Allosteric activation of functionally asymmetric RAF kinase dimers.

Although RAF kinases are critical for controlling cell growth, their mechanism of activation is incompletely understood. Recently, dimerization was shown to be important for activation. Here we show that the dimer is functionally asymmetric with one kinase functioning as an activator to stimulate activity of the partner, receiver kinase. The activator kinase did not require kinase activity but did require N-terminal phosphorylation that functioned allosterically to induce cis-autophosphorylation of the receiver kinase. Based on modeling of the hydrophobic spine assembly, we also engineered a constitutively active mutant that was independent of Ras, dimerization, and activation-loop phosphorylation. As N-terminal phosphorylation of BRAF is constitutive, BRAF initially functions to activate CRAF. N-terminal phosphorylation of CRAF was dependent on MEK, suggesting a feedback mechanism and explaining a key difference between BRAF and CRAF. Our work illuminates distinct steps in RAF activation that function to assemble the active conformation of the RAF kinase.

Regulation of RAF protein kinases in ERK signalling.

RAF family kinases were among the first oncoproteins to be described more than 30 years ago. They primarily act as signalling relays downstream of RAS, and their close ties to cancer have fuelled a large number of studies. However, we still lack a systems-level understanding of their regulation and mode of action. The recent discovery that the catalytic activity of RAF depends on an allosteric mechanism driven by kinase domain dimerization is providing a vital new piece of information towards a comprehensive model of RAF function. The fact that current RAF inhibitors unexpectedly induce ERK signalling by stimulating RAF dimerization also calls for a deeper structural characterization of this family of kinases.

ARAF mutations confer resistance to the RAF inhibitor belvarafenib in melanoma.

Although RAF monomer inhibitors (type I.5, BRAF(V600)) are clinically approved for the treatment of BRAFV600-mutant melanoma, they are ineffective in non-BRAFV600 mutant cells1-3. Belvarafenib is a potent and selective RAF dimer (type II) inhibitor that exhibits clinical activity in patients with BRAFV600E- and NRAS-mutant melanomas. Here we report the first-in-human phase I study investigating the maximum tolerated dose, and assessing the safety and preliminary efficacy of belvarafenib in BRAFV600E- and RAS-mutated advanced solid tumours (NCT02405065, NCT03118817). By generating belvarafenib-resistant NRAS-mutant melanoma cells and analysing circulating tumour DNA from patients treated with belvarafenib, we identified new recurrent mutations in ARAF within the kinase domain. ARAF mutants conferred resistance to belvarafenib in both a dimer- and a kinase activity-dependent manner. Belvarafenib induced ARAF mutant dimers, and dimers containing mutant ARAF were active in the presence of inhibitor. ARAF mutations may serve as a general resistance mechanism for RAF dimer inhibitors as the mutants exhibit reduced sensitivity to a panel of type II RAF inhibitors. The combination of RAF plus MEK inhibition may be used to delay ARAF-driven resistance and suggests a rational combination for clinical use. Together, our findings reveal specific and compensatory functions for the ARAF isoform and implicate ARAF mutations as a driver of resistance to RAF dimer inhibitors.

Structural basis for the action of the drug trametinib at KSR-bound MEK.

The MAPK/ERK kinase MEK is a shared effector of the frequent cancer drivers KRAS and BRAF that has long been pursued as a drug target in oncology1, and more recently in immunotherapy2,3 and ageing4. However, many MEK inhibitors are limited owing to on-target toxicities5-7 and drug resistance8-10. Accordingly, a molecular understanding of the structure and function of MEK within physiological complexes could provide a template for the design of safer and more effective therapies. Here we report X-ray crystal structures of MEK bound to the scaffold KSR (kinase suppressor of RAS) with various MEK inhibitors, including the clinical drug trametinib. The structures reveal an unexpected mode of binding in which trametinib directly engages KSR at the MEK interface. In the bound complex, KSR remodels the prototypical allosteric pocket of the MEK inhibitor, thereby affecting binding and kinetics, including the drug-residence time. Moreover, trametinib binds KSR-MEK but disrupts the related RAF-MEK complex through a mechanism that exploits evolutionarily conserved interface residues that distinguish these sub-complexes. On the basis of these insights, we created trametiglue, which limits adaptive resistance to MEK inhibition by enhancing interfacial binding. Our results reveal the plasticity of an interface pocket within MEK sub-complexes and have implications for the design of next-generation drugs that target the RAS pathway.

Regulation of RAF family kinases: new insights from recent structural and biochemical studies.

The RAF kinases are required for signal transduction through the RAS-RAF-MEK-ERK pathway, and their activity is frequently up-regulated in human cancer and the RASopathy developmental syndromes. Due to their complex activation process, developing drugs that effectively target RAF function has been a challenging endeavor, highlighting the need for a more detailed understanding of RAF regulation. This review will focus on recent structural and biochemical studies that have provided 'snapshots' into the RAF regulatory cycle, revealing structures of the autoinhibited BRAF monomer, active BRAF and CRAF homodimers, as well as HSP90/CDC37 chaperone complexes containing CRAF or BRAFV600E. In addition, we will describe the insights obtained regarding how BRAF transitions between its regulatory states and examine the roles that various BRAF domains and 14-3-3 dimers play in both maintaining BRAF as an autoinhibited monomer and in facilitating its transition to an active dimer. We will also address the function of the HSP90/CDC37 chaperone complex in stabilizing the protein levels of CRAF and certain oncogenic BRAF mutants, and in serving as a platform for RAF dephosphorylation mediated by the PP5 protein phosphatase. Finally, we will discuss the regulatory differences observed between BRAF and CRAF and how these differences impact the function of BRAF and CRAF as drivers of human disease.

Raf promotes dimerization of the Ras G-domain with increased allosteric connections.

Ras dimerization is critical for Raf activation. Here we show that the Ras binding domain of Raf (Raf-RBD) induces robust Ras dimerization at low surface densities on supported lipid bilayers and, to a lesser extent, in solution as observed by size exclusion chromatography and confirmed by SAXS. Community network analysis based on molecular dynamics simulations shows robust allosteric connections linking the two Raf-RBD D113 residues located in the Galectin scaffold protein binding site of each Raf-RBD molecule and 85 Å apart on opposite ends of the dimer complex. Our results suggest that Raf-RBD binding and Ras dimerization are concerted events that lead to a high-affinity signaling complex at the membrane that we propose is an essential unit in the macromolecular assembly of higher order Ras/Raf/Galectin complexes important for signaling through the Ras/Raf/MEK/ERK pathway.

Small molecule stabilization of the KSR inactive state antagonizes oncogenic Ras signalling.

Deregulation of the Ras-mitogen activated protein kinase (MAPK) pathway is an early event in many different cancers and a key driver of resistance to targeted therapies. Sustained signalling through this pathway is caused most often by mutations in K-Ras, which biochemically favours the stabilization of active RAF signalling complexes. Kinase suppressor of Ras (KSR) is a MAPK scaffold that is subject to allosteric regulation through dimerization with RAF. Direct targeting of KSR could have important therapeutic implications for cancer; however, testing this hypothesis has been difficult owing to a lack of small-molecule antagonists of KSR function. Guided by KSR mutations that selectively suppress oncogenic, but not wild-type, Ras signalling, we developed a class of compounds that stabilize a previously unrecognized inactive state of KSR. These compounds, exemplified by APS-2-79, modulate KSR-dependent MAPK signalling by antagonizing RAF heterodimerization as well as the conformational changes required for phosphorylation and activation of KSR-bound MEK (mitogen-activated protein kinase kinase). Furthermore, APS-2-79 increased the potency of several MEK inhibitors specifically within Ras-mutant cell lines by antagonizing release of negative feedback signalling, demonstrating the potential of targeting KSR to improve the efficacy of current MAPK inhibitors. These results reveal conformational switching in KSR as a druggable regulator of oncogenic Ras, and further suggest co-targeting of enzymatic and scaffolding activities within Ras-MAPK signalling complexes as a therapeutic strategy for overcoming Ras-driven cancers.

SHOC2 complex-driven RAF dimerization selectively contributes to ERK pathway dynamics.

Despite the crucial role of RAF kinases in cell signaling and disease, we still lack a complete understanding of their regulation. Heterodimerization of RAF kinases as well as dephosphorylation of a conserved "S259" inhibitory site are important steps for RAF activation but the precise mechanisms and dynamics remain unclear. A ternary complex comprised of SHOC2, MRAS, and PP1 (SHOC2 complex) functions as a RAF S259 holophosphatase and gain-of-function mutations in SHOC2, MRAS, and PP1 that promote complex formation are found in Noonan syndrome. Here we show that SHOC2 complex-mediated S259 RAF dephosphorylation is critically required for growth factor-induced RAF heterodimerization as well as for MEK dissociation from BRAF. We also uncover SHOC2-independent mechanisms of RAF and ERK pathway activation that rely on N-region phosphorylation of CRAF. In DLD-1 cells stimulated with EGF, SHOC2 function is essential for a rapid transient phase of ERK activation, but is not required for a slow, sustained phase that is instead driven by palmitoylated H/N-RAS proteins and CRAF. Whereas redundant SHOC2-dependent and -independent mechanisms of RAF and ERK activation make SHOC2 dispensable for proliferation in 2D, KRAS mutant cells preferentially rely on SHOC2 for ERK signaling under anchorage-independent conditions. Our study highlights a context-dependent contribution of SHOC2 to ERK pathway dynamics that is preferentially engaged by KRAS oncogenic signaling and provides a biochemical framework for selective ERK pathway inhibition by targeting the SHOC2 holophosphatase.

Inhibition of RAF dimers: it takes two to tango.

The RAS-regulated RAF-MEK1/2-ERK1/2 pathway promotes cell proliferation and survival and RAS and BRAF proteins are commonly mutated in cancer. This has fuelled the development of small molecule kinase inhibitors including ATP-competitive RAF inhibitors. Type I and type I½ ATP-competitive RAF inhibitors are effective in BRAFV600E/K-mutant cancer cells. However, in RAS-mutant cells these compounds instead promote RAS-dependent dimerisation and paradoxical activation of wild-type RAF proteins. RAF dimerisation is mediated by two key regions within each RAF protein; the RKTR motif of the αC-helix and the NtA-region of the dimer partner. Dimer formation requires the adoption of a closed, active kinase conformation which can be induced by RAS-dependent activation of RAF or by the binding of type I and I½ RAF inhibitors. Binding of type I or I½ RAF inhibitors to one dimer partner reduces the binding affinity of the other, thereby leaving a single dimer partner uninhibited and able to activate MEK. To overcome this paradox two classes of drug are currently under development; type II pan-RAF inhibitors that induce RAF dimer formation but bind both dimer partners thus allowing effective inhibition of both wild-type RAF dimer partners and monomeric active class I mutant RAF, and the recently developed "paradox breakers" which interrupt BRAF dimerisation through disruption of the αC-helix. Here we review the regulation of RAF proteins, including RAF dimers, and the progress towards effective targeting of the wild-type RAF proteins.

Effects of Raf dimerization and its inhibition on normal and disease-associated Raf signaling.

Raf kinases are essential for normal Ras-Raf-MEK-ERK pathway signaling, and activating mutations in components of this pathway are associated with a variety of human cancers, as well as the related developmental disorders Noonan, LEOPARD, and cardiofaciocutaneous syndromes. Although the Raf kinases are known to dimerize during normal and disease-associated Raf signaling, the functional significance of Raf dimerization has not been fully elucidated. Here, using mutational analysis and a peptide inhibitor, we show that dimerization is required for normal Ras-dependent Raf activation and for the biological function of disease-associated Raf mutants with moderate, low, or impaired kinase activity. However, dimerization is not needed for the function of B-Raf mutants with high catalytic activity, such as V600E-B-Raf. Importantly, we find that a dimer interface peptide can effectively block Raf dimerization and inhibit Raf signaling when dimerization is required for Raf function, thus identifying the Raf dimer interface as a therapeutic target.

An Effective MM/GBSA Protocol for Absolute Binding Free Energy Calculations: A Case Study on SARS-CoV-2 Spike Protein and the Human ACE2 Receptor.

The binding free energy calculation of protein-ligand complexes is necessary for research into virus-host interactions and the relevant applications in drug discovery. However, many current computational methods of such calculations are either inefficient or inaccurate in practice. Utilizing implicit solvent models in the molecular mechanics generalized Born surface area (MM/GBSA) framework allows for efficient calculations without significant loss of accuracy. Here, GBNSR6, a new flavor of the generalized Born model, is employed in the MM/GBSA framework for measuring the binding affinity between SARS-CoV-2 spike protein and the human ACE2 receptor. A computational protocol is developed based on the widely studied Ras-Raf complex, which has similar binding free energy to SARS-CoV-2/ACE2. Two options for representing the dielectric boundary of the complexes are evaluated: one based on the standard Bondi radii and the other based on a newly developed set of atomic radii (OPT1), optimized specifically for protein-ligand binding. Predictions based on the two radii sets provide upper and lower bounds on the experimental references: -14.7(ΔGbindBondi)<-10.6(ΔGbindExp.)<-4.1(ΔGbindOPT1) kcal/mol. The consensus estimates of the two bounds show quantitative agreement with the experiment values. This work also presents a novel truncation method and computational strategies for efficient entropy calculations with normal mode analysis. Interestingly, it is observed that a significant decrease in the number of snapshots does not affect the accuracy of entropy calculation, while it does lower computation time appreciably. The proposed MM/GBSA protocol can be used to study the binding mechanism of new variants of SARS-CoV-2, as well as other relevant structures.

Conformational Plasticity of Cyclic Ras-Inhibitor Peptides Defines Cell Permeabilization Activity.

Cyclorasins 9A5 and 9A54 are 11-mer cyclic peptides that inhibit the Ras-Raf protein interaction. The peptides share a cell-penetrating peptide (CPP)-like motif; however, only cyclorasin 9A5 can permeabilize cells to exhibit strong cell-based activity. To unveil the structural origin underlying their distinct cellular permeabilization activities, we compared the three-dimensional structures of cyclorasins 9A5 and 9A54 in water and in the less polar solvent dimethyl sulfoxide (DMSO) by solution NMR. We found that cyclorasin 9A5 changes its extended conformation in water to a compact amphipathic structure with converged aromatic residues surrounded by Arg residues in DMSO, which might contribute to its cell permeabilization activity. However, cyclorasin 9A54 cannot adopt this amphipathic structure, due to the steric hindrance between two neighboring bulky amino-acid sidechains, Tle-2 and dVal-3. We also found that the bulkiness of the sidechains at positions 2 and 3 negatively affects the cell permeabilization activities, indicating that the conformational plasticity that allows the peptides to form the amphipathic structure is important for their cell permeabilization activities.

Metabolic stress regulates ERK activity by controlling KSR-RAF heterodimerization.

Altered cell metabolism is a hallmark of cancer, and targeting specific metabolic nodes is considered an attractive strategy for cancer therapy. In this study, we evaluate the effects of metabolic stressors on the deregulated ERK pathway in melanoma cells bearing activating mutations of the NRAS or BRAF oncogenes. We report that metabolic stressors promote the dimerization of KSR proteins with CRAF in NRAS-mutant cells, and with oncogenic BRAF in BRAFV600E-mutant cells, thereby enhancing ERK pathway activation. Despite this similarity, the two genomic subtypes react differently when a higher level of metabolic stress is induced. In NRAS-mutant cells, the ERK pathway is even more stimulated, while it is strongly downregulated in BRAFV600E-mutant cells. We demonstrate that this is caused by the dissociation of mutant BRAF from KSR and is mediated by activated AMPK. Both types of ERK regulation nevertheless lead to cell cycle arrest. Besides studying the effects of the metabolic stressors on ERK pathway activity, we also present data suggesting that for efficient therapies of both genomic melanoma subtypes, specific metabolic targeting is necessary.

New tools for carbohydrate sulfation analysis: heparan sulfate 2-O-sulfotransferase (HS2ST) is a target for small-molecule protein kinase inhibitors.

Sulfation of carbohydrate residues occurs on a variety of glycans destined for secretion, and this modification is essential for efficient matrix-based signal transduction. Heparan sulfate (HS) glycosaminoglycans control physiological functions ranging from blood coagulation to cell proliferation. HS biosynthesis involves membrane-bound Golgi sulfotransferases, including HS 2-O-sulfotransferase (HS2ST), which transfers sulfate from the cofactor PAPS (3'-phosphoadenosine 5'-phosphosulfate) to the 2-O position of α-l-iduronate in the maturing polysaccharide chain. The current lack of simple non-radioactive enzyme assays that can be used to quantify the levels of carbohydrate sulfation hampers kinetic analysis of this process and the discovery of HS2ST inhibitors. In the present paper, we describe a new procedure for thermal shift analysis of purified HS2ST. Using this approach, we quantify HS2ST-catalysed oligosaccharide sulfation using a novel synthetic fluorescent substrate and screen the Published Kinase Inhibitor Set, to evaluate compounds that inhibit catalysis. We report the susceptibility of HS2ST to a variety of cell-permeable compounds in vitro, including polyanionic polar molecules, the protein kinase inhibitor rottlerin and oxindole-based RAF kinase inhibitors. In a related study, published back-to-back with the present study, we demonstrated that tyrosyl protein sulfotranferases are also inhibited by a variety of protein kinase inhibitors. We propose that appropriately validated small-molecule compounds could become new tools for rapid inhibition of glycan (and protein) sulfation in cells, and that protein kinase inhibitors might be repurposed or redesigned for the specific inhibition of HS2ST.

Targeting the Raf kinases in human cancer: the Raf dimer dilemma.

The Raf protein kinases are key intermediates in cellular signal transduction, functioning as direct effectors of the Ras GTPases and as the initiating kinases in the ERK cascade. In human cancer, Raf activity is frequently dysregulated due to mutations in the Raf family member B-Raf or to alterations in upstream Raf regulators, including Ras and receptor tyrosine kinases. First-generation Raf inhibitors, such as vemurafenib and dabrafenib, have yielded dramatic responses in malignant melanomas containing B-Raf mutations; however, their overall usefulness has been limited by both intrinsic and acquired drug resistance. In particular, issues related to the dimerisation of the Raf kinases can impact the efficacy of these compounds and are a primary cause of drug resistance. Here, we will review the importance of Raf dimerisation in cell signalling as well as its effects on Raf inhibitor therapy, and we will present the new strategies that are being pursued to overcome the 'Raf Dimer Dilemma'.

Intrinsic protein disorder in oncogenic KRAS signaling.

How Ras, and in particular its most abundant oncogenic isoform K-Ras4B, is activated and signals in proliferating cells, poses some of the most challenging questions in cancer cell biology. In this paper, we ask how intrinsically disordered regions in K-Ras4B and its effectors help promote proliferative signaling. Conformational disorder allows spanning long distances, supports hinge motions, promotes anchoring in membranes, permits segments to fulfil multiple roles, and broadly is crucial for activation mechanisms and intensified oncogenic signaling. Here, we provide an overview illustrating some of the key mechanisms through which conformational disorder can promote oncogenesis, with K-Ras4B signaling serving as an example. We discuss (1) GTP-bound KRas4B activation through membrane attachment; (2) how farnesylation and palmitoylation can promote isoform functional specificity; (3) calmodulin binding and PI3K activation; (4) how Ras activates its RASSF5 cofactor, thereby stimulating signaling of the Hippo pathway and repressing proliferation; and (5) how intrinsically disordered segments in Raf help its attachment to the membrane and activation. Collectively, we provide the first inclusive review of the roles of intrinsic protein disorder in oncogenic Ras-driven signaling. We believe that a broad picture helps to grasp and formulate key mechanisms in Ras cancer biology and assists in therapeutic intervention.

Advanced Glycation End Products (AGE) Potently Induce Autophagy through Activation of RAF Protein Kinase and Nuclear Factor κB (NF-κB).

Advanced glycation end products (AGE) accumulate in diabetic patients and aging people because of high amounts of three- or four-carbon sugars derived from glucose, thereby causing multiple consequences, including inflammation, apoptosis, obesity, and age-related disorders. It is important to understand the mechanism of AGE-mediated signaling leading to the activation of autophagy (self-eating) that might result in obesity. We detected AGE as one of the potent inducers of autophagy compared with doxorubicin and TNF. AGE-mediated autophagy is inhibited by suppression of PI3K and potentiated by the autophagosome maturation blocker bafilomycin. It increases autophagy in different cell types, and that correlates with the expression of its receptor, receptor for AGE. LC3B, the marker for autophagosomes, is shown to increase upon AGE stimulation. AGE-mediated autophagy is partially suppressed by inhibitor of NF-κB, PKC, or ERK alone and significantly in combination. AGE increases sterol regulatory element binding protein activity, which leads to an increase in lipogenesis. Although AGE-mediated lipogenesis is affected by autophagy inhibitors, AGE-mediated autophagy is not influenced by lipogenesis inhibitors, suggesting that the turnover of lipid droplets overcomes the autophagic clearance. For the first time, we provide data showing that AGE induces several cell signaling cascades, like NF-κB, PKC, ERK, and MAPK, that are involved in autophagy and simultaneously help with the accumulation of lipid droplets that are not cleared effectively by autophagy, therefore causing obesity.

Potent Pan-Raf and Receptor Tyrosine Kinase Inhibitors Based on a Cyclopropyl Formamide Fragment Overcome Resistance.

While selective BRafV600E inhibitors have been proven effective clinically, acquired resistance rapidly develops through reactivation of the mitogen-activated protein kinase (MAPK) pathway. Simultaneous targeting of multiple nodes in the pathway offers the prospect of enhanced efficacy as well as reduced potential for acquired resistance. Replacement pyridine group of Y-1 by a cyclopropyl formamide group afforded I-01 as a novel multitargeted kinase inhibitor template. I-01 displayed enzyme potency against Pan-Raf and receptor tyrosine kinases (RTKs). Based on the binding mode of I-01, analogues I-02-I-18 were designed and synthesized. The most promising compound I-16 potently inhibits all subtypes of Rafs with IC50 values of 3.49 (BRafV600E), 8.86 (ARaf), 5.78 (BRafWT), and 1.65 nM (CRaf), respectively. I-16 not only exhibit comparable antiproliferative activities with positive control compounds against HepG2, SW579, MV4-11, and COLO205 cell lines, but also suppress the proliferation of melanoma SK-MEL-2 harboring overexpressed BRafWT with IC50 values of 0.93 µM. The Western blot results for the ERK inhibition in human melanoma SK-MEL-2 cell lines show that I-16 inhibits the proliferation of SK-MEL-2 cell lines without paradoxical activation of ERK, which support the hypothesis that the inhibition of Pan-Raf and RTKs might be a tractable strategy to overcome the resistance of melanoma induced by the therapy with the current selective BRafV600E inhibitors.

Tyrosine phosphorylation of RAS by ABL allosterically enhances effector binding.

RAS proteins are signal transduction gatekeepers that mediate cell growth, survival, and differentiation through interactions with multiple effector proteins. The RAS effector RAS- and RAB-interacting protein 1 (RIN1) activates its own downstream effectors, the small GTPase RAB5 and the tyrosine kinase Abelson tyrosine-protein kinase (ABL), to modulate endocytosis and cytoskeleton remodeling. To identify ABL substrates downstream of RAS-to-RIN1 signaling, we examined human HEK293T cells overexpressing components of this pathway. Proteomic analysis revealed several novel phosphotyrosine peptides, including Harvey rat sarcoma oncogene (HRAS)-pTyr(137). Here we report that ABL phosphorylates tyrosine 137 of H-, K-, and NRAS. Increased RIN1 levels enhanced HRAS-Tyr(137) phosphorylation by nearly 5-fold, suggesting that RAS-stimulated RIN1 can drive ABL-mediated RAS modification in a feedback circuit. Tyr(137) is well conserved among RAS orthologs and is part of a transprotein H-bond network. Crystal structures of HRAS(Y137F) and HRAS(Y137E) revealed conformation changes radiating from the mutated residue. Although consistent with Tyr(137) participation in allosteric control of HRAS function, the mutations did not alter intrinsic GTP hydrolysis rates in vitro. HRAS-Tyr(137) phosphorylation enhanced HRAS signaling capacity in cells, however, as reflected by a 4-fold increase in the association of phosphorylated HRAS(G12V) with its effector protein RAF proto-oncogene serine/threonine protein kinase 1 (RAF1). These data suggest that RAS phosphorylation at Tyr(137) allosterically alters protein conformation and effector binding, providing a mechanism for effector-initiated modulation of RAS signaling.

RAF inhibitors transactivate RAF dimers and ERK signalling in cells with wild-type BRAF.

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.