title: 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.
authors.affiliation: Program in Molecular Pharmacology and Chemistry and Department of Medicine, Memorial Sloan-Kettering Cancer Center, New York, New York 10065, USA.
authors.forename: Poulikos I
authors.initials: PI
authors.lastname: Poulikakos
authors.forename: Chao
authors.initials: C
authors.lastname: Zhang
authors.forename: Gideon
authors.initials: G
authors.lastname: Bollag
authors.forename: Kevan M
authors.initials: KM
authors.lastname: Shokat
authors.forename: Neal
authors.initials: N
authors.lastname: Rosen
mesh_terms: D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D020134:Catalytic Domain; D002460:Cell Line; D045744:Cell Line, Tumor; D004789:Enzyme Activation / Q000187:drug effects; D048049:Extracellular Signal-Regulated MAP Kinases / Q000378:metabolism*; D006801:Humans; D007211:Indoles / Q000494:pharmacology; D020935:MAP Kinase Signaling System / Q000187:drug effects*; D051379:Mice; D020929:Mitogen-Activated Protein Kinase Kinases / Q000378:metabolism; D008954:Models, Biological; D009369:Neoplasms / Q000188:drug therapy / Q000201:enzymology / Q000235:genetics / Q000378:metabolism; D010766:Phosphorylation; D011485:Protein Binding; D047428:Protein Kinase Inhibitors / Q000378:metabolism / Q000494:pharmacology* / Q000627:therapeutic use; D055503:Protein Multimerization; D048493:Proto-Oncogene Proteins B-raf / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D013449:Sulfonamides / Q000494:pharmacology; D015533:Transcriptional Activation / Q000187:drug effects*; D048490:raf Kinases / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D018631:ras Proteins / Q000235:genetics / Q000378:metabolism
chemical_list: D007211:Indoles; C528407:PLX 4720; D047428:Protein Kinase Inhibitors; D013449:Sulfonamides; D000255:Adenosine Triphosphate; D048493:Proto-Oncogene Proteins B-raf; D048490:raf Kinases; D048049:Extracellular Signal-Regulated MAP Kinases; D020929:Mitogen-Activated Protein Kinase Kinases; D018631:ras Proteins