stimulate formation of the complex (B).
A Model of Paradoxical CRAF Activation by BRAF
It is unclear why PLX4720 only induces weak binding of BRAF to CRAF, but this may stem from its unique property of displacing the α-C helix of BRAF when it binds (Tsai et al., 2008
) and suggests that this helix is important for BRAF binding to CRAF, something that will only be resolved when the BRAF:CRAF crystal structure is solved. We have attempted to identify other proteins that may be required to stabilize the BRAF–CRAF complexes. Our unpublished mutagenesis data suggests that 14-3-3 is required to stabilize these drug-induced complexes (data not shown) and this is consistent with previous observations demonstrating that 14-3-3 mediates BRAF binding to CRAF (Garnett et al., 2005; Rushworth et al., 2006
). Although this appears to contradict our observation that dephosphorylation does not disrupt the complex, because 14-3-3 binds to BRAF and CRAF in a phosphorylation-dependent manner, we presume that 14-3-3 protects these sites from dephosphorylation. We have also used RNAi to examine the potential role of other proteins implicated in BRAF-CRAF complex formation or pathway activation, including the scaffold proteins KSR, Sprouty2 and RKTG and the small G protein RHEB, but our preliminary results have not revealed obvious roles for these proteins. Our studies have parallels to the recently described heterodimers between DRAF and KSR in Drosophila
(Rajakulendran et al., 2009
). Notably, flies have only one RAF isoform and it appears to be an ortholog of BRAF rather than ARAF or CRAF. Our inability to demonstrate an obvious role for KSR in mediating BRAF binding to CRAF or CRAF activation by BRAF suggests that the mechanism underlying dimerization here may be different from those described in flies, but clearly additional studies are required to investigate further the role of scaffold proteins in mediating the phenomena we report.
In contrast to the BRAF-selective inhibitors, the pan-RAF inhibitors appear to induce paradoxical activation of CRAF. They induce BRAF binding to CRAF and CRAF activation, but do not activate MEK–ERK signaling. We posit that this is because these agents target both BRAF and CRAF. Thus, although their inhibition of BRAF will stimulate CRAF activation, they will simultaneously inhibit CRAF (C). This model is supported by our observation that T421N
CRAF converts sorafenib from a pathway inhibitor to a pathway activator and we argue that the paradoxical activation of CRAF by these inhibitors is mediated by BRAF, rather than disrupted feedback inhibition as previously suggested (Hall-Jackson et al., 1999
Recently, paradoxical activation of PKB/AKT and PKC
was also described (Cameron et al., 2009; Okuzumi et al., 2009
). While ATP-competitive inhibition can block kinase function, they do not block the upstream events that activate the target kinase. For instance, PKB/AKT inhibitors block the function of this kinase, but occupation of the ATP-pocket by these inhibitors was sufficient to induce the priming phosphorylation usually required for its full activation (Okuzumi et al., 2009
). Inhibitor binding to PKC
has been shown to have a similar effect (Cameron et al., 2009
). Importantly, the paradoxical activation of PKB/AKT and PKC
did not result in pathway activation because of the continued presence of the inhibitors (Frye and Johnson, 2009
). In contrast, although BRAF inhibitors also block BRAF kinase activity, this relieves auto-inhibition and results in BRAF hyperphosphorylation, BRAF binding to CRAF, pathway activation and oncogenesis, all presumably because BRAF can heterodimerize with CRAF. Our study also highlights the critical difference between BRAF-selective and pan-RAF drugs. Whereas BRAF-selective drugs cause pathway activation in a RAS-dependent manner, this does not occur with pan-RAF drugs.
Our results provide important insight into the genetics of human cancer. Excluding V600 mutants, D594 mutants are the third most common in BRAF in cancer (34 out of 443 cases or ~7.7%; www.sanger.ac.uk/genetics/CGP/cosmic/
). Furthermore, as mentioned in the Introduction, while BRAF
mutations are generally mutually exclusive, 4 of the 34 (11.8%) tumors with D594 mutations also have mutations in RAS. This is a highly significant enrichment for the coincidence of these mutations (p < 10−9
; Fisher's Exact Test) and suggests a functional interaction. We now provide strong circumstantial evidence of such an interaction using transgenic mice. By themselves, D594A
Braf and G12D
Kras do not induce melanoma, but they cooperate to induce rapid onset melanoma. This highly significant result (p < 0.0002) provides a rational explanation for the coincidence of these mutations in human cancer. Furthermore, we show that the BRAF inhibitors also hyperactivate this pathway in growth factor stimulated cells, providing an explanation of why kinase dead BRAF mutations are not always coincident with RAS mutations; presumably in some tumors the cooperating mutation is upstream of RAS.
Our results also suggest several potential mechanisms by which resistance to RAF targeting drugs could develop in patients. BRAF mutant tumors could become resistant to BRAF-selective drugs, if they acquire a mutation in RAS or an upstream component that activates RAS, or if the drugs select a population of cells harboring pre-existing mutations in RAS. Theoretically this would cause BRAF-mediated CRAF activation, which may not only induce resistance, but could potentially promote tumor growth. In line with this, increased expression of CRAF can mediate acquired resistance to pan-RAF drugs in BRAF mutant cancer cells in vitro (Montagut et al., 2008
), establishing that CRAF can mediate resistance under some circumstances. Our in vitro studies also suggest that a potential mechanism of resistance in patients with RAS mutant tumors being treated with pan-RAF drugs is acquisition (or selection for cells with pre-existing mutations) of a CRAF mutation such as a gatekeeper mutant that prevents drug binding. Again this would potentially result in BRAF-mediated activation of CRAF (D) and possibly accelerated tumor growth.
Although our studies are restricted to cell lines and transgenic mice, they do have important immediate clinical implications. They strongly argue that BRAF-selective inhibitors should not be administered to patients with RAS mutant tumors, because long-term use could accelerate tumor growth. Intriguingly, 10%–15% of patients treated with BRAF-selective drugs develop squamous cell carcinoma (SCC)(Flaherty et al., 2009; Schwartz et al., 2009
). Although MEK–ERK signaling has not yet been implicated in this response, 22% of SCCs harbour oncogenic mutations in RAS
, 8% NRAS
, 5% KRAS
), raising the intriguing possibility that the BRAF-selective drugs act as tumor promoters in premalignant skin cells harboring existing mutations in RAS and/or activation of upstream components that activate RAS.
While sorafenib is equipotent for wild-type and V600E
BRAF (Wilhelm et al., 2004
), the BRAF inhibitors we used are approximately 10-fold more active against V600E
BRAF (King et al., 2006; Tsai et al., 2008
). Nevertheless, our data establish that they target wild-type BRAF in RAS mutant cells. The problem of mutant v.s. wild-type protein specificity is likely to be difficult to resolve, because whereas full inhibition of V600E
BRAF may be necessary for clinical response in BRAF mutant tumors, activation of only a small proportion of wild-type BRAF could be sufficient to activate the pathway in RAS mutant cells. Thus, to achieve efficacy against V600E
BRAF but avoid activation of wild-type BRAF in RAS mutant cells, the drugs will need to be exquisitely selective for the mutant protein. Alternatively, pan-RAF drugs may be effective because they will target both V600E
BRAF and CRAF activated by BRAF in RAS mutant tumors. Furthermore, our data suggest that CRAF or MEK selective drugs should be used in RAS mutant tumors, because they do not induce BRAF-CRAF complexes and will not activate the pathway if the tumors acquire mutations such as T421N
CRAF that block drug binding. Perhaps RAF and MEK inhibitors should be combined to provide the best responses and prevent emergence of resistance, but these issues need to be balanced against the urgency of the clinical problem being addressed.
In summary, we show that inhibition of BRAF in RAS mutant cancer cells leads to MEK hyperactivation through CRAF. We have elucidated another mechanism by which BRAF activates MEK–ERK signaling, not only to drive tumorigenesis and tumor progression, but also potentially to allow development of de novo or acquired resistance to RAF-targeted therapies. Clearly, BRAF is a remarkably versatile oncogene that can promote MEK–ERK activation and tumor progression through several mechanisms and these will require different therapeutic strategies for effective disease management. Notably, many of the mutations that occur in other kinases in cancer are also predicted to cause inactivation (www.sanger.ac.uk/genetics/CGP/cosmic/
). Our data raise the possibility that these could also act as idiosyncratic gain-of-function mutations that drive tumorigenesis. This study also raises important clinical questions and highlights the importance of fully understanding how signaling networks function to fully comprehend how patients may respond to targeted drugs. They also highlight the importance of genetic screening for patients, not only to identify those who are likely to respond, but to exclude those who could experience adverse effects and thereby ensure successful implementation of personalized medicine.