Unexpected Off-Targets and Paradoxical Pathway Activation by

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Unexpected Off-Targets and Paradoxical Pathway Activation by Kinase Inhibitors Oliver Hantschel* Swiss Institute for Experimental Cancer Research (ISREC), School of Life Sciences, École Polytechnique Fédérale de Lausanne (EPFL), CH-1015 Lausanne, Switzerland ABSTRACT: Protein kinase inhibitors are an increasingly important class of targeted anticancer therapeutics. More than two dozen new drugs of this class have entered routine clinical use over the past decade. This review article focuses on how the development of methods to study the kinomeand proteome-wide selectivity of kinase inhibitors, in conjunction with advances in the structural understanding of kinase inhibitor binding modes, has resulted in a better appreciation of the mechanism of action of clinical kinase inhibitors. I provide examples of how this has led to the discovery of unexpected off-target effects, intriguing cases in which kinase inhibitors may cause pathway activation, and new mechanisms responsible for resistance to kinase inhibitors. Finally, I illustrate that although certain kinase targets may be pharmacologically easily tractable, a better understanding of the regulation and biology of the targets is required to generate drugs that are efficacious in cancer patients.

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in cancer and other diseases remain untargeted, and the biology of a large portion of the kinome remains poorly studied.7,8 Kinase Inhibitor Resistance. A common lesson learned is that although most of these drugs have been in clinical use for only the past 5 years or less, drug resistance has developed quickly. Several molecular mechanisms of resistance have been identified, among which point mutations in the ATP-binding pocket are a universal common mechanism.9,10 In particular, mutations of the gatekeeper residue responsible for critical interactions with most of the approved kinase inhibitors were first observed in BCR-ABL (T315I mutation) and EGFR (T790M mutation) in patients treated with imatinib and gefitinib or erlotinib,11,12 but also in patients treated with various inhibitors targeting KIT, ALK, PDGFR, RET, FLT3, and BRAF.2 To cope with this phenomenon, drugs with a different binding mode were developed: these drugs target kinases bearing the resistance-inducing point mutations. The prime examples are the BCR-ABL inhibitors nilotinib, dasatinib, and bosutinib, all of which inhibit kinases with point mutations that result in imatinib resistance but not those with the T315I mutation. In addition, ponatinib also efficiently inhibits BCRABL T315I in CML patients.13−17 Other recent examples include ceritinib, which was approved for ALK-positive nonsmall cell lung cancer (NSCLC) that is resistant to crizotinib,18 and the combination of the BRAF inhibitor dabrafenib with trametinib, which inhibits the downstream MEK kinases and has been shown to limit the development of

he U.S. Food and Drug Administration (FDA) has approved 24 additional kinase inhibitors since the approval of the BCR-ABL kinase inhibitor imatinib in 2001, and several hundred kinase inhibitors have entered clinical trials (Table 1). The three FDA-approved allosteric mTOR kinase inhibitors (sirolimus, everolimus, and temsirolimus), all of which are rapamycin analogues, are not discussed here, because of their distinct chemical structure and mechanism of action. There are excellent reviews of these compounds elsewhere.1 Kinase inhibitors are mainly used for the treatment of hematological cancers and solid tumors but also for the treatment of rheumatoid arthritis and idiopathic pulmonary fibrosis (Table 1).2 Together with humanized monoclonal antibodies, many of which target the extracellular domain of receptor tyrosine kinase receptors or their ligands, kinase inhibitors now constitute a major fraction of all newly approved drugs.3,4 The 25 kinase inhibitors in clinical use all bind to part of the ATP binding site and have to overcome competition by the high cellular ATP concentrations. These drugs have a limited number of primary targets, which are predominantly tyrosine kinases: BCR-ABL, EGF-, and VEGF-receptor family members are targeted by five drugs each and therefore constitute the three largest subgroups. In addition, various receptor tyrosine kinases (KIT, PDGFR, ALK, MET, and RET), cytoplasmic tyrosine kinases (SRC, BTK, and JAK), and the serine/threonine kinases BRAF and MEK1, are the main targets of the approved kinase inhibitors (Table 1).2 The selectivity of these inhibitors ranges from potently inhibiting only a few kinases to inhibiting several dozen kinases.5,6 Collectively, drugs that target fewer than 20 kinases are currently in clinical use. However, the majority of the more than 200 kinases of the kinome that are known to be involved © XXXX American Chemical Society

Special Issue: New Frontiers in Kinases Received: October 30, 2014 Accepted: December 19, 2014

A

DOI: 10.1021/cb500886n ACS Chem. Biol. XXXX, XXX, XXX−XXX

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ACS Chemical Biology Table 1. List of FDA-Approved Kinase Inhibitors name

trade name

year of FDA approval

imatinib

Gleevec

2001

gefitinib erlotinib sorafenib

Iressa Tarceva Nexavar

2003 2004 2005

dasatinib sunitinib

Sprycel Sutent

2006 2006

lapatinib nilotinib crizotinib ruxolitinib vandetanib

Tykerb Tasigna Xalkori Jakafi Caprelsa

2007 2007 2011 2011 2011

vemurafenib axitinib bosutinib cabozantinib pazopanib

Zelboraf Inlyta Bosulif Cometriq Votrient

2011 2012 2012 2012 2012

ponatinib regorafenib

Iclusig Stivarga

2012 2012

tofacitinib afatinib dabrafenib ibrutinib trametinib ceritinib nintedanib

Xeljanz Gilotrif Tafinlar Imbruvica Mekinist Zykadia Ofev

2012 2013 2013 2013 2013 2014 2014

clinically important target(s) BCR-ABL, KIT, PDGFR EGFR EGFR VEGFR, PDGFR, RAF BCR-ABL PDGFR, KIT, VEGFR EGFR, ERBB2 BCR-ABL ALK JAK2 RET, VEGFR, EGFR BRAF VEGFR BCR-ABL RET, MET VEGFR, FGFR, PDGFR BCR-ABL VEGFR, KIT, PDGFR JAK3 EGFR ALK BTK MEK ALK VEGFR, FGFR, PDGFR

approved disease indications

PDB entry for structure of drug-kinase complex

chronic myeloid leukemia, B-cell acute lymphoblastic leukemia, gastrointestinal stromal tumor, mastocytosis nonsmall cell lung cancer nonsmall cell lung cancer, pancreatic cancer renal cell carcinoma, hepatocellular carcinoma

1IEP, 1OPJ

chronic myeloid leukemia, B-cell acute lymphoblastic leukemia renal cell carcinoma, gastrointestinal stromal tumor, pancreatic cancer breast cancer chronic myeloid leukemia ALK-positive nonsmall cell lung cancer myelofibrosis thyroid cancer

2GQG 4AGD 3BBT 3CS9 2XP2 not available 2IVU

melanoma renal cell carcinoma chronic myeloid leukemia thyroid cancer renal cell carcinoma, sarcomas

3OG7 4AGC, 4AG8 3UE4 not available not available

chronic myeloid leukemia, B-cell acute lymphoblastic leukemia colorectal carcinoma, gastrointestinal stromal tumor

3OXZ, 3OY3 not available

rheumatoid arthritis nonsmall cell lung cancer melanoma mantle cell lymphoma, chronic lymphoid leukemia melanoma ALK-positive nonsmall cell lung cancer after crizotinib resistance idiopathic pulmonary fibrosis

3LXK 4G5J not available not available not available 4MKC 3C7Q

resistance in melanoma patients.19 Several in vitro and cellular models were developed to retrospectively model and predict resistance mechanisms.20,21 Kinases As Drug Targets. The 518 human protein kinases were once considered undruggable owing to the size of this family of enzymes and the highly conserved nature of the ATPbinding site.22 Early pharmacological kinase inhibitors reached high nanomolar potencies but lacked selectivity.23 The important and often essential roles of kinases in the developing organism and in normal homeostatic signaling raised further concerns that the inhibition of protein kinases, even if better selectivity could be achieved, might have detrimental side effects in humans. It is perhaps fortunate for the field that the first approved inhibitor, imatinib, displayed remarkable selectivity24,25 and is very well tolerated by most patients.26,27 By contrast, some of its successors showed a much more problematic side-effect profile, possibly because of their lack of selectivity.28,29 The crystal structures of various kinase domains with hundreds of different kinase-inhibiting compounds have improved our understanding of the molecular mechanism-ofaction of kinase inhibitors over the past 15 years.30,31 In parallel, methods to assess kinase inhibitor selectivity needed to be developed. This central information is critical to the understanding of kinase inhibitor action. Importantly, off-target inhibition may cause not only acute but also chronic side effects that may affect quality-of-life and limit therapeutic success. Particularly carefully studied in this respect are the BCR-ABL

2ITY 1M17 4ASD

inhibitors because the chronic nature of the disease, the high overall survival and the year-long exposure of patients to these drugs require particular care with respect to drug tolerance. Still, this is a difficult field because the pharmacokinetics, pharmacodynamics, and other factors also need to be taken into account and strongly influence the occurrence of certain side effects.28 Therefore, only a few observed side effects could be attributed to the inhibition of specific off-target kinases. Assaying the Selectivity of Kinase Inhibitors. Kinase inhibitors were initially tested on rather small panels consisting of only a few kinases,26 which did not allow firm conclusions regarding the global selectivity of these drugs. Over the past decade, several in vitro screening methods have been developed and are reviewed in more detail elsewhere.32−34 In general, one can distinguish methods that assess the physical association, that is, binding of an inhibitor to a large collection of kinases, from methods that assess inhibition of kinase activity. A powerful and unbiased but simple chemical proteomics technique to measure drug selectivity is drug-affinity purification, in which an immobilized drug derivative is incubated with cell lysates and the bound proteins are identified by mass spectrometry (MS).25,35 One complication is that chemical modification of the drug-of-interest is often required to allow its covalent capture, which may result in a reduction in the activity of the drug derivative. To limit the issues associated with compound modification, a large fraction of the expressed kinome can be enriched with beads covered with broad selectivity kinase inhibitors (Kinobeads technology), after B


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Figure 1. Unexpected off targets of kinase inhibitors. Structures of kinase inhibitors (magenta sticks) bound their on-target kinase domains (golden cartoons) are shown in the left panels. The right panels show these kinase inhibitors (magenta sticks) bound to the unexpected off-targets (blue cartoons). The chemical structures of the inhibitors are shown in the middle panels. The following PDB entries or models were used for the structural representations: (a) JAK2-fedratinib; molecular dynamics model; see ref 49, BRD4-fedratinib; 4PS5; (b) ALK-(R)-crizotinib, 2XP2; MTH1-(S)-crizotinib, 4C9X; (c) ABL-imatinib, 1OPJ; NQO2-imatinib, 3FW1.

covers >80% of the human kinome. The kinases are expressed as bacteriophage fusion particles and are one-by-one bound to broad-specificity inhibitor-coated microbeads. Competition with the test compound is used to derive apparent binding affinity constants and compound selectivity maps. A landmark publication in 2005 analyzed the selectivity profile of 20 kinase inhibitors for 119 kinases.36 Owing to the simplicity and quantitative nature of this assay, it has been applied for further large-scale comparative studies of kinase inhibitor specificity.

which competition with the (unmodified) drug of interest is performed followed by MS analysis.24 Although this approach does not require chemical modification of the drug-of-interest, some targets might be missed, as they are not captured by the Kinobeads. In parallel, a second affinity-based kinase inhibitor profiling technology has been developed (KINOMEscan technology). Currently, this technology uses a library of up to 450 kinase domains and disease-relevant kinase mutations and therefore C


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hematological and solid tumors because it epigenetically modulates the expression of important cancer drivers, such as the Myc, Aurora-B, and Bcl2 family members.44,45 Recently developed inhibitors of BRD4, such as JQ1 and I-BET151, are entering clinical development.46 A recent paper reported inhibition of the interaction of the first bromodomain of BRD4 with an acetyl-lysine histone peptide by several kinase inhibitors and an independent study reported cocrystallization of several kinase inhibitors with BRD4 (Figure 1A).47,48 Kinase inhibitors in late-stage clinical development, including the PLK inhibitor BI-2536, the RSK inhibitor BI-D1870, and the JAK2 inhibitor fedratinib (former names: TG-101348 and SAR302503), were of particular interest. All kinase inhibitors inhibited the BRD4 binding with nanomolar IC50 values.47 Fedratinib inhibits a broader spectrum of kinases than the FDA-approved JAK2 inhibitor, ruxolitinib, and will be discussed in more detail in the last section of this article.49 These results showed that the dual effects of inhibition of kinase signaling pathways and epigenetic control of gene expression could be combined in a single agent (Figure 1A). This demonstrates a new paradigm for rational polypharmacological targeting. Analysis of the inhibitor-binding modes using cocrystal structures combined with structure−activity relationship studies to identify design features may enable the rational design of dual kinase-bromodomain inhibitors in the future. Crizotinib Enantiomers Inhibit Different Enzymes. Crizotinib is a potent inhibitor of several receptor tyrosine kinases, including ALK and MET50 and was FDA-approved in 2011 for the treatment of patients with locally advanced or metastatic ALK-positive NSCLC. Drug-affinity purification coupled to MS demonstrated that the (R)-enantiomer of crizotinib is a kinase inhibitor, whereas the major target of (S)crizotinib is the MTH1 enzyme (Figure 1B). MTH1 is involved in nucleotide pool homeostasis and cleaves oxidized nucleotides, thereby preventing the generation of point mutations that would be caused by the incorporation of such nucleotides into DNA. Inhibition of MTH1 by (S)-crizotinib impaired the growth of KRAS-driven tumor cell lines by inducing DNA single-strand breaks and impairing tumor growth in a colon carcinoma xenograft model.51 In a parallel study, MTH1 was validated as an anticancer target in vivo, and potent and selective inhibitors were developed.52 It is important to note that many preparations that are sold as (R)-crizotinib contain varying degrees of (S)-crizotinib. This leads to significantly different IC50 values for ALK kinase inhibition depending on the batch.51 This underlines the necessity of careful quality control of the kinase inhibitors that can be obtained commercially but also nicely illustrates how the use of an enantiomer of a kinase inhibitor, often used as a negative control for chemical proteomics experiments, can lead to important discoveries. Oxidoreductase NQO2 Inhibited by BCR-ABL Inhibitors. Chemical proteomic profiling has demonstrated interactions of the BCR-ABL kinase inhibitors imatinib and nilotinib but not dasatinib, with NQO2 (NAD(P)H:quinone oxidoreductase 2).24,25 NQO2 is a flavoprotein involved in xenobiotic metabolism, including the reduction of quinones to hydrochinones.53 Imatinib and nilotinib inhibited the NQO2 oxidoreductase activity at concentrations similar to those at which their therapeutic effects affect their kinase targets.54 A cocrystal structure of the complex of NQO2 with imatinib showed that imatinib acted as a competitive inhibitor that occupies the substrate binding site of NQO2 (Figure 1C).

Specifically, in 2008, the inhibitory profiles of the 38 mostutilized kinase inhibitors, including all FDA-approved kinase inhibitors, were tested against 317 kinases,37 and a study in 2011 assayed 72 inhibitors against a panel of 442 kinases.6 As an alternative to these affinity-based methods, large-scale kinase assay panels to monitor the inhibition of kinase activity have been developed. These are also available on a large scale, illustrated by a study that profiled 300 kinases for inhibition by 178 kinase inhibitors.38 Various vendors offer in vitro kinase assays panels on a scale of ∼80% of the kinome, currently including ∼400 distinct kinases. Parallel kinase assays are initially performed in the presence of 1−3 fixed concentrations of a kinase inhibitor, which offers a global view on inhibitor selectivity. Collectively, among the FDA-approved kinase inhibitors only a few, such as lapatinib and imatinib, are highly selective, whereas most drugs inhibit between 10 and 100 kinases offtarget with varying potency.6,17 A comparative analysis of both affinity-based and activity-based inhibitor-selectivity panels showed a good overlap but also demonstrated the limitations and complementarity of these methods.39,40 Although the readout of the large-scale kinase panels is more direct because kinase inhibition rather than compound binding is assessed, it also has a few limitations. Selectivity in in vitro assays at low ATP concentration can be different than in cells.41 Allosteric inhibitors may evade detection because the full-length kinases are not often used. Furthermore, regulatory mechanisms may not be recapitulated due to the expression system or the lack of certain post-translational modifications. The coverage is not kinome-wide and important off-target kinases that are difficult to express and purify may evade detection. An important limitation also arises from other (nonkinase) targets of the kinase inhibitors not being included in the panels and therefore not being identified. These limitations emphasize the importance of the chemical proteomics techniques with their unbiased manner of identifying drug targets using MS. Still, the presence of kinase drug targets in multiprotein complexes and with regulatory subunits that may modulate binding affinities make the identification of targets more complex. Besides measuring binding/inhibition of a given drug to its on- and off-targets with the methods described above, measurements of the dissociative half-life or drug-target residence time has emerged as an important parameter to assess drug selectivity and evaluate possible adverse events in vivo.42 Drug-target residence time measurements take binding kinetics of drugs into account and therefore provide timedependent information on drug selectivity. Importantly, a slow off-rate may sustain on-target inhibition over time, while an offtarget is rapidly reactivated if its off-rate is much faster than for the on-target-drug complex, despite identical binding affinities of the drug to both on- and off-target. Still, recent evidence emphasized the complexity of this issue by showing that drugtarget residence time is only a useful parameter if pharmacokinetic elimination is faster than binding dissociation.43 Three examples of unexpected off-target proteins of kinase inhibitors illustrate the necessity of comprehensively understanding the selectivity of kinase inhibitors. Bromodomain Interactions Inhibited by Kinase Inhibitors. BRD4 belongs to a group of epigenetic transcriptional regulators that contain bromodomains, prototypic protein−protein interaction domains that bind the acetylated lysine residues in histone tails. BRD4 is an emerging target in D


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conformations (Figure 2A) has also been observed for other inhibitors. A recent paper reported a selective and potent Erk1/ 2 inhibitor, SCH772984, with a unique on-target binding mode that was dependent on specific conformational changes in the Gly-rich loop and resulted in the formation of a new binding pocket. This inhibitor demonstrated two strongly altered modes of binding to the atypical kinase haspin, which has only weak sequence homology to ERK1/2 and to JNK1, which is closely related to ERK1/2 (Figure 2B).56 Similarly, the pyrrolo-pyrimidine and pyrazole ring planes of ruxolitinib are rotated 180 deg once bound to JAK2,49 as opposed to their orientation when bound to Src (Figure 2C).57 Furthermore, the cyclopentane ring of ruxolitinib points toward the N-lobe and the nitrile group to the C-lobe once bound to JAK2, whereas the opposite orientation was observed in the Src structure (Figure 2C).49,57 It is important to note that the alternative inhibitor conformations when bound to off-target proteins are often of lower affinity than those for the conformations that bind to their intended target kinases. However, conformational plasticity is not a general phenomenon. For example, dasatinib binds with a virtually identical conformation to its targets Abl, Lyn, Src, Btk, Bmx, EphA4, and p38α (Figure 2D). All targets share a threonine residue in the gatekeeper position, which has been established as a major determinant of dasatinib selectivity.58 Furthermore, with the exception of EphA4 and p38α, they have a high sequence homology, which may contribute to the very similar binding conformation. On the General Utility of Knowing Off-Targets. The identification of kinase inhibitor off-targets has contributed to a better understanding of drug action but has also proven very useful for structural biology and as a means of identifying possible secondary medical uses of approved drugs. The selectivity profiling of the BCR-ABL inhibitor bosutinib identified CaMKII and HER3 among several other off-target proteins.40 This helped to obtain high-resolution crystal structures of these two protein kinases in complex with bosutinib.59,60 After its FDA-approval, the BCR-ABL inhibitor dasatinib was shown to potently inhibit Btk as a major target.58 The parallel identification of Btk, as a functionally critical kinase that is activated in subtypes of diffuse large B-cell lymphoma (DLBCL), led to the demonstration that growth of DLBCL cells could be inhibited by dasatinib.61 This proof-of-concept evidence was an important step for the development of more specific Btk inhibitors, among which ibrutinib was FDAapproved for mantle cell lymphoma and CLL in 2013.62,63 Ibrutinb is an irreversible Btk inhibitor that forms a covalent bond with a cysteine residue in the active site of Btk. This residue is common to only 11 other human kinases, which explains ibrutinib’s high selectivity.64 Together with its prolonged pharmacokinetic profile and outstanding potency, this observation has renewed interest in irreversible kinase inhibitors.65 It is now well-documented that drug-bound kinases can also have unexpected effects on cell signaling, despite inhibition of the targeted kinase’s own enzymatic activity. This will be discussed in the next two sections. Active Conformations of Inhibitor-Bound Kinases. Based on their binding mode, type 1 and type 2 kinase inhibitors can be distinguished.31 Type 1 inhibitors are characterized by locking the kinase domain in its active conformation. In contrast, type 2 inhibitors induce an inactive conformation, produced by inducing the ’DFG-out’ conformation, in which the Asp-residue of the DFG (Asp-Phe-Gly)-

Interestingly, the ability of imatinib, and to a lesser extent of nilotinib, to undergo cis−trans rotational isomerization was responsible for this effect. NQO2 bound the more compact cis conformation of the drug, whereas the trans rotamer is responsible for potent inhibition of imatinib’s kinase targets (Figure 1C).54 The cis rotamer of imatinib was also observed when bound to the Syk tyrosine kinase (see below and Figure 2A).55 Despite the potent inhibition of NQO2 activity by imatinib and nilotinib in vitro and in cells, the physiological consequences of this off-target inhibition remain unclear. Conformational Plasticity of Kinase Inhibitors. The conformational plasticity of imatinib, which binds to NQO2 and Syk, as opposed to BCR-ABL in dramatically different

Figure 2. Conformations of kinase inhibitors binding to different kinases. For all panels, the kinase domains of kinase inhibitor−kinase complex structures were superimposed. For graphical clarity only the conformations of the kinase inhibitors are shown in stick representation. The following PDB entries were used for the structural representations. (a) Imatinib-ABL, 1OPJ; imatinib-SYK, 1XBB; (b) SCH772984-ERK1/2, 4QTA and 4QTB; SCH772984-haspin, 4QTC; SCH772984-JNK1, 4QTD; (c) Ruxolitinib-JAK2, molecular dynamics model, see ref 49; ruxolitinib-SRC, 4U5J; (d) Dasatinib-ABL, 2GQG; dasatinib-SRC, 3G5D; dasatinib-LYN, 2ZVA; dasatinib-BTK, 3K54; dasatinib-BMX, 3SXR; dasatinib-p38α, 3LFA; dasatinib-EPHA4, 2Y6O. E


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Figure 3. Paradoxical pathway activation by RAF kinase inhibitors. (a) The oncogenic BRAF V600E mutant kinase drives activation of the MEKERK kinase pathway (left), which is inhibited in the presence of BRAF inhibitors, such as vemurafenib (right). (b) Oncognic NRAS mutations (indicated by the red star) drive MEK-ERK activation in BRAF wild-type (wt) cells (left). Paradoxically, pathway activation is sustained in the presence of vemurafenib by heterodimerization of BRAF with CRAF, despite inhibition of BRAF activity (right). (c) Likewise, a kinase-dead mutant of BRAF is also able to sustain MEK-ERK pathway activation.

ylation by phosphatases.71 Similarly, binding of BCR-ABL kinase inhibitors dramatically affects the intramolecular protein−protein interactions of BCR-ABL72 and strongly remodels the BCR-ABL protein−protein interaction network.73 Paradoxical Pathway Activation by RAF Kinase Inhibitors. RAF kinases activate MEK-ERK kinase signaling, a central pathway involved in cell proliferation. Recurrent mutations in BRAF were identified in the majority of malignant melanomas and at a lower frequency in other human cancers.74 Common mutations of Val-600 causes RAS-independent constitutive activation of BRAF and leads to the development of melanomas in mouse models.75,76 These findings triggered the development of BRAF kinase inhibitors. Vemurafenib (PLX4032) was FDA-approved for the treatment of melanoma and is the first approved serine-/threonine-kinase inhibitor.77 Despite impressive clinical responses in melanoma patients, acquired drug resistance can occur as the result of activating point mutations in the downstream MEK1 kinase or other mechanisms.78 An intriguing mechanism for the intrinsic resistance was discovered when BRAF inhibitors with different mechanisms-of-action were assayed for MAPK pathway inhibition in both NRAS wild-type (wt) and mutant cell lines that also expressed either wt or mutant BRAF.79 Paradoxically, BRAF-selective type 1 inhibitors, such as vemurafenib, activated MEK and ERK kinases but only in cell lines with the mutated form of NRAS or those expressing the wt BRAF (Figure 3A, B). Furthermore, even kinase-inactive BRAF mutants showed this paradoxical signaling behavior and drove melanomagenesis in mice (Figure 3C).79 Two independent studies verified these surprising results with other BRAF inhibitors and larger panels of cell lines.80,81 The underlying molecular mechanisms are complex and have not yet been entirely clarified, but a key factor involves the asymmetric homodimerization of BRAF or heterodimerization with ARAF, CRAF, or KSR, which is a

sequence motif at the N-terminal end of the activation loop is rotated out of the active site.66 The activation loop can adopt different conformations, thus regulating the access of the substrate to the catalytic cleft.67 In addition to the DFG-motif, the overall conformation of the activation loop is primarily in an open conformation in the type 1 inhibitor complexes, whereas different closed conformations are observed for the type 2 inhibitor complexes. Opening and closing of the activation loop is often regulated by inter- or intramolecular protein−protein interactions impinging on the kinase domain. These interactions include the binding of cyclins to cyclindependent kinase or the interaction of the SH2 domains of ABL or FES with their kinase domains.67−69 An open conformation of the activation loop enables substrate binding and is often stabilized by the phosphorylation of one or several residues. Activation loop phosphorylation can be measured in cell lysates and intact cells using phospho-specific antibodies: this has become a standard method to monitor kinase activation. Upon type 1 inhibitor binding, the activation loop is found in an open conformation accessible for autophosphorylation and/ or phosphorylation by an upstream kinase. In many cases, this leads to the paradoxical effect of strong activation loop phosphorylation despite inhibition of the catalytic activity of the kinase by the inhibitor. Under these circumstances, the activation loop phosphorylation can no longer be used as a suitable biomarker.56,69,70 Another example is that certain highly specific Akt1 inhibitors cause paradoxical hyperphosphorylation not only of Thr-308, the activation loop phosphorylation site in Akt1, but also of Ser-473 in Akt1’s Cterminal hydrophobic motif.71 Strikingly, the inhibitor-bound Akt1 showed increased membrane binding mediated by its own PH domain. Akt might consequently be more susceptible to phosphorylation by kinases or less susceptible to dephosphorF


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that was discussed in detail above, fedratinib inhibited BCRABL and several other kinases.49,102 The phase III clinical trial of fedratinib was recently suspended because of the occurrence of Wernicke’s encephalopathy. This disease is known to be induced by thiamine deficiency. In cell lines, it was demonstrated that fedratinib potently inhibits the human thiamine transporter (hTHTR2). Inhibition of thiamine uptake was found to be unique to fedratinib and was not observed with the other JAK2 inhibitors.103 In addition to these pharmacological setbacks, a series of recent papers has demonstrated the essential role of JAK2 signaling for normal hematopoiesis. Conditional deletion of JAK2 in adult mice leads to their rapid death associated with hematopoietic stem cell exhaustion that results in a lethal pancytopenia.104−106 Under certain experimental settings, JAK2 deletion even accelerated progression of other oncoproteindriven leukemias in mouse models.104 These findings underline the important role of JAK2 in normal homeostasis and indicated that the therapeutic window of JAK2 inhibitors may be very narrow. Some of the above problems may be circumvented by the development of more mutation-selective drugs. This in turn will require a better understanding of JAK2 kinase regulation. Importantly, the V617F mutation and other oncogenic JAK2 mutations are located on the pseudokinase domain (termed JH2 domain). The mechanism by which these mutations activate the JAK2 tyrosine kinase domain (termed JH1 domain) was unknown, but some recent findings shed some light on this. First, it was shown that the JH2 (pseudokinase) domain displayed dual-specificity kinase activity and autophosphorylated a tyrosine and a serine residue. The phosphorylation of these sites is important in maintaining the (JH1) kinase in the autoinhibited state.107 Second, the crystal structure of the JH2 domain was solved, and the oncogenic mutations, including the V617F mutation, were shown to substantially reduce JH2 activity that result in lower autophosphorylation and higher JH1 activity.108 Third, the relationship of JH1 and JH2 domains was revealed by the crystal structure and a molecular dynamics based model of the Tyk2 and JAK2 JH2-JH1 domain units.109,110 The JH2 and JH1 domains form a large intramolecular interface. Oncogenic mutations map in proximity to this interface, further supporting the role of the JH2 domain as an autoinhibitory domain.109,110 These novel insights into JAK2 regulation will certainly offer novel opportunities to overcome some of the limitations of the current JAK2-targeting strategies, including drugs that are more selective for oncogenic mutations and opportunities to target pathway components up- or downstream of JAK kinases. Outlook. With the current approval rate and the number of kinase inhibitors in clinical trials, up to 50 kinase inhibitors might be in clinical use by the end of this decade for the treatment of cancer and possibly for autoimmune, neurodegenerative, and infectious diseases. Because acquired resistance has been detected with all of the approved kinase inhibitors, finding suitable combinations of kinase inhibitors, as well as combinations with conventional cancer therapies, is a major challenge that is already a field of intense research.111 Technical platforms to test hundreds of drug combinations on hundreds of cell lines or cells from cancer patients exist. Combined with genomics, transcriptomics, and proteomics, these techniques will provide a solid experimental basis for systems pharmacology to model and predict combinations with particular efficacy that can then be tested in animal models and clinical trials.112−114 Furthermore,

pseudokinase and Ras-MAPK scaffolding protein (Figure 3B).82−85 Furthermore, BRAF-MEK1 complexes were observed in KRAS mutant cells with the wt but not the mutant form of BRAF.86 According to these models, vemurafenib binding to one of the protomers of RAF kinase dimers allosterically drives the activation of the other RAF kinase protomer. In general, competitive inhibitors that stabilize a rigid closed conformation of the kinase domain were found to be effective inducers of RAF dimerization.87 RAF dimerization is also necessary for the physiological RAS-induced RAF activation and for the action of signaling of oncogenic BRAF mutants with lower or impaired kinase activity. In contrast, dimerization is not required for the function of high-activity BRAF mutants, such as the V600E mutant protein.86,88 To overcome the limitations associated with BRAF inhibitor resistance, inhibitors of the downstream MEK kinases were developed. The MEK1/2 inhibitor trametinib gained approval in May 2013 for use as a single-agent therapy for V600E metastatic melanoma and 10 months later was approved for use in combination with vemurafenib.89 Similarly to BRAF homo- and heterodimerization, the formation of asymmetric dimers of kinase domains has been recognized as a key allosteric mechanism that regulates the activation of the EGF-receptor family of kinases.90,91 By analogy to the paradoxical BRAF activation, the broadselectivity kinase inhibitor bosutinib40 enhances the ability of HER3 to act as an allosteric activator of EGFR by increasing the affinity of the HER3-EGFR heterodimer.60 The described mechanisms in which inhibitor-bound kinase domains gain to the capacity to allosterically activate kinases in the context of a dimer are reminiscent of the mechanisms by which certain pseudokinases activate catalytically competent kinases. One example is the pseudokinase STRAD, which activates the LKB1 kinase in a complex with the MO25 scaffold.92 There are increasing numbers of other examples.93 JAK2: A Difficult Target Only at Second Glance. The last example illustrates further challenges in targeting kinases: JAK kinases (JAK1/2/3 and Tyk2) are activated downstream of a variety of cytokine receptors and result in the activation of STAT transcription factors.94 In 2005, mutations in JAK2, most commonly the V617F mutation, which constitutively activated the tyrosine kinase activity of JAK2, were found in patients with myeloproliferative neoplasms.95,96 Expression of JAK2-V617F in mouse models faithfully recapitulated the hallmarks of the human disease.97 Therefore, it was expected that inhibition of JAK2 signaling would lead to strong clinical responses in patients with these diseases. Ruxolitinib became the first-inclass JAK2 inhibitor and was FDA-approved in 2011.98 Interestingly, although the drug led to reductions in splenomegaly and improvement in disease-related symptoms and quality of life, patients that did or did not have JAK2 mutations responded equally well.99,100 Longer-term follow-up has led to doubts regarding any disease-modifying role of ruxolitinib and thereby triggered debates on the clinical utility of JAK2 inhibitors.101 To better understand the mechanism of action of the JAK2 kinase inhibitors, the selectivity and binding mode of ruxolitinib as well as fedratinib (formerly named SAR302503 or TG101348), the second-in-class JAK2 inhibitor, were determined. Whereas both inhibitors are type 1 binders, they occupy overlapping but distinct sites in the catalytic cleft of JAK2.49 Fedratinib showed a much broader kinase inhibition profile than ruxolitinib.49,6 In addition to the cross-inhibition of BRD4 G


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P., and Zuercher, W. J. (2013) A public−private partnership to unlock the untargeted kinome. Nat. Chem. Biol. 9, 3−6. (9) Levitzki, A. (2013) Tyrosine kinase inhibitors: Views of selectivity, sensitivity, and clinical performance. Annu. Rev. Pharmacol. Toxicol. 53, 161−185. (10) Wheeler, D. L., Dunn, E. F., and Harari, P. M. (2010) Understanding resistance to EGFR inhibitors-impact on future treatment strategies. Nat. Rev. Clin. Oncol. 7, 493−507. (11) Gorre, M. E., Mohammed, M., Ellwood, K., Hsu, N., Paquette, R., Rao, P. N., and Sawyers, C. L. (2001) Clinical resistance to STI-571 cancer therapy caused by BCR-ABL gene mutation or amplification. Science 293, 876−880. (12) Pao, W., Miller, V. A., Politi, K. A., Riely, G. J., Somwar, R., Zakowski, M. F., Kris, M. G., and Varmus, H. (2005) Acquired resistance of lung adenocarcinomas to gefitinib or erlotinib is associated with a second mutation in the EGFR kinase domain. PLoS Med. 2, e73. (13) Weisberg, E., Manley, P. W., Breitenstein, W., Bruggen, J., Cowan-Jacob, S. W., Ray, A., Huntly, B., Fabbro, D., Fendrich, G., Hall-Meyers, E., Kung, A. L., Mestan, J., Daley, G. Q., Callahan, L., Catley, L., Cavazza, C., Mohammed, A., Neuberg, D., Wright, R. D., Gilliland, D. G., and Griffin, J. D. (2005) Characterization of AMN107, a selective inhibitor of native and mutant Bcr-Abl. Cancer Cell 7, 129−141. (14) Shah, N. P., Tran, C., Lee, F. Y., Chen, P., Norris, D., and Sawyers, C. L. (2004) Overriding imatinib resistance with a novel ABL kinase inhibitor. Science 305, 399−401. (15) Golas, J. M., Arndt, K., Etienne, C., Lucas, J., Nardin, D., Gibbons, J., Frost, P., Ye, F., Boschelli, D. H., and Boschelli, F. (2003) SKI-606, a 4-anilino-3-quinolinecarbonitrile dual inhibitor of Src and Abl kinases, is a potent antiproliferative agent against chronic myelogenous leukemia cells in culture and causes regression of K562 xenografts in nude mice. Cancer Res. 63, 375−381. (16) O’Hare, T., Shakespeare, W. C., Zhu, X., Eide, C. A., Rivera, V. M., Wang, F., Adrian, L. T., Zhou, T., Huang, W.-S., Xu, Q., Metcalf, C. A., Tyner, J. W., Loriaux, M. M., Corbin, A. S., Wardwell, S., Ning, Y., Keats, J. A., Wang, Y., Sundaramoorthi, R., Thomas, M., Zhou, D., Snodgrass, J., Commodore, L., Sawyer, T. K., Dalgarno, D. C., Deininger, M. W. N., Druker, B. J., and Clackson, T. (2009) AP24534, a Pan-BCR-ABL inhibitor for chronic myeloid leukemia, potently inhibits the T315I mutant and overcomes mutation-based resistance. Cancer Cell 16, 401−412. (17) Hantschel, O., Grebien, F., and Superti-Furga, G. (2012) The growing arsenal of ATP-competitive and allosteric inhibitors of BCRABL. Cancer Res. 72, 4890−4895. (18) Shaw, A. T., Kim, D. W., Mehra, R., Tan, D. S., Felip, E., Chow, L. Q., Camidge, D. R., Vansteenkiste, J., Sharma, S., De Pas, T., Riely, G. J., Solomon, B. J., Wolf, J., Thomas, M., Schuler, M., Liu, G., Santoro, A., Lau, Y. Y., Goldwasser, M., Boral, A. L., and Engelman, J. A. (2014) Ceritinib in ALK-rearranged non-small-cell lung cancer. N. Engl. J. Med. 370, 1189−1197. (19) Flaherty, K. T., Infante, J. R., Daud, A., Gonzalez, R., Kefford, R. F., Sosman, J., Hamid, O., Schuchter, L., Cebon, J., Ibrahim, N., Kudchadkar, R., Burris, H. A., 3rd, Falchook, G., Algazi, A., Lewis, K., Long, G. V., Puzanov, I., Lebowitz, P., Singh, A., Little, S., Sun, P., Allred, A., Ouellet, D., Kim, K. B., Patel, K., and Weber, J. (2012) Combined BRAF and MEK inhibition in melanoma with BRAF V600 mutations. N. Engl. J. Med. 367, 1694−1703. (20) Azam, M., Latek, R. R., and Daley, G. Q. (2003) Mechanisms of autoinhibition and STI-571/Imatinib resistance revealed by mutagenesis of BCR-ABL. Cell 112, 831−843. (21) Kasap, C., Elemento, O., and Kapoor, T. M. (2014) DrugTargetSeqR: A genomics- and CRISPR-Cas9-based method to analyze drug targets. Nat. Chem. Biol. 10, 626−628. (22) Evan, G. (2008) The future of cancer therapy: an interview with Gerard Evan. Dis Model Mech 1, 90−93. (23) Gazit, A., Yaish, P., Gilon, C., and Levitzki, A. (1989) Tyrphostins I: Synthesis and biological activity of protein tyrosine kinase inhibitors. J. Med. Chem. 32, 2344−2352.

the large and growing data sets of the kinome-wide selectivity of hundreds of compounds will identify further pharmacological rules for kinase selectivity and allow better predictions to prevent inhibition of certain off-target proteins.47,115,116 Academic research will further profit from new wellcharacterized and potent kinase inhibitors that will be developed by the pharmaceutical industry. Those can be used as important tools to provide insight into signaling pathways, the plasticity of cancer cell networks, cancer stem cell biology, and tumor cell−microenvironment interactions. It is noteworthy that the use of low selectivity kinase inhibitors has often lead to wrong interpretations on the role of a kinase in a particular signaling pathway or cellular process.102 On the molecular level, there is already a good set of methods available to predict the binding mode of novel kinase inhibitors using molecular dynamics simulations.117,118 The increasing understanding of inhibitor binding modes, inhibitor selectivity, and mechanisms of inhibitor resistance has brought us closer to the goal of producing rationally designed drugs, as elegantly illustrated by the example of the discovery of nilotinib and ponatinib.13,16 Finally, it can be expected that additional unexpected off-target proteins and unconventional modes-ofaction of kinase inhibitors that go beyond to what is described in this review will be discovered and enrich our insight into kinase-inhibitor protein interactions.

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AUTHOR INFORMATION

Corresponding Author

*Tel: +41 21 69 37251. Fax: +41 21 69 37210. E-mail: oliver. hantschel@epfl.ch. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS

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REFERENCES

I thank the ISREC Foundation, Anna Fuller Fund, Swiss National Science Foundation (grant No. 31003A_140913), Swiss Cancer League (grant No. KLS-3132-02-2013) and National Centre of Competence in Research (NCCR) Chemical Biology for financial support of my laboratory.

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