Key Points
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The oncoprotein BRAF is frequently activated due to genetic alterations in tumours promoting deregulation of RAF–MEK–ERK signalling. Targeting BRAF with inhibitors is a validated therapeutic strategy for a substantial proportion of cancer patients.
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RAF inhibitors alone or in combination with MEK inhibitors have elicited dramatic responses and prolonged the survival of patients with melanoma whose tumours harbour mutationally activated BRAF-V600. However, their effectiveness is limited by the development of drug resistance, frequently by mechanisms that promote reactivation of RAF–ERK signalling in the presence of the drug.
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In BRAF-V600 tumours other than melanoma, or in tumours carrying BRAF alterations other than the BRAF-V600 mutation, current clinical RAF inhibitors have shown modest effectiveness.
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RAF inhibitors have unique biochemical properties that account for their wide therapeutic window, on-target toxicities and major mechanisms of resistance.
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RAF dimerization is a common mechanism of both intrinsic and acquired resistance to current clinical RAF inhibitors vemurafenib and dabrafenib, which stabilize the αC-helix of RAF kinase in the OUT position. These compounds effectively inhibit monomeric mutant BRAF but fail to inhibit dimeric RAF.
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Structurally, inhibitor resistance due to RAF dimerization is the result of negative allostery for inhibitor binding to the second protomer of the RAF dimer, once the first is bound to an inhibitor.
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Next-generation RAF inhibitors that stabilize the αC-helix of RAF kinase in the active (IN) position will inhibit RAF monomers and dimers, but they are predicted to have a narrow therapeutic window due to inhibition of wild-type BRAF in normal cells. Thus, combinatorial approaches with current clinical inhibitors may be beneficial.
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Paradoxical pathway activation (allosteric priming) is a critical adverse event observed with most RAF inhibitors in the presence of RAS. Its effect on downstream signalling is currently ameliorated with the combined use of MEK inhibitors.
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Several structurally diverse, next-generation RAF inhibitors are under preclinical or clinical development and may be effective in BRAF-mutant tumours that are resistant to current clinical RAF inhibitors.
Abstract
The discovery that a subset of human tumours is dependent on mutationally deregulated BRAF kinase intensified the development of RAF inhibitors to be used as potential therapeutics. The US Food and Drug Administration (FDA)-approved second-generation RAF inhibitors vemurafenib and dabrafenib have elicited remarkable responses and improved survival of patients with BRAF-V600E/K melanoma, but their effectiveness is limited by resistance. Beyond melanoma, current clinical RAF inhibitors show modest efficacy when used for colorectal and thyroid BRAF-V600E tumours or for tumours harbouring BRAF alterations other than the V600 mutation. Accumulated experimental and clinical evidence indicates that the complex biochemical mechanisms of RAF kinase signalling account both for the effectiveness of RAF inhibitors and for the various mechanisms of tumour resistance to them. Recently, a number of next-generation RAF inhibitors, with diverse structural and biochemical properties, have entered preclinical and clinical development. In this Review, we discuss the current understanding of RAF kinase regulation, mechanisms of inhibitor action and related clinical resistance to these drugs. The recent elucidation of critical structural and biochemical aspects of RAF inhibitor action, combined with the availability of a number of structurally diverse RAF inhibitors currently in preclinical and clinical development, will enable the design of more effective RAF inhibitors and RAF-inhibitor-based therapeutic strategies, tailored to different clinical contexts.
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Acknowledgements
The authors are grateful to Bogos Agianian for generating the movies. E.G. is supported by NIH grant R01CA178394 and awards from the Melanoma Research Alliance, the Pershing Square Sohn Cancer Research Alliance, the Irma T. Hirschl Trust, the Gabrielle's Angels Foundation for Cancer Research, and the Sidney Kimmel Foundation for Cancer Research. P.I.P. is supported by NIH grant R01CA204314, the Sidney Kimmel Foundation for Cancer Research, the Melanoma Research Foundation, the Dermatology Foundation and the Melanoma Research Alliance.
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Z.K., E.G. and P.I.P. contributed equally to researching the literature, to writing the article and to reviewing and editing the manuscript before submission.
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The authors declare no competing financial interests.
Supplementary information
Supplementary S1 (movie)
αC-OUT RAF inhibitors fail to inhibit dimeric RAF (9,713 KB) Vemurafenib binds the first protomer within the RAF dimer and stabilizes the αC-helix in the OUT position. However, the same movement is not sterically possible in the second protomer without disrupting the dimer. The αC-helix-IN conformation of the second protomer prevents binding of a second vemurafenib, leading to persistence of cellular RAF activity. (MOV 9713 kb)
Supplementary S2 (movie)
αC-IN RAF inhibitors potently inhibit dimeric RAF (13,593 KB) In contrast to vemurafenib (and other αC-OUT RAF inhibitors), RAF inhibitors that stabilize the αC-helix in the IN position, such as AZ628, occupy and inhibit both protomers within the dimer, thus effectively suppressing cellular RAF activity. (MOV 13593 kb)
Glossary
- Langerhans cell histiocytosis
-
(LCH). A myeloid neoplasia characterized by inflammatory lesions containing pathological dendritic cells, frequently harbouring the BRAF-V600E mutation.
- Protomer
-
The structural unit of an oligomeric protein; in the case of wild-type BRAF, a functional BRAF dimer is composed of two protomers.
- Steric clashes
-
When atoms from different residues come into close proximity, the resultant repulsion between the atoms leads to a change in conformation.
- Negative allostery
-
Also known as allosteric inhibition, occurs when binding of one ligand to a substrate (in this case a BRAF protomer) decreases the affinity of another ligand at a different site (that is, the other protomer).
- Therapeutic window
-
The range of concentrations of a drug in the patient that provides safe effective therapy. The therapeutic window is wide when the minimum toxic concentration is much higher than the minimum effective concentration.
- Allosteric priming
-
A phenomenon recently observed with several small-molecule inhibitors, by which binding of the inhibitor induces the active conformation of the target kinase, which results in its increased interaction with upstream regulators and consequent kinase priming and activating phosphorylation.
- Steric effects
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A phenomenon arising as a result of the fact that each residue and its atoms occupy a certain amount of space in a defined conformation.
- Residence time
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The period for which the target kinase is occupied by the small-molecule inhibitor.
- Off-rate
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The rate of dissociation of the small-molecule inhibitor from the kinase.
- Cross-resistance
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The development of tumour resistance to a potential therapy after treatment of a patient with a different therapeutic agent.
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Karoulia, Z., Gavathiotis, E. & Poulikakos, P. New perspectives for targeting RAF kinase in human cancer. Nat Rev Cancer 17, 676–691 (2017). https://doi.org/10.1038/nrc.2017.79
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DOI: https://doi.org/10.1038/nrc.2017.79
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