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. Author manuscript; available in PMC: 2019 Jun 5.
Published in final edited form as: J Invest Dermatol. 2010 Jun 24;130(11):2669–2671. doi: 10.1038/jid.2010.177

The Wrath of RAFs: Rogue Behavior of B-RAF Kinase Inhibitors

Fred M Kaplan 1, Michael J Mastrangelo 2,3, Andrew E Aplin 1,3
PMCID: PMC6550481  NIHMSID: NIHMS1028575  PMID: 20574441

TO THE EDITOR

The melanoma field has been invigorated by the recent presentation of phase I clinical trial results using an inhibitor, PLX4032, that elicits mutant B-RAF selective effects (Flaherty et al., 2009). The majority of mutant B-RAF melanoma patients who were administered PLX4032 showed clinical responses within 2 weeks and experienced an approximately 7–8 month progression-free survival benefit. In a field with a dearth of effective treatments for advanced disease, the rapid progression from identification of B-RAF mutations in 2002 (Davies et al., 2002) to development of a clinically effective drug against mutant B-RAF is a strong argument for the development of targeted therapies. However, a series of recent publications have uncovered novel mechanisms that paradoxically activate the RAF pathway in the presence of clinically relevant B-RAF inhibitors (Halaban et al., 2010; Hatzivassiliou et al., 2010; Heidorn et al., 2010; Poulikakos et al., 2010). Furthermore, they highlight that patient selection is likely to be critical to prevent adverse effects of RAF inhibitors in a subset of melanoma patients.

In the canonical receptor tyrosine kinase signaling pathway, RAF serine/threonine kinases are recruited to the membrane by RAS and activated by phosphorylation. Three RAF isoenzymes exist: A-RAF, B-RAF, and C-RAF. RAFs form both homodimers and heterodimers but, notably, it is the heterodimer complex that exhibits increased activity even when one of the RAF protomers in the complex is kinase-dead (Rushworth et al., 2006; Ritt et al., 2010). RAFs activate the MAPK/ERK kinase (MEK)/extracellular signal-regulated kinase 1/2 (ERK1/2) pathway, which promotes proliferation, migration, and survival in tumor cells (Michaloglou et al., 2008). B-RAF mutations are found in approximately 50% of melanomas; the most frequent mutation encoding a valine to glutamic acid substitution at amino-acid 600 (B-RAFV600E) results in a constitutively active B-RAF kinase (Davies et al., 2002). An inhibitor, PLX4720 (a close structural analog of PLX4032), potently inhibits the growth of B-RAFV600E melanoma cells in vitro and in tumor xenograft models (Tsai et al., 2008).

Activating RAS mutations are present in approximately 15–25% of melanomas (N-RAS 20%, K-RAS 2%) in a mutually exclusive manner to B-RAFV600E mutations (http://www.sanger.ac.uk/genetics/CGP/cosmic). Four recent papers show that several structurally distinct B-RAF inhibitors including PLX4032/4720 induce a paradoxical activation of MEK/ERK1/2 signaling in mutant N-RAS melanoma cells (Halaban et al., 2010; Hatzivassiliou et al., 2010; Heidorn et al., 2010; Poulikakos et al., 2010). Similar effects are also observed in some wild-type B-RAF/wild-type RAS melanoma cells, presumably due to high basal levels of active RAS, and in mutant K-RAS cell lines. Activation of RAF signaling by RAF inhibitors has been observed previously (Hall-Jackson et al., 1999; King et al., 2006), but now the underlying mechanisms have been delineated.

The overarching model is that GTP-loaded RAS promotes RAF dimerization and that, within RAF dimer complexes, a drug-inactivated RAF isoenzyme transactivates a C-RAF partner. The activated C-RAF partner, in turn, phosphorylates and activates the MEK/ERK1/2 pathway (Figure 1). Some differences in the underlying mechanisms between the studies are described, most notably the target isoform of the RAF inhibitor. Conversely, Poulikakos et al. (2010) and Hatzivassiliou et al. (2010) show that PLX4720/4032 activation occurs through the formation of C-RAF/C-RAF dimers and can occur in the absence of B-RAF. By contrast in the Heidorn et al. (2010) model, C-RAF is activated by an inactive B-RAF. Consistent with this second model, a naturally occurring kinase-deficient B-RAF mutant (B-RAFD594V), which is found in a small subset of melanomas, interacts with C-RAF and activates MEK/ERK1/2 signaling. The differences are based on results obtained with “gatekeeper” mutations that sterically prevent inhibitor binding to the active site in RAF. Poulikakos et al. (2010) and Hatzivassiliou et al. (2010) show that the “gate-keeper” threonine 421 to asparagine of C-RAF (C-RAFT421N) prevents the cross-activation of C-RAF by preventing the drug-induced translocation of C-RAF to the plasma membrane. Alternatively, Heidorn et al. (2010) show that ERK1/2 activation is prevented by a gatekeeper mutation in B-RAF (B-RAFT529N). Alikely difference may be related to the specific drugs used. PLX4720 induces a shift in the aC-helix of B-RAF and actually destabilizes the interaction between B-RAF and C-RAF (Hatzivassiliou et al., 2010). In agreement with this, Halaban et al. (2010) did not detect B-RAF/C-RAF heterodimers in the presence of PLX4032. By contrast, other ATP competitive inhibitors, such as 885-A and GDC-0879, stabilized the interaction between B-RAF and C-RAF (Hatzivassiliou et al., 2010; Heidorn et al., 2010). It is noteworthy that although PLX4032/4720 was originally described as a selective mutant B-RAF inhibitor, recent analysis shows it also inhibits both C-RAF and A-RAF in in vitro kinase assays (Hatzivassiliou et al., 2010; Poulikakos et al., 2010). The mutant selective effects observed in cells and patients are likely due to the lower affinity of mutant B-RAF for ATP compared to wild-type forms of B-RAF and C-RAF (Hatzivassiliou et al., 2010).

Figure 1. Model figure for B-RAF inhibitor-mediated activation of the C-RAF/MEK/ERK pathway in non-mutant B-RAF melanoma cells.

Figure 1.

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In wild-type and mutant N-RAS cells, B-RAF and C-RAF are recruited to the plasma membrane and associate with activated RAS (RAS-GTP). Formation of B-RAF/C-RAF heterodimers or C-RAF/C-RAF homodimers leads to activation of the MEK/ERK1/2 pathway. Treatment with ATP-competitive RAF inhibitors promotes the formation of RAF dimers. In one scenario, binding of the RAF inhibitor to B-RAF leads to the formation of B-RAF/C-RAF heterodimers that contains an inactivated B-RAF and a hyperactivated C-RAF. In the second scenario, binding of the RAF inhibitor to C-RAF results in the formation of C-RAF/C-RAF homodimers that contain one inactivated C-RAF and one hyperactivated C-RAF. In either scenario, MEK/ERK1/2 signaling is hyperactivated. We note that some experiments with PLX4032/4720 indicate RAS-independent mechanisms of activation, adding further complexity to this model.

A second difference is the involvement of RAS. Hatzivassiliou et al. (2010) and Heidorn et al. (2010) use knockdown and dominant negative approaches to show that the 885-A-and GDC-0879-induced activation of MEK is dependent on RAS. Although Poulikakos et al. (2010) show that mutant RAS enhances PLX4032-induced activation of MEK, they additionally show that an N-terminal deletion mutation of C-RAF, which is independent of RAS, is activated by PLX4032/4720. Similarly, Halaban et al. (2010) show that C-RAF with a mutation in the RAS binding domain remains activatable by PLX4032. These studies again point to subtle differences in the mechanisms of activation between the inhibitors used and indicate that both RAS-dependent and RAS-independent mechanisms likely contribute to PLX4032/4720-induced activation.

Although mechanistic details differ, the underlying message is that in the wrong genotypic setting, B-RAF inhibitors lead to inappropriate activation of MEK/ERK1/2 signaling. This inappropriate signaling has some profound effects on cellular homeostasis. Several wild-type B-RAF and RAS or wild-type B-RAF and mutant RAS cell lines showed hyperproliferation upon treatment with inhibitors PLX4720 and GDC-8079 (Hatzivassiliou et al., 2010). In early stage primary melanomas, treatment with PLX4720/4032 resulted in increased cellular motility (Halaban et al., 2010). Furthermore, the progression to melanoma was observed in melanocytes from transgenic mice expressing both kinase-defective B-RAF (B-RAFD954A) and constitutively activated KRAS (KRASG12D) (Heidorn et al., 2010).

Overall, these studies emphasize the importance of genotypically stratifying melanoma patients before enrollment on B-RAF inhibitor trials to reduce unwanted side effects. The inappropriate activation of RAF kinase activity provides a mechanistic basis for why approximately 20% of patients enrolled in clinical trials for PLX4032 developed keratoacanthomas and squamous cells carcinomas (Flaherty et al., 2009). They also provide guidance to the design of novel therapeutics to inhibit oncogenic B-RAF kinase activity with reduced side effects to the patients.

ACKNOWLEDGMENTS

The work in the Aplin Lab was supported by grants from the National Institutes of Health (GM067893, CA125103), the American Cancer Society (RSG-08-03-01-CSM), and the Pennsylvania Department of Health (AF0301). We thank Dr Michele Weiss for the figure illustration.

Abbreviations:

ERK1/2

extracellular signal-regulated kinase 1/2

MEK

MAPK/ERK kinase

Footnotes

CONFLICT OF INTEREST

The authors state no conflict of interest.

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